Acetylcholinesterase-derived peptides and uses thereof
The invention relates to a cell growth and/or differentiation regulatory peptide comprising a sequence of about 9 to about 150 amino acids derived from acetylcholinesterase amino acid sequence, preferably from the C-terminal region of acetylcholinesterase. The invention also relates to pharmaceutical compositions comprising the peptides, particularly for use in promoting survival of stem cells, promoting differentiation of stem cells, promoting growth of stem cells and/or promoting the growth-enhancing effect of a growth factor on stem cells, alone, or in combination with other growth factors. Of particular interest is the use of the peptides in the treatment of thrombocytopenia, post-irradiation conditions, post-chemotherapy conditions, or conditions following massive blood loss and promotion of neural progenitors in use for cell therapies aimed at restoring neural functions in diseased individuals. Further, the invention relates to antibodies against the peptides, inter alia for diagnostic use, for example, the diagnosis of stress-induced male infertility. The invention also relates to in vitro and in vivo methods for screening of drugs that affect the central nervous system, and are potential modulators of interactions between the “readthrough” form of acetylcholinesterase, AChE-R, the intracellular receptor RACK1 and the kinase PKC.
This application is a continuation-in-part (CIP) of U.S. patent application Ser. No. 09/998,042, filed Nov. 30, 2001, which is a continuation-in-part of PCT international application No. PCT/IL00/00311, filed May 31, 2000, which claims priority of Israeli Patent Application No. 130224, filed May 31, 1999 and Israeli Patent Application No. 131707, filed Sep. 2, 1999, incorporated herein by reference.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This work was supported by the US Army Medical Research and Material Command DAMD 17-99-9547 (July 1999-August 2004) and the Defense Advance Research Project Agency DARPA N66001-01-C-8015 (May 2001-May 2004). The US Government has certain rights in this invention.FIELD OF THE INVENTION
The invention is directed to the field of stem cell survival and expansion. Specifically, the invention is directed at the stem cell survival and expansion effects of peptides derived from acetylcholinesterase. In addition, the invention relates to a system for screening of nervous system drugs that are directed to central nervous system conditions or disorders. More specifically, the invention relates to the screening of modulators of the AChE-R-PKCβII-RACK1 complex.BACKGROUND OF THE INVENTION
Stress insults evoke a plethora of responses in the organism, affecting the functioning of various systems.
In the hematopoietic system, stress insults are associated with rapid and significant changes in blood cell composition. For example, following massive blood loss, or after surgery, the hematopoietic system responds within hours, by an elevation of the white blood cell and platelet counts. However, the mechanisms responsible for initiating this adjustment are not fully understood. Glucocorticoid hormones, known to be elevated under stress, play a leading role in the adaptive reaction of the bone marrow in response to stress. Glucocorticoid hormones induce absolute increases in all hematopoietic lineages, especially myeloid cells. This involves a cascade of events culminating in changes in the proliferation, differentiation and apoptotic events characteristic of each of the hematopoietic cell lineages [Lansdorp (1995) Exp. Hematol. 23, 187-91]. Also, significant changes occur under glucocorticoid hormones in the levels of hematopoietic growth factors controlling the proliferation of stem cells from which blood cells develop.
Hematopoietic stem cells (HSCs) are pluripotent, in that they give rise to all blood cell lineages. These cells migrate during ontogeny to settle in the bone marrow as a permanent self-renewing source of blood cells. Under normal conditions the vast majority of HSCs are non-dividing, but under conditions of development or stress they can undergo clonal expansion and self-renewal [Keller and Snodgrass (1990) J. Exp. Med. 171, 1407-18]. A large number of cytokines and growth factors, such as stem cell factor (SCF), thrombopoietin (TPO), and FLT-3 ligand, are thought to mediate the proliferative capacity of HSCs, through specific receptors, c-kit, c-mpl and flt3/flk-2, respectively. Alone, their capacity to stimulate proliferation is limited. For example, SCF can maintain survival for a few days in vitro, but not the self-renewal of HSCs (Li and Johnson, Blood 84, 408-14, 1994). However, when used in combination, these growth factors acquire a potent co-stimulatory effect. The early phase of adaptation of the hematopoietic system to stress (first 24 hr), requires coordinator(s), such as leu-enkephalin, which modulate the effects of growth factors on stem cells. However, leu-enkephalin is present in the circulation only immediately following the stress insult, whereas the modulation of hematopoiesis continues long after that phase. Therefore, additional long-acting modulators remain to be identified.
The enzyme acetylcholinesterase (AChE) is expressed in brain tissue, but also in most, if not all, of the mammalian hematopoietic cell lineages. AChE is expressed in many parts of the vertebrate embryo, with a developmentally regulated pattern in specific cell types and tissues during the embryonic and adult stages. AChE diversity is noted in several pathological states, such as Alzheimer's disease, where AChE activity was shown to decrease, not only in the primary site of the disease, the brain, but also in the hematopoietic system.
It has now surprisingly been found that the C-terminal peptides of AChE-S and AChE-R have independent biological activities. Specifically, it has been found that these peptides promote stem cell survival. It has also been found that these peptides promote stem cell expansion, when used in combination with growth factors. Further, it has been found that such peptides are capable of augmenting hematopoiesis in vivo.
In the central nervous system (CNS), physiological stress induces rapid and robust signaling processes in mammalian brain neurons. These processes are known to suppress long term potentiation (LTP) [Vereker, E. et al. (2000) J Neurosci, 20, 6811-9], augment long term depression (LTD) [Xu, L. et al. (1997) Nature, 387, 497-500], and induce release of synaptic vesicles, potentiating neurotransmission [Stevens, C. F. and Sullivan, J. M. (1998) Neuron, 21, 885-93]. At the long term, stress-induced signaling attenuates the stress response, enabling the organism to be less excessively affected by a stressful event. This induces neuronal dendrite branching [Sousa, N. et al. (2000) Neuroscience, 97, 253-66] and synapse re-organization [McEwen, B. S. (1999) Ann Rev Neurosci, 22, 105-22]. However, the molecular pathway(s) leading from short to long term processes and which enable the adjustment to stressful stimuli, are not yet known.
Ample information suggests the involvement of specific protein kinases in at least some of these stress-induced processes. The enzymatic activity of certain subtypes of protein kinase C (PKC) [Coussens, L. et al. (1986) Science, 233, 859-66] was shown to be subject to changes (i.e. biochemical activation, membrane translocation) under physiological [Hu, G. Y. et al. (1987) Nature, 328, 426-9], biochemical [Macek, T. A. et al. (1998) J Neurosci, 18, 6138-46] and cytoarchitectural [Tint, I. S. et al. (1992) Proc Natl Acad Sci USA, 89, 8160-4] responses at the cellular and organismal levels. A relevant mediator of the stress-related changes in PKC activities is likely to be largely absent from brain neurons under normal conditions, but should be induced rapidly and for long periods following stress insults.
A relevant putative mediator of the stress-related changes in PKC activities should be intracellular in its location and capable of activating or translocating active PKC within neuronal perikarya. The “readthrough” acetylcholinesterase variant AChE-R is a promising candidate for this role [Soreq, H. and Seidman, S. (2001) Nat Rev Neurosci, 2, 294-302]. Brain AChE-R is exceedingly rare in the adult, non-stressed brain. Various stress insults induce AChE-R overproduction through alternative splicing, creating a different C-terminal domain from that of synaptic AChE (AChE-S). AChE-R levels rise rapidly under acute psychological stress [Kaufer, D. et al. (1998) Nature, 393, 373-7] or chemical neurotoxication [Shapira, M. et al. (2000) Hum Mol Genet, 9, 1273-81] and stay elevated for over two weeks following head injury [Shohami, E. et al. (2000) J Mol Med, 78, 228-36]. Being a secretory protein, AChE-R fulfills the extracellular function of reducing the stress-induced acetylcholine levels. In parallel, it accumulates in neuronal cell bodies [Sternfeld, M. et al. (2000) Proc Natl Acad Sci USA, 97, 8647-52], where acetylcholine hydrolysis is unlikely. Transgenic mice overexpressing neuronal AChE-R, but not the normally abundant synaptic variant AChE-S, display reduced levels of stress-associated neuropathologies [Sternfeld et al. (2000) id ibid.]. This suggests distinct stress-related function(s) for the AChE-R protein. Intriguingly, the unique C-terminal domain of AChE-R does not participate in acetylcholine hydrolysis for which the core domain, common to all of the AChE variants is sufficient [Duval, N. et al. (1992) J Cell Biol, 118, 641-53].
Using a yeast two-hybrid screen, the inventors discovered that the C-terminal domain of AChE-R forms a tight complex with RACK1 [PCT/IL00/00311]. Interestingly, the inventors have shown that PKCβII is also part of this complex (
In search for the marker of the transition between short and long term processes following stress stimuli, the inventors have demonstrated that interaction with AChE-R activates PKCβII and facilitates its translocation into densely packed neuronal clusters (
In view of these unprecedented results, it this an object of this invention to provide a method for screening of nervous system drugs that modulate the trimeric complex AChE-R/PKC/RACK1 interactions.SUMMARY OF THE INVENTION
This invention is directed at a cell growth and/or differentiation regulatory peptide comprising a sequence of about 9 to about 150 amino acids derived from Acetylcholinesterase amino acid sequence. The said sequence preferably contains a region predicted to be rich in beta-pleated sheet structure and turns. Also preferably, the said sequence contains a predicted amphipathic helix structure. In another embodiment of the peptide of the invention, the said sequence is derived from the C-terminal region of acetylcholinesterase.
The said sequence is preferably derived from the readthrough or synaptic variant of acetylcholinesterase, preferably from the mature form thereof. The said sequence is preferably about 20 to about 70 amino acids in length. More preferably, the said sequence is SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ ID: No.8. Still more preferably, the said peptide is SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ ID: No.8.
A peptide of the invention which is a cyclic peptide is also considered to be within the scope of the invention.
The peptide of the invention is preferably synthetic and preferably comprises the amino acid sequence of SEQ ID: No. 1, SEQ ID: No. 2, SEQ ID: No. 3, SEQ ID: No.7 or SEQ ID: No.8. More preferably, the peptide has the amino acid sequence denoted by SEQ ID: No. 1, 2, 3, 7 or 8. The peptide is preferably linear and synthetic.
In one embodiment, the invention provides a peptide capable of promoting cell survival and/or differentiation and comprising the amino acid sequence denoted by SEQ ID: No. 1, SEQ ID: No. 2 or SEQ ID: No. 3, and functional analogues and derivatives thereof.
In another embodiment, the invention provides a peptide of the invention which is a hematopoietic stem cell growth and/or differentiation regulatory peptide. Preferably, said peptide is capable of promoting stem cell survival and/or myeloid and megakaryocytic differentiation and comprises the amino acid sequence denoted by SEQ ID: No. 1, SEQ ID: No. 2 or SEQ ID: No. 3, or functional analogues and derivatives thereof. Said peptide may be either linear or cyclic, and is preferably synthetic.
In a still further embodiment, the invention provides a peptide thereof for use in ex vivo or in vivo expansion of hematopoietic stem cells and of neural progenitors.
The invention also provides a peptide thereof for use in ex vivo or in vivo promotion of megakaryocytic differentiation of hematopoietic stem cells.
The invention also relates to a pharmaceutical composition comprising a synthetic peptide of any one of the preceding embodiments of the invention.
The pharmaceutical composition preferably comprises a synthetic peptide comprising the amino acid sequence of SEQ ID: No. 1, SEQ ID: No. 2, or SEQ ID: No. 3. More preferably, the pharmaceutical composition comprises a synthetic peptide having the amino acid sequence denoted by SEQ ID: No. 1, SEQ ID: No. 2, or SEQ ID: No. 3. The peptide contained within the pharmaceutical composition may be either linear or cyclic, and is preferably synthetic.
The invention provides a pharmaceutical composition according to the invention, for regulating hematopoietic stem cell growth, promoting survival of stem cells, differentiation of stem cells, promoting growth of stem cells, and/or promoting the growth-enhancing effect a growth factor on stem cells. The growth factor is preferably GM-CSF, SCF, TPO, EGF or bFGF.
In a preferred embodiment of the invention, the stem cells are embryonic stem cells, nerve stem cells, epithelial stem cells, mesenchymal stem cells, or hematopoietic stem cells.
The invention provides a pharmaceutical composition comprising a peptide according to the invention for the treatment of thrombocytopenia, post-irradiation condition, post-chemotherapy condition, or condition following massive blood loss. The invention also provides said pharmaceutical composition for use in inducing synthesis of acetylcholinesterase mRNA and/or promoting the formation of hematon bodies.
In a further embodiment, the invention provides an antibody directed against a peptide of the invention. The antibody is preferably provided for use in diagnosing elevated glucocorticoid level; bone marrow stress, abnormality, dysfunction, or stressed condition, or of increased platelet count or of brain infarct risk in a mammal, or stress-induced male infertility. The antibody is preferably directed at a peptide comprising SEQ ID: No. 1, SEQ ID: No. 2, or SEQ ID: No. 3. More preferably, the antibody is directed at a peptide which is selected from SEQ ID: No. 1, SEQ ID: No. 2, or SEQ ID: No. 3.
In yet another embodiment, the invention provides a method for the diagnosis of elevated glucocorticoid level; bone marrow stress, abnormality, dysfunction or stressed condition, or of increased platelet count or of brain infarct risk in a mammal, comprising obtaining a sample from said mammal, contacting said sample with an antibody of the invention, removing unbound antibody, and detecting the extent of reaction between said antibody and acetylcholinesterase or a fragment thereof present in said sample. The said sample is preferably a serum or bone marrow sample.
In a specifically preferred embodiment, the invention provides a method for the diagnosis of stress-induced male infertility comprising obtaining a sperm cell sample from said male, smearing and drying said sperm cells, contacting said cells with an antibody of the invention, removing unbound antibody and detecting the extent of reaction between said antibody and acetylcholinesterase or a fragment thereof present in said sperm cell.
In another specifically preferred embodiment the invention provides a method for the diagnosis of stress-induced male infertility further comprising the step of determining the pattern of expression of acetylcholinesterase or a fragment thereof in said sperm cell.
In yet another embodiment, the invention provides a method for the diagnosis of stress induced male infertility for use in fertility counseling.
Another aspect of the invention relates to a method of screening for a candidate drug or substance (hereinafter the “test drug”) that affects the central nervous system, wherein said test drug is a modulator of the interaction between AChE-R/RACK1/PKC, and which screening method comprises the steps of:
- a. providing a reaction mixture comprising the AChE-R variant of AChE or any functional fragment thereof, the cognate receptor for activated kinase C (RACK1) and the protein kinase C βII (PKCβII);
- b. contacting said mixture with a test drug under suitable conditions for said interaction; and
- c. determining the effect of the test drug on an end-point indication, wherein said effect is indicative of modulation of said interaction by the test drug.
In this screening method, the said modulator inhibits or enhances the interaction between AChE-R/RACK1/PKC.
The reaction mixture may be a cell mixture or a cell-free mixture, and may optionally further comprise solutions, buffers and compounds which provide suitable conditions for interaction between AChE-R/RACK1/PKC and the detection of an end-point indication for said interaction. The modification of said end-point indicates modulation of the interaction between AChE-R/RACK1/PKC by said test drug.
In one embodiment of this screening method, the reaction mixture is a cell-free mixture.
In this embodiment, the screening method comprises the steps of:
- a. providing a cell-free mixture comprising the AChE-R variant of AChE or any functional fragment thereof, RACK1 and PKCβII;
- b. contacting said mixture with the test drug under conditions suitable for an in vitro interaction; and
- c. determining the effect of the test drug on co-precipitation of PKCβII and RACK1 with the AChE-R or fragment thereof as an end-point indication, whereby the absence or increase of said co-precipitation indicates modulation of formation of a complex between AChE-R/RACK1/PKC by the test drug.
The cell-free mixture may comprise any one of AChE-R variant of AChE or any functional fragment thereof, RACK1 and PKCβII, which are provided as purified recombinant proteins or as a cell lysate of cells expressing said proteins.
The said AChE-R variant of AChE may be a fusion protein comprising AChE-R or functional fragment thereof and any one of GST (Glutathion-S-Transferase) and GFP (Green Fluorescent Protein).
In another embodiment of this screening method, the said reaction mixture is a cell mixture, particularly a transfected cell culture, and more particularly a transfected mammalian cell culture.
In this embodiment, the screening method comprises the steps of:
- a. providing a transfected cell culture expressing the AChE-R variant of AChE or functional fragment thereof, the cognate receptor for activated kinase C (RACK1) and the PKCβII;
- b. contacting said transfected cell culture with the test drug;
- c. detecting the interaction between AChE-R/RACK1/PKC in the presence of the test drug by searching for an end-point indication, whereby inhibition of said end-point indicates inhibition of complex formation between AChE-R/RACK1/PKC by said test drug.
The transfected cell to be used may be transfected by:
- a. an expression vector comprising a nucleotide sequence coding for the AChE-R variant of AChE or a functional fragment thereof;
- b. optionally, constructs comprising a nucleic acid sequence coding for any one of the cognate receptor for activated kinase C (RACK1) and the PKCβII.
The end-point indication may be the sub-cellular translocation of catalytically active PKCβII, which can be detected by a visually detectable signal.
Alternatively, the end-point indication may be co-precipitation of PKCβII and RACK1 with the AChE-R or functional fragment thereof leading to a detectable signal, whereby modification of said detectable signal in the presence of the test drug indicates modulation of the formation of a complex between AChE-R/RACK1/PKC by said test drug.
In a yet further embodiment of the screening method, the modulator of the interaction between AChE-R/RACK1/PKC also modulates the expression of RACK1 and/or PKCβII.
A further aspect of the present invention is a method for the in vivo screening of candidate drugs that affect the central nervous system, wherein said drug is a modulator of an interaction between AChE-R/RACK1/PKC, and which screening method comprises the steps of:
- a. providing an AChE-R transgenic animal;
- b. administering the test drug to said animal;
- c. sacrificing the animal and dissecting its brain to give samples for preparation of brain extracts or for immunohistochemistry;
- d. detecting the expression of RACK1 or PKCβII in said brain samples; and
- e. determining the effect of the test drug on an end-point indication, wherein said effect is indicative of the in vivo modulation of said interaction by the test drug;
The end-point indication of this in vivo screening method is the expression of RACK1 and PKCβII in the brain, which can be detected by a visually detectable signal.
Preferred transgenic animals are Xenopus and mammals, such as mice, cows, goats, pigs and sheep. Most preferably, the transgenic animal is an AChE-R transgenic mouse, which has been described in Sternfeld et al. (2000) [Sternfeld et al. (2000) id ibid.] herein incorporated by reference.
In one embodiment of the in vivo screening method, the RACK1 or PKCβII expression can be detected by means that can detect RNA or protein.
In one specific embodiment, the RNA detection is performed by means appropriate for RNA detection, said means selected from the group consisting of RT-PCR, Northern Blot, in situ hybridization, RNAse protection and S1 nuclease analysis.
In another specific embodiment, the protein detection is performed by means appropriate for protein detection, said means selected from the group consisting of Western Blot and immunohistochemistry.
The evaluation and screening methods of the invention are suitable for assessing and screening for any test drug, e.g. test drugs selected from protein based, carbohydrates based, lipid based, nucleic acid based, natural organic based, synthetically derived organic based, antibody based and metal based substances. In preferred embodiments, the protein or antibody based substance may products of combinatorial libraries.
The drugs to be evaluated by the methods of the invention can be any candidate or known drugs, e.g. drugs for the treatment of anxiety conditions, post-traumatic stress, Alzheimer's disease, muscle malfunctioning, neurodegenerative disorders, damage resulting from exposure to xenobiotics, panic, neuromuscular disorders, Parkinson's disease, Huntington's chorea, muscle fatigue, multiple chemical sensitivity, autism, multiple sclerosis and Sjogren's disease.
In yet a further aspect, the invention provides for a method for the treatment of stress-associated conditions or disorders, for a subject in need of such treatment, said method comprising:
- a. providing a composition comprising as active ingredient a modulator of an interaction between AChE-R/RACK1/PKC;
- b. administering a therapeutic effective amount of said composition to said subject;
wherein said modulator is selected by the drug screening methods provided by the invention.
Lastly, the invention provides a kit for the diagnosis of stress-induced male infertility, comprising:
- (a) an antibody specific for the AChE-R variant;
- (b) means for collecting a sperm sample;
- (c) slides for smearing the sperm sample;
- (d) means for detecting the antibody;
- (e) negative control slides.
All the above and other characteristics and advantages of the invention will be further understood through the following illustrative and non-limitative description of preferred embodiments thereof, with reference to the appended drawings.BRIEF DESCRIPTION OF THE FIGURES
Arrow indicates transcription start site, triangles, conserved transcription factor binding motifs, boxes, exons 1, 5, 6 and intron 4′ as indicated, white boxes, exons 2-4 and introns 2′-3- as indicated; GRE half site (hs).
Flow cytometry of the recovered cells, using anti CD34 and anti CD45 antibodies.
Cytochemical staining of enriched CD34+ cells for AChE catalytic activity in the presence of inhibitors for BuChE and AChE. The inhibitors are iso-OMPA (ISO) and BW284C51 (BW). Nuclear staining (right) of the different forms of AChE was performed in the presence of different concentrations of Hydrocortisone, the AChEmRNA signal is shown as % of normal (N).
Upper panel shows cytochemical staining of enriched CD34+ cells for AChE catalytic activity in the presence of different concentrations of Hydrocortisone. The lower panel shows in situ hybridization for detection of the different forms of AChE under different concentrations of Hydrocortisone.
Cells were fixed and permeabilized with Fix and Perm (Caltag, Calif., US) and stained with monoclonal antibodies to CD34 conjugated to pycoerythrin indicated as FL-2 and with highly specific rabbit anti-ARP antibodies followed by anti rabbit antibodies conjugated to fluorescein isothiocyanate, indicated as Fl-1. CD34+ cells were gated and 40,000 cells were analyzed for ARP expression indicated as percentage of positive cells.
Transformed bone marrow endothelial cells were incubated in a serum free medium (SFM) with 2nM of ARP, with or without endothelial growth factors (bFGF 20 ng/ml and EGF 10 ng/ml), for 48 hrs. Cell proliferation was determined by the level of BrdU incorporation measured by 5-Bromo-2′-deoxy-Uridine Labeling and Detection Kit III. Each column shows the average value of four wells +/− the standard error of the mean.
Left—In situ hybridization of representative CD34+ cells treated with ARP. Right—Average labeling densities of AChE mRNA splice variants (S, E, R) versus ARP concentration (top), as pixels (pi)×103.
FIGS. 7A-C: ARP induces stem cell proliferation as measured by BrdU incorporation.
FIGS. 8A-B: Redundant properties of ARP and SCF.
FIGS. 12A-C: ARP has short- and long-term hematological effects in vivo.
Schaffer collaterals-CA1 synaptic pathway on hippocampal slices from an injected mice with ARP (i.p. 0.1 mg/Kg body weight) or with P-BAN as control, were tested after LTP induction. The changes in the slope (sl) of the post synaptic field potential was followed for 3 hrs (indicated by Time=T in minutes).
Amino acid homology between RACK1 and the sequence established from the rat neonatal aorta ARP- two-hybrid positive clone (boxed), the synthetic peptides which inhibit PKC binding to RACK1 and are therefore homologous, at least in part, to the binding site of PKC to RACK1.
Abbreviations: Prot.=protease; Ovl.=overlay; Detec. Ab.=detection antibody; Ponc.=Ponceau; Hom.=homogenates; N-ter.=N-terminus; n.=none.
Homogenates from mouse hippocampus (hip) and cortex (crt) [composed of 3 stressed (str), 4-6 and 3 control (cont), 1-3 mice] were separated on a denaturing gel and analyzed by immunoblot with anti-RACK1 antibody. Jurkatt cells (Jc) homogenate was separated as well.
Top: CMV-based vector encoding pGARP, a GFP fusion protein with ARP1. Bottom: The drawing on the side represents the experimental concept.
- 1. Homogenates: Shown are immunolabeled RACK1 and PKCβII (but not ARP1) in non-transfected COS cell homogenates (−). In the presence of transfected GARP (+), COS cells show a band in the position correspondent to ARP.
- 2. Anti-GFP: Immunoprecipitation with anti-GFP antibodies precipitates PKCβII, ARP and RACK1 in pGARP transfected but not in non-transfected COS cells.
Abbreviations: Transfec.=transfection; Detec. Ab.=detection antibody
RACK1 and PKCβII co-immunoprecipitate with anti-AChE antibodies. The schematic on the left represents the experimental concept.
- 1. Homogenates: PKCβII and RACK1 are immunodetected in homogenates of COS cells, which do not express AChE, and PKCβII, RACK1 and AChE are detected in PC12 cell homogenates.
- 2. Anti-AChE N-terminus: Dissolved immunoprecipitation complexes created with antibodies to the N-terminus of AChE display no signals in COS cells, but are positive for all three partner proteins in PC12 cells, demonstrating AChE requirement for the creation of these complexes.
Abbreviations: Detec. Ab.=detection antibody; Hom.=homogenates; Nter.=N-terminus; Prot.G=protein G.
Shown are parietal cortex sections stained with cresyl violet or with anti-RACK1 or anti-AChE-R antibodies, in lower and higher magnifications. Note uneven labeling patterns of both proteins in the cytoplasm and proximal processes of individual pyramidal neurons in the parietal cortex and hippocampus CA1 (insets). Note RACK1 and AChE-R expression increases in layers 5 (arrows) of the parietal cortex under stress.
Abbreviations: CV=cresyl violet; N. Str.=no stress; Str.=stress.
Abbreviations: Cont.=control; Tg.=transgenic.
Abbreviations: Cont.=control; Mrg.=merge; Tg.=transgenic.DETAILED DESCRIPTION OF THE INVENTION
A number of terms as used herein are defined herein below
- AChE, Acetylcholinesterase;
- AChE-R, “readthrough” form of AChE
- AChE-S, “synaptic” form of AChE
- AS, antisense;
- AS-ODN, antisense oligodeoxynucleotide;
- ARP, acetylcholinesterase “readthrough” peptide;
- ASP, acetylcholinesterase “synaptic” peptide;
- BFU, burst-forming units;
- B, erythroid bursts;
- Blast or bl, blast cell (colonies);
- BuChE, Butyrylcholinesterase;
- CFU, colony-forming units;
- CSF, colony-stimulating factor;
- “derived from” acetylcholinesterase, the term “derived from”, when referring to acetylcholinesterase or to equivalent terms, in the context of this application is intended to mean amino acid sequence corresponding to amino acid sequence of acetylcholinesterase protein, or to predicted amino acid sequence of the open reading frame of an acetylcholinesterase splice variant mRNA. This includes sequences identical to acetylcholinesterase sequence, and sequences having one or more deletions, additions, and/or substitutions, preferably of conservative nature, i.e., changes that are not expected to change the overall structure of the peptide. However, any change that does not affect the function or activity of the peptide or that increases same is contemplated to be within the scope of the invention;
- EAC, early-acting cytokines, these are preferably IL-3, IL-6, Flt3, thrombopoietin, but may also comprise others such as soluble IL-6 receptor;
- GEMM, granulocyte-erythrocyte-macrophage-megakaryocyte (colonies);
- GM, granulocyte-macrophage (colonies);
- GST, glutathione-S-transferase;
- HSC, hematopoietic stem cells;
- Mix, mixed hematopoietic colonies;
- MK, Megakaryocyte (colonies);
- ODN, oligodeoxynucleotide;
- ORF, open reading frame;
- PKC, protein kinase C
- RACK1, receptor for the activated kinase C, member of the WD40 family protein
- RT, room temperature;
- UCB, umbilical chord blood;
- UTR, untranslated terminal region;
- WBC, white blood cells.
This invention relates to peptides derived from the open reading frame of the acetylcholinesterase mRNAs. The peptides of the invention are useful, inter alia, in upregulating AChE mRNA, enhancing growth and/or differentiation of stem cells. In addition, this invention relates to the trimeric complex AChE-R-PKCβII-RACK1, and to methods of screening substances that can affect the interactions between these three molecules both in vitro and in vivo.
A number of methods of the art of molecular biology are not detailed herein, as they are well known to the person of skill in the art. Such methods include site-directed mutagenesis, PCR cloning, expression of cDNAs, analysis of recombinant proteins or peptides, transformation of bacterial and yeast cells, transfection of mammalian cells, and the like. Textbooks describing such methods are e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology, by F. M. Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al. (eds.) 3rd ed. John Wiley & Sons; ISBN: 0471137812, 1995. These publications are incorporated herein in their entirety by reference. Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art. See e.g., Current Protocols in Immunology, Coligan et al. (eds), John Wiley & Sons. Inc., New York, N.Y.
AChE pre-mRNA undergoes alternative splicing, which generates, at the translational level, three variants of the AChE protein [Ben Aziz Aloya et al. (1993) Proc. Natl. Acad. Sci. USA 90, 2471-5]. The best known transcript is AChE-S mRNA, formed by splicing of exon 4 to exon 6, which yields the principal “synaptic” isoform, found in brain and muscle [Soreq et al. (1990) Proc. Natl. Acad. Sci. USA 87, 9688-92]. Translation of this mRNA, results in a C-terminal extension of the common core domain of 534 amino acid residues by a 40 residue peptide, containing the cysteine that is involved in dimerization. The second splicing option, which generates the erythrocytic transcript (AChE-E), is based on splicing of exon 4 to exon 5, which encodes a different 40 residue peptide at the C-terminus. This latter peptide is subsequently cleaved at residue 14 (number 557 from the N-terminus) and forms a glycophospholipid linkage that may be integrated into erythrocyte membranes [Kerem et al. (1993) J. Biol. Chem. 268, 180-184,] and anchor AChE to their surfaces. The third splicing option involves continuous transcription through pseudointron I4, to yield the E1-E2-E3-E4-I4-E5 mRNA transcript. This transcript has been detected in embryonic and tumor cells (AChE-R, the so called “readthrough” form), as well as in stressed brain. Translation of AChE-R mRNA results in a 26 residue hydrophilic C-terminal extension devoid of cysteine [Li et al. (1991) J. Biol. Chem. 266, 23083-90]. In human AChE-transgenic Xenopus tadpoles, this isoform is secreted largely as soluble monomers, unlike the AChE-S isoform which accumulates in synapses.
All three variants share the same 534 residue core domain. When recombinant AChE-R and AChE-S were produced in micro- injected Xenopus oocytes and subjected to immunoblot analysis, a certain degree of natural C-terminal truncation occurred which created fast-migrating bands, with a molecular weight similar to that of the core protein, i.e. without the C-terminus, in both preparations [Sternfeld et al. (1998a) J. Neurosci. 18, 1240-9].
It has now surprisingly been found that a peptide derived from Acetylcholinesterase has independent biological activities. The invention is directed to a peptide comprising sequence derived from the open reading frame of an acetylcholinesterase mRNA splice variant. In a preferred embodiment of the invention, the splice variant is AChE-R or AChE-S. The peptide of the invention comprises preferably from about 9 to about 150, preferably about 100 amino acids, of the AChE mRNA open reading frame. More preferably, the peptide of the invention is derived from said AChE mRNA open reading frame.
The peptide of the invention preferably comprises a major region predicted to be rich in turns and β-pleated sheets. Also preferably, the peptide of the invention comprises an amphipathic helix structure, preferably unilaterally hydrophobic, which is preferably of a length of about 10 to about 30, more preferably abut 17 amino acid residues. Further preferably, the peptide of the invention is of low immunogenicity. This may be tested by injecting the peptide to an experimental animal, in the presence of adjuvants as known in the art. A peptide of low immunogenicity will not elicit antibodies unless conjugated or otherwise modified. Further details of antibody generation are disclosed herein below, e.g., in the Experimental Procedures section.
In a preferred embodiment of the invention, the peptide comprises AChE sequence derived from the C-terminus thereof. The peptide sequence is preferably derived from between about 9 to about 150, preferably about 100 amino acids from the C-terminus of the AChE translation product. More preferably, the peptide sequence is derived from the C-terminus, preferably from between 9 to about 100 amino acids of the C-terminus, of the mature AChE protein.
Preferably, the peptide comprises amino acid sequence corresponding to AChE intron 4, exon 4, exon 5, and/or exon 6 sequences. More preferably, the peptide comprises intron 4 and exon 5 sequences, or exon 4 and exon 6 sequence, or exon 5 and exon 6 sequences. Thus, in a preferred embodiment of the invention, the peptide is derived from the AChE-R or AChE-S mRNA open reading frames. Preferably, the peptide is derived from the C-terminus of the protein coded by said open reading frames, and more preferably, the peptide is derived from the C-terminus of the mature protein coded by said AChE-R or AChE-S mRNAs. In a preferred embodiment of the invention, the peptide comprises about 50, more preferably about 30, and most preferably about 26 amino acids of the mature C-terminus of AChE-R or AChE-S encoded protein.
The peptide of the invention comprises more preferably between 20-70 amino acids of the C-terminus of a mature AChE protein, which is preferably the protein encoded by the AChE-R or AChE-S mRNAs.
In a more preferred embodiment, the peptide comprises the C-terminal 26 amino acids of the mature translation product of AChE-R mRNA, which is the amino acid sequence N-terminus
GMQGPAGSGWEEGSGSPPGVTPLFSP C-terminus, also denoted herein as SEQ ID: No. 1. A peptide having said 26 C-terminal amino acids is denoted herein as ARP, which is the most preferred embodiment of the invention. Further preferred embodiment is the peptide having 53 amino acids comprising of the 26 amino acids derived from the C-terminus of the protein encoded by the AChE-R mRNA and consecutive sequence into the core domain, also denoted herein as SEQ ID: No. 7.
In another preferred embodiment, the peptide of the invention comprises the C-terminus between about 30 and about 70 amino acids of the protein encoded by the AChE-S mRNA. More preferably, the peptide comprises about 40 amino acids of the mature C-terminus of the protein encoded by the AChE-S mRNA. The peptide having 40 amino acids derived from the C-terminus of the protein encoded by the AChE-S mRNA is denoted herein as ASP, or SEQ ID: No. 2. Another preferred embodiment is the non-helical region of SEQ ID: No. 2, i.e., the 27 C-terminal amino acids thereof, also denoted herein as SEQ ID: No. 3. Further preferred embodiment is the peptide having 67 amino acids comprising of the 40 amino acids derived from the C-terminus of the protein encoded by the AChE-S mRNA and consecutive sequence into the core domain, also denoted herein as SEQ ID: No. 8.
The peptide of the invention may comprise sequences derived from sources other than the above-described, so long as the function of said peptide is not substantially affected. The additional sequences are generally zero to 300 amino acids in length, and may comprise sequences derived from acetylcholinesterase, growth factors, enzymes, structural proteins, or non-natural sequences.
Acetylcholinesterase sequences that may be added may e.g., be taken from the above-described exons 4, 5, and/or 6, and/or intron 4 sequences, for the purposes of creating an internally linked peptide dimer. Dimers may possess enhanced activity [Corcoran et al. (1998) Eur. Cytokine Netw. 9, 255-62]. Dimer formation of the peptide of the invention may be achieved e.g., by inclusion of cysteines in the peptide sequence. This may be achieved either by choosing cysteines in the natural acetylcholinesterase-derived sequence of the peptide, or by adding cysteine residues to the peptide sequence, or by adding to the peptide sequence such amino acid sequences that contain cysteine residues, preferably amino acid sequences which are known to form dimers.
Dimerization may also be achieved by fusing the desired peptide sequence to an IgG backbone [Corcoran et al (1998) id ibid.].
The peptide of the invention may comprise also growth factor sequences. This may be useful e.g., when it is desired to enhance survival and growth of hematopoietic stem cells. The growth factors are in that case preferably selected from early-acting factors, such as IL3, IL6, TPO and/or FLT3. When it is desired to enhance expansion of hematopoietic stem cells, the amino acid sequence of growth factors such as GM-CSF or preferably SCF, may be added to the peptide sequence.
Alternatively, the peptide of the invention may be useful when it is desired to enhance the growth of endothelial cells. The growth factors are in that case preferably selected from endothelial growth factors, such as EGF and bFGF.
Where known, the active peptide of such growth factors may be used instead of the entire growth factor sequence. Of course, dimerization of the peptide of the invention may also enhance growth factor activity and/or binding affinity.
In some cases, it may be desired to promote differentiation of a cell derived from a hematopoietic source. Such differentiation may be especially desired if said cell has acquired growth-factor independent uncontrolled growth characteristics. The peptide of the invention may in such cases be fused to a toxin, which upon entering the cell, may exert cytostatic effects, in addition to the differentiation-promoting effect of the peptide of the invention. Fusions of peptides to toxins have been described in many researches and review articles, see e.g. [Pastan et al. (1991) Science 254:1173-7].
One of the effects of the peptide of the invention is targeted to bone marrow stem cells. Therefore, when it is desired to provide the peptide of the invention for use in treating a patient in need of such treatment, it may be desirable to target the peptide of the invention to the cells desired. This may be achieved by fusing the peptide to a sequence capable of binding to a bone marrow stem cell surface marker. One example for such a marker is the CD34 antigen. Consequently, the peptide of the invention may comprise CD34-ligand sequence. Alternatively, the peptide of the invention may comprise anti-CD34 single chain sequences. The preparation of antibodies and single-chain antibodies is well known in the art, see also below.
When it is desired to add amino acid sequences or domains to the peptide of the invention, it may be desired to separate the acetylcholinesterase-derived part of the sequences from the additional sequences by way of a linker sequence. Linker sequences may consist mainly of amino acids that do not provide spatial constraints, such as glycine and preferably alanine. An example for a flexible peptide linker sequence is described in e.g., [White et al. (1999) J. Immunol. 162, 2671-6].
The peptide of the invention comprises most preferably SEQ ID: No. 1 or 2. However, it is to be understood that the invention pertains to any peptide comprising sequence structurally similar to AChE sequence with substantially equal or greater activity. Changes in the structure of the peptide comprise one or more deletions, additions, or substitutions. The number of deletions or additions, which may occur at any point in the sequence, including within the acetylcholinesterase-derived sequence, will generally be less than 25%, preferably less than 10% of the total amino acid number. This figure does not include additions as described above, e.g., addition of sequences coding for growth factor sequences.
Preferred substitutions are changes that would not be expected to alter the secondary structure of the peptide, i.e., conservative changes. The following list shows amino acids that may be exchanged (left side) for the original amino acids (right side).
Amino acids can also be grouped according to their essential features, such as charge, size of the side chain, and the like. The following list shows groups of similar amino acids. Preferred substitutions would exchange an amino acid present in one group with an amino acid from the same group.
- 1. Small aliphatic, nonpolar: Ala, Ser, Thr Pro, Gly;
- 2. Polar negatively charged residues and their amides: Asp, Asn, Glu, Gln;
- 3. Polar positively charged residues: His, Arg, Lys;
- 4. Large aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys;
- 5. Large aromatic residues: Phe, Tyr, Trp.
Further comments on amino acid substitutions and protein structure may be found in additional references [Schulz et al. (1987) Principles of Protein Structure, Springer-Verlag, New York, N.Y.; Creighton, T. E. (1983) Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco].
The preferred conservative amino acid substitutions as detailed above are expected to substantially maintain or increase the function or activity of the peptide of the invention, as detailed herein below. Of course, any amino acid substitutions, additions, or deletions are considered to be within the scope of the invention where the resulting peptide is a peptide of the invention, i.e., a peptide which is substantially equal or superior in terms of function to the preferred peptide of the invention.
The peptide of the invention may be further modified to improve its function, affinity, or stability. For instance, cyclization may be used to impart greater stability and/or overall improved performance upon the peptide. A number of different cyclization methods have been developed, including side chain cyclization and backbone cyclization. These methods are well documented in the prior art [Yu et al. (1999) Bioorg. Med. Chem. 7, 161-75; Patel et al. (1999) J. Pept. Res. 53, 68-74; Valero et al. (1999) J. Pept. Res. 53, 56-67; Romanovskis et al. (1998) J. Pept. Res. 52, 356-74; Crozet et al. (1998) Mol. Divers. 3, 261-76; Rivier et al. (1998) J. Med. Chem. 41, 5012-9; Panzone et al. (1998) J. Antibiot. (Tokyo) 51, 872-9; Giblin et al. (1998) Proc. Natl. Acad. Sci. USA 95, 12814-8; Limal et al., (1998) J. Pept. Res. 52:121-9; U.S. Pat. No. (USP) 5,444,150]
A preferred method of cyclization involves stabilization of an amphipathic alpha-helix by using para-substituted amino acid derivatives of a benzene ring [Yu et al. (1999) id ibid.]. Another preferred method of cyclization is backbone cyclization [Reissmann et al. (1994-95) Biomed. Pept. Proteins Nucleic Acids 1:51-6, and references therein]. A relatively new method of cyclization which involves backbone-to side chain connections may also be used [Reissmann et al. (1994-95) id ibid.].
Other modifications as known in the art may be carried out. For instance, it may be desirable to link polyethyleneglycol (PEG) groups to the peptide. Such groups may impart enhanced stability upon the peptide. Another effect of these groups may be lowered immunogenicity. This feature of PEG-linked peptides may be particularly desirable when the peptide of the invention is to be used in vivo. Preparation of PEG-linked peptides has been described [Guerra et al. (1998) Pharm. Res. 15:1822-7].
The present invention provides AChE-derived peptides. A therapeutic or research-associated use of these tools may necessitate their introduction into or interaction with tissue cultured cells or cells of a living organism. For this purpose, or for assisting in the penetrance of such peptides into the brain through the blood-brain barrier, it is desired to improve membrane permeability of the peptides. The principle of derivatization with lipophilic structures may be used in creating peptides with enhanced membrane permeability. For instance, the sequence of a known membranotropic peptide may be added to the sequence of the peptide of the invention [Soukchareun et al. (1998) Bioconjug. Chem. 9, 466-75]. Further, the peptide may be derivatized by partly lipophilic structures such as Palmityl or Geraniol groups. For instance, lauroyl derivatives of peptides have been described by Muranishi et al., Pharm. Research 8, 649, 1991. Further modifications of peptides comprise the oxidation of methionine residues to thereby create sulfoxide groups [Zacharia et al. (1991) Eur. J. Pharmacol. 203, p. 353]. Zacharia and coworkers also describe peptide or derivatives wherein the relatively hydrophobic peptide bond is replaced by its ketomethylene isoester (COCH2). These and other modifications known to the person of skill in the art of protein and peptide chemistry enhance membrane permeability.
Another way of enhancing membrane permeability is the use of receptors, such as virus receptors, on cell surfaces in order to induce cellular uptake of the peptide or protein. This mechanism is used frequently by viruses, which bind specifically to certain cell surface molecules. Upon binding, the cell takes the virus up into its interior. The cell surface molecule is called a virus receptor. For instance, the integrin molecules CAR and AdV have been described as virus receptors for Adenovirus [Hemmi et al. (1998) Hum. Gene Ther. 9, 2363-73] and references therein. The CD4, GPR1, GPR15, and STRL33 molecules have been identified as receptors/co-receptors for HIV [Edinger et al. (1998) Virology 249, 367-78].
Thus, conjugating peptides, proteins or oligonucleotides to molecules that are known to bind to cell surface receptors will enhance membrane permeability of said peptides, proteins or oligonucleotides. Examples for suitable groups for forming conjugates are sugars, vitamins, hormones, cytokines, transferrin, asialoglycoprotein, and the like molecules. Low et al., U.S. Pat. No. 5,108,921, describe the use of these molecules for the purpose of enhancing membrane permeability of peptides, proteins and oligonucleotides, and the preparation of said conjugates. Of course, as one type of the cells targeted by the peptide of the invention are hematopoietic stem cells, it is advantageous to chose a cell surface protein that will occur preferably on such cells, such as a hematopoietic stem cell surface marker, preferably the CD34 molecule.
Alternatively, membrane permeability may be enhanced targeting the peptide to ubiquitous cell surface structures. Low and coworkers (see above) teach that molecules such as folate or biotin may be used to target a conjugate molecule to a multitude of cells in an organism, because of the abundant and unspecific expression of the receptors for these molecules.
The above use of cell surface proteins for enhancing membrane permeability of a peptide of the invention may also be used in targeting said peptide of the invention to certain cell types or tissues. For instance, if it is desired to target cancer cells, it is preferable to use a cell surface protein that is expressed more abundantly on the surface of those cells. Examples are the folate receptor, the mucin antigens MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC7, the glycoprotein antigens KSA, carcinoembryonic antigen, prostate-specific membrane antigen (PSMA), HER-2/neu, and human chorionic gonadotropin-beta. Wang et al. (1998) teaches the use of folate to target cancer cells [Wang and Low (1998) J Control Release 53(1-3), 39-48] and Zhang et al. (1998) teaches the relative abundance of each of the other antigens noted above in various types of cancer and in normal cells [Zhang et al. (1998) Clin. Cancer Res. 4, 2669-76]. As the peptide of the invention preferably acts to promote differentiation of hematopoietic stem cells, other markers may be used, as advantageous in each particular case. The above-noted CD34 antigen, or the CD41 and CD33 antigens, may be used as useful markers for targeting a peptide of the invention to uncontrollably growing cells that are derived from hematopoietic stem cells.
The protein, peptide or oligonucleotide of the invention may therefore, using the above-described conjugation techniques, be targeted to certain cell type as desired. For instance, if it is desired to enhance differentiation in cells of the lymphocytic lineage, a peptide of the invention may be targeted at such cells, for instance, by using the MHC class II molecules that are expressed on these cells. This may be achieved by coupling an antibody, or the antigen-binding site thereof, directed against the constant region of said MHC class II molecule to the protein or peptide of the invention. Further, numerous cell surface receptors for various cytokines and other cell communication molecules have been described, and many of these molecules are expressed in more or less tissue- or cell-type restricted fashion. Thus, for instance, when it is desired to target a subgroup of T cells, the CD4 T cell surface molecule may be used for producing the conjugate of the invention. CD4-binding molecules are provided by the HIV virus, whose surface antigen gp42 is capable of specifically binding to the CD4 molecule.
The peptides of the invention may be introduced into cells by the use of a viral vector. The use of vaccinia vector for this purpose is detailed in Chapter 16 of the above-noted Current Protocols in Molecular Biology. The use of Adenovirus vectors has been described [Teoh et al. (1998) Blood 92, 4591-4601; Narumi et al. (1998) Cell. Mol. Biol. 19, 936-941; Pederson et al. (1998) J. Gastrointest. Surg. 2, 283-91; Guang-Lin et al. (1998) Transplant. Proc. 30, 2923-4; Nishida et al. (1998) Spine 23, 2437-42; Schwarzenberger et al. (1998) J. Immunol. 161, 6383-9; Cao et al. (1998) J. Immunol. 161, 6238-44]. Retroviral transfer of antisense sequences has been described [Daniel et al. (1998) J. Biomed. Sci. 5, 383-94].
When using viruses as vectors, the viral surface proteins are generally used to target the virus. As many viruses, such as the above Adenovirus, are rather unspecific in their cellular tropism, it may be desirable to impart further specificity by using a cell-type or tissue-specific promoter. Griscelli et al. (1998) teach the use of the ventricle-specific cardiac myosin light chain 2 promoter for heart-specific targeting of a gene whose transfer is mediated by Adenovirus [Griscelli et al. (1998) Hum. Gene Ther. 9, 1919-28]. The peptide of the invention is preferably targeted to hematopoietic progenitor cells. Promoters and transcription factor binding motifs involved in hematopoietic-specific transcription have been described in a number of articles. It is also possible to isolate and use in the practice of the invention a promoter of a known gene which is specifically expressed in hematopoietic stem cells, such as the SZF1 gene [Liu et al. (1999) Exp. Hematol. 27, 313-25], or the CD34 gene [U.S. Pat. No. 5,556,954]. Further examples of transcription factor binding motifs and promoters involved in hematopoietic stem cell specific transcription have been described [Onyango et al. (1999) Exp. Hematol. 27, 313-25; Meng et al. (1999) Blood 93, 500-8; Nony et al. (1998) J. Biol. Chem. 273, 32910-9; Ye et al. (1998) Hum. Gene Ther. 9, 2197-205].
Isolation of 5′ gene sequences and of the promoter contained therein may be carried out by routine procedures, e.g., using cosmid or P1 phage libraries, hybridization using cDNA sequences, and isolation of genomic 5′ gene fragments and testing same for promoter activity, e.g., using the Chloramphenicolacetyltransferase or luciferase genes as reporter genes. Such techniques have been described [Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley and Sons, New York; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor; Jiang et al. (1999) J. Biol. Chem. 274, 7893-900; Lennon et al. (1997) Immunogenetics 45, 266-73; Grombacher et al. (1996) DNA Cell. Biol. 15, 581-8; Scherer et al. (1996) Hum. Genet. 97, 114-6; Dirks et al. (1989) J. Interferon Res. 9, 125-33].
Alternatively, or in addition, the viral vector may be engineered to express an additional protein on its surface, or the surface protein of the viral vector may be changed to incorporate a desired peptide sequence. The viral vector may thus be engineered to express one or more additional epitopes which may be used to target said viral vector. For instance, cytokine epitopes, MHC class II-, CD34-, CD33-, CD41-binding peptides, or epitopes derived from homing molecules, may be used to target the viral vector in accordance with the teaching of the invention.
The peptide of the invention possesses one or more of the following activities:
- stem cell survival promoting activity;
- stem cell expansion promoting activity;
- neural progenitor expansion promoting activity;
- endothelial cell expansion promoting activity;
- stem cell-derived cell differentiation promoting activity;
- AChE mRNA inducing activity in stem cells;
- SCF substitution activity for hematopoietic stem cells;
- rescue of GM-CSF activity in hematopoietic stem cells in the presence of anti-AChE antisense oligonucleotides;
- induction of DNA synthesis activity in GM-CSF treated hematopoietic stem cells;
- enhancement of growth factor activity of GM-CSF, SCF, TPO EGF and bFGF.
- interaction with the RACK1 protein, and therefore potential involvement in intracellular signal transduction pathways.
The activity of the peptide according to the invention may be investigated by assays involving stem cell survival and growth. Such assays, which are known to the person of skill in the art, may be conducted either in vivo or in vitro. The term “stem cells” as used herein comprises stem cells of various origins and potency. It includes the totipotent embryonic stem cells, but also nerve, epithelial, and mesenchymal stem cells.
Preferred stem cells are embryonic stem cells, nerve stem cells, epithelial stem cells, mesenchymal stem cells, and hematopoietic stem cells. Particularly preferred embodiments of assays for the activities of the peptide of the invention are given in the Examples section herein under. It is to be understood that the examples, which refer to hematopoietic stem cells, may be carried out in a similar manner, following identical principles of method and analysis, with other stem cells, as mentioned above.
Embryonic stem cells may be obtained from totipotent cells of an embryo, according to procedures known in the art. A number of publications describes embryonic stem cells of various origins and their obtention [Robertson E. in: Teratocarcinoma and Embryonic stem cells: A practical Approach, Robertson, E. (ed.), IRL Press, Oxford, p. 71 (1987); Dushnik-Levinson and Benvenisty (1995) Biol. Neonate 67, 77; Thomson et al. (1998) Science 282, 1145]. In vitro aggregation of embryonic stem cells may result in the formation of embryoid bodies. The cells of embryoid bodies are regionally partially differentiated, including cells of mesoderm, ectoderm, and endoderm lineages. The growth and differentiation of embryonic stem cells, or cells derived from embryoid bodies, or the formation of embryoid bodies, are processes that may be influenced by a peptide of the invention. The preferred influence of a peptide of the invention is stimulation of growth and/or differentiation of an embryonic stem cell.
Nerve stem cells may be obtained and characterized as known in the art [Morrison et al. (1999) Cell 96, 737-49]. Epithelial stem cells may be obtained and characterized as known in the art [Cotsarelis et al. (1999) Exp. Dermatol. 8, 80-8]. Mesenchymal stem cells may be obtained and characterized as known in the art [Pittenger et al. (1999) Science 284, 143-7; Horwitz et al. (1999) Nat. Med. 5, 309-13; Ghilzon et al. (1998) Leuk. Lymphoma 32, 211-21; Bruder et al. (1998) Clin Orthop. 355 Suppl, S247-56].
Hematopoietic stem cells may be prepared from sources such as bone marrow or umbilical chord blood. The procedures for obtaining such cells have been described in a number of publications known to those of skill in the art [U.S. Pat. Nos. 5,610,056, 5,728,581, 5,668,104, 5,199,942; and Ahmed et al. (1999) Stem Cells 17:92-9]. Crude stem cell cultures are often contaminated with non-pluripotent cells. It is therefore preferred to purify these cells, using a stem cell marker. Any stem cell marker, preferably a stem cell surface marker, as known in the art may be used. The preferred stem cell marker is CD34. Cells are purified using an agent capable of specifically binding the stem cell marker. Such agents may be e.g., ligands, synthetic compounds including peptides, or antibodies. A preferred agent is a specific anti-stem cell marker antibody. A preferred antibody is the anti-CD34 antibody. While antibodies may be prepared by methods known well to those of skill in the art (see below), a preferred anti-CD34 antibody is the CD34-PE, available from Becton Dickinson, Immunocytometry System Inc., Mountain View, Calif., USA. Stem cell marker enriched cells may be purified in a number of ways. For instance, the stem cell marker carrying cells may be labeled, using an antibody directed against the stem cell marker. The antibody is either labeled itself or is reacted with a second, labeled antibody. Examples of antibodies that may be used are the above CD34-PE (Phycoerythrin-labeled), or a combination of anti-CD34 (e.g., the said CD34-PE) and FITC-RaMIg. FITC-RaMIgs are Fluorescein-isothiocyanate-labeled rabbit anti-mouse IgG antibodies. They should of course be used only if the first antibody is a mouse (usually monoclonal) antibody. FITC-RaMIg or similar labeled antibodies with the desired specificities are available from several companies, including Sigma, St. Lois, Mo., USA, and PIERCE, Rockford, Ill. 61105, USA.
Using the above stem cell surface marker binding agent, CD34+ cells may be purified in a variety of ways, including panning, fluorescence-activated cell sorting, or by using magnetic beads. Panning involves adsorbing cells binding a certain antibody to a surface covered with this antibody, and thus enriching these cells [Hoogenboom et al. (1999) Eur. J. Biochem. 260, 774-784]. The use of magnetic beads involves magnetic beads that carry the antibody. After the cells are bound to the bead via the antibody, a magnetic force is applied to separate the beads from the rest of the culture solution, thereby enriching the bound cells. Magnetic beads are commercially available, e.g., from Dynal, Norway. Antibodies and other agents may be bound to bead by a variety of techniques known to the person of skill in the art, e.g., via chemical cross-linking. Cross-linking agents and references regarding the procedure of cross-linking are disclosed, e.g., in the Life Science Products catalog of the above PIERCE.
Hematopoietic stem cells are then cultured as known in the art, see e.g., Current Protocols in Immunology is published by John Wiley & Sons. The concentration of the cells is kept initially low, i.e., at about 50,000 to about 250,000 cells per ml. A preferred medium is IMDM [Bruserud et al. (1999) J. Hematother. 8, 63-73]. The medium preferably comprises autologous plasma, in an amount of between 5 and 30%, preferably 10%. Other additions to the culture media are as known in the art, preferably 2 mM L-glutamine, 20 IU/ml Heparin, and antibiotics Penicillin, Streptomycin, and Amphotericin B. Penicillin and Streptomycin are added to a final concentration of preferably 5 μg to 2 mg per ml, more preferably 100 μg/ml. Amphotericin B is added at a final concentration of 1 to 100 μM, preferably 20 μM.
To the culture of hematopoietic stem cells may be added early growth factors, including IL-3, IL-6, either alone or in combination with soluble IL-6 receptor, Thrombopoietin, stem cell factor, granulocyte-macrophage colony-stimulating factor, FLT-3 or combinations thereof.
The preferred concentrations of these agents may be inferred from the example section herein below, or alternatively, from the above U.S. Pat. Nos. 5,610,056, 5,728,581, 5,668,104, 5,199,942, and Ahmed et al., Stem Cells 17:92-9, 1999.
Alternatively, to the culture of endothelial cells there may be added growth factors, including EGF and bFGF.
A peptide according to the invention is added to the stem cell cultures, together with, or at different times than the above growth factors. The preferred peptide concentration is about 1 ng/ml to about 1 500 ng/ml, more preferred about 50 to about 100 ng/ml.
Growth factors and the peptide according to the invention may be supplemented preferably every 24 hours to about every ten days, more preferably every 3-5 days. The cultures are grown for about 14 hours to about three months, more preferably for about 24 hours to about one month.
Cultures of stem cells may then be analyzed by cytochemical staining, by cell proliferation assay, viable cell count, cell phenotyping, for CD34, CD33, and CD 41 markers, and growth of progenitor colonies, using established techniques, preferably as described herein below. Cell survival may also be evaluated by determining dead cell counts, e.g., using apoptosis-specific reagents. Such reagents in kit form are available from many companies, including Clontech Laboratories UK Ltd., Hampshire RG24 8NE, UK, InterGen Energy Inc., Boston, Mass. 02114, USA, R&D Systems, Minneapolis, Minn. 55413, USA, and Boehringer Mannheim/Hoffmann-La Roche Ltd, 4070 Basel, Switzerland.
Stem cell growth may be quantified in a variety of ways, such as viable cell count using trypan blue exclusion (see herein below), or measuring DNA replication. A large number of reagents and systems are commercially available for the purposes of quantifying DNA replication. For instance, incorporation of the nucleotide analog Bromo-deoxy-Uridine (BrdU) may serve as an indicator of DNA replication (see herein below). However, also radioactive nucleotide analogs, such as 3H-Thymidine, may be added to the culture medium and their incorporation into DNA measured by liquid scintillation counting. Techniques for measuring DNA replication are well known in the art and are described in many textbooks and articles.
Peptides of the invention, when used either alone or in combination with the above early cytokines, will promote stem cell survival significantly. Preferably stem cell survival is promoted by about 10%, more preferably by about 100%, and still more preferably by about 1000 or more percent, when compared to control cultures lacking the peptide of the invention.
It is understood by the skilled person that promotion of stem cell survival by a peptide of the invention will usually be highest in terms of percentage in the absence of other growth factors. Further, the percentage of growth promotion by the peptide of the invention depends upon the growth factor(s) used in combination with the peptide of the invention, on the culture conditions, and on the source and type of stem cells used for the assay.
In another assay, the peptide of the invention may be tested for its ability to promote stem cell differentiation. For instance, hematopoietic stem cells expanded by growth in a medium comprising a peptide of the invention will display increased ability to differentiate into megakaryocytic (MK) and myeloid cells. The differentiation of cells may be assayed from about three days in culture to about three months, preferably from about one week in culture to about three weeks, and more preferably at about two weeks in culture medium comprising a peptide of the invention. The differentiated morphology of the cells may be analyzed microscopically, including analysis by light microscopy, electron microscopy. Alternatively, the differentiation of stem cells may conveniently be analyzed using cell surface differentiation markers, such as cluster of differentiation (CD) markers. A preferred CD marker for myeloid differentiation of hematopoietic stem cells is the CD33 marker. A preferred marker for megakaryocytic differentiation is the CD41 marker. Analysis of these markers may be carried out as known in the art, e.g., using antibodies specific for the desired marker, either labeled or in conjunction with labeled second antibodies. The label may then be detected by enzymatic reaction, fluorescence microscopy, fluorescence-activated cell sorting, or the like techniques.
The activity of the peptide of the invention upon differentiation of stem cells in culture may thus be determined by the analysis of differentiation-associated markers. A peptide of the invention having differentiation-promoting activity will significantly enhance the number of cells in a culture that express morphology of differentiated cells, or that express a differentiation marker. The enhancement (as compared to control cultures lacking the peptide of the invention) is preferably at least about 30%, more preferably at least 150%, still more preferably between at least 100 and at least 1000%.
The AChE production inducing activity of the peptide invention may be tested by culturing stem cells or stem cell-derived cells in the presence and in the absence of the peptide of the invention. The amount of AChE in these cells may then be quantified by techniques known in the art, such as immunochemical or cytochemical staining, AChE enzyme assay, and the like. For detection by immunochemical staining, anti-AChE antibodies may be used; however, also AChE-specific substrates can be employed (see herein below). The addition of AChE-specific enzyme inhibitors such as iso-OMPA in this assay may serve as a control reaction [Keymer et al. (1999) Eur. J. Neurosci. 11, 1049-57].
The above-detailed activities of the peptide of the invention make it useful in the treatment of various disorders or conditions in which growth enhancement of stem cells is desired. In a preferred embodiment, the condition is thrombocytopenia or a post-irradiation condition, or a chemotherapy condition, or a condition of massive blood loss.
Thrombocytopenia is a severe complication encountered in cancer patients treated with high dose chemotherapy followed by autologous or allogenic transplantation of bone marrow transplantation (BMT), peripheral blood stem cell (PBSC) transplantation or cord blood transplantation (CBT).
While engraftment of the myeloid and erythroid lineages occurs within 2-3 weeks, there is often a prolonged period of profound thrombocytopenia due to insufficient platelet production. Such patients require multiple platelet transfusions and hospitalization and are at risk of life threatening hemorrhages for a few weeks up to several months.
Megakaryocytopoiesis and platelet production depend on the availability of adequate numbers of cytokine responsive progenitor cells in the graft, while long term thrombopoiesis results from the engraftment of multipotent stem cells. It was thought that the recently discovered physiological regulator of thrombopoiesis, thrombopoietin (TPO) or the truncated cloned MGDF (MK growth and development factor) would enhance platelet recovery following aggressive chemotherapy. Numerous studies showed that this exciting new cytokine had potent early and late acting activities. However, in phase II clinical trials, use of TPO before or after BMT had no impact on the duration of severe thrombocytopenia nor did it reduce the requirement for platelet transfusion post-transplant or in AML patients post-chemotherapy. The lack of significant clinical activity of TPO is probably related to the paucity of target cells, the MK progenitors (MK-P). To complicate matters, MGDF has recently been retracted due to immunogenicity in healthy donors who developed antibodies and became severely thrombocytopenic.
IL-11, which has a limited positive effect on thrombopoiesis, is currently being administered in cancer patients to support platelet production following chemotherapy. IL-11 is not MK lineage specific and not effective in BMT patients with protracted thrombocytopenia. Insufficient MK precursor engraftment and delayed platelet recovery may be due to BM stromal and endothelial cell damage. These cells are responsible for the production of cytokines and provide the appropriate environment for megakaryocytopoiesis.
Currently, platelet transfusions, which have tripled in number over the past 10 years, remain the only means to prevent hemorrhage in severely thrombocytopenic patients. They are extremely costly and provide only a limited mode of treatment, due rapid allo-immunization and the development of refractoriness.
Expansion in vitro of hematopoietic stem cells and precursors for the purpose of transplantation is a new and exciting alternative approach to the treatment of thrombocytopenia. The development of recombinant human hematopoietic growth factors and sophisticated techniques for hematopoietic stem cell selection has facilitated the use of cellular therapy by ex vivo manipulation of hematopoietic cells before clinical use.
This novel modality, now referred to as “ex vivo expansion” aims at the expansion of HSC and progenitors for transplantation. Animal studies have shown shorter periods of cytopenias (including thrombocytopenia) following lethal irradiation of animals transplanted with BM cells that were expanded with cytokines IL-3, IL-1 and GM-CSF or TPO, KL, IL-1a and IL-3.
The feasibility of expanding MK progenitors from BM with new cytokine combinations and their subsequent transplantation to shorten the period of protracted thrombocytopenia following BMT in patients is starting to be explored. Facilitated platelet engraftment in patients with received T cell depleted donor BM pre-incubated with IL-3 and GM-CSF to stimulated the ex vivo expansion of early and late MK precursors has been shown. Also the peptides of the present invention may be used for inducing such expansion.
Therefore, a preferred embodiment of the invention is the use of a peptide of the invention in expansion of hematopoietic stem cells. This may be particularly useful in medical applications. As the amount of hematopoietic stem cells available for purposes such as transplantation is typically very limited, there is a need for the expansion of hematopoietic progenitor cells for a number of clinical uses such as gene therapy, augmentation of bone marrow transplantation (BMT) and replacement of BMT. Such expansion may be either ex-vivo or in-vivo. U.S. Pat. No. 5,861,315, and references therein, which are incorporated herein in their entirety by reference, describes methods for the expansion of hematopoietic stem cells, using a combination of the cytokines IL-6 and soluble IL-6 receptor. The combination of IL-6 and soluble IL-receptor is also said to sustain undifferentiated growth of embryonic stem cells.
Thus, in a preferred embodiment of the invention, a peptide of the invention is used for the ex uivo expansion of hematopoietic stem cells. The isolation, culture, expansion, and transplantation of such stem cell cultures has been described in many prior art articles [U.S. Pat. No. 5,861,315; Contassot et al. (1998) Bone Marrow Transplant. 22, 1097-102; Ahmed et al. (1999) id ibid.].
In a preferred embodiment, mononuclear cells (MNC) obtained from human umbilical cord blood are separated by Ficoll-Hypaque density gradient centrifugation after depletion of phagocytes with Silica. CD34+ cells are purified as described hereinabove and below, preferably using magnetic beads coated with anti-CD34 antibody. Purified CD34+ cells are then incubated in suspension culture containing alpha-medium 20% fetal bovine serum 1% fraction V BSA. When cultured without serum, the culture preferably contains 2% pure BSA, 10 μg/ml insulin, 200 μg/ml transferrin, 10 μM beta-mercaptoethanol, and 40 μg/ml low-density lipoprotein instead of FBS and BSA. At regular time points, the distance of which is about three days to two weeks, the culture is diluted with fresh medium in an about 1:1 ratio. For assaying the number of hematopoietic progenitor cells, a sample is removed from the culture and the cells cultured in a clonal methylcellulose assay, as previously described [Nakahata et al. (1982) J. Clin. Invest. 70, 1324-1328; U.S. Pat. No. 5,861,315]. The culture contained alpha-medium, 0.9% methylcellulose, 30% FBS, 1% BSA, 50 μM beta-mercaptoethanol, and cytokines. Methylcellulose cultures free of serum contain 1% BSA, 300 μg/ml human transferrin, 160 μg/ml soybean lecithin, and 96 μg/ml cholesterol instead of BSA and FBS. Cytokines to be added to the cultures include one or more of SCF, IL-1, IL-3, IL-6, soluble IL-6 receptor, IL-11, EPO, TPO, GM-CSF. Examples of preferred cytokines or combinations thereof are:
Examples of preferred concentrations are e.g., IL3, 5 ng/mL, IL-6, 50 ng/mL, TPO 1 ng/mL, SCF, 10-100 ng/mL, FLT-3 ligand (FLT3), 50 ng/mL, GM-CSF, 50 ng/mL, sIL-6R, 1280 ng/ml [U.S. Pat. No. 5,861,315; Ahmed et al. (1999) id ibid.], see also Examples, particularly Example 3, and “Experimental procedures” section herein below. The peptide of the invention is added to the cultures either concomitantly, before, or after the cytokines, preferably in a concentration of about 50 ng/ml. However, it will be appreciated by the person of skill in the art that the optimal concentration of the peptide of the invention may vary according to its length, structure, and modification, and according to the combination of cytokines it is used with. Cytokines and the peptide of the invention are replenished as described herein below, preferably about every 2 to 7 days, more preferably about every 4 days. The development of progenitors may be scored according to known criteria [U.S. Pat. No. 5,861,315; Koike et al. (1998) Exp. Med. 168, 879-890; Tanaka et al. (1992) Blood 80, 1743-1749; Nakahata et al. (1982) J. Clin. Invest. 70, 1324-1328]. The peptide of the invention preferably has megakaryocyte-differentiation promoting properties, as detailed in Example 3 herein below. The activity of the peptide of the invention in promoting differentiation of stem cells into megakaryocytes is particularly advantageous when ex-vivo expanded stem cells are used for the purpose of bone marrow transplantation, or when transplanting such cells after a bone marrow transplantation. In these circumstances, thrombocytopenia is a frequent and sometimes fatal occurrence [Ahmed et al. (1999) id ibid].
In a further embodiment, the peptide of the invention can be used for promotion of the expansion of committed neural progenitors in vivo in a developing embryo. Furthermore, the peptide of the invention can be used for promotion of the expansion of committed neural progenitors ex vivo, using cultured embryonic stem cells and/or embryoid bodies derived thereof.
The peptide of the invention may be used in free form or as salt, e.g., as metal salt, including sodium, potassium, lithium or calcium salt, or as a salt with an organic base, or as a salt with a mineral acid, including sulfuric acid, hydrochloric acid or phosphoric acid, or with an organic acid e.g., acetic acid or maleic acid. Generally, any pharmaceutically acceptable salt of the peptide of the invention may be used.
The peptide of the invention may be used as such or in the form of a composition. A composition will generally contain salts, preferably in physiological concentration, such as PBS (phosphate-buffered saline), or sodium chloride (0.9% w/v), and a buffering agent, such as phosphate buffer in the above PBS. The preparation of pharmaceutical compositions is well known in the art, see e.g., U.S. Pat. Nos. 5,736,519, 5,733,877, 5,554,378, 5,439,688, 5,418,219, 5,354,900, 5,298,246, 5,164,372, 4,900,549, 4,755,383, 4,639,435, 4,457,917, and 4,064,236. The peptide of the present invention, or a pharmacologically acceptable salt thereof is preferably mixed with an excipient, carrier, diluent, and optionally, a preservative or the like pharmacologically acceptable vehicles as known in the art, see e.g., the above US patents. Examples of excipients include, glucose, mannitol, inositol, sucrose, lactose, fructose, starch, corn starch, microcrystalline cellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, polyvinyl-pyrrolidone and the like. Optionally, a thickener may be added, such as a natural gum, a cellulose derivative, an acrylic or vinyl polymer, or the like.
The pharmaceutical composition is provided in solid, liquid or semi-solid form. A solid preparation may be prepared by blending the above components to provide a powdery composition. Alternatively, the pharmaceutical composition is provided as lyophilized preparation. The liquid preparation is provided preferably as aqueous solution, aqueous suspension, oil suspension or microcapsule composition. A semi-solid composition is provided preferably as hydrous or oily gel or ointment. About 0.001 to 60 w/v %, preferably about 0.05 to 25 w/v % of peptide is provided in the composition.
A solid composition may be prepared by mixing an excipient with a solution of the peptide of the invention, gradually adding a small quantity of water, and kneading the mixture. After drying, preferably in vacuo, the mixture is pulverized. A liquid composition may be prepared by dissolving, suspending or emulsifying the peptide of the invention in water, a buffer solution or the like. An oil suspension may be prepared by suspending or emulsifying the peptide of the invention or protein in an oleaginous base, such as sesame oil, olive oil, corn oil, soybean oil, cottonseed oil, peanut oil, lanolin, petroleum jelly, paraffin, Isopar, silicone oil, fatty acids of 6 to 30 carbon atoms or the corresponding glycerol or alcohol esters. Buffers include Sorensen buffer (Ergeb. Physiol., 12, 393 1912), Clark-Lubs buffer (J. Bact., 2, (1), 109 and 191, 1917), Macllvaine buffer (J. Biol. Chem., 49, 183, 1921), Michaelis buffer (Die Wasserstoffinonenkonzentration, p. 186, 1914), and Kolthoff buffer (Biochem. Z., 179, 410, 1926).
A composition may be prepared as a hydrous gel, e.g. for transnasal administration. A hydrous gel base is dissolved or dispersed in aqueous solution containing a buffer, and the peptide of the invention, and the solution warmed or cooled to give a stable gel.
Preferably, the peptide of the invention is administered through intravenous, intramuscular or subcutaneous administration. Oral administration is expected to be less effective, because the peptide may be digested before being taken up. Of course, this consideration may apply less to a peptide of the invention which is modified, e.g., by being cyclic peptide, by containing non-naturally occurring amino acids, such as D-amino acids, or other modification which enhance the resistance of the peptide to biodegradation. Decomposition in the digestive tract may be lessened by use of certain compositions, for instance, by confining the peptide of the invention in microcapsules such as liposomes. The pharmaceutical composition of the invention may also be administered to other mucous membranes. The pharmaceutical composition is then provided in the form of a suppository, nasal spray or sublingual tablet. The dosage of the peptide of the invention may depend upon the condition to be treated, the patient's age, bodyweight, and the route of administration, and will be determined by the attending physician. Doses ranging from 0.1 μg/kg to 100 mg/kg, preferably from 0.5 μg/kg to 5 mg/kg, more preferably 0.1 μg/kg to 1 mg/kg, most preferably about 100 μg/kg.
The uptake of a peptide of the invention may be facilitated by a number of methods. For instance, a non-toxic derivative of the cholera toxin B subunit, or of the structurally related subunit B of the heal-labile enterotoxin of enterotoxic Escherichia coli may be added to the composition, see U.S. Pat. No. 5,554,378.
In another embodiment, the peptide of the invention is provided in a pharmaceutical composition comprising a biodegradable polymer selected from poly-1,4-butylene succinate, poly-2,3-butylene succinate, poly-1,4-butylene fumarate and poly-2,3-butylene succinate, incorporating the peptide of the invention as the pamoate, tannate, stearate or palmitate thereof. Such compositions are described e.g., in U.S. Pat. No. 5,439,688.
In another embodiment, a composition of the invention is a fat emulsion. The fat emulsion may be prepared by adding to a fat or oil about 0.1-2.4 w/w of emulsifier such as a phospholipid, an emulsifying aid, a stabilizer, mixing mechanically, aided by heating and/or removing solvents, adding water and isotonic agent, and optionally, adjusting adding the pH agent, isotonic agent. The mixture is then homogenized. Preferably, such fat emulsions contain an electric charge adjusting agent, such as acidic phospholipids, fatty acids, bilic acids, and salts thereof Acidic phospholipids include phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. Bilic acids include deoxycholic acid, and taurocholic acid. The preparation of such pharmaceutical compositions is described in U.S. Pat. No. 5,733,877.
The invention also comprises anti-AChE antibodies and the use thereof for the diagnosis of pathological conditions, particularly hematopoietic pathological conditions.
The antibodies of the invention may also be used for diagnosis of a stress-induced male infertility.
Polyclonal antibodies may be generated in rabbits, chicken, mice, rats, sheep, or similar mammals. For generation of antibodies against a peptide of the invention, the peptide is produced by recombinant DNA technology in mammalian cells, as described in the above general references for molecular biology. Alternatively, the peptide may be synthetically produced by organic chemistry. The peptide may also be produced in bacterial or insect cells as detailed in the above-noted Current Protocols in Molecular Biology, Chapter 16.
The peptide is purified from the cells in which it has been produced. Peptide purification methods are known to the person of skill in the art and are detailed e.g., in the above-noted Current Protocols in Molecular Biology, Chapter 16, and in Current Protocols in Protein Science, Wiley and Sons Inc. Chapters 5 and 6. Advantageously, the peptide may be produced as a fusion with a second protein, such as Glutathione-S-transferase or the like, or a sequence tag, such as the histidine tag sequence. The use of fusion or tagged proteins simplifies the purification procedure, as detailed in the above-noted Current Protocols in Molecular Biology, Chapter 16, and in the instructions for the His-tag protein expression and purification kit, as available from Qiagen GmbH, 40724 Hilden, Germany.
If the protein or peptide has been expressed as a fusion protein, it may be desirable to cleave the fusion partner before using the protein for the generation of antibodies, in order to avoid generation of antibodies against the fusion partner. The cleavage of fusion partners and the isolation of the desired protein are described in the above-noted Current Protocols in molecular Biology, Chapter 16. Vectors, protocols and reagents for expressing and purifying maltose-binding protein fused recombinant proteins are also available commercially.
When producing a peptide of the invention, it may be desirable not to remove the fusion partner, as the fusion protein may stimulate the production of antibodies against the peptide. Generally, this consideration may be relevant when generating antibodies from peptides that are less than 50 amino acids in length. In particular, it has been found that the ARP peptide, when injected, is virtually non-immunogenic. A Keyhole Limpet hemocyanin (KLH)-conjugated ARP peptide was found to elicit antibodies unable to detect ARP or acetylcholinesterase. Antibodies capable of detecting ARP were successfully generated using a Glutathione-S-transferase-ARP fusion protein (detailed herein below). Accordingly, in a preferred embodiment of the invention, antibodies are elicited using a conjugate or fusion protein of the peptide of the invention as antigen. A preferred fusion partner is Glutathione-S-transferase.
As noted further above, the peptide may also be synthesized by chemical methods known in the art of chemistry.
The generation of polyclonal antibodies against proteins is described in Chapter 2 of Current Protocols in Immunology, Wiley and Sons Inc. The generation of antibodies against peptides may necessitate some changes in protocol, because of the generally lower antigenicity of peptides when compared to proteins. The generation of polyclonal antibodies against peptides is described in the above-noted Current Protocols in Immunology, Chapter 9, and exemplified herein below.
Monoclonal antibodies may be prepared from B cells taken from the spleen or lymph nodes of immunized animals, in particular rats or mice, by fusion with immortalized B cells under conditions which favor the growth of hybrid cells. For fusion of murine B cells, the cell line Ag-8 is preferred.
The technique of generating monoclonal antibodies is described in many articles and textbooks, such as the above-noted Chapter 2 of Current Protocols in Immunology. Chapter 9 therein describes the immunization, with peptides, of animals. Spleen or lymph node cells of these animals may be used in the same way as spleen or lymph node cells of protein- immunized animals, for the generation of monoclonal antibodies as described in Chapter 2 therein.
The techniques used in generating monoclonal antibodies are further described in Kohler and Milstein, Nature 256, 495-497, 1975 and in U.S. Pat. No. 4,376,110.
In the preparation of antibodies from a gene bank of human antibodies the hypervariable regions thereof are replaced by almost random sequences [U.S. Pat. No. 5,840,479]. This method of antibody generation is preferred if it is difficult to immunize an animal with a given peptide or protein. The peptide of the invention may be poorly immunogenic, even as a conjugate. The antibodies described in U.S. Pat. No. 5,840,479 are further preferred if it is desired to use antibodies with a structure similar to human antibodies, for instance, when antibodies are desired that have low immunogenicity in humans.
Once a suitable antibody has been identified, it may be desired to change the properties thereof. For instance, a chimeric antibody may achieve higher yields in production. Chimeric antibodies wherein the constant regions are replaced with constant regions of human antibodies are further desired when it is desired that the antibody be of low immunogenicity in humans. The generation of chimeric antibodies has been described in a number of publications [Cabilly et al. (1984) Proc. Natl. Acad. Sci. USA 81, 3273; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Boulianne et al. (1984) Nature 312, 643; EP 125023; EP 171496; EP 173494; EP 184187; WO 86/01533; WO 87/02671; Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor].
The term “antibody” is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab′)2, which are capable of binding antigen. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody [Wahl et al. (1983) J. Nucl. Med. 24, 316-325].
It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of the peptide of the invention and of intact AChE or its isoforms, according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).
An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term “epitope” is meant to refer to that portion of any molecule capable of being bound by an antibody that can also be recognized by that antibody. Epitopes or “antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.
An “antigen” is a molecule or a portion of a molecule capable of being bound by an antibody, which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
The antibodies, including fragments of antibodies, useful in the present invention, may be used to quantitatively or qualitatively detect the peptide of the invention, in a sample. This can be accomplished by immunofluorescence techniques employing a fluorescently or color-labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.
The antibodies. (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a peptide of the invention. In situ detection may be accomplished by removing a histological specimen from a mammal, and providing the labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the peptide, but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Such assays for the peptide of the invention typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, cells which have been incubated in tissue culture, or alternatively, human sperm cells, in the presence of a labeled antibody capable of identifying the peptide, and detecting the antibody by any of a number of techniques well known in the art.
The biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers followed by treatment with a detectably labeled antibody in accordance with the present invention, as noted above. The solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by conventional means.
By “solid phase support”, “solid phase carrier”, “solid support”, “solid carrier”, “support” or “carrier” is intended any support or carrier capable of binding antigen or antibodies. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrier configuration may be spherical, as in a bead, cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
The binding activity of a given lot of antibody, of the invention as noted above, may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.
One of the ways in which an antibody in accordance with the present invention can be detectably labeled is by linking the same to an enzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactive labeling the antibodies or antibody fragments, it is possible to detect receptor tyrosine phosphatase (R-PTPase) through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S. et al., North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled “An Introduction to Radioimmune Assay and Related Techniques” by Chard, T., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a g counter or a scintillation counter or by autoradiography.
It is also possible to label an antibody in accordance with the present invention with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can be then detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals such as 152E, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (ETPA).
The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
An antibody molecule of the present invention may be adapted for utilization in an immunometric assay, also known as a “two-site” or “sandwich” assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
Typical, and preferred, immunometric assays include “forward” assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a “reporter molecule”). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.
In another type of “sandwich” assay, which may also be useful with the antigens of the present invention, the so-called “simultaneous” and “reverse” assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional “forward” sandwich assay.
In the “reverse” assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the “simultaneous” and “forward” assays.
The present invention provides an immunoassay for the detection and quantification of a peptide of the invention. The creation of immunoassays, such as RIA or ELISA, has been described in many articles, textbooks, and other publications. Reference is made to WO 97/03998, p. 48, line 4 to p. 52, line 27. Immunoassays of the invention may be if two general types: Firstly, immunoassays using an immobilized peptide of the invention, may be used. Secondly, immunoassays using immobilized antibodies directed against an epitope of a peptide of the invention may be used to quantify a peptide of the invention.
In a preferred embodiment of the invention, the assay is an immunoblot assay. The sample, e.g., a serum sample, is diluted, e.g., 1:10, in order to avoid overloading. The sample is then loaded onto a polyacrylamide gel, optionally a gradient gel, and electrophoresed. Synthetic or recombinantly produced peptide of the invention, preferably SEQ ID: No. 1, SEQ ID: No. 2, or SEQ ID: No. 3, may be added in separate lanes or spiked to the sample lanes, as positive controls. The gel is then blotted, preferably onto a Nitrocellulose or Nylon membrane. The blot is reacted with antibodies against the peptide of the invention, preferably antibodies reactive with SEQ ID: No. 1, 2 or 3. A more preferred antibody is the rabbit anti-GST-ARP antibody as described herein. Bound antibody may then be detected by antibodies reactive with the antibody of the invention, e.g., anti-rabbit immunoglobulins. These immunoglobulins are preferably labeled, e.g., by Peroxidase conjugation. The detection of the label is then carried out according to methods known in then art. Preferably, peroxidase-conjugated immunoglobulins are detected using the ECL™ detection system (Amersham Pharmacia Biotech, UK).
As described above, a preferred sample is serum. However, other body fluids may be used, including cerebrospinal fluid, liquor, saliva, and the like. Another specifically preferred sample is sperm cells. Also, liquid extracts of body tissue may be analyzed. Alternatively, body tissue may be analyzed without extraction using cytochemical staining or immunostaining as described herein.
A preferred body fluid is cerebrospinal fluid. For instance, increased levels of AChE or of a peptide of the invention in cerebrospinal fluid, may be indicative of elevated blood cortisol levels, and may further be indicative of stress.
Such assays as hereinabove described may find use in diagnostics, as the level of the peptide of the invention may need to be evaluated in a number of conditions. For instance, such assays may be useful in order to monitor the effect of treatment of a patient with a peptide of the invention. Furthermore, such assays may be used in determining psychological stress (see Example 8 herein below). Still further, such assays may be useful as an indicator of stress to the bone marrow. Stressed bone marrow will up-regulate ARP, which is a peptide of the invention, as detailed herein under in the Examples section. Finally, such assays may be useful in the diagnosis of a number of disorders where growth or expansion of hematopoietic stem cells is adversely effected, or alternatively, uncontrolled growth of hematopoietic stem cells occurs.
Thus, in a preferred embodiment, the invention provides a method for the diagnosis of elevated glucocorticoid level, bone marrow stress, abnormality, dysfunction or stressed condition, or of increased platelet count or of brain infarct risk in a mammal, comprising obtaining a sample from said mammal, contacting said sample with an antibody of the invention, removing unbound antibody, and detecting the extent of reaction between said antibody and acetylcholinesterase or a fragment thereof present in said sample. The sample is preferably serum or a bone marrow sample.
An additional aspect of the present invention relates to a method for diagnosis of stress induced male infertility.
As demonstrated in Example 10, elevated serum corticosterone levels and spermatid overexpression of AChE-R were observed in FVB/N mice subjected to acute psychological stress or injected with synthetic ARP of the present invention. Reduced seminal gland weight and lower sperm counts and motility were observed in AChE-R transgenic mice with massive AChE-R excess as compared with matched controls. ARP immunostaining labeled mature mouse sperm in stressed and ARP injected mice but meiotic pachytene spermatocytes in AChE-R transgenics.
Most interestingly, in humans, ARP labeling covered sperm head and midpiece in controls but was concentrated at the midpiece in couple infertility specimens. Excess AChE-R and its C-terminal peptide ARP may thus suppress male fertility through both autonomous system regulation and direct sperm interactions.
The intensified midpiece labeling of sperm cells from subjects with unexplained couple infertility emphasizes the physiological relevance of the animal models of the invention. Although stress in such patients may be treatment-induced [Negro-Vilar et al. (1993) Enviromental Health Prespectives Supplements 101(Suppl 2), 59-64], its effects may nevertheless be significant. As exposure to anticholinesterases induces similar AChE-R accumulation [Kaufer et al. (1998) id ibid], the findings of the present invention may also explain the impaired sperm properties and resultant male infertility that are associated with exposure to agricultural insecticides [Rivier et al. (1986) Science 231, 607-609]. Moreover, the presumed co-localization with sperm mitochondria may explain at least part of the impairments observed in spermatogenesis and sperm motility through interference with mitochondrial metabolism. Several potential means of preventing such effects and improving fertility include transcriptional suppression of stress-induced AChE overexpression, prevention of the stress-associated shift in alternative splicing from AChE-S to AChE-R (Kaufer et al. (1998) id ibid.) or antisense destruction of the vulnerable AChE-R mRNA transcript [Grisaru et al. (1999) ibid; Shohami et al. (2000) id ibid.]. AChE-R and ARP thus present previously unrecognized targets for studying, analyzing and treating stress-induced male infertility.
Thus, in a preferred embodiment the invention provides a method for the diagnosis of stress induced male infertility, comprising obtaining a sperm cell sample from said male, smearing and air drying said sperm cell and contacting said sperm cell with the antibody of the invention. Preferably, using the immunohystochemical staining as described above.
Additionally, the invention provides a method for the diagnosis of stress induced male infertility, further comprising the step of determining the pattern of the AChE-R expression in said sperm cell.
In yet another embodiment, the invention provides a method for the diagnosis of stress induced male infertility for use in fertility counseling.
Using the non-biased yeast two-hybrid screening approach, the inventors found two potential links explaining the function of intracellular, specially intraneural, AChE-R (see Example 13). Tight, co-immunoprecipitable and naturally co-localized complexes of AChE-R appeared to connect the C-terminal domain ARP1 with PKCβII and its intracellular shuttling protein RACK1 (Example 14). Intensified labeling and neuronal mobilization of both RACK1 and PKCβII was observed in the stress-protected transgenic mice constitutively overexpressing AChE-R (
A variety of RACK1 clones were identified with the capacity to induce intense β-gal staining in the yeast cell context, strengthening the notion that AChE-R/RACK1 interactions are both common and tight in the embryonic brain from which the screened library originated (Example 13 and data not shown). The cell biology tests point at neuronal accumulation and/or subcellular mobilization as the outcome of these interactions under in vivo stress or transgenic overexpression. The RACK1 region which was found to be essential for AChE-R interaction consists of two anti-parallel four strand “blades” which together cover ca. 30% of the of the RACK1 perimeter. The large number of conserved WD domain residues in this region of the protein may highlight the requirement for correct blade folding of RACK1 as essential for ARP1 interactions. The AChE-R-induced changes in the intensity of RACK1/PKCβII interactions, may also be physiologically relevant.
In addition to its primary function of acetylcholine hydrolysis, AChE was shown to initiate adhesive cell-cell interactions through its core domain and promote mammalian neurite extension in a manner similar to that of its non-enzyme membrane protein homolog neuroligin [reviewed by Soreq and Seidman, (2001) id ibid.]. Nevertheless, the neuritogenic activities of distinct AChE variants appeared to depend on their unique C-termini, which to date were not found to share sequence homologies with other proteins. In hematopoietic cells, the 26 C-terminal residues of the stress-induced AChE-R protein exert proliferative and growth factor activities, as described in Examples 4, 7 and 8 of the present application, and also in [Grisaru et al. (2001) Molecular Medicine, 7, 93-105]. However, it was not yet known whether these activities depend on extra- or intracellular interactions. Also, neither the molecular mechanism(s) nor the nervous system relevance of such activities had yet been explored. The present invention additionally reveals that, at least part of the C-terminus specific non-catalytic effects of AChE-R, are intracellular and PKCβII mediated.
As described in Examples 16, 17 and 19, co-immunoprecipitable and naturally co-localized complexes of AChE-R appeared to connect the C-terminal domain ARP1 with PKCβII and its intracellular shuttling protein RACK1. Intensified labeling and neuronal mobilization of both RACK1 and PKCβII was observed in the stress-protected transgenic mice constitutively overexpressing AChE-R. Transgenic AChE-R overexpression translocated RACK1 to the perikaryal circumference and intensified PKCβII clustering in pyramidal neurons, and activated PKC suggesting functional relevance for AChE-R overexpression in the stress-suppressing cascades of neuronal signal transduction.
As shown by Examples 16 and 17, AChE-R interaction with PKCβII is indirect and is mediated by the PKCβII shuttling protein RACK1. RACK1 is a member of the WD family of proteins. WD proteins can simultaneously bind different partners to various regions in their multi-blade rings [Smith et al. (1999) id ibid.], which provides flexibility and combinatorial diversity. Thus, RACK1 is a scaffold for cell-cell interaction proteins such as β-integrin [Liliental and Chang (1998) J Biol Chem, 273, 2379-831, members of signaling cascades like cAMP phosphodiesterase [Yarwood et al. (1999) J Biol Chem, 274, 14909-17], C2-containing proteins such as phospholipase C-γl [Disatnik et al. (1994) Proc Natl Acad Sci USA, 91, 559-63], src kinase [Chang et al. (1998) Mol Cell Biol, 18, 3245-56] and/or PH domain-containing proteins such as the β-adrenergic receptor [Rodriguez et al. (1999) Biochemistry, 38, 13787-94]. Interaction between RACK1 and AChE-R would likely compete with other associations, changing the subcellular balance between these variable complexes. Alternatively, or in addition, AChE-R/RACK1 interactions may promote the formation of additional triple complexes with other proteins. Diverse links between AChE and other signaling molecules may, in turn, explain its capacity to exert various non-catalytic intracellular functions, a possibility which awaits further investigation.
PKCβII and RACK1 are readily available in neurons, albeit in inactive and possibly, non-associated or, loosely attached compositions. The present inventors have now found that stress responses and the subsequent accumulation of AChE-R [Kaufer et al. (1998) id ibid.] facilitate the formation of triple, tightly bound AChE-R/RACK1/PKCβII complexes.
Thus a further aspect of the invention relates to a method for the screening of drugs for treatment of the nervous system. According to this aspect, a test drug is selected by a screening method for a candidate substance which is a modulator of the interactions between AChE-R/RACK1/PKC. Of particular interest is a drug which is specifically aimed at affecting CNS properties and treating CNS-associated disorders. This screening method comprises the steps of: (a) providing a reaction mixture comprising the AChE-R variant of acetylcholinesterase or any functional fragment thereof, the cognate receptor for activated kinase C (RACK1) and the protein kinase C βII (PKCβII); (b) contacting said mixture with a test drug under suitable conditions for said interaction; and (c) determining the effect of the test drug on an end-point indication. The effect of the test drug on the end-point is indicative of modulation of said interaction by the test drug.
In general, the present invention is directed to screening methods for identifying modulators of particular signal pathways. The interaction of the three complex participants (AChE-R/RACK1/PKC) is observed in the presence and absence of a candidate modulator. More particularly, the screening assay according to the invention is conducted by assessing the interaction between AChE-R, PKC and RACK1 either by measuring binding directly or by measuring a physiological or metabolic effect. The measurement is made in the presence and in the absence of a candidate modulator. Successful candidates which agonize the signal, effect an increase in a metabolic or physiologic output, whereas antagonists effect a decrease in a selected metabolic or physiological end point.
Depending on the assay system chosen, the interaction and its modification can be observed in a variety of ways, including intracellular binding assays affecting an observable parameter; either a physiological readout, such as change in subcellular distribution, or an in-vitro co-precipitation.
In one specific embodiment, the reaction mixture used by the method of the invention may be a cell mixture or a cell-free mixture.
It is known to the man of skill in the art that protein-protein interactions are susceptible to conditions such as pH, ion concentration, temperature, and any other factors that may interfere with such interactions. Thus, according to a specific embodiment, this reaction mixture may optionally further comprise solutions, buffers and compounds which provide suitable conditions for interaction between AChE-R/RACK1/PKC and the detection of an end-point indication for said interaction.
In yet another specific embodiment the screened modulator may either inhibit or enhance the interaction between AChE-R/RACK1/PKC. Therefore, modification of the end-point indicates modulation of the interaction between AChE-R/RACK1/PKC by the test drug. For example, decrease or absence of the end point, in the presence of the test drug, indicates inhibition of the interaction between AChE-R/RACK1/PKC. Alternatively, presence or any increase of the end-point indicates enhancement of the interaction between AChE-R/RACK1/PKC by the test drug.
In a particular embodiment, the reaction mixture used by this screening method of the present invention is a cell-free mixture. According to this embodiment, the screening method comprises the steps of: (a) providing a cell free mixture comprising the AChE-R variant of acetylcholinesterase or any functional fragment thereof, RACK1 and PKCβII; (b) contacting said mixture with a test drug under conditions suitable for an in vitro interaction; and (c) determining the effect of the test substance on co-precipitation of PKCβII and RACK1 with the AChE-R or fragment thereof as an end-point indication. Absence of said co-precipitation indicates inhibition of formation of a complex between AChE-R/RACK1/PKC by the test drug, whereas increase in said co-precipitation indicates enhancement of this complex formation.
In a particular embodiment, the cell-free mixture used by the method of the present invention comprises any one of AChE-R variant of acetylcholinesterase or any functional fragment thereof, RACK1 and PKCβII. These proteins may be provided as purified recombinant proteins or alternatively, as a cell lysate of cells expressing these proteins.
According to a specifically preferred embodiment, the AChE-R variant of acetylcholinesterase comprised within the cell free mixture may be a fusion protein. A “fusion protein” as used herein is a recombinant protein made of segments, which are naturally not normally fused in the same manner. As a non limiting example such fusion protein may comprise AChE-R or functional fragment thereof and any one of GST (Glutathion-S-Transferase) and GFP (Green Fluorescent Protein, as exemplified in Example 14). Fusion protein of any of the three complex precipitant proteins may alternatively comprise a protein such as MBP (maltose-binding protein). Thus, a fusion product of the AChE-R molecule with any one of GFP and GST, is a continuous protein molecule having sequences fused by a typical peptide bond, typically made as a single translation product and exhibiting properties derived from each source peptide.
In yet another alternative embodiment, the reaction mixture used by the present invention is a cell mixture. More particularly, the cell mixture may be a transfected cell culture. According to this embodiment, the screening method comprises the steps of: (a) providing transfected cell culture expressing the AChE-R variant of acetylcholinesterase or functional fragment thereof, the cognate receptor for activated kinase C (RACK1) and the PKCβII; (b) contacting said transfected cell culture with a test substance; (c) detecting the interaction between AChE-R/RACK1/PKC in the presence of the test drug by searching for an end-point indication, whereby modification of said end-point indicates modulation of complex formation between AChE-R/RACK1/PKC by the test drug.
According to a particular embodiment, the transfected cell may be transfected by: (a) an expression vector comprising a nucleotide sequence coding for the AChE-R variant of AChE or functional fragment thereof; and/or optionally (b), constructs comprising a nucleic acid sequence coding for any one of the cognate receptor for activated kinase C (RACK1) and the PKCβII.
“Expression Vectors”, as used herein, encompass vectors such as plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system. Such system typically includes a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of RNA expression, a sequence that encodes a suitable ribosome binding site, RNA splice junctions, sequences that terminate transcription and translation and so forth. Expression vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell.
The term “operably linked” is used herein for indicating that a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Plasmids are the most commonly used form of vector but other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. (1985 and supplements) Cloning Vectors: a Laboratory Manual, Elsevier, N.Y.; and Rodriquez, et al. (eds.) (1988) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, which are incorporated herein by reference.
Accordingly, the term control and regulatory elements includes promoters, terminators and other expression control elements. Such regulatory elements are described in Goeddel [Goeddel et al. (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.]. For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding any of the three desired proteins used by the screening method of this invention.
In general, such vectors contain in addition specific genes, which are capable of providing phenotypic selection in transformed cells. A variety of selectable markers can be incorporated into any construct. For example, a selectable marker which confers a selectable phenotype such as drug resistance, nutritional auxotrophy, resistance to a cytotoxic agent or expression of a surface protein, can be used. The expression vector of the invention may further comprise a tag sequence. Such sequences enable the detection and isolation of the recombinant protein. As a non-limiting example such tag sequences may be any one of HA, c-myc, GST, GFP and His-6.
The use of prokaryotic and eukaryotic viral expression vectors to express the genes coding for the AChE-R and optionally for the PKCβII and the RACK1 according to the present invention, is also contemplated.
The vector is introduced into a host cell by methods known to those of skilled in the art. Introduction of the vector into the host cell can be accomplished by any method that introduces the construct into the cell, including, for example, calcium phosphate precipitation, microinjection, electroporation or transformation. See, e.g., Ausubel, F. M. ed. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York.
“Cells” or “transfected cells” are terms used in the present invention. It is understood that such terms refer not only to the particular subject cells but to the progeny or potential progeny of such a cell. Because certain modification may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
“Transfected cell” as used herein refers to cells which can be stably or transiently transfected with vectors constructed using recombinant DNA techniques. A drug resistance or other selectable marker is intended in part to facilitate the selection of the transformants. Additionally, the presence of a selectable marker, such as drug resistance marker may be of use in keeping contaminating microorganisms from multiplying in the culture medium. Such a pure culture of the transfected cell would be obtained by culturing the cells under conditions which require the induced phenotype for survival.
As used herein, the term “transfection” means the introduction of a nucleic acid, e.g., an expression vector, into recipient cells by nucleic acid-mediated gene transfer.
It is to be appreciated that the cell or the transfected cell used by the screening method of the present invention may further contain endogenously or exogenously expressible possible interacting molecules essential for activation of said pathway or said complex formation.
One preferred end-point indication for the “cell mixture” and preferably the transfected cell based screening method, may be the subcellular translocation of catalytically active PKCβII. Subcellular distribution of PKCβII can be detected by a visually detectable signal, generated for example, by using in situ hybridization or immunohystochemistry. A visually detectable signal may be for example a fluorescent signal (
In general, PKC is mainly referred to as a morphologically active kinase. However, PKCβII has been shown to be associated with oxidative [Paola et al., (2000) Biochem Biophys Res Commun, 268, 642-6] and ischemic stresses [Cardell and Wieloch, (1993) J Neurochem, 61, 1308-14] and essential for fear conditioning [Weeber et al., (2000) J Neurosci, 20, 5906-14]. Intriguingly, genomic disruption of the glucocorticoid receptor, which upregulates AChE-R production [Grisaru et al. (2001) ibid] abolished anxiety responses [Tronche et al. (1999) Nat Genet, 23, 99-103]. The current findings of the present invention therefore propose a chain of events that may assist in overcoming traumatic stress responses. This cascade initiates with glucocorticoid hormone release, proceeds with transcriptional activation and alternative splicing to elevate AChE-R levels and results in RACK1 and PKCβII mobilization. AChE-R thus emerges as a modulator, and PKCβII as an initiator of long-term morphological responses to stress. Relevant processes include the reported PKC-induced inhibition of pre-synaptic metabotropic glutamate receptors [Macek et al. (1998) J Neurosci, 18, 6138-46], coupling to the cAMP response element binding protein in CA1 neurons [Roberson et al. (1999) J Neurosci, 19, 4337-48] and the PKC-regulated release of vesicle pools [Stevens and Sullivan, (1998) Neuron, 21, 885-93]. The increase in neuronal PKCβII in AChE-R transgenic mice further proposes that when such changes become permanent they can confer stress protection, whereas the PKCβII disruption study indicates that chronic impairments in this cascade may be detrimental. Nevertheless, excessive AChE-R production may also be detrimental, at least for recovery from closed head injury [Shohami et al. (2000) id ibid.].
Similarly to the “cell-free mixture”-based screening method, another preferred end-point indication for the “cell mixture” based screening method, may be co-precipitation of PKCβII and RACK1 with the AChE-R or functional fragment thereof. Co-precipitation of these interacting molecules leads to a detectable signal, whereby modification of said detectable signal in the presence of the test drug indicates modulation of the formation of a complex between AChE-R/RACK1/PKC by said test drug.
In yet another specifically preferred embodiment, the cell or the transfected cell used by the screening method of the invention may be a prokaryotic or eukaryotic cell, particularly a bacterial cell, yeast cell, an insect cell, a plant cell or preferably a mammalian cell. Most preferred are cells selected from the group consisting of COS and PC12 cells.
Based on the results described in Examples 18 to 22, AChE-R and/or PKCβII levels should be also tested in patients with post-traumatic stress disorder [McEwen, (1999) Annu Rev Neurosci, 22, 105-22], post-stroke phenomena and inherited susceptibility to processes in which PKCβII plays a major role, e.g. panic attacks [Gorman et al., (2000) Am J Psychiatry, 157, 493-505], where fear conditioning is intimately involved. Likewise, the dissociation between RACK1 and PKCβII under ethanol exposure [Ron et al. (2000) J Biol Chem, 274, 27039-46] may be relevant to the stress-suppressing effect of alcohol. In addition, it would be interesting to test PKCβII levels and subcellular localization in patients hypersensitive to anticholinesterases (e.g Alzheimer's disease drugs) which also induce AChE-R overproduction [Kaufer et al. (1998) ibid; Shapira et al. (2000) Hum Mol Genet, 9, 1273-81].
Therefore, a candidate drug which modulates the interaction between AChE-R/RACK1/PKCβII, may affect fear conditions, panic and traumatic stress responses.
In a yet further embodiment of the screening method, the modulator of the interaction between AChE-R/RACK1/PKC also modulates the expression and intracellular distribution of RACK1 and/or PKCβII.
Triple AChE-R/RACK1/PKCβII complexes are likely involved with the neuronal redistribution of PKCβII in brain development [Gallicano et al., (1997) Bioassays 19, 29-36], aging [Battaini et al. (1999) Exp Neurol 159, 559-64] and neurodegeneration [McNamara et al. (1999) J Neurochem 72, 1735-43], all of which involve considerable modulations in AChE-R levels. AChE-R is further expressed in other RACK1/PKCβII producing tissues, including epithelial, muscle, hematopoietic, and germ cells [Soreq and Seidman, (2001) id ibid] where its capacity to induce PKCβII-mediated changes should be examined.
As shown by Examples 19-21, AChE-R/RACK1/PKCβII complexes are mobilized from their resting state intracellular location, translocating RACK1 to the perikaryal circumference and PKCβII into densely packed clusters. Modified properties and location of PKCβII in stress-responding neurons may possibly change the stress-induced kinase activation, phosphodiesterase mobilization, or other processes. Several RACK1 reports have addressed the effect of PKC-RACK interactions on PKC activity. Addition of RACK1 to PKC in the presence of PKC activators did not significantly change PKC activity [Chang et al. (1998) id ibid.], yet PKC activators induced RACK1 interaction with PKC, Src [Chang et al. (2001) id ibid.] and integrin β1 [Liliental and Chang (1998) id ibid.], presumably by inducing a conformational change in RACK1 which exposes it to the binding of other proteins. PKC activation is also known to induce the MAPK pathway associated with stress responses [Gil et al. (2000) id ibid; Kaneki et al. (1999) id ibid.].
The presence of PKCβII in the deep cortical layers and in the stratum oriens and stratum radiatum of CA1 in hippocampus (
A yet further aspect of the present invention is a method for the in vivo screening of candidate drugs that affect the central nervous system, wherein said drug is a modulator of an interaction between AChE-R/RACK1/PKC. This screening method comprises the steps of: (a) providing an AChE-R transgenic animal; (b) administering the test drug to said animal; (c) sacrificing the animal and dissecting its brain to give samples for preparation of brain extracts or for immunohistochemistry; (d) detecting the expression of RACK1 or PKCβII in said brain samples; and (e) determining the effect of the test drug on an end-point indication, wherein said effect is indicative of the in vivo modulation of said interaction by the test drug. The end-point indication of this screening method is the expression of RACK1 and PKCβII in the brain.
The preferred transgenic animals are Xenopus and mammals, such as mice, cows, goats, pigs and sheep. In said transgenic animals, the transgene is a recombinant expression vector containing a promoter controlling the transcription of the sequence encoding the “readthrough” form of AChE, and wherein said promoter is selected from the group of eukaryotic host cell compatible promoters consisting of CMV, CMV-like, AChE and AChE-like promoter. Most preferably, the transgenic animal is an AChE-R transgenic mouse, which has been described in Sternfeld et al. (2000) [Sternfeld et al. (2000) id ibid].
The brain of the transgenic animal should be dissected in a manner suitable for either RNA or protein analysis, either in tissue sections or in solution. Method for appropriate dissection of tissues for sections and immunohistochemistry or RNA staining are known to the man of skill in the art, and have been described in numerous publications, e.g. Wilkinson and Nieto (1993) Methods in Enzymology, 222, 361-372; Marti et al. (1995) Development 121, 2537-2547. Similarly, methods for dissection in order to prepare tissue extracts have been described by e.g. Sternfeld et al. (2000) [Sternfeld et al. (2000) id ibid].
In the above-described method, RACK1 and PKCβII expression can be detected through procedures specific for detecting the expression of RNA or protein.
In one specific embodiment, the RNA detection is performed by means appropriate for RNA detection, said means selected from the group consisting of RT-PCR, Northern Blot, in situ hybridization, RNAse protection and S1 nuclease analysis. These and other techniques for RNA detection have been described in [Sambrook et al. (1989) id ibid.], herein incorporated by reference.
In another specific embodiment, the protein detection is performed by means appropriate for protein detection, said means selected from the group consisting of Western Blot and immunohistochemistry. These and similar methods for protein detection have been described in Current Protocols in Immunology, Coligan et al. (eds), John Wiley & Sons. Inc., New York.
Transgenic AChE-R -filled neurons with both punctiform PKCβII and RACK1 labeling are mostly relevant to stress-response inhibitory pathways [Herman and Cullinan, (1997) Trends Neurosci, 20, 78-84]. These were located in cortical upper layers, the hippocampal CA1 region, the lateral septum and the basolateral amygdala. PKCβII punctiform patterns also appeared in a subset of basolateral amygdala neurons, which are generally considered excitatory to psychological stress. However, the existence in this region of stress-inhibitory neurons, has been discussed with regards to the regulation of fearful behavior [Davis et al., (1994) Trends Neurosci, 17, 208-14]. These neurons presumably suppress other basolateral amygdala neurons that are stress-excitatory, consistent with the limited stress-related neuropathology hallmarks in the brain of AChE-R transgenic mice [Sternfeld et al. (2000) id ibid.]. The neuronal location of AChE-R/RACK1/PKCβII complexes further suggests relevance to the PKC-activated down regulation of transient K+ channels in dendrites of hippocampal CA1 pyramidal neurons [Hoffman and Johnston, (1998) J Neurosci, 18, 3521-8]. Such downregulation may contribute to the reduced stress overload of AChE-R overexpressing mice.
It is to be understood that the term modulator is used throughout this specification as any substance, being it a drug or a compound, capable of enhancing or diminishing the interactions within the complex AChE-R/RACK1/PKC. The modulator may be able to interact with the complex, or with each separate component of the complex, or even with any combination of two components of the complex, in such a way that it may strengthen or weaken its interactions. Alternatively, the modulator may affect, i.e., induce or repress, augment or diminish, the expression of at least one of the components of the complex. In addition, another effect of the modulator may be to trigger a change in the intracellular localization of at least one of the components of the complex. In all of the above-exemplified situations, the component of the complex might be in the complex or not, to be affected by the competence of the modulator. Thus the modulator may be able to affect each molecule when not in the complex, and thereby inhibit complex formation.
It is noteworthy that each of the components in the triple AChE-R/RACK1/PKCβII complexes represents one out of several options. Thus, other PKC isoforms most likely interact with different shuttling proteins, with distinct and different effects on the complex physiological phenomena that follow traumatic experiences. Likewise, RACK1 operates as a shuttling vector for many other proteins, and at least part of these interactions (e.g. phosphodiesterase) are likely to compete with the currently described one and ameliorate its consequences. Finally, the AChE-R protein is one out of three diverse AChE isoforms, each with its own C-terminal peptide and possibly different interactions.
The test drug for the screening and evaluation methods of the invention may be any substance selected from the group consisting of protein based, carbohydrates based, lipid based, nucleic acid based, natural organic based, synthetically derived organic based, antibody based and metal based substances.
As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, double-stranded polynucleotides and single-stranded such as sense or anti-sense. More particularly, anti-sense directed against the AChE-R variant may attenuate said complex formation.
Among successful candidate drugs or substances will be peptides which mimic regions on either of the three complex forming proteins, as well as non-peptidic small molecules. Due to their ease of identification, these peptides are particularly useful in alternate forms of the screening assays that detect modification and modulation of the interaction between AChE-R/RACK1/PKC. Although the assay methods disclosed may not all be suitable for direct screening of large chemical libraries, they do enable a sophisticated screening of candidates that can be combined with other techniques for selecting leads.
In a particular embodiment a protein or antibody based substance may be a product of a combinatorial library. Thus, the invention is also directed to methods to screen libraries of candidate modulators using the above-described methods and to peptides representative of sites on any of the three complexed proteins, which are themselves useful in these assays as well as in other applications involving the relevant interaction.
The drugs to be evaluated by the methods of the invention can be any candidate or known drugs, e.g. drugs for the treatment of anxiety conditions, post-traumatic stress, Alzheimer's disease, muscle malfunctioning, neurodegenerative disorders, damage resulting from exposure to xenobiotics, panic, neuromuscular disorders, Parkinson's disease, Huntington's chorea, muscle fatigue, multiple chemical sensitivity, autism, multiple sclerosis and Sjogren's disease.
In yet a further aspect, the invention provides for a method for the treatment of stress-associated conditions or disorders, for a subject in need of such treatment, said method comprising:
- a. providing a composition comprising as active ingredient a modulator of an interaction between AChE-R/RACK1/PKC;
- b. administering a therapeutic effective amount of said composition to said subject;
wherein said modulator is selected according to the drug screening methods provided by the invention.
“Treatment” refers to therapeutic treatment. Those in need of treatment include those already with a disease or disorder, whether at clinical or pre-clinical stage.
Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.EXAMPLES
Cell source: UCB was collected, following informed consent of the parents and with the approval of the Sourasky Medical Center Ethics Committee, as previously described [Grisaru et al. (1999a) Am. J. Obstet. Gynecol, 180, 1240-1243]. Following 1:1 (v/v) dilution in Iscove's modified Dulbecco medium (IMDM, Beit Haemek, Israel), mononuclear cells were separated using 3% gelatin (Difco, Detroit, Mich.) and Ficoll-Hypaque gradients (<1.077 g/ml; Pharmacia, Uppsala, Sweden) [Pick et al. (1998) Br. J. Haematol. 103, 639-50]. CD34+ cells were enriched using CD34 immunoglobulin-coated magnetic beads (CD34 progenitor cell selection system, Dynal, Norway). CD34+ cells analysis was performed by flow cytometry (Becton Dickinson Immunocytochemistry System Inc., San Jose, Calif.), using CD34-PE (Becton Dickinson Immunocytometry System, Inc.) and CD45-FITC (Dako, Glostrup, Denmark) monoclonal antibodies. May-Grünwald-Giemsa staining revealed stem cell morphology.
Liquid cultures: UCB CD34+ cells were set for liquid cultures at a concentration of 105/mL in IMDM, containing 10% autologous plasma, 2 mM L-glutamine (Sigma Chemical Co., St Louis, Mo.), penicillin (100 mg/mL), streptomycin (100 mg/mL), amphotericin B (2×10−5 M) (Sigma Chemical Co.), and heparin (20 IU/mL, Gibco, Grand Island, N.Y.), all in a fully humidified atmosphere at 37° C. and 5% CO2. The following elements were added where noted:
- 1. Hematopoietic growth factors: Interleukin 3 (IL3, 5 ng/mL, Immunex, Seattle, Wash.), interleukin 6 (IL6, 50 ng/mL, R&D Systems, Minneapolis, Minn.), TPO (1 ng/mL, R&D Systems), stem cell factor (SCF, 10 ng/mL, R&D Systems), FLT-3 ligand (FLT3, 50 ng/mL, R&D Systems), granulocyte-macrophage colony stimulating factor (GM-CSF, 50 ng/mL, Biogenesis Ltd, Bournemouth, UK) and combinations of the above, for 24 hr to 28 days (supplemented every 4 days).
- 2. Endothelial Growth factors: Epidermal growth factor (EGF 10 ng/ml, Sigma Chemical Co.), basic Fibroblast growth factor (bFGF 20 ng/ml, Sigma Chemical Co.) incubated in a serum free medium (SFM) for 48hr.
- 3. Stress mimicking conditions: Hydrocortisone sodium succinate (Abic Ltd., Netanya, Israel) at concentrations equivalent to normal, intermediate and stress serum cortisol levels (0.1, 0.6 and 1.2 μM, respectively (De Vroede et al., Arch. Des. Child 78, 544-7, 1998), for 24 hr.
- 4. Antisense oligonucleotides: 3′-terminal 2′-O-Methylated 15- and 20-mer oligodeoxynucleotides in the antisense (AS) orientation, targeted against the common sequence domain in human AChEmRNA and BuChE mRNA, as control, were used for 24 hr, as detailed elsewhere [Grisaru et al. (1999b) Mol. Cell. Biol. 19, 788-95].
- 5. AChE C-terminal peptides: The following C-terminal peptides of the AChE synaptic (ASP, also denoted SEQ ID: No. 2) and readthrough (ARP, also denoted SEQ ID: No. 1) isoforms were synthesized using a 433A peptide synthesizer (PE Applied Biosystems, Inc., Norwalk, Conn.).
ASP:1-DTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL-40, also denoted as SEQ ID: No. 2.
ARP: 1-GMQGPAGSGWEEGSGSPPGVTPLFSP-26, also denoted as SEQ ID: No. 1.
Length and integrity of the peptide preparations were ensured following purification by HPLC, using a D-6000 chromatography data station (Hitachi Instruments, Inc., San Jose, Calif.). The working concentrations were 50 and 100 ng/mL (supplemented every 4 days) for liquid cultures grown between 24 hr to 28 days.
Culture analyses: Twenty-four hr liquid cultures served for cytochemical staining, in situ hybridization, and cell proliferation assay by 5-bromo-2′-deoxy-Uridine (BrdU) incorporation [Grisaru et al. (1999b) id ibid.]. Twenty-eight day liquid cultures were sampled every 6-8 days for viable cell counting (using trypan blue dye exclusion), cell phenotyping (CD34+, CD33+ and CD41+ quantification using flow cytometry, with CD34-PE, CD33-FITC (Immunotech/Coulter, Hialeah, Fla.), CD41-FITC (Immunoquality Products, Groningen, Netherlands) and CD45-FITC monoclonal antibodies), and growth of progenitor colony (granulocyte-macrophage and megakaryocytic), using previously described techniques (Pick et al. (1998) id ibid.).
Cytochemical staining: Staining of AChE activity was essentially as detailed elsewhere [Grisaru et al. (1999b) id ibid], on non-fixed liquid cultures following 300×g centrifugation on collagen-coated cover slips placed on the bottom of the culture well, in the presence of 10−5 M iso-OMPA (ISO) or BW284C51 (BW), selective inhibitors of BuChE and AChE, respectively [see also Keymer et al.(1999) Eur. J. Neurosci. 11, 1049-57]. Nuclear staining was with 4′,6-diamidino-2-phenylindole [DAPI, see e.g., Peterson et al. (1999) Genetics 152, 427-439].
Animals and tissue collection: Male FVB/N mice (2-6 months old) were sacrificed 24 hr following 4 successive days of a forced swim session as detailed [Kaufer et al. (1998) id ibid.], or a single injection of ARP (34 nmol/kg weight), or phosphate buffer (PB) for control. ARP, a 26 amino acid residue peptide synthesized according to the C-terminal sequence of human AChE-R [Grisaru et al. (1999b) id ibid.] was HPLC purified and mass-spectrometry analyzed for purity. Control naive mice and hAChE-R transgenics were sacrificed with no prior treatment.
Blood samples were allowed to clot 1 hr at room temperature and overnight at 4° C., followed by centrifugation and serum collection.
Testes and seminal vesicles were excised and weighed, fixed in 4% paraformaldehyde or Bouin's fixative for histological staining or kept at −70° C. for protein extraction.
Sperm cells were collected from one cauda epididymis shredded in 1 ml saline.
Evaluation of serum corticosterone levels: concentrations were determined by radioimmunoassay.
Epididymal sperm motility was assessed by visually determining percent motile sperm. Sperm concentration was measured using the Makler chamber (company, city, state).
In situ hybridization: In situ hybridization procedures, were performed on cultured cells and human fetal tissues, as detailed elsewhere [Grisaru et al. (1999b) id ibid; Kaufer et al. (1998) id ibid.]. Cultured cells were centrifuged at 300×g and fixed, using 4% paraformaldehyde, to collagen-coated cover slips placed on the bottom of the culture well. Tissues from fetal hematopoietic organs (AGM, liver, spleen and bone marrow) were obtained in each of the selected gestational stages, from 2-3 normal aborted human fetuses. The project was approved by the Sourasky Medical Center Ethics Committee, and written informed consent was obtained from the parents. 5′-Biotinylated, 2′-O-methylated AChEcRNA probes complementary to 3′-alternative human AChE exons were employed. Detection and quantification of the various AChEmRNA transcripts in fetal tissues were performed as previously described [Grisaru et al. (1999b) id ibid.]. Confocal microscopy scans of the culture-derived cells were obtained using a MRC-1024 Bio-Rad confocal microscope (Hemel Hempsted Herts., UK). A projection was built from each cell image and specific criteria were set for size and intensity of the Fast Red fluorescence. Image-Pro 3.0 software (Media Cybernetics, Silver Spring, Md., USA) was used to analyze the signals obtained. ANOVA (Analysis of Variance) test was used for calculation of p values.
Human sperm smears: Air dried sperm smears of ejaculates collected from male donors or infertility patients were stained with the anti-ARP antibody as mentioned above.
Confocal microscopy: An MRC-1024 Bio-Rad confocal microscope equipped with an inverted microscope and a 63×/2.4 oil immersion objective was used to scan the fast red precipitate used for ARP and ASP immunodetection. Fast red was excited at 488 nm, emission was measured using a 580df32 filter. Sections were scanned every 0.35 μm, and a three-dimensional projection was created from all sections.
For imaging of AChE-R, RACK1 and PKC, brain slices were scanned using a Bio-Rad MRC-1024 scanhead (Hemel Hempsted Herts.,UK) coupled to an inverted Zeiss Axiovert 135M microscope with a 40× oil immersion objective (N.A. 1.3). Excitation wavelength was 488 nm (using 10% of a 100 mW laser power). Fluorescence emission was measured using a 580df32 bandpass interference filter (580 nm±16 nm) for detecting tetra-methyl-rhodamine and a 525/40 filter for detecting fluorescein. The confocal iris was set to 3 mm. Conditions of scanning took into consideration the overlap of fluorescein fluorescence into the rhodamine filter (as were determined by control experiments). Images were then further processed using Image pro Plus 4.01 program (version 4.0, Media Cybernetics, Silver Spring, Md.).
DNA sequence analysis: The reverse sequence of the 7q22 cosmid (accession no. AF002993) containing the human ACHE gene and its upstream sequences, was searched for consensus motifs for binding transcription factors which regulate hematopoietic expression, using the MatInspector program with core similarity of 1, or the Findpatterns program of the University of Wisconsin GCG software package (Quandt et al., Nucleic Acids Res. 23, 4878-84, 1995).
Immunoblot: Mouse serum was diluted 1:10. ARP, ASP, recombinant AChE-S (Sigma Chemical Co.) and recombinant AChE-R extracted from transfected COS cells served as positive controls. Protein electrophoresis in SDS gradient (4-20%) polyacrylamide gels (Bio-Rad Laboratories, Hercules, Calif.) was followed by immunodetection using the rabbit anti-GST-ARP antibodies, Peroxidase-conjugated anti-rabbit immunoglobulins and ECL™ detection (Amersham Pharmacia Biotech, UK).
Methods for Culturing Murine Hematopoietic Cells:
Blood: 1 ml of murine blood is collected by cardiac puncture and immediately deposited into pediatric vaccutainer tubes containing Na citrate. Blood counts are performed using the AcT diff Coulter counter (Coulter-Beckman).
Bone marrow: The tibia and femurs are surgically removed, cleaned and both ends of the bone cut open. The bones are placed in small tissue culture plates containing 2-5 ml of medium composed of RPMI, antibiotics and 10% heat inactivated fetal calf serum (complete medium) supplemented with heparin (5 U/ml) to prevent clotting. The BM contents are flushed out into the medium using a 25 gauge needle. Cells are passed up and down through the syringe three times to guarantee a single cell suspension.
Spleen: The spleen is surgically removed and cleaned and placed in 5 ml of medium composed of complete RPMI with heparin (5 U/ml) as described above. Both ends of the spleen are cut open and the spleen cells are expressed from the organ capsule by squeezing down on the spleen with the back barrel of a 5 cc sterile syringe. A single cell suspension is prepared by passing the cells up and down three times through a 5 ml syringe.
The hematopoietic cells are washed by the addition of an additional 5 ml of medium composed of complete RPMI with heparin, counted by manual hemocytometry and pelleted at 1500 RPM for 10 minutes. The cell pellets are resuspended at a concentration of 5×106-10−7 cells/ml in ice cold freezing medium containing 50% DMSO and 30% RPMI (as above). Cells are allowed to remain on ice for up to 5 minutes and subsequently placed at −20° C. for 30 minutes. Cells are then transferred to −80° C. for a period of up to 6 months; for longer storage, cells are transferred to −180° C.
Hematopoietic Progenitor Cultures—
For all colony assays duplicate samples of 1-2×105 cells are placed into 1 ml of medium plus the appropriate supplements for each cell lineage in small round tissue culture dishes (2.5 cm). These small dishes are placed into a larger TC dish (10 cm) together with a third small dish containing sterile water to prevent evaporation in the cultures. Cells are cultured at 37° C. in a fully humidified atmosphere containing 5% CO2.
Two hundred thousand (2×105) cells are placed into 1 ml of Alpha medium containing antibiotics, 30% methyl cellulose, 10% FCS and 2 ng/ml recombinant murine (r-mu) erythropoietin, and both BFU-E and the smaller CFU-E are counted after 10 days.
CFU-GM and CFU-GEMM
One hundred thousand (1×105) cells are placed into 1 ml of Alpha medium containing antibiotics, 3% agar, 10% FCS and 5 ng/ml or both r-mu IL-3 and r-mu GM-CSF. CFU-GM, CFU-GEMM and CFU-bl are counted after 12 days.
Two hundred thousand (2×105) cells are placed into 1 ml of Alpha medium containing antibiotics, 30% methyl cellulose, 10% FCS and 5 ng/ml. r-mu thrombopoietin and 10 ng/ml stem cell factor. CFU-MK and BFU-MK are counted by staining the cells for AChE after 12 days.
Animal Models and In Vivo Experiments—transgenic—
FVB/N mouse pedigrees expressing human AChE variants were described elsewhere, as were the biochemical methods for measuring AChE activity [Sternfeld et al. (1998b) J. Physiol. Paris, 92, 249-55]. The confined swim protocol for exerting acute psychological stress was performed as detailed [Kaufer et al.(1998) id ibid.]. Immediately following the stress, the treated mice were injected intraperitoneally with 100 ng ARP or 0.03 ng AS1, both per gram body weight. Another group of non-stressed mice were injected either with normal saline or ARP. Twenty-four hours later, the animals were sacrificed and peripheral blood was collected in EDTA covered tubes (Becton Dickinson Immunocytochemistry System, Inc., San Jose, Calif.) prepared with 25 units of heparin sodium USP (Kamada LTD, Kibbutz Beit-Kama, Israel). Whole blood AChE activity was analyzed, and WBC and platelet counts determined, using an Ac-T diff hematology analyzer (Beckman Coulter, Inc., Fullerton, Calif.).
Cytochemical and Immunohistochemical Staining
Staining of AChE activity was as detailed above. For immunohistochemistry, murine bone marrow smears were fixed with 4% paraformaldehyde (10 minutes, room temperature, RT); permeabilized with buffer containing 20 mM HEPES (pH 7.4), 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2 and 0.5% Triton X-100 (4 minutes on ice); washed twice with PBS (5 min each, RT); incubated in 1% H2O2 in methanol (15 min RT); and washed twice in PBS. Non-specific sites were blocked by incubating in 5% horse serum in PBS (20 min, RT). Labeling was in a humidified chamber with 1:50 dilution of affinity purified rabbit antiserum prepared against GST-fused recombinant ARP (1 hr, RT). Following 3 washes with PBS, smears were incubated with 1:100 biotinylated goat anti-rabbit Ig (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) (30 min, RT). After 3 PBS washes, a mixture of biotin and avidin-peroxidase was added (30 min, ABC Elite Kit, Vector Labs, Burlingame, Calif.) and reacted with diaminobenzidine-hydrogen peroxide mixture (Sigma Chemical Co., St. Louis, Mo., 10 min), followed by counterstaining with Meyer's hematoxylin mixture (Sigma Chemical Co., St. Louis, Mo.), and immunomounting.
Immunohistochemistry and Nuclear Staining in Testis Sections
Detection of the AChE core protein and/or its variant C-terminal peptides was performed on 7 μm thick paraffin embedded testis sections with either anti-ASP (C-16; Santa Cruz Biotechnology, Santa Cruz, Ca) or anti-ARP polyclonal antibodies (Sternfeld et al. (2000) id ibid.]. PCNA was detected using a dedicated staining kit (Zymed Laboratories, San-Francisco, Calif.); nuclear staining and counterstaining was with DAPI and hematoxylin, respectively (Sigma, St. Louis Mo.).
Expression of Recombinant ARP
The sequence, coding the C-terminal region of I4 (i.e., the “readthrough” variant of acetylcholinesterase, comprising the ARP peptide sequence), was amplified by PCR using the following oligonucleotide primers:
- GCT GGA TCC ATC GAG GGG CGA GGT ATG CAG GGG CCA GCG GGC (14-up), also denoted as SEQ ID: No. 4,
- and TAT AAG CTT CTA GGG GGA GAA GAG AGG GGT (I4-down), also denoted as SEQ ID: No. 5, and introduced into pGEX-KG (ATCC accession No. ATCC77103, see also Anal. Biochem. 192:262-267, 1991) plasmid.
GST and I4-GST fusion protein were purified from the supernatant of E. coli lysate by affinity chromatography on glutathione-Sepharose (Pharmacia), eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, dialyzed to 0.1 M ammonium acetate buffer, pH 7.0, aliquoted and lyophilized. The stability and identity of the protein was confirmed by SDS-PAGE. The following protease inhibitors were used during the preparation: aprotinin (10 microgram/ml), benzamidine (5 mM), Pefabloc SC (0.2 mM), and EDTA (1 mM). Prior to affinity chromatography, the E. coli lysate was incubated for 20 min at 37° C. with 0.2 mM Mg-ATP in order to dissociate the fusion proteins from contamination of bacterial proteins. The procedure was performed according to Pharmacia recommendations.
Two New Zealand female rabbits were immunized subcutaneously with 0.3 mg fusion protein in complete Freund's adjuvant, and then reimmunized monthly with 0.2 mg fusion protein in incomplete Freund's adjuvant. Blood samples were taken 10 days after the immunization. The specific antibodies in the sera were detected by ELISA on immobilized fusion protein, in the presence of excess of soluble GST (20 microgram/ml). The reacting sera were chosen for antibody purification. The immobilized I4-GST, GST and E. coli lysate were prepared using Affigel 10 (Bio-Rad) according to the manufacturer's recommendations.
Crude IgG fraction was prepared from the serum by 50% saturation (NH4)2SO4 precipitation and dialyzed in 100 mM Tris-HCl, pH 8.0. In order to get rid of anti-GST antibodies, the IgG fraction was incubated with GST beads (Affigel 10, Bio-Rad) overnight at 4° C. The bound material was eluted with 4.5 M MgCl2. The procedure was repeated with the unbound material several times, until no antibodies were eluted from GST beads. In order to get rid of antibodies against possible contamination of bacterial proteins, the same procedure was performed with immobilized heat-shocked E. coli lysate proteins.
The unbound material was then applied to I4-GST beads (Affigel 10, Bio-Rad), incubated 2 hr at room temperature or overnight at 4° C., and the bound material was eluted with 3.5 M MgCl2. The eluted antibodies were dialyzed against 10 mM Tris-HCl, pH 8.0, and then against PBS, containing 0.025% NaN3.
A fragment of human AChE-R cDNA (nt 1796-1865 of hAChE, accession number M55040, followed by nt 1-111 from the genomic hAChE I4-E5 domain (Accession No. S71129, stop codon in position 86) was used as “bait” for the two-hybrid screen. Cloning into the EcoR1/SmaI sites of pGBK-T7 (Clontech, Palo Alto, Calif.), yielding the plasmid pGBK-ARP1. Cloning of the “bait” sequence into the Bspl2OI/XbaI sites of pEGFP-C2 (Clontech) yielded the pGARP vector. The AChE-R expressing plasmid used for transfections has been described in detail [Seidman et al.(1995) Mol Cell Biol., 15, 2993-3002].
The “bait” EcoRI/HpaI fragment of AChE-R cDNA encodes the 51 amino acid long C-terminal fragment of AChE-R fused to the DNA-binding domain (BD) (amino acids 1-147) of the yeast GAL4 transcriptional activator. An amplified and CsCl gradient-purified human fetal brain cDNA library cloned into the AD vector [Chien et al. (1991) Proc. Natl. Acad. Sci USA 88, 9578-9582] encodes for a fusion protein with the yeast GAL4 activation domain AD, (amino acids 768-881). The AH109 yeast strain (Clontech) was sequentially transformed with the pGBK-ARP1 plasmid, and with 10-25 μg of the library DNA, using the Yeastmaker transformation system (Clontech). A total number of 240,000 independent clones were screened.
Preparation of Recombinant RACK1
A plasmid overexpressing MBP-RACK1 in E. coli pDEM31, a derivative of pMAL-c2 (New England Biolabs, Beverly, Mass.) [Rodriguez et al. (1999) id ibid.], was a kind gift from Dr. Daria Mochly-Rosen, Stanford. The pDEM31 vector expresses in E. coli recombinant RACK1 fused to the maltose binding protein, which was purified on an amylose affinity column (New England Biolabs). The 36 kDa RACK1 protein was released by proteolysis with factor Xa (New England Biolabs).
Cell Transfection Experiments
PC12 cells were transiently transfected with the plasmid encoding AChE-R, using Lipofectamine Plus (Life Technologies, Paisley, UK). Cells were lysed 24 hours following transfection in lysis buffer (0.1M phosphate buffer pH 7.4, 1% Triton X-100, and Complete mini protease inhibitor cocktail (Roche, Mannheim, Germany)). Cell debris was removed by centrifugation at 12,000×g for 10 min.
Protein samples containing recombinant RACK1 were separated by SDS-PAGE. Following blotting, the nitrocellulose membrane was incubated in a blocking solution (3% non fat dried milk, 2% BSA, 0.2% Tween-20 in Tris buffered saline (TBS, 0.1M tris pH 7.4, 1.7M NaCl)) for 1 hour. Overlay was in 6 ml of 1:20 diluted clear supernatant from homogenates of PC12 cells expressing either human AChE-R [Grifman et al. (1998) Proc Natl Acad Sci USA 95, 13935-40]. The final protein concentration was 2mg/mL, in 50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 0.1% BSA, 0.1% polyethylene glycol (PEG), 12 mM beta-mercaptoethanol and Complete mini protease inhibitors cocktail (Roche), in a final concentration of 0.05% Triton X-100. Following incubation (lh, room temperature), unbound material was removed by 3 brief washes and three 5 min washes in 0.05% Tween-20 in TBS. Following fixation with 4% paraformaldehyde (30 min, at 4° C.), bound AChE was detected using goat polyclonal antibodies targeted to the N-terminal domain of hAChE (Cat. No. sc-6431, Santa Cruz Biotechnology, Santa Cruz, Calif.; dilution 1:500).
Clear supernatants of PC12 or COS cell homogenates (200 μL, 1.5 mg protein/mL) were prepared by manual homogenization, followed by 30 min centrifugation at 12,000×g, 4° C. Supernatants were diluted 5-fold with NET buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% gelatin and Complete mini protease inhibitors cocktail (Roche)), in a final concentration of 0.05% Triton X-100). Goat polyclonal antibodies (Santa Cruz) targeted to the N-terminal domain of hAChE (10 μL, 200 μg/mL) were added for overnight rotation at 4° C. 75 μL of Protein G MicroBeads (Miltenyi Biotec, Bergisch Galdbach, Germany) was added and incubation continued for another h. Mixtures were loaded on MACS magnetic separation columns (Miltenyi Biotec), washed 3 times with 200 μL of TBS buffer containing 0.05% Tween-20 and eluted with gel loading buffer. Elutes were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, Calif.), blotted and incubated with the noted detection antibodies. For immunoprecipitation, dissected mouse brain regions were homogenized in nine volumes of the lysis buffer. Homogenates were passed several times through a 21 G needle. Insoluble debris was removed by a 30 min 12,000×g centrifugation. Homogenates were kept frozen at −70° C. until use.
Laboratorv Animals and Stress Experiments
Male 6-8 weeks old FVB/N mice were subjected to saline injection (0.2 ml, intraperitoneal) which induces mild psychological stress in this stress-sensitive strain. Stressed mice, control and AChE-R transgenic mice [Sternfeld et al. (2000) id ibid] were sacrificed 24 h post-injection. To prepare brain sections, four mice from each line were deeply anesthetized with Pental (pentobarbitone sodium 200 mg/ml, CTS Chemical industries, Petach Tikva, Israel) at a dose of 100 mg/Kg and transcardially perfused with 4% (vol/vol) paraformaldehyde. Brains were post-fixed by immersion in 4% (vol/vol) paraformaldehyde (overnight, 2-8° C.) and incubated in 12% (vol/vol) sucrose in 0.1M phosphate buffered saline (PBS). Coronal cryostat sections (30 μm) were floated in PBS and kept at −20° C. in 40% (vol/vol) ethylene glycol and 1% polyvinylpyrrolidone in 0.1 M potassium acetate (pH 6.5) until staining.
Antibodies and Working Dilutions
Immunohistochemical analyses were essentially as previously described [Shoham and Ebstein (1997) Exp. Neurol. 147, 361-76; Sternfeld et al. (2000) id ibid.], using rabbit anti-ARP [Sternfeld et al. (2000) id ibid.] 1:100, rabbit anti PKCβII (Cat. No. sc-210, Santa Cruz) 1:100, rabbit anti PKCβII (Sigma-Rehovot, Israel) 1:250 and mouse anti RACK1 (Cat. No. R20620, Transduction Laboratories, San Diego, Calif.) 1:200. Immunoblot analyses were with rabbit anti-N-terminus AChE antibodies (Cat. No. N-19, Santa Cruz), 1:500; mouse monoclonal antibody against all isoforms of PKC of mouse, rat and human origin (Cat. No. sc-80, Santa Cruz), dilution 1:100, or mouse monoclonal antibody against RACK1 (R20620,Transduction Laboratories), dilution 1:2500.
Sections were incubated with the primary antibody and then with biotin-conjugated donkey anti-rabbit antibody (Cat No. AP132B, Chemicon, Temecula, Calif.; 1 hour, room temp., overnight at 2-8° C.) and extravidin-peroxidase (Sigma). RACK1 staining was further preceded by trypsin type II treatment (Sigma), 1 μg/ml with calcium chloride 0.001% for 2 min, at room temp., which required the addition of 0.001% soybean trypsin inhibitor (Sigma) during staining. Detection was with horseradish peroxidase-conjugated goat anti-mouse antibody (1:100 dilution, Sigma). Pre-incubation of anti-RACK1 with 10 μM RACK1 for 1 h at room temp totally eliminated staining with anti-RACK1, demonstrating specificity. For all antibodies, staining was intensified with 0.075% diaminobenzidine and 0.05% nickel ammonium sulfate.
Fluorescence Double Labeling of RACK1, ARP and PKCβII
RACK1 and ARP: Primary staining solutions contained 0.001% trypsin inhibitor (Sigma type IIS), 0.3% Triton X100, 0.05% Tween 20, 2% normal goat serum, 2% normal donkey serum, rabbit anti-ARP1 (1:100) and mouse anti-RACK1 (1:100). The secondary antibody solution contained 0.3% Triton X100, 0.05% Tween 20, 2% normal goat serum, 2% normal donkey serum, donkey-anti-rabbit conjugated to fluorescein (Chemicon, AP182F) diluted 1:100 and goat-anti-mouse conjugated with tetra-methyl-rhodamine (Sigma, T7782) diluted 1:800. Sections were mounted on SuperFrost slides (Menzel Glaser, Freiburg, Germany), air-dried, covered in ImmuMount (Shandon, Pittsburgh, Pa.) and covered for microscopy.
PKCβII and ARP1: The primary staining solution contained 0.3% Triton X100, 0.05% Tween 20, 2% normal goat serum, 2% normal donkey serum, rabbit anti-ARP (1:100) and mouse-anti-PKCβII (Sigma, P8083), diluted 1:500. Secondary antibody solutions and preparation for microscopy were as specified above for ARP and RACK1.Example 1 Hydrocortisone Elevates ACHE Gene Expression in Hematopoietic Stem Cells
This example relates to ACHE gene expression in hematopoietic stem cells and the influence of hydrocortisone thereon. The inventors have searched the extended promoter of the human ACHE gene (cosmid accession no. AF002993) for consensus motifs that may bind stress-associated and hematopoietic transcription factors. Two such clusters, one located 17 Kb upstream from the transcription start site, and another positioned at the first intron, were found to include motifs for AP1, NFkB, EGR-1 (as identified by a matrix search against the TransFac database, Heinemeyer et al., Nucleic Acids Res. 26, 364-370, 1998), interleukin-6 (IL6), with the consensus sequence CTGGG/AAA, glucocorticoid responsive element (GRE) half palindromic site, TGTTCT, and Stat-5, TTCCCAGAA or TT(C/A)(C/T)N(A/G) (G/T)AA (
The functional effects of the glucocorticoid-binding motifs in the ACHE upstream sequence (
Enriched CD34+ cells were subjected to cytochemical staining for AChE catalytic activity in the presence of 10−5 M iso-OMPA (
The assumption that hematopoietic ACHE gene expression is modulated under stress was tested in CD34+ cells cultured for 24 hr with increasing doses of hydrocortisone. Treated cells were subjected to cytochemical staining for AChE activity as well as to high resolution in situ hybridization followed by confocal microscopic quantification of labeling density. This method provides an accurate and credible tool for the examination of transcriptional responses in the heterogeneous population of primary HSCs from different individuals. Hybridizations were performed for each of the three transcripts of human AChEmRNA presented in
The expression of ARP in CD34+ hematopoietic cells was evaluated by flow cytometry in whole cord blood and bone marrow from a patient with immune thrombocytopenic purpura (ITP), as demonstrated in
These findings demonstrate higher expression of AChE-R in proliferating hematopoietic progenitors from either newborns or individuals suffering from over-proliferation of blood cells.Example 3 Readthrough AChE is Overproduced in the Myeloidogenic Mid-gestation Liver
To study the relevance of each of the AChEmRNA transcripts during development of the hematopoietic organs, in situ hybridization was performed on paraffin-embedded sections taken from human fetuses at different gestational ages. Consistent with the embryonic spatiotemporal shifts in blood cell forming tissues, we observed changes in the labeling intensity with the various probes used, in the aorta-gonad-mesonephric region (AGM), liver, spleen and bone marrow cells.
For the AChE-S and AChE-E probes, expression levels were distributed similarly in liver and spleen. For example, labeling intensity for both these probes was high in mid-gestation liver and spleen, when the principal hematopoietic activity was erythropoiesis, and labeling of both decreased steadily from the 9th week onward, as myelopoiesis became more prevalent. In contrast, the AChE-R transcript was detected only during the 16 week transition from erythro- to myelopoiesis in the mid-gestation liver and not in the spleen (Porcellini et al., Int. J. Cell Cloning 1, 92-104, 1983). The unique expression pattern of AChE-R mRNA and its apparent correlation with myeloidogenesis demonstrated that AChE-R acts as a selective hematopoietic element.Example 4 ARP Sustains Cell Expansion and Differentiation
The predicted secondary structure of peptides ARP and ASP was analyzed.
Both the “synaptic” exon 6-derived and the “readthrough” pseudointron 4-derived peptides (ASP, ARP) include a major region predicted to be rich in turns and β-pleated sheets; in addition, the longer ASP peptide is predicted to contain a unilaterally hydrophobic α-helical domain with amphipathic properties (
The effect of ARP on cell proliferation was further analyzed using BrdU incorporation assay. BrdU incorporation was measured by 5-Bromo-2′-deoxy-Uridine Labeling and Detection Kit III, Roche.
As shown in Table 1, ARP, but not ASP, facilitates BrdU incorporation into cord blood progenitors. ARP induces similar effects on BrdU incorporation into CD34+ progenitors from the peripheral blood of adult donors.
Shown are BrdU incorporation values following the noted incubation times.
In addition, the effect of ARP was examined in transformed bone marrow endothelial cells (Schweitzer et al., Lab Invest vol. 76, 5-36:1997). Cells were incubated in a serum free medium (SFM) with 2 nM of ARP, with or without endothelial growth factors (bFGF 20 ng/ml and EGF 10 ng/ml), for 48 hrs. As shown in
Thus, ARP may serve as a proliferating factor for endothelial cells, having the most dramatic effect when working in synergy with the “classical” endothelial growth factors.
Addition of ARP alone increased the number of viable cells more than 10-fold (
The compatibility of the ARP-supported expansion with differentiation was tested by quantifying the myeloid (CD33+) and megakaryocytic (MK, CD41+) cells after 2 weeks in liquid culture (Table 2). Cells expanded under the influence of ARP displayed increased ability to differentiate into MK and myeloid progeny. Moreover, ARP potentiated the effect of TPO to enhance the number of MKs. Surprisingly, the TPO-potentiating effect of ARP was found to be more pronounced than the TPO-potentiating effect of stem cell factor (SCF). SCF is a known TPO potentiating agent for stem cells (Deutsch et al., Med. Oncol. 13, 31-42, 1996). ARP also facilitated the capacity of GM-CSF and SCF to support myelopoiesis. Thus, the effects of ARP over MK (p=0.08, paired Student's t-Test) and myeloid (p=0.04) expansion are independent. Further, the effect of ARP on the megakaryocytopoietic capacity of TPO and SCF (p=0.03) is synergistic. The myeloid potentiation capacity of ARP over that of GM-CSF and SCF is additive.
aPresented are fold expansions (and, where noted, SEMs) of 2-week primary cultures of CD34+ cells from 1 to 3 individuals grown with the noted cytokines.
ARP, AChE C-terminal “readthrough” peptide;
GM-CSF, granulocyte-macrophage colony stimulating factor;
SCF, stem cell factor;
Potentiated expansion values are highlighted in bold letters.
ARP was added in concentrations of 50 or 100 ng/ml to cultured CD34+ cells and the levels of the various AChEmRNA transcripts after 24 hr were examined, by in situ hybridization combined with confocal microscopy analysis.
Antisense Suppression of AChE-R Production
The ARP-induced enhancement of ACHE gene expression suggested that the AChE-R protein and not necessarily ARP, may be responsible for the sustained viability and the significant expansion of HSCs. To distinguish between these two possibilities, the inventors employed antisense oligodeoxynucleotides (AS-ODN) to selectively suppress AChE-R mRNA levels, reduce the intracellular production of AChE-R and test, under these conditions, the proliferative effects of GM-CSF with or without ARP. AChE-R mRNA includes a 1,094 bp 3′-untranslated region (UTR), with 62% G,C content. This marks it as a more vulnerable molecule to nucleolytic degradation than AChE-S mRNA, which includes a 219 bp UTR with 66% G,C. A.
To selectively reduce AChE-R mRNA levels in HSCs, extremely low doses (20 pM) of anti-AChEmRNA AS-ODNs [Grisaru et al. (1999b) id ibid.] were employed. AS1 and AS3 are 2′-O-methyl-protected AS-ODNs targeted to ACHE exon 2, which is common for AChE-S and AChE-R mRNA. An irrelevant AS-ODN (ASB) targeted to BuChE mRNA served as a control (
ARP Retrieves the Antisense-suppressed Cell Proliferation Effect of GM-CSF
Cell proliferation was evaluated by measuring BrdU incorporation following 16 hr incubation in the presence of 20 pM of the noted AS-ODNs with or without 50 ng/ml ARP and/or GM-CSF.
To determine whether the ARP expansion effects could replace any of the known growth factors, the inventors tested ARP alone or combined with known growth factors, on long-term CD34+ cell cultures.
In summary, CD34+ cultures grown without growth factors for 28 days displayed typical fibroblast morphology (
To test the number of progenitors and differentiation routes available to ARP-treated cells, the inventors subjected the above cultures to a second expansion phase. Cells removed once a week from the primary liquid cultures were grown in the absence of ARP in a semi-solid substrate. In the absence of the growth factor mixture, there was no secondary expansion. IL3 and GM-CSF were used to induce granulocyte-macrophage (GM) expansion and TPO and SCF was used for megakaryocyte (MK) expansion. During this second expansion phase, blood cell progenitors that had previously been treated with early-acting cytokines developed into either GM or MK colonies (
These tests provide evidence for maintenance of all type of progenitors in ARP-treated cultures.
aCultures were seeded at 50,000 cells/well. Shown are cells per culture × 10−3 on day 14; 1 of 3 reproducible experiments as in the figure above.
bThese are also CD34+ positive early cells with expansion potential.
In order to study the possible involvement of ARP in the embryonic development process, the anti-ARP and anti-ASP antibodies have been used to label structures in the embryonic mouse cortex. These antibodies can label either cell types that produce AChE-R and AChE-S, or cells that have binding sites for the ARP and ASP peptides.
In the dorsolateral neurocortex, neurons migrate from the lateral cortical stream toward the outer perimeter of the brain.
BrdU Labeling in Developing Brain
In order to study the effect of ARP on mitotic activity of developing mouse brain, a BrdU incorporation analysis was performed. The C-terminal peptides ARP or ASP were injected 0.1 mg/Kg into a pregnant mouse; 24 hr later, the BrdU was injected and after 1 hr the embryo was isolated and fixed for examination. To show specificity of the results for the peptides, the experiments were performed in the presence of antisense oligonucleotides. The AS3 ODN, 2 nM, was injected into a pregnant mouse and 5 hr later the BrdU. After 1 hr the embryo was isolated and fixed. Controls were saline injection or an ODN with the inverse sequence of the AS3. Labeled neurons (positive for BrdU incorporation) were counted to assess neuronal proliferation.
Embryonic neuroepithelial cells have one of two fates: (1) to continue proliferation and migration up and down from the ventricular zone up to the cortical plate, or (2) to quit the proliferative cycle and initiate terminal differentiation. The balance between these two processes determines the number of proliferating neuronal progenitors as well as the thickness of the cortical plate where post-mitotic neurons accumulate (see scheme at
As shown in
AS3, an ODN directed toward a sequence common to both AChE-S and AChE-R, much more effectively suppresses the mRNA of AChE-R than that of AChE-S [Shohami et al. (2000) J. Mol. Med. 78, 228-36]. The suppression of AChE-R is correlated with an increased thickness of the layer of post-mitotic neurons compare to control sections (reproducible outcome from one of three animals used for each treatment).
Immunolabeling of ARP in Treated Brain
To confirm that the antisense treatment suppresses the level of ARP production and that this occurs through destruction of AChE-R mRNA, in situ hybridization and immunolabeling were performed.
ARP Accumulates in the Serum Under Stress and Facilitates the Stress-induced Hematopoietic Responses in vivo
To find out whether the ARP peptide occurs naturally in blood and if its levels increase under psychological stress, FVB/N mice (n=12) were subjected to confined swim protocol for exerting acute psychological stress as detailed elsewhere [Kaufer et al.(1998) id ibid.]. Serum samples removed 24 hr later were subjected to gradient gel electrophoresis.
To determine the in viuo capacity of ARP to affect hematopoietic expansion under acute psychological trauma, mice were injected immediately after the stress protocol with 0.1 mg/kg ARP or 30 ng/kg AS1. Another group of mice were not subjected to stress and were injected intraperitoneally with normal saline (n=6) or ARP (n=4). 24 hours later, the animals were sacrificed and whole blood obtained for AChE activity and white blood cells. Bone marrow smears were subjected to immunohistochemical labeling with an affinity purified rabbit antiserum prepared against GST-fused recombinant ARP.
Persistent AChE-R Overproduction Increases Platelet and WBC Counts in a Dose-dependent Manner
A series of AChE transgenic mouse pedigrees [Sternfeld et al. (1998b) id ibid.] was employed, to reveal if chronic increases in AChE-R would confer persistent changes in blood cell composition. Blood AChE levels, platelet and WBC counts were determined in FVB/N mice (Control, n=22) as compared to transgenic FVB/N mice carrying the AChE-S (TG-S, n=12), AChE-R (TG-R70 and TG-R45, n=9 and 6, respectively) or inert-inactivated AChE-S (AChE-Sin, n=3) transgene.
ARP Accumulation in the Serum Under Stress
The intense labeling of ARP in the unfractionated mouse serum removed 24 hr following stress treatment revealed more pronounced increases in this peptide than in its native protein AChE-R. This may reflect elevated proteolytic activity under stress. Combined with the absence of cleavage sites for common proteases within the ARP sequence, this further explains the reproducible series of proteolytic degradation products of serum AChE-R which were intensified in the stressed serum samples. The physiological implications of this finding are that AChE catalytic activity measurements are underestimates of the extent of its overproduction in the blood under stress. Likewise, measuring acetylcholine hydrolysis may underestimate the actual amounts of the AChE protein and its degradation products in the brain or muscle. The reported decreases of AChE activity in Alzheimer's disease may hence mislead researchers and clinicians alike by masking the accumulation of morphologically active AChE-derived peptides with long-term effects.
ARP Accumulation in Human Blood Plasma Under Lipopolysaccharide Exposure
To test whether ARP accumulation can be observed in different stress causing situations (as demonstrated above), ARP expression and AChE activity were analyzed in human serum following exposure to bacterial lipopolysaccharide (LPS) as a model for bacterial infection.
Twenty volunteers, ages 19 to 30 years old were i.v. injected with a placebo or endotoxin (Salmonella abortus equi., 0.8 ng/Kg body weight). Blood was collected at baseline, and at hourly intervals up to 10 hr post-injection.
ARP levels and AChE activity were analyzed, as shown hereunder. In addition, the emotional and behavioral states of those twenty volunteers were assessed, as well as rectal temperature, heart rate and plasma levels of cytokines and cortisol (not shown).
Interestingly during this experiment, it has been found by the inventors that in conjunction with the accumulation of ARP, endotoxin induced a significant increase in rectal temperature and elevation in cortisol and cytokines levels (data not shown).
Moreover, a significant endotoxin-induced increase in anxiety level was observed at 1-2 hr post-injection but not later as well as significant increase in depressed mood, which was evident at 3-4 hr post injection.
These endotoxin-induced emotional and cognitive disturbances in healthy volunteers were associated with increased plasma levels of AChE-R and cortisol (data not shown).
However, the observed correlations between depressed mood and cortisol secretion, as well as between depressed mood and cytokine secretion, suggesting that AChE-R and cortisol are independently associated with endotoxin-induced increase in depressed mood.
Mass Spectroscopy of Gel-eluted Band
To verify the identity of the plasma-accumulated short peptide that immunoreacted with the anti-ARP antibodies, larger plasma samples (180 μg/lane) were electrophoresed. A Poinceau-stained band that co-migrated with the ARP Ab-positive band was cut out of the gel and subjected to electron spray mass spectrometry. As shown in
ARP Modulations Potentiate the in vivo Hematopoietic Responses to Stress
While ARP alone did not exert immediate effects on mouse blood cell composition, its injection under stress enhanced ARP labeling in bone marrow cells and induced an elevation in WBC counts within 24 hr. This suggests that acute stress modifies the number and/or state of ARP-responsive elements on hematopoietic cells. Anti-ARP antibodies labeled primarily small cells in ARP-treated stressed animals, whereas the limited labeling in untreated stressed animals and in AS1-treated stressed animals only appeared in relatively larger cells. This indicates labeling of the stem cells which expanded during the 24 hr post-stress. The similar patterns of the in viuo effects on bone-marrow ARP labeling and WBC counts with the ex vivo expansion effects on CD34+ cells implies that stress-induced increases in AChE-R may be causally related to the post-stress elevation in WBC counts (Goldberg et al., Folia Biol. 36, 319-31, 1990).
Transgenic animal models used here provide an opportunity for testing the chronic effects of elevations of different AChE variants. While AChE-S had no apparent effect on either platelet or WBC counts, AChE-R modulations exerted dose dependent changes: 2.5-fold excess in blood AChE-R activity, similar to the AChE-R elevation noted in the mouse brain under stress [Kaufer et al. (1998) id ibid] sufficed to significantly elevate platelet counts. The more dramatic 130-fold excess in blood AChE-R levels of the robust-producing transgenic pedigree [Sternfeld et al. (1998b) id ibid.] elevated both platelet and WBC counts. This finding, and the in vivo accumulation of ARP under stress, raise the possibility that the increased risk for brain infarcts following acute stress or exposure to anticholinesterases (Harmsen et al., Stroke 21, 223-9, 1990, Schultz et al., Anesthesiology 79, 114-21, 1993) is associated with the increased platelet counts due to AChE-R overproduction. This calls for a search for AChE-R overproduction in Alzheimer's disease patients, where ARP may increase platelet counts and cause the cerebral infarcts, characteristic of this disease (Inestrosa et al., Neurosci Lett 163, 8-10, 1993, Snowdon et al., Jama 277, 813-7, 1997). Anti-ARP antibodies provide a novel diagnostic tool for testing this option (and for risk assessment) and AS-ODN treatment may offer an attractive protocol for prevention of such adverse responses.
The significance of ARP extends beyond the hematopoietic system. There is evidence for cross-talk between hematopoietic cells at different stages of differentiation and bone-marrow stromal or endothelial cells. Stroma influences cytokine production and is responsible for maintaining steady-state hematopoiesis and its adjustment under stress (Gupta et al., Blood 91, 3724-33, 1998). It has been proposed that primitive CD34+ progenitors provide a soluble positive feedback signal to induce cytokine production by either stromal or endothelial cells (Jazwiec et al., Leukemia 12, 1210-20, 1998). ARP may play such a role, with important implications for ex vivo stem cell expansion, cancer treatment and gene therapy. In the mammalian brain, ARP may further affect the stress-associated plasticity of neuron and glia properties, consistent with previous findings of the inventors of morphogenic activities for AChE-R in transfected glia (Karpel et al., J. Neurochem. 66, 114-23, 1996).
The stem cell survival and proliferative effects of ARP denote a previously unforeseen activity that is particular to the AChE-R protein yet distinct from the acetylcholine hydrolysis and cell-cell adhesion capacity characteristic of the core domain common to all AChE isoforms. The pronounced expression of AChE-R during early embryogenesis, further demonstrate the involvement of ARP in inducing the proliferation of other embryonic stem cells. Moreover, neural stem cells were shown to produce a variety of blood cell types in vivo (Bjornson et al. Science 283, 534-7, 1999).
The findings presented here suggest that ARP is involved in the induction of growth and expansion capacities of pluripotent stem cells from multi-tissue origins. The unique properties of this peptide and equivalent peptides can contribute toward the development of diverse human differentiating cell sources for biomedical and research purposes.Example 11 AChE-R Effects on Hippocampal LTP Suggest Causal Involvement in Neuronal Stress Responses
At the molecular level, psychological stress notably leads to fast yet long lasting modulation of gene expression. As for the genes concerning the cholinergic system, it has been shown that within one hour from acute stress, long lasting changes in cholinergic gene expression are facilitated [Kaufer et al. (1998) id ibid.]. This particularly refers to drastic elevation in the levels of the normally rare “readthrough” variant of acetylcholinesterase (AChE-R), coupled with down-regulation of acetylcholine synthesizing and packaging proteins, the enzyme ChAT and the associated vesicular acetylcholine transporter (vAChT). This feedback response presumably contributes to reduce ACh levels following stress. Another outcome of stress responses involves a sudden increase in proteolytic activities. This leads, among other effects, to the cleavage of the C-terminal peptide (ARP) from the “readthrough” core enzyme. Immunodetection using anti-ARP antibodies reveals an increase in AChE-R degradation products in the cerebrospinal fluid of patients under stress [Kaufer (2000) PhD thesis, Hebrew University of Jerusalem, Jerusalem]. Moreover as shown above, the injection of synthetic ARP by itself induces proliferation of hematopoietic progenitor cells and over-expression of bone marrow AChE-R within 24 hr [Grisaru et al. (2001) Molecular Medicine, 7, 93-105]. These recent observations raised the intriguing possibility that ARP also possesses physiological and behavioral functions. To test this working hypothesis, the effects on LTP of confined swim stress (1 hr after induction), were compared with those induced by ARP injection (24 hr post-treatment) and with transgenic mice over-expressing AChE-R.
Differential Properties of AChE Variants in Synaptic Plasticity—Stress Effects
The “readthrough” AChE variant is the sole AChE variant that is up-regulated under psychological stress. Therefore, the possibility that the immediate recovery from psychological stress, in light of the over-expression of the AChE “readthrough” form will affect the pattern of LTP, was explored.
Stress was induced by forcing mice to swim twice for 4 min, with 4 min interval, and 1 hr later slices were taken for LTP experiments. The Schaffer collaterals-CA1 synapse pathway was tested. Basal field potentials were recorded for 15 min at 0.033 Hz. LTP was then induced by 3 consecutive tetanic stimulations, each of 1-sec duration, at 50 Hz with 20 sec inter-stimulus intervals. After tetanization, the change in the slope of the post-synaptic field potential (PSP) was followed for up to 3 hrs.
As shown in
Therefore, stress changes the onset of LTP, lagging the early phase, yet achieving a subsequent stable potentiation.
Transgenic mice over-expressing the “readthrough” isoform AChE-R enabled direct examination of the question whether stress affects LTP, via elevation of AChE-R. Slices were prepared from adult control and transgenic mice, 3 to 5 months old, and LTP experiments were performed as described above.
As shown in
Injected ARP Effects
The option that ARP (the AChE Readthrough Peptide) serves as a stress signal was next examined. In case that ARP participates in signaling stress conditions by elevating the “readthrough” isoform in the CNS, as in the hematopoietic system, it would be expected to induce the LTP pattern that was observed under stress conditions or in the AChE-R transgenic mice (
In conclusion, these findings point at the proteolytic cleavage of ARP as a causally involved step in the synaptic responses to stress and suggesting existence of ARP binding sites in the hippocampus.
Moreover, these findings point at the possible mechanisms by which ARP might mediate such effects.Example 12 Testicular Overproduction of the Stress-associated “Readthrough” Acetylcholinesterase Variant Impairs Sperm Properties
Suppressed male fertility is often attributed to stressful lifestyle, however, the protein(s) mediating such impairments are not yet known. Since ARP accumulation was observed under different stress situations, the contribution and the involvement of AChE-R in stress-induced infertility was next examined.
Testicular AChE-R is Overexpressed in Psychologically Stressed Mice
The corticosterone levels and AChE activities were examined in testicular homogenates from FVB/N mice that were subjected to repeated acute psychological stress (4 successive daily sessions of confined swim). As shown in
To study the pattern of AChE-R expression in stressed vs. untreated mice testis, in situ hybridization using a cRNA probe selective for the AChE-R mRNA transcript was performed on sections of testicular tubules from untreated FVB/N mice or from FVB/N mice subjected to 4 constitutive daily treatments of confined swim stress. As shown in
Similarly, Immunolabeling with an antibody selective for ARP, displayed no detectable staining in control mice, yet stained internal spermatid cell layers in tubuli of stressed mice (
Impaired Sperm Qualities Under AChE-R Excess Suggest Functional Significance to ARP
To determine the in vivo capacity of ARP to affect different biochemical and physiological male fertility properties, the effect of injection of ARP or chronic expression of AChE-R (AChE-R transgenic mice) on different parameters was examined.
Twenty-four hours following injection of mice with 85 μg/kg ARP (but not PBS), blood corticosterone levels were doubled as compared with FVB/N mice or AChE-R transgenics (Table 4). While the mechanism(s) for such short-term glucocorticoid increases are yet unknown, this finding suggested that ARP might be independently involved in activating peripheral stress responses. Seminal gland weight was substantially reduced in AChE-R transgenics, but not in ARP injected mice, reinforcing the distinction between ARP and AChE-R effects; however, sperm counts were lower both in ARP-injected and in AChE-R transgenics than in untreated FVB/N mice. This did not reflect changes in cell division as the numbers of PCNA-positive cells in testicular tubules remained unchanged (Table 4). Intriguingly, sperm cells displayed significantly reduced motility in both ARP injected mice and AChE-R transgenics (Table 4) suggesting that ARP exerts rapid yet long lasting impairments of sperm properties.
aAll tested animals were FVB/N adult mice, 2 to 4 month-old males (numbers shown in parentheses). Controls were untreated and PBS injected.
b24-hr post i.p. injection of 34 nmol/Kg ARP.
cLine 45 [Sternfeld et al. (1998a) id ibid].
dAverage labeled cells or labeled areas for the noted (in parentheses) number of tubular sections and the identified antibodies.
Asterisks note significant difference (p < 0.05, Wilcoxon-Mann-Whitnety) from controls.
Corticosterone elevation initiates at the hypothalamic—pituitary-adrenal (HPA) axis, activated by calcium increases under psychological stress [Kaufer et al. (1999) Current Opinion in Neurology 12, 739-743]. The observed corticosterone and AChE-R overexpression following ARP injection suggest an HPA-activating, auto-regulatory function for ARP. AChE-R accumulation would induce the cell-cell or cell-substrate signaling capacities established for AChE [Grisaru et al.(1999b) id ibid.]. The normally rare AChE-R isoform differs from the major synaptic AChE-S variant in such properties, for example in cultured glia (Karpel et al. J. Neurochem., 66, 114-123, 1996), supporting causal involvement for ARP in morphogenic functions. This calls for identifying the yet unknown brain protein partner(s) of ARP and the signal transduction mechanisms it activates.
Both Stress and ARP Injection Enhance ARP Immunolabeling of Spermatid Heads
To subcellularly localize the AChE-R isoform, immunolabeled mature spermatids from the central cavity of testicular tubules were subjected to confocal microscopy (
Loss of ARP Immunolabeling in Human Sperm Heads from Subjects with Unexplained Couple Infertility
To test the validity of the predictions based on the mice results, for human sperm properties, the inventors performed immunolabeling of ARP in smeared sperm cells from individuals with reported unexplained couple infertility. Healthy cells from sperm donations served as controls. Normal specimens were primarily co-stained in sperm head and midpiece regions. In contrast, sperm samples from male partners of couples with unexplained infertility displayed large fractions of cells labeling limited to the midpiece and unlabeled heads (
While reduced seminal gland weight could probably be attributed to the long-lasting effects of stress, the intensified labeling of developing sperm cells with anti-ARP antibodies suggests direct ARP effects on spermatogenesis and/or sperm properties. Focal perinuclear labeling of pachytene spermatocytes from AChE-R transgenics was associated with an apparent suppression of spermatogenesis manifested in reduced sperm counts. Transient excess, such as that induced following repeated acute stress or ARP injection, caused more limited impairments in spermatogenesis yet impaired sperm functioning most effectively. That this was associated with ARP accumulation was indicated from the modified ARP labeling of sperm heads in mice or midpiece and men.
Anti-ARP antibodies could label both ARP binding and AChE-R production sites. In stressed or ARP-affected mouse spermatids, the punctuated head labeling appeared reminiscent of the mitochondrial distribution in the region surrounding spermatid heads. This assumption was supported by the intriguing labeling patterns in human sperm from infertile couples, where staining was most intense in the midpiece region which is enriched in mitochondria in primate sperm.
In summary, these results significantly indicate impaired sperm properties under overproduction of the stress-associated “readthrough” isoform of acetylcholin- esterase, AChE-R and its naturally cleaved C-terminal peptide ARP.
Thus, excess AChE-R and its C-terminal peptide ARP may suppress male fertility through both autonomous system regulation and direct sperm interactions.Example 13 Detection of ARP Binding Proteins by Using the Yeast Two-hybrid System
In order to study the possible signaling pathway through which ARP can exert its intracellular signaling leading to the observed proliferation and differentiation, screening for detection of ARP binding proteins was performed using the yeast 2-hybrid system.
The yeast 2-hybrid system is based on that transcription factors, such as GAL4, consist of two discrete modular domains: the DNA-binding domain (DNA-BD) and the activation domain (AD). A “bait” gene is expressed as a fusion to the DNA-BD, while a cDNA library is expressed as a fusion to the AD (Chien et al. (1991) id ibid; Fields et al. Trends Genet 10, 286-92, 1994). When the fusion proteins interact, the DNA-BD and AD brought into close proximity, thus reconstituting GAL4 and activating transcription of a reporter gene (
The bait is cloned into the DNA-BD vector where it is expressed as a fusion to amino acids 1-147 of the yeast GAL4 protein. A second gene or cDNA library is cloned into the AD vector, where it is expressed as a fusion to amino acids 768-881 of the yeast GAL4 protein. When the fusion proteins interact, the DNA-BD and AD domains are brought into close proximity and can activate transcription of reporter genes.
In order to identify AChE C-terminal peptide interacting proteins, the GAL4-based two-hybrid system was used. Sequences that encode AChE C-terminal peptides ARP and ASP were cloned into the DNA-BD pGBKT7 vector (Clonetech), (
Summary of the Yeast 2-hybrid Preliminary Screens
Four preliminary yeast two-hybrid screens were performed, the outcomes of which are summarized below several points suggest that certain positive clones are meaningful.
- 1. The number of ARP positives in the developing rat aorta seems to be considerably higher than in the adult tissue, this is in line with the embryonic expression pattern of AChE-R.
- 2. Several positive clones appear more then once, representing independent cDNA chains of variable lengths.
3. In certain cases, the positives are logical candidates for AChE interactions (see below).
During the first library screenings, 8 candidate partners emerged. Of these, a literature survey pointed to 2 candidates as most promising.
Fragment of AChE-ARP used for the two-hybrid screen
Underlined is the actual ARP-peptide
PLEVRRGLRAQACAFWNRFLPKLLSATGMQGPAGSGWEEGSGSPPGVTPLFSP, also denoted as SEQ ID: No. 7.
Receptor for Activated Protein Kinase C (RACK)—2 clones
H. sapiens melanoma antigen, family D, 1 (MAGED1)—2 clones
H. sapiens guanine nucleotide-binding protein g(i)/g(s)/g(t) β subunit 2 (transducin β chain 2)
H. sapiens duplicate spinal muscular atrophy—2 clones
H. sapiens peptidase D (PEPD)
M. musculus Eph receptor A6 (Epha6)
H. sapiens succinate dehydrogenase iron-protein subunit (sdhB) gene
H. sapiens mitogen-activated protein kinase 7 (MAPK7)
H. sapiens HLA-B associated transcript-3 (D6S52E)
mitochondrial intermediate peptidase (MIP)
Fragment of AChE-ASP used for the two-hybrid screen
Underlined is the actual ASP-peptide: PLEVRRGLRAQACAFWNRFLPKLLSATDTLDEAERQWKAEFHRWSSYMV HWKNQFDHYSKQDRCSDL, also denoted as SEQ ID NO 8.
Receptor for Activated Protein Kinase C (RACK)—2 clones (also showed up in the ARP screen, probably binds to the common part).
H. sapiens C-terminal binding protein 2 (CTBP2).
H. sapiens activating signal cointegrator 1.
H. sapiens colon carcinoma laminin-binding protein.
H. sapiens clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J) (CLU).
H. sapiens mRNA for silencer element.
H. sapiens heme-regulated initiation factor 2-β kinase (HRI).
Identification of RACK1 as ARP and ASP Interacting Molecule
One of the isolated partners, RACK1, was further analyzed for its ARP interactions. RACK1 is a cytoplasmic G protein homologue, which serves as a protein kinase C receptor.
Interaction between RACK1 and AChE may therefore be the link between AChE and other signaling molecules through which it exerts its non-catalytic intracellular functions.
Amino acid sequence alignment of RACK1 with the sequence obtained from the two-hybrid positive clone shows close to 100% homology (
An in vitro overlay assay was combined with protein blot analysis to test for RACK1 interaction with the full AChE-R protein. RACK1 was expressed in E. coli and purified by affinity chromatography from E. coli as a fusion with maltose binding protein (MBP), and subsequently released from the fusion protein by proteolytic cleavage using factor Xa. Both cleaved and uncleaved preparations were used for the overlay assay. RACK1 samples were submitted to electrophoresis on a 4-10% denaturing polyacrylamide gel, blotted on a NC membrane, which was then stained with Ponceau (
In order to study the expression of RACK1 in stress situation, homogenates from mouse hippocampus and cortex (composed of 3 stressed, 4-6 and 3 control, 1-3 mice) were separated on a denaturing gel and analyzed by immunoblot with anti-RACK1 antibody (
pGARP, a vector that encodes a fusion protein between green fluorescent protein (GFP) and ARP1 under the CMV promoter, was used to test whether ARP1 further promotes triple complex formation with PKCβII and RACK1 in mammalian cells (
Both COS and PC12 cells express RACK1 and PKCβII constitutively, whereas only PC12 expresses AChE-R, as observed in immunoblots of the soluble fraction of cell homogenates (
The in uivo relevance of AChE-R/RACK1 interactions was explored in normal and post-stress mouse brain. Immunoreactive RACK1 was observed in the cytoplasm and closely proximal processes of pyramidal neurons, in layers 3 and 5 of the frontal and parietal cortex, in both superficial and deep layers of the piriform cortex, and in regions CA1 and CA3 of the hippocampus. A subset of these neurons also overexpresses AChE-R under acute psychological stress (
Hippocampal homogenates from AChE-R overexpressing transgenics [Sternfeld et al. (2000) id ibid.] were tested to investigate whether AChE-R overproduction would modulate the levels, properties and/or neuronal localization of its partner proteins RACK1 and PKCβII. The results show that the hippocampal homogenates displayed significant increases (compared to the levels in control FVB/N stress-prone mice) of neuronal AChE-R and RACK1, as well as a faster migrating PKCβII band that was only faintly detected in the hippocampus of a control animal (
Of the three target proteins, RACK1 and AChE-R appeared more widely distributed and could be detected in numerous brain regions (
In control mice, PKCβII antibodies displayed diffuse staining [Weeber et al. (2000) id ibid.] in sub-regions of layers 5,6 in the cortex, in the stratum oriens and stratum radiatum layers of the hippocampus CA1 field, in the striatum matrix, and in the substantia nigra pars reticulata. Axonal bundles including the nigro-striatal tract were also labeled (data not shown). Another and novel staining pattern consisted of dense PKCβII clusters in neuronal perikaria and in the axonal stems. This punctiform pattern appeared in upper layers of the parietal, temporal and piriform cortex, dorsal striatum, basolateral amygdala, hippocampal CA1 and lateral septum. In general, cells in AChE-R transgenic mice that displayed prominent AChE-R labeling were positive for RACK1 and presented PKCβII punctiform staining (
In samples obtained from AChE-R transgenic mice, anti-PKCβII antibodies detected diffuse and axonal staining patterns similar to those observed in the parental FVB/N strain. However, the punctuated pattern was altered. Stronger and denser clusters of PKCβII staining were located on the perikaryal circumference of a larger fraction of hippocampal CA1 neurons (
Confocal micrographs of upper layer neurons from the parieto-temporal cortex double labeled with antibodies against AChE-R and RACK1 or PKCβII displayed distinct yet overlapping distributions for the three partner proteins within neuronal perikarya in compound field projections. As expected, AChE-R labeling was conspicuously more intense in AChE-R transgenics than in FVB/N controls (compare in
Proteins destined to be secreted are initially concentrated near the nucleus, where their processing takes place, whereas proteins that are associated with the perikaryal cytoskeleton, plasma membrane and/or proximal process structures are distributed more peripherally in the cell. In cortical neurons from control mice, both AChE-R and RACK1 immunostaining formed perinuclear accumulations (compare in
1. An antibody directed against the ARP peptide as denoted by SEQ ID NO:1, which antibody is specific for any one of the AChE-R variant of acetylcholinesterase and a C-terminal peptide derived there from.
2. The antibody according to claim 2, for use in the diagnosis of stress-induced male infertility.
3. A method for the diagnosis of stress-induced male infertility comprising obtaining a sperm cell sample from said male; smearing and drying said sample; contacting said dried sample with an antibody specific for the AChE-R variant of acetylcholinesterase; removing unbound antibody; detecting the extent of reaction between said antibody and the AChE-R variant of acetylcholinesterase or a fragment thereof present in said sample; determining the pattern of expression of the AChE-R variant of acetylcholinesterase or a fragment thereof, in said sperm cells; whereby the absence of AChE-R from sperm heads together with intense presence in the midpiece region indicates the presence of stress-induced male infertility.
4. The method according to claim 3, for use in fertility counseling.
5. A kit for the diagnosis of stress-induced male infertility, comprising:
- (a) an antibody specific for the AChE-R variant;
- (b) means for collecting a sperm sample;
- (c) slides for smearing the sperm sample;
- (d) means for detecting the antibody;
- (e) negative control slides.
International Classification: G01N 33/567 (20060101); C07K 16/40 (20060101);