IN VIVO ANIMAL MODEL FOR STUDYING ALZHEIMER'S DISEASE

Abstract: In vivo mouse model for studying Alzheimer's disease is produced by introducing Trisomy 16 mouse embryonic central nervous system (CNS) tissues to the brain of a live host mouse. The neuropathology in the introduced Trisomy 16 CNS tissues closely parallels that of human Alzheimer's disease, so that the host mouse with such introduced tissues provides a potentially useful living animal model in which to study the development of Alzheimer's disease and in which to test potential preventative or curative treatments.

Description: Title ; IN VIVO ANIMAL MODEL FOR STUDYING ALZHEIMER'S DISEASE . Field of Invention This invention relates to Alzheimer's disease (AD) and concerns a novel approach for investigating the development of the disease and for testing potential preventative and curative treatments. Background to the Invention Alzheimer's disease is characterised by amyloid plaques and neurofibrillary tangles (NFT), which are located primarily in the neocortex and hippocampus (see references 1,2). Since similar neuropathological changes are observed in trisomy 21 (ie having 3 chromosome 21s) Down Syndrome (see references 3,4), it is thought the AD pathology may be due to abnormal expression of gene(s) located on human chromosome 21. In particular, the gene encoding the amyloid precursor protein (APP) has been mapped to chromosome 21 (see references 5,6). The APP gene, together with several single copy sequences including D21S16 that are localised on human chromosome 21, have been mapped to mouse chromosome 16 (see references 7,8). Consequently, it is of interest to ask whether over-expression of genes on chromosome 16 in the trisomic mouse (having 3 chromosome 16s) would produce similar pathological changes to those observed in human AD. Although reliable strategies exist for breeding Trisomy 16 mice (see references 9, 10), the consequences of developmental over-expression of genes on chromosome 16 have not been previously investigated due to the lethal effects of this aneuploidy during gestation (see reference 10). The present invention concerns an approach which facilitates long term study of Trisomy 16 mice tissues by providing an in vivo model which is found to follow human neuropathology closely, making it possible to study the neuropathology and other aspects of Alzheimer's disease. Summary of the Invention According to one aspect of the invention there is provided a method of producing an in vivo mouse model for studying Alzheimer's disease, comprising introducing Trisomy 16 mouse embryonic central nervous system (CNS) tissues to the brain of a live host mouse. In another aspect the invention provides a method of studying Alzheimer's disease comprising introducing Trisomy 16 mouse embryonic CNS tissues to the brain of a live host mouse, and studying the mouse. Experiments described below indicate that the neuropathology in the introduced Trisomy 16 CNS tissues closely parallels that of human Alzheimer's disease, so that the host mouse with such introduced tissues provides a potentially useful living animal model in hich to study the development of Alzheimer's disease and in which to test potential preventative and curative treatments. The mouse model may be studied in various ways in order to derive information on AD. For example, the introduced CNS tissues may be examined (following sacrifice of the mouse) after various different time intervals, providing information on neuropathological changes with time as the disease develops. Other tissues, eg from other parts of the brain, of the host may also be examined in this way, enabling a study of the interaction between the mouse and introduced tissues. As a further possibility, the mouse may be treated (generally after introduction of the Trisomy 16 tissues) by various candidate potential therapeutic (curative or preventative) stratagies that it is thought may inhibit the development or reverse the pathological manifestations of AD. By studying the effects of such strategies, and comparing them with controls, the invention canienable screening and assessment of such strategies prior to use in humans. Hence, in a further aspect the invention provides a method of assisting the treatment or prevention of Alzheimer's disease, comprising introducing Trisomy 16 mouse embryonic CNS tissues to the brain of a live host mouse, and studying the mouse. Techniques for breeding Trisomy 16 mice are known: see for example references 9 and 10. For example male offspring from matings of homozygous Robertsonian translocations Kb (9:16) 9 Rma and Rb (11:16) 2H may be mated with females of the CFLP strain possessing acrocentric chromosomes only, to yield a Rb9 Rma/Rb2H x CFLP cross. Trisomy 16 embryos may then be selected from normal littermates. Insead of using translocations 9 and 11, other long arm translocations involving chromosome 16 may also be used in this technique, including Rb(16:17), (6:16) and (13:16). In certain circumstances it is desirable for the host mouse to have a similar genetic background to that of the Trisomy 16 embryos, to reduce the risk of i munorejection: with embryos produced as described above, the host is conveniently mice of the CFLP strain in such circumstances. However, in other circumstances genetically dissimilar hosts may be used to study immunological factors in the development of AD-like pathology. The introduced tissues may be any of a wide range of CNS tissues that are associated with the AD neuropathology, including cortex, hippocampus, basal forebrain nucleii, cerebellum and basal ganglia. The CNS tissues are conveniently introduced to the host by grafting as solid pieces into cortical cavities or ventricular spaces, or as dissociated cell suspensions injected directly into the host neuropil. Cell suspension grafts of CNS tissues may also be used. The invention also includes within its scope a mouse which has had Trisomy 16 mouse emlpryonic CNS tissues introduced to the brain thereof while alive. The invention will be further described, by way of illustration, in the following Example and by reference to the accompanying figures in which: Figure la illustrates genes located on human chromosome 21 and also on mouse chromosome 16. Figure lb illustrates gene dosage for trisomic (lanes 1- 4) and control (lanes 5-8) tissues confirmed by Southern blot analysis. Figure lc illustrates karyotyping of triso ic liver tissues to confirm Robertsonian translocations (arrows) by a method modified for chorionic villi (see reference 32). Figure 2 illustrates histological characterisation of trisomic tissue grafts using (a) Cresyl violet staining of Nissl substance to reveal the distribution of cells, (b) Thioflavin-S as a traditional marker of amyloid deposits, and immunocytochemical staining using polyclonal antisera raised against (c) the synthetic beta A4 protein (see reference 12) and (d) purified paired helical filaments (PHF). Scale bars in a,b = lOum; c,d = lOOum insets in c and d = lOum. Figure 3 illustrates immunocytochemical co-localisation of the beta A4 protein and PHF undertaken on trisomic grafts, with (a) demonstrating cells immunoreactive for the beta A4 protein and visualised with 3-3 diaminobenzidine, (b) depicting the same section immunoreactive for PHF and visualised with a rhodamine conjugated secondary antibody (Miles), (c) showing cells in a different Trisoimc graft section immunoreactive for beta A4 protein and visualised with a rhodamine conjugated secondary antibody (Miles) and (d) showing immunoreactive co-localisation of the same cells positive for the polyclonal alpha - antichymotrypsin antiserum (see reference 13) and visualised with a FITC conjugated secondary antibody (Miles). Scale bar = lOum, with all Figures at the same magnification. Figure 4 illustrates immunoreactive cells, demonstrating the presence of Tau 6.423 within a hippocampal Trisomy 16 graft (Figure 4a) and in post-mortem Alzheimer's disease tissue (Figure 4b). Figure 5 illustrates the co-localisation of the A4 protein (Figure 5a) and Tau 6.423 (Figure 5b) within the same cell of a Trisomy 16 hippocampal graft, visualised by fluorescent immunocytochemistry. Example Referring first to Figure la, this illustrates genes located on human chromosome 21 and also on mouse chromosome 16, and is adapted from reference 31. Litters containing Trisomy 16 mice were generated by a breeding regime in which male offspring from matings of homozygous Robertsonian translocations Rb(9:16)9Rma and Rb(ll:16)2H were mated with females of the CFLP strain possesing acrocentric chromosomes only, to yield a Rb9Rma/Rb2H x CFLP cross. Although the Trisomy 16 embryo may be visually selected from its normal littermates, confirmation of the assignment of tissues to either the trisomic or the normal control transplantation group was undertaken by Southern blot analysis (Figure lb) and karyotyping (Figure lc). In Figure lb, hybridisation with «. l.lkb fragment of the APP cDNA revealed increased dosage for the trisomic tissues at a 3.2 kb band. A constant band (cb) was detected. Cortical and hippocampal tissues from seven E15-E17 trisomic and eighteen normal littermates were grafted as solid pieces into frontal and retrosplenial cortical cavities of young adult female mice of the CFLP strain (see reference 11). Animals were sacrificed 4-6 months post transplantation and the tissues processed for immunocytochemical analysis using buffered 4% formalin and paraffin wax embedding. Neocortical and hippocampal tissues from post mortem Alzheimer brain and a single E18 Trisomy 16 embryo were post fixed and embedded by the same procedure. Tissue sections were cut at 8 um. The results of histological characterisation and immunocytochemical localisation of proteins are shown in Figures 2 and 3. Immunoreactivity within Figures 2c and d was visualised using the chromogen 3-3 diaminobenzidine (Sigma). Histological assessment showed poor survival of neocortical grafts transplanted to the frontal cortex of host animals, irrespective of whether they were derived from trisomic or control donor embryos. In contrast, healthy hippocampal grafts were obtained from both groups of donors. Nissl body staining revealed no gross differences in either numbers or distribution of cells within the hippocampal grafts between the two groups. However, within the trisomic grafts, large cells judged to be neuronal by their morphology, tended to form aggregrates (Figure 2a). No differences were seen in the cellular composition of adjacent host parenchyma. Thioflavin-S, a histological marker for amyloid, showed positive staining within a few cells and around some blood vessels in the trisomic hippocampal grafts (Figure 2b). Antisera raised against the amyloid beta A4 protein (see reference 12), alpha - antichymotrypsin (see reference 13) and purified paired helical filament (PHF) preparations (antibody A128) (see reference 14), and a monoclonal antibody that is specific for a form of tau bound within the pronase resistant core of the PHF (Tau 6.423) (see reference 15), were intitially tested for their specificity on human AD tissue. All antibodies revealed numerous densely stained cells within the trisomic hippocampal tissues (Figures 2c,d, 3 and 4a). Whereas all viable trisomic grafts were seen to contain at least some densely immunoreactive cells, the number of such cells was never more than 1-5% of the total number of cells as determined from the Nissl stained sections. No similar staining was observed within any of the control grafts, within host brains or within Trisomy 16 embryos. The localisation of immunoreactive product was predominantly intracellular and co-localisation has been demonstrated for beta A4 both with the A128 antiserum and with alpha - antichymotrypsin (Figure 3). In areas where beta A4, alpha - antichymotrypsin and A128 immunoreactive cell bodies formed aggregrates, there was a fine filamentous extracellular staining in the vicinity of the cell soma. In addition, occasional beta A4 and alpha - antichymotrypsin immunoreactivity was seen in the walls of small blood vessels. By contrast, Tau 6.423 immunoreactivity was exclusively intracellular (Figure 4). The amyloid A4 protein was first isolated from the neuropathological plaque core (see references 16-18). It has been found to share amino-acid sequence homology with, and to be antigenically related to, the amyloid protein isolated from AD cerebral vasculature (beta-protein) (see references 12, 19, 20). Consequently the common protein is now known as beta A4. Although the gene encoding the beta A4 protein has been mapped on chromosome 21 (see references 5, 6) to the region 21qll-q22 (see references 21-23), genetic familial linkage studies have shown recombination events occurring between the amyloid beta A4 gene and the disease locus (see reference 24), thus making it unlikely that a mutation in the amyloid beta A4 gene causes familial AD. The serine protease inhibitor alpha _.- antichymotrypsin is predominantly located in association with amyloid deposits in the cores of neuritic plaques and around blood vessels in the AD brain (see reference 13). While amyloid plaques may also be observed in Creutzfeldt-Jakob disease, the presence of alpha -antichymotrypsin immunoreactivity only in the AD pathology has made this protein a distinctive marker for distinguishing the two neurodegenerative disorders (see references 13). In AD cortex, alpha _.- antichymotrypsin immunoreactivity is also observed intracellularly within both neurones and glia (see reference 25) . The major ultrastructural components of the neurofibrillary tangle are paired helical filaments (see reference 1). The precise chemical composition of these abnormally expressed filaments has still to be determined. Although a number of histological studies have shown labelling of neurofibrillary tangles by antibodies to microtubule-associated protein tau (see reference 26), ubiquitin (see reference 27) and amyloid beta A4, only tau protein has been established biochemically and ultrastructurally as a constituent of the protease resistant core of the PHF (see reference 15). Indeed Tau 6.423, which selectively recognises isolated stripped PHFs and the tau released from PHFs, shows no cross-reactivity with other tau proteins found in control brain tissues (see references 15). The presence of this particular epitope in the cytoplasmi.c granular inclusions of the Trisomy 16 hippocampal grafts reported here, and its absence in control grafts and host brain tissues, strongly suggest that a highly distinctive element of the molecular pathology of AD is captured by this model. This interpretation is further supported by the recent demonstration that precisely similar intracellular granular inclusions, in which there is co-localisation of Tau 6.423 (selective for intracellular tangles) and beta A4 markers (Figure 5), occur in AD but not in control brain. The demonstration of immunoreactivity within the hippocampal Trisomy 16 grafts for the proteins associated with AD suggests we are observing the same neurodegenerative features as those causative of AD. This view is supported by the fact that, whereas amyloid and NFTs are observed in a variety of neurodegenerative disorders, the distribution of alpha - antichymotrypsin and the presence of Tau 6.423 immunoreactivity is specific to AD (see references 13, 15). The observation of immunoreactivity for proteins associated with AD neuropathology within mature hippocampal grafts of Trisomy 16 mice, together with the absence of staining in the embryos, indicates the involvement of a slowly developing neuropathological process. Co-localisation of immunoreactivity for beta A4 amyloid, constituents of PHF and alpha -antichymotrypsin suggests that these distinct proteins are being produced within the same cell. A striking feature of the immunoreac ivity observed within this animal model is the intracellular distribution of immunoreactive product for beta A4 and the NFT. This is in apparent contrast to the traditional descriptions of post mortem AD in which the amyloid deposition is predominantly located in extracellular plaque cores with the NFTs being intracellular. However, the observation of intracellular beta A4 immunoreactivity accords with recent evidence that beta A4 protein may also constitute a component of the PHF within tangle-bearing neurones (see reference 28). Moreover, the occurence of beta A4 and Tau 6.423 immunoreactivity localised in granular inclusions within cells in both the Trisomy 16 grafts and the AD brain suggests the existence of an intracellular pathological pathway in which an antigenically distinct form of Beta A4 amyloid (or its precursor protein) and tau protein both accumulate first in cytoplasmic granular inclusions and then in intracellular tangles. Whether PHF formation represents a later stage" of the trisomy 16 model is as yet unknown. Many of the proteins that are abnormally expressed in AD, such as tau, ubiquitin, alpha - antichymotrypsin and other protease inhibitors (see references 13, 26, 27, 29, 30), are not located on chromosome 21. However, the demonstration of Alzheimer-like pathology in the mouse Trisomy 16 grafts strongly suggests that proteins central to the causation of AD are indeed encoded on this chromosome. Due to the rarity of post mortem studies of newly diagnosed AD, it has not been possible to study the onset and progression of the disease in a controlled and systematic way in humans, and there have hitherto been no adequate animal models. It is proposed that Trisomy 16 murine grafts provide a powerful system in which to study the processes of neurodegeneration, and to assess the viability of alternative therapeutic strategies to inhibit the development or reverse the pathological manifestations of Alzheimer's disease. Typically, this will involve producing the mouse models by introducing Trisomy 16 mouse embroyonic CNS tissues to the brains of live host mice, treating the mice with a candidate curative or preventative agent, sacrifycing mice after suitable time intervals, examining the mice, typically brain tissues thereof, to assess the degree of development of AD, and comparing the results with those from controls. REFERENCES 1 . Kidd , M. ( 1963 ) Nature, 197 : 192-193 . 2. Wisniewski, H.M. et al . (1976) J. Neurol. Sci . , 27: 173-181. 3. Burger, P.C. and Vogel, F.S. (1973) Am. J. Pathol., 73:457-468 4. Oliver, C and Holland, J. (1986) Psychol. Med., 16: 307-322. 5. Goldgaber, D. et al. (1987) Science, 235:877-880. 6. Kang, J. et al. (1987) Nature, 325:733-736. 7. Lovett, M. et al. (1987) Biochem. Biophys. Res. Comm. 144:1069-1075. 8. Coyle, J.T. et al. (1988) Trends Neurosci., 11:390- 391. 9. Gropp, A. et al. (1975) Cytogenet. Cell Genet. 14:42- 62. 10. Gearhart et al. (1986) Brain Res. Bull. 16:789-801. 11. Stenevi, U. et al . (1985) In A. Bjorklund and U. Stenevi (Eds.), Neural Grafting in the Mammalian CNS, Elsevier, Amsterdam, pp. 41-49. 12. Masters, C.L. et al. (1985b) EMBO J., 4:2757-2763. 13. Abraham, C. et al . (1988) Cell, 52:487-501. 14. Sparkman, D.R. and White, C.L. Ill (1989) in G.D. Milner, et al. (Eds.), Familial Alzheimer's Disease - Molecular Genetics and Clincal Perspectives, Marcel Dekker Inc., N.Y., pp.269-285. 15. Wischik, CM. et al. (1988) Proc. Natl. Acad. Sci. USA 85:4506-4510. 16. Masters, C.L. et al. (1985a) Proc. Natl. Acad. Sci. 82:4245-4249. 17. Roher, A. et al. (1986) Proc. Natl. Acad. Sci. USA, 83:2662-2666. 18. Selkoe, D.J. et al . (1986) J. Neuroc em. 46:1820- 1834. 19. Glenner, G.G. and Wong, C.W. (1984) Biochem. Biophys. Res. Comm. , 120:885-890. 20. Wong, C.W. et al. (1985) Proc. Natl. Acad. Sci. USA, 82:8729-8732. 21. Robakis, N.K. et al. (1987) Lancet, i:384-385. 22. Tanzi, R.E. et al. (1987) Science, 235:880-884. 23. Zabel, B.U. et al. (1987) Cytogenet. Cell Genet. 46:725-726. 24. Van Broeckhoven, C. et al. (1987) Nature, 329:153- 155. 25. Abraham, C. et al. (1990) Neurobiol. Aging 11:123- 129. 26. Brion, J.P. et al. (1985) Archiv. Biol. 95:229-235. 27. Mori, H. et al. (1987) Science 235:1641-1644. 28. Hyman, B.T. et al- (1989) Neurosci. Lett. 101:352- 355. 29. Rabin, M. et al. (1986) Somatic Cell. Mol. Genet. 12:209-214. 30. Van Nostrand, W.E. et al. (1989) Nature 341:546-549. 31. Epstein, C.J. (1988) Disc. Neurosci., 5:127-134. 32. Waters, J.J and Bartlett, D.J. (1988) Clin. Cytogen. Bull., 2:30-31.

Claims: CLAIMS 1. A method of producing an in vivo mouse model for studying Alzheimer's disease, comprising introducing Trisomy 16 mouse embryonic central nervous system tissues to the brain of a live host mouse. 2. A method of studying Alzheimer's disease, comprising introducing Trisomy 16 mouse embryoniccentral nervous system tissues to the brain of a live host mouse, and studying the mouse. 3. A method of assisting the treatment or prevention of Alzheimer's disease, comprising introducing Trisomy 16 mouse embryonic central nervous system tissues to the brain of a live host mouse, and studying the mouse. 4. A method according to claim 2 or 3, further comprising treating the mouse by a potential therapeutic strategy. 5. A method accoriding to cliam 2, 3 or 4, wherein the mouse is sacrificed a suitable length of time after- introduction of the Trisomy 16 tissues, and tissues from the brain of the mouse are examined. 6. A method according to any one of the preceding claims, wherein the Trisomy 16 tissues are obtained from Trisomy 16 mice embryos obtained by mating female mice of the CFLP strain with male offspring from matings of mice with Robertsonian translocations involving chromosome 16. 7. A method- according to claim 6, wherein the host mouse is of the CFLP strain. 8. A method according to any one of the preceding claims, wherein the Trisomy 16 tissues are selected from one ox- more of the following: cortex, hippocampus, basal forebrain nucleii, cerebellum and basal ganglia. 9. A method according to any one of the preceding claims, wherein the Trisomy 16 tissues are introduced to the host mouse by grafting solid pieces or cell suspensions into cortical cavities or ventricular spaces, or by injecting dissociated cell suspensions into the host neuropil. 10. A mouse which has has Trisomy 16 mouse embryonic central nervous system tissues introduced to the brain thereof.

Inventors: DUNNETT STEPHEN BRUCE, RICHARDS SARAH-JANE, DUNNETT, STEPHEN, BRUCE, RICHARDS, SARAH-JANE
Applicants: MEDICAL RES COUNCIL, MEDICAL RESEARCH COUNCIL
Application year: 1990
Main classification: G01N33
IPC codes: A01K67, G01N33