EP1420635A1 - Transgenes modell zur behandlung von neurodegenerativen erkrankungen - Google Patents

Transgenes modell zur behandlung von neurodegenerativen erkrankungen

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EP1420635A1
EP1420635A1 EP01964267A EP01964267A EP1420635A1 EP 1420635 A1 EP1420635 A1 EP 1420635A1 EP 01964267 A EP01964267 A EP 01964267A EP 01964267 A EP01964267 A EP 01964267A EP 1420635 A1 EP1420635 A1 EP 1420635A1
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synuclein
transgenic
happ
mice
mouse
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Eliezer Masliah
Edward Rockenstein
Margaret E. Mallory
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University of California
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University of California
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Definitions

  • This invention relates to transgenic mice for use in the study of neurodegenerative diseases.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • LBD Lewy Body disease
  • AD Alzheimer's disease
  • each disease is associated with the degeneration of neurons, interneuronal synaptic connections and eventually cell death, the depletion of neurotransmitters, and abnormal accumulation of misfolded proteins, the precursors of which participate in normal central nervous system function.
  • ⁇ -synuclein also known as NACP (Non-amyloid component precursor).
  • NACP Non-amyloid component precursor
  • NAC Non-amyloid component precursor
  • LBD neurodegenerative disorders denominated Lewy body disease
  • Patients with LBD are characterized by dementia, parkinsonism, psychiatric alterations, deposition of amyloid, and formation of Lewy bodies (LBs) with filamentous characteristics.
  • LBs are the pathogenic hallmark of both PD and LBD.
  • the neuritic plaques that are the classic pathological hallmark of AD are composed essentially of A ⁇ , a 39-43 amino acid (aa) proteolytic product of the Alzheimer's amyloid precursor protein (APP), and NAC, a 35 aa proteolytic fragment of the NACP protein. Both A ⁇ and NAC were first identified in amyloid plaques as proteolytic fragments of their full
  • mutations are denoted by the wild type amino acid, the number of the amino acid at which the mutation is found and the amino acid that is found in the mutant form of the protein.
  • mutations seem to alter the processing of APP preferentially to the A ⁇ 2 proteolytic fragment, which has a propensity to form aggregates that are pathogenic.
  • mutations cannot account for the majority of Alzheimer's patients in whom the disease arises spontaneously.
  • APP is expressed abundantly in synapses under normal conditions, is well conserved across species, and has been implicated in neural plasticity, learning, and memory.
  • APP has three alternative splice variants in which exons 7 and 8 (hAPP695), exon 8 (hAPP751 ) or no exons (hAPP770) are spliced out of the full length transcript.
  • the ratio of the three forms varies between regions of the brain and in normal vs. disease states; however, no definitive pattern of normal vs. abnormal has been determined
  • proteolytic fragments A ⁇ 39 ⁇ 3 under normal conditions. Disease results from an imbalance in the production of the fragments, biasing the overproduction of the proteolytic fragments,
  • ⁇ -Synuclein is part of a large family of proteins including ⁇ - and ⁇ -synuclein and synoretin. As with APP, ⁇ -synuclein is expressed in the normal state in synapses and is
  • a ⁇ is secreted from neurons and accumulates in extracellular amyloid plaques. Additionally A ⁇ can be detected inside neurons. ⁇ -Synuclein accumulates in intraneuronal inclusions called Lewy bodies. Although the two proteins are typically found together in extracellular neuritic AD plaques, they are also occasionally found together in intracellular inclusions.
  • Aggregates may be formed in nerve cell cultures by overexpression of ⁇ - synuclein. Overexpression of wild-type APP does not cause formation of extracellular protein aggregates in culture; however, overexpression of the Swedish APP mutation, the most pathogenic of the APP mutations, does. Aggregates may also be formed in
  • transgenic APP failed to show extensive AD-type neuropathology (Kammesheidt et al., 1992; Lamb et al., 1993; Mucke et al., 1994; Higgins et al., 1995; Andraa et al., 1996). This was likely due to low levels of protein expression. Games et al. (1995) were able to generate a transgenic mouse that showed some AD-type pathology due to the high level of expression of a mutant APP (V717F) driven by a PDGF- ⁇ promoter. The transgenic animals did exhibit deposits of human A ⁇ in the hippocampus, corpus callosum and the cerebral cortex, but in no other regions of the brain. Plaques were observed, but there were no neurofibrilary tangles. The author stated that such results were expected as neurofibrilary tangles did not exist in rodents.
  • mice were found to have normal learning and memory at 3 months, but showed impairment by 9 or 10 months. Plaques were observed in the cortical and
  • a ⁇ 2 fragment was toxic to the cells, but no plaques were formed, suggesting a plaque independent role for A ⁇ 1-42 in the progression of AD.
  • transgenic animals expressing APP were found to display a variety of neurological problems including learning deficits, disturbed behavior and seizures. Again, the severity of neurological dysfunction seemed to be tied to the level of expression of the APP protein. Differences could also be attributed to the regions of the brain in which APP was expressed due to differences in promotors, integration into the genome, etc. As with the physiological symptoms though, neurological dysfunction in these animals resembled some aspects, but not all, of those seen in AD, PD and LBD. The lack of an animal model
  • mice in which the mutant gene was expressed were found to be lethal in inbred FVB/N or C57BL/6J mice (Carlson, et al., 1997). Lines of FVB/N mice
  • mice expressing ⁇ -synuclein under control of the PDGF ⁇ promotor have also been generated for use as a model of PD and LBD (Masliah et al., 2000). Neuronal expression of ⁇ -synuclein resulted in the progressive accumulation of ⁇ -synuclein and the
  • mice demonstrated motor impairments which were associated with loss of dopaminergic terminals in the basal ganglia. However, no amyloid plaques, fibrillary tangles or cell death were observed.
  • mice have also been generated.
  • promotors e.g. PDGF ⁇ , Thy-1
  • both wild type and mutant forms A30P and A53T
  • none of the mice provide a completely accurate model of PD.
  • differences were seen in the amount and location of protein expressed, depending on the promoter used and the site of insertion of the transgene into the genome. For example, when expression from the h ⁇ -synuclein transgene was driven by the PDGF- ⁇ promotor, the
  • mice expressing mutant versions of h ⁇ -synuclein developed hallmarks of disease earlier than those expressing the wild type version of the protein. Additionally, neurological dysfunction was dependent on the regions of the brain in which the protein was expressed.
  • the invention is a transgenic mouse model with transgenes for expression of human ⁇ -amyloid peptides or hAPP and h ⁇ -synuclein.
  • the transgenes may be wild type, mutant or truncated forms of the genes.
  • the transgenes may be expressed under any of a number of promoters including the PDGF- ⁇ , Thy1 and prion (PrP) promoters.
  • An intron such as the SV40 intron, may be included in the transgene construct or the promotor, as in the case of the Thy1 promoter, the intron of the promoter may be used.
  • the mice of the present invention are generated by crossbreeding of mice carrying
  • transgenic mouse lines overexpressing h ⁇ -synuclein (Masliah et al., 2000) and either wild- type or mutant forms of hAPP (Mucke et al, 2000) to elucidate the in vivo mechanism of amyloidogenesis in AD, PD and LBD. Additionally, there are a number of other hAPP transgenic mice available as referenced. Available mice include those expressing APP
  • Thy1 promoter under control of the mouse or human Thy1 promoter (Andraa et al, 1996), the PDGF- ⁇ promoter (Games et al., 1995) with orwithout an SV40 intron, and the PrP (Borchelt et al.,
  • mice are well studied and characterized regarding regions of the brain and time frame in which the transgenes are expressed.
  • transgenes can readily be generated by crossing two mice that overexpress either hAPP or h ⁇ - synuclein in regions of the brain most effected by AD, PD or LBD as desired. Ideally the mice crossed should carry only one copy of the transgene such that littermates expressing both, either or neither transgene can be compared to each other. Overexpression of transgenes can be detected by any of a number of methods well known to one skilled in the art including, but not limited to, ribonuclease protection assay (RPA), Western blot, immunofluorescence, or like analysis, where the signal obtained is above background cross-reactivity with mouse RNA or protein found in non-transgenic littermates.
  • RPA ribonuclease protection assay
  • the bigenic PDGF- ⁇ - ⁇ -synuclein-PDGF- ⁇ -hAPP mice of the invention have been shown to express high levels of APP which is properly processed into A ⁇ fragments, in addition to exhibiting extracellular amyloidosis and neurofibrilary tangles which, in an age-
  • hAPP bigenic mice resembled the Lewy body variant of AD. Bigenic mice developed
  • the onset of motor deficits began at 6 months with the bigenic mice, rather than at 12 months as is typically seen with the PDGF- ⁇ -hAPP mice.
  • the bigenic mice showed the most prominent age-dependent degeneration of cholinergic neurons and presynaptic terminals as compared to their singly- or non-transgenic littermates. Although synaptic degeneration was observed in the singly transgenic mice, neuronal cell death was not observed in the singly transgenic littermates.
  • hAPP- ⁇ -synuclein mice also had abundant ⁇ -amyloid plaques and an even greater number of ⁇ -synuclein-immunoreactive neuronal inclusions.
  • the present invention is a method for the evaluation of the in vivo effects of ⁇ -synuclein with APP, in combination, on amyloidogenesis and neurodegeneration. Evaluation of the effects of the proteins in the development and progression of disease may be assessed by functional, pathological or biochemical assays,
  • synuclein and hAPP/A ⁇ have distinct, as well as overlapping, pathogenic effects on the integrity and function of the brain. While h ⁇ -synuclein did not affect the A ⁇ -dependent development of neuritic plaques or the overall A ⁇ content in the brain, it worsened hAPP/A ⁇ -dependent cognitive deficits and neurodegeneration in specific brain regions. These findings indicate that h ⁇ -synuclein can enhance the toxicity of A ⁇ through plaque- independent mechanisms. They correlate well with clinical observations suggesting that people with the Lewy body variant of AD have a more rapid cognitive decline than people with pure AD.
  • hAPP/A ⁇ Overexpression of hAPP/A ⁇ , in turn, promoted the intraneuronal accumulation of h ⁇ -synuclein in transgenic mice and accelerated the development of motor deficits.
  • drugs aimed at blocking the accumulation of A ⁇ or h ⁇ -synuclein will likely benefit a broader spectrum of neurodegenerative disorders than previously anticipated.
  • the bigenic mouse provides a better predictive model of the potential for an intervention to be useful to a patient.
  • FIGURE 2. RNase protection assay of total RNA from brains of transgenic and non-
  • transgenic mice Lane U represents undigested transcripts. The right hand side notation of the figure indicate the size of the digested samples. Sample lanes represent analysis of three separate mice each from Non-Tg, non-transgenic mice (i.e. control); Thy1 -hAPPtg, transgenic mice; Thy 1 - ⁇ -syn'tg , transgenic mice; and double tg, transgenic mice containing both the human (h)APP751 and the ⁇ -synuclein genes.
  • mice that express different levels of each protein By using mice that express different levels of each protein, one can obtain a variety of models with different rates of onset and severity of disease. These will mimic the differences seen in
  • Transgenic mice expressing hAPP or ⁇ -synuclein can be divided into two general categories of low expressing and high expressing strains. The classes can be further subdivided into mice that express wild type proteins that are less toxic than mutant proteins. For example, all mice expressing APP will eventually develop some plaques and
  • mice expressing the Swedish double mutation at aa 670/671 will result in disease with fewer plaques in age matched animals.
  • different mutations and promoters will manifest various severity of disease in ⁇ -synuclein transgenic mice.
  • Mice expressing h ⁇ -synuclein under the control of a Thy-1 promoter accumulate protein in the synapses and neurons throughout the brain, including the thalmus, basal ganglia, substantia nigra and brainstem.
  • mice expressing ⁇ -synuclein under the control of the PDGF- ⁇ promoter accumulate protein in synapses in the neocortex, limbic system and olfactory regions, as well as in inclusion bodies in neurons in deeper layers of the neurocortex.
  • Another strain displayed ⁇ -synuclein expression in glial cells mimicking multiple system atrophy.
  • mice expressing hAPP and h ⁇ -synuclein in regions of the brain most severely effected in LBD could be crossed to serve as a model for LBD.
  • transgenic mice are selected based on criteria including level and brain region of protein expression, age of development of symptoms, neurological disorders and effect of background strain. Unlike transgenic mice that are generated by the insertion of
  • mice of the invention are bigenic mice generated by the combination of mice with well known
  • Models are designed to most closely fit the disease of interest based on the criteria among those listed above.
  • the expression of one protein is not disrupted by the expression of the other.
  • the enhancement of the pathology of ⁇ -synuclein by co- expression of hAPP is now known.
  • One skilled in the art may make rational decisions in combining known strains of mice to develop bigenic strains that most closely mimic the
  • Transgenic mice are achieved routinely in the art using the technique of microinjection, as described in U.S. Patent No. 4,736,866 issued to Leder et al., and as provided by B. Hogan et al. (1986).
  • U.S. Patent 5,574,206 issued to Jolicoeur particularly describes the creation of transgenic mice bearing functional HIV genes and their use in the modeling and study of HIV-mediated diseases. These references are herein incorporated by reference.
  • h ⁇ -synuclein and hAPP Coexpression of h ⁇ -synuclein and hAPP in vivo.
  • Transgenic mice in which neuronal expression of h ⁇ -synuclein (Masliah et al., 2000) or FAD-mutant hAPP (Mucke et al., 2000) is directed by the PDGF ⁇ chain promoter have been described. Additionally, a number of other APP mice have been described including, but not limited to, those described in the present application. Lines with high levels of neuronal h ⁇ -synuclein (line D) or A ⁇ (line J9) production were selected.
  • EXAMPLE 3 Overexpression of human ⁇ -synuclein in bigenic mice enhances Alzheimer's disease-like neuropathology. It has been established that both ⁇ -synuclein and NAC are integral components of plaques formed in in vitro models of Alzheimer's disease. In order to determine how these molecules contribute to amyloidogenesis in vivo, specifically in the PDAPP bigenic mouse models, brain sections from effected bigenic mice were treated with formic acid and immunostained with antibodies against ⁇ -synuclein and NAC.
  • ⁇ -synuclein is critical for plaque formation in vivo and as such, constitutes a reasonable target for the design of anti-amyloidogenic compounds useful for in vivo therapies of Alzheimer's disease and other neurodegenerative conditions involving amyloid formation.
  • EXAMPLE 4 Neurological deficits in h ⁇ -synuclein/hAPP mice. Motor deficits were assessed with the rotarod test (FIG. 1). Consistent with previous observations, h ⁇ -synuclein mice developed age-dependent motor deficits. At 6 months of age, h ⁇ -synuclein/hAPP mice already showed deficits relative to nontransgenic controls (P ⁇ 0.03 by repeated measures ANOVA), whereas h ⁇ -synuclein mice still performed normally. At 12 months of age, both h ⁇ -synuclein and h ⁇ -synuclein/hAPP mice showed deficits in this test compared with nontransgenic controls (P ⁇ 0.001 by repeated measures ANOVA).
  • hAPP mice had no significant motor deficits at either age. The severity of motor deficits was similar in 6- month-old h ⁇ -synuclein/hAPP and 12-month-old h ⁇ -synuclein and h ⁇ -synuclein/hAPP mice, suggesting that hAPP/A ⁇ accelerates the development of h ⁇ -synuclein-dependent motor deficits.
  • PDGF-hAPP mice have previously been shown to develop cognitive deficits. To determine the effects of h ⁇ -synuclein and hAPP/A ⁇ on spatial learning and memory, mice were assessed in a water maze test (Morris, 1984).
  • mice In the sessions during which the target platform was visible, all groups of mice were able to locate the platform equally well, indicating that the motor deficits of h ⁇ -synuclein/hAPP do not preclude normal performance in the water maze test.
  • mice In the hidden platform sessions, mice have to use their memory of the spatial relationship of the platform to visual cues outside of the maze to locate the submerged platform.
  • h ⁇ -synuclein/hAPP mice showed the most significant learning deficits, whereas h ⁇ -synuclein mice and nontransgenic controls performed normally.
  • hAPP mice tended to perform better than h ⁇ -synuclein/hAPP mice in the hidden platform sessions, but the difference was not statistically significant.
  • the probe trial during which the platform is removed provides a putative measure of spatial memory retention.
  • h ⁇ -synuclein/hAPP mice and hAPP mice showed significantly less preference for the target quadrant than nontransgenic controls, suggesting impaired memory retention, whereas h ⁇ -synuclein mice were not impaired.
  • Lewy body diseases which also combine motor and cognitive deficits.
  • Synaptophysin-immunoreactive presynaptic terminals of defined signal intensity (SIPT) in the neocortex of 4- to 20-month-old mice (n 15-19/genotype) were measured.
  • SIPT defined signal intensity
  • SIPT levels in the neocortex were highest in nontransgenic controls (25.6 ⁇ 0.9) and lowest in h ⁇ -synuclein/hAPP mice (18.3 ⁇ 4.8, P ⁇ 0.05 by Tukey-Kramer test), with values for hAPP mice (20.9 ⁇ 1.7) and h ⁇ -synuclein mice (23.0 ⁇ 0.9) falling in between.
  • SIPT levels in the caudate/putamen at 20 months of age were normal in nontransgenic mice (27.1 ⁇ 1.5) and hAPP mice (25.1 ⁇ 3.6), but decreased in h ⁇ -synuclein mice (21.0 ⁇ 0.9) and h ⁇ -synuclein/hAPP mice (20.1 ⁇ 1.7) (P ⁇ 0.05 vs nontransgenic controls by Tukey-Kramer test).
  • EXAMPLE 6 A ⁇ promotes accumulation of h ⁇ -synuclein. Accumulation of h ⁇ -synuclein within neurons is a hallmark of Lewy body diseases, including the Lewy body variant of AD. Age- dependent accumulation of h ⁇ -synuclein in neurons of h ⁇ -synuclein singly transgenic mice has been previously observed (Masliah, 2000). Between 4 and 20 months of age, the number of neuronal inclusions in the neocortex was on average 1.6-fold higher in h ⁇ - synuclein/hAPP mice than in age-matched h ⁇ -synuclein mice (15.3 ⁇ 1.1 vs.
  • a ⁇ , ⁇ and A ⁇ also differed in their effect on the intracellular accumulation of ⁇ -synuclein in neuronal cell cultures when added to the culture medium.
  • a ⁇ 2 strongly increased the intracellular accumulation of ⁇ -synuclein, but A ⁇ o did not.
  • RNA from hemibrains or from dissected brain regions was isolated and then analyzed by a solution hybridization RNase protection assay and resolved on a acrylamide/urea/Tris/borate/EDTA gel ( Figure 2).
  • Lane U represents undigested transcripts of the ⁇ -synuclein gene ( ⁇ -syn), the A ⁇ precursor gene (APP) and actin
  • Sample lanes represent analysis of three separate mice each from Non-Tg, non-transgenic mice (i.e. control); Thy1-hAPPtg, transgenic mice expressing human (h)APP751 cDNA under the regulatory control of the murine (m)Thy-1 gene; They1- ⁇ - syn'tg, transgenic mice expressing human ⁇ -synuclein under the regulatory control of the muring (m)Thy-1 gene; and double tg, transgenic mice containing both the human (h)APP751 and the ⁇ -synuclein genes under the regulatory control of the murine
  • EXAMPLE 8 Crossbreeding ofha-synuclein or PDAPP-J9M tg mice with ma- or m ⁇ -synuclein KO mice.
  • knock out mice having the genes encoding ⁇ -synuclein or ⁇ - synuclein deleted (Lexicon Genetics Inc., The Woodlands, TX) were crossed with heterozygous PDAPP-J9M tg mice.
  • ⁇ -synuclein and ⁇ -synuclein on amyloid deposition was evaluated in both the homozygous KO mice (PDAPP+/+;m ⁇ -syn-/-) and in the mice hemizygous for synuclein (PDAPP+/+;m ⁇ -syn+/-) utilizing the same rotorod and neuropathological analysis used in previous examples.
  • Lewy body diseases are common neurodegenerative disorders, second only to AD. They comprise a heterogeneous group of diseases including PD, diffuse Lewy body disease, and the Lewy body variant of AD.
  • Lewy body disease typically has Lewy bodies, parkinsonism and cognitive impairments. Approximately 25% of patients with AD develop frank parkinsonism, and h ⁇ -synuclein-immunoreactive Lewy body-like inclusions develop in most cases of sporadic AD and FAD, as well as in Down's syndrome, which is associated with early-onset AD. Moreover, Lewy bodies contain hAPP. These associations indicate that hAPP/A ⁇ plays a role in the formation of Lewy bodies and the development of Lewy body diseases.
  • h ⁇ -synuclein mice demonstrate that h ⁇ -synuclein and hAPP/Ab have distinct, as well as convergent, pathogenic effects on the integrity and function of the brain. While h ⁇ -synuclein did not affect the A ⁇ -dependent development of neuritic plaques or the overall A ⁇ content in the brain, it worsened hAPP/A ⁇ -dependent cognitive deficits and neurodegeneration in specific brain regions. These findings indicate that h ⁇ -synuclein enhances the toxicity of A ⁇ through plaque- independent mechanisms. These data explain clinical observations suggesting that people with the Lewy body variant of AD have a more rapid cognitive decline than people with pure AD.
  • hAPP/A ⁇ Overexpression of hAPP/A ⁇ , in turn, promoted the intraneuronal accumulation of h ⁇ -synuclein in transgenic mice and accelerated the development of motor deficits. These effects were most likely mediated by A ⁇ or another hAPP product, in vitro studies indicate that A ⁇ is the culprit. In view of these pathogenic interactions between A ⁇ and h ⁇ -synuclein, drugs aimed at blocking the accumulation of Ab or h ⁇ -synuclein will benefit a broader spectrum of neurodegenerative disorders than previously anticipated.
  • Oxidative stress induces amyloid-like aggregate formation of ⁇ ACP/ ⁇ -synuclein in vitro. Neuroreport 10:717-21.

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