JP2004538013A - Development of genetic recombination model for medical treatment of neurodegenerative diseases - Google Patents

Abstract

Alzheimer's disease, Parkinson's disease and Lewy body disease are the most common neurodegenerative disorders in the elderly. The present invention is a transgenic mouse containing two transgenes, human α-synuclein and human amyloid precursor protein, which cause the formation of neuritic plaque, Lewy bodies and neurodegeneration found in the above-mentioned diseases. This transgenic mouse is an accurate model of the disease by both functional and pathological assays.

Description

【Technical field】
[0001]
(Field of the invention)
The present invention relates to transgenic mice used in the study of neurodegenerative diseases.
[0002]
(Government interests)
This invention was made with government support from the National Institutes of Health under grant number AG10869.
[Background Art]
[0003]
(background)
Alzheimer's disease (AD), Parkinson's disease (PD) and Lewy body disease (LBD) are the most common neurodegenerative disorders in the elderly. Although their outbreaks continue to increase and cause significant social health problems, so far these diseases cannot be cured or prevented. Although there is a genetic component for these diseases, in most cases they occur spontaneously in the population without mutations. Nerve damage results from the formation of protein aggregates composed of proteins normally expressed in the brain. It is not known what converts non-toxic proteins into their toxic state.
[0004]
Recent epidemiological studies have demonstrated a close clinical relationship between AD and PD, with about 30% of Alzheimer's disease patients also having PD. Compared to other elderly populations, AD patients generally develop comorbid PD. In addition, PD patients with dementia usually develop classic AD.
[0005]
Although each neurodegenerative disease appears to be biased in specific brain regions and cell populations, resulting in a distinct pathology, PD, AD and LBD also share common features. Patients with familial AD, Down syndrome and sporadic AD grow Lewy bodies on the amygdala, a typical neuropathological feature of PD. In addition, each disease is associated with degeneration of neurons, synaptic connections and ultimately cell death in neurons, loss of neurotransmitters, and abnormalities in abnormally folded proteins whose precursors are involved in normal central nervous system function. Related to the accumulation.
[0006]
Biochemical studies have confirmed a link between AD, PD and LBD. The central component of Lewy bodies is α-synuclein, also known as NACP (non-amyloid component precursor). The proteolytic fragment of NACP, known as NAC, is an important component of amyloid plaque in AD. This suggests that α-synuclein plays a role in the pathogenesis of both AD and PD.
[0007]
These findings have led to the development of a new classification of a neurodegenerative disorder called Lewy body disease (LBD). LBD patients are characterized by dementia, Parkinson's syndrome, mental changes, deposition of amyloid, the formation of Lewy bodies (LB) with fibrous features. LB is a hallmark of PD and LBD pathology. While there are some animal models that mimic certain aspects of AD and other animal models that mimic certain aspects of PD, there are no models that combine the characteristics of AD and PD, such as those found in LBD.
[0008]
The neuritic plaques that are characteristic of the typical pathology of AD are mainly Aβ, a 39-43 amino acid (aa) proteolytic product of Alzheimer's amyloid precursor protein (APP), and 35 aa proteolytic fragments of the NACP protein. NAC. Both Aβ and NAC were first identified in amyloid plaques as proteolytic fragments of their full-length proteins, for which full-length cDNAs have been identified and cloned. The cloning of APP (Kang et al., 1987) has led to a surge in research, in which a number of mutations in AD have been found, K670N / M671L (Swedish mutation), V717I (London mutation), Familial forms including V717F (Indiana mutation), presenilin 1 and presinilin 2 have been associated. (Note: mutations are indicated by the wild-type amino acid, the number of the amino acid in which the mutation was found, and the amino acid found in the mutant form of the protein). These mutations cause the processing of APP to alter Aβ, which tends to form pathogenic aggregates._1-42It will change to proteolytic fragment preference. However, such mutations cannot account for the majority of Alzheimer's patients whose disease occurs spontaneously.
[0009]
APP is highly expressed in synapses in normal conditions, is well conserved across species, and is involved in neural flexibility, learning, and memory. APP has three alternative splicing variants in which exons 7 and 8 (hAPP695), exon 8 (hAPP751) or no exons (hAPP770) have been spliced from full-length transcripts. Although the proportions of the three types differ between regions of the brain and between normal and diseased states, a clear pattern of normal and abnormal has not been determined (Rockenstein et al., 1995). It is not known what regulates the different splicing of transcripts, but evidence suggests that splicing in neurons can be regulated by factors that affect neuronal differentiation and activity. In addition, APP is normally under the proteolytic fragment Aβ_39-43Is processed. Diseases are caused by proteolytic fragments, especially aggregates (ie, Aβ_1-42) Can be initiated but due to an imbalance in the production of fragments that is biased towards overproduction.
[0010]
α-Synuclein is part of a large family of proteins including β- and γ-synuclein and sinoretin. Like APP, α-synuclein is expressed in synapses in the normal state and is thought to play a role in neural flexibility, learning, and memory. Mutations in human (h) α-synuclein that promote aggregation of α-synuclein have been identified (A30P and A53T), and they have been implicated in the rare form of the autosomal dominant form of PD. The mechanism by which these mutations increase the tendency of α-synuclein to aggregate is unknown.
[0011]
Many cases of AD and PD occur spontaneously, despite the fact that numerous mutations can be found in APP and α-synuclein in the population. The most common sporadic forms of these diseases are associated with abnormal accumulation of Aβ and α-synuclein, respectively. However, the reasons for the excessive accumulation of these proteins are unknown. Aβ is secreted from neurons and accumulates in extracellular amyloid plaques. In addition, Aβ is detected inside neurons. Alpha-synuclein accumulates in inclusion bodies within neurons called Lewy bodies. Although the two proteins are typically found together in extracellular neural AD plaques, they are also sometimes found together in intracellular inclusions.
[0012]
The processing of non-toxic precursor proteins into their proteolytic products has been studied for analysis both in tissue culture systems and with purified proteins. Aggregates can form in neuronal cell cultures due to overexpression of α-synuclein. Overexpression of wild-type APP does not cause formation of extracellular protein aggregates in culture, but does cause overexpression of the Swedish mutation, which is the most pathogenic of APP mutations. Aggregates also form in cells overexpressing α-synuclein in response to oxidative stress in cell culture, suggesting that it is important in disease development. Furthermore, the purified Aβ and NAC peptide can form aggregates in vitro under the same temperature and pH conditions alone or as a mixture.
[0013]
Attempts to construct animal models for AD, PD and LBD have been disappointing. Initially, animals expressing the recombinant APP did not 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 is probably due to low levels of protein expression. Games et al. (1995) were able to create transgenic mice exhibiting certain AD-type pathologies, because high levels of expression of mutant APP (V717F) driven by the PDGF-β promoter. Transgenic animals showed deposition of human Aβ in the hippocampus, corpus callosum and cerebral cortex, but not in other areas of the brain. Plaques were observed, but no neurofibrillary tangles. The authors stated that such a result was expected because neurofibrillary tangles were absent in rodents.
[0014]
Genetically engineered mice expressing the Swedish double mutation (670/671) or the Swedish mutation coupled to the London mutation (V717I) under the control of the Thy1 expression cassette have also been generated (Struchler-Pierrat et al., 1997). Year). The age of onset of plaques varied from 6 months to over 2 years in each mouse species and appeared to correlate well with protein expression levels. In animals with both Swedish and Indiana mutations, lower expression levels were required to induce disease-related pathological changes. However, none of the animals manifested the disease completely and accurately. Plaque formation with neuronal changes, dystrophic cholinergic fibers and inflammation was observed in hippocampus and neocortex, but not in other brain regions. In another study using transgenic mice expressing the Swedish mutation of APP under the PrP-promoter (Hsiao et al., 1996), the mice were found to have normal learning and memory at 3 months. However, the disorder appeared by 9 or 10 months. Plaques were observed in cortical and limbic regions in these transgenic mice and Aβ_{1-42 / 43}A 14-fold increase was observed.
[0015]
Aβ_1-42Is known to be one of the strong causative factors in AD, Mucke et al. (2000) describe a toxic fragment of APP, Aβ_1-42Genetically engineered mice that expressed only E. coli were prepared. Aβ_1-42Expression of the fragment was cytotoxic, but no plaque was formed and Aβ in the progression of AD_1-42Suggests a plaque-independent role for.
[0016]
In addition, transgenic animals expressing APP have been found to exhibit various neurological abnormalities, including learning deficits, impaired behavior and seizures. Again, the severity of neuronal dysfunction appeared to be linked to APP protein expression levels. Differences can also be attributed to regions of the brain where APP is expressed due to differences in promoters, integration into the genome, and the like. Similar to the physiological symptoms, the neurological dysfunction in these animals was similar in some, but not all, of those seen in AD, PD and LBD. The lack of an animal model for the disease is clearly not due to lack of effort, but rather because it cannot elicit all the changes that occur in the disease state.
[0017]
The expression effect of the transgenic sequence also greatly depended on the strain of mouse in which the mutant gene was expressed. For example, the concentration of APP producing plaques in outbred transgenic lines was found to be lethal in inbred FVB / N or C57BL / 6J mice (Carlson et al., 1997). Lines of FVB / N mice that express sufficiently low levels of APP695 to survive have CNS disorders including neophobia, impaired spatial alternations, mainly with decreased cerebral glucose utilization and astrocytosis. (Hsiao et al., 1995). A similar syndrome is known to occur in about 20% of non-transgenic mice of the same species, suggesting that APP promotes a pre-existing tendency in the strain.
[0018]
Attempts have been made to generate a more accurate model of AD by crossing strains of transgenic mice to alter disease symptoms. Overexpression of the fibroblast growth factor 2 gene (FGF2) in transgenic mice overexpressing APP renders APP more lethal, but does not alter the symptoms seen. Holtzman et al. (2000) generated a large number of bigenic mice by mating transgenic mice expressing the V717F variant of APP with mice expressing various forms of apolipoprotein E (apoE). I let it. The ε4 allele of apo E was the first genetic risk factor identified for sporadic AD and late-onset familial AD (FAD) (Strittmatter et al., 1993). Subsequently, the ε3 allele of apo E was found to be a weaker risk factor for the disease. ApoE variants, especially the ε4 allele expression, along with the V717F variant version of APP in mice, increased the incidence of fibril deposition, neuronal plaques and neurodegeneration. Similar pathological features of AD were simply observed in old age in mice expressing only the V717F variant of APP. The combination of transgene expression did not change the observed pathology of the disease, but simply changed the time frame in which the pathology was observed. Expression of the V717F mutant version of APP in ApoE knockout mice did not result in neurodegeneration. This study shows a clear role for apoE in the pathology of AD, but did not provide a more accurate model of the disease than single transgenic mice.
[0019]
Transgenic mice expressing α-synuclein under the control of the PDGFβ promoter have also been created for use as models for PD and LBD (Masliah et al., 2000). Neuronal expression of α-synuclein resulted in a progressive accumulation of α-synuclein and the development of inclusions in neurons in the neocortex, hippocampus and substantia nigra. In addition, electron dense nuclear deposition and cytoplasmic encapsulation were observed. The mouse exhibited a dyskinesia associated with a dopaminergic terminal deletion in the basal ganglia. However, no amyloid plaques, fibrotic changes or cell death were observed.
[0020]
Numerous α-synuclein transgenic mice have also been generated. However, despite the use of both wild-type and mutant forms of α-synuclein (A30P and A53T), as well as different promoters (eg, PDGFβ, Thy-1), all mice were completely accurate in PD. Does not bring a simple model. Similar to various APP transgenic mice, differences were found in protein expression levels and expression positions depending on the promoter used and the insertion site of the transgene into the genome. For example, when expression from the hα-synuclein transgene was driven by the PDGFβ promoter, the expression pattern of hα-synuclein was most similar to that seen in normal brain or diffuse LBD. Expression of hα-synuclein using the Thy-1 promoter resulted in higher expression of the transgene in a pattern most similar to that seen in PD. Higher expression resulted in a more severe condition. Mice expressing a mutant version of hα-synuclein clearly demonstrated disease characteristics earlier than those expressing a wild-type version of the protein. In addition, neuronal dysfunction depends on the area of the brain where the protein is expressed.
[0021]
Disease states are often the result of multiple changes in an organism, rather than changes in the expression levels of a single protein or a single mutation in a gene. Although each of the transgenic animals described above has certain characteristics of AD, PD and / or LBD, none of them could provide a complete model for any disease. No single animal was found with neurofibrillary tangles, a typical indicator of AD. Recognition that the pathology and development of AD, PD and LBD overlap, emphasizes the need for animal models that exhibit a more complete set of disease states and clinical signs.
DISCLOSURE OF THE INVENTION
[Means for Solving the Problems]
[0022]
(Summary of the Invention)
The present invention is a transgenic mouse model having a transgene for expressing human β-amyloid peptide or hAPP and hα-synuclein. The transgene can be a wild-type, mutant or truncated version of the gene. The transgene can be expressed under any of a number of promoters, including PDGF-β, Thy1 and prion (PrP) promoters. An intron, such as the SV40 intron, may be included in the transgene construct or promoter, and the promoter intron may be used, as in the case of the Thy1 promoter.
[0023]
The mice of the present invention are generated by crossing mice having a single gene for α-synuclein or hAPP. Applicants have generated a number of transgenic mouse strains that overexpress hα-synuclein (Masliah et al., 2000) and a large number of either wild-type or mutant hAPPs (Mucke et al., 2000) to produce AD, PD And the in vivo mechanism of amyloid production in LBD. Further, as referenced, there are numerous other hAPP transgenic mice available. Available mice include those that express APP under the control of the mouse or human Thy1 promoter (Andraa et al., 1996), PDGF-β promoters with or without the SV40 intron (Games et al., 1995), and PrP (Borchelt et al., 1996; Hsia et al., 1999). The mice have been well studied and characterized for the brain region and time frame in which the transgene is expressed. Bigenic mice can easily be generated by crossing two mice that overexpress either hAPP or hα-synuclein in the region of the brain that is best achieved by AD, PD or LBD, if desired. . Ideally, bred mice will have only the transgene so that litters that express both transgenes, express either transgene, or express neither transgene can be compared to each other. You need to have one copy. Overexpression of the transgene can be by any of a number of methods well known to those of skill in the art, including but not limited to ribonuclease protection assay (RPA), Western blot, immunofluorescence, or other assays. , Where the signal obtained is above background cross-reactivity with mouse RNA or protein found in non-transgenic littermates.
[0024]
The bigene PDGF-β-α-synuclein-PDGF-β-hAPP mice of the present invention have extracellular amyloidosis and neurogenesis similar to senescent plaques morphologically found in AD in an age and brain region specific manner. In addition to exhibiting fibrotic changes, it has been shown to express APP properly processed to Aβ fragments. Expression of hα-synuclein did not appear to alter hAPP expression. However, there was more extensive accumulation of hα-synuclein in bigenic mice compared to single hα-synuclein mice.
[0025]
Functional and morphological changes in PDGF-β-α-synuclein-PDGF-β-hAPP bigenic mice resembled Lewy body variants of AD. Bigenic mice produced physiological and pathological features and reduced motor function, more closely resembling human disease states, than single transgenic mice. The effect of the combination of the expression of both genes is synergistic. The development of fibrous α-synuclein inclusions, a feature not previously observed in rodents but a standard feature of PD and LBD, was seen. Synaptic degeneration was observed in both single transgenic mice, but no neuronal cell death was observed in bigenic mice. Bigenic mice have severe deficits in learning and memory, and PDGF-β-α-synuclein single transgenic mice, which show only minor if motor deficits occur, Motor deficits occurred. The onset of motor deficits began at 6 months in bigenic mice, rather than the 12 months typically seen in PDGF-β-hAPP mice. Bigenic mice exhibited the most pronounced age-dependent degeneration of cholinergic neurons and presynaptic terminals compared to single transgenic littermates or non-transgenic littermates. Synaptic degeneration was observed in single transgenic mice, but neuronal cell death was not observed in single transgenic littermates. The hAPP-α-synuclein mice also had large amounts of β-amyloid plaques and, rather, a larger number of α-synuclein-immunoreactive neuronal inclusions. Ultrastructurally, these inclusions were often fibrous in double transgenic mice, but amorphous in single transgenic α-synuclein mice. This is the first demonstration of the presence of filamentous Lewy body-like inclusions that were previously thought to be absent in rodents.
[0026]
Furthermore, the present invention is a method for evaluating the effect of a combination of α-synuclein and APP on amyloid production and neurodegeneration in vivo. Assessment of the effect of the protein on the development and progression of the disease can be determined by functional, pathological or biochemical assays, ideally with only a single transgene for either α-synuclein or hAPP or It can be evaluated in comparison with littermates without the transgene. Studies in the bigeneic α-synuclein-hAPP mouse have shown that hα-synuclein and hAPP / Aβ have distinct and overlapping pathogenic effects on brain integrity and function. Although hα-synuclein does not affect the development of Aβ-dependent neuronal plaques or overall Aβ content in the brain, it exacerbates hAPP / Aβ-dependent cognitive deficits and neurodegeneration in certain brain regions. These findings indicate that hα-synuclein can enhance Aβ toxicity through a plaque-independent mechanism. They correlate well with clinical findings, suggesting that people with Lewy body mutations in AD have more acute cognitive decline than people with pure AD. Overexpression of hAPP / Aβ in turn promoted intraneuronal accumulation of hα-synuclein in transgenic mice and accelerated the development of motor deficits. In view of these pathogenic interactions between Aβ and hα-synuclein, drugs aimed at blocking the accumulation of Aβ or hα-synuclein have a broader spectrum of neurodegenerative disorders than previously expected. It will bring benefits. In addition, bigenic mice provide a better predictive model for potential interventions that are useful for patients.
BEST MODE FOR CARRYING OUT THE INVENTION
[0027]
(Detailed description and preferred embodiments)
The mechanism by which altered expression and processing of α-synuclein or APP leads to neurodegenerative disorders such as AD, PD and LBD remains somewhat unknown. It is generally understood that abnormal protein aggregation is a common feature in these disorders, and that either full-length α-synuclein or its protein fragments and Aβ are the major components of these protein aggregates. In many pathological conditions, multiple changes are necessary for the development of a complete medical condition. Animal models with a single mutation in either APP or α-synuclein have not yielded accurate models of AD, PD or LBD. However, bigenic PDGF-β-α-synuclein-PDGF-β-hAPP mice exhibit characteristics of these diseases that were not previously observed in any of the single transgenic animals. By expressing both proteins, an accurate model of the disease is obtained. By using mice that express different levels of each protein, various models can be obtained that have different rates of disease incidence and severity. These would mimic the differences found in humans.
[0028]
Transgenic mice expressing hAPP or α-synuclein can be divided into two general categories: low-expressing strains and high-expressing strains. This classification can be further subdivided into mice expressing wild-type proteins that are less toxic than the mutant proteins. For example, all mice expressing APP will eventually develop some plaque and some disease. However, the rate of plaque development and resulting disease will be the fastest in mice expressing the Swedish double mutation at aa670 / 671. Expression of hAPP with a single mutation at aa717 will result in disease with fewer plaques in age-matched animals. Similarly, different mutations and promoters will show varying degrees of disease in α-synuclein transgenic mice. Mice expressing hα-synuclein under control of the Thy-1 promoter accumulate proteins in synapses and neurons throughout the brain, including the thalamus, basal ganglia, substantia nigra, and brain stem. One strain of mice expressing α-synuclein under the control of PDGF-β has proteins in synapses in the neocortex, limbic and olfactory as well as in inclusion bodies of neurons in the deeper layers of the neurocortex. To accumulate. Other lines showed α-synuclein expression in glial cells, mimicking multisystem atrophy.
[0029]
In order for proteins to work synergistically to produce conditions not seen in single transgenic mice, it is necessary to turn to a line of mice that express those proteins in the same brain region. Aβ_1-42Production is clearly the driving force in the development of an LBD-like state in bigenic mice. Expression of hAPP increases accumulation of hα-synuclein. It also increases the severity of the disease and results in early onset of the disease in hα-synuclein transgenic mice. Hα-synuclein transgenic mice without overt disease develop disease when crossed with transgenic mice expressing an amyloidogenic version of APP. Mating of hα-synuclein mice without overt disease with transgenic mice expressing a non-amyloidogenic version of hAPP does not exacerbate the disease development process. The spontaneous forms of AD, PD and LBD have a relatively slow onset, suggesting low levels of mutant protein expression, so that crossing two strains without significant disease alone requires human It does not provide a model for the spontaneous onset of the disease that is often found. Similarly, transgenic animals expressing APP earlier than α-synuclein mimic AD patients who later develop dementia. Transgenic animals expressing low levels of both proteins mimic the spontaneous onset of their disease, slow in AD, PD and LBD. The brain region of expression also provides the incentive to cross certain transgenic lines. For example, mice expressing hAPP and hα-synuclein in the most severely affected brain regions in LBD could be bred to provide a model for LBD.
[0030]
Existing transgenic mice are selected based on criteria such as the level of protein expression and brain area, age at onset of symptoms, neuropathy and background strain effects, and the like. Unlike transgenic mice, which are generated by inserting the construct into a mouse embryo that can result in a large number of phenotypes, the mice of the present invention use two genes generated by a combination of mice with well-known characteristics. Mouse. The model is designed to best fit the disease of interest based on the criteria listed above. The expression of one protein is not disturbed by the other. The worsening of the pathology of α-synuclein due to co-expression of hAPP is not known. One skilled in the art can make rational decisions on combinations of known strains of mice to create a bigenic strain that most closely mimics the disease state of interest.
Embodiment 1
[0031]
Creation of transgenic α-synuclein and hAPP mice. Transgenic mice are described in U.S. Pat. No. 4,736,866 issued to Leder et al. It is routinely made in the art using the microinjection method provided by Hogan et al. (1986). U.S. Pat. No. 5,574,206 issued to Jolicoeur specifically describes the generation of transgenic mice with functional HIV genes and their use in modeling and studying HIV-mediated diseases. These documents are incorporated herein by reference.
Embodiment 2
[0032]
Co-expression of hα-synuclein and hAPP in vivo. Transgenic mice have been described in which neuronal expression of hα-synuclein (Masliah et al., 2000) or FAD-mutated hAPP (Mucke et al., 2000) is driven by the PDGF β chain promoter. In addition, a number of other APP mice have been described, including but not limited to those described in this application. High levels of neuronal hα-synuclein (line D) or Aβ (line J9) producing lines were selected. Mating between heterozygous hα-synuclein and heterozygous hAPP mice from these lines resulted in four groups of littermates: hα-synuclein mice (n = 26), hAPP mice (n = 32), hα-synuclein / hAPP mice (n = 21) and non-transgenic controls (n = 31) were given. Levels of transgene-derived mRNA in the brain were similar in single transgenic and double transgenic mice, indicating that co-expression of hα-synuclein and hAPP did not alter transgene expression ( (Fig. 2). At the protein level, expression of hα-synuclein did not affect hAPP expression in double transgenic mice. However, brain hα-synuclein levels tend to be higher in hα-synuclein / hAPP mice than in hα-synuclein mice, and may reflect the effect of Aβ on hα-synuclein accumulation.
Embodiment 3
[0033]
Overexpression of human .ALPHA.-synuclein exacerbates Alzheimer's disease-like neuropathology in bigenic mice. Both α-synuclein and NAC have been demonstrated to be integral components of plaques formed in in vitro models of Alzheimer's disease. To determine how these molecules contribute to amyloidogenesis in vivo, particularly in the PDAPP bigene mouse model, brain sections from the resulting bigene mice were treated with formic acid and treated with α- Immunostaining with antibodies to synuclein and NAC. Analysis of the results confirms that in PDAPP bigenic mice, NAC is present in the amyloid core, while α-synuclein is found in dystrophic neurites, as an in vitro model of Alzheimer's disease. Furthermore, neuropathological studies of PDAPP digenic mice overexpressing α-synuclein revealed extensive stellate gliosis in the neocortex and hippocampus with increased amyloid deposition, and increased α-synuclein expression resulted in increased amyloid production. Was again confirmed to play an important role in the pathogenesis of neurodegenerative conditions associated with. Thus, α-synuclein is crucial for plaque formation in vivo, and as such is a rational for the design of anti-amyloidogenic compounds useful in the treatment of Alzheimer's disease and other neurodegenerative conditions, including amyloidogenesis. It was finally proved that the target was composed.
Embodiment 4
[0034]
Nerve deficiency in hα-synuclein / hAPP mice. Motion deficits were evaluated in a rotating bar test (FIG. 1). In agreement with previous observations, hα-synuclein mice developed age-dependent motor deficits. At 6 months of age, hα-synuclein / hAPP mice already showed deficiency relative to non-transgenic controls (P <0.03 according to repeated measures ANOVA), but hα-synuclein mice still performed normally . At 12 months of age, both hα-synuclein mice and hα-synuclein / hAPP mice showed deficits in this test compared to non-transgenic controls (P <0.001 according to repeated measures ANOVA). . hAPP mice did not have significant motor deficits at any age. The severity of motor deficits is similar in 6-month-old hα-synuclein / hAPP mice and 12-month-old hα-synuclein mice and hα-synuclein / hAPP mice, where hAPP / Aβ produces hα-synuclein-dependent motor defects. To promote.
[0035]
PDGF-hAPP mice have previously been shown to develop cognitive deficits. To determine the effects of hα-synuclein and hAPP / Aβ on spatial learning and memory, mice were evaluated in the water maze test (Morris, 1984). In sessions where the target platform was visible, all groups of mice were able to find the platform equally well, indicating that hα-synuclein / hAPP motor deficits did not interfere with normal performance in the water maze test. . In sessions where the platform is hidden, the mouse must use the visible cues outside the maze and the memory of its spatial relationship to locate the sinking platform. In these sessions, hα-synuclein / hAPP mice showed the most significant learning deficits, while hα-synuclein mice and non-transgenic controls performed normally. hAPP mice tended to perform better than hα-synuclein / hAPP mice in the hidden platform session, but the differences were not statistically significant. Scrutiny tests that move the platform in the meantime provide an estimate of spatial memory retention. In a scrutiny test, hα-synuclein / hAPP and hAPP mice showed significantly less execution in the quadrant of interest than non-transgenic controls, suggesting impaired memory retention, while hα-synuclein mice Was not impaired.
[0036]
These results indicate that motor deficits in hα-synuclein / hAPP mice are mainly caused by hα-synuclein, whereas deficits in spatial learning and memory are mainly caused by hAPP / Aβ. Similar effects of hα-synuclein and hAPP would cause Lewy body disease, also combined with motor and cognitive deficits.
Embodiment 5
[0037]
Age-dependent neurodegeneration. Degeneration of cholinergic neurons in the basal ganglia of Meynert results in significant acquisition deficits in the water maze test in rodents. It is also a potentially important determinant of cognitive decline in AD and Lewy body disease. Choline acetyltransferase (ChAT) mediates the synthesis of acetylcholine and serves as a marker for cholinergic neurons. The density of ChAT immunoreactive neurons in the basal ganglia of the transgenic model (n = 12-13 / genotype, age range: 4-20 months) was measured. In hα-synuclein / hAPP mice, simple regression analysis found a high significant inverse correlation between aging and the number of cholinergic neurons (R = 0.828, P = 0.0005). ). Such a trend toward correlation is also present in hα-synuclein mice (R = 0.538, P = 0.071) and hAPP mice (R = 0.511, P = 0.089), but non- Absent in transgenic controls (R = 0.127, P = 0.695). The greatest loss of cholinergic neurons was observed in hα-synuclein / hAPP mice, while a more modest loss was found in aged hAPP mice. These results are consistent with the finding that cholinergic deficiency is more severe in humans with a Lewy body variant of AD than with pure AD. The hα-synuclein / hAPP and hAPP mice also have a significant loss of cholinergic neurons in the caudate nucleus / putamen, which is significant in the caudate nucleus of AD patients with or without Lewy bodies. Is consistent with a decrease in ChAT activity.
[0038]
People with AD or Lewy body variants of AD typically also have significant synaptic loss in the neocortex, which reflects a decrease in synaptophysin immunoreactivity. Synaptophysin immunoreactive presynaptic terminals (SIPT) at defined signal intensities were measured in the neocortex of 4-20 month old mice (n = 15-19 / genotype). In hAPP mice (R = 0.744, P = 0.0006) and hα-synuclein / hAPP mice (R = 0.524, P = 0.021), a significant difference between aging and neocortical SIPT levels There was an inverse correlation, but not in hα-synuclein mice (R = 0.479, P = 0.071) or non-transgenic controls (R = 0.151, P = 0.564). At 20 months of age, SIPT levels in the neocortex (% occupied area, mean ± SD, n = 3-4 / genotype) were highest in non-transgenic controls (25.6 ± 0.9) and hα -Synuclein / hAPP mice (18.3 ± 4.8, p <0.05 by Tukey-Kramer test), lowest, hAPP mice (20.9 ± 1.7) and hα-synuclein mice (23 ± 0. The value for 9) was in the middle. In contrast, at 20 months of age (mean ± SD, n = 3-4 / genotype), the SIPT levels in the tail / putamum were higher in non-transgenic mice (27.1 ± 1.5) and hAPP. Normal in mice (25.1 ± 3.6) but hα-synuclein mice (21.0 ± 0.9) and hα-synuclein / hAPP mice (20.1 ± 1.7) (Tukey-Kramer assay At P <0.05 relative to the non-genetically modified control.
[0039]
These results indicate that age-dependent loss of cholinergic neurons in hα-synuclein / hAPP mice is primarily dependent on hAPP / Aβ, with hα-synuclein having some additional effects in the basal ganglia. Suggests. Age-dependent loss of SIPT in the neocortex of these mice is mainly due to hAPP / Aβ, with a small contribution of hα-synuclein, whereas loss of SIPT in the basal ganglia is almost All due to hα-synuclein.
Embodiment 6
[0040]
Aβ promotes accumulation of hα-synuclein. Accumulation of hα-synuclein in neurons is characteristic of Lewy body disease, including Lewy body variants of AD. Age-dependent accumulation of hα-synuclein in neurons of hα-synuclein single transgenic mice has already been observed (Masliah, 2000). Between 4 and 20 months of age, the number of inclusion bodies of neurons in the neocortex was 1.6 times higher on average in hα-synuclein / hAPP mice than in age-matched hα-synuclein mice ( mm²15.3 ± 1.1 vs 9.5 ± 0.9, mean ± SEM, P = 0.0002 by unpaired two-tailed Student's t-test, n = 28-32 / genotype). This is illustrated in 12 month old mice. Inclusion body numbers increased with age in both hα-synuclein mice and hα-synuclein / hAPP mice. These results indicate that hAPP / Aβ promotes accumulation of hα-synuclein in neurons.
[0041]
Ultrastructural analysis shows that all intraneuronal inclusions in hα-synuclein mice are amorphous and electron-dense, whereas many of the intraneuronal inclusions in hα-synuclein / hAPP are fibrous, typically with fibrous elements. Increasing similarity to Lewy bodies in human diseases, including: The fibrous inclusions in the neuronal cytoplasm were decorated with gold particles when brain sections of hα-synuclein / hAPP mice were analyzed by immunogold electron microscopy with anti-hα-synuclein or anti-Aβ. These results suggest that Aβ promotes in vivo fibrosis of hα-synuclein by direct interaction. Furthermore, in support of this possibility, Aβ-immunoreactive granular deposits were detected by confocal microscopic analysis in hα-synuclein-positive intraneuronal inclusions in hα-synuclein / hAPP mice.
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To further evaluate whether the changes observed in hα-synuclein / hAPP mice were indeed arising from the pathogenic interaction between Aβ and hα-synuclein, in vivo analysis was performed using two in vitro models. Extended to In a cell-free system, both human Aβ in freshly lysed and aggregated (aged)_1-42Strongly promoted the production of high molecular weight polymers of hα-synuclein, consistent with previous observations. In contrast, less fibrous Aβ_1-40Did not affect in vitro aggregation of hα-synuclein. Apparently, Aβ_1-42And Aβ_1-40Also differed in their effect on the intracellular accumulation of α-synuclein in neuronal cell cultures when added to the culture medium. Aβ_1-42Strongly increased the intracellular accumulation of α-synuclein, while Aβ_1-40Did not increase.
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Aβ in endoplasmic reticulum and intermediate compartment_1-42Overproduction can interfere with the processing of hα-synuclein, which promotes its accumulation. However, cell culture data indicate that Aβ_1-42Extracellular exposure to Aβ is sufficient to induce intracellular accumulation of hα-synuclein, whereas Aβ_1-40Exposure shows that this is not the case. In this sense, the extracellular Aβ of the cell_1-42Incubation disrupts lysosomal membrane integrity, whereas Aβ_1-40It is interesting to note that incubation of cells into cells does not. Also, the association of soluble hα-synuclein with planar lipid bilayers results in extensive bilayer disruption. Aβ_1-42And hα-synuclein lead to endosomal-lysosomal membrane leakage and Aβ in the cell matrix_1-42And a direct interaction between hα-synuclein. Such an interaction promotes intracellular accumulation of hα-synuclein. Aβ_1-42This effect on hα-synuclein is probably mediated by direct fibrous interactions between these molecules or by free radicals. Aβ exerts oxidative stress and studies have shown that oxidative cross-linking of hα-synuclein contributes to the formation of Lewy bodies (Hashimoto et al., 1999; Lee et al., 2000).
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In contrast to the prominent effects of hAPP / Aβ in hα-synuclein-related pathologies, hα-synuclein expression is due to extracellular deposition of Aβ into plaques or 8-, 12- and 20-month-old hα-synuclein / The incidence of plaque-associated neurite dystrophy in hAPP mice was not significantly altered compared to age-matched hAPP mice (n = 3-4 per genotype and age). 12 month old hAPP and hα-synuclein / hAPP mice have similar cerebral levels of Aβ as measured by ELISA (mg / g brain hemisphere, mean ± SD, n = 4-11 / genotype)._1-40(4.3 ± 0.88 vs 4.6 ± 0.6) and Aβ_1-42(248 ± 48 vs. 275 ± 41).
Embodiment 7
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Total RNA was isolated from hemispheres or from dissected brain regions, then analyzed in a solution hybridization RNase protection assay and separated on an acrylamide / urea / Tris / borate / EDTA gel (FIG. 2). . Lane U represents undigested transcripts of α-synuclein gene (α-syn), Aβ precursor gene (APP) and actin (actin). The note on the right hand side of the figure indicates the size of the digested sample. Sample lanes represent the analysis of three separate mice, non-Tg, non-transgenic mice (ie, controls); Thy1-hAPPtg, human (h) under the regulatory control of the mouse (m) Thy-1 gene, respectively. ) A transgenic mouse expressing APP751 cDNA; a Thy1-α-syn'tg, a transgenic mouse expressing human α-synuclein under the control of the mouse (m) Thy-1 gene; and a double tg, mouse ( m) Transgenic mice containing both human (h) APP751 and the α-synuclein gene under Thy-1 gene regulatory control.
Embodiment 8
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Cross breeding of hα-synuclein or PDAPP-J9Mtg mice with mα- or mβ-synuclein KO mice. To further confirm that the deletion of α-synuclein results in suppression of Aβ deposition in digenic mice, the deletion of α-synuclein or β-synuclein Knockout (KO) mice (Lexicon Genetics Inc., The Woodlands, TX) carrying the encoding gene were bred to heterozygous PDAPP-J9Mtg mice. The following lines were created: 1) (PDAPP − / −; mα-syn + / +), 2) (PDAPP − / −; mα-syn +/−), 3) (PDAPP +/−; mα-syn + / +). ), 4) (PDAPP +/−; mα-syn +/−) and 5) (PDAPP − / −; mα-syn − / −). Mouse genotype was determined by PCR using terminal DNA. Subsequently, the (PDAPP +/−; mα-syn +/−) was crossed to produce the following progeny: 1) (PDAPP + / +; mα-syn + / +), 2) (PDAPP + / +; mα-syn + //), and 3) (PDAPP + / +; mα-syn − / −). The same mating protocol was followed for generation of bigene hα-synuclein or PDAPP-J9M and mβ-synuclein KO mice.
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The effect of α-synuclein and β-synuclein on amyloid deposition was determined using the same rolling bar and neuropathological analysis used in the previous example, using homozygous KO mice (PDAPP + / +; mα-syn − / −). ) And synuclein were evaluated in both hemizygous mice (PDAPP + / +; mα-syn +/−).
Embodiment 9
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Cross breeding of hα-synuclein or PDAPP-J9Mtg mice with mice overexpressing hβ-synuclein. Highly expressing heterozygous β-synuclein mice were bred with heterozygous α-synuclein or PDAPP-J9Mtg mice following standard crossbreeding protocols (Mucke et al., 1995), the contents of which are incorporated herein by reference. 1) non-tg littermates (PDAPP − / −; hα− or βsyn − / −), 2) digenes (PDAPP +/− and hβ-synuclein +/−), 3) digenes (hα-synuclein +/− and hβ-synuclein +/−), 4) single tg hAPP, and 5) single tg hα-synuclein or β-synuclein. Mouse genotype was determined by PCR using terminal DNA.
[0049]
Since the original strain of α-synuclein tg mice showed motor deficits when subjected to a rotating rod, the bigenic hybrids were evaluated in a similar manner. Analysis of rolling stick behavior revealed that overexpression of β-synuclein reduced motor deficits in α-synuclein tg mice. Subsequent neuropathological analysis indicated that β-synuclein overexpression resulted in a decrease in neuronal inclusions. Overexpression of β-synuclein has also been shown to result in reduced neuronal loss in α-synuclein tg mice. These results further confirm that β-synuclein is active in vivo and that it constitutes a potential anti-Parkinson and anti-Alzheimer therapeutic.
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The hα-synuclein / hAPP digenic mice have cognitive and motor degeneration, loss of cholinergic neurons and SIPT, extensive amyloid plaques, and fibrous inclusions within hα-synuclein immunoreactive neurons. All these features are also found in the Lewy body variants of AD. Collectively, Lewy body disease is the second most common neurodegenerative disease except for AD alone. They comprise a heterogeneous group of diseases, including PD, pervasive Lewy body disease, and Lewy body variants of AD. Patients with Lewy body disease typically have Lewy bodies, Parkinsonism and cognitive impairment. Approximately 25% of patients with AD develop apparent Parkinson's syndrome, and hα-synuclein immunoreactive Lewy body-like inclusions have sporadic AD as in Down's syndrome associated with early-onset AD. And in most cases of FAD. Also, Lewy bodies include hAPP. These associations indicate that hAPP / Aβ plays a role in the formation of Lewy bodies and the development of Lewy body disease.
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Studies in hAPP / hα-synuclein mice show that hα-synuclein and hAPP / Ab have a definitive, as well as intensive, pathogenic effect on brain integrity and function. . hα-synuclein did not affect the development of Aβ-dependent neuritic plaques or the overall Aβ content in the brain, but exacerbated hAPP / Aβ-dependent cognitive deficits and neurodegeneration in certain brain regions. These findings indicate that hα-synuclein enhances Aβ toxicity through a plaque-independent mechanism. These data explain the clinical findings that suggest that people with the Lewy body variant of AD exhibit cognitive decline more rapidly than people with pure AD. Overexpression of hAPP / Aβ then promoted the intraneuronal accumulation of hα-synuclein in transgenic mice and accelerated the development of motor deficits. These effects are most likely mediated by Aβ or other hAPP products, and in vitro studies have shown that Aβ is the culprit. In view of these pathogenic interactions between Aβ and hα-synuclein, agents aimed at blocking the accumulation of Ab or hα-synuclein may benefit a wider range of neurodegenerative diseases than previously expected. There will be.
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[Brief description of the drawings]
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FIG. 1. Characterization of motor deficits in hSYN and hAPPtg mice on a rotating bar. Single tghSYN mice showed significant motor deficits as compared to non-transgenic littermates. Similarly, bigenic hSYN / hAPP mice exhibited levels of dysfunction, similar to single tghSYN mice. Mice expressing only hAPP were not different from non-tg controls. N = 6-8 per genotype. 12 months old.
FIG. 2. RNase protection assay of total RNA from transgenic and non-transgenic mouse brains. Lane U shows undigested transcript. The note on the right hand side of the figure indicates the size of the digested sample. Sample lanes represent the analysis of three separate mice, non-Tg, non-transgenic mice (ie, controls); Thyl-hAPPtg, transgenic mice; Thyl-α-syn'tg, transgenic mice, respectively. Mice; and double tg, transgenic mice containing both human (h) APP751 and α-synuclein genes.

Claims (17)

Genetically modified mice that include:
A first recombinant nucleotide sequence encoding a human amyloid precursor protein (hAPP) integrated into the mouse genome and operably linked to a first promoter; and integrated into the mouse genome. A second recombinant nucleotide sequence encoding human (h) α-synuclein operably linked to a second promoter;
The first and second transgenic nucleotide sequences are expressed, and as a result of the expression, the transgenic mouse has developed a neurodegenerative disease. The first promoter comprises a platelet-derived growth factor β (PDGF-β) promoter optionally operably linked to a simian virus (SV) 40 intron, a Thy-1 promoter or a prion (Prp) promoter. 2. The transgenic mouse of claim 1 comprising the transgenic mouse. The transgenic mouse of claim 1, wherein the second promoter comprises a PDGF-β promoter, optionally operably linked to an SV40 intron, a Thy-1 promoter, or a Prp promoter. 2. The transgenic mouse of claim 1, wherein the protein encoded by the first and second transgenic nucleotide sequences is overexpressed compared to a non-transgenic mouse of the same species. 2. The transgenic mouse of claim 1, wherein the nucleotide sequence of hAPP comprises an intron between exons 6-9 of hAPP and encodes hAPP770, hAPP751 or hAPP695. hAPP has a double mutation involving a lysine to asparagine change at amino acid (aa) 670 and a methionine to leucine change at aa 671 or a single mutation at aa717 including a valine change to phenylalanine or isoleucine. The transgenic mouse of claim 1, which is a mutant hAPP comprising the mouse. 2. The transgenic mouse of claim 1, wherein the nucleotide sequence encoding hAPP encodes only the portion of APP comprising Aβ _1-42 . The transgenic mouse of claim 1, wherein the nucleotide sequence of α-synuclein comprises the coding sequence of α-synuclein. The hα-synuclein is a mutant hα-synuclein comprising a mutant hα-synuclein cDNA encoding a protein comprising a change from alanine to proline at aa30 or from alanine to threonine at aa53. 1 transgenic mouse. Genetically modified mice that include:
Human amyloid precursor protein operably linked to the platelet-derived growth factor β (PDGF-β) promoter integrated into the mouse genome and operably linked to the Simian Virus (SV) 40 intron A first recombinant nucleotide sequence encoding (hAPP);
A second recombinant nucleotide encoding a human (h) α-synuclein operably linked to a PDGF-β promoter operably linked to the SV40 intron and integrated into the mouse genome Array;
The first and second transgenic nucleotide sequences are expressed, resulting in a neurodegenerative disease in the transgenic mouse. 11. The transgenic mouse of claim 10, wherein the protein encoded by the first and second transgenic nucleotide sequences is overexpressed compared to a non-transgenic mouse of the same species. 11. The transgenic mouse of claim 10, wherein the hAPP comprises a coding sequence that includes an intron between exons 6-9. 11. The transgenic mouse of claim 10, wherein the hAPP comprises a valine to isoleucine mutation at amino acid 717. 11. The transgenic mouse of claim 10, wherein the hα-synuclein comprises a coding sequence for hα-synuclein. 11. The transgenic mouse of claim 10, wherein the neurodegenerative disease comprises the generation of intraneuronal inclusions characteristic of Lewy body disease. 11. The transgenic mouse of claim 10, wherein the neurodegenerative disease comprises the generation of fibrous Lewy body-like inclusions, death of neurons or the development of (and?) Motor deficits. A method for screening a therapeutic agent for the prevention or treatment of a neurological disease, comprising administering to a transgenic mouse a therapeutic intervention, comprising:
A first recombinant nucleotide sequence encoding a human amyloid precursor protein (hAPP) integrated into the mouse genome and operably linked to a first promoter;
A second recombinant nucleotide sequence encoding human (h) α-synuclein integrated into the mouse genome and operably linked to a second promoter;
The first and second transgenic nucleotide sequences are expressed, and the expression results in the transgenic mouse developing a neurodegenerative disease.

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