JP2003509071A - A novel animal model of Alzheimer's disease showing amyloid plaques and mitochondrial dysfunction - Google Patents

Abstract

  (57) [Summary] The present invention relates to transgenic animal models, and more particularly to Alzheimer's disease animal models. The present invention relates to a novel animal model of Alzheimer's disease that exhibits amyloid plaques and mitochondrial dysfunction.

Description

Detailed Description of the Invention

The present invention relates to transgenic animal models, and more particularly to animal models of Alzheimer's disease. The present invention relates to a novel animal model of Alzheimer's disease that exhibits amyloid plaques and mitochondrial dysfunction.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects the majority of the elderly. At the clinical level, the disease is characterized by memory loss and cognitive decline, and at the neuropathic level, intracellular neurofibrillary fibril accumulation and β-amyloid peptide (A-β) formation of amyloid plaques. It has been characterized as extracellular accumulation (Yanker et al., 1996). In addition to these manifestations, there are numerous other abnormal changes, including impaired immune and inflammatory systems and increased oxygen stress and activation of apoptotic machinery that ultimately induce cell death.

Amyloid plaques are mainly composed of A-β peptides with 40-42 residues produced during the proteolytic process of β-amyloid peptide precursor protein (APP). Extracellular accumulation of A-β is highly specific for AD and related disorders. These are the early and invariant features of all AD, including the familial form (FAD). FAD develops relatively early (between 40 and 60 years) with 5% (1
9 FAD cases with FAD mutations (6 single or double pseudosense mutations), 50-70% of FAD cases (over 50 families) with presenilin 1 (P
S1) mutation (currently 40 different mutations have been identified), less FA
In case D, it was due to mutations in the presenilin 2 (PS2) gene (2 pseudosense mutations in 8 families) (for a review, see Price and Sisodia, 1998). Mutations in these three genes have been shown to induce changes in APP proteolysis that induce overexpression of A-β and early onset of pathological conditions and symptoms similar to the sporadic form of AD.

In addition to mutations in the APP, PS1 and PS2 genes, other factors contributing to AD have been demonstrated in humans, especially those with mitochondrial dysfunction (Beal, 19
98). Therefore, different investigational approaches in AD-affected individuals have demonstrated that mitochondrial dysfunction is important in the development of this disease.

[0005]   Therefore, according to the first approach, mitochondrial cytochrome C oxy
Human A
It has been found in the brains of D-affected patients (Parker et al., 1989; Parker et al.
, 1994; Mutisya et al., 1994; Kish et al., 1992). Mitocon
Mutations in the doria COX gene have also been found in late forms of AD (Davi
s et al., 1996, 1997). Mitochondrial COX activity is derived from patients with AD
Platelet mitochondria were characterized by a deficiency of their mitochondrial DNA.
52% reduction in cytoplasmic hybrid cells fused to the cell line SH-SY5Y. Mitco
This reduction in ndria activity leads to the overproduction of free radicals, Ca ²⁺ The basal concentration of A. (Sheehan et al., 1997).

In a second, equally important approach, mitochondrial dysfunction has a role in apoptosis-induced cell death in AD patients, in particular:
An important role by Bax identified at the lesion site in AD patients has been demonstrated (Bax
Opens a large mitochondrial channel and, as demonstrated in cultured cells, C
It is a mitochondrial protein of the Bcl-2 family known to induce cell death by inducing mitochondria to release apoptotic molecules including cytochrome C, which is a substrate of OX and activates caspases in the cytoplasm. ). In fact, Bax is enriched in neurons with senile plaques (the location of bax correlates with the location of A-β accumulation in adjacent sections of the brain at hippocampal level in AD patients) and early neurofibrillary degeneration, which is Bax. Play a role in the formation of neurofibrillary degeneration in AD patients (MacGibbon et al., 1997; Tortos).
a et al., 1998; Nagy et al., 1997).

In vitro evidence of A-β neurotoxicity has been clearly shown in the literature to be associated with the production of oxidative free radicals (Pappola et al., 1998).
. However, this finding does not appear to solve the question of whether oxidative stress is due to mitochondrial dysfunction, or whether oxidative stress may appear in the brain associated with A-β accumulation in vivo. This is a finding found in numerous publications, since it is a finding and / or suggestion that oxidative stress-mediated neurotoxicity of A-β is actually a finding only in vitro (not in vivo). Is. Furthermore, the in vivo toxicity of A-β has been challenged in numerous reports (Papolla et al., 1998).

While A-β accumulation is known to be associated with mitochondrial dysfunction in human brain tissue, a link between mitochondrial impairment and A-β accumulation in transgenic animal models of AD has been demonstrated. No findings are currently reported. Mutation A
Two recent publications on transgenic animals expressing PP (animals developed by Hsiao et al. 1996) show superoxide dismutase (SOD) and hemooxygenase-1 (HO-1) associated with A-β accumulation.
And lipid peroxidase and hydroxynonenal (HNE) have been reported (Smith et al., 1998; Papolla et al., 1998). However, all these markers are oxidative stress markers that are present downstream of mitochondrial dysfunction and are not specific for this dysfunction. In addition, some of these markers used in the previously described studies (ie SOD and HO-1
) Is not an oxidative factor that induces neuronal death, but is rather known to be an antioxidant that is associated with a defense mechanism that leads to reduced oxidative damage and cell death.

To the extent that presenilin is considered, mutations in PS1 have been reported to induce apoptosis in cultured cells and in the brain of transgenic mice expressing mutant PS1 in vivo (Chiu et al., 1999). But this is PS
It has not been shown that apoptosis induced by the 1 mutation is mediated by mitochondrial dysfunction. This is because apoptosis may be the result of mitochondrial dysfunction or another mechanism of cell death that is not necessarily associated with mitochondrial dysfunction, eg inhibition of protease activity of interleukin converting enzyme 1-β (ICE). Have shown that it inhibits Bax-induced changes existing in the downstream of mitochondria (for example, DNA degradation), but does not affect mitochondrial changes (deficiency of mitochondrial membrane potential and generation of free radicals) ( Xiang et al., 1996). Recent in vitro studies have demonstrated that PS1 mutations are associated with free radical overproduction in impaired calcium homeostasis and loss of mitochondrial membrane potential (Begley et al., 1).
999; Guo et al., 1996, 1997, 1998). However, these studies show mitochondrial dysfunction and A-β accumulation in transgenic mice expressing mutant PS1 because the transgenic mice used in these studies for the preparation of synaptosomes do not undergo A-β accumulation. It does not show any association between. Therefore, in these references the mutation PS1 (Chui et al., 1999)
Or mutated APP (Hsiao et al., 1996; Irizzary et al., 1997a
, 1997b; Johnson-Wood et al., 1997) or co-expressing mutant APP and mutant PS1 (Borchlt et al., 1997; Ho).
lcomb et al., 1998) There is no evidence of a relationship between in vivo mitochondrial dysfunction and A-β accumulation in the brain of transgenic mice.

Therefore, it was demonstrated for the first time by the present invention that amyloid accumulation is associated with in vivo mitochondrial dysfunction in the brain of transgenic animals.
In addition, the present invention also investigates a new animal model of AD that is more representative and reproduces the neuropathology encountered in humans.

Accordingly, the present invention firstly relates to a transgenic animal model of Alzheimer's disease that exhibits both amyloid plaques and mitochondrial dysfunction. Advantageously, this model co-expresses β-amyloid peptide precursor (APP) and presenilin, preferably PS ₁ .

A transgenic animal is understood to be any non-human animal whose genome has been modified. The modification of the genome may result from a change or modification of one or more genes by knocking in or knocking out. This modification may be due to conventional mutagenic effects or changes in factors, or may be caused by site-directed mutagenesis as described in "Materials and Methods".

The modification of the genome may also result from the insertion or replacement of the gene in the wild type or mutant form.

[0014]   It is advantageous to modify the genome in germline stem cells, preferably in their pronucleus.

In the context of the present invention, the animal model is advantageously a mammal. In particular, this can be a mouse, rat, or rabbit obtained using conventional transformation techniques. As an example showing one of the transformation procedures, the microinjection method of the expression cassette containing the modified gene in the two pronuclei before fertilization was described as described in “Materials and Methods”.

In this regard, the animal model of the invention is obtained by injection of an expression cassette containing a nucleic acid. This nucleic acid is preferably DNA, and may be genomic DNA (gDNA) or complementary DNA (cDNA).

In the model of the invention, the DNA encodes any gene that may be part of the AD pathogenesis process. Advantageously, the gene encoded by the DNA is associated with the mechanism of A-β peptide production in its amyloidogenic form.

In particular, the DNA encodes mutant forms of APP and / or presenilin (especially PS1) such that cells of the animal model co-express the two mutant proteins.

The mutation in the APP gene may be one of the various mutations currently described in the literature. Preferably, the mutations in the APP gene are selected alone or in combination from "Swedish" (S), "London" (L) and "Dutch" (D) mutations.

These mutations are well described in the literature and are generally characterized by the following modifications.

[0021]

[Table 1]

The London-type mutation also includes all substitutions other than isoleucine at position 717 for APP770 (such as V717G and V717F).

The APPs that can be used in the present invention are different isoforms (especially 695, 751).
, And 770) or truncated forms such as the APP99 isoform.

The mutation in the PS1 gene can be one of the 40 mutations currently described in the literature. Preferably, the mutation of PS1 gene is M146L, A246E, C410Y.
, H163R, L226V, L235P mutations and the like, alone and in combination.

[0025]   The M146 mutation is preferred for the preparation of the model of the invention.

In the model of the present invention, the DNA is placed under the control of expressible sequences, especially transcription promoter sequences.

The promoter sequences which may be mentioned are, in particular, the HMG promoter (Gautier).
Et al., 1989) and the PDGF promoter (Sasahan et al., 1991),
The Thy-1 promoter (Luthi et al.) And the prion gene promoter (Scott et al., 1992).

In a particularly preferred embodiment of the invention, the animal model comprises an APP gene with S, D, and L mutations (this gene is under the control of the PDGF promoter) and a PS1 gene with the M146L mutation. (This gene is under the control of the HMG promoter).

The animal model of the present invention is very advantageous because it is a very representative model of AD. This is because this model develops amyloid plaques from 6 months of age (which greatly reduces the time required for animal reproduction), and the mutant APP and PS1 proteins are significantly higher than the endogenous levels (respectively endogenous). Sex APP and PS1 levels at least 3-5 times and 2-3 times).

Therefore, the results described in the examples show that transgenic mice expressing the mutant APP and the mutant PS1 in the same figure develop Alzheimer's disease-type neuropathology (ie, fiber form A-β accumulation, abnormal axis). Shows neurodegenerative changes in the cord structure type and activation of inflammatory central nervous system cells (such as astrocytes)).

Particularly preferably, this model exhibits mitochondrial dysfunction, which is also found in AD patients, in addition to amyloid plaques.

The results described in the examples show the association of mitochondrial dysfunction in the neuropathology of these transgenic AD mice. With respect to the findings currently published in the field of mitochondria and Alzheimer's disease, these results indicate that two important mitochondrial markers (ie, Bax and cytochrome C) cause mitochondrial dysfunction in the brain of transgenic AD mice of the present invention. This is the first proof of research. Finally, these results show that axon structures closely related to A-β accumulation in the brains of transgenic AD mice in the same way as in AD patient brains.
It is demonstrated that ax and cytochrome C are expressed.

In the present invention, mitochondrial dysfunction is understood to be alteration, overexpression, or inhibition of mitochondrial protein expression. These proteins (preferably having subcellular mitochondrial location) include the Bcl-2 family (Ba
x, Bak, and Bad) and the Bcl-2 family of anti-apoptotic proteins (such as Bcl-2 and Bcl-xL), or any other mitochondrial protein that does not belong to the Bcl-2 family and plays a role in apoptosis (eg. Cytochrome C and AIF) or proteins recently recorded to be present in mitochondria and which may play a role in apoptosis (such as Aralar or BMCP1) are included.

The mitochondrial proteins whose expression is modified and which may preferably be mentioned are in particular the Bax and / or cytochrome C proteins.

The present invention also relates to the use of an animal model for the identification of compounds, as described above, which can be used for the treatment of neurodegenerative diseases, preferably Alzheimer's disease.

Therefore, as a result of its advantageous property of reproducing the characteristics of AD very faithfully, this model is used for comparison with known models and is particularly well suited for the treatment of AD, especially human AD. Compounds suitable for can be identified.

These compounds are chemical molecules, peptide or protein molecules, antibodies or chimeric molecules and can also be antisense DNA or ribozymes.

The identified compounds can be used as such or as drugs in combination with pharmaceutically acceptable excipients to obtain a pharmaceutical composition. The excipient is
In particular, a sterilized isotonic solution (monosodium phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc. or a mixture of these salts) or in some cases sterile water or a saline solution. It may be a dry (especially lyophilized) composition which upon addition may be made into an injectable solution. Injections may be effective by routes such as fixative, topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal.

The identification of the compounds described above is carried out by contacting the animal model of the invention with the administration of a compound or mixture of compounds suspected of acting, and in particular various biochemical and / or biochemical and / or brain levels of the model. Based on measurements of compound effects on histological changes (eg, those described in the Methods and Results section, including A-β accumulation production levels, changes related to neurodegeneration, changes in mitochondrial molecule expression, etc.).

The present invention also relates to cells harvested from the above animal models and their use for the identification of compounds that can be used in the treatment of neurodegenerative diseases, preferably Alzheimer's disease.

The identification of the above-mentioned compounds is carried out by contacting a compound or a mixture of compounds with which the action of cells extracted from the animal model of the present invention is estimated and various parameters (cell death,
It is based on the measurement of A-β peptide production, and the effect of compounds on whole cell, cell homogenate, or subcellular fraction levels on mitochondrial activity such as free radical production, respiratory chain, mitochondrial potential, etc.

The results described in the examples demonstrate the advantages of the model of the invention, in particular this trans for the development of therapeutic strategies such as mitochondrial drugs that are resistant to mitochondrial dysfunction and nerve death induced by mitochondrial dysfunction. Clearly supports the use of the transgenic model.

The invention is described in more detail with the aid of the following examples, which are to be considered exemplary and do not limit the invention.

Description of Table I The table summarizes the results obtained from a double transgenic mouse model showing markers of A-β accumulation and apoptosis mediated by mitochondrial dysfunction in axon structures.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows various transgenic mouse strains: 1 (AA LD2 (B6),
2 (APP LD2 (FVB)), 3 (NT), 4 (Thy-1 Kozak)
APP ₇₅₁ SL (28)), 5 (Thy-1 Kozak APP ₇₅₁
SL (26)), 6 (Thy-1 Kozak APP ₇₅₁ SDL (100
1)), 7 (PDGF APP ₆₉₅ SDL (46)), 8 (HMG APP
SDL 20 (76)) APP, β-secretase fragment, and A-
It is a figure which shows the comparison of (beta).

FIG. 2A is a human A-β form for analyzing the expression of APP, β-secretase 12 kDa fragment, and A-β in brain homogenates of transgenic mice of different ages with single or double mutations. FIG. 6 shows the use of the antibody WO-2 specific for Escherichia coli. Although the APP expression level does not change with aging of transgenic mice, the accumulation of A-β clearly increases with aging.

[0047]   FIG. 2B is a diagram showing an analysis of expression of human M146LPS1.

FIG. 3 shows a single mutation (APP mutation) (b1, b2) or a double mutation (APP / P).
S1) Plate showing expression of human APP in the hippocampus and cortex of transgenic mice carrying the mutation (c1, c2). High levels of APP expression in hippocampal (b1, c1) and cortical (b2, c2) neurons of these two transgenic mouse lines, when undetectable, correspond to non-transgenic control animals (a1, a2) It should be noted that APP expression levels in brain regions are very low. In the same way, expression of human PS1 resulted in single mutation (
PS1 mutation) (e1, e2) or double mutation (APP / PS1 mutation) (f1, f
Obvious in the same brain region in transgenic mice with 2) and undetectable in non-transgenic control animals (d1, d2).

4 and 5 are 6 months old (FIG. 4) with a double mutation (APP / PS1 mutation).
And A-β accumulation in the brain of 12-month-old (FIG. 5) transgenic mice. Specific for different epitopes of A-beta peptide some antibodies: 6E10 (Fig. 4A and 5A) of the αβ _{1- 17,} Dako of .alpha..beta _6-17 (Fig. 4B and 5B), 4G8 (figure .alpha..beta _17-24 4C and 5C), QCB of αβ _1-42
(FIGS. 4D and 5D), and immunohistochemical detection using total αβ FCA18 (FIGS. 4A and 5A).

FIG. 6 shows histological staining of hippocampal levels of 12-month-old transgenic mice carrying the double mutation (APP / PS1 mutation) with Thioflavin S (FIG. 6A) and Congo red (FIG. 6B). It is a photograph showing a section. Evidence for a fibrous morphology of A-β accumulation in the brain of these mice. Note the altered morphology of A-β accumulation: globular (arrow) or irregular morphology (arrowhead) in transgenic mice carrying the double mutation.

FIG. 7 is a diagram showing the progress of A-β accumulation immunolabeled with the anti-Aβ antibody 4G8 in a transgenic mouse having a double mutation (APP / PS1 mutation). A
-The density of β accumulation was 9 and 12 months old mice (B1, B2, B3, respectively).
And even more pronounced at 12 months of age (C1, C2, C3) compared to A1, A2, A3). A-β accumulation is mainly present in a restricted brain region (mainly hippocampal platform) in juvenile mice (A1 to A2), whereas whole hippocampal body of 12-month-old mice (C1 to C12). Also note that it is present in the cortical area. Figures A2, B2, C2 and A3, B3, C3 are shown in Figures A1, B1, C respectively.
1 and the regions distinguished by black frames in FIGS. A2, B2, and C2 are shown at a higher magnification. Hi: hippocampus, Ctx: cortex.

FIG. 8: Double transgenic APP ₆₉₅ S plotted against age.
6 μm thick hemi-brain section from DL x PS1 M1476L mouse (Franklin and Paxinos stereotaxic atlas with bregma axial level -3.4)
It is a figure which shows the A- (beta) accumulation number immunolabeled with the anti-A (beta) antibody 4G8 in the inside. Accumulation was quantified using an image analysis system attached to a color camera and microscope (Q600, LEICA).

FIG. 9 is a diagram showing a regional distribution of A-β accumulation in the brain of a 12-month-old transgenic mouse having a double mutation (APP / PS1 mutation). 25 μm sections corresponding to 6 representative levels in the axial direction of the mouse brain were immunolabeled with anti-Aβ antibody 4G8. Note the very large accumulation of A-β in the hippocampal body and all cortical areas.

FIG. 10 shows double mutation (APP) visualized by Aβ immunohistochemistry (antibody AG8) performed on 25 μm thick hemibrain sections (Bregma axial level-3.4). / PS1 mutation) is a diagram showing the quantification of the amount of A-β in a 12-month-old transgenic mouse having the same. The total surface of the hippocampus and cortex reaches 3% and 1% or more, respectively (FIG. 10B), and the most abundant regions of these two brain structures reach 9% and 5% or more, respectively (FIG. 10C), and reach the amount of A-β. Attention is necessary. On the other hand, the amount of A-β in the subcortical region is less than 0.5%. 1: Dorsal cortex including especially primary visual cortex and auditory cortex 2: Ventral cortex including especially olfactory and entorhinal cortex 3: Hippocampus 4: Remaining region of hemi-brain (subcortical structure)

FIG. 11 shows that immunoreactive axon structures are present in the hippocampus of transgenic mice carrying the double mutation (APP / PS1 mutation) (4), non-transgenic control mice (1), and 1. One APP mutation (APP mutation) (2) or P
It is a figure which shows that it does not exist in the hippocampus of the transgenic mouse which has S1 mutation (PS1 mutation) (3). In transgenic mice carrying the double mutation (APP / PS1 mutation), the APP immunoreactive axon structure showed whole cortical regions (including entorhinal cortex (a) and cingulate cortex (d)) as shown in FIG. 11B. It is also present in the dentate gyrus and CA1 and CA3 hippocampal regions (b, c). The areas separated by black boxes in Figures b, b1 and b3 were visualized at higher magnification on b1, b2 and b3 and b4 respectively. Also note the very high level of human APP protein expression in the same neuronal cell body (arrow b2). The arrows 1, 2, 3, and 4 respectively indicate the dentate gyrus direction.

FIG. 12 shows that immunoreactive axon structures are present in the hippocampus of transgenic mice carrying the double mutation (APP / PS1 mutation) (4), non-transgenic control mice and one APP mutation ( APP mutation) or PS1 mutation (PS
It is a figure showing that it does not exist in the hippocampus of the transgenic mouse having 1 mutation) (3). In transgenic mice carrying the double mutation (APP / PS1 mutation), the PS1 immunoreactive axon structure has the entire cortical region (including entorhinal cortex (a) and cingulate cortex (d)) as described in FIG. 12B. Dentate gyrus and CA1 and hippocampal region C
It is also present in A1 and CA3 (b, c, e). The areas separated by black boxes in Figures c, d, and e were visualized at higher magnification in c ', d', and e ', respectively. Also note the very high level of human PS1 protein expression in the same neuronal cell bodies in e ′ and d ′ (arrows).

FIG. 13 shows that δ-catenin immunoreactive axon structures are present in the hippocampus (Hi) and cortex (Ctx) of transgenic mice carrying the double mutation (APP / PS1 mutation) (b1, b2). FIG. 3 shows that the non-transgenic control mice do not exist in the same brain structure (a1, a2). The areas separated by black frames in Figures b1 and b2 were visualized at higher magnification on b1a, b1b and b2a, b2b, respectively. Similar to APP and PS1 immunoreactive axon structures, δ-catenin reactive axon structures appear to be classified into plaques of various sizes (ie small, medium, large).

FIG. 14 shows the synaptophysin immunoreactive axon structure in transgenic mice (FIG. 14A) with a double mutation (APP / PS1 mutation) and humans with AD (FIG. 14B). Note that in double immunolabeled brain sections, both synaptophysin immunoreactive axon structures (brown immunolabels) were strongly surrounded by A-β accumulation (blue immunolabels).

FIG. 15 shows SMI immunoreactive phosphorylated neurofilaments in transgenic mice with the double mutation (APP / PS1 mutation) and humans with AD. Note that in the double immunolabeled brain section, both SMI immunoreactive phosphorylated neurofilaments (brown immunolabel) were strongly surrounded by A-β accumulation (blue immunolabel).

FIG. 16 shows axons containing dystrophic axons located around or inside A-β accumulation (blue immunolabeling) in the brain of transgenic mice carrying the double mutation (APP / PS1 mutation). FIG. 3 shows structure and tau-1 immunoreactivity (brown immunolabeling) enriched in nerve cell bodies (arrow heads). Neither dystrophic axons immunoreactive with tau-1 protein nor cell bodies were present in non-transgenic control mice.

FIG. 17 shows activated GFA present in transgenic mice with double mutation (APP / PS1 mutation) (FIG. 17A) and humans with AD (17B).
It is a figure which shows P immunoreactive astrocytes (brown immunolabeling). Note that in the double immunolabeled brain section, both activated astrocytes were somewhat surrounded by A-β accumulation (blue immunolabel).

18A-18H show neurons in the dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal region (FIGS. 18B, 18F) as well as non-transgenic control mice (FIGS. 18A-18D) and double mutations. Bax in the entorhinal cortex of transgenic mice (APP / PS1 mutation) (FIGS. 18E to 18H).
It is a figure which shows expression. Note that the Bax immunoreactive axon structure (arrow head) is present in double transgenic mice, but not in non-transgenic control mice.

18I-18L are identical in the dentate gyrus (FIGS. 18K and 18L) and CA3 hippocampal region (FIGS. 18I and 18J) of transgenic mice carrying the double mutation (APP / PS1 mutation). Bax expression (brown) and A in sections
FIG. 3 is a diagram showing a double immunolabel capable of visualizing −β accumulation (blue). Note that Bax immunoreactivity is associated with aberrant axonal structures such as dystrophic axons that are closely associated with A-β accumulation (arrow). Bax immunoreactivity is also thought to be strongly immunolabeled at the perinuclear cytoplasmic level and is enriched in some abnormal cell bodies resembling so-called "dark" neurons (black arrowheads). Bax immunoreactivity is also evident in some glial cells suggesting activated astrocytes (white arrowheads).

FIG. 19 shows control human subjects (FIG. 19A) and AD patients (FIG. 19B-
FIG. 19F) shows Bax immunoreactive axon structures in the frontal cortex from (F). In AD patients rather than control subjects, there is Bax immunoreactive axons structures such as dystrophic axons (arrows) (brown immunolabeling), very strong in that some of the cytoplasm and Kinkurainobe Chobutsu in Note that this is similar to neurofibrillary degeneration (arrowhead), as it appears to be closely associated with A-β accumulation (blue immunolabeling) and aberrant cell bodies indicating the labeled region.

20A-20B show cytochrome C expression in the CA1 hippocampal region from non-transgenic mice (FIG. 20A) and transgenic mice with double mutation (APP / PS1 mutation) (FIG. 20B). It is a photograph. Note that cytochrome C immunoreactive axon structures (arrows) are present in double transgenic mice, but not in non-transgenic control mice.

20C-20H show cytochrome C expression (brown) and A-β in dentate gyrus of transgenic mice carrying the double mutation (APP / PS1 mutation), hippocampal regions CA1 and CA3 in identical sections. It is a figure which shows the double immunolabel which can visualize accumulation (blue). Cytochrome C immunoreactivity is associated with A-β accumulation (b, c, d, e.
, And f) are closely associated with abnormal axonal structures such as dystrophic axons. Cytochrome C immunoreactivity is also enriched in certain abnormal cell bodies that appear to be strongly immunolabeled at the perinuclear cytoplasmic level. High-power photographs in g and h support a close association between cytochrome C immunoreactive cell structure and A-β accumulation (arrows in g and h).

FIG. 21 shows control human subjects (FIG. 21A) and AD patients (FIG. 21B-
FIG. 21F) shows cytochrome C immunoreactive axon structures in the frontal cortex from (F). In AD patients, but not control subjects, there are cytochrome C immunoreactive axon structures (brown immunolabels) such as dystrophic axons (arrows), some of which are highly expressed in the cytoplasm and proximal extensions. Note that this is similar to neurofibrillary degeneration (arrowheads), as it appears to be closely associated with A-β accumulation (blue immunolabeling) and aberrant cell bodies showing a strongly labeled region.

FIG. 22 shows visualization of Bax expression by red rhodamine fluorescence (FIGS. 22A-C) and green fluorescence fluorescein (respectively in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). FIG. 22D-F) shows dual immunofluorescent labels that allow visualization of APP, PS1 or SMI expression. Bax and APP and Bax and PS within the same neural structure
Note that 1 is present (coexistence in the same neuron and plaque) and that in the control Bax and SMI immunofluorescence coexist in or around the plaque but not in the cell body. Double immunofluorescent labeling (FIGS. 22G-22H) shows the coexistence of cytochrome C and δ-catenin in a large number (but not all) of neuronal structures in double transgenic mice. Photos 22I and 22J show Bax and APP
Shows the co-existence of a double immunofluorescent label and axon structures in or around the plaque in brain tissue of human AD patients.

FIG. 23 shows visualization of Bax expression by red CY3 fluorescence (FIGS. 23A-23C) and green fluorescence fluorescein (respectively in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). 23D and 23E
, And 23F) are dual immunofluorescent labels capable of visualizing APP, PS1 or cytochrome C expression. Bax and APP, Bax and PS
1 shows confirmation of coexistence of Bax and cytochrome C.

A / Materials and Methods 1. PS1 Mutagenesis Human PS1 was transfected with Sculptor ^{™ +} (Amersham, France).
Mutagenesis was performed using an in vitro mutagenesis system. The PS1 coding region containing the Kozak consensus motif was added to the Bluscript vector (Stratage).
ne), subcloned and used the oligonucleotides containing the desired mutations and introduced the mutations according to the protocol provided by the manufacturer. Variant sequences were evaluated by sequence analysis.

2. Generation and Identification of PS1 M146L Transgenic Mice To construct the transgene, the cDNA encoding mutant human PS1 was subcloned between the SmaI and BamHI restriction sites of the polylinker of the transgenic expression vector HMG (Czech). Et al., 1997). For microinjection, the vector sequences were removed by restriction with the enzyme NotI and the fragment containing the expression cassette was purified by gel electrophoresis. The purified fragment was treated with 10 mM Tris-HCl (pH 7.4) and 0.1 mM EDT.
Diluted in A to a final concentration of 2.5 ng / μl and injected into one of two pronuclei of fertilized mouse embryos. The surviving embryos were immediately transplanted into the fallopian tubes of adoptive mothers (pseudopregnancy).
The presence of the transgene in newborns was identified by either PCR or Southern analysis. Oligomers corresponding to human PS1 (5'-TAA TTG GTC CA
T AAA AGG C-3 ′; 5′-GCA CAG AAA GGG AG
PCR was performed using T CAC AAG-3 ') to amplify a fragment of size 550 bp. In the case of Southern blot, the first from the HMG expression cassette
The 1.2 kb PstI-SalI fragment from this intron (which was radiolabelled with αP-32-dCTP) was used as a probe for transgene detection and the endogenous HMG gene as an internal control. . Taken together, these analyzes can rule out the possibility of any major rearrangement or deletion of the transgene in the primary and its progeny.

3. Construction of vector for APP transgene expression 3.1 PDGF APP ₆₉₅ SDL According to standard procedures (Ausubel et al., Modern molecular biology protocol),
The plasmid containing the PDGF expression cassette was linearized using the restriction endonuclease SnaBI to create blunt ends for subcloning the APP cDNA.

Mutagenesis of APP has been previously described (Czech et al., 1997). The cDNA encoding human APP ₆₉₅ SDL was cloned into the restriction endonuclease Sma.
It was excised from the cloning plasmid using I and Cla. The sticky ends generated by ClaI were made blunt by treatment with DNA polymerase Klenow fragment. The properties and integrity of the PDGF APP ₆₉₅ SDL constructs were evaluated by analysis of restriction patterns and the binding between the fusion DNA fragments was checked by partial sequencing.

The plasmid preparation kit (Qiagen) was used for the preparation of supercoiled DNA. The complete transgene was purified by digestion with the restriction enzyme NotI and separation of the transgene fragment by electrophoresis as described above. Use an aliquot for microinjection with a floating filter (Millip
ore, membrane type: VS; 0.025 μm, TE buffer (10 mM Tris)
It was dialyzed against (pH 7.4), 0.1 mM EDTA and filtered (Spin
-X; Costar; polyacetate film; 0.22 μm). The DNA was diluted to a final concentration of 1-2 μg / μl for microinjection.

3.2 APP ₇₅₁ SDL APP mutagenesis has been previously described (Czech et al., 1997) and exon-8 containing SmaI / BglI into a Bluescript vector containing the mutation.
The mutated APP sequence was converted to APP ₇₅₁ cDNA by insertion of the I APP fragment.
Moved to A. Transgenic expression construct APP ₇₅₁ SDL Thy-1
In order to prepare Escherichia coli, the APP cDNA derived from the SmaI (-95) site to the ClaI (2699) site was prepared, and the modified pBlue containing SalI site at each site of the insertion site.
It was cloned into the script vector. The vector was digested with SalI and the insert was cloned into the mouse Thy-1 vector using the XhoI site (L
Uthi et al. Neuroscience, 17, 4688-4699). The correct orientation was assessed by restriction analysis and the construct was sequenced at the ligation site. For microinjection, the cassette was linearized with NotI-PvuI restriction and the fragment containing the transgene was purified.

3.3 Mutagenesis of the APP ₇₅₁ Kozak SL APP has been previously described (Czech et al., 1997) and exon-8 containing SmaI / BglI into a Bluescript vector containing the mutation.
The mutated APP sequence was converted to APP ₇₅₁ cDNA by insertion of the I APP fragment.
Moved to A. To optimize the translation initiation site of APP, an optimized Kozak consensus sequence was introduced by site-directed mutagenesis by PCR. PCR
The combinations of oligonucleotides for were the following: sense oligo (start region): ccc ggg t cc acc atg ctg ccc ggt tt.
gg (underlined in Kozak sequence), antisense oligo: ttc agg gt
a gac ttc ttg gc. The PCR product was transferred to pCR2 (Invtroge
nA, France) and then sequenced to obtain AmaI and Ac
It was subcloned into the Bluescript vector containing the APP ₇₅₁ SL cDNA using cI to remove the 5'UTR of APP and introduce the Kozak consensus sequence. Transgenic APP ₇₅₁ Kozak SL Thy-1
To generate the construct, the 3'extended ClaI-extended APP cDNA (269
9) was subcloned into a modified Bluescript vector containing SalI sites on both sides of the insert. Digest vector with SalI and insert insert with Xh
It was cloned into the mouse Thy-1 vector using the oI site (Luthi et al., J. Neuroscience, 17, 4688-4699). The correct orientation was assessed by restriction analysis and the construct was sequenced at the ligation site. For microinjection, the cassette was linearized with NotI-PvuI restriction and the fragment containing the transgene was purified.

3.4 Generation of Transgenic Animals Transgenic using standard procedures previously described (eg, “Manipulation of Mouse Embryos”, Hogan et al., CSH Press, Cold Spring Harbor, NY). Genetic animals were acquired and identified.

[0078]   4. Western blot analysis   Transgenic mice and non-transgenic control mice (
Brain tissue derived from litters was treated with a protease inhibitor (Completete on ice). ^TM , Boehringer-Mannheim, Germany).
Homogenized in 32M sucrose solution. Cell debris at 1500 g for 5 minutes (
It was removed by centrifugation at 4 ° C. The protein concentration in the supernatant was
It was measured using the quality test (Pierce, USA). For PSI detection,
25 μg protein extract with 8 mM urea and 50 mM dithiothreitol
Incubate for 20 minutes in Laemmli loading buffer containing 56 ° C
It was For the detection of APP and A-β, 25 μg protein extract was added at 95 ° C.
Denatured in 30 μl of standard Lemmli loading buffer for 10 minutes. Po
Separation of proteins by polyacrylamide gel electrophoresis (SDS-PAGE)
did. Proteins are filtered with nitrocellulose (Amersham, Franc)
ce), heat the filter in PBS for 5 minutes to increase the sensitivity, and immediately
TBST solution of 5% (w / V) skim milk powder (850 mM Tris-HCl (pH
8.1), 150 mM NaCl, 0.05% (V / V) Tween 20)
Saturated and incubated overnight at 4 ° C. with a solution of primary antibody in TBST buffer only
. The antibody was bound to a horseradish peroxidase-conjugated anti-IgG antibody (Amersha
m, France) and the chemiluminescent detection system (Amersh) according to the manufacturer's instructions.
Am, France). Primary antibody MAB1 for detection of PS1
563 (Chemicon, USA) at a 1 / 10,000 dilution
And the antibody WO-2 was used at a concentration of 0.1 μg / ml for the detection of A-β.

5. Antibodies used for immunohistochemistry The following primary antibodies (Abs) were used: Mouse anti-APP monoclonal antibody (1: 100; 22C11, Boehr.
Inger), biotinylated mouse anti-Aβ _17-24 monoclonal antibody (1: 200, clone 4G8, Senetek), biotinylated mouse anti-Aβ _1-17 monoclonal antibody (1: 200, clone 6E10, Sentek), mouse anti-Aβ _{6- 17} monoclonal antibodies (1: 100, clone 6F / 3
D, Dako), rabbit anti-Aβ _1-42 polyclonal antibody (1: 300, QCB), rabbit anti-Aβ _total polyclonal antibody (1: 1000, FCA18, "C
Hecler), rat anti-PSl monoclonal antibody (1:50, Chemicon), rabbit anti-δ-catenin polyclonal antibody (1: 2000, RPR), rabbit anti-GFAP polyclonal antibody (1: 3000, Dako), rabbit anti-synaptophysin polyclonal antibody (1 : 100, Dako), mouse phosphorylated anti-neurofilament (PNF) monoclonal antibody (SMI
312, 1: 100, Sternberger), mouse anti-Tau-1 (Tau, microtubule-associated protein) monoclonal antibody (5 μg / ml, Boehringer, Mannheim), rabbit anti-Bax polyclonal antibody (P19, 1: 300, Santa C).
ruz), a mouse anti-cytochrome C monoclonal antibody (clone 7H8.2C12, 1,
: 200, Pharmingen).

The secondary antibody used (1: 400, Vector) was a mouse anti-IgG antibody (H + L) in experiments using mouse primary antibody, and a rabbit anti-IgG antibody in experiments using primary rabbit and rat antibody. It was a rat anti-IgG antibody (H + L). In some immunofluorescence labeling experiments, the secondary antibody used was the fluorescent dye CY.
It was a rabbit anti-IgG antibody conjugated to 3 (1/400, Vector).

6. Animal Manipulation Animals were stored under controlled temperature and humidity conditions and subjected to a 12 hour day / 12 hour night cycle (lights on 7:00 EST). Animals had free access to food and water. Meets the standards of the Guide to Experimental Animal Care and Use (American Council for Scientific Research ILAR) with the consent of the Rhone Planroller Ethics Committee on Animal Care and Use, but respects French regulations and EEC directives. Experiments were conducted on animals.

[0082]   The animals used for the neurohistopathology study are listed in the table below.

[0083]

[Table 2]

7. Neural Histopathology 7.1 Preparation of Brain Tissue. Anesthetize the mouse under heavy anesthesia (Pentobarbital: 60
mg / ml / kg i. p. , Ketamine: 40 mg / ml / kg, i. p. ),
Physiological serum and paraformaldehyde (4% PBS solution) were perfused through the heart. The brain was then removed and fixed in the same fixative solution at 4 ° C for 24 hours. After fixation, brains were separated into right and left hemi-brain, the latter subjected to a standard paraffin embedding protocol.

Blocks of paraffin-embedded left hemi-brain from transgenic and non-transgenic mice and post-mortem human brain tissue (frontal cortex) from AD patients and control subjects (provided by Dr. JP Brion, Belgium). , Using a microtome (LEICA RM 2155, France) to a thickness of 6 μm (serial section). Tissue compartments corresponding to the right hemi-brain from transgenic and non-transgenic mice were cut to a thickness of 25 μm.

For each immunohistochemistry experiment, brain sections were first dewaxed with xylene and 100
Dehydrated in% ethanol. The sections were then incubated in H202 (1% methanol solution) to block endogenous peroxidase activity, rinsed with ethanol and citrate buffer (10 mM sodium citrate pH 6) and finally in citrate solution. And put it in the microwave for 2 x 5 minutes. For the experiment to immunolabel A-β protein, the sections were subjected to a further step (ie incubation for 3 minutes in 80% pseudo-acid).

7.2 Thioflavin S To block the nuclear fluorescence, sections were taken with Mayer hematoxylin solution (Sig.
ma, France), after incubation for 10 minutes, 1% thioflavin S (S
(IGMA, France). The sections were observed with a microscope equipped with a fluorescence system (Axioscop Zeiss, France) using a FITC filter.

[0088]   7.3. Congo red.

Congo red stained sections were analyzed using a polarization system (amyloid staining kit, Accustain, Sigma, France). Only the red accumulation of birefringence (relative to the rotation of the polarizer) is considered as Congo red positive accumulation.

7.4. Immunoenzyme labeling After incubation for 30 minutes in blocking buffer (10% normal goat serum (Chemicon) in PBS containing 0.1% Triton (Sigma)), dewaxed brain sections were incubated in primary antibody solution. (Overnight at 4 ° C). After rinsing several times, the sections were manufactured by the manufacturer (Vectastin ABC kit, Vect
Or Laboratories, Burlingame, CA), followed by contact with biotinylated secondary antibody (2 hours at ambient temperature) and avidin-biotin peroxidase complex. As a chromogen of the peroxidase enzyme,
3-3'-diaminobenzidine was used. For preabsorption experiments, anti-Bax antibody (
Antibody P19, Santa Cruz) was synthesized with synthetic Bax peptide (control peptide P19, Santa Cruz) (test concentration of peptide: 0.002, 0.
02, and 0.2 mg / ml) for at least 12 hours before use according to the immunohistochemistry protocol described above. Using the same protocol, anti-cytochrome C antibody (7H8.2C12, Pharminge
n) was incubated with exogenous purified cytochrome C from horse or rat heart (test concentrations of purified protein: 0.01 and 0.1 mg / ml).

7.5. Quantification of amyloid amount Aβ immunolabeling (biotinylated mouse anti-Aβ17-24, monoclonal antibody 4G
8, Senetek) accumulation of A-β in 25 μm hemi-brain sections with diaminobenzidine as chromogen and image analysis system equipped with a color camera and microscope (
Quantification using the Q600 system, LEICA). Record each anatomical region of interest and define a threshold for anatomical detection of mean gray level (and distinguish between specific label and background noise) corresponding to immunolabeling of A-β accumulation. did. The experimenter manually checked each field to eliminate artifacts manually. In each mouse, A-beta amount was measured in mouse stereotactic atlas of the bregma body axis level -3.4 (Franklin and Paxinos). The amount of A-β is defined as the percentage of A-β immunolabeled parasitism with respect to the total area of the brain area analyzed (ie hippocampus, cortex and the rest of the section (subcortical area)).

7.6. Double Immunoenzyme Labeling Immunohistochemical double labeling experiments were performed by a two step protocol. Briefly, the sections were treated with a primary antibody (eg anti-G) according to the above protocol.
Immunolabeling was performed in the first step with FAP, anti-synaptophysin, anti-Bax, or anti-cytochrome C), and the antibody was visualized by the brown labeling obtained with diaminobenzidine, the enzyme substrate for horseradish peroxidase. Then, the same section was subjected to primary anti-Aβ _17-24 antibody (mouse monoclonal antibody 4G8, Sen).
etek) and the antibody was visualized by a blue label obtained using the horseradish peroxidase substrate SG (SG peroxidase substrate kit, Vector).

7.7. Double immunofluorescent labeling In particular, Bax is APP, PS1, cytochrome C in the axon structure associated with A-β accumulation in transgenic mice carrying the double mutation (APP / PS1 mutation).
Alternatively, dual immunofluorescent labeling was performed in the same experiment to show whether it co-localized with SMI. Briefly, sections were first incubated in anti-Bax antibody and then conjugated to CY3 biotinylated secondary rabbit anti-IgG antibody (1: 400, C
hemicon) or using any of the secondary rabbit anti-IgG antibodies and a signal amplification kit (streptavidin-peroxidase / tetramethylrhodaminetyramide complex) according to the manufacturer's instructions (New England Nuclear). did. For secondary immunofluorescent labeling, sections were serially incubated with primary anti-APP antibody, anti-PS1 antibody, anti-cytochrome C antibody, or anti-SMI antibody, followed by biotinylated secondary mouse or rat anti-IgG antibody. . Finally, the sections were manufactured by the manufacturer (New England Nuclear).
Were visualized using a second signal amplification system (streptavidin-peroxidase / tetramethylrhodamine tyramide complex) according to the instructions in. The sections were CY3 or Rhodamine filters (excitation 550 for Bax immunofluorescence labeling).
nm, emission 570 nm) and, in the case of the second immunofluorescent label, observed under a microscope using a fluorescence filter (excitation 495 nm, emission 517 nm). In the same way, in a double immunofluorescence labeling experiment performed to show whether cytochrome C co-exists with the δ-catenin protein, the sections were first incubated in anti-cytochrome C antibody and then the rhodamine fluorescence system was used. Visualized using. In the second step, these sections were incubated in anti-δ-catenin antibody and visualized using the fluorescein fluorescence system as described above.

Examples Example 1: Analysis of transgene expression levels 1.1 Comparison of transgene expression levels in different transgenic mice expressing mutant APP. Brain homogenates from different transgenic mouse lines (different expression constructs) were analyzed for APP expression by Western blotting using WO-2 monoclonal antibody. This antibody recognizes human APP and A-β, but does not recognize endogenous mouse APP, so it is specifically displayed by this antibody. Therefore, no signal was detected in the lane corresponding to the non-transgenic mouse (FIG. 1, lane 3). Lanes 1 and 2 of FIG. 1 were previously shown to express high levels of the APP transgene (M
oechard et al., 1999a) corresponding to brain extract from transgenic mice. Lane 7 transgenic PDGF APP ₆₉₅ SDL mice
Used with mice carrying the S1 M146 transgene, a double transgenic mouse line was generated. Moderate APP expression levels were observed in mice with the HMG promoter in the construct (Czech et al., 1997). AP
Note that in mice transgenic for the Swedish mutation of P, the beta secretase fragment (12 kDa) was increased compared to the complete APP holoprotein.

1.2 Western Blot Analysis of Transgene Expression and Processing in the Brain of APP and Presenilin Double Transgenic Mice The transgene expression of each individual has two transgenes in mice (obtained by mating) To find out if they were modified, brain homogenates from double transgenic mice of different ages were analyzed for APP and A-β expression by Western blotting using WO-2 monoclonal antibody (
the above). The intensity of the bands corresponding to the complete 100 kDa APP sequence and the 12 kDa amino-terminal fragment was not different between PDGF-APP ₆₉₅ SDL (monotransgenic) and APP ₆₉₅ SDL x PS1M146L double transgenic mice. (Also comparing Figure 2A with Lane 7 of Figure 1). Moreover, the age of transgenic mice has no effect on the expression of the APP transgene or metabolism at the β-cleavage level. However, A-β
Is highly accumulated at 9 months and this accumulation starts at 6 months of age. This increase in A-β is highly correlated with the onset of amyloid plaque formation in the brain of transgenic mice (see below). This suggests that the increase in A-β detected by Western blotting may correspond to A-β accumulated in amyloid plaques.

Another Western blot analysis was performed on brain homogenates to identify whether human PS1 protein was expressed in double transgenic and non-transgenic (littermate) control mice. A monoclonal antibody against the amino terminal portion of PS1 was used. This antibody is specific for human PS1; the endogenous PS1 protein signal is not detected in brain homogenates from single mutant APP mice (FIG. 2B, lanes 1 and 2). A characteristic amino-terminal PS1 fragment was detected in the brain of PS1M146L expressing mice (FIG. 2B,
Lanes 3-7), complete PS1146L homoprotein appears (approximately 51 kDa).
), Which is presumably as previously described in transgenic rats and mice overexpressing PS1 (Czech et al., 1998; Thinakar).
an et al., 1996) due to saturation of presenilin processing. Expression and processing of human PS1 in double transgenic mice does not change with age of animals.

In conclusion, the expression level of each transgene (APP or PS1) is
Similar for mono- and double transgenic mice.

Example 2: One (PDGF APP ₆₉₅ SDL or HMG PS1M146
L) or di (PDGF APP ₆₉₅ SDL x HMG PS1M146L) Location and regional expression of transgenes in mice Immunohistochemical experiments for APP using mouse monoclonal antibody against APP protein (antibody 22C11), single transgenic (
PDGF APP ₆₉₅ SDL) and double transgenic (PDGF A
PP ₆₉₅ SDL × HMG PS1M146L) mice were demonstrated to express APP protein more strongly than non-transgenic control mice (FIG. 3). This result, two transgenic mouse strains that are confirmed to express human APP protein encoded by the _{APP 6} 95 SDL transgene. Human APP is exclusively present in neurons. Its expression pattern is similar in the two transgenic mouse strains, containing multiple brain structures,
The highest expression levels were found in the neuronal layer of the hippocampal body (Fig. 3), followed by similar expression levels in cortical regions such as the entorhinal cortex and amygdala. The transgene expression levels are low in the subcortical region, and human APP protein is not detected at either glial or vascular levels.

In the same way, immunohistochemical experiments on PS1 using a rat monoclonal antibody against human PS1 protein (an antibody that specifically recognizes human PS1) showed that it was single transgenic (HMG PS1M146L) and double. Transgenic (PDGF APP ₆₉₅ SDL x HMG PS1M146
L) It was shown that some immunolabeled glial cells were occasionally found, although the mice express human PS1 protein in the brain at specific locations in neurons (FIG. 3). The expression pattern of human PS1 protein is similar in the two transgenic mouse lines. The expression levels in cortical structures such as the hippocampal body (FIG. 3) and different areas of the cortex and their subcortical areas are evaluated. The protein is not expressed in white matter. In both the human APP protein and the human PS1 protein, neither the expression pattern nor the expression level change with age (6 to 12 weeks of age).

Example 3: Demonstration that the process of amyloid accumulation is accelerated in the brain of double transgenic mice (PDGF APP ₆₉₅ SDL x HMG PS1M146L) with both mutant APP protein and mutant pS1 protein. To show whether the transgenic mouse model accumulates A-β in the brain, various anti-Aβ antibodies (see Materials and Methods) and AD
Conventional histological markers (thioflavin S and Congo red) that stain A-β accumulation in human tissues from patients were used.

Using a set of A-β accumulation markers, we used single transgenic mice (ie PDGF APP ₆₉₅ SDL or HMG PS1M.
146L) showed that no amyloid accumulation was observed in the brain at 3, 6, 9, 12, and 15 months of age. On the other hand, double transgenic (P
In the DGF APP ₆₉₅ SDL × HMG PS1M146L) mouse, A-β accumulation was observed from the age of 6 months. The various anti-Aβ antibodies used all show the presence of this amyloid protein accumulation process (FIGS. 4 and 5). These A-β accumulations are stained with thioflavin S and Congo red to also confirm their fiber formation (FIG. 6).

Example 4: Double transgenic (PDGF APP ₆₉₅ SDL x HMG
Progression and regional pattern of amyloid accumulation in PS1M146L) mice Double transgenic mice (PDGF APP ₆₉₅ SDL x HMG P
S1M146L) for the purpose of assessing the regional distribution pattern of amyloid progression and accumulation due to transgene expression of S1M146L).
The number of β accumulations and the amount of amyloid in 25 μm thick sections at 12 months of age were quantified using anti-Aβ antibody 4G8. For quantification of amyloid levels, we focused on the cortex and hippocampus because these two brain structures are involved in the neuropathology of transgenic mice and AD-affected patients in an early and predominant manner. is there.

The number of A-β accumulated was 6 as shown qualitatively and quantitatively in FIGS. 7 and 8, respectively.
Higher in 12 month old mice than in 9 and 9 month olds. A-β at 6 and 9 months of age
Accumulation is present in the limited brain region corresponding to the hippocampus and the back of the hippocampal region CA1 (FIG. 7). In contrast, at 12 months of age, more A-β accumulation is present throughout the hippocampal and cortical areas along the axial length of the brain (FIGS. 7 and 9). 1
In 2-month-old mice, A-β accumulation is only occasionally present in some subcortical tissues, such as the inner capsule, lateral thalamus, and basal ganglia. Accumulation is absent in both the cerebellum and spinal cord. Quantitative analysis of the amount of amyloid in 12-month-old mice (
As shown in FIG. 10), the percentage of Aβ immunolabeling region for the entire analysis region was
It is shown to reach values of 3.1, 0.8-1.1 and less than 0.5 for the cortical and the rest of the hemi-brain respectively. Amyloid load reaches values above 9% and 5% in the most abundant regions of the hippocampus and cortex, respectively. These amounts of amyloid are similar (6-12%) to those previously described in human AD brain (Hyman et al., 1993).

Example 5: Double transgenic (PDGF APP ₆₉₅ SDL x HMG
PS1M146L) Axon structure in mice Transgenic mice with advanced A-β accumulation show large or intermediate size accumulation, while different markers of dystrophic axons (APP (FIG. 11), PS
Photographs of one immunolabeling with 1 (FIG. 12) and δ-catenin (FIG. 13), on the other hand using anti-Aβ antibody 4G8 and axon structure or anti-synaptophysin antibody or anti-SMI antibody Photographs of dual immunolabels visualizing A-β accumulation demonstrate the inclusion of multiple axon structures in the exact same brain section (FIGS. 14 and 15, respectively). As shown in FIGS. 14 and 15, synaptophysin immunoreactive and SMI immunoreactive axon structures are present in the brains of double transgenic mice and are closely associated with A-β accumulation. Furthermore, this result is
It shows that the axon structure is very similar to the structure described in human AD brain.

To show whether neurofibrillary degeneration is also present in the brains of double transgenic mice, anti-Aβ antibody 4G in exactly the same brain section for the purpose of visualizing A-β accumulation.
8 and nerve fiber degeneration or anti-Tau-I antibody was used for double immunolabeling. As shown in FIG. 16, anti-Tau-I antibody labels axon structures such as axons that cover A-β accumulation and abnormal cell bodies. These two immunolabeling structure types are
It is present in brain regions (hippocampus and cortex) where A-β accumulation is abundant, but not in subcortical regions where A-β accumulation is low but not present.

Taken together, in addition to the phenomenon of amyloid accumulation, these results clearly show that degeneration occurs within the cognitive structure of double transgenic mice in a manner similar to AD brain.

Example 6: Double transgenic (PDGF APP ₆₉₅ SDL x HMG
PS1M146L) Presence of Activated Astrocytes Within or Around Amyloid Accumulation in Mice Since A-β accumulation in AD brain tissue is associated with glial cells (inflammatory cells of the central nervous system) A study was conducted to determine if accumulation occurs in the neural network of our transgenic mice as it progresses. GFAP immunoreactive astrocytes are used in non-transgenic control mice and transgenic mice that do not develop A-β accumulation (eg, single transgenic PDGF).
Neither APP ₆₉₅ SDL nor HMG PS1M146L mice). In contrast, all double transgenic mice with A-β accumulation in the brain have GFAP immunoreactive astrocytes that are closely associated with A-β accumulation (Figure 17).

Example 7: Double transgenic (PDGF APP ₆₉₅ SDL x HMG
PS1M146L) Demonstration of mitochondrial dysfunction in mice One immunolabeling was performed to visualize the Bax protein itself, and a double immunolabeling was performed to visualize Bax protein and A-β accumulation in exactly the same brain section. It was This was done with the aim of establishing an association between Bax expression and amyloid plaques.

The results of the single labeling show that Bax protein is expressed in multiple brain tissues (neural layer of hippocampal body and all cortical regions (FIGS. 18A-H)) in both non-transgenic control and transgenic mice. It has been shown. But,
Single transgenic (PDGF APP ₆₉₅ SDL x HMG PS1M1
46L) mice or double transgenic (PDGF APP ₆₉₅ SDL x HMG PS1M146L rather than non-transgenic control mice)
) Only mice have B in cell structure (dystrophic axons (FIGS. 18A-H), neuronal cell bodies resembling early nerve fiber degeneration, and occasionally activated glial cells (see below)).
It shows ax immunoreactivity. Interestingly, these abnormal Bax immunoreactive cell structures are exclusive to brain regions rich in A-β accumulation, such as the dentate gyrus, hippocampal regions CA1 and CA3, hippocampus, and cingulate and entorhinal cortex. Exists in.

Double immunolabeling with anti-Bax and anti-Aβ antibodies suggests that Bax immunoreactivity in axon structures and activated glial cells is closely associated with A-β accumulation in double transgenic animals. Substantially confirmed (Fig. 18I-L). These Bax immunoreactive cell structures were never detected in the brain of non-transgenic or single transgenic animals. The Bax immunolabeling in these studies is specific, as shown by preabsorption experiments with blocking peptides.

The result of one immunolabeling of cytochrome C indicates that the cytochrome C protein is expressed in the neuronal cell body in four groups of test animals (non-transgenic controls and one and double transgenic animals-Table I). Was shown. Cytochrome C
The distribution pattern of immunoreactive neurons is similar to that described for Bax. Interestingly, in the case of Bax, cytochrome C had a cellular structure only in double transgenic mice (such as axons and abnormal neuronal cell bodies (FIG. 20)).
It is shown to be expressed in. These cytochrome C immunoreactive cell structures are never found in either single transgenic or non-transgenic control mice (Figure 20). In double transgenic mice,
Cytochrome C immunoreactive axon structures, such as Bax immunoreactive axon structures, have A-β
Brain tissue in which accumulation is found (ie predominantly dentate gyrus, hippocampal areas CA1 and CA3
And the cingulate cortex (FIG. 20)). Double immunolabeling of cytochrome C and A-β accumulation indicates that cytochrome C immunoreactive axon structures are closely associated with amyloid plaques in these double transgenic animals (FIG. 20). Cytochrome C immunolabeling as well as Bax immunolabeling is specific, as shown in preabsorption experiments using purified cytochrome C protein.

In parallel with experiments performed on transgenic mice, Bax / Aβ and cytochrome C on post-mortem human brain tissue from control individuals and AD patients.
/ Aβ double immunolabeling was performed. The results obtained (FIGS. 19 and 21) show that human A
In the D brain, we show that Bax immunoreactive or cytochrome C immunoreactive axon structures, such as dystrophic axons and early nerve fiber neurodegeneration, are present in and around amyloid plaques. Bax immunoreactive and cytochrome C immunoreactive abnormal neural structures were not found in control human tissues.

To confirm the presence of Bax and cytochrome C proteins in aberrant axon structures in the brains of double transgenic mice, Bax covered APP, PS1 in the same axon structure covering A-β accumulation. , And double immunofluorescence labeling showing coexistence with cytochrome C were also performed (FIGS. 22 and 23). Finally, cytochrome C was shown to co-exist with δ-catenin in these axon structures (Figure 22).

The fact that Bax and cytochrome C are overexpressed in axon structures (such as axons and abnormal neuronal cell bodies) in the brain of human AD brains and double transgenic mice is due to the fact that AD patients and this transgenic mouse are overexpressed. The involvement of these proteins in the regulation of early events associated with nerve death in the model is shown.

These findings in mice indicate that, firstly, two different mitochondrial proteins known to induce cell death in culture, namely Bax and cytochrome C, transduce the progression of A-β accumulation. It shows that it changes in the transgenic mouse. This altered expression of Bax and cytochrome C resides in these two mitochondrial markers accumulating in aberrant neural structures closely associated with amyloid plaques.

The key point is that the Bax immunoreactive and cytochrome C immunoreactive axon structures in double transgenic mice are similar to those found in the human AD brain, which is identical. It shows that a mitochondrial dysfunction type is found in human AD patients.

[0117]

[Table 3]

[0118]

[References]

[0119]

[Table 4]

[Brief description of drawings]

FIG. 1 shows a comparison of APP, β-secretase fragment, and A-β of various transgenic mouse strains.

FIG. 2A: APP, β-secretase 12 kDa fragment, and A-β in brain homogenates of transgenic mice with single or double mutations at different ages.
FIG. 5 shows the use of antibody WO-2 directed specifically against the human A-β form to analyze the expression of

FIG. 2B   FIG. 6 shows an analysis of the expression of human M146LPS1.

FIG. 3 is a plate showing the expression of human APP in the hippocampus and cortex of transgenic mice carrying a single mutation (APP mutation) (b1, b2) or a double mutation (APP / PS1) mutation (c1, c2). is there.

FIG. 4A is an example of A-β accumulation in the brain of a 6-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Immunohistochemical detection using antibodies specifically directed against different epitopes of the A-β peptide: αβ _1-17 6E10.

FIG. 4B is an example of A-β accumulation in the brain of a 6-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Immunohistochemical detection using Dako of αβ _6-17 , in particular antibodies directed against different epitopes of the A-β peptide.

FIG. 4C is an example of A-β accumulation in the brain of a 6-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Immunohistochemical detection using 4G8, in particular antibodies directed against different epitopes of the A-β peptide: αβ _17-24 .

FIG. 4D is an example of A-β accumulation in the brain of 6-month-old transgenic mice carrying the double mutation (APP / PS1 mutation). Immunohistochemical detection using QCB of antibodies specifically directed against different epitopes of the A-β peptide: αβ _1-42 .

FIG. 4E is an example of A-β accumulation in the brain of a 6-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Antibodies directed specifically against different epitopes of the A-β peptide: immunohistochemical detection using FCA18 for all αβ.

FIG. 5A is an example of A-β accumulation in the brain of a 12-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Immunohistochemical detection using antibodies specifically directed against different epitopes of the A-β peptide: αβ _1-17 6E10.

FIG. 5B is an example of A-β accumulation in the brain of a 12-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Immunohistochemical detection using Dako of αβ _6-17 , in particular antibodies directed against different epitopes of the A-β peptide.

FIG. 5C is an example of A-β accumulation in the brain of a 12-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Immunohistochemical detection using 4G8, in particular antibodies directed against different epitopes of the A-β peptide: αβ _17-24 .

FIG. 5D is an example of A-β accumulation in the brain of a 12-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Immunohistochemical detection using QCB of antibodies specifically directed against different epitopes of the A-β peptide: αβ _1-42 .

FIG. 5E is an example of A-β accumulation in the brain of a 12-month-old transgenic mouse having a double mutation (APP / PS1 mutation). Antibodies directed specifically against different epitopes of the A-β peptide: immunohistochemical detection using FCA18 for all αβ.

FIG. 6A is a photograph showing a histological section stained with thioflavin S at the level of hippocampal formation in a 12-month-old transgenic mouse having a double mutation (APP / PS1 mutation).

FIG. 6B is a photograph showing histological sections stained with Congo red at the level of hippocampal formation of 12-month-old transgenic mice having a double mutation (APP / PS1 mutation).

FIG. 7 shows the progress of A-β accumulation immunolabeled with anti-Aβ antibody 4G8 in a transgenic mouse having a double mutation (APP / PS1 mutation).

FIG. 8. Double transgenic APP ₆₉₅ SDL x plotted against age.
Anti-A in 6 μm thick hemi-brain sections (Franklin and Paxinos stereotaxic at Bregma axial level -3.4) from PS1 M1476L mice.
It is a figure which shows the A- (beta) accumulation number immunolabeled with (beta) antibody 4G8.

FIG. 9 is a diagram showing a regional distribution of A-β accumulation in the brain of a 12-month-old transgenic mouse having a double mutation (APP / PS1 mutation).

FIG. 10A: Double mutation (APP / P) visualized by Aβ immunohistochemistry (antibody AG8) performed on 25 μm thick hemi-brain sections (Bregma axial level-3.4).
It is a figure which shows the quantification of the amount of A- (beta) in the 12-month-old transgenic mouse which has (S1 mutation).

FIG. 10B: Double mutations (APP / P visualized by Aβ immunohistochemistry (antibody AG8) performed on 25 μm thick hemi-brain sections (Bregma axial level-3.4).
It is a figure which shows the quantification of the amount of A- (beta) in the 12-month-old transgenic mouse which has (S1 mutation).

FIG. 10C: Double mutation (APP / P) visualized by Aβ immunohistochemistry (antibody AG8) performed on 25 μm thick hemi-brain sections (Bregma axial level-3.4).
It is a figure which shows the quantification of the amount of A- (beta) in the 12-month-old transgenic mouse which has (S1 mutation).

FIG. 11A: Immunoreactive axon structures are present in the hippocampus of transgenic mice carrying the double mutation (APP / PS1 mutation) (4), non-transgenic control mice (1), and one APP mutation. (APP mutation) (2) or PS1 mutation (
It is a figure which shows that it does not exist in the hippocampus of the transgenic mouse which has a PS1 mutation) (3).

FIG. 11B: Immunoreactive axon structures are present in the hippocampus of transgenic mice carrying the double mutation (APP / PS1 mutation) (4), non-transgenic control mice (1), and one APP mutation. (APP mutation) (2) or PS1 mutation (
It is a figure which shows that it does not exist in the hippocampus of the transgenic mouse which has a PS1 mutation) (3).

FIG. 12A: Immunoreactive axon structures are present in the hippocampus of transgenic mice carrying the double mutation (APP / PS1 mutation) (4), non-transgenic control mice and one APP mutation (APP mutation). Or PS1 mutation (PS1 mutation) (
It is a figure which shows that it does not exist in the hippocampus of the transgenic mouse which has 3).

FIG. 12B: Immunoreactive axon structures are present in the hippocampus of transgenic mice carrying the double mutation (APP / PS1 mutation) (4), non-transgenic control mice and one APP mutation (APP mutation). Or PS1 mutation (PS1 mutation) (
It is a figure which shows that it does not exist in the hippocampus of the transgenic mouse which has 3).

FIG. 13: δ-catenin immunoreactive axon structures are present in the hippocampus (Hi) and cortex (Ctx) of transgenic mice carrying the double mutation (APP / PS1 mutation) (b).
FIG. 1 is a diagram showing that the non-transgenic control mice do not exist in the same brain structure (a1, a2).

FIG. 14A is a diagram showing a synaptophysin immunoreactive axon structure in a transgenic mouse having a double mutation (APP / PS1 mutation).

FIG. 14B shows synaptophysin immunoreactive axon structures in humans with AD having a double mutation (APP / PS1 mutation).

FIG. 15: Transgenic mouse with double mutation (APP / PS1 mutation) and A
FIG. 5 shows SMI immunoreactive phosphorylated neurofilaments in humans with D.

FIG. 16: Axon structure and nerves containing dystrophic axons located around or inside A-β accumulation (blue immunolabeling) in the brain of transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure which shows tau-1 immunoreactivity (brown immunolabeling) concentrated in the cell body (arrow head).

FIG. 17A is a diagram showing activated GFAP immunoreactive astrocytes (brown immunolabeling) present in a transgenic mouse having a double mutation (APP / PS1 mutation).

FIG. 17B shows activated GFAP immunoreactive astrocytes (brown immunolabeling) present in humans with AD having the double mutation (APP / PS1 mutation).

18A: Dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal area (FIGS. 18B, 18F).
FIG. 18 shows Bax expression in the entorhinal cortex of neurons of FIG. 18A and non-transgenic control mice (FIGS. 18A to 18D) and transgenic mice having a double mutation (APP / PS1 mutation) (FIGS. 18E to 18H).

18B: Dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal region (FIGS. 18B, 18F).
FIG. 18 shows Bax expression in the entorhinal cortex of neurons of FIG. 18A and non-transgenic control mice (FIGS. 18A to 18D) and transgenic mice having a double mutation (APP / PS1 mutation) (FIGS. 18E to 18H).

18C: Dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal region (FIGS. 18B, 18F).
FIG. 18 shows Bax expression in the entorhinal cortex of neurons of FIG. 18A and non-transgenic control mice (FIGS. 18A to 18D) and transgenic mice having a double mutation (APP / PS1 mutation) (FIGS. 18E to 18H).

18D: Dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal region (FIGS. 18B, 18F).
FIG. 18 shows Bax expression in the entorhinal cortex of neurons of FIG. 18A and non-transgenic control mice (FIGS. 18A to 18D) and transgenic mice having a double mutation (APP / PS1 mutation) (FIGS. 18E to 18H).

18E: Dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal region (FIGS. 18B, 18F).
FIG. 18 shows Bax expression in the entorhinal cortex of neurons of FIG. 18A and non-transgenic control mice (FIGS. 18A to 18D) and transgenic mice having a double mutation (APP / PS1 mutation) (FIGS. 18E to 18H).

18F: Dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal region (FIGS. 18B, 18F).
FIG. 18 shows Bax expression in the entorhinal cortex of neurons of FIG. 18A and non-transgenic control mice (FIGS. 18A to 18D) and transgenic mice having a double mutation (APP / PS1 mutation) (FIGS. 18E to 18H).

18G: Dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal region (FIGS. 18B, 18F).
FIG. 18 shows Bax expression in the entorhinal cortex of neurons of FIG. 18A and non-transgenic control mice (FIGS. 18A to 18D) and transgenic mice having a double mutation (APP / PS1 mutation) (FIGS. 18E to 18H).

18H: Dentate gyrus (FIGS. 18C, 18G) and CA3 hippocampal region (FIGS. 18B, 18F).
FIG. 18 shows Bax expression in the entorhinal cortex of neurons of FIG. 18A and non-transgenic control mice (FIGS. 18A to 18D) and transgenic mice having a double mutation (APP / PS1 mutation) (FIGS. 18E to 18H).

FIG. 18I: Dentate gyrus (FIGS. 18K and 18L) and CA3 hippocampal region (FIGS. 18I and 18J) of transgenic mice carrying the double mutation (APP / PS1 mutation).
FIG. 8 shows a dual immunolabeling visualization of Bax expression (brown) and A-β accumulation (blue) in exactly the same section of FIG.

FIG. 18J: Dentate gyrus (FIGS. 18K and 18L) and CA3 hippocampus region (FIGS. 18I and 18J) of transgenic mice with a double mutation (APP / PS1 mutation).
FIG. 8 shows a dual immunolabeling visualization of Bax expression (brown) and A-β accumulation (blue) in exactly the same section of FIG.

FIG. 18K: Dentate gyrus (FIGS. 18K and 18L) and CA3 hippocampus region (FIGS. 18I and 18J) of transgenic mice carrying the double mutation (APP / PS1 mutation).
FIG. 8 shows a dual immunolabeling visualization of Bax expression (brown) and A-β accumulation (blue) in exactly the same section of FIG.

FIG. 18L: Dentate gyrus (FIGS. 18K and 18L) and CA3 hippocampus region (FIGS. 18I and 18J) of transgenic mice carrying the double mutation (APP / PS1 mutation).
FIG. 8 shows a dual immunolabeling visualization of Bax expression (brown) and A-β accumulation (blue) in exactly the same section of FIG.

FIG. 19A shows Bax immunoreactive axon structures in the frontal cortex from control human subjects.

FIG. 19B   FIG. 3 shows Bax immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 19C   FIG. 3 shows Bax immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 19D   FIG. 3 shows Bax immunoreactive axon structures in the frontal cortex from AD patients.

[FIG. 19E]   FIG. 3 shows Bax immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 19F   FIG. 3 shows Bax immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 20A is a photograph showing cytochrome C expression in the CA1 hippocampus region from a non-transgenic mouse (FIG. 20A) and a transgenic mouse having a double mutation (APP / PS1 mutation) (FIG. 20B).

FIG. 20B is a photograph showing cytochrome C expression in the CA1 hippocampus region from a non-transgenic mouse (FIG. 20A) and a transgenic mouse having a double mutation (APP / PS1 mutation) (FIG. 20B).

FIG. 20C: Visualization of cytochrome C expression (brown) and A-β accumulation (blue) in identical sections of dentate gyrus, hippocampal regions CA1 and CA3 of transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure which shows a double immunolabel which can be activated.

FIG. 20D: Visualization of cytochrome C expression (brown) and A-β accumulation (blue) in identical sections of dentate gyrus, hippocampal regions CA1 and CA3 of transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure which shows a double immunolabel which can be activated.

FIG. 20E. Visualization of cytochrome C expression (brown) and A-β accumulation (blue) in identical sections of dentate gyrus, hippocampal regions CA1 and CA3 of transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure which shows a double immunolabel which can be activated.

FIG. 20F Visualize cytochrome C expression (brown) and A-β accumulation (blue) in identical sections of dentate gyrus, hippocampal regions CA1 and CA3 of transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure which shows a double immunolabel which can be activated.

FIG. 20G Visualize cytochrome C expression (brown) and A-β accumulation (blue) in identical sections of dentate gyrus, hippocampal regions CA1 and CA3 of transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure which shows a double immunolabel which can be activated.

FIG. 20H Visualize cytochrome C expression (brown) and A-β accumulation (blue) in identical sections of dentate gyrus, hippocampal regions CA1 and CA3 of transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure which shows a double immunolabel which can be activated.

FIG. 21A shows cytochrome C immunoreactive axon structures in the frontal cortex from a control human subject.

FIG. 21B shows cytochrome C immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 21C shows cytochrome C immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 21D shows cytochrome C immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 21E shows cytochrome C immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 21F shows cytochrome C immunoreactive axon structures in the frontal cortex from AD patients.

FIG. 22A shows a double immunofluorescent label capable of visualizing Bax expression by red rhodamine fluorescence in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation).

FIG. 22B shows a double immunofluorescent label capable of visualizing Bax expression by red rhodamine fluorescence in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation).

FIG. 22C shows a dual immunofluorescent label capable of visualizing Bax expression by red rhodamine fluorescence in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation).

FIG. 22D shows a dual immunofluorescent label capable of visualizing APP, PS1, or SMI expression by green fluorescent fluorescein in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure.

FIG. 22E shows a double immunofluorescent label capable of visualizing APP, PS1 or SMI expression by green fluorescent fluorescein in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure.

FIG. 22F shows a dual immunofluorescent label capable of visualizing APP, PS1, or SMI expression by green fluorescent fluorescein in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). It is a figure.

22G: Visualization of Bax expression by red rhodamine fluorescence (FIGS. 22A-C) and green fluorescence fluorescein (FIGS. 22D- respectively) in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). APP by F)
FIG. 22G shows a dual immunofluorescent label capable of visualizing PS1, PS1, or SMI expression, wherein the dual immunofluorescent labels (FIGS. 22G-22H) are very numerous (not all) in double transgenic mice. The coexistence of cytochrome C and δ-catenin is shown in the neuronal structure of).

FIG. 22H. Visualization of Bax expression by red rhodamine fluorescence (FIGS. 22A-C) and green fluorescence fluorescein (FIGS. 22D- respectively) in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). APP by F)
FIG. 22G shows a dual immunofluorescent label capable of visualizing PS1, PS1, or SMI expression, wherein the dual immunofluorescent labels (FIGS. 22G-22H) are very numerous (not all) in double transgenic mice. The coexistence of cytochrome C and δ-catenin is shown in the neuronal structure of).

22I shows dual immunofluorescent labeling of Bax and APP and the coexistence of axonal structures in or around the plaque in brain tissue of human AD patients.

FIG. 22J shows coexistence of dual immunofluorescent labeling of Bax and APP and axon structures in or around plaques in brain tissue of human AD patients.

FIG. 23A is a diagram showing a double immunofluorescent label capable of visualizing Bax expression by red CY3 fluorescence in exactly the same brain section from a transgenic mouse having a double mutation (APP / PS1 mutation).

FIG. 23B is a diagram showing a double immunofluorescent label capable of visualizing Bax expression by red CY3 fluorescence in exactly the same brain section derived from a transgenic mouse having a double mutation (APP / PS1 mutation).

FIG. 23C is a diagram showing a double immunofluorescent label capable of visualizing Bax expression by red CY3 fluorescence in exactly the same brain section from a transgenic mouse having a double mutation (APP / PS1 mutation).

FIG. 23D shows a dual immunofluorescent label capable of visualizing APP, PS1 or cytochrome C expression by green fluorescent fluorescein in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). FIG.

FIG. 23E shows a dual immunofluorescent label capable of visualizing APP, PS1 or cytochrome C expression by green fluorescent fluorescein in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). FIG.

FIG. 23F shows a dual immunofluorescent label capable of visualization of APP, PS1 or cytochrome C expression by green fluorescent fluorescein in identical brain sections from transgenic mice carrying the double mutation (APP / PS1 mutation). FIG.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Imperato, Assunta             France, F-92210 Saint-Cloud,             Abunyu Pozo Dei Borgo 5 (72) Inventor Bonisi, Brno             France, F-91230, Monzilleron,             Lieu de Cialum-1 (72) Inventor Tremp, Guyunter             France, F-91120, Palaiseau, Les             Gidence Dou Parc Dardonnay 6 (72) Inventor Chietsk, Kristian             France, F-75007, Paris, Cite             Dow Larma 4 F-term (reference) 4B065 AA91X AB01 AC20 BA02                       CA46

Claims (12)

[Claims] 1. A non-human transgenic animal model of Alzheimer's disease showing amyloid plaques and mitochondrial dysfunction. 2. The animal model according to claim 1, which co-expresses β-amyloid peptide precursor (APP) and presenilin, preferably presenilin 1 (PS1). 3. The animal model according to claim 2, which co-expresses a mutant form of APP and / or PS1. 4. The animal model according to claim 3, wherein the mutation in the APP gene is selected from “Swedish”, “London”, and “Dutch” mutations, alone or in combination. 5. The mutation in the PS1 gene is M146L, A246E, C
4. The animal model of claim 3, selected from the 410Y, H163R, L286V, and L235P mutations alone or in combination. 6. The animal model according to claim 5, which is a M146L mutation. 7. The animal model according to claim 1, wherein the mitochondrial dysfunction is alteration, modification, overexpression, or inhibition of mitochondrial protein expression. 8. The animal model according to claim 7, wherein the protein is a mitochondrial protein. 9. The animal model according to claim 8, wherein the protein is BAX and / or cytochrome C protein. 10. Use of an animal model according to claims 1 to 9 for identifying compounds which can be used for the treatment of neurodegenerative diseases, preferably Alzheimer's disease. 11. A cell collected from the animal model according to any one of claims 1 to 9. 12. Use of the cells according to claim 11 for identifying compounds which can be used for the treatment of neurodegenerative diseases, preferably Alzheimer's disease.