ZA200602670B - Transgenic animals with serious disorders related to alzheimer's disease - Google Patents

Abstract

The invention relates to non-human transgenic animals with serious disorders related to Alzheimer's disease. The animals can be used for the discovery of compounds for the treatment of Alzheimer's disease.

Description

~:300r/ 02670

The present application relates to transgenic animals which are models of Alzheimer’s disease (AD). It also relates to the use of these animals.

Alzheimer’s disease 1s a progressive neurodegenerative disease which affects a large proportion of the elderly population. This disease is characterized in clinical terms by a loss of memory and a decline in cognitive functions and, in neuropathological terms, by a pronounced loss of neurones and the presence in the brain of intracellular neurofibrillar deposits and of extracellular deposits of P-amyloid peptide (AB) forming amyloid plaques.

Amyloid plaques are mainly made up of AB peptides containing 40 or 42 residues, which are generated during the proteolytic process for the BAB peptide precursor (APP). The extracellular deposits of AB are very specific for disorders related to Alzheimer’s disease. They represent the early and invariable characteristic of all forms of Alzheimer’s disease, including the familial forms (FAD). The familial forms appear relatively early (between 30 and 60 years old) and are due to mutations in the APP gene in 5% of FAD cases, with eight single or double missense mutations identified, in the presenilin 1 (PS1l) gene in 50 to 70% of FAD cases, with more than 100 different mutations identified to date, and in the presenilin 2 gene in more rare FAD cases, with two missense mutations described. It has been shown that mutations in these three genes induce changes in the proteolysis of APP, which lead to an overproduction of AB, especially of the long form AB42, and to the early appearance of the

REPLACEMENT SHEET (RULE 26)

* pathological conditions and of symptoms similar to those of the sporadic forms of Alzheimer’s disease.

Animal models intended to represent certain characteristics of the pathology of Alzheimer’s disease have already been described in the literature.

They are, firstly, transgenic mice carrying mutations in the APP gene. They develop pathological conditions similar to Alzheimer’s disease from one year old. Thus, the PDAPP mouse, overexpressing human APP carrying the mutation V717F, develops Af deposits in the brain with age, but shows no neuronal loss beyond the positioning of the plaques themselves (Irizarry et al., 1997,

J.Neurosc. 17(18): 7053-7059). This phenomenon will be referred to as “plaque effect”.

Similarly, the Tg (HuAPP695. K670N-M6711) 2576 mouse, expressing the human isoform APP K670N-M6711. (APPSw for

Swedish mutation), exhibits amyloid-type deposits but shows no neuronal loss (Irizarry et al., 1987, J.

Neuropathol. Exp. Neurol 56: 695-973).

In a study by Calhoun et al. (1998, Nature 395: 755- 25 .756), a neuronal loss was shown in certain brain regions in the vicinity of the amyloid plaques, in

APP23 transgenic mice 14-18 months old expressing a mutated isoform of human APP. This observation is controversial since the loss 1s small and occurs in relatively old animals and especially in the vicinity of plaques, which might correspond to the previously observed “plaque effect”. In addition, 1t 1s not mentioned, or hardly at all, in a recent commentary which underlines that current animal models do not exhibit complete similarity with all the known characteristics of the pathological conditions of

Alzheimer’s disease, inter alia the neuronal loss (Trojanowski, 2002, Am.J. Pathol.l160: 409-411).

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: Furthermore, transgenic mice carrying mutations in the

PS1 gene are known. They do not appear to develop any pathological condition of Alzheimer’s disease type, but exhibit a high amount of AB42 peptide (twofold increase compared to wild-type PS1l) which is recognized as being highly pathogenic.

In addition, in the transgenic animal models described which carry FAD mutations P264L or M146L in the mouse

PS1 gene (“knock-in”), the mutated PS1 protein is not stably expressed (Siman et al., 2000, J.Neurosci., 20: 8717-8726; Flood et al., 2002, Neurobiol. Aging 23: 335-348; Rozhmahel et al., 2002, 23: 187-194). These mice also exhibit a high amount of AB42 peptide.

Application WO 02/0008407 describes such transgenic mice in which the gene encoding presenilin has been mutated by introducing a P264L mutation.

Due to the role of the PS1 protein in the formation of the AB42 forms, double transgenic mice carrying mutations in the APP and PS1 genes have also been produced. Like the single transgenics described above, these mice exhibit AB deposits but exhibit no neuronal loss (Takeuchi et al., 2000, Am.J.Pathol. 157: 331- 339).

Thus, the existing animal models of Alzheimer’s disease are not satisfactory since they fail to reproduce a neuronal loss which is, however, a major characteristic of neurodegenerative diseases, including Alzheimer’s disease.

The applicant has therefore endeavoured to produce animals exhibiting major characteristics of Alzheimer’s disease, including neuronal loss.

~ 4 - ) It has shown that it is possible to obtain such animals by introducing specific mutations into the gene encoding the PS1 protein in mice, and by crossing them with mice overexpressing the human APP gene.

A first aspect of the invention therefore concerns a nonhuman animal exhibiting, advantageously in its genome, at least one nucleic acid sequence encoding presenilin 1 carrying at least one of the two mutations corresponding to the mutations M233T and L235P on the mouse PS1 protein.

Advantageously, such an animal carries both mutations.

Preferably, the PS1 protein carrying the mutations

M233T and L235P is of murine origin.

Particularly preferably, the mutated presenilin 1 protein is endogenous.

Thus, an animal according to the present invention advantageously produces a protein comprising the sequence SEQ ID N° 2. It preferably produces a protein having the sequence SEQ ID N° 3. It advantageously comprises in its genome the nucleic acid sequence

SEQ ID N° 1 or the sequence SEQ ID N° 8.

The sequences SEQ ID N° 1, SEQ ID N° 2 and SEQ ID N° 8 result respectively from mutations introduced into the wild-type sequences SEQ ID N° 4, SEQ ID N° 5 and SEQ ID

N° 9. The sequence SEQ ID N° 5 is that of residues 229 to 237 of the mouse wild-type presenilin 1 protein. The sequence SEQ ID N° 9 is that of the wild-type exon 7 of the mouse gene encoding the presenilin 1 protein, i.e. non mutated.

Advantageously, an animal according to the present invention coexpresses APP, preferably human APP. Such a

* gene may comprise one or more FAD mutations. Thus, the mutations in the APP gene may be one of the various mutations described to date in the literature. The mutations in the APP gene may be chosen from the “Swedish” (8S), “London” (L) and “Dutch” (D) mutations, alone or in combination.

These mutations are well described in the literature and are characterized in general by the following modifications: position mutation mutation mutation with respect to K 670 N E 693 Q Vv 717 1

APP770 and and/or

M 671 L A 692 G ~ with respect to K 651 N E 674 Q V 698 1

APP751 and and/or

BN M 652 L A 673 G with respect to K 585 N E 618 © V 642 1

APP6S5 and and/or

M 596 L A 617 G LL with respect to E 22 Q V 46 1 the A-P peptide and/or (R42) A 21 G

Also included in the London mutation are all the substitutions other than with isoleucine which are located at position 717 with respect to APP770, such as, for example, the mutations V 717 G and V 717 F.

It is understood that the APP which can be used in the context of the invention may be in various isoforms, and in particular in the forms 695, 751 and 770 or in a truncated form, such as, for example, the isoform

APP99, excluding the Swedish mutation for the latter.

CL SSSA V4 i ) Advantageously, said animal also comprises, advantageously in its genome, a nucleic acid sequence encoding all or part of the gene encoding APP751.

Advantageously, the APP751 protein is of human origin.

It preferably exhibits the mutations K670N and M671L (Swedish) and V717I (London).

In the context of the present invention, the APP gene is advantageously placed under the control of sequences which allow strong expression thereof in neurones, and in particular of transcription-promoting sequences, such as an exogenous promoter. By way of promoter sequences, mention may most particularly be made of the

HMG promoter {Gautier et al. (1989), Nucleic Acids Res 17: 20, 8389), and also the PDGF promoter (Sasahara et al. (1991), Cell 64, 217-27), the Thy-1 promoter (Luthi et al. (1997), J Neurosci 17, 4688-99) and the Prion gene promoter (Scott et al. (1992), Protein Sci 1, 986- 97).

According to a particularly advantageous embodiment of the invention, the animal model comprises the APP gene having the S, D and/or L mutations, placed under the control of the Thyl promoter.

Thus, an animal according to the present invention preferably produces a protein comprising the sequence

SEQ ID N° 7. It may exhibit the nucleic acid sequence

SEQ ID N° 6.

Preferably, it is a transgenic mouse derived from crossing between a transgenic mouse ThyAPP (TG53) carrying a nucleic acid sequence encoding the human protein APP751SL and a transgenic mouse carrying a nucleic acid sequence encoding the mouse PS1 protein carrying the mutations M233T and L235P.

- 7 = ) The animals according to the present invention reproduce, for the first time, one of the most important characteristics of neurodegenerative diseases, which is early neuronal loss.

They show, moreover, the other characteristics conventionally described for these pathological conditions. The animals exhibit accelerated depositing of amyloid plaques, clearly visible from 2 months of age, and notably so from 6 months of age.

They also exhibit a ratio of the forms AB42 to total

AP, AP4az2/aP, of greater than approximately 0.9, from 2% months old. Such a ratio 1s very high compared to that described in the literature for other transgenic mice.

The neuronal loss, which is already visible in 6-month- old mice, is clearly pronounced at 10 months.

PKR (Double strand RNA-dependent Protein Kinase) 1s a stress-activated kinase which phosphorylates eIF2Z, involved in apoptosis.

PKR is detected in the hippocampus (the structure where the neuronal loss takes place) of 10-month-old

APPxPS1KI mice according to the invention. It 1s not detected in the hippocampus of 12-month-old

APPxPS1M1461 transgenic mice in which, moreover, no neuronal loss is observed.

The novel characteristics of the animals according to the present invention make them study models which are more complete and representative of the disorders observed in patients suffering from Alzheimer’s disease, than those already described. These animals are therefore particularly suitable for demonstrating the neuroprotective properties of compounds intended

: for the treatment of neurodegenerative diseases, preferably Alzheimer’s disease.

Preferably, the animals according to the present invention have the mutant alleles of psl in the homozygous state and those of APP in the heterozygous state. However, the same characteristics of said animal can be described in an animal having one of the two mutated psl alleles in the heterozygous state and those cf APP in the heterozygous state, with, however, a phenotype which is less marked or which appears later.

Another advantage of the animals according to the present invention is that the amount of mutated PS1 protein expressed by this transgenic mouse is equivalent to the amount of endogenous PS1 protein normally expressed by a normal (nontransgenic) mouse, expressing a nonmutated PS1. This characteristic makes it an advantageous study model - without overexpression of the PS1 protein - for demonstrating compounds intended for the treatment of neurodegenerative diseases.

These compounds may in particular be compounds which have an action on the regulation of the PS1l gene at the transcriptional, post-transcriptional, translational or post-translational level, or on the PS1 protein itself by modifying or regulating one or more of its properties, or which have a similar action on the interaction partners or the targets of the PS1 protein, or as compounds which have an action on the regulation of APP and, more broadly, any molecules downstream of the signals initiated by PS1 and APP during the neurodegenerative process.

In the context of the present invention, the animals are advantageously mammals, such as rodents. in particular they are a mouse, a rat or a rabbit.

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The mice and the constructs for obtaining them are obtained by methods known to those skilled in the art.

They may be obtained according to conventional transgenesis techniques. By way of example illustrating one of the methods of transgenesis, mention may be made of the method of electroporation of a gene construct containing the modified genes into mouse embryonic stem cells and, after selection, transfer of the cells carrying the desired genetic event into a recipient blastocyst, as described in the examples. In this regard, the mutated PS1 animals according to the invention are obtained by electroporation of an expression cassette comprising a nucleic acid.

Preferably, this nucleic acid is a DNA which may be a genomic DNA (gDNA) or a complementary DNA (cDNA).

The modification of the genome may be the result of an alteration or a modification of one or more genes by “knock-in”. This modification may be due to the action of conventional altering or mutagenic agents or else perhaps carried out by site-directed mutagenesis. In the present invention, as regards the mutated psl gene, it preferably involves a homologous recombination with a targeting vector carrying the transgene mutated beforehand by site-directed mutagenesis as described in the examples which follow.

The animals expressing the mutated APP protein are cbtained by microinjection of a gene construct into the nucleus of a zygote.

The double transgenic animals are obtained by crossing mutated psl animals and mutated APP animals.

The animals according to the present invention may advantageously be used for demonstrating the

) neuroprotective properties of compounds intended for the treatment of neurodegenerative diseases, and preferably Alzheimer’s disease. These compounds may be chemical molecules, peptide or protein molecules, antibodies, chimeric molecules and also antisense RNAs or ribozymes. The compounds demonstrated may be used as medicinal products, as they are or in combination with a pharmaceutically acceptable vehicle in order to obtain a pharmaceutical composition. They may in particular be isotonic, sterile saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of such salts), or dry, in particular lyophilized, compositions which, through the addition, where appropriate, of sterilized water or of physiological saline, make it possible to constitute injectable solutes. The injections may be given stereotactically, topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally, etc.

Another subject of the invention therefore relates to a method for demonstrating compounds intended for the treatment of neurodegenerative diseases, comprising at least the following steps: - administering the test compound or a mixture of test compounds to animals according to the present invention, and - observing the evolution of one or more characteristic markers reproducing the neuropathology observed in humans.

Another subject of the invention relates to a method for demonstrating compounds intended for the treatment of neurodegenerative diseases, comprising at least the following steps:

- bringing cells extracted from the animals according to the present invention into contact with a compound or a mixture of compounds, and = measuring the effect(s) of the compounds on whole cells, in cell homogenates or on a subcellular fraction.

Another subject of the invention relates to any biological product derived from one of the two animals of the invention, and also to their uses for demonstrating compounds intended for the treatment of neurodegenerative diseases, preferably Alzheimer’s disease. The term “biological product” means in particular cells, protein extracts, DNA, RNA or else antibodies.

Thus, a subject of the present invention is cells or cell lines derived from an animal as described above, in particular embryonic stem cells.

A subject of the invention is also a mouse PS1 protein carrying the amino acid mutations M to T, and L to P, respectively at positions 233 and 235. Advantageously, such a protein comprises the sequence SEQ ID N° 2.

Preferably, it has the sequence SEQ ID N° 3.

Another subject of the present invention is a nucleic acid encoding the mouse PS1 protein carrying the amino acid mutations M to T, and L to P, respectively at positions 233 and 235.

Advantageously, such a nucleic acid according to the claim comprises the sequence SEQ ID N° 1 or the sequence SEQ ID N° 8.

: - £UCE/02670

K A subject of the present invention is also the sequences complementary to these nucleic acids and vectors comprising these nucleic acids or the sequences complementary thereto.

Another aspect of the invention concerns the use of these proteins for demonstrating the neuroprotective properties of compounds intended for the treatment of neurodegenerative diseases.

The present invention is illustrated by the following examples, without it being limited, however, to only these examples.

In these examples, the results described demonstrate the advantage of the PS1KI mice and clearly support the preferred use of the PS1KIxAPP model in therapeutic strategies since it has the advantage of representing the main characteristics of the neurodegenerative diseases known to date.

FIGURE LEGENDS

Figure 1 A: Diagrammatic representation of the structure of the murine psl gene and of the main restriction sites around the wild-type exon 7 (upper line) and the targeting vector used (middle line). The nucleotide base changes to generate the codon mutations

M233T and L235P, mutations in exon 7 ("y, are represented in the dotted frame. The mutated allele

PS1KI containing the neomycin (Neo) resistance cassette is represented on the lower line. The position of the 230 bp probe used to identify the newborns is also indicated.

Figure 1 B: Southern blot using the 230 bp probe to distinguish the wild-type WT alleles (band at 9.2 kb) and the heterozygous PS1KI (He, double band) and

~ 13 - ) homozygous PS1KI (Ho band at 7.4 kb) alleles in various mice.

Figure 1 C: Immunoblot of the C-terminal fragment of

PS1 showing that the levels of expression of the PS1 protein are not altered by the presence of the mutations of the PS1KI allele.

Figures 2 A, 2B and 2C: Quantification, respectively, of total AB, of AB42 and of the total AB/AB42 ratio, at 2.5, 4, 6 and 10 months old.

Figure 3: Acceleration of the process of deposition of the AP peptide in the APP751SLxPS1KI Ho mice. Plate illustrating the regional distribution of the extracellular deposits of the AB peptide in the brain at 6 months. The images represent the AB immunolabeling (Ab 4G8) in 3 APP751SL mice (figs. 3A, 3B and 3C) and 3

APP751SLxPS1KI Ho mice (figs. 3D, 3E and 3F). The immunolabeling demcnstrates the appearance at 6 months of the first deposits, which are still rare, in the cortex (Cx) and in the hippocampus (Hp) of the APP751SL mice. In comparison, in the APP751SLxPS1KI Ho mice of the same age, the number of deposits 1s greatly increased in these regions. It should be noted that, in these mice, deposits are already present in notable amount in the thalamus (T).

Figure 4: Progression with age of the process of deposition of the AP peptide. Plate illustrating the regional distribution of the AP deposits in the brain at 10 months. The images correspond to 2 APP751SL mice (figs. 4A and 4B) and 2 APP751SLxPSIKI Ho mice (figs. 4C and 4D). In the APP751SL mice, the immunolabeling demonstrates a considerable increase in the number and in the size of the deposits in the cortex (Cx) and the hippocampus (Hp) at 10 months, compared to © months of age, and the appearance of the first deposits in the

) thalamus (see fig. 3). The density and the size of the deposits are also greater at 10 months in the cortex, the hippocampus and the thalamus of the APP751SLxPS1KI

Ho mice. It should be noted that, in these mice, a small number of deposits can be detected in the striatum (St).

Figure 5: Process of neuronal death in CAl in the

APP751SLxPS1KI Ho mice. Plate illustrating affected pyramidal neurones in the hippocampus of 10-month-old

APP751SLxPS1KI Ho mice. The images represent Cresyl violet staining, at low magnification, in the hippocampus in 2 PS1KI Ho mice (figs. 5A and 5B), 2

APP7518L mice (figs. 5C and 5D) and 2 APP751SLxPS1KI Ho mice (figs. 5 E and 5F). The density and the thickness of the pyramidal cell layers in the hippocampus are qualitatively comparable in the 10-month-old APP751SL mice and PS1KI Ho mice. On the other hand, at the same age, they are clearly decreased in the APP751SLxPSI1KI

Ho mice, in particular in layer 1 of Ammon’s horn (CAl). It should be noted that the number of small cells stained blue (glial type cells) appears to be increased in the hippocampus of the APP751SLxPS1KI Ho mice.

Figure 6: Process of neuronal death in CAl in the

APP751SLxPS1KI Ho mice. Plate illustrating affected neurones in CAl at 10 months old via the use of other neuronal markers, methyl green and BIP immunolabeling.

The images represent the methyl green staining, at high magnification in CAl, in a nontransgenic mouse (fig. 6A), a PKS1KI Ho mouse (fig. 6B), an APP751SL mouse (fig. 6C) and an APP751SLxPS1KI Ho mouse (fig. 6D).

They represent the BIP immunolabeling at high magnification in CAl, in a PS1KI Ho mouse (fig. 6E) and an APP751SLxPS1KI Ho mouse (fig. ©6F). Compared to the nontransgenic, PS1KI Ho and APP751SL mice, the number of neuronal cells stained with methyl green is clearly

’ decreased in the CAl region of the APP751SLxPS1KI Ho mouse. The detection of a considerable number of stained glial type cells in the hippocampal parenchyma of this double transgenic mouse should be noted. The

BIP immunolabeling also confirms the considerable loss of pyramidal neurones in CAl in the 10-month-old

APP751SLxPS1KI Ho mouse.

Figure 7: Neuronal death in CAl1 and intracellular deposition of the AB peptide. Plate illustrating the two pathological processes, affected neurones and abnormal intracerebral accumulation of the Ap peptide at 10 months old. The images represent, at high magnification in CAl, the AB immunolabeling in 2 APP751 mice (figs. 7A and 7B) and 2 APP751SLxPS1KI Ho mice (figs. 7E and TF) . They represent, at high magnification in CAl, the Cresyl violet staining in the

APP751 mice (figs. 7C and 7D), the APPT751SLxPS1KI Ho mice (figs. 7G and 7H) and 2 PS1KI Ho mice (7I and 7J).

At 10 months old, both in the single APP7515L mice and in the APP751SLxPS1KI Ho doubles, the extracellular deposits of BAP are observed mainly on either side of the layer of neurones in CAl. On the other hand, in CAl (characterized by a pronounced effect on neurones all along the layer in the APP751SLxPS1KI Ho mice, figs. 7C and 7D), the AB immunolabeling with a granular appearance (corresponding to the abnormal intraneuronal accumulation of the AB peptide, see arrows) appears more intense in the APP751SLxPS1KI Ho mice. This is also true at 6 months old (see fig. 8).

Figure 8: Early onset of the process of neuronal death in CAl in the APP751SLxPS1KI Ho mice. Plate illustrating the CAl region of the hippocampus at 6 months old. The images represent, at high magnification in CAl, the AP immunolabeling in 3 APP751 mice (8A, 8B and 8C) and 3 APP751SLxPS1KI Ho mice (figs. 8G, 8H and 8I). They represent the Cresyl violet staining in the

; APP751 mice (figs. 8D, 8E and 8F) and the

APP751SLxPS1KI Ho mice (8J, 8K and 8L). At 6 months old, the CAl1 region of the hippocampus, in an

APP751SLxPS1KI Ho mouse, is characterized by an already considerable number of extracellular deposits of Ap (fig. 8I), an intense intracellular granular labeling of AB (see arrows) and a loss of neurones stained with

Cresyl violet associated with an increase in the number of glial type cells (fig. 8L). For the other two

APP751SLxPS1KI Ho mice, the layer of CAl neurones stained with Cresyl violet appears to be hardly disorganized (fig. 8J) or not at all (fig. 8K). It should be noted that, for these two mice, the intracellular AP immunolabeling appears less intense and more diffuse (figs. 8G and 8H) than in the 3rd mouse (fig. 8I).

EXAMPLES

Example 1: Construction of the targeting vector carrying the mutations M233T and L235P. The aim was to introduce two mutations into exon7 of the mouse PS1 gene, leading to alteration of residue M233 to T and residue L235 to P. The two new codons correspond to mutations identified in early onset Alzheimer patients (FAD) .

A line of PS1 knock-in (PS1KI) mice was generated using a 2-step mutagenesis strategy similar to that described in Kwok et al. (1997 Neuroreport 8; 157-42) and

Champion et al. (1996, Neuroreport 7, 1582-4).

The strategy was aimed at constructing a targeting vector carrying nucleic acid changes in codons 233 and 235 of the murine psl gene (see fig. 1A).

Succinctly, a 17 kb genomic fragment of the mouse PS1 gene was isolated by screening a 129SvJ mouse genomic

’ DNA library constructed in a lambda bacteriophage (Stratagene, catalogue # 946313). Analysis by digestion with restriction enzymes, sequencing, and comparison with the available partial sequences of the murine PS1l gene (Mitsuda et al. 1997, JBC 272, 23489-97) indicated that this fragment contained the region intron5 to exon 11 of the mouse PSI gene. A 9.8 Kb BamHI-HindIIl subfragment containing a portion of intron 5, exon 6, intron 6, exon 7 and a portion of intron 7 was subcloned into the plasmid pGEM-11Zf (+) (Promega,

France) (fig. 1A). The mutagenesis of the 2 codons was carried out using the Gene Editor kit (Promega) on the

DNA fragment containing exon 7 and was confirmed by nucleotide sequencing.

The long (5’) arm of the homologous recombination vector was obtained by cloning the 7 Kb BamHI-Xbal fragment containing exon 6. The short (3) arm was itself generated by subcloning the 1.8 Kb XbalI-EcoRI fragment containing exon 7 which has been subjected to mutagenesis. A positive selection cassette (pMCI-Neo cassette) was introduced into the Xbal site located in intron 6 at position -470 bp, positioned 5’ of exon 7 (see fig. 1A).

Example 2: Production of ES cells comprising PS1KI

The targeting vector, described in example 1, was linearized by digestion with NotI and electroporated into the embryonic stem (ES) cell line CK35 provided by

Dr Charles Babinet, Pasteur Institute, Paris, France.

The cells were cultured as previously described (W. Wurtz and A. Joyner, Gene Targeting: 2A Practical

Approach by Alexandra L. Joyner (Editor). Oxford

University Press; 2nd edition (February 15, 2000)).

- 18 =- ) 430 cellular clones liable to be carrying the homologous recombination were selected in the presence of G418. The genomic DNA of these clones was analyzed by Southern blotting as previously described (Sambrook,

Fritsch and Maniatis, Molecular Cloning; A Laboratory

Manual, Cold Spring Harbor Laboratory Press, pnd edition, 1989) using a PS1 probe located outside the long arm recombination domain (fig. 1A). Four cellular clones carrying the desired mutations in the PSI gene could thus be identified. These cellular clones were used to establish a PS1KI transgenic mouse line.

Example 3: Construction of the PS1KI mouse line

The clone 18C5 was injected into blastocysts of C57B1/6 mice.

Five of the chimeric mice obtained showed transmission of the psl mutant allele to the germinal line (and therefore to their descendance).

From these founders, the PS1KI mouse line was established on a pure 129SV genetic background and on a mixed 129SV-C57Bl/6 background.

The presence of the mutated PS1KI allele in the heterozygous (He) or homozygous (Ho) state was determined by Southern blotting with the 230 bp psl probe (fig. 1B). The mutant mice are viable and fertile.

Example 4: Assaying of PS1 in the PS1KI line

After euthanasia, the brain of the mice was removed and weighed. One hemisphere was conserved for immunohistochemistry (post-fixation) and the other was frozen and then homogenized individually on ice using a

Potter homogenizer, in 2 ml of a buffer solution: 0.32 M sucrose, 4 mM Tris-HCl, pH 7.4, containing a cocktail of protease inhibitors (Complete™, Roche

Diagnostics). The protein concentration was determined

- 19 -~ k by the BCA method (Pierce). The homogenate was conserved at -80°C.

For the detection of PSl, 25 pug of brain protein extract were incubated at 56°C for 20 min in Laemmli loading buffer containing 8M urea and 50 mM dithiothreitol. The proteins were fractionated Dy

NuPAGE 4-12% Bis-Tris polyacrylamide gel electrophoresis (SDS-PAGE) in MES (2-(N- morpholino) ethanesulfonic acid) buffer. After transfer of the proteins onto a nitrocellulose filter (Amersham,

France), the filter was heated in PBS for 5 min in order to increase the sensitivity, and immediately saturated with 5% (w/v) of powdered skimmed milk in a

PRST (0.05% PRS (V/V), Tween 20) buffer for 1 h and incubated overnight at 4°C with primary antibody in

PBST buffer alone. Binding of the antibody was detected with an anti-IgG (anti-mouse) antibody conjugated to horseradish peroxidase (Amersham, France) at a dilution of 1/10 000 in PBST, followed by a system of detection by chemiluminescence (Amersham, France) according to the manufacturer’s instructions. For the detection of

PS1, the primary antibody MAB1563 (Chemicon, USA) was used at a 1/10 000 dilution. For the semi-quantitative analysis, the luminescence signals were digitized with a GeneGnome 16 bit CCD camera (Syngene, Cambridge,

England) and analyzed with the Genetools software (Syngene). The linearity of the signal was verified by means of standard curves established with samples of 2.5 to 10 pg of homogenate per lane.

This analysis by immunoblotting made it possible to determine that the levels of expression of the C- terminal fragment of mutated PS1 remain normal and are not decreased in the PS1KI233/235 mouse (fig. 1C).

Example 5: Production of the PS1KIxAPP line by crossing the PS1KI and APP lines

PS1KI mice (described in examples 1 to 4) were crossed with a line of transgenic mice overexpressing the human form of the APP;s; CDNA carrying the Swedish (mutation

K670N; M671L) and London (V717I) FAD mutations, under the control of the Thy-1 promoter. The mice overexpressing the human form of the APP;5; CDNA carrying the mutations were obtained as described in patent application WO 01/20977.

In all the following experiments, mice having the same genetic backgrounds were used to minimize any effect due to variations in genetic background.

Example 6: Assaying of the total Af} and AB42 amyloid peptide by the immunoelectrochemiluminescence method

To quantify the overall pool of AB in the brain (soluble forms and aggregated or insoluble forms), aliquots of brain homogenate were treated with 2 volumes of a 9M solution of guanidine hydrochloride (GH) in 50 mM Tris, pH 7.4. The homogenates were mixed for 1 h, with 3 periods of sonication of 15 min, followed by centrifugation at 50 000 g at 4°C for 2 h.

The guanidine extracts were diluted to 1/20 in 20 mM

Tris-HCl buffer, pH 7.6, containing 150 mM NaCl, 0.5%

BSA (w/v) and 0.05% Tween 20 (w/v). The concentration of the AP peptide in the fractions was then determined by dimmunoelectrochemiluminescence (Yang et al., 1994,

Biotechnology (NY) 12(2), 193-194) using 2 anti-A peptide mouse monoclonal antibodies (4G8 and 6E10) and the Origen M8 Analyzer reader (IGEN Eurcpe Inc.

Oxford), following a protocol modified according to

Khorkova et al. (J. Neurosci. Methods 82, 159-166 (1998) ).

The monoclonai antibody 4G8 (Senetek PLC), which recognizes the residues 17-24 epitope of the Af

B peptide, is ruthenylated by means of the TAG-NHS ester according to the supplier’s protocol (IGEN Europe Inc.,

Oxford). Ru-4G8 and the biotinylated antibody 6E10, epitope 1-10 of the APB peptide (Senetek PLC), are brought into contact with the soluble fraction of brain and the Ru-4G8/AB/6El0-biot tripartite complexes are quantified using the Origen reader. A range of synthetic peptide AB (Bachem) is used to calibrate each experiment. The amount of peptide AB is calculated in nanograms per g of initial weight of brain tissue.

To measure specifically the forms of AB peptide which end at position 42 (AB42), the antibody ©6E10 was replaced with the monoclonal antibody 22F9, which binds specifically to the AB42 C-terminal end (Wirths et al., 2002, Brain Pathol. 12, 275-286).

In conclusion, the presence of the psl knock-in (PS1-

KI) gene leads to: - An acceleration in the accumulation of AB (fig. 2A) and AB42 (fig. 2B) in the brain, with an even more pronounced effect when the

PS1KI allele 1s present in the homozygous state (gene-dose effect). The effect of

PS1KI (Ho) 1s more accentuated than with the transgenic mouse overexpressing PS1M146L previously described in application We 01/20977. - A massive increase in the proportion of Af peptide exhibiting a P42 end, which represents the vast majority of the AB when the PSI1KI mutation 1s in the homozygous state, as shown in fig. 2C (AB42/total AP ratio equal to 0.92, at 2% months old, vs 0.25 in the absence of

PS1KI and an intermediate value 0.70 in the presence of just one PSIKI allele: gene-dose

) effect). It 1s recognized in the literature that the species of AP peptide which finish at the P42 end represent the most pathological forms of the peptide. The PS1KIxAPP line therefore represents a model which is particularly enriched in pathological forms.

Example 7: Analysis of the deposits of AJ peptide by immunohistochemistry

For the immunohistochemistry/histology experiments, after having been removed and then post-fixed in 4% paraformaldehyde, the half-brains are cryoprotected overnight at 4°C in a 0.2M sodium phosphate buffer (NaH,PO,4.2H,0/Na,HPO,4.12H,0, pH 7.4) containing 20% (P/V) sucrose. They are then frozen for 1 min in isopentane kept at a temperature of -30°C in dry ice. 25 pm thick sections, cut on a cryostat thermostated at -30°C (LEICA CM3000), are finally placed in a 0.02M PBS buffer and then conserved at 4°C.

Immunoenzymatic detection of the AP peptide was carried out, on these sections, by means of the revelation system involving the formation of avidin-biotin- peroxidase complexes (ABC) in which the horseradish peroxidase coupled to avidin is biotinylated. Briefly, after incubation for 30 min in blocking buffer (normal goat serum (Chemicon) at 10% in PBS containing 0.1% triton (Sigma)), the brain sections are placed in contact with a 0.3% H,0, solution in order to eliminate the endoperoxidases present in the tissue. These sections are then incubated in the primary antibody solution containing 0.3% triton and 2% normal serum (overnight at 4°C). The anti-AB primary antibody (4G8,

Senetek) (monoclonal antibody directed against residues 17-24 of the AP peptide) used is biotinyiated. After rinsing, the sections are therefore brought directly into contact with the ABC complex for 1 hour according

) to the manufacturer’s instructions (Vectastin ABC Kit,

Vector Laboratories, Burlingame, CA) . 3,3" -

Diaminobenzidine was used as chromogene for the peroxidase enzyme.

Thus, the acceleration of the abnormal accumulation of the AP peptide in the brain of the APP751SLxPS1KI Ho double transgenic mice, previously detected by biochemical assays on half-brain homogenates, was confirmed by immunohistochemistry. Specifically, microscopic analysis of the AB immunolabeling obtained on a half-brain section demonstrated the existence of an accelerated process of deposition of the AB peptide in the brain parenchyma of these mice. In fact, while the first deposits appear in the cortex and the hippocampus around the age of 6 months in the APP751SL mice (fig. 3), they can be detected from the age of 2 months in the APP751SLxPS1KI double transgenics in the homozygous state. Compared to the APP751SL single transgenics, the density of the AP deposits is clearly greater in the hippocampus and in the cortex in the 6- month-old double transgenics (APP751SLxPS1KI Ho). In addition, the deposits are more widely distributed; in particular, deposits are already detected in the thalamus and also the pons (fig. 3).

With age, in particular at 10 months old, the density and also the size of the deposits are increased in the brain of the APP751SL single transgenic mice (fig. 4).

The distribution of these deposits 1s also broader since they are present in the thalamus. In the 10- month-old APP751SLxPS1KI Ho double transgenics, a similar progression of the process of deposition of the

AB peptide is observed in the hippocampus, the cortex, the thalamus and the pons. The first deposits can be detected in a limited number in the striatum (fig. 4).

On the other hand, the cerebellum remains spared by the process of AP deposition. It should be noted that, in

* the brain of the 10-month-old PS1KI Ho mice (n = 4), no deposition of the AP peptide is detected.

Example 8: Analysis of neuronal loss by histology and immunohistochemistry

The presence of a very high proportion of pathological

AB42 peptide led to an analysis of whether, in the

APP751SLxPS1KI Ho line, besides the acceleration of the process of deposition of the APB peptide, a neuronal loss develops with age. For this, 3 types of staining making it possible to visualize the disappearance of neuronal cells on brain tissue sections were carried out: a) histology with Cresyl violet, which stains the

Nissl bodies (cytoplasmic organelles associated with ribosomes of the rough endoplasmic reticulum) and makes it possible to demonstrate on brain sections all neuronal and glial cells; b) histology with methyl green, which stains the DNA of all cells; c) immunohistochemistry with BIP, which reveals the expression in the cells of a resident chaperone protein of the endoplasmic reticulum.

For the Cresyl violet staining, the brain tissue sections are mounted on gelatinized slides and then incubated for 10 minutes in a solution of Cresyl violet (C 1791, Sigma) at 0.5% in distilled water. After rinsing in acidic medium, the sections are finally dehydrated. : For the methyl green staining, the sections are mounted on gelatinized slides, incubated for 10 minutes in a solution of methyl green (M5015 from Sigma) at 1% in distilled water, rinsed, and then dehydrated.

For the BIP immunohistochemistry (polyclonal antibody,

SPA-826, Stressgen), the protocol is identical to that applied for the AP peptide immunohistochemistry (see

- 25 = * above), except for the additional incubation (1 hy ambient temperature) of the sections in a solution of biotinylated secondary antibody (anti-rabbit IgG antibody made in goat, Vector) before they are incubated in the ABC complex.

Microscopic analysis demonstrated, through the use of various histological/immunohistochemical markers, a decrease in the thickness of the pyramidal cell layer of the hippocampus, in particular of CAl, in the brain of the APP751SLxPS1KI Ho mice (n = 3/3) (figs. 5 and 6). This decrease indicates the existence of a process of neuronal death which is already well established at the age of 10 months. At 6 months, neuronal death is present in the brain of 1/3 mice, suggesting the early onset of a neurotoxic process (fig. 8). Analysis in parallel in the hippocampus, and in particular in CAl, of the 2 pathological processes, namely abnormal accumulation of the AB peptide in the brain and affected neurones, suggests a more probable role in the neurotoxic process of the intracellular accumulation of

AB (phenomenon already described in the Thy- 1APP751SLxPS1 M146L mice) than of its accumulation in extracellular deposits (fig. 7). In fact, the neurones still present in CAl exhibit an abnormally high expression of the AP peptide. In addition, the effect on neurones in CAl is clearly present in regions lacking extracellular deposits. The existence of a probable gene-dose effect in the process of neuronal death in CAl should be noted. An effect on neurones was also found in very old (> 15 months) APP751SLxPS1KI mice having only one PS1KI allele.

Claims (28)

- 26 - ~ 2UC8/02670 “ CLAIMS

1. A nonhuman animal exhibiting a nucleic acid sequence encoding a presenilin 1 protein carrying at least one of the mutations corresponding to the mutations M233T and L235P on the mouse PSl protein.

2. The animal as claimed in claim 1, characterized in that the mutation(s) 1s(are) in the homozygous state.

3. The animal as claimed in either of claims 1 and 2, characterized in that it carries both mutations.

4. The animal as claimed in one of claims 1 to 3, also comprising a nucleic acid sequence encoding all or part of the gene encoding APP.

5. The animal as claimed in one of claims 1 to 4, characterized in that it expresses an amount of mutated PS1 comparable to the amount of endogenous PS1 of an animal expressing a nonmutated PS1.

6. The animal as claimed in one of claims 1 to 5, characterized in that the presenilin 1 protein carrying the mutations M233T and L235P is of murine origin.

7. The animal as claimed in one of claims 4 to 6, characterized in that the APP protein is APP751 and is of human origin.

8. The animal as claimed in one of claims 4 to 7, characterized in that the APP751 protein is of human origin and exhibits the Swedish and London mutations.

“ 9. The animal as claimed in one of claims 1 to 8, characterized in that it is a rodent.

10. The animal as claimed in one of claims 1 to 9, characterized in that it 1s a mouse, a rat or a rabbit.

11. The animal as claimed in one of claims 1 to 10, characterized in that it produces a protein comprising the sequence SEQ ID N° 2.

12. The animal as claimed in one of claims 1 to 11, characterized in that it exhibits in its genome the nucleic acid sequence SEQ ID N° 1.

13. The animal as claimed in one of claims 4 to 12Z, characterized in that the expression of the gene encoding APP is under the control of an exogenous promoter.

14. The animal as claimed in one of claims 1 to 13, characterized in that it exhibits a neuronal loss.

15. The animal as claimed in one of claims 1 to 12, characterized in that it exhibits an Abetad2/total Abeta ratio of greater than approximately 0.9.

16. The animal as claimed in one of claims 1 to 15, characterized in that the mutated presenilin 1 protein 1s endogenous.

17. A cell or cell line derived from an animal as claimed in one of claims 1 to 16.

18. A cell or cell line comprising a nucleic acid sequence encoding the murine presenilin 1 gene in an M233T- and L235P-mutated form.

19. An embryonic stem cell derived from an animal as claimed in one of claims 1 to 16.

20. A mouse PS1 protein carrying the amino acid 5) mutations M to T, and L to P, respectively at positions 233 and 235.

21. A protein as claimed in claim 20, characterized in that it comprises the sequence SEQ ID N° 2.

22. A nucleic acid encoding the mouse PS1 protein carrying the amino acid mutations M to T, and L to P, respectively at positions 233 and 235.

23. The nucleic acid as claimed in claim 22, characterized in that it comprises the sequence SEQ ID N° 1.

24. A vector comprising a nucleic acid as claimed in claim 23.

25. The use of an animal as claimed in one of claims 1 to 16, for demonstrating compounds intended to treat Alzheimer’s disease.

26. A method for demonstrating compounds intended for the treatment of neurodegenerative diseases, comprising the following steps: - administering the test compound or a mixture of test compounds to an animal as claimed in one of claims 1 to 16, and ~ observing the evolution of one or more characteristic markers reproducing the neuropathology observed in humans.

: 27. A method for demonstrating compounds intended for the treatment of neurodegenerative diseases, comprising the following steps: - bringing cells extracted from an animal as claimed in one of claims 1 to 16 into contact with a compound or a mixture of compounds, and - measuring the effect (s) of the compounds on whole cells, in cell homogenates or on a subcellular fraction.

28. The use of a protein as claimed in either of claims 20 and 21, for demonstrating compounds intended for the treatment of Alzheimer’s disease.