WO2016112961A1 - Inactivations conditionnelles de caspase 2 et procédés associés - Google Patents

Inactivations conditionnelles de caspase 2 et procédés associés Download PDF

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WO2016112961A1
WO2016112961A1 PCT/EP2015/050512 EP2015050512W WO2016112961A1 WO 2016112961 A1 WO2016112961 A1 WO 2016112961A1 EP 2015050512 W EP2015050512 W EP 2015050512W WO 2016112961 A1 WO2016112961 A1 WO 2016112961A1
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caspase
human mammal
tissue
site
gene
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Etienne Jacotot
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
Universite Paris Diderot Paris 7
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/641—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22055—Caspase-2 (3.4.22.55)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the invention relates to a non-human mammal, preferably a mouse, having a Caspase-2 gene conditional knockout system.
  • It also relates to methods for producing a Caspase-2 knockout non-human mammal, and Caspase-2 knockout non-human mammals obtained by said methods. It also relates to isolated cells, tissues and/or cellular fractions from said non-human mammals. It also relates to nucleic acid sequences and vectors suitable for producing a Caspase-2 knockout non-human mammal.
  • Caspase-2 (Ich-1, Nedd2) is the most conserved caspase in mammalians. It was found to play roles in apoptotic and non apoptotic processes, including tumor suppression and aging. Regarding apoptotic processes, Caspase-2 has been shown to be required for apoptosis induced by cytotoxic drugs and DNA-damaging agents, TRAIL, heat shock, cytoskeletal disruption, neonatal brain ischemia, optic nerve injury, ⁇ -amyloid- induced neuronal death, dendrite spine loss and cognitive dysfunction in J20 APP (Alzheimer- like) mice, serum-deprivation-induced neuronal death, and in oocyte cell death in response to nutrient deprivation.
  • cytotoxic drugs and DNA-damaging agents TRAIL
  • heat shock cytoskeletal disruption
  • neonatal brain ischemia optic nerve injury
  • ⁇ -amyloid- induced neuronal death dendrite spine loss and cognitive dysfunction
  • J20 APP Al
  • Caspase-2 contains a caspase activation and recruitment domain (CARD), which is a protein-protein interaction domain required for the dimerization of pro-caspase-2 molecules and for its activation.
  • CARD caspase activation and recruitment domain
  • RAIDD (RIP-associaied ICH-l/CED-3-homologous protein with a death domain) contains a CARD and has been shown to function as an adaptor for Caspase-2.
  • Caspase-2 zymogen contains basal enzyme activity that is enhanced by dimerization (via the CARD) and further proteolytic processing into subunits (pl 9 and pl 2). Structural studies on human Caspase-2 indicate that it forms a pl9/pl2 homodimer in solution with two active sites.
  • Caspase-2 zymogen is a dimer. As a consequence of its ability to readily dimerize in solution, Caspase-2 can autoactivate when overexpressed in cultured cells.
  • the activation complex for Caspase-2 has been proposed to be the PIDDosome, comprising Caspase 2, RAIDD and PIDD.
  • PIDDosome comprising Caspase 2, RAIDD and PIDD.
  • studies in PIDD null mice suggest that non-neuronal death does not require PIDD.
  • phosphorylation of Ser-140 in the prodomain of Caspase-2 has been shown to inhibit Caspase-2 activation.
  • Caspase-2 The cleavage of Caspase-2 is not required for its initial activation. Rather it appears that autocatalytic processing of Caspase-2 stabilizes the active enzyme. This manner of activation by dimerization supports caspase-2 being a member of the initiator caspase group. However, unlike other initiator caspases, Caspase-2 does not directly activate executioner caspases. Caspase-2 has been shown to cleave Caspase-7, but only at lower than cellular pH and therefore it may not be a physiologically relevant cleavage event. Instead, Caspase-2 activates the executioner caspases indirectly by inducing mitochondrial outer membrane permeabilization (MOMP).
  • MOMP mitochondrial outer membrane permeabilization
  • Caspase-2 appears to induce MOMP through cleavage and activation of the pro-apoptotic Bcl-2 family protein Bid. Bid, in turn induces release of cytochrome c from mitochondria and subsequent apoptosis. Thus Caspase-2 appears to be activated upstream of the mitochondrial pathway. Activated Caspase-2 cleaves additional cellular targets including, Golgin 160, Mdm2, and ICAD.
  • Caspase-2 is involved in a wide range of cellular mechanisms, and its study in vivo is of great importance for the bio-medical community. For that purpose constitutive Caspase-2 knockout mice have been reported in the litterature.
  • mice are useful for studying and identifying compositions which improve conditions where Caspase-2 plays a role, including in a non- limitative manner: neonatal brain injury, Alzheimer disease, pore-forming bacteria-related pathological conditions including pneumonia, osteomyelitis, endocarditis, blood stream infections, toxic shock syndromes and other toxin-mediated diseases, brain and heart ischemia, aging, cancer, and anti-cancer agents toxicity.
  • Caspase-2 importance in apoptosis is also considered as somewhat contentious by some authors as constitutive Caspase-2 1 (Casp2 1 ) mice are viable and fertile with only minor apoptotic defects in some cell types (Bergeron L, Perez GI, Macdonald G et al; Defects in regulation of apoptosis in caspase-2-deficient mice; Genes Dev; 1998;12: 1304-1314).
  • Caspase-2 null mice without profound phenotypes suggest either that these Caspases do not have a major role in development or that there are compensatory changes in other caspases, which obscure the function of the targeted caspase.
  • Studies with Casp2 ⁇ ' ⁇ mice also indicated that Caspase-2 may have important regulatory functions in non-apoptotic contexts, including aging, oxidative stress, genomic stability, and tumor suppression.
  • constitutive Caspase-2 knockout mice are gene deficient in all cells of the body, including cells in the vascular bed and immune system, and throughout fetal/postnatal development. This complicates the use of such mice especially as compensatory cell death pathways have been demonstrated in constitutive Caspase-2 KO mice in response to brain injury.
  • the invention relates to conditional Caspase-2 knock-out non-human mammals, most preferably mice, and to methods for inactivating Caspase-2 in specific tissues and at specific time during development and life of said transgenic non-human mammals using a conditional knockin/knockout technology, including CreLoxP, Flip-FLP recombinase, and Tet-on/off technology.
  • a conditional knockin/knockout technology including CreLoxP, Flip-FLP recombinase, and Tet-on/off technology.
  • the invention relates to a non-human mammal in which the Caspase-2 gene, or a fragment thereof, is flanked with sequences which are recognized by a recombinase, and which can thus be deleted in chosen tissues and time by recombinase treatment or expression.
  • expression of the recombinase can be obtained in a timely specific fashion by crossing the conditional knockout mice with either mice expressing the rceombinase (e.g. Cre) under the control of a gene promotor having a perinatal or juvenile, optionally tissue-specific, selective expression, or mice expressing the recombinase under the control of an inducible promoter (e.g. Tetracycline or Tamoxifen-dependent).
  • an inducible promoter e.g. Tetracycline or Tamoxifen-dependent
  • a first aspect of the invention is a non-human mammal having a Caspase-2 gene conditional knockout system.
  • the invention further contemplates isolated cells, tissues and cellular fractions that have been obtained from said non-human mammal.
  • a second aspect of the invention relates to a method for producing a Caspase- 2 knock-out non-human mammal, an isolated cell, tissue and/or cellular fractions thereof, comprising at least a step of:
  • the invention relates to a Caspase-2 gene conditional knockout non-human mammal obtained by the method described above, and to an isolated cell, tissue and/or cellular fractions thereof.
  • the invention relates to a nucleic acid sequence suitable for producing a Caspase-2 gene conditional knockout non-human mammal, as defined above.
  • the resulting animals including isolated cells, tissues and/or cellular fractions, can be then used to identify agents that affect animal or cellular functions via direct or indirect interaction with the Caspase-2 protein and/or its Casp2 gene.
  • the invention relates to a method for screening candidate compounds which modulate the activity of Caspase-2 in a mammal.
  • the invention relates to a method for screening candidate compounds suitable for treating and/or preventing and/or reducing the likelihood of occurence of disorders associated with Caspase-2, in a mammal, comprising the steps of:
  • Figure 1A Schematic representation of targeting strategy selected. Diagram is not depicted to scale. Hatched rectangles represent Casp2 coding sequences, grey rectangles indicate non-coding exon portions, solid lines represent chromosome sequences. The neomycine positive selection cassette is indicated. loxP sites are represented by triangles and FRT sites by double triangles. The initiation (ATG) and Stop (Stop) codons are indicated. The size of the flanked Casp2 sequence to be deleted is specified. The strategy results in the conditional deletion of 1000 bp of coding sequences encoding for the Casp2 active site. The splicing of exon 5 to exon 7 leads to a frame shift resulting in a premature stop codon.
  • Figure 1B-1C PCR identification of the homologous recombination event at the 3' end.
  • the figure indicates the PCR screening strategy for the 3' homologous recombination event. Green arrows illustrate the primer's localisation.
  • FIG. 2 Southern blot analysis for 5' homologous recombination in ES cells. Schematic representation of the wild-type Casp2 allele and the recombined allele with the relevant restriction sites for the Southern blot analysis is shown. The strategy for the 5' Southern blot analysis is indicated. The genomic DNA of the tested ES cell clones was compared with wild-type DNA (C57bl6J). The digested DNAs were blotted on nylon membrane and hybridised with the 5' probe detecting the Stul-fragment to screen for 5' homologous recombination event.
  • FIG. 3 Southern blot analysis for 3' homologous recombination in ES cells. Schematic representation of the wild-type Casp2 allele and the recombined allele with the relevant restriction sites for the Southern blot analysis shown. The strategy for the Southern blot detection of the 3 '-targeting event is indicated. The genomic DNA of the tested ES cell clones was compared with wild-type DNA (C57bl6J). The digested DNAs were blotted on nylon membrane and hybridised with external 3 '-probe to screen for 3' homologous recombination event.
  • Figure 4 Scheme of Cre- or Flp-excision at the recombined Casp2 locus.
  • FIG. 5 PCR genotyping of the Casp2 conditional Knock-out mouse line. Schematic representation of the Casp2 wild-type and conditional Knock-out alleles with the binding sites of the screening primers is shown. PCR screening was conducted using genomic DNA from tail biopsies of wild-type C57BL/6 mice (WT), heterozygous conditional Knock-out mice (Het Flp) and homozygous conditional Knock-out mic (HO Flp) as template. PCR amplification without DNA (H 2 0) was used as a negative control. PCR picture was obtained after loading of PCR reactions on LabChip® system from Caliper LifeSciences Figure 6: Southern blot analysis of the heterozygous casp2 conditional mice.
  • the genomic DNA of the heterozygous conditional Knock-out animals (Het Flp) was compared to wild-type genomic DNA (WT).
  • the Avrll digested DNAs were blotted on nylon membrane and hybridised with external 3' probe.
  • Figure 7 After treatment with recombinant Pen-CRE proteins, the LoxP +/+ cells show a reduction in Caspase-2 protein expression. Quantification of Casp2 fluorescence intensity in the y-axis (ratio intensity/surface casp2 vs intensity/surface tubulin). The control (Co) is compared to the CRE-treated cells (Cre).
  • FIG. 8 Cre-mediated Casp2 knock-out reduces ⁇ synaptotoxicity in primary casp2flox/flox hippocampal neurons. Histograms show quatification of synaptotoxicity after triple immunofluorescence staining using Phalloidin, anti-Basson, and anti-MAP2.
  • hippocampal neurons embryonic day 16 from wt mice (a) or Casp2flox/flox mice (b) were cultured for 3 weeks in microfluidic chambers, treated or not for 5 days with 1 ⁇ PEN-CRE, and subjected to treatment with 100 nM oligomeric ⁇ for 3 hours. Then cell were fixed and dendritic spine was analysed and quantified by fluorecence microscopy. Kruskal-Wallis **; post-hoc Dunn's Test: * only for Co.+ ⁇ .
  • Conditional knock-out methods allow gene deletion in a tissue or time specific manner. This is done by introducing short sequences, such as loxP or FLP sites around the gene or sequence of interest. These sequences will be introduced into the germ-line via the same mechanism as a knock-out.
  • This germ-line can then be crossed to another germline containing a recombinase, such as a Cre-recombinase (http://www.ncbi.nlm.nih.gov/protein/AAV84941.1) which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites (see for reference: Austin et al; 1981 Cell, 25:729-736; Hoess et al.; 1982 Proc Natl Acad Sci. USA 79:3398-3402 ; Craig ; 1988 Annu Rev Genet 22:77-105 ; Hoess & Abremski 1990; The Cre-lox recombination system.
  • a recombinase such as a Cre-recombinase (http://www.ncbi.nlm.nih.gov/protein/AAV84941.1) which is a viral enzyme that can recognize these sequences, recombines them and deletes
  • the C domain (catalytic site) is similar in structure to the domain in the Integrase family of enzymes isolated from lambda phage.
  • Tet-on/off methods also known as Tetracycline-controlled transcriptional activation methods
  • Tetracycline-controlled transcriptional activation methods are also known in the Art and have been developed for controlled gene expression systems (see for reference: Schoonig et al ; Transgenic Res., 2012 Tet- Transgenic Rodents: a comprehensive, up-to date database DOI 10.1007/sl 1248-012- 9660-9).
  • the inventors were able to functionally inactivate the Caspase-2 gene in a non- human mammal, in a conditional manner and in specific tissues, including neurons and/or brain.
  • conditional Caspase-2 knock-outs either in predetermined tissues or systematically, will allow to address more directly the exact role of Caspase-2 in apoptotic and non-apoptotic conditions.
  • a first aspect of the invention is a non-human mammal, most preferably a mouse, having a Caspase-2 gene conditional knockout system.
  • said Caspase-2 gene conditional knockout system can be tissue specific, which encompasses a non-human mammal having a Caspase-2 gene knock-out in which the Caspase-2 gene, or a fragment thereof, and the corresponding Caspase-2 protein, is absent or modified in at least one tissue, but not on others.
  • the flanked sequence within the Caspase-2 gene is an exon or a group of exons.
  • said exon or group of exons contains a sequence coding for the catalytic site of Caspase-2, and the flanking sequence is a recognition site for at least one recombinase, such as Cre-Lox recombination and FLP-FRT recombination.
  • nucleic acid sequence of the Mus Musculus Caspase-2 locus (NM_007610) is represented by sequence SEQ ID N°21.
  • sequence SEQ ID N°20 The corresponding translated Caspase-2 protein, herein after described as the Caspase-2 long isoform, is represented by sequence SEQ ID N°20.
  • the Casp2 gene includes also nine exons, all of which are also provided for reference, including Exon la, Id, le, 2, 3b, 4, 5, 6a, and 6b, 7, 8 and 9, which are respectively of sequences SEQ ID N°2 to 13.
  • the invention relates to transgenic non-human mammals, in particular mice, in which the Caspase-2 gene can be conditionally inactivated, either in predetermined tissues or systematically.
  • a "non-human mammal” is any non-human mammal generally used as animal models in pharmaceutical and scientific research and development, including monkey, porcine, canine, or rodent animal models, more preferably a rodent, and most preferably a mouse.
  • a non-human mammal of the invention is a C57BL/6 mouse.
  • a transgenic mouse in which a critical portion of the Casp2 gene is flanked by loxP sites has been produced, opening the possibility to produce non-human mammals in which the Casp2 gene is inactivated in specific tissues and/or at different times during development by homologous recombination, including Cre-mediated deletion.
  • Cre-mediated deletion in said transgenic mice can be achieved through virus-mediated Cre expression, through breeding mice engineered to express inducible Cre with Casp2 flox/flox mice, or through treatment of Casp2 flox/flox cells with Cre, including recombinant cell-permeable Cre.
  • the new particular mouse strain that has been obtained EJA-1 (C57B16/Casp2 flox/flox ) can be combined with any Cre strain available and allows the development of both inducible and cell specific (neurons and non-neurons) strains.
  • This mouse line will be of interest for the whole bio-medical community.
  • the invention relates to a non-human mammal, preferably of murine origin, having a Caspase-2 gene conditional knockout system.
  • the invention relates to a non-human mammal in which the Caspase-2 gene can be conditionally inactivated using homologous recombination, wherein said Caspase-2 gene or a fragment thereof is flanked by recognition-sites for at least one recombinase, such as a Cre recombinase.
  • conditional Caspase-2 gene knockout is suitable for the obtention of a non- human mammal having a Caspase-2 knock-out inducible phenotype.
  • a "Caspase-2 knock out inducible phenotype” includes reduced sensitivity to cell death or cell compartment dysfunction or degeneration.
  • a Casp2 ⁇ ⁇ phenotype induced during the neonatal/post-natal time is also expected to be neuroprotective against perinatal ischemia, hypoxia-ischemia, excitotoxicity, hyperoxia, perinatal arterial stroke.
  • a Casp2 _/ ⁇ phenotype induced during adult ou ageing time is expected to modify oxydative stress response, ageing, autophagy, and to protect heart and brain against the consequences of cardiac ischemia, and heart failure, and to protect kidney against ischemia.
  • the Caspase-2 conditional knockout non-human mammal is suitable for expressing at least one non- functional Caspase-2 protein, or alternatively for not expressing, or in limited amounts, a functional Caspase-2 protein, in comparison to the wild-type non-human mammal.
  • non-functional Caspase-2 protein includes in particular a Caspase-2 having reduced cleavage activity, or even no cleavage activity at all.
  • ⁇ nonfunctional Caspase-2 protein may also have reduced specificity for the amino-acids surrounding the Aspartic acid residue.
  • a non- functional Caspase-2 protein may be obtained by modifying the reading frame, which will lead to the emergence of a novel, non-naturally occuring STOP codon.
  • the "catalytic site” of Caspase-2 includes the site that is responsible for the proteolytic cleavage after an Aspartic acid residue (termed the PI position), and which is encoded at least by the Caspase-2 exon 6, and more particularly Caspase-2 exon 6b of sequence SEQ ID N°10.
  • the invention also relates to a Caspase-2 conditional knockout non-human mammal as defined above, bearing at least one mutation in the Caspase-2 gene, in particular in or around exon 6, including in or around exon 6a and exon 6b, and preferably in or around exon 6b of sequence SEQ ID N°10 which encompasses the catalytic site.
  • a conditional Caspase-2 knockout may be tissue-specific.
  • the invention also relates to a non-human mammal as defined above, having a tissue-specific Caspase-2 gene conditional knockout system
  • tissue-specific conditional Caspase-2 knockout non- human mammal refers to a “conditional Caspase-2 knockout” non-human mammal for which the Caspase-2 gene, can be inactivated only in specific tissues (including organs), and not on others.
  • tissue-specific Caspase-2 knockout non- human mammal refers to a “conditional Caspase-2 knockout” non-human mammal for which the Caspase-2 gene has been inactivated in specific tissues (including organs), and not on others.
  • the Caspase-2 gene may be inactivated in a tissue selected from: brain, central nervous system (CNS), neurons, microglia, oligodendroglial cell lineage.
  • a tissue selected from: brain, central nervous system (CNS), neurons, microglia, oligodendroglial cell lineage.
  • a Caspase-2 conditional knockout non- human mammal has at least one Caspase-2 gene comprising the following sequence:
  • [CASP2] includes at least one Caspase-2 exon, or a fragment thereof ; and wherein xl and x2 each independently represents an integer equal or superior to 1 ;
  • [Site 1] and [Site 2] are nucleic acid sequences, identical or different, which include at least one recognition-site for at least one recombinase.
  • At least one Caspase-2 gene includes one copy of said Caspase-2 gene, or two copies of said Caspase-2 gene.
  • Caspase-2 conditional knockout non-human mammals of the invention may be either homozygous or heterozygous for said Caspase-2 gene. In the context of in vivo studies, it is preferable that said non-human mammals should be homozygous.
  • Caspase-2 conditional knockout non-human mammals of the invention include preferably non-human mammals wherein both alleles of the Caspase-2 gene include a conditional knock-out DNA construct.
  • the [CASP2] sequence may include at least one Caspase-2 exon, or a fragment thereof, which includes part of the catalytic site of Caspase-2.
  • a Caspase-2 conditional knockout non- human mammal has at least one Caspase-2 gene as defined above, wherein [CASP2] includes at least Caspase-2 exon 6b of sequence SEQ ID N°10, or a fragment thereof.
  • [Site 1] and [Site 2] are recognized by the same at least one recombinase, and suitable for homologous recombination by said recombinase.
  • a Caspase-2 conditional knockout non- human mammal has at least one Caspase-2 gene(s) comprising the following sequence:
  • [CASP2] includes at least one Caspase-2 exon 6b, or a fragment thereof; and wherein x1 and x2 each independently represents an integer equal or superior to 1;
  • [Site 1] and [Site 2] include at least one recognition-site for at least one recombinase.
  • [Site 1] and [Site 2] are selected from the list consisting of: recognition sites suitable for Cre-Lox recombination and/or FLP-FRT recombination, and consist preferably of a LoxP site, such as for instance the canonical LoxP site of sequence SEQ ID N°24 and/or a consensus LoxP site of sequence SEQ ID N°25.
  • LoxP sites are also considered by the invention including LoxP sites of sequence SEQ ID N°26 to 34.
  • Lax P locus of X-over PI
  • the site includes a central asymmetric 8 bp sequence, variable except for the middle two bases, flanked by two sets of, preferably palindromic, 13 bp sequences.
  • LoxP sites which are suitable for the invention are detailed following table with their corresponding SEQ ID:
  • N represents any base including A, TAJ, C or G, and in particular A, T, C or
  • LoxP sites which are suitable for the invention are detailled in Langer (2002 Nucleic Acids res; vol 30 N°14 p3067-3077; Transgenic Res); Schoonig et al. (2012; Tet-Transgenic Rodents: a comprehensive, up-to date database; DOI 10.1007/sl 1248-012-9660-9) and Missirlis et al. ((2006). "A high- throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination". BMC Genomics 7: 73).
  • LoxP sites suitable for the invention can be determined by the man skilled in the Art.
  • [Site 1] and [Site 2] can be inserted into a non- coding region.
  • [Site 1] and [Site 2] may advantageously be part of a Caspase-2 intron.
  • [Site 1] and [Site 2] can be inserted into a Caspase-2 intron such as intron 4 and/or intron 6
  • a Caspase-2 conditional knockout non- human mammal has at least one Caspase-2 gene comprising the following sequence:
  • [CASP2] includes at least one Caspase-2 exon 6b of sequence SEQ ID N°10, or a fragment thereof; and characterized in that [Site 1] and [Site 2] are nucleic acid sequences which include at least one recognition-site for at least one recombinase suitable for Cre- Lox recombination and/or FLP-FRT recombination, in particular Cre-Lox recombination, and preferably a LoxP site, such as the LoxP sites of sequence SEQ ID N°24 to 34.
  • the invention relates to a Caspase-2 conditional knockout non-human mammal having at least one Caspase-2 gene comprising sequence SEQ ID N°23.
  • the Caspase-2 gene can be conditionally inactived, either in a systemic or in a tissue-specific way, by bringing into contact the Caspase-2 conditional knock-out gene with the corresponding recombinase.
  • such recombination event can be triggered in order to produce a Caspase-2 knockout non-human mammal.
  • the invention also provides a method for producing a Caspase-2 knockout non-human mammal, comprising at least a step of:
  • Step b) may further include at least one of the following steps:
  • a method for producing a Caspase-2 knockout non-human mammal may comprise at least the steps of :
  • the recombinase includes at least one cell-penetrating domain and/or at least one Nuclear Localization Signal (or NLS).
  • a recombinase suitable for the invention can be the Cre recombinase of sequence SEQ ID °1.
  • the recombinase is combined to a cell-penetrating peptide, including a cell-penetrating peptide selected from HIV-Tat protein, or Penetratin.
  • the cell-penetrating peptide can be inserted at the N-terminal or C-terminal end of the recombinase, optionally including a linker domain.
  • the recombinase comprises at least one Nuclear Localization Signal site (or NLS).
  • a Cre recombinase can be selected from Penetratin-CRE (Ozyme RP-15) and Tat-Cre (Ozyme RP-7).
  • the concentration of the recombinase, in particular Cre recombinase can vary based on the biological material to be treated which includes concentrations from 0.1 ⁇ to 100 ⁇ , in particular from 0.1 ⁇ to 10 ⁇ , or even 0.5 ⁇ to 5 ⁇ , which includes 0.5 ⁇ , ⁇ , 1.5 ⁇ , 3 ⁇ , or 5 ⁇ .
  • a medium suitable for said recombinase is as described in Example 2 and in the Material & Methods section, which includes DMEM supplemented with foetal calf serum, including DMEM/FCS5%/B27/N2 (Invitrogen) or Neurobasal ® medium supplemented with B27/Glutamax/PS (Invitrogen).
  • step b) consists of bringing into contact said non-human mammal, isolated cell, tissue and/or cellular fraction thereof, with a recombinase; or alternatively a micro-organism and/or a virus capable of inducing the production of said recombinase, wherein said microorganism and/or virus is selected from the group consisting of: adenovirus (AAV), CMV, or lentivirus.
  • AAV adenovirus
  • CMV CMV
  • lentivirus lentivirus
  • step b) consists of breeding said non- human mammal with a second non- human mammal capable of expressing said recombinase, wherein the expression of said recombinase is under the control of at least one tissue-specific promoter.
  • tissue-specific promoter in the sense of the invention, encompasses any promoter which is more or less activated in specific tissues.
  • tissue-specific promoters include the NG-2 promoter (for oligodendrocyte precursor cells or OPCs) and the Camk2a promoter (for neurons).
  • tissue-specific promoters are known in the Art, as shown for instance in:
  • Novel NG2- CreERT2 knock-in mice demonstrate heterogeneous differentiation potential of NG2 glia during development;
  • step b) may consist of bringing into contact said recombinase with said specific tissue only, and not in a systematic way.
  • the expression of the corresponding recombinase can thus be modulated in a tissue-specific manner, in order to obtain a tissue-specific conditional Caspase-2 knock-out.
  • the tissue-specific promoter is a neuron-specific promoter.
  • EJA-1 mice comprising a Caspase-2 gene or fragment thereof flanked by Cre-Lox sites can be crossed with mice expressing Cre linked to NG-2 promoter (to get casp2 deletion in OPCs) or with mice expressing Cre linked to Camk2a promoter (to get Casp2 deletion in neurons).
  • the invention further relates to a Caspase-2 knockout non-human mammal, and/or a tissue-specific Caspase-2 knockout non-human mammal obtained by the method defined above.
  • such Caspase-2 knockout non-human mammals can be characterized by the presence of at least one copy of the knock-out Caspase-2 gene bearing at least one recombination site for said recombinase.
  • the invention further encompasses an isolated cell, tissue and/or cellular fraction from a non-human mammal having a Caspase-2 gene conditional knockout system, or alternatively a Caspase-2 knockout non-human mammal as obtained by the method defined above.
  • Isolated cell, tissue and/or cellular fraction may be obtained according to any protocol known in the Art.
  • the invention explicitly relates to an isolated cell, tissue and/or cellular fraction including at least one Caspase-2 gene comprising the following sequence:
  • [CASP2] includes at least one Caspase-2 exon, or a fragment thereof; and wherein xl and x2 each independently represents an integer equal or superior to 1;
  • [Site 1] and [Site 2] are nucleic acid sequences, identical or different, which include at least one recognition-site for at least one recombinase.
  • an isolated cell, tissue, and/or cellular fraction includes a sample selected from: microglial and macroglial cells including isolated neurons, oligodendrocytes, astrocytes and macrophages, lymphocytes, muscle-derived cells, cardiomyocytes, fibroblasts, epithelial cells, osteoblasts, cerebrospinal fluid, fractionated or unfractionated blood sample including serum, or isolate mitochondria from any of these cells or cytosols, and other cell lines.
  • a tissue refers to any sample that can be isolated from a non-human mammal, and which still maintains at least part of its cellular organization.
  • a tissue may include an organ.
  • a "cellular fraction” refers to any sample or cell organelle (i.e. mitochondria, endoplasmic reticulum, lysosomes, nucleus) or cell compartment (i.e. axones, dendrites, membrane fractions, cytosol), including a plurality of cells that can be isolated from a non- human mammal or cell culture thereof, and that has been obtained through cell fractionation, including for instance differential centrifugation and FACS.
  • nucleic acid sequences and vectors useful for obtaining conditional Caspase-2 knock-out mutants as defined above are further provided.
  • the invention relates to a nucleic acid sequence comprising or consisting of sequence SEQ ID N°23 or SEQ ID N°24, and/or a nucleic acid sequence comprising or consisting of:
  • [CASP2] includes at least one Caspase-2 gene, or fragment thereof ; and wherein xl and x2 represent an integer equal or superior to 1 ;
  • [Site 1] and [Site 2] are nucleic acid sequences, identical or different, which include at least one recognition-site for at least one recombinase.
  • the invention relates to a nucleic acid sequence comprising or consisting of:
  • [CASP2] includes at least one Caspase-2 exon, or fragment thereof ; and wherein xl and x2 represent an integer equal or superior to 1 ;
  • [Site 1] and [Site 2] are nucleic acid sequences, identical or different, which include at least one recognition-site for at least one recombinase.
  • a Caspase-2 exon or fragment thereof includes exon la, la, le, 2, 3b, 4, 5, 6a, and/or 6b, which are respectively of sequences SEQ ID N°2 to 13.
  • a caspase-2 exon may include caspase-2 exon 6, or a fragment thereof, which includes exon 6a and exon 6b, and preferably exon 6b of sequence SEQ ID N°10.
  • the nucleic acid sequence comprises or consists of: wherein [CASP2] includes at least one Caspase-2 exon 6b of sequence SEQ ID N°10, or a fragment thereof; and characterized in that [Site 1] and [Site 2] are nucleic acid sequences which include at least one recognition-site for at least one recombinase suitable for Cre- Lox recombination and/or FLP-FRT recombination, preferably a LoxP site.
  • [Site 1] and [Site 2] are selected from the list consisting of: recognition sites suitable for Cre-Lox recombination and/or FLP-FRT recombination, in particular Cre-Lox recombination, and consist preferably of a LoxP site.
  • [Site 1] and [Site 2] can comprise or consist of SEQ ID N°24.
  • [Site 1] and [Site 2] can be inserted into a non-coding region.
  • [Site 1] and [Site 2] may advantageously be part of a Caspase-2 intron, such as intron 4 or intron 6.
  • nucleic acid sequences may be in the DNA or in the RNA form, and preferably DNA. Such nucleic acid sequences may be in the form of, or incorporated into, vectors.
  • a “vector” refers to a nucleic acid sequence, generally a DNA molecule, which is suitable for introducing a specific gene or fragment thereof into a target cell.
  • vectors may be in the form of a plasmid or a nucleic acid probe, either as a DNA or as a RNA construct.
  • vectors of the invention are in the form of a plasmid and in the DNA form.
  • Such vectors may further include one or more additional elements selected from: cloning sites, selectable markers and/or reporter genes.
  • the present invention encompasses methods for screening, candidate compounds which modulate the activity of Caspase-2.
  • Caspase-2 Because the activity and expression of Caspase-2 have been associated with a wide range of physiological and/or pathological conditions, those methods are also useful for identifying compounds for treating and/or preventing and/or reducing the likelihood of occurence of disorders associated with Caspase-2, in particular selected from: apoptosis induced by cytotoxic drugs and DNA-damaging agents, TRAIL, heat shock, cytoskeletal disruption, neonatal brain ischemia and/or injury, optic nerve injury, ⁇ -amyloid-related disorders including ⁇ -amyloid-induced neuronal death, prion-induced neuronal death, synuclein alpha induced cell dysfunction or degeneration or death, dendrite spine loss and cognitive dysfunction in J20 APP (Alzheimer- like) mice, serum-deprivation-induced neuronal death, and in oocyte cell death in response to nutrient deprivation, Alzheimer's disease, pore-forming bacteria-related pathological conditions including pneumonia, osteomyelitis, endocarditis
  • the invention also relates to methods for screening, candidate compounds which are suitable for treating and/or preventing and/or reducing the likelihood of occurence of disorders associated with Caspase-2.
  • the invention relates to a method for screening candidate compounds suitable for treating and/or preventing and/or reducing the likelihood of occurence of disorders associated with Caspase-2, in a mammal, comprising a step of:
  • a biological value that is indicative of the occurence of a disorder associated with Caspase-2 will depend on the disorder that is specifically considered.
  • a biological value may encompass any biochemical or clinical data that can be determined on the non-human mammal, isolated cell, tissue, and/or cellular fraction thereof.
  • a biological value may include the level of expression or activity of Caspase-2, or of a Caspase-2 dependent protein; or alternatively the level of expression of a Caspase-2 dependent gene.
  • said candidate compound is able to treat, prevent and/or reduce the likelihood of occurence of disorders associated with Caspase-2, if the biological value measured at step c) is modulated, and preferably decreased, in comparison to the biological value assessed in the reference value
  • Step c) may comprise or consist of determining the level of expression of Caspase-2, or a nucleic acid encoding it; and/or determining the occurence of an interaction between said compound and at least one selected from : Caspase-2 ; a nucleic acid encoding Caspase-2 ; a Caspase-2 gene.
  • Step c) may also consist of, or include a step of, measuring the activity of
  • said candidate compound is able to modulate the activity or expression of Caspase-2 if the activity or expression measured at step c) is modulated in comparison to the activity or expression assessed in the reference value.
  • McStay & Green Measuring apoptosis: caspase inhibitors and activity assays
  • Cold Spring Harb Protoc; 2014(8):799-806 Affinity-based probes can also be used for isolating caspases, such as the ones disclosed in McStay & Green ("Identification of active caspases using affinity-based probes"; Cold Spring Harb Protoc; 2014(8):856-860).
  • a disorder associated with Caspase-2 may include ⁇ -amyloid-related disorders, in particular selected from ⁇ -amyloid-induced neuronal death and ⁇ -amyloid mediated synaptotoxicity.
  • a biological value may include the level of dendrite spine loss on said mammal, cell, tissue and/or cellular fraction thereof.
  • a “reference value” corresponds to the reference biological value measured on a reference sample (i.e. isolated cell, tissue and/or cellular fraction) and/or a reference mammal. Such reference may be established prior to the administration of said candidate compound, or alternatively on a mammal having a distinct Caspase-2 phenotype (i.e. a non- human mammal having a Caspase-2 / + phenotype).
  • a “modulation of the biological value ", including a “modulation of the activity of Caspase-2” may include both an increase or a decrease of said biological value and/or activity, in comparison to the reference value.
  • the reference value may be determined on the same non-human mammal, but with a distinct Caspase-2 phenotype.
  • the invention relates to a method for screening candidate compounds suitable for treating and/or preventing and/or reducing the likelihood of occurence of disorders associated with Caspase-2, in a mammal, comprising at least the steps of:
  • said non-human mammal, isolated cell, tissue and/or cellular fraction thereof prior to homologous recombination may be used for establishing said reference value.
  • the candidate compound may then be provided to said non-human mammal, isolated cell, tissue and/or cellular fraction thereof after homologous recombination.
  • said recombinase may also be administered in a tissue-specific manner.
  • EJA-1 mice can be crossed with mice expressing tamoxifen- inducible Cre-recombinase for producing a tamoxifen-inducible casp2-/- strain.
  • EXAMPLE 1 Deletion of the caspase-2 gene using Cre/loxP recombination.
  • Genomic region of interest containing the murine Casp2 locus was isolated by
  • PCR from C57BL/6 ES cell genomic DNA were subcloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, California). The resulting sequenced clones (containing intron 4 to intron 6) were used to construct the targeting vector. Briefly, a 1-kb region comprising exon 6 and Casp2S-specific exon was flanked by a Neo cassette (FRT site-PGK promoter-Neo cDNA-FRT site-ZoxP site) and a distal loxP site in order to have access to the constitutive and conditional knock-out lines by deleting Casp2 exon 6 and Casp2S specific exon.
  • Neo cassette FRT site-PGK promoter-Neo cDNA-FRT site-ZoxP site
  • Linearized targeting vector was transfected into C57BL/6 ES cells (genOway, Lyon, France) according to genOway's electroporation procedures (ie 108 ES cells in presence of 100 ⁇ g of linearized plasmid, 260Volt, 500 ⁇ ). Positive selection was started 48 hours after electroporation, by addition of 200 ⁇ g/ml of G418 (150 ⁇ g/ml of active component, Life Technologies, Inc.). 1042 resistant clones were isolated and amplified in 96-well plates. Duplicates of 96-well plates were made. The set of plates containing ES cell clones amplified on gelatin were genotyped by both PCR and Southern blot analysis.
  • primer pair For PCR analysis, one primer pair was designed to amplify sequences spanning the 3' homology region. This primer pair was designed to specifically amplify the targeted locus:
  • Targeted locus was confirmed by Southern blot analysis using internal and external probes on both 3' and 5' ends. 21 clones were identified as correctly targeted at the Casp2 locus.
  • the ES cells used in the injection experiment were originally derived from a C57BL/6 mouse strain which have black color. These cells were injected into blastocysts derived from an albino C57BL/6 strain (C57BL/6J-Tyrc-2J/J), which have a white coat color. The resulting offspring was thus chimeras of two different cell types (ES cell- derived cells and host blastocyst-derived cells) and the degree of chimerism was easily monitored by the percentage of light and dark patches on these animals.
  • Clones were microinjected into albino C57BL/6J-Tyrc-2J/J blastocysts, and gave rise to male chimeras with a significant ES cell contribution (as determined by a black coat color). Mice were bred to C57BL/6 mice expressing Flp recombinase to remove the Neo cassette (Casp2 lox mice).
  • Wild-type allele gives rise to 607-bp product and floxed allele gives rise to a 724-bp product.
  • Heterozygous conditional Knock-out mice identified by PCR were further verified by Southern blot analysis of Avrll-digested genomic DNA using a 3' external probe (size: 407 bp).
  • the external 3' probe is generated by PCR on genomic DNA using the following primer pairs:
  • the mouse Casp2 gene is located on chromosome 6B2.1 and extends over 17.5 kb.
  • Thegene features are as follows.
  • the mouse Casp2 gene includes 9 exons.
  • the ATG translation initiation and the STOP codons are located in exons la and 9, respectively.
  • a 139 bp 5'-UTR and a 2030 bp 3 '-UTR have been located.
  • the gene encodes for a 452 amino acid open reading frame.
  • the gene was cloned and the conditional targeting vector constructed and inserted into C57B1/6 embryonic stem (ES) cells through homologous recombination.
  • the targeted ES cell clones was injected into blastocysts to create Floxed chimeras.
  • Homozygous Floxed Caspase-2 mice were then produced.
  • the deletion includes the exon 6b (catalytic sequence) as well as a region in intron 6 containing the putative exon specific for the short isoform, disrupting the production of both biologically active forms of Caspase-2.
  • FIG. 1A A cartography of the Casp2 endogenous locus, compared to a Casp2 recombinant locus (after homologous recombination with the CRE recombinase) is disclosed in figure 1A. Illustration of the cloning strategy for the construction of the targeting vector is disclosed in figure IB. 5' and 3' homologous recombination in C57BL/6 ES cells and corresponding controls is further illustrated in figures 2 and 3.
  • the recombined Casp2 locus is also illustrated after breeding of cloned mice to C57BL/6 mice expressing a Flp recombinase to remove the control Neo cassette, in order to obtain Casp2 lox mice (see figure 4). Correct excision of the Neo cassete with corresponding controls is determined using PCR genotyping, as shown in figure 5.
  • Heterozygous casp2 conditional mice can be further controlled by southern blot analysis, and compared to wild type mice.
  • Figure 6 provides evidence for heterozygous Casp2 conditional mice, and thus the presence of two alleles (4.8 and 7.6 kb).
  • Cre-mediated deletion of Caspase-2 provides neuroprotection in primary neurons isolated from EJA-l flox/flox mice.
  • Primary neurons were generated from embryos of EJA-l flox/flox mice and treated with recombinant Tat-CRE of sequence SEQ ID N°l.
  • Penetratin-CRE Ozyme RP-15
  • Tat-Cre Ozyme RP-7
  • the cells are plated in a complete medium, and then treated with a dilute recombinase in a complete medium without serum; the treatment is of about 3 to 4 hours before changing the DMEM/FCS5%/B27/N2 medium (Invitrogen).
  • the cells are cultivated in a Neurobasal/B27/Glutamax/PS medium (Invitrogen) and then treated with a diluted Cre in the same medium. Afterwards the NB/B27/Glutamax/PS medium is changed following treatment at 12h, 24h, 48h and 4 days following treatment. Cells are then fixed 7 days later.
  • primary neurons are infected with an adenovirus or lentivirus expressing CRE-GFP to induce expression of Cre-recombinase, using either one of the protocols disclosed in Ahmed et al. ("Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors"; BMC Neuroscience; (2004); 5 :4).
  • Adenoviruses include Ad-Cre-GFP pre-packed Ready-to-use human Adenoviruses of the type 5, or AAV5, (dEl/E3) sold by Vector Biolabs (Cat. No. 1700).
  • the messenger RNA and protein are extracted and analysed by qRT-PCR and western blot. Following infection of primary neurons with Adenovirus expressing CRE- GFP or treatment with recombinant Pen-CRE proteins, the LoxP +/+ cells show a reduction in Caspase-2 mRNA and protein expression.
  • Hippocampus were micro-dissected from E16 embryos of C57B16/J wt mice (Rene Janvier, France) or C57B16/J-Casp2 flox/flox mice in cold Gey's Balanced Salt Solution (GBSS, Sigma) supplemented with 0.1% glucose (Life technologies).
  • Dissected structures were digested with papain (20U/ML in DMEM, Sigma; St. Louis, MO, USA) and mechanically dissociated in the presence of DNAse. Hippocampal cells were then rinsed and re-suspended in DMEM (Life Technologies, Inc., Gaithersburg, MD, USA) to a final density of 18 million cells/ml in Neurobasal (Life technologies) and Glutamax (0.1% LifeTechnologies) supplemented with B27 (1/50) and penicilline/sptreptomycine 1% (Gibco).
  • This cell suspension was then used to fill the reservoirs of microfluidic chambers, as described (Peyrin et al, 2011 Lab Chips 11(21):3663; Deleglise et al, 2014 Neuropathologica Acta Comm. 2: 145).
  • Microfluidic chips were placed in plastic Petri dishes containing H20-EDTA to prevent evaporation and incubated at 37°C in a humid 5% C02 atmosphere. The culture medium was renewed every seven days.
  • Oligomeric and fibrillar forms of ⁇ 1-42 were produced according to (Stine WB, Dahlgren KN, Krafft GA, LaDu MJ (2003) In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J Biol Chem 278(13): 11612-11622) and controlled by electron microscopy (Deleglise B, Magnifico S, Duplus E, Vaur P, Soubeyre V, Belle M, Vignes M, Viovy JL, Jacotot E, Peyrin JM, Brugg B. ⁇ -amyloid induces a dying-back process and remote trans- synaptic alterations in a micro fluidic-based reconstructed neuronal network. Acta Neuropathol Commun. 2014 Sep 25;2(1): 145).
  • lyophilized peptides were solubilized at 1 mM in 1, 1, 1, 3, 3, 3,- hexafiuoro-2-propanol (HFIP, Sigma Aldrich). After 30 min of incubation at RT, HFIP was evaporated for 12h under chemical hood and peptides were dried for lh (Speed Vac 4°C).Then, ⁇ peptide stock solution were obtained by resolubilization at 5 mM in dimethylsulfoxide (DMSO, Sigma Aldrich). To obtain oligomers, ⁇ stock solution was diluted in cold phenol- free DMEM-F12 medium (Life technologies) to a final concentration of 100 ⁇ ⁇ 1-42. The solution was incubated 24h at 4°C and centrifuged at 20 000 g (10 min; 4°C) before supernatant (soluble ⁇ fraction) collection and storage at -80°C.
  • HFIP 1, 1, 1, 3, 3, 3, 3,- hexafiuoro-2-propano
  • hippocampal cell were treated for 3h with 100 nm of ⁇ 1-42 oligomers.
  • Cells were then fixed in 4% paraformaldehyde (PFA, Sigma; St. Louis, MO, USA), permeabilized with Triton X-100 (0.2%) in PBS containing BSA (0.1%), and labelled with the following antibodies: rabbit polyclonal anti-MAP-2 (AB5622; 1 :400, MILLIPORE), mouse monoclonal Anti-Bassoon SAP7F407; 1 :400, Enzo LifeSciences).
  • Species-specific secondary antibodies coupled to Alexa 350, 488 and 500 were used (1/500, Life Technologies, Inc., Gaithersburg, MD, USA).
  • Phalloidin conjugated to Alexa Fluor 555 (1 :500, EnzoLifeTechnologies) was added with secondary antibodies to stain F-actin. Images were acquired with an Axio- observer Zl (Zeiss, Germany) fitted with a cooled CCD camera (CoolsnapHQ2, Ropert Scientific). The microscope was controlled with Metamorph and Micro-manager softwares. Images were processed with Image J software before being used for quantification. S.Immunofluorescence
  • Cultures were fixed were fixed in 4% paraformaldehyde (PFA, Sigma; St. Louis, MO, USA) for 20 minutes at room temperature. Cultures cells were then washed twice with PBS+Azide 0.1% for 5 minutes and permeabilized for 10 minutes with 0.2% Triton X-100 and 0. 1% BSA (serum albumine de bovin, sigma) in PBS + Azide 0,1%. Then the saturation was realized by the incubation during 30 minutes in PBS+Azide 0.1%+BSA1%. Next the cultures cells were incubated in PBS+Azide 0.1%+BSA1% during 30min for saturation. Primary antibodies were then added and the samples incubated at 4°C overnight in PBS.
  • PFA paraformaldehyde
  • Anti-P-tubulin isotype III (mouse monoclonal 1 :400, SIGMA T5076) Anti-Cre Recombinase (mouse monoclonal 1 :250, abeam ab24607); Anti- Caspase-2(H19, rabbit polyclonal 1 :200, SIGMA sc-623) ; anti-MAP-2 (Anti- Microtubule- Associated Protein-2 (rabbit polyclonal 1 :400,MILLIPORE AB5622)), Anti-Bassoon (mouse monoclonal 1 :400, EnzoLifeSciences SAP7F407).
  • Species-specific secondary antibodies coupled to Alexa 350, 488 and 500 were used (1/500, Life Technologies, Inc., Gaithersburg, MD, USA) to visualize bound primary antibodies.
  • Phalloidin conjugated to Alexa Fluor 555(1 :500, Enzo LifeTechnologies) was added with secondary antibodies and stains F-actin
  • Images were acquired with an Axio-observer Zl (Zeiss, Germany) fitted with a cooled CCD camera (CoolsnapHQ2, Ropert Scientific). The microscope was controlled with Metamorph and Micro-manager software. Images were analyzed using ImageJ software.
  • E16 hippocampal neurons isolated from Casp2 flox/flox mice were cultured for seven days in microfluidic devices and treated with 2 ⁇ Pen-CRE. After 3 days, celle were fixed and immuno-labelled with the casp2 antibody HI 9 and tubulin. Histogram shows immunofluorescence quantification of Casp2 (ratio intensity/surface casp2 vs intensity/surface tubulin).
  • Caspase-2 is necessary for Amyloid P(l-42)-induced apoptosis in vitro (Troy et al; 2000).
  • CRE-mediated casp2 deletion in EJA-l flox/flox neurons primary hippocampal neurons isolated from EJA-l flox/flox mice, were pre-exposed or not (mock) for three days with recombinant Tat-CRE, and then treated with Amyloid ⁇ (1-42) oligomers ( ⁇ ). at 6 hour post- ⁇ treatment, synaptoprotection was observed. At 24 hours post- ⁇ treatment, axonal degeneration was found to be reduced when compared to mock-treated neurons.
  • OGD oxygen glucose deprivation
  • conditional Caspase-2 knock-out mice which have been obtained are particularly efficient for screening purposes, and for the selection of candidate compounds which are able to modulate the activity of Caspase-2 in vivo, in particular for the selection of candidate compounds which are able to treat or prevent the occurence of a disorder associated with ⁇ -mediated synaptotoxicity.

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Abstract

L'invention concerne un mammifère non humain ayant un système d'inactivation conditionnelle du gène caspase 2, et des cellules isolées, ainsi que des tissus et/ou des fractions cellulaire desdits tissus.
PCT/EP2015/050512 2015-01-13 2015-01-13 Inactivations conditionnelles de caspase 2 et procédés associés WO2016112961A1 (fr)

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