Caspase-7 Expanded Function and Intrinsic Expression Level Underlies Strain-Specific Brain Phenotype of Caspase-3-Null Mice

Caspase-7 Expanded Function and Intrinsic Expression Level Underlies Strain-Specific Brain Phenotype of Caspase-3-Null Mice

The Journal of Neuroscience, November 3, 2004 • 24(44):9977–9984 • 9977 Development/Plasticity/Repair Caspase-7 Expanded Function and Intrinsic Expression Level Underlies Strain-Specific Brain Phenotype of Caspase-3-Null Mice Caroline Houde,1 Kathleen G. Banks,2 Nathalie Coulombe,3 Dita Rasper,3 Erich Grimm,3 Sophie Roy,1,4 Elizabeth M. Simpson,2 and Donald W. Nicholson1,4 1Biochemistry Department, McGill University, Montreal, Quebec H3G 1Y6, Canada, 2Centre for Molecular Medicine and Therapeutics, British Columbia Institute for Children’s and Women’s Health, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada, 3Merck Frosst Canada and Company, Pointe-Claire–Dorval, Quebec H9R 4P8, Canada, and 4Merck Research Laboratories, San Diego, California 92121 Caspase-3-deficient mice of the 129S1/SvImJ (129) strain show severe brain development defects resulting in brain overgrowth and perinatal lethality, whereas on the C57BL/6J (B6) background, these mice develop normally. We therefore sought to identify the strain- Ϫ Ϫ dependent ameliorating gene. We biochemically isolated caspase-7 from B6-caspase-3-null (Casp3 / ) tissues as being the enzyme with caspase-3-like properties and capability of performing a caspase-3 surrogate function, apoptotic DNA fragmentation. Moreover, we show that, in contrast to the human enzymes, mouse caspase-7 is as efficient as caspase-3 at cleaving and thus inactivating ICAD (inhibitor of caspase-activated DNase), the inhibitor of apoptotic DNA fragmentation. Low levels of caspase-7 expression and activation correlate with Ϫ Ϫ Ϫ Ϫ lack of DNA fragmentation in 129-Casp3 / apoptotic precursor neurons, whereas B6-Casp3 / cells, which can fragment their DNA, show higher levels of caspase-7 expression and activation. The amount of caspase-7 activation in apoptotic precursor neurons is inde- pendent of the presence of caspase-3. Together, our findings demonstrate for the first time a strong correlation between caspase-7 Ϫ Ϫ activity, normal brain development, and apoptotic DNA fragmentation in Casp3 / mice. Key words: apoptosis; brain development; strain-specific phenotype; caspase-3 compensation; caspase-7; DNA fragmentation Introduction A balance between the proliferation of neural progenitors and Apoptosis, or programmed cell death, is mediated by the activa- apoptotic cell death is essential for proper brain development tion of caspases (Casp), a family of cysteine proteases present as (Kuan et al., 2000). In the mouse, targeted deletion of caspase-3 proenzymes in all cells and activated by cleavage and reorganiza- (Kuida et al., 1996), caspase-9 (Hakem et al., 1998; Kuida et al., tion of their subunits after an intracellular or extracellular apo- 1998), or apaf-1 (a coactivator of caspase-9) (Cecconi et al., 1998; ptotic signal (Nicholson, 1999). Caspases orchestrate the orga- Yoshida et al., 1998) genes, yet not of other caspases, results in nized death of the cell by cleaving a small but specific brain development defects. In all cases, supernumerary cells in complement of protein substrates. One of the best characterized the forebrain and midbrain cause neural tissue protrusions morphological changes resulting from caspase substrate cleavage through the skull as a result of defects in apoptotic cell death. is DNA fragmentation at internucleosomal intervals (Wyllie, Apaf Ϫ/Ϫ embryos do not survive past embryonic day 16.5 (E16.5) 1980; Nagata et al., 2003). Caspase-activated DNase (CAD), the when they are from a mixed 129/Sv-CP-NMRI genetic back- DNase responsible for apoptotic DNA fragmentation, is trans- ground (Cecconi et al., 1998), but some embryos do survive for a lated in the cytoplasm where it is stabilized by its cognate chap- few days when on a mixed 129/O1a-C57BL/6 or 129/Ola-CD1 erone and inhibitor ICAD (Sakahira et al., 2000). Once activated, background (Yoshida et al., 1998). Casp3 Ϫ/Ϫ and Casp9 Ϫ/Ϫ mice the well characterized executioner caspase, caspase-3, cleaves of a mixed 129/Sv-C57BL/6 or 129/O1a-C57BL/6J background ICAD, releasing inhibition of CAD, which then translocates to are born, albeit at a lower frequency than the expected Mendelian the nucleus where it fragments DNA (Sakahira et al., 1998). ratio. Most live-born pups die within a few weeks, but a few survive and age with no apparent gross abnormalities. Variabili- ties in phenotype might be reflective of strain-specific modifier Received May 12, 2004; revised Sept. 16, 2004; accepted Sept. 17, 2004. C.H. was supported by the Canadian Institutes of Health Research. E.M.S. is supported by a Merck Frosst grant to genes that affect the apoptotic program. the Centre for Molecular Medicine and Therapeutics and is a holder of a Canada Research Chair in Genetics and We report here that the Casp3-deletion phenotype is strain Behavior. We thank Dr. S. Nagata for the mICAD construct. specific. The C57BL16J (B6) strain is resistant to loss of caspase-3, CorrespondenceshouldbeaddressedtoD.W.Nicholson,MerckResearchLaboratories–SanDiego,3535General whereas the 129S1/SvImJ (129) strain develops severe brain de- Atomics Court, MRL SDB1, San Diego, CA 92121. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.3356-04.2004 fects. Using a biochemical approach to investigate the molecular Copyright © 2004 Society for Neuroscience 0270-6474/04/249977-08$15.00/0 mechanism responsible for the resistance of the B6 strain to the 9978 • J. Neurosci., November 3, 2004 • 24(44):9977–9984 Houde et al. • Caspase-7 Role in Casp3-Null Mouse Brain Development loss of caspase-3, we identified caspase-7 as having similar prop- zerland). Lysates were cleared by 10 min centrifugation at 10,000 ϫ g at erties to caspase-3 in the mouse. Although caspase-7 is expressed 4°C. Cleavage activity at the caspase-3 aspartate-glutamate-valine- in both strains during brain development, its expression level is aspartate recognition site (DEVDase) was measured for 96 and 48 ␮gof lower in 129 compared with B6 brains. Lower levels of caspase-7 proteins for each tissue after pretreatment of the extracts with granzyme expression and activation in precursor neurons from 129- B for 5 min at 37°C. For E9.5 and E12.5 heads and brains, DTT was Ϫ Ϫ omitted, and they were homogenized using a plastic pellet pestle for 1.5 Casp3 / mice correlate with lack of DNA fragmentation, Ϫ/Ϫ ml tubes (Kontes, Vineland, NJ). whereas B6-Casp3 cells with higher intrinsic caspase-7 levels DEVDase activity assay. Protein extracts, purified recombinant enzymes, can fragment their DNA. This is the first report supporting a role or chromatography fractions were mixed with a final concentration of for caspase-7 in brain development and DNA fragmentation in 10 ␮M Ac-DEVD-AMC (acetyl-aspartate-glutamate-valine-aspartate- the absence of caspase-3. amino-methyl-coomasin) (Biomol, Plymouth Meeting, PA) in a final volume of 200 ␮l in buffer consisting of 50 mM HEPES-KOH, 2 mM Materials and Methods EDTA, 10% glycerol (v/v), 0.1% 3-[(3-cholamidopropyl) dimethylam- Congenic derivation and genotyping. Mice deleted for Casp3 were gener- monio]-1-propanesulfonate (CHAPS), and 5 mM DTT added fresh. The ated as described previously (Casp3tm1Mr1) (Keramaris et al., 2000). caspase-3 inhibitor M-791 (Merck Frosst Canada) was added at concen- Briefly, a targeting vector containing both the lacZ-PGKneo (␤-galacto- trations ranging from 2.5 ␮M to 10 pM. Liberation of AMC resulting from sidase–phosphglycerate kinase–neomycin phosphotransferase) and the cleavage at the caspase-3 consensus site DEVD was scored by the Cyto- bacterial diphtheria toxin ␣-subunit gene driven by the PGK promoter Fluor (PerSeptive Biosystems, Framingham, MA) for 60 min at room was constructed to remove most of exon 5, intron 5, and exon 6. Germ- temperature (excitation, 380 nm; emission, 460 nm). line chimeras were generated by injecting the targeted RW4 embryonic Chromatography. Livers, kidneys, and spleens were homogenized with stem cells into C57BL/6J blastocysts, and PCR was used to confirm ho- an 8 mm probe on the Polytron Brinkmann homogenizer on ice in ICE ϩ Ϫ mozygous disruption of the Casp3 gene. Two male Casp3 / at back- III buffer (50 mM HEPES-KOH, pH 7.0, 2 mM EDTA, 10% sucrose, 0.1% cross generation (N7) on C57BL/6NTac were backcrossed onto both B6 CHAPS, and 5 mM DTT added fresh). Cells were broken by 25 stokes in and 129. Experimental animals were backcrossed 4–10 generations onto a ground glass dounce. Lysates were cleared by two centrifugations at the B6 strain and 4–14 generations onto the 129 strain. Genotyping for 10,000 ϫ g for 30 min and an ultra-centrifugation at 100,000 ϫ g for 1 hr. the Casp3 wild-type (WT) and knock-out (KO) alleles was performed on Extracts were activated by granzyme B pretreatment before loading into DNA from adult tail or ear biopsy or embryo tail, paw, or yolk sac by PCR a Mono-Q HR 5/5 anion exchange column (Pharmacia, Uppsala, Sweden). using the previously described oligonucleotide pairs (Vanderluit et al., Elution was performed with a NaCl gradient ranging from 0 to 0.8 M. Frac- 2000). Specifically, the WT Casp3 allele 5Ј primer 5Ј-AAGCTG- tions of 1 ml were collected, of which 10 ␮l were tested for DEVDase activity. TCTTCGTCCAGTGAG-3Ј (oEMS1051) corresponding to base pairs Biotin labeling. DEVDase activity-containing ion exchange chroma- 3122–3142, and the 3Ј primer 5Ј-CTAAGTTAACCAAACTGAGC- tography fractions were pooled. The sample was separated into two; ACCGC-3Ј (oEMS905) corresponding to base pairs 3475–3451, gener- one-half was labeled with 10 ␮M Biotin-DEVD-AOMK (Merck Frosst ated a band of 353 bp. Primers for the KO allele used the same 5Ј primer Canada and Co.) for 30 min at 37°C, and the other half was incubated (oEMS1051) as the WT gene, and the 3Ј primer 5Ј-GTCGATC- with DMSO only.

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