Calpain Mediates Excitotoxic DNA Fragmentation via Mitochondrial Pathways in Adult Brains
2005; Elsevier BV; Volume: 280; Issue: 16 Linguagem: Inglês
10.1074/jbc.m414552200
ISSN1083-351X
AutoresJiro Takano, Masanori Tomioka, Satoshi Tsubuki, Makoto Higuchi, Nobuhisa Iwata, Shigeyoshi Itohara, Masatoshi Maki, Takaomi C. Saido,
Tópico(s)Connexins and lens biology
ResumoCalpain has been implicated in excitotoxic neurode-generation, but its mechanism of action particularly in adult brains remains unclear. We generated mutant mice lacking or overexpressing calpastatin, the only solely calpain-specific inhibitor ever identified or synthesized. Modulation of calpastatin expression caused no defect in the mice under normal conditions, indicating that calpastatin functions as a negative regulator of calpain only under pathological conditions. Kainate-evoked excitotoxicity in hippocampus resulted in proteolytic activation of a proapoptotic Bcl-2 subfamily member (Bid), nuclear translocation of mitochondria-derived DNA fragmentation factors (apoptosis-inducing factor and endonuclease G), DNA fragmentation, and nuclear condensation in pyramidal neurons. These apoptotic responses were significantly augmented by calpastatin deficiency. Consistently calpastatin overexpression suppressed them. No evidence of caspase-3 activation was detected. Our results demonstrated that calpain mediates excitotoxic signals through mobilization of proapoptotic factors in a caspase-independent manner. These mutant mice will serve as useful tools for investigating calpain involvement in various diseases. Calpain has been implicated in excitotoxic neurode-generation, but its mechanism of action particularly in adult brains remains unclear. We generated mutant mice lacking or overexpressing calpastatin, the only solely calpain-specific inhibitor ever identified or synthesized. Modulation of calpastatin expression caused no defect in the mice under normal conditions, indicating that calpastatin functions as a negative regulator of calpain only under pathological conditions. Kainate-evoked excitotoxicity in hippocampus resulted in proteolytic activation of a proapoptotic Bcl-2 subfamily member (Bid), nuclear translocation of mitochondria-derived DNA fragmentation factors (apoptosis-inducing factor and endonuclease G), DNA fragmentation, and nuclear condensation in pyramidal neurons. These apoptotic responses were significantly augmented by calpastatin deficiency. Consistently calpastatin overexpression suppressed them. No evidence of caspase-3 activation was detected. Our results demonstrated that calpain mediates excitotoxic signals through mobilization of proapoptotic factors in a caspase-independent manner. These mutant mice will serve as useful tools for investigating calpain involvement in various diseases. The phenomenon of excitotoxicity is related to the key pathological processes that are involved not only in acute neuronal cell death evoked by surgical insults such as ischemia and spinal cord injury but also in chronic neurodegeneration that arises in neurological disorders such as Alzheimer disease (1Lipton S.A. Rosenberg P.A. N. Engl. J. Med. 1994; 330: 613-622Crossref PubMed Scopus (2521) Google Scholar, 2Mattson M.P. Neuromol. Med. 2003; 3: 65-94Crossref PubMed Scopus (406) Google Scholar, 3Rao S.D. Weiss J.H. Trends Neurosci. 2004; 27: 17-23Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 4Young C. Tenkova T. Dikranian K. Olney J.W. Curr. Mol. Med. 2004; 4: 77-85Crossref PubMed Scopus (54) Google Scholar). Recent clinical evidence for the role of excitotoxicity in the pathogenesis of Alzheimer disease was highlighted by the fact that memantine, a glutamate receptor blocker, alleviates the cognitive defects of Alzheimer patients (5Reisberg B. Doody R. Stoffler A. Schmitt F. Ferris S. Mobius H.J. N. Engl. J. Med. 2003; 348: 1333-1341Crossref PubMed Scopus (1706) Google Scholar). Elucidation of the mechanism of excitotoxic neuronal degeneration will therefore contribute to development of strategies for the treatment of both acute and chronic neuronal disorders. Several pieces of experimental evidence (see below) led us to focus our attention in the present study on the role of calpain, a calcium-activated neutral protease, in excitotoxic neuronal death. Because excitotoxicity primarily involves an unphysiological elevation of intraneuronal calcium concentrations, the selective limited proteolysis of intracellular proteins by calpain activated in this way could be responsible for irreversibly modulating substrate functions (6Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar). This effect could be more damaging than other reversible calcium-dependent processes such as phosphorylation and dephosphorylation.The involvement of calpain in neuronal cell death has been implicated in various neuropathological circumstances including ischemia, spinal cord injury, multiple sclerosis, Alzheimer disease, Parkinson's disease, and polyglutamine diseases (7Saito K. Elce J.S. Hamos J.E. Nixon R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2268-2632Crossref Scopus (526) Google Scholar, 8Yokota M. Saido T.C. Tani E. Kawashima S. Suzuki K. Stroke. 1995; 26: 1901-1907Crossref PubMed Scopus (65) Google Scholar, 9Shields D.C. Schaecher K.E. Saido T.C. Banik N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11486-11491Crossref PubMed Scopus (147) Google Scholar, 10Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (901) Google Scholar, 11Kim Y.J. Yi Y. Sapp E. Wang Y. Cuiffo B. Kegel K.B. Qin Z.H. Aronin N. DiFiglia M. Proc. 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Driscoll M. Tavenmarakis N. Nature. 2002; 419: 939-944Crossref PubMed Scopus (253) Google Scholar). Although a number of calpain substrates including cytoskeletal proteins, kinases, and kinase modulators have been identified (16Wang K.K. Yuen P.W. Calpain: Pharmacology and Toxicology of Calcium-dependent Protease. Taylor & Francis, Philadelphia, PA1999: 77-101Google Scholar), the detailed mechanism of the action of calpain, particularly in vivo, has remained elusive. One of the drawbacks to addressing this issue lies in the redundancy of the protease; there are at least two molecular species of ubiquitous calpain, the μ-calpain and m-calpain isoforms, which require micromolar and millimolar calcium, respectively, for activation in biochemical terms (6Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar, 17Inomata M. Hayashi M. Nakamura M. Imahori K. Kawashima S. J. Biochem. (Tokyo). 1985; 98: 407-416Crossref PubMed Scopus (42) Google Scholar). Each of these isoforms is a heterodimer composed of a distinct 80-kDa catalytic subunit and an identical 30-kDa regulatory subunit. Therefore, the structure of the large subunit determines the biochemical properties of the isoforms. Interestingly both the large and small subunits possess multiple calcium-binding sites (18Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar, 19Strobl S. Fernandez-Catalan C. Braun M. Huber R. Masumoto H. Nakagawa K. Irie A. Sorimachi H. Bourenkow G. Bartunik H. Suzuki K. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 588-592Crossref PubMed Scopus (314) Google Scholar), indicating that the two subunits probably interact with each other in a dynamic manner in the presence of calcium. Most researchers familiar with the biochemical properties of these calpain isoforms had presumed that μ-calpain, which can be activated by submicromolar levels of calcium in the presence of phospholipids (20Coolican S.A. Hathaway D.R. J. Biol. Chem. 1984; 259: 11627-11630Abstract Full Text PDF PubMed Google Scholar, 21Saido T.C. Shibata M. Takenawa T. Murofushi H. Suzuki K. J. Biol. Chem. 1992; 267: 24585-24590Abstract Full Text PDF PubMed Google Scholar), rather than m-calpain, would be the main participant in physiological processes, but this presumption has been entirely upset by findings from studies using reverse genetic approaches.While genetic deletion of the commonly shared 30-kDa subunit resulted in disappearance of both μ- and m-calpains leading to embryonic lethality (22Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell. Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (292) Google Scholar), deletion of the μ-calpain large subunit apparently led to no severe phenotypes (23Azam M. Andrabi S.S. Sahr K.E. Kamath L. Kuliopulos A. Chishti A.H. Mol. Cell. Biol. 2001; 21: 2213-2220Crossref PubMed Scopus (214) Google Scholar), indicating that m-calpain is likely to play an essential role in early developmental processes. Consistently genetic deficiency of the m-calpain 80-kDa subunit resulted in a lethal phenotype, 1J. Takano and T. C. Saido, unpublished observation. 1J. Takano and T. C. Saido, unpublished observation. although it remains possible that μ- and m-calpains may function in a cooperative manner under certain circumstances. These reverse genetic observations are contradictory to the biochemistry-based analysis of calpain isoform functions as stated above. The mechanism of m-calpain activation in vivo remains unknown but may be associated with cell adhesion because m-calpain was shown to colocalize with adhesion molecules (24Beckerle M.C. Burridge K. DeMartino G.N. Croall D.E. Cell. 1987; 51: 569-577Abstract Full Text PDF PubMed Scopus (210) Google Scholar) and because calpain has been identified only in multicellular organisms (6Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar). In any case, this complexity of subunit composition and function of calpain isoforms together with the coexistence of these isoforms in various cell types including neurons (25Murachi T. Trends Biochem. Sci. 1983; 8: 167-169Abstract Full Text PDF Scopus (269) Google Scholar) makes the reverse genetic approaches targeting calpain subunits less powerful than expected in addressing the pathophysiological roles of calpain in vivo. Another technical problem in calpain research is that there have been no solely calpain-specific synthetic inhibitors; most, if not all, of the synthetic "calpain inhibitors" thus far used inhibit other cysteine proteases represented by cathepsin B as potently as calpain (26Rock K.L. Gramm C.G. Rothstein K.C. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2178) Google Scholar), making it difficult to draw definitive conclusions from experiments using synthetic inhibitors.To overcome these drawbacks and to gain new insights into the pathophysiological roles of calpain, we focused our attention in the present study on calpastatin. This protein is composed of ∼700 amino acid residues and has been biochemically characterized as a potent stoichiometric inhibitor of both μ-calpain and m-calpain in an identical manner (27Goll D.E Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2329) Google Scholar). One molecule of calpastatin inhibits four molecules of calpain. The binding affinity of calpastatin to calpain is so high in the presence of calcium that the Ki values are extremely low (28Maki M. Takano E. Osawa T. Ooi T. Murachi T. Hatanaka M. J. Biol. Chem. 1988; 263: 10254-10261Abstract Full Text PDF PubMed Google Scholar). Calpastatin is the only solely calpain-specific inhibitor that has ever been identified or synthesized to our knowledge. In addition, there is no molecular redundancy unlike the family of caspase inhibitors. Therefore, manipulation of calpastatin expression levels in vivo should have profound and specific effects on the proteolytic activity of both calpain isoforms and thus should shed light on the function of ubiquitous calpain as a whole regardless of calpain isoform redundancy, although there is the problem that a calpastatin deficiency might result in a lethal phenotype due to the overactivation of calpain. We therefore adopted two different strategies that involved developing calpastatin-deficient mice and calpastatin-overexpressing mice by reverse genetic methods and analyzed calpain activation and subsequent cellular events evoked by excitotoxicity. Surprisingly and luckily to us, genetic modulation of calpastatin expression levels did not cause any defect in the development, fertility, morphology, or life span of the mice under normal conditions, indicating that calpastatin is a negative regulator of calpain only under pathological conditions. This suggests that calpastatin knock-out and transgenic mice will serve as ideal models to examine whether and how calpain participates in various pathological processes. We also attempted to induce typical caspase-dependent apoptosis in adult mouse brains. Failure to achieve this due to a drastic postdevelopmental reduction of caspase-3 expression in brain tissues will also be discussed.MATERIALS AND METHODSTargeting of Calpastatin Gene—A genomic DNA clone of mouse calpastatin was isolated from the 129/Sv mouse strain taken from a bacterial artificial chromosome library as described previously (29Takano J. Watanabe M. Hitomi K. Maki M. J. Biochem. (Tokyo). 2000; 128: 83-92Crossref PubMed Scopus (57) Google Scholar). The targeting vector was generated using the following DNA fragments: a 1.5-kb SphI fragment from intron 4 to exon 6, three tandem repeats of 250-bp SV40 early mRNA polyadenylation signals to terminate transcription (30Maxwell I.H. Harrison G.S. Wood W.M. Maxwell F. BioTechniques. 1989; 7: 276-280PubMed Google Scholar), a 5.5-kb SphI-ScaI calpastatin gene fragment from intron 6 to intron 8, a 2.0-kb pgk-neo gene cassette (for positive selection), and a 1.0-kb SacI-NotI diphtheria toxin A fragment cassette from pMC1DT-A (for negative selection) (31Yagi T. Ikawa Y. Yoshida K. Shigetani Y. Takeda N. Mabuchi I. Yamamoto T. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9918-9922Crossref PubMed Scopus (210) Google Scholar, 32Gomi H. Yokoyama T. Fujimoto K. Ikeda T. Katoh A. Itoh T. Itohara S. Neuron. 1995; 14: 29-41Abstract Full Text PDF PubMed Scopus (229) Google Scholar). Polyadenylation signals were placed in exon 6 to inhibit transcription of the calpain-inhibitory domains (30Maxwell I.H. Harrison G.S. Wood W.M. Maxwell F. BioTechniques. 1989; 7: 276-280PubMed Google Scholar).E14 cells derived from strain 129/Ola mouse blastocysts were used as an embryonic stem cell line (33Hooper M. Hardy K. Handyside A. Hunter S. Monk M. Nature. 1987; 326: 292-295Crossref PubMed Scopus (912) Google Scholar). Cell culture and the targeting experiments were carried out as described previously (34Itohara S. Mombaerts P. Lafaille J. Iacomini J. Nelson A. Clarke A.R. Hooper M.L. Farr A. Tonegawa S. Cell. 1993; 72: 337-348Abstract Full Text PDF PubMed Scopus (458) Google Scholar). In brief, the embryonic stem cells were electroporated with a Gene Pulser (800 V and 3 millifarads with a 0.4-cm electrode distance, Bio-Rad) using 30 μg of linearized targeting vector generated by NotI treatment. Clones selected with G418 at 150 μg/ml were screened and identified by hybridization with 5′ and 3′ external probes following BamHI digestion of genomic DNA (Fig. 1, A and B). Chimeric mice were generated as described previously by Bradley et al. (35Bradley A. Evans M. Kaufman M.H. Robertson E. Nature. 1984; 309: 255-256Crossref PubMed Scopus (1087) Google Scholar). Embryonic stem cells were microinjected into C57BL/6J blastocysts at 3.5 days postcoitum. After injection, the embryos were transferred into the uteri of pseudopregnant ICR mice. Mice heterozygous for the mutation were further intercrossed to obtain the chimera to C57BL/6J mice. The genotypes of the mice were determined by Southern blot analysis of genomic DNA prepared from their tails. The heterozygous mice were then backcrossed with C57BL/6 mice five to six times. To obtain homozygotes, the resulting heterozygotes were intercrossed. All mice were maintained by the Research Resource Center, RIKEN Brain Science Institute; all animal experiments were carried out according to the guidelines for animal experimentation in RIKEN.Calpastatin Transgenic Mice—The human calpastatin cDNA (36Asada K. Ishino Y. Shimada M. Shimojo T. Endo M. Kimizuka F. Kato I. Maki M. Hatanaka M. Murachi T. J. Enzyme Inhib. 1989; 3: 49-56Crossref PubMed Scopus (59) Google Scholar) was cloned into pNN265 from which the NotI fragment was subcloned into pMM403 containing the calcium/calmodulin-dependent protein kinase-II α subunit promoter (37Mayford M. Wang J. Kandel E.R. O'Dell T.J. Cell. 1995; 81: 891-904Abstract Full Text PDF PubMed Scopus (447) Google Scholar). The SfiI-linearized DNA construct was microinjected into the pronucleus of C57BL/6Cr zygotes, which were transferred into foster females to create calpastatin transgenic founders. The genotype was determined by PCR with specific primers 5′-CATGAACCACAGACAGCTTGGTTGAC-3′ and 5′-GGAGGATTTGATATTCACCTGGCCCG-3′ that generate a 350-bp product. We confirmed that the transgene-derived human calpastatin was robustly expressed in hippocampus and neocortex. The details of the characterization of the mice are described elsewhere (38Higuchi M. Tomioka M Takano J. Shirotani K. Iwata N. Masumoto H. Maki M. Itohara S. Saido T.C. J. Biol. Chem. 2005; 280: 15229-15237Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar).Northern Blotting and Reverse Transcription-PCR—Polyadenylated RNA was isolated from half-cortex brain samples using a Micro-Fast Track™ 2.0 kit (Invitrogen). Samples were electrophoresed in 1% formaldehyde agarose gels and transferred to nylon membranes. The membranes were probed with [α-32P]dCTP-labeled fragments of calpastatin and μ-calpain cDNAs. Specific cDNA probes encoding the inhibitory domain IV of mouse calpastatin and encoding domain IV of mouse μ-calpain, respectively, were used. The gels were stained with ethidium bromide, and membranes were reprobed with an actin cDNA (Sigma) to confirm the quantity of DNAs loaded.Reverse transcription (RT) 2The abbreviations used are: RT, reverse transcription; AIF, apoptosis-inducing factor; EndoG, endonuclease G; MAP2, microtubule-associated protein 2; ICAD, inhibitor of caspase-activated DNase; CAD, caspase-activated DNase; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; tBid, truncated Bid. -PCR was carried out using polyadenylated RNA obtained as described above by methods described previously (29Takano J. Watanabe M. Hitomi K. Maki M. J. Biochem. (Tokyo). 2000; 128: 83-92Crossref PubMed Scopus (57) Google Scholar). PCR products were electrophoresed on 5% polyacrylamide gels. Gels were stained with ethidium bromide (1 μg/ml), and then DNA fragments were visualized under short wave (254 nm) UV illumination and photographed. For direct sequencing of the short DNA PCR products, DNA fragments were excised from the polyacrylamide gels under long wave (365 nm) UV illumination. Nucleotide sequences were determined with an automated fluorescent sequencer, ABI PRISM 3700 (PerkinElmer Life Sciences), using a BigDye terminator cycle sequencing ready reaction kit (PerkinElmer Life Sciences).Calpastatin Activity Assay—Cortical tissues (120 mg) from wild-type, calpastatin–/–, and calpastatin transgenic mice were homogenized in 2.5 ml of 20 mm Tris-HCl buffer (pH 7.5) containing 5 mm EDTA and 0.1% Triton X-100 at 100 °C and then ultracentrifuged at 4 °C. The supernatants were subjected to ion-exchange chromatography using a DEAD-Toyopearl 650S (Tosoh) column (0.5 × 5 cm) with a linear gradient (0–0.5 m) of NaCl and then to calpastatin activity assays using 14C-labeled casein (100 μg; 4200 dpm) and bovine m-calpain (0.45 unit) as described previously (39Nagao S. Saido T.C. Akita Y. Tsuchiya T. Suzuki K. Kawashima S. J. Biochem. 1994; 115: 1178-1184Crossref PubMed Scopus (39) Google Scholar). The activity to inhibit the m-calpain activity was defined as calpastatin activity.Subcellular Fractionation—Hippocampal tissues taken from the brains perfused with ice-cold phosphate-buffered saline (PBS) were homogenized by 20 strokes of a Dounce homogenizer in 10 volumes of ice-cold buffer M (210 mm mannitol, 70 mm sucrose, 10 mm HEPES (pH 7.5), 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1× protease inhibitor mixture, EDTA-free (Roche Applied Science). Homogenates were centrifuged twice at 1500 × g for 5 min at 4 °C to remove unlysed cells and nuclei. The supernatants were centrifuged at 10,000 × g for 10 min at 4 °C. Pellets were saved as the mitochondrial (high density membrane) fractions. The supernatants were centrifuged at 100,000 × g for 1 h at 4 °C and saved as the cytosolic (S100) fractions. The high density membrane fractions were lysed in buffer MX (buffer M with 1% Triton X-100) for immunoblotting. Protein concentrations were assayed with the Micro-BCA protein assay kit (Pierce) using bovine serum albumin as a standard.Western Blotting—Protein samples denatured in SDS sample buffer (0.25 m Tris-HCl (pH 6.8), 1% SDS, 25% glycerol, 5% 2-mercaptoethanol) were subjected to SDS-PAGE in Tris-Tricine buffer and blotted onto polyvinylidene difluoride membranes. Blotted membranes were blocked with 5% skim milk in PBS containing 0.1% Tween 20 (PBST) for 1 h and then incubated with primary antibodies for 16 h at 4 °C. After three washes in PBST, the membranes were incubated with anti-mouse or anti-rabbit IgG, horseradish peroxidase-linked F(ab′)2 fragment (Amersham Biosciences) for 2 h. Immunoreactive signals were detected with the ECL Advance Western blotting detection kit (Amersham Biosciences) and quantified with a LuminoImager, LAS-3000, and Science Lab 2001 Image Gauge software (Fuji Photo Film) as described previously (40Iwata N. Mizukami H. Shirotani K. Takaki Y. Muramatsu S. Lu B. Gerard N.P. Gerard C. Ozawa K. Saido T.C. J. Neurosci. 2004; 24: 991-998Crossref PubMed Scopus (213) Google Scholar).Primary Antibodies—An antipeptidic antibody against the carboxyl terminus of mouse calpastatin was generated using a synthetic peptide, CKKTEEVSKPKAKEDARHS, conjugated to keyhole limpet hemocyanin as described previously (41Saido T.C. Nagao S. Shiramine M. Tsukaguchi M. Sorimachi H. Murofushi H. Tsuchiya T. Ito H. Suzuki K. J. Biochem. (Tokyo). 1992; 111: 81-86Crossref PubMed Scopus (130) Google Scholar, 42Saido T.C. Iwatsubo T. Mann D.M. Shimada H. Ihara Y. Kawashima S. Neuron. 1995; 14: 457-466Abstract Full Text PDF PubMed Scopus (501) Google Scholar). An antibody specific to the calpain cleavage site of the amino-terminal 135-kDa fragment of αII-spectrin was produced according to Manya et al. (43Manya H. Inomata M. Fujimori T. Dohmae N. Sato Y. Takio K. Nabeshima Y. Endo T. J. Biol. Chem. 2002; 277: 35503-35508Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Antibodies to m-calpain, inhibitor of caspase-activated DNase (ICAD), and caspase-activated DNase (CAD) were described previously (44Saido T.C. Nagao S. Shiramine M. Tsukaguchi M. Yoshizawa T. Sorimachi H. Ito H. Tsuchiya T. Kawashima S. Suzuki K. FEBS Lett. 1994; 346: 263-267Crossref PubMed Scopus (63) Google Scholar, 45Sakahira H. Enari M. Nagata S. J. Biol. Chem. 1999; 274: 15740-15744Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The following antibodies were purchased as indicated: mouse monoclonal anti-microtubule-associated protein 2 (MAP2) (clone AP20, Leinco Technology), αII-spectrin (clone AA6, Biohit), and cytochrome c oxidase subunit I (clone 1D6, Invitrogen) antibodies; a goat polyclonal antibody to caspase-3 (K-17, Santa Cruz Biotechnology); and rabbit polyclonal antibodies to activated caspase-3 (Asp-175, Cell Signaling Technology), endonuclease G (EndoG) (AB3639, Chemicon), apoptosis-inducing factor (AIF) (AB16501, Chemicon), and Bid (AR-52, Alexis).Intrahippocampal Injection—Stereotaxic administration of kainate into the hippocampus was performed as described previously (46Tomioka M. Shirotani K. Iwata N. Lee H.J. Yang F. Cole G.M. Seyama Y. Saido T.C. Mol. Brain. Res. 2002; 108: 18-32Crossref PubMed Scopus (25) Google Scholar). Briefly adult male mice were deeply anesthetized with sodium pentobarbital, placed in a stereotaxic apparatus, and given a unilateral injection of 0 or 0.1 nmol of kainate in 0.3 μl of PBS into the hippocampal CA1 region using a 26S-gauge needle equipped with a 0.5-μl motorized syringe (Hamilton). The coordinates of the injection were anterior-posterior –2.3 mm, medial-lateral –1.5 mm, and dorsal-ventral –1.6 mm from the bregma. Two minutes after the needle insertion, kainate was injected at a constant flow rate of 0.05 μl/min. The needle remained in place for an additional 2 min to prevent reflux of fluid.Histochemistry—Twenty-four hours after injection of kainate or PBS, mice were anesthetized with sodium pentobarbital (50 mg/kg of body weight) and perfused through the heart with ice-cold PBS followed by 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). The brains were removed, postfixed in the same fixative overnight at 4 °C, and embedded in paraffin. Coronal sections (4 μm thick) were then dehydrated and stained with 1% Cresyl Violet to confirm the injection site. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was carried out using an in situ cell death detection kit (Roche Applied Science) according to the manufacturer's instruction. For quantification of cell death, we counted the number of TUNEL-positive cells and propidium iodide-stained nuclei in hippocampus. The immunohistochemical analysis was performed with the tyramide signal amplification method using a tyramide signal amplification fluorescence system (PerkinElmer Life Sciences) as described previously (40Iwata N. Mizukami H. Shirotani K. Takaki Y. Muramatsu S. Lu B. Gerard N.P. Gerard C. Ozawa K. Saido T.C. J. Neurosci. 2004; 24: 991-998Crossref PubMed Scopus (213) Google Scholar). Quantification of immunoreactivity was carried out using MetaMorph (version 6.1) imaging software (Universal Imaging Corp.) as described by Iwata et al. (40Iwata N. Mizukami H. Shirotani K. Takaki Y. Muramatsu S. Lu B. Gerard N.P. Gerard C. Ozawa K. Saido T.C. J. Neurosci. 2004; 24: 991-998Crossref PubMed Scopus (213) Google Scholar, 47Iwata N. Takaki Y. Fukami S. Tsubuki S. Saido T.C. J. Neurosci. Res. 2002; 70: 493-500Crossref PubMed Scopus (166) Google Scholar).Statistical Analysis—The statistical significance was examined by Student's t test after confirming the equality between the group variances or by Fisher's protected least significant difference when it was necessary. A probability level of p < 0.05 was accepted as statistically significant in all of the studies.RESULTSFig. 1A summarizes the strategy used to generate calpastatin knock-out mice. The targeting vector carrying three tandem polyadenylation sequences was inserted into exon 6 to terminate transcription through homologous recombination (30Maxwell I.H. Harrison G.S. Wood W.M. Maxwell F. BioTechniques. 1989; 7: 276-280PubMed Google Scholar) for the following reasons. (i) Every exon is composed of 3× multiple numbers of base pairs, making a frameshift strategy inadequate (29Takano J. Watanabe M. Hitomi K. Maki M. J. Biochem. (Tokyo). 2000; 128: 83-92Crossref PubMed Scopus (57) Google Scholar). (ii) The calpastatin gene possesses multiple sites for initiation and termination of transcription (48De Tullio R. Sparatore B. Salamino F. Melloni E. Pontremoli S. FEBS Lett. 1998; 422: 113-117Crossref PubMed Scopus (41) Google Scholar). (iii) A neighboring adipocyte-derived leucine aminopeptidase gene overlaps with part of the calpastatin gene corresponding to the fourth inhibitory domain (49Hattori A. Matsumoto K. Mizutani S. Tsujimoto M. J. Biochem. (Tokyo). 2001; 130: 235-241Crossref PubMed Scopus (44) Google Scholar). (iv) The gene region encoding calpain-inhibitory domains starts at exon 9 (29Takano J. Watanabe M. Hitomi K. Maki M. J. Biochem. (Tokyo). 2000; 128: 83-92Crossref PubMed Scopus (57) Google Scholar). Southern blot analyses of BamHI-digested genomic DNA using 5′ and 3′ probes designated in Fig. 1A indicate that the gene targeting was successfully achieved as intended (Fig. 1B). We then performed Northern blotting, Western blotting, RT-PCR, and immunohistochemistry to confirm the amounts of calpastatin gene products in heterozygous (+/–) and homozygous (–/–) mice as compared with wild-type mice (+/+) (Fig. 1, C–F). The amount of calpastatin mRNA and corresponding anti-calpastatin antibody-immunoreactive protein decreased in a gene dose-dependent manner, whereas the levels of μ-calpain, m-calpain, and αII-spectrin (fodrin) remained unchanged (Fig. 1, C and D).Calpastatin appeared as doublet bands in Western blotting due to the presence of distinct initiation sites for transcription in the calpastatin gene (50Cong M. Thompson V.F. Goll D.E. Antin P.B. J. Biol. Chem. 1998; 273: 660-666Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Interestingly a trace amount of anti-calpastatin antibody immunoreactivity with lower molecular weight was detected in knock-out mi
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