Inhibition of Autophagy Prevents Hippocampal Pyramidal Neuron Death after Hypoxic-Ischemic Injury
2008; Elsevier BV; Volume: 172; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2008.070876
ISSN1525-2191
AutoresMasato Koike, Masahiro Shibata, Masao Tadakoshi, Kunihito Gotoh, Masaaki Komatsu, Satoshi Waguri, Nobutaka Kawahara, Keisuke Kuida, Shigekazu Nagata, Eiki Kominami, Keiji Tanaka, Yasuo Uchiyama,
Tópico(s)MicroRNA in disease regulation
ResumoNeonatal hypoxic/ischemic (H/I) brain injury causes neurological impairment, including cognitive and motor dysfunction as well as seizures. However, the molecular mechanisms regulating neuron death after H/I injury are poorly defined and remain controversial. Here we show that Atg7, a gene essential for autophagy induction, is a critical mediator of H/I-induced neuron death. Neonatal mice subjected to H/I injury show dramatically increased autophagosome formation and extensive hippocampal neuron death that is regulated by both caspase-3-dependent and -independent execution. Mice deficient in Atg7 show nearly complete protection from both H/I-induced caspase-3 activation and neuron death indicating that Atg7 is critically positioned upstream of multiple neuronal death executioner pathways. Adult H/I brain injury also produces a significant increase in autophagy, but unlike neonatal H/I, neuron death is almost exclusively caspase-3-independent. These data suggest that autophagy plays an essential role in triggering neuronal death execution after H/I injury and Atg7 represents an attractive therapeutic target for minimizing the neurological deficits associated with H/I brain injury. Neonatal hypoxic/ischemic (H/I) brain injury causes neurological impairment, including cognitive and motor dysfunction as well as seizures. However, the molecular mechanisms regulating neuron death after H/I injury are poorly defined and remain controversial. Here we show that Atg7, a gene essential for autophagy induction, is a critical mediator of H/I-induced neuron death. Neonatal mice subjected to H/I injury show dramatically increased autophagosome formation and extensive hippocampal neuron death that is regulated by both caspase-3-dependent and -independent execution. Mice deficient in Atg7 show nearly complete protection from both H/I-induced caspase-3 activation and neuron death indicating that Atg7 is critically positioned upstream of multiple neuronal death executioner pathways. Adult H/I brain injury also produces a significant increase in autophagy, but unlike neonatal H/I, neuron death is almost exclusively caspase-3-independent. These data suggest that autophagy plays an essential role in triggering neuronal death execution after H/I injury and Atg7 represents an attractive therapeutic target for minimizing the neurological deficits associated with H/I brain injury. 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15: 1001-1011Crossref PubMed Google Scholar The induction of autophagy has been shown in neonatal and adult mouse cortex and striatum after H/I injury,10Zhu C Wang X Xu F Bahr BA Shibata M Uchiyama Y Hagberg H Blomgren K The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia.Cell Death Differ. 2005; 12: 162-176Crossref PubMed Scopus (360) Google Scholar, 28Zhu C Xu F Wang X Shibata M Uchiyama Y Blomgren K Hagberg H Different apoptotic mechanisms are activated in male and female brains after neonatal hypoxia-ischaemia.J Neurochem. 2006; 96: 1016-1027Crossref PubMed Scopus (222) Google Scholar and LC3, a marker protein for autophagy, is required for autophagosome formation via its conversion from LC3-I to LC3-II.29Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T Kominami E Ohsumi Y Yoshimori T LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5305) Google Scholar However, it is unclear if autophagy participates as a pro- or anti-death factor in the execution of neuron death after H/I injury.30Levine B Yuan J Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 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In such damaged neurons autophagy was strongly induced, and this ischemic neuronal death was rescued by an Atg7 deficiency. These data indicate that autophagy plays an essential role in the execution of pyramidal neuron death in the neonatal mouse hippocampus after H/I injury. The experiments described below were performed in compliance with the regulations of Osaka University Graduate School of Medicine's Guidelines for the Care and Use of Laboratory Animals. Neonatal and adult C57BL/6J mice at postnatal day 7 (P7) and 8 weeks of age were obtained from CLEA Japan (Tokyo, Japan) or Charles River Laboratories Japan (Yokohama, Japan). Nestin-Cre transgenic mice [strain name: B6.Cg(SJL)-Tg(nestin-Cre)1Kln/J]32Tronche F Kellendonk C Kretz O Gass P Anlag K Orban PC Bock R Klein R Schutz G Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.Nat Genet. 1999; 23: 99-103Crossref PubMed Scopus (1402) Google Scholar were purchased from the Jackson Laboratory (Bar Harbor, ME). Atg7flox/flox mice,16Komatsu M Waguri S Ueno T Iwata J Murata S Tanida I Ezaki J Mizushima N Ohsumi Y Uchiyama Y Kominami E Tanaka K Chiba T Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1855) Google ScholarCAD homozygous (−/−) mice,33Kawane K Fukuyama H Yoshida H Nagase H Ohsawa Y Uchiyama Y Okada K Iida T Nagata S Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation.Nat Immunol. 2003; 4: 138-144Crossref PubMed Scopus (193) Google Scholar and caspase-3 heterozygous (+/−) mice backcrossed onto a C57BL/6 background for at least 10 generations34Kuida K Zheng TS Na S Kuan C Yang D Karasuyama H Rakic P Flavell RA Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.Nature. 1996; 384: 368-372Crossref PubMed Scopus (1695) Google Scholar, 35Leonard JR Klocke BJ D'Sa C Flavell RA Roth KA Strain-dependent neurodevelopmental abnormalities in caspase-3-deficient mice.J Neuropathol Exp Neurol. 2002; 61: 673-677Crossref PubMed Scopus (112) Google Scholar were transferred to the Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, and were housed in a pathogen-free facility. Atg7flox/flox mice were bred with nestin-Cre transgenic mice to produce Atg7flox/flox; nestin-Cre mice.36Komatsu M Waguri S Chiba T Murata S Iwata J Tanida I Ueno T Koike M Uchiyama Y Kominami E Tanaka K Loss of autophagy in the central nervous system causes neurodegeneration in mice.Nature. 2006; 441: 880-884Crossref PubMed Scopus (2754) Google ScholarCaspase-3+/− mice were crossed to obtain a mixture of +/+, +/−, and −/− pups. To determine the genotypes of these lines, genomic DNA was prepared from pup tail snips and analyzed by polymerase chain reaction (PCR). The following primer sets were used for genotyping: for the Cre transgene, 5′-TTTGCCTGCATTACCGGTCGATGCAAC-3′ and 5′-TGCCCCTGTTTCACTATCCAGGTTACGGA-3′; wild-type and Atg7flox alleles, 5′-TGGCTGCTACTTCTGCAATGATGT-3′ and 5′-CAGGACAGAGACCATCAGCTCCAC-3′16Komatsu M Waguri S Ueno T Iwata J Murata S Tanida I Ezaki J Mizushima N Ohsumi Y Uchiyama Y Kominami E Tanaka K Chiba T Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1855) Google Scholar; wild-type and mutant CAD alleles, 5′-AAAAGAACAGTCGGGACTGC-3′, 5′-GATTCGCCAGCGCATCGCCTT-3,′ and 5′-TTCACACCAGGAGACATCTG-3′33Kawane K Fukuyama H Yoshida H Nagase H Ohsawa Y Uchiyama Y Okada K Iida T Nagata S Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation.Nat Immunol. 2003; 4: 138-144Crossref PubMed Scopus (193) Google Scholar; wild-type and mutant caspase-3 alleles, 5′-AGGGTCCCATTTGCATAGCTTTGC-3′, 5′-GCGAGTGAGAATGTGCATAAATTC-3,′ and 5′-TGCTAAAGCGCATGCTCCAGACTG-3′.34Kuida K Zheng TS Na S Kuan C Yang D Karasuyama H Rakic P Flavell RA Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.Nature. 1996; 384: 368-372Crossref PubMed Scopus (1695) Google Scholar Neonatal and adult H/I brain injury was induced in mice on P7 and at 8 weeks of age, respectively, essentially according to the Rice-Vannucci model,31Rice III, JE Vannucci RC Brierley JB The influence of immaturity on hypoxic-ischemic brain damage in the rat.Ann Neurol. 1981; 9: 131-141Crossref PubMed Scopus (1887) Google Scholar with minor modifications.10Zhu C Wang X Xu F Bahr BA Shibata M Uchiyama Y Hagberg H Blomgren K The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia.Cell Death Differ. 2005; 12: 162-176Crossref PubMed Scopus (360) Google Scholar After the mice were deeply anesthetized with isoflurane (2%), the left common carotid artery was dissected and ligated with silk sutures (6/0). After the surgical procedure, the pups and adult mice were allowed to recover for 1 hour. They were then placed in chambers maintained at 37°C through which 8% humidified oxygen (balance, nitrogen) flowed for 40 or 45 minutes (neonate) or 35 or 40 minutes (adult). After hypoxic exposure, the pups and adult mice were returned to their dams and the plastic cages, respectively. The animals were allowed to recover for 3, 8, 24, or 72 hours, or 7 days (neonate). At each stage, brains were processed for biochemical and morphological analyses. This procedure resulted in brain injury in the ipsilateral hemisphere, consisting of cerebral infarction mainly in the hippocampus.9Sheldon RA Sedik C Ferriero DM Strain-related brain injury in neonatal mice subjected to hypoxia-ischemia.Brain Res. 1998; 810: 114-122Crossref PubMed Scopus (219) Google Scholar, 13Ness JM Harvey CA Strasser A Bouillet P Klocke BJ Roth KA Selective involvement of BH3-only Bcl-2 family members Bim and Bad in neonatal hypoxia-ischemia.Brain Res. 2006; 1099: 150-159Crossref PubMed Scopus (55) Google Scholar Control littermates were neither operated on nor subjected to hypoxia. The preparation of rabbit antibodies against rat LC321Koike M Shibata M Waguri S Yoshimura K Tanida I Kominami E Gotow T Peters C von Figura K Mizushima N Saftig P Uchiyama Y Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease).Am J Pathol. 2005; 167: 1713-1728Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 37Lu Z Dono K Gotoh K Shibata M Koike M Marubashi S Miyamoto A Takeda Y Nagano H Umeshita K Uchiyama Y Monden M Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation.Arch Histol Cytol. 2005; 68: 71-80Crossref PubMed Scopus (41) Google Scholar and Atg738Tanida I Mizushima N Kiyooka M Ohsumi M Ueno T Ohsumi Y Kominami E Apg7p/Cvt2p: a novel protein-activating enzyme essential for autophagy.Mol Biol Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (322) Google Scholar was described previously. Rabbit polyclonal antibodies against ubiquitin (DAKO, Glostrup, Denmark), caspase-3 (Cell Signaling, Danvers, MA), cleaved caspase-3 (Asp175; Cell Signaling), and cleaved caspase-7 (Asp198; Cell Signaling), and mouse monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 6C5; Ambion, Austin, TX), caspase-7 (clone 10-1-62; BD Biosciences, San Jose, CA), and neuronal nuclei (NeuN) (Chemicon, Temecula, CA) were obtained commercially. After H/I injury (n = 3 to 5 for each procedure and each stage), mice were deeply anesthetized with pentobarbital (25 mg/kg i.p.) and fixed by cardiac perfusion with 4% paraformaldehyde buffered with 0.1 mol/L phosphate buffer (pH 7.2) containing 4% sucrose for light microscopy and with 2% paraformaldehyde-2% glutaraldehyde buffered with 0.1 mol/L phosphate buffer for ordinary electron microscopy.21Koike M Shibata M Waguri S Yoshimura K Tanida I Kominami E Gotow T Peters C von Figura K Mizushima N Saftig P Uchiyama Y Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease).Am J Pathol. 2005; 167: 1713-1728Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 39Koike M Nakanishi H Saftig P Ezaki J Isahara K Ohsawa Y Schulz-Schaeffer W Watanabe T Waguri S Kametaka S Shibata M Yamamoto K Kominami E Peters C von Figura K Uchiyama Y Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.J Neurosci. 2000; 20: 6898-6906Crossref PubMed Google Scholar, 40Koike M Shibata M Ohsawa Y Nakanishi H Koga T Kametaka S Waguri S Momoi T Kominami E Peters C Figura K Saftig P Uchiyama Y Involvement of two different cell death pathways in retinal atrophy of cathepsin D-deficient mice.Mol Cell Neurosci. 2003; 22: 146-161Crossref PubMed Scopus (122) Google Scholar For light microscopy, brain tissues were quickly removed from the mice and further immersed in the same fixative for 2 hours at 4°C. Samples processed for paraffin embedding were cut into 5-μm sections with a semimotorized rotary microtome (RM2245; Leica, Nussloch, Germany) and placed on silane-coated glass slides. Samples for cryosections were embedded in OCT compound (Miles, Elkhart, IN) after cryoprotection with 15% and 30% sucrose solutions and cut into 10-μm sections with a cryostat (CM3050,; Leica). The sections were placed on silane-coated glass slides and stored at −80°C until used. For routine histological studies, paraffin sections or cryosections were stained with hematoxylin and eosin (H&E). Samples for electron microscopy were postfixed with 2% OsO4 with 0.1 mol/L phosphate buffer, block-stained in 1% uranyl acetate, dehydrated with a graded series of alcohol, and embedded in Epon 812 (TAAB, Reading, UK). For light microscopic observations, semithin sections were cut at 1 μm with an ultramicrotome (Ultracut N; Reichert-Nissei, Tokyo, Japan) and stained with toluidine blue. For electron microscopy, silver sections were cut with an ultramicrotome, stained with uranyl acetate and lead citrate, and observed with an electron microscope (H-7100; Hitachi, Tokyo, Japan). For double-immunofluorescent staining, cryosections were first incubated with anti-LC321Koike M Shibata M Waguri S Yoshimura K Tanida I Kominami E Gotow T Peters C von Figura K Mizushima N Saftig P Uchiyama Y Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease).Am J Pathol. 2005; 167: 1713-1728Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 37Lu Z Dono K Gotoh K Shibata M Koike M Marubashi S Miyamoto A Takeda Y Nagano H Umeshita K Uchiyama Y Monden M Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation.Arch Histol Cytol. 2005; 68: 71-80Crossref PubMed Scopus (41) Google Scholar (1:100) or anti-cleaved caspases-3 or -7 (1:100) at 4°C overnight, followed by goat anti-rabbit IgG coupled with Alexa Fluor 488 (Invitrogen, Grand Island, NY) for 1 hour at room temperature. For LC3 detection, further incubations were performed with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and finally with streptavidin coupled with Alexa Fluor 488 for 1 hour at room temperature. After immunostaining, terminal dUTP nick-end labeling (TUNEL) staining was performed as described below. The sections were then viewed under a confocal laser-scanning microscope (LSM 5 Pascal: Carl Zeiss, Jena, Germany; or FV1000: Olympus, Tokyo, Japan). For NeuN staining, deparaffinized sections or cryosections were autoclaved for 20 minutes in 10 mmol/L Na-citrate buffer (pH 6.1)36Komatsu M Waguri S Chiba T Murata S Iwata J Tanida I Ueno T Koike M Uchiyama Y Kominami E Tanaka K Loss of autophagy in the central nervous system causes neurodegeneration in mice.Nature. 2006; 441: 880-884Crossref PubMed Scopus (2754) Google Scholar before the incubation with NeuN (1:1000). For ubiquitin staining, sections were incubated with anti-ubiquitin (1:1000) without pretreatment. These sections were further incubated with biotinylated goat anti-rabbit or mouse IgG for 1 hour, and finally with peroxidase-conjugated streptavidin (Vector Laboratories) for 1 hour at room temperature. Staining for peroxidase was performed using 0.0125% 3,3′-diaminobenzidine tetrahydrochloride and 0.002% H2O2 in 0.05 mol/L Tris-HCl buffer (pH 7.6) for 10 minutes. TUNEL staining was applied to deparaffinized sections or cryosections as previously described.27Nitatori T Sato N Waguri S Karasawa Y Araki H Shibanai K Kominami E Uchiyama Y Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis.J Neurosci. 1995; 15: 1001-1011Crossref PubMed Google Scholar, 40Koike M Shibata M Ohsawa Y Nakanishi H Koga T Kametaka S Waguri S Momoi T Kominami E Peters C Figura K Saftig P Uchiyama Y Involvement of two different cell death pathways in retinal atrophy of cathepsin D-deficient mice.Mol Cell Neurosci. 2003; 22: 146-161Crossref PubMed Scopus (122) Google Scholar, 41Gavrieli Y Sherman Y Ben-Sasson SA Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (9100) Google Scholar Briefly, sections with or without a 10-minute proteinase K (20 μg/ml) pretreatment at room temperature were incubated with 100 U/ml terminal deoxynucleotidyl transferase (TdT) and 10 nmol/L biotinylated 16-2′-dUTP (Roche Diagnostics, Basel, Switzerland) in TdT buffer (100 mmol/L sodium cacodylate, pH 7.0, 1 mmol/L cobalt chloride, 50 μg/ml gelatin) in a humid atmosphere at 37°C for 1 hour, followed by further incubation with Alexa Fluor 594 or peroxidase-conjugated streptavidin (Vector Laboratories) for 1 hour at room temperature. Staining for peroxidase was performed as above. In the case of double-immunofluorescent staining, an in situ cell death detection kit, TMR red (Roche Diagnostics), was used according to the manufacturer's recommended protocol after immunohistochemistry. The number of neurons with pyknotic nuclei in sections stained by H&E and that of neurons positive for TUNEL or cleaved caspase-3, or double-positive for TUNEL and cleaved caspase-3 in sections doubly stained for TUNEL and cleaved caspase-3, respectively, were counted in the whole pyramidal regions of the hippocampus located in the same portion (bregma: −1.82 mm of adult C57BL/6J mice42Paxinos G Franklin BJK The Mouse Brain in Stereotaxic Coordinates. Academic Press, San Diego2001Google Scholar) by light microscopy (magnification, ×200) (three sections per animal). Data from three animals at each stage were averaged. H/I injury and neuroprotection were evaluated by the damaged area and the number of TUNEL-positive (dead) neurons in the pyramidal layers of the hippocampus 3 days after H/I injury using brain sections corresponding to the same portion of the hippocampus as mentioned above. The total and damaged areas in the whole pyramidal layers of the ipsilateral hippocampus from each animal were measured using a morphological analysis software version 2.5 (Mac SCOPE; Mitani Corp., Fukui, Japan). The damaged area was defined as a region of the pyramidal layers that was NeuN-negative (see Figure 7D). The degree of H/I injury 3 days after ischemic insult was classified into four grades: the number of TUNEL-positive neurons in the pyramidal layers of the hippocampus per section was less than 10 for grade 0, 11 to 100 for grade I, 101 to 200 for grade II, and more than 201 for grade III. The percentage of mice showing each grade was further calculated in the cases of Atg7-deficient mice (n = 17) and their littermate controls (n = 32). The damaged area in the hippocampal pyramidal layers was also examined 7 days after H/I injury, in which time clear-cut shrinkage occurred in the ipsilateral hippocampus of control littermate mice (Atg7flo/flox mice), resulting in loss of ipsilateral hippocampal areas. To quantitate these damaged areas after neonatal HI, the areas of lesioned and unlesioned hippocampi were measured and compared within the same section, using the same software as mentioned above.13Ness JM Harvey CA Strasser A Bouillet P Klocke BJ Roth KA Selective involvement of BH3-only Bcl-2 family members Bim and Bad in neonatal hypoxia-ischemia.Brain Res. 2006; 1099: 150-159Crossref PubMed Scopus (55) Google Scholar The area loss of the lesioned ipsilateral hippocampus was calculated as a percentage of the intact contralateral hippocampus and the area loss was determined for each animal by measurement of at least three sections according to the method mentioned above. The degree of hippocampal damage was measured using Atg7-deficient (n = 11) and their littermate control (n = 12) mice. Anesthetized mice were sacrificed by decapitation at 3, 8, 24, or 72 hours after H/I injury (n = 5 per group), and untreated control animals were sacrificed on P7 or at 8 weeks (n = 6 per group). The brains were rapidly dissected on a bed of ice. The left and right hippocampi were separately excised from the mice, frozen in liquid nitrogen, and stored at −80°C until used. Genomic DNA was isolated according to a previously described method.40Koike M Shibata M Ohsawa Y Nakanishi H Koga T Kametaka S Waguri S Momoi T Kominami E Peters C Figura K Saftig P Uchiyama Y Involvement of two different cell death pathways in retinal atrophy of cathepsin D-deficient mice.Mol Cell Neurosci. 2003; 22: 146-161Crossref PubMed Scopus (122) Google Scholar Briefly, each tissue was separately homogenized gently in 1 ml of a lysis buffer consisting of 4 mol/L guanidine thiocyanate and 0.1 mol/L Tris-HCl, pH 7.0, and incubated at room temperature for 1 hour. Then, 20 μl of QIAEX II suspension (Qiagen, Hilden, Germany) was added to each sample, and the samples were vortexed, incubated at room temperature for 1 hour, and finally spun at 12,500 × g for 2 minutes. The pellets were washed by resuspending them three times in a NEW Wash solution (GENECLEAN kit; Bio 101, Vista, CA) consisting of 50% ethanol, 100 mmol/L NaCl, 10 mmol/L Tris/HCl, and 1 mmol/L ethylenediaminetetraacetic acid, pH 7.5, followed by resuspension in a TE buffer consisting of 10 mmol/L Tris/HCl and 1 mmol/L ethylenediaminetetraacetic acid, pH 8.0. Samples were incubated at room temperature for 10 minutes and spun at 12,500 × g for 2 minutes. The s
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