Development-dependent Regulation Of Molecular Chaperones After Hypoxia-ischemia

Background: Transient brain hypoxia-ischemia(HI) leads to delayed neuronal death and neurological deficits in both neonates and adults. However, the susceptibility of brain to HI differs by maturity of neuron. Although some investigators report that immature brains are less vulnerable to HI than mature brains, the underlying mechanisms are unknown. Our group has been focusing on misfolding, aggregation, and accumulation of proteins as a factor affecting neurotoxicity. Molecular chaperones play critical roles in these pathways, and their expression influence the resistance of cells to stress. In this study, the expression of common cytosolic and endoplasmic reticulum(ER) chaperone after HI in post-natal 7(P7) and 26(P26) days rat brains was observed to clarify the mechanism of the age-dependent vulnerability of brains to HI.Part I: The different expression of molecular chaperones m RNA and protein after hypoxia-ischemia between different ageMethods: Hypoxia-Ischemia model: Post-natal Sprague-Dawley rats at P7 and P26 were anesthetized with halothane, the left common carotid artery was exposed and ligated, and incision was sutured, thereafter the pups or rats were returned to their dams or original cages. Following 1 h of recovery, the rats were placed in a hypoxic chamber through which humidified 8% oxygen with the balance of nitrogen flowed for 60 min for groups P7, and for 30 min for group P26. The brains with HI were collected at 30 min, 4, 12 and 24 h after HI. Sham operated control brains were also collected at 4 h in the same way as the HI rats. In situ hybridization: Brains were removed under RNAse free conditions and frozen by immersion in dry ice cooled 2-methylbutane. Coronal sections two levels(bregma 0.70 mm and-4.16 mm) were cryosectioned at 12 μm onto glass slides, and stored at-80 °C. Sections were fixed in 4% formaldehyde-PBS and acetylated in 0.25% acetic anhydride and 0.1 M TEA–HCl(p H 8), then dehydrated with ethanol and chroloform. A 248 bp Apa I–Xho I fragment from the 3′end of rat Hsp70 c DNA and full-length GRP78 c DNA were subcloned into p GEM7 zf for use as a probe. Plasmids containing partial sequences of Hsp60 were provided by Dr. Kokame(National Cardiovascular Center Reserch Institute, Japan). 35 S labeled anti-sense riboprobes generated by in vitro transcription using the Promega Riboprobe? System. The denatured probe was added to a solution containing 100 μg/ml salmon sperm DNA, 250 μg/ml each of yeast total RNA and t RNA, 50% formamide, 20 m M Tris–HCl(7.4), 1 m M EDTA, 300 m M Na Cl, 10% dextran sulfate, and 1×Denhardts. The hybridization solution was added to the sections and hybridized at 55 °C overnight. After hybridization, the sections were treated with 20 μg/ml RNAse A for 30 min at 37 °C, and washed with 0.1x SSC containing DTT at 65 °C. Sections were dehydrated with ethanol and exposed Kodak Bio Max MR films. Western blotting: Brains was homogenized using a Dounce homogenizer in lysis buffer:15 m M ris-HCl p H 7.6, 0.25 M sucrose, 1 m M Mg Cl2, 2.5 m M EDTA, 1 m M EGTA, 1 m M DTT, 1 μg/ml pepstatin A, 2.5 μg/ml aproptonin, 10 μg/ml leupeptin, 1 m M PMSF, 0.1 m M Na3VO4, 25 m M Na F, and 2 m M Na4P2O7. The homogenates were centrifuged to get subcellar fractions. The 800 x g precipitate containing nuclei(P1), 10,000 x g precipitate containing mitchondria and synaptosomes(P2), and 165,000 x g precipitate containing microsomes(P3) and cytosol(S3) fractions were obtained. Protein concentration was determined by a microbicinchoninic acid(BCA) method. Samples were denatured at 95oC with 1x sample buffer containing SDS and DTT. Equal amounts of protein from each fraction were electrophoresed(8% SDS-PAGE) and western blotted. All antibodies were purchased from Stressgen, and used at the manufacture recommended concentration. The blots were developed using the ECL detection method, and images of immunoblots were analyzed with Kodark ID image analysis software. Data are presented mean±SEM. Statistical analyses are one-way ANOVAs with post-hoc Tukey HSD t-tests. * denotes P<0.01. Immunohistochemistry: Immunohistochemistry and double-label fluorescence confocal microscopy were performed as previously described.Results:(1) Hsp70 m RNA is induced after HI dracitically in P26 rat brain, but weakly in P7. Induction of Hsp70 m RNA was seen in the ipsilateral hemisphere throughout the cortical, striatal, hippocampal and subcortical areas, and occurred as early as 0.5h, peaked at 4h, and remained elevated at 24 h of recovery at both striatal and hippocampal levels after HI. This occurred mainly in the ipsilateral hemispheres of P26 rat brains. Compared with P26 brain regions, induction of Hsp70 m RNA in P7 brains was particularly weak in the ipsilateral and was not detectable in the contralateral brains after HI. Hsp60 m RNA was induced significantly in P26,but the induction was negligible in P7 brain sections after HI. ER GRP78 m RNA was also induced moderately,but significantly in P26 and minimally in P7 brain regions at 4 or 24 h of recovery after HI.(2) Hsp70 protein expression is increased after HI dracitically in P26 rat brain, but weakly in P7. Consistent with Hsp70 m RNA expression,Hsp70 protein was consistently induced after HI dramatically in P26 brain samples, but only slightly in P7 brain samples. Hsp 60 protein did not reach to the statistically significance during the measured recovery time points after HI. Similarly, the level of GRP78 protein was not significantly changed in both P7 and P26 brain samples after HI.(3) Nuclear translocation of HSF1 after HI. The HSF1 protein redistribution between nuclear and cytosolic fractions was further studied in both P7 and P26 brain samples after HI. Western blotting revealed that HSF1 was redistributed from the cytosolic S3 fraction to the nuclear P1 fraction during 0.5,4,and 24 h recovery periods, which occurred markedly in P26, but minimally in P7 brain samples after HI.(4) The double staining confocal microscopy of brain sections with HSF1(red color) and HSP70 antibodies(green color) shows the level of the P26 HSF1 protein immunoreactivity was increased in the nuclei if the CA1, DG and necortical regions slightly at 30 min, and reaching a plateau for 4-24 h after HI. Correspondingly, the level of the P26 HSP70 protein immunolabeling was upregulated, but in a relatively delayed fashion in the cytoplasm of the CA1, DG and neocortical regions, e.g, slightly at 4h and markedly at 24 h after 24 h after HI.In comparison with those in P26 brain sections, the level and pattern of the HSF1 protein immunoreactivities in P7 brain sections were not altered in the CA1, DG and Cx regions after HI. Consistently, in P7 brain sections, HSP70 protein was also not significantly induced in neurons. And HSP70 protein is induced in microglia of P7 brain but not astrocyte.Conclusions:(1) Although mature brains are more vulnerable to HI than inmature brains, Hsp70 m RNA induction in mature brain is significantly more drastic than in inmature brain.(2) Compare to the expression of HSP70 m RNA, the induction of HSP70 protein was delayed after HI.(3) HSP70 protein is induced in microglia of P7 brain but not astrocyte. 4. HSF1 occurred nuclear translocation after HI in mature brain.Part II: The residential levels of molecular chaperones and folding enzymeMethods: P7 and P26 Sprague-Dawley rats. Western blottin g:Equal protein amounts in P7 and P26 were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels(SDS-PAGE) and then transferred to Immobilon-P membranes.The membranes were incubated with 3% BSA in TBS for 30 min, and then overnight at 4℃ with HSF1, HSC70, HSP40, GRP78, GPR94, PDI and HSP60 antibody.After washing, the membranes were further incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies for 1h at room temperature.The β-actin levels on the immunoblots were also labeled as endogenous protein loading controls.The immnoblots were developed using enhance chemiluminescence and developed on Kodak X-Omat LS film.Densitometry was performed with Kodak ID image analysis software.Results: The HSF1 level was significantly higher in P26 than P7 brain samples. The level of cytoplasmic residential chaperones HSC70 and HSP40 were virtually the same between P7 and P26 brain samples.The level of residential ER molecular chaperones and folding enzymes GRP78,GRP94 and PDI were slightly but significantly lower in P26 compared to P7 brain samples.The mitochondrial matrix HSP60 level was significantly higher in P26 than P7 brain samples.Conclusions:(1) Compare to the immature brains, HSF1 is significantly higher in mature brains.(2) HSC70 and HSP40 are almostly the same level in immature and mature brains.(3) Compare to the immature brains, HSP60 is higher in mature brains.4. Compare to the immature brains, GRP78, GRP94 and PDI are lower in mature brains.