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Down-regulation of parkin protein in transient focal cerebral ischemia: A link between stroke and degenerative disease?

ABSTRACT: Ubiquitylated protein aggregates are characteristic features of neurodegenerative disorders that are also found in acute pathological states of the brain such as stroke. Many of the proteins connected to neurodegenerative diseases play a role in the ubiquitin-proteasomal pathway. Mutation of one of these proteins, the E3 ubiquitin ligase parkin, is the cause of autosomal recessive juvenile Parkinson's disease. Here we show that transient focal cerebral ischemia of 1-h duration induces marked depletion of parkin protein levels, to 60%, 36%, 33%, and 25% of controls after 1, 3, 6, and 24 h of reperfusion, but that ischemia does not cause lower protein levels of E2 ubiquitin-conjugating enzymes Ubc6, Ubc7, or Ubc9. After 3 h of reperfusion, when parkin protein levels were already reduced to <40% of control, ATP levels were almost completely recovered from ischemia and we did not observe DNA fragmentation, suggesting that parkin depletion preceded development of neuronal cell death. Up-regulation of the expression of parkin has been shown to protect cells from injury induced by endoplasmic reticulum (ER) dysfunction, and this form of cellular stress is also triggered by transient cerebral ischemia. However, in contrast to observations in neuroblastoma cells, we saw no up-regulation of parkin expression in primary neuronal cell cultures after induction of ER dysfunction. Our data thus suggest that ischemia-induced depletion of parkin protein may contribute to the pathological process resulting in cell injury by increasing the sensitivity of neurons to ER dysfunction and the aggregation of ubiquitylated proteins during the reperfusion period.

SUBMITTER: Mengesdorf T

PROVIDER: S-EPMC137541 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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Down-regulation of parkin protein in transient focal cerebral ischemia: A link between stroke and degenerative disease?

Mengesdorf Thorsten T Jensen Poul H PH Mies Gunter G Aufenberg Christoph C Paschen Wulf W

Proceedings of the National Academy of Sciences of the United States of America 20021101 23

Ubiquitylated protein aggregates are characteristic features of neurodegenerative disorders that are also found in acute pathological states of the brain such as stroke. Many of the proteins connected to neurodegenerative diseases play a role in the ubiquitin-proteasomal pathway. Mutation of one of these proteins, the E3 ubiquitin ligase parkin, is the cause of autosomal recessive juvenile Parkinson's disease. Here we show that transient focal cerebral ischemia of 1-h duration induces marked dep ...[more]

PMID: 12415119

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Project description:Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases. Experiment Overall Design: affy platform; replicate samples; two treated samples, two placebo. Experiment Overall Design: MIAME compliance: All samples were run in commercial arrays from Affymetrix, using Rat Genome U34A GeneChip arrays as described in the Affymetrix web site. The sample information is described below. Likewise, the Affymetrix protocol is detailed and referenced above. These descriptions include all information currently considered under the MIAME supportive guidelines, with which the JHMI Microarray Core Facility abides in all its procedures. Experiment Overall Design: Methodology. Expression profiling using DNA microarrays (One round amplification): Experiment Overall Design: Experimental Design and Sample Information. Transient focal cerebral ischemia was induced in ovariectomized female rats with and without replacement with 17beta-estradiol by intraluminal middle cerebral artery occlusion (MCAO), as described in the manuscript. At 6 and 24 hours after 2-hour MCAO, total RNA was prepared from the ischemic and contralateral cerebral cortex and striatum between coronal levels 2 and 3 mm relative to Bregma, which represent the core of MCA territory. RNA samples from 3 animals were combined into one pooled sample and hybridized to Affymetrix RG-U34A GeneChip, as described below. Each microarray experiment was performed in duplicate using two independent pooled samples per group (6 animals per group at each time point, 2 pooled samples for each brain region per time point). Experiment Overall Design: Microarray Hybridization Protocol. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN). RNA from control and experimental tissue are processed using the RNA amplification protocol described by Affymetrix (Affymetrix Expression Manual). Briefly, 5 micrograms of total RNA were used to synthesize first strand cDNA using oligonucleotide probes with 24 oligo-dT plus T7 promoter as primer (Proligo LLC, Boulder, Colorado) and the SuperScript Choice System (Invitrogen, Carlsbad, California). Following the double stranded cDNA synthesis, the product was purified by phenol-chloroform extraction, and biotinilated anti-sense cRNA was generated through in vitro transcription using the BioArray RNA High Yield Transcript Labeling kit (ENZO Life Sciences Inc., Farmingdale, New York). 15 ug of the biotinilated cRNA were fragmented at 94 C for 35 min (100mM Tris-acetate, pH 8.2, 500mM KOAc, 150mM MgOAC), and 10ug of total fragmented cRNA were hybridized to the Affymetrix U34A GeneChip arrays for 16hr at 45 C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was then used to wash and stain the Chips, removing the non-hybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinilated cRNA. The staining was then amplified using goat IgG as blocking reagent and biotinilated anti-streptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate. Experiment Overall Design: Quality Control. To ascertain the quality control of the total RNA from the samples, we used the Agilent Bioanalyzer, Lab on a Chip technology, and confirmed the rRNA ratios and clean run patterns of the samples. Likewise, this technology is used to confirm the quality of the RNA in the form of cRNA and fragmented cRNA. To assess the QC of the hybridization, GeneChip image, and comparison between chips, we confirmed the following parameters: scaling factor values within comparable range; low background values (50-95); high percentage of present calls (between 30 and 55%); consistent 3â/5â ratios of GAPDH as representation of housekeeping genes, and presence or absence of Bio B and C as internal spike controls.

Dataset Information

Down-regulation of parkin protein in transient focal cerebral ischemia: A link between stroke and degenerative disease?

Publications

Down-regulation of parkin protein in transient focal cerebral ischemia: A link between stroke and degenerative disease?

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