Proteomics Screening Of Candidates Involved In Injury And Protection Of Brain Ischemia

Cerebral vascular diseases, nervous system diseases due to disabilities of cerebral vessels, are among those most popular diseases with high incidence, mortality rate, disabilities rate and recurrence rate. Ischemic stroke is the most popular type of cerebral vascular diseases, accounting for 70%-80% of them. Ischemic stroke is characterized by complex spatial and temporal events evolving over hours or even days. In the core ischemic territory, blood flow deficits, low ATP levels and energy stores, ionic disruption and metabolic failure are severe, and cell death progresses in minutes. However, the peripheral zones within the flow compromised territory—the ischemic penumbra—suffer milder insults due to residual perfusion from collateral blood vessels. Since relatively slow progress of active cell death in penumbra, it is reasonable to develop clinic therapy aiming at this area. The present study compared the proteome of hippocampal CA1 and its nature internal control CA3 neighboring subregions with distinct susceptibility to transient global ischemia, assuming that the proteomic difference indicates injury, protection and regeneration molecular mechanisms. There are at least three fundamental mechanisms leading to cell death during ischemic brain injury: excitotoxicity and ionic imbalance, oxidative/nitrosative stress, and apoptotic-like cell death. These mechanisms demonstrate overlapping and redundant features. They mediate injury within neurons, glia and vascular elements, and at the subcellular level, they impact the function of mitochondria, nuclei, cell membranes, endoplasmic reticula and lysosomes. Some known molecules are key factors in these pathways, such as ion channels, mitochondrial apoptotic components and nucleic transcription factors. Mitochondria and nuclei are the most important organelles involved in the above mechanisms. Mitochondria contain a number of proapoptotic molecules, including cytochrome c, secondary mitochondrial activator of caspase(Smac/Diablo), endonulease, apoptosis inducing-factor(AIF), and so on. Formation of permeable transition pore on mitochondrial membranes as an intial step of apoptosis makes this organelle the core of cell fate. Nuclei are not only the targets of injury, but the origin of protein synthesis involved in death and survival pathways as well. Up to now, mitochondria are considered mainly as the upstream of nuclei. Our present study profiled the mitochondrial proteomic variation after stroke to find new target molecules involved in cell injury and protection mechanisms. Two dimensional electrophoresis (2DE), the favourite of most proteomics researchers, has unique priority of separating and visualizing thousands of protein spots simultaneously by cheap apparatus and reagents. The "Silver Blue" staining method developed after Neuhoff's was announced to have achieved the sensitivity of nanogram, assuring more than a thousands of spots routinely after 1 mg of loading. We have obtained about 2000 spots of lmg tissue protein with much better reproducibility than silver staining. The biological mass spectrometry we've chosen were matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF MS. The TOF/TOF MS can deduce protein identity by de novo sequencing of the further dissociated ions after MALDI, which has much more hits than MALDI-TOF-MS. There have been a number of genomic and proteomic investigation on brain ischemia (whole brain, hippocampus or cultured cortex), employing oligonucleotide array, expressed sequencing tags (EST) cDNA array, high-throughput immunoblot, 2-DE tandem isotope coded affinity tags(ICAT), surface enhanced laser dissociation/ionization time-of-flight mass spectrometry (SELDI-TOF). 2DE-MS has been implemented to identify proteins differentially expressed in the CA1 and CA3 regions of the rat hippocampus and to assess changes in protein expression following a 6-h exposure to intermittent hypoxia. Proteomic investigation on CA1 and CA3 regions or mitochondria enduring ischemic stroke has not been tried as much as we know. The present study was mainly based upon 2-DE tandem mass spectrometry to pan out hippocampal subregions specific or mitochondrial targets involved in stroke, which was verified by immunological technique later.Rats after sham control operation and four-vessel occlusion (4-VO) of ischemia-reperfusion injury were transcardiacally perfused and fixed. Neurons were visualized by nissel staining on frozen hippocampus slices to examine the survival extent after operation at time points of 24 hours and 7 days. After injury, CA1 neurons began to die after 24hr (181.72±1.50), and collapsed broadly after 7 days (28.74±0.93), much less than the sham control (203.70±2.72) with statistical significance. Nevertheless CA3 neurons kept intact all along (table 1). Research on CA1 proteome 7 days after stroke when broad cell death had occurred was useless to reveal the molecular mechanisms before death. We chose 24hour after stroke when injury effect on neuron had begun before their death as the time point versus sham control of observation. Brain coronal sections were acutely obtained from sham control animals, followed by dissociation of hippocampal subregions CA1 and CA3. Tissue was prepared before two dimensional electrophoresis, gel staining and image scanning to obtain gel images with good separation and reproducibility. Differentially displayed proteins between sham control CA1 and CA3 were revealed by analysis of professional software. 8 of those 9 picked protein spots were successfully sequenced by MALDI-TOF/TOF-MS. Proteins differentially expressed between the sham control and 24hr stroke groups were also uncovered in the same way, and 21 of those 23 picked protein spots were successfully sequenced. Hippocampal mitochondria were separated from rat of sham control group and 24h after stroke group, and then examined by electron microscopy. The mitochondria were fixed, sliced, stained and observed, A large proportion of (70-80%) extracts were mitochondria with intact membrane and cristae. Mitochondrial proteins differentially expressed between the sham control and 24hr stroke groups were also uncovered in the same way, except for MALDI-TOF-MS instead of MALDI-TOF/TOF-MS, and 13 of those 24 picked protein spots were successfully sequenced. Among the sequenced mitochondrial proteins, prohibitin was up regulated after stroke and validated by immunoblot to scale its dynamics (1, 6, 12, 24hour after stroke). Immunoblot results shew that accumulation of mitochondrial prohibitin didn't change lhour after stroke, but was up regulated 6hours after stroke (one-ANOVA, p p＜0.05, n=3).The present study mined according to uniquely area specific susceptibility and key role of mitochondria in cell injury for molecular targets involved in stroke, mainly based upon the proteomic technologies of 2-DE tandem MS-TOF(/TOF)-MS. We found difference of proteome between sham control CA1 and CA3. The 8 successfully identified proteins were GFAP d (glial fibrillary acidic protein delta), Tpil (triose phosphate isomerase 1), aplysia ras-related homolog A2, rCRMP-3 (rat Collapsin response mediator proteins-3), HMGB1 (High mobility group box 1), platelet-activating factor acetylhydrolase isoform lb alpha2, protein similar to biliverdin reductase, glutamine synthetase 1。Glutamine synthetase 1 was more highly expressed in CA3, the others were more in CA1. rCRMP-3 may be a marker of postischemic neurogenesis. Tpil may contribute to higher susceptibility of CA1 for its higher expression in this area. Glutamine synthetase 1 can reasonably eliminate excess neurotoxic glutamate in CA3 to protect this area from stroke injury. We found differentially expressed proteins in CA1 after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated unnamed protein product (HSP60 family), CKB (creatine kinase B), LASPI(LIM and SH3 protein 1), ATP synthase, brain abundant, membrane attached signal protein 1, protein similar to biliverdin reductase, and up regulated PGAM1 (phosphoglycerate mutase 1), stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein), alpha B-crystallin, and DJ-1 protein. Creatine kinase B was not reported to be involved in ischemic area. We speculate that its role as a metabolic enzyme contribute to energy deficiency there. ATP synthase is the core enzyme to synthesize ATP, so its down regulation reasonably causes energy deficiency in ischemic area. Upregulation of PGAM1 indicated the increased glycolysis or energy deficiency in ischemic area; itself can be a potential therapeutic target. Whether the antiapoptotic role of alpha B-crystallin in myocarcial ischemia is also present in brain requires further investigation. Serum DJ-1 has already been considered as early marker of brain ischemia stroke, although its detailed local function in ischemic area remain unclear. We found differentially expressed proteins in CA3 after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated nucleoside diphosphate kinase and upregulated voltage-dependent anion channel-like protein, brain creatine kinase, guanine deaminase, SPNA2 (brain Spectrin alpha chain), alpha synuclein, ATP synthase beta subunit, Rat Liver F1-Atpase Chain B. VDAC is included in the PTPC (permeability transition pore complex) which is proved to allow some proapoptotic components to release, so VDAC may have some effect on progress of brain ischemia. There was no report about role of nucleoside diphosphate kinase and so on in brain ischemia as much as we know. Some mitochondrial proteins in hippocampus were found to be differentially expressed after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated HCNP (Hippocampal cholinergic neurostimulating peptide), PDHE1-A type I (Pyruvate dehydrogenase E1 component alpha subunit), LASP-1 (LIM and SH3 domain protein 1), ATP synthase beta subunit, Alpha-internexin, GSTp (Glutathione S-transferase pi), GMF-beta (Glia maturation factor beta), and upregulated Heat shock 70 kDa protein 1A/1B, Alpha-tubulin 3, Prohibitin, Synapsin-2, Heat shock cognate 71 kDa protein, and Guanine nucleotide-binding protein subunit beta 1. PDH (Pyruvate dehydrogenase) is key enzyme in TCA cycle (tricarboxylic acid cycle). Downregulation of PDH and ATP synthase would centainly interrupt the energy supply. GSTp previously implicated as stroke related gene's product may play protective role through inhibition of JNK and STAT pathways. Preceding research on prohibitin was primarily settled in productive organs and their derived carcinomas. Prohibitin was proved to be a key component of Rb-E2F pathway which plays the central role in mitosis. Recent studies have proved importance of aberrant cell cycle reentry before various nervous system diseases. Prohibitin has been shown to be involved in oxidative stress injury, which may due to its shuttle between mitochondria and nuclei. Our research can provided a new proof of crosstalk between those two organelles by prohibitin and its role in ischemic hypoxic brain diseases such as ischemic stroke. To verify the above hypothesis, we need to interfere the cellular expression and mitochondrial accumulation of prohibitin, and observe the survival of neurons and expression and activity of prohibitin related signal molecules...