Differential Proteomic Analysis Of Cerebral Synaptosomes Isolated From Vascular Cognitive Impairment Rats

With the aging of population, the number of people suffering from dementia will increase gradually. However, the treatment strategies for dementia were of limited value, and once the diagnosis was established, the symptoms would deteriorate and be irreversible, which brought a heavy economic burden to the social and family. Vascular cognitive impairment (VCI) was deemed to be a common type of dementia, and it was one of the minority types of dementia which could be treatable and preventable. To prevent and delay the occurrence and development of VCI had been global economic issues and focuses, which clinicians and researchers paid attention to. Chronic cerebral hypoperfusion (CCH) was referred to as an ischemic cerebrovascular disease. All factors leading to hypoperfusion can resulted in progressive or permanent cognitive impairment. CCH had been proposed to play a pivotal role in VCI and Alzheimer's disease (AD). CCH was an emerging concern and increasing evidence showed that it might be responsible for the cognitive deficits associated with VCI. Necrosis, loss and apoptosis of neurons were implicated in the pathogenesis of VCI associated with CCH.Besides, CCH triggered cleavage of the amyloid precursor protein (APP) into Aβ-sized fragments and resulted in progressive accumulation of Aβpeptides detected, which reproduced features of Aβbiogenesis characteristic of sporadic Alzheimer's disease. There was evidence demonstrated that synapse damage occurred in chronic cerebral hypoperfusion. However, the specific mechanisms involved in the conversions from CCH to VCI were still obscure. In this study, we identified the differentially expressed proteins in prefrontal cortex and hippocampus synaptosomes in VCI rats induced by CCH by proteomics, and explored the changes of synaptic function involved in the VCI pathogenesis at molecular level.Recent researches showed that synapse loss in AD victims existed before cognition declined, and the degree of the synapse loss correlated with the cognitive severity. On the ground of the above, it was deemed that the synaptic dysfunction was a leading factor of AD and scholars were inclined to regard the synaptic failure as one of the initial factors. Synapse was the basic unit of neurotransmitter release, and tens of synaptic proteins were implicated in the process. The synapse related proteins'changes could generate the dysfunction of synapse, cognitive deficits and the death of neurons. As yet, with respect to the alternation of synaptic function that contributed to VCI insulted by CCH had not been delineated. Synaptosomes were employed to study the function of synapse. Synaptosomes had integrated structure; postsynaptic membrane connected with the presynaptic membrane and the cleft was retained. Synaptosomes were equivalent to intact cells, and the neurotransmitter stayed the same as before. Synaptosomes contained the synaptic related proteins chiefly, rarely the others. The synaptosomes lysate was composed of sub-celluar components such as cytoplasm, mitochondria, plasma membrane and synaptic vesicle. Synaptosomes played a pivotal role in the basic research of celluar or molecular function of synapse, which improved specify. Differential proteomics was applied to compare proteins between pathological tissue (cell or body liquid) and the control or the different stage one in the way of quantity, expression level and modification. It helped to discover the disease related proteins and provided an approach to the pathogenesis and biomarkers for diagnosis or treatment. Recent years, mass data of ptoteomic analysis of the brain in AD were obtained. It was demonstrated that individual proteins between post mortem brain tissues from persons with AD, and those from age-matched nondemented control (NC) tissues were differentially expressed by means of quantitative proteomic analysis. In severely injured brain regions,76 proteins were differentially expressed in AD hippocampus compared with NC,62 proteins were differentially expressed in temporal cortex, and 39 proteins were differentially expressed in entorhinal cortex. Thrity-four proteins were differentially expressed in AD cerebellum compared with NC。In the present study, we utilized permanent occlusion of bilateral common carotid arteries (2VO) to induce the model of VCI resulted from CCH. According to the alternations of the ultrastructure and the pilot studies, we deduced that the turning point of the reversible cerebral injury was within 1 month. Sucrose gradient centrifugation was employed to extract prefrontal cortex and hippocampus synaptosomes at 2 week and 4 week of the operation of 2VO. And then we utilized 2D-DIGE combined with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to determine the differentially expressed proteins of synaptosomes between model and the control one.Main research contents were as follows(1) Observation of the ultrastructure of VCI rats induced by CCHWe observed the ultrastructure of the rats at different stages (1m,2m,2m, and 4m). In prefrontal cortex we found that there was a lot of lipofuscin in 1 month, and the number declined in 2 month; then it was similar to the sham-treated control group. While the number of dark neuron reach the top in 2 month and then fall. In hippocampus we observed that lysosome in the cytoplasm of neuron increased as time; dark neuron teemed and lipofuscin presented in large numbers in 3 month; nuclei became pyknotic in 4 month. It suggested that to some extent prefrontal cortex can resist the ischemia and anoxia induced by 2VO at 1 month, while as the ischemia time prolonging, the compensation became invalid, and in hippocampus the CCH might be mainly involved in the apoptosis. In sum, the changes of lipofuscin and lysosome viewed by electron microscopy suggested that brain damage in initial stage of the chronic forebrain ischemia was reversible. Tanaka et al. found that CCH leaded to reversible alternations in central monoaminergic neuronal function within three weeks of ligation. Preliminary works done by our research group members indicated that the regional cerebral blood flow (rCBF) declined in both young and adult rats at 1 month, and the rats showed behavior changes; significant decrease of synaptophysin in hippocampus and dentate gyrus detected by immunohistochemistry staining; the positive cells number of MAP-2 and NF200 in cortex and hippocampus stained by immunohistochemistry was on the decline with the extension of ischemia, but there was a reversible change of the morphology of MAP-2 and NF200 in the first month; HE staining showed that fibers in subcortical white matter were loosen, myelin fractured and demylinizating in the 2VO rats from one month on, which got ingravescent as time went on; image-analysis system proved that white matter started to show myelinoclasis and present vesicle-like alternations at 1 month. From the above, we deduced that the turning point of reversible cerebral damage was within 1 month.(2) Proteomic analysis of synaptosomes isolated from different brain regions of VCI rats induced by CCHThe synaptosomes were extracted from the prefrontal cortex and hippocampus at 2 week and 4 week after the ligation respectively.2D-DIGE combined with MALDI-TOF MS was applied to determine the proteins that were differentially expressed in synaptosomes of prefrontal cortex and hippocampus.|AR|>1.3, P< 0.05 were set to select the differential protein spots. Nine proteins in synaptosomes were identified by mass spectrum. Six were mitoenergetic proteins:1) ATP synthase beta subunit.2) Proteins in the electron transport chain:NADH dehydrogenenase (ubiquinone) Fe-S protein 1 (NDUFS1) and ubiquinol-cytochrome c reductase core protein 1 (UQCRC1).3) Proteins involved in Krebs cycle: dihydrolipoamide acetyltrasnsferase (PDC-E2), isocitrate dehydrogenase 3 (NAD+) alpha precursor (IDH3A) and malate dehydrogenenase (MDH). Besides, hsp70, G protein a subunit (Ga) and voltage-dependent anion channel 1 (VDAC1) were also verificated. The variation trends of proteins in synaptosomes were as follows:the 6 mitoenergetic proteins and hsp 70 were elevated at 2 week after the operation, and subsided to baseline at 4 week. Ga was downregulated at 2 week synaptosomes in prefrontal cortex. VDAC1 of prefrontal cortical synaptosomes was upregulated at 4 week.The changes in the 9 proteins in synaptosomes of VCI rats at different stages were analyzed. The mitoenergetic proteins (enzymes in Krebs cycle, proteins in the electron transport chain—NDUFS1, UQCRC1—and ATP synthase beta subunit) were elevated at 2 week and dropped back to the level of the control, which indicated that the metabolic reserve in synaptosomes was aroused within the turning point (4 week), and NDUFS1, UQCRC1 was reffered to the generation of reactive oxygen leading to dysfunction of mitochondia and further synapse oxidative damage and apoptosis. Whether the transient decline of Gαat 2 week affected cognition or not needed further investigation. Our results implied that metabolic reserve existed in turning point from CCH to VCI, and the effect anti-oxidative stress and anti-apoptosis of hsp70 and Ga might transiently existed, which probably help cognitive save. The disappearance of the protective mechanism mentioned above and the VDAC1 stimulating apoptosis may be implicated in the pathogenesis of VCI.