| Stroke is the leading cause of disability and the third leading cause of death in adults worldwide and the mechanism which disrupts brain function and causes neuron death remains poorly understood.The main research contents are as follows:(1)In the chapter2,to searched for more potential molecules that regulate cell death in stroke,we performed mouse transient focal cerebral ischemia for 3 h or sham operation.To explored the proteomics profile after ischemia 3 h,we extracted the mouse affected brain area protein,the productions from both groups were separated on the SDS-PAGE and stained with coomassie blue.Surprisingly,compared to the control group,the protein bands on the gel with estimated size of 48 k Da were decreased in the ischemic group.We then analyzed the protein components in these bands using a LTQ Orbitrap Velos Rro Mass Spectrometry approach peptide sequences,were found to match Enolase1(ENO1).We next probed the ENO1 dynamic protein level with antibody against ENO1,consistent with Coomassie blue,ENO1 expression level was significant decreased after ischemia 3 h.To examined the brain distribution of ENO1,we performed RT-PCR to detected the m RNA levels of ENO1 in various regions,which including the brainstem,cortex,hippocampus,olfactory bulb,and cerebellum.In the brain,ENO1 was expressed ubiquitously in all brain regions.Furthermore,m RNA levels of ENO1 were expressed during the first week after birth toward adulthood.Next,we also carried out immunoblotting to test the protein expression levels of ENO1 in various regions.Consistent with the ENO1 m RNA levels expression,ENO1 was expressed general in all brain regions.Furthermore,ENO1 was highly expressed during the first week after birth,and then expression raised gradually toward adulthood.To confirmed whether neuron expression ENO1,we tested mouse primary cultured hippocampal neurons at Day in vitro 14(DIV14)with antibodies against ENO1 and MAP2(a Neuronal Marker).Our results demonstrated that ENO1 was enriched in cultured neurons preparations.We next investigated the subcellular localization of ENO1 in neuron,and the results showed that ENO1 colocalized with green fluorescent protein(GFP)labeled soma and dendrite.(2)In the chapter3,we next assessed whether OGD induce ENO1 expression change in cultured hippocampus neurons.Using immunoblotting,we quantified the ENO1 expression level after OGD 1-3 h.The cultured hippocampal neurons OGD 1 h or 2 h significantly increased the ENO1 expression level compared to the controls,respectively,and OGD 3 h significantly decreased ENO1 expression level.To examined the ENO1 subcellular distribution in cultured neuron after OGD,we measured the ENO1 in these neurons with quantitative immunostaining.The cultured neurons OGD 2 h significantly increased ENO1 in the cytoplasm,while cultured neurons treated with OGD for 3 h or 6 h resulted in a significant decreased in the cytoplasmic fluorescence intensity,respectively,which consistent with immunoblotting.As ENO1 expression level dynamic changed in focal cerebral ischemia mouse and in cultured neurons after OGD treated.We next assessed whether the overexpression of ENO1 in neurons protect neuron from death.We transfected ENO1-V5 plasmid and GFP plasmid in cultured hippocampal neurons at DIV 10 and OGD 1 h or 2 h at DIV14.Using immunocytochemistry,we quantified the length of dendrite and number of spine on the transfected(GFP-positive)neurons.The overexpression of ENO1 in the cultured hippocampal neurons significantly protected the dendrites and post-synaptic spine density compared to the control,as indicated by the number of spine-positive puncta along the transfected dendrites after OGD stimulation.The average length of dendrite and spine density between control and ENO1 overexpression neurons were not significantly changed,thus,overexpression of ENO1 in cultured hippocampal neurons protected spine loss and dendrite damages during OGD.Furthermore,the enzymatic product of ENO1,phosphoenolpyruvate(PEP),was also synchronously changed along with the dynamic ENO1 level.The neuronal injury caused by OGD treatment in vitro or ischemia in vivo was mitigated by the application of PEP.Moreover,deletion of the ENO1(homolog of mammalian ENO1)do not affected anoxia-starvation(AS)-induced worm death in C.elegans.(3)In the chapter4,to systematically profile proteome and phosphoproteome after cerebral ischemia onset,we took mice hippocampal of focal cerebral ischemia model and monitored changes in protein abundance using a combination of stable isotope labeling and high-resolution liquid chromatography-tandem mass spectrometry(LC-MS/MS).We identified 28 proteins that were dysregulated,184 phosphorylation sites of 135 proteins were upregulated and 689 phosphorylation sites of 420 proteins were downregulated at 2 h after focal cerebral ischemia.Moreover,RNA sequencing(RNA-Seq)shows that 415 genes were upregulated and 222 genes were downregulated.These proteins fall into a variety of functional categories,including those regulating synaptic structure,neurotransmission and excitatory receptors.Moreover,phosp horylation of synaptotagmin-1(syt1)at T112 site increased neuronal injury during OGD 2 h in vitro,deletion of the SNT-1(homolog of mammalian syt1)significantly decreased AS-induced worm death in C.elegans.(4)In the chapter5,frozen mouse brain slices at 4 h post-MCAO ischemia were employed for positive selection,and sham slices were used for negative selection to exclude nonspecific binding aptamers.To identify the aptamer candidates,the ss DNA of the 10 th pool was sequenced.One of the highly abundant sequences,LCW17 were selected to be synthesized and labeled with FAM for further research.(5)In the chapter6,To identify the target protein of aptamer LCW17,the homogenate protein of ipsilateral hemisphere 4 h post-ischemia was extracted and subjected to a pull-down experiment with biotin-labeled aptamer LCW17,respectively.The results showed a high level of Vigilin in the LCW17 binding band.To further verify the LCW17-binding target,we tested if aptamer LCW17 could directly bind to purified recombinant his-tagged Vigilin.Various amounts of aptamer LCW17 were used to conduct a pull-down assay.We next characterized the binding affinity of LCW17 to Vigilin and the equilibrium dissociation constant(Kd)was calculated as 25± 3 n M.We incubated FAM-labeled aptamer LCW17 with tissue slices obtained from mice across a time course of 2 h,3 h and 4 h of ischemia.We found a significant time-dependent increased in the sum of integrated intensity of the fluorescent clusters.Next,the conditional medium of both control and OGD treatment were harvested and subsequently precleaned with a centrifuge followed by immunoblotting.Results showed that the cultured medium with different duration of OGD treatments contained higher protein Vigilin levels than those of controls,respectively.At last,we injected 60 pmol Cy5-labeled aptamers into the brain of 3 h ischemia mouse.The biodistribution of the aptamers in the brain was imaged ex vivo at 1 h post-injection.Obviously,after stereotactic injection of aptamer Cy5-LCW17,the intensity of the fluorescence in the CA1 area of the ischemic brain is about twice of the sham.In the control experiment,after stereotactic injection of 60 pmol Cy5-Libray,the fluorescence signal is very low.Taken together,our data revealed that ENO1 plays a novel and protective role in cerebral ischemia-induced neuronal injury,highlighting a potential of ENO1 as a therapeutic target of neuronal protection from cerebral ischemia.Global phosp hoproteome showed that phosphorylation of syt1 at T112 site increased neuronal injury during OGD 2 h in vitro,deletion of the SNT-1(homolog of mammalian syt1)significantly decreased AS-induced worm death in C.elegans.Moreover,ischemic brain slice-based aptamer selection will enable identification of more probes and potential target molecules for diagnosis and therapy of ischemic stroke.Aptamer LCW17 and Vigilin may potentially be applied to define the molecular mechanism underlying ischemic stroke,as well as its diagnosis. |
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