| Stroke remians a vexing public health problem leading to the high rates of death and disability.Currently,thrombolysis with tissue plasminogen activator(tPA)remains the only approved agent for treating ischemic stroke patients,although many approaches have been tried in the attempt to reduce the devastating impact of stroke.Unfortunately,however,less than 6 % of all ischemic stroke patients are treated with tPA and most stroke victims receive only supportive care,due to the narrow therapeutic window,risk of symptomatic intracerebral hemorrhage,perceived lack of efficacy in certain high-risk subgroups,and a limited pool of neurological and stroke expertise in the community.Thus,there is a great need to investigate options to treat those patients,representing the vast majority,in whom tPA or interventional approaches are not feasible or contraindicated.Neuroprotection is an alternative approach to thrombolysis.Over the past two decades,many molecular targets have been identified in order to achieve neuroprotection.Disappointingly,in clinical trials,promising pre-clinical studies of neuroprotectants have not been translated into positive outcomes.Although reasons for the failures are exceedingly complex,narrow therapeutic window,single target and disregard for repair may be critical,as reticular pathophysiological events occur in the peri-infarct area over days to weeks,including inflammation,oxidant stress,apoptosis and neuroplasticity-mediated circuit reorganization. Epigenetics refers to changes in a chromosome that affect gene activity and expression,but can also be used to describe any heritable phenotypic change that does not derive from a modification of the genome.As critical components involved in the process of epigenetics,Histone deacetylases(HDACs)modulate gene expression and cellular signaling,such as inflammation,oxidant stress,apoptosis and neuroplasticity-mediated circuit reorganization.HDACs deacetylate conserved lysine amino acids on histones,leading to chromatin modification,and therefore,silence gene transcription.HDAC inhibitors have been shown to be neuroprotective when administrated in the acute phase of stroke.However,the role of HDACs in the delayed phase of stroke rocovery remians unclear.Here,we investigated the following questions:(1)Whether a new time window for stroke functional recovery existed and HDACs are involved in the pathophysiological process of stroke recovery?(2)The mechanisms underlying epigenetic modulation in promoting stroke recovery?Charpter 1.HDAC2 is the key mediator for the secondary functional loss after stroke.To investigate whether a new time window for stroke functional recovery existed,we produced a photothrombotic stroke in mice and daily detected motor function by measuring the number of foot faults in the grid-walking task and forelimb symmetry in the cylinder task 1-14 d after stroke.Interestingly,a marked functional recovery occurred 2-4 d compared to 1 d,motor function impairment was again exacerbated 5-7 d compared to 3 d and gradually improved from 8 d after stroke,indicating a typical secondary functional loss phase from 5 to 7 d after stroke.To investigate whether epigenetic mechanisms contribute to secondary functional loss after stroke,Western blot was performed to assess the histone acetylation level in the peri-infarct tissue.Ischemia caused a substantial decrease in histone acetylation in the peri-infarct cortex of photothrombotic stroke to motor cortex in mice at 6 d after stroke.Thus,HDACs may contribute to the secondary function loss after stroke.Histone acetylation is a dynamic process controlled by the opposing actions of two large families of enzymes:the histone acetyltransferases(HATs)and HDACs.In this study,we focus on the role of HDACs in stroke functional recovery To investigate the role of HDACs in the secondary functional loss phase,we infused a pan-HDAC inhibitor TSA daily into the peri-infarct cortex of conscious mice via an implanted microcannula 2-4,5-7,or 8-10 d after stroke and tested motor function at 3 d before stroke and 24 h after the last TSA injection.These TSA treatments significantly increased acetyl-H4 level in the peri-infarctcortex,compared to vehicle.Surprisingly,treatment with TSA 5-7 d but not 2-4 or 8-10 d after stroke significantly ameliorated ischemia-induced motor function impairment,suggesting that HDACs plays a critical role in the secondary functional loss phase.HDACs have been divided into classes I(HDAC 1-3,8),II(HDAC 4-7,9and10),?(Sirtuins)and ?(HDAC 11).To examine effect of class-specific HDACs inhibitors,we daily infused MGCD0103,a selective inhibitor of the class I HDACs,including HDAC1,HDAC2 and HDAC3,and TMP269,a selective inhibitor of the class ?a HDACs,including HDAC4,HDAC5,HDAC7 and HDAC9,into the periinfarct cortex of conscious mice 5-7 d and measured motor function at 8 d after stroke.Only MGCD0103 ameliorated ischemia-induced functional impairment,although both of drugs inhibited the activities of related HDACs.Moreover,treatment with MGCD0103 5-7 d after stroke(10 mg/kg/d,i.p.)significantly improved motor function of the rats subjected to transient middle cerebral artery occlusion(tMCAO)for 120 min.Thus,the class I but not class II HDACs mediate the secondary functional loss after stroke.Furthermore,administration of SAHA,a selective inhibitor of HDAC1 and HDAC2,which has been approved by FDA to treat cancer,5-7 d after stroke promoted functional recovery from stroke to a similar extent as that observed in the stroke mice treated with MGCD0103,suggesting that HDAC1 and HDAC2 contribute to the secondary functional loss after stroke.HDAC2 is an isoform negatively regulating cell survival and neuronal plasticity.In contrast,HDAC1 gain of function has potent protection against neurotoxicity in vivo model for ischemia.Thus,HDAC2 may be a key mediator for the secondary functional loss after stroke.Accordingly,we infused adeno-associated virus vector AAV-CAG-EGFP-Cre into the peri-infarct cortex of HDAC2flox/flox mice immediately after photothrombotic stroke,and found that this treatment significantly improved motor function at 8 d after stroke.Moreover,we generated an adenovirus vector selectively expressing HDAC2(Ad-HDAC2-Flag)and its control expressing inactive HDAC2(Adinactive-HDAC2-Flag),and infused them into the peri-infarct cortex of mice immediately after stroke and measured motor function at 8 d after stroke.AdHDAC2-Flag substantially worsened the stroke-induced functional impairment 8 d after stroke,compared to its control.Collectively,HDAC2 in the peri-infarct cortex plays a critical role for the secondary functional loss after stroke.To determine whether HDAC2 is a unique target for effect of HDACs inhibitors on motor function and avoid the developmental abnormality that occurs following deletion of HDAC2 during development,HDAC2 conditional knockout(HDAC2CKO)mice were generated by crossing HDAC2flox/flox mice with EMX1-Cre transgenic mice.We infused TSA daily into the peri-infarct cortex of conscious HDAC2 CKO mice and WT littermates 5-7 d after stroke,and tested motor function at 8 d after stroke.Compared to WT mice,HDAC2 CKO mice displayed significantly decreased number of foot faults and improved forelimb symmetry,further suggesting that HDAC2 loss of function enhanced motor function recovery from stroke.Importantly,treatment with TSA was ineffective in HDAC2 CKO mice,although the drug significantly ameliorated stroke-induced functional impairment in WT mice.Moreover,we infused Ad-HDAC2-Flag or its control Ad-inactiveHDAC2-Flag into the peri-infarct cortex of HDAC2 CKO mice immediately and measured motor function at 8 d after stroke.Ad-HDAC2-Flag significantly worsened the stroke-induced functional impairment 8 d after stroke but had no effect on shamoperated mice,suggesting that the functional rescue effect of HDAC2 CKO mice is HDAC2 activity-dependent.Together,these findings support the notion that HDAC2 rather than other HDACs contributes to the secondary functional loss after stroke.To investigate the long-lasting effect of inhibiting HDACs in the secondary functional loss phase,We infused MGCD0103 into the peri-infarct cortex of conscious mice from 5 d to 7 d and detected motor function by measuring the number of foot faults in the grid-walking task and forelimb symmetry in the cylinder task at 11,18 and 25 d after stroke.Mice subjected to photothrombotic stroke in the forelmib motor cortex showed a marked impairment of motor function and MGCD0103-treated mice displayed significantly better motor function than vehicletreated mice did during 11-25 d after stroke,indicating an early and sustained functional recovery.Furthermore,we generated a lentiviral vector containing shRNA of HDAC2,and infused it into the peri-infarct cortex of mice immediately after stroke and detected motor function at 11,18,and 25 d after stroke.LV-HDAC2-shRNA-GFP substantially ameliorated stroke-induced functional impairment during 11-25 d after stroke,compared to its control LV-GFP.Moreover,we generated an adenovirus vector selectively expressing HDAC2(Ad-HDAC2-Flag)and its control expressing inactive HDAC2(Ad-inactive-HDAC2-Flag),and infused them into the peri-infarct cortex of mice immediately after stroke and detected motor function 11,18 and 25 d after stroke.Ad-HDAC2-Flag substantially worsened the stroke-induced functional impairment 11-25 d after stroke,compared to Ad-inactive-HDAC2-Flag.Collectively,our findings demonstrate that HDAC2 is a crucial target negatively controlling early and sustained behavioral recovery from stroke.Moreover,we generated HDAC2 conditional knockout(HDAC2 CKO)mice,in which,HDAC2 was deleted in all neural progenitor cells,by crossing HDAC2flox/flox mice with Nestin-Cre transgenic mice.We then infused TSA,a panHDAC inhibitor,into the peri-infarct cortex of conscious HDAC2 CKO mice and HDAC2flox/flox littermates 5-7 d after stroke,and tested motor function at 11 d after stroke.Compared to HDAC2flox/flox mice,HDAC2 CKO mice displayed significantly decreased number of foot faults and improved forelimb symmetry,suggesting that conditional knockout of HDAC2 promotes motor function recovery from stroke.Importantly,treatment with TSA was ineffective in HDAC2 CKO mice,although the drug significantly ameliorated stroke-induced functional impairment in HDAC2flox/flox littermates,indicating unique significance of HDAC2 in promoting early and sustained stroke functional recovery.Charpter 2.The mechanisms underlying class I HDACs inhibitor MGCD0103 opens a new time window for stroke treatment.In this charpter,we mainly explore the molecular,structural and electrophysiological mechanisms underlying class I HDACs inhibitor MGCD0103 in promoting stroke recovery.We observed up-regulation of HDAC2 after stroke by immunofluorescence analyses and activity assay.To know how ischemia upregulates HDAC2,we examined effects of free radicals and inflammatory factors,key pathophysiological mechanisms underlying secondary injury after stroke.We treated the cultured cortical neurons by H2O2 combined with peroxynitrite(ONOO-)or by inflammatory factors TNFa combined with IL-1? and matrix metalloproteinase9(MMP9),and found that they significantly increased HDAC2 expression.Although N-methyl-D-aspartate(NMDA)type of glutamate receptor(NMDAR)overactivation is crucial for neuronal death,NMDA did not change HDAC2 expression in the cultured neurons.Thus,ischemia-induced production of free radicals and inflammatory factors may account for HDAC2 up-regulation after stroke.Infarct area reduction may directly contribute to the functional recovery after stroke,it remains unknown whether delayed administration of MGCD0103 after stroke promotes functional recovery via reducing infarct volume.To investigate this issue,we assessed stroke volume at 8 d after photothrombotic stroke in mice treated with vehicle or MGCD0103 during 5-7 d after stroke.Stroke volumes were similar between mice treated with vehicle and MGCD0103.Moreover,stroke volume at 8 d after rat MCAO was also assessed when vehicle or MGCD0103 was treated during5-7 d after stroke.No difference was observed between groups.The above results suggest that MGCD0103 rescues secondary functional loss after stroke probably via other mechanisms rather than reducing infarct size.To explore the mechanisms underlying HDAC inhibitor-produced functional rescue,we performed RNA sequencing of peri-infarct cortex extracts of both MGCD0103-and vehicle-treated animals 5-7 d after stroke.At an expression fold change cutoff of 2.0,we found 1148 differentially expressed genes(DEGs)between MGCD0103-and vehicle-treated cortices,among which 417 genes showed higher expression and 731 genes showed lower expression.Upon generation of pathway and gene ontology analyses,we noticed these DEGs between vehicle-and MGCD0103-treated cortex to be implicated in biological processes related to inflammatory response,superoxide anion generation,neuronal plasticity and others.Neuronal plasticity can lead to the remapping of function from damaged areas to peri-infarct surviving tissue.We thus detected several key proteins of neuronal plasticity to be significantly changed in MGCD0103-treated cortex,including Noval,a regulator of neuronal miRNA function,Unc5 c,a netrin receptor,Bestrophin 1(Best1),an anion channel contributes to GABA tonic inhibition(Itonic),and Discl,a regulator of mitochondrial dynamics controlling the morphogenesis of neuronal dendrites and axons.Immunoblotting analysis of independent MGCD0103-and vehicle-treated samples confirmed the expression changes.Furthermore,the genes with higher expression also showed higher acetylation in their promoter regions These findings suggest that neuroplasticity in the peri-infact area at the level of gene expression was enhanced when treated with MGCD0103.To investigate whether increased neuroplasticity-related gene expression contributes to structural plasticity,we measured the number of dendritic spines and dendritic branchings by Golgi staining.Stroke caused substantial de creases in dendritic spine density,suggesting a spine loss.Treatment with MGCD0103 during5-7 d after stroke reversed the stroke-induced spine loss.Stroke also caused a significant reduction of dendritic length and branchings,and MGCD0103 significantly increased dendritic complexity compared to vehicle.Collectively,these results indicate that MGCD0103 promotes structural neuroplasticity acompanied by epigenetically increased neuroplasticity-related gene expression after stroke.Dendritic spines are the postsynaptic sites of excitatory synapses.To further ascertain whether structural plasticity might also be accompanied by increased functional plasticity,we performed whole-cell voltage clamp recordings to assess mEPSCs in peri-infarct pyramidal neurons in acute brain slices derived from poststroke mice and the same site pyramidal neurons from sham controls.Treatment with MGCD0103 during 5-7 d after stroke reversed the stroke-induced mEPSC frequency decrease,however,no significant difference in amplitude was observed between groups,in line with the observation that MGCD0103 treatment increases the number of synapses in the peri-infarct cortex.Thus,in addition to epigenetically enhanced neuroplasticity-related gene expression,MGCD0103 treatment also lead to increased structual plasticity and synaptic transmission in the peri-infarct area.Excitation-inhibition imbalance also plays an important role in mediating stroke recovery.More importantly,RNA sequencing of peri-infarct cortex extracts revealed that MGCD0103 reversed stroke-induced Best1 up-regulation,which was further confirmed by western blot analysis of the peri-infarct cortex tissue,implicating class I HDACs in the regulation of GABAergic signaling.Accordingly,we collected interstitial fluid samples from peri-infarct cortex of different groups by microdialysis.LC-MS/MS analysis of the samples revealed that stroke led to significant increases in both extracellular glutamate and GABA,and treatment with MGCD0103 5-7 d after stroke reversed the ischemia-induced extracellular level of GABA but not of glutamate.Extracellular concentration of GABA is critical in determining tonic conductance and tonically active extrasynaptic GABAA receptors set an excitability threshold for neurons and control LTP.Therefore,we performed whole-cell voltageclamp recordings in the peri-infarct cortex in in vitro brain slices prepared at 8 d after stroke.We found that a significant increase in Itonic in pyramidal neurons after stroke,consistent with previous findings,and more importantly,treatment with MGCD0103 5-7 d after stroke reversed the ischemia-induced Itonic.Moreover,MGCD0103 did not affect phasic inhibitory currents,resting membrane and GABA reversal potentials.Thus,inhibiting class I HDACs may enhance excitability in the peri-infarct cortex by regulating Itonic.Reducing excessive GABA-mediated Itonic and enhancing excitability in surviving neurons promotes functional recovery after stroke.Collectively,our findings suggest that inhibiting class I HDACs enhances functional neuroplasticity of peri-infarct cortex after stroke.Finally,we investigated the role of class I HDACs in cell survival and neuroinflammation.We infused MGCD0103 into the peri-infarct cortex of conscious mice 5-7 d after stroke.Next day,we measured the number of surviving neurons and amoeboid microglia,an indicator of phagocytic activity,and important regulators of cell survival and neuroinflammation,including brain-derived neurotrophic factor(BDNF),tumor necrosis factor-alpha(TNF?),interleukin-1-beta(IL-1?).As expected,treatment with MGCD0103 significantly increased the number of surviving neurons and BDNF level,and substantially decreased the number of amoeboid microglia,and reversed stroke-induced up-regulation of TNFa and IL-1?in the peri-infarct cortex.Thus,besides substantially increased neuroplasticity,inhibiting class I HDACs may promote functional recovery after stroke via enhancing survival and alleviating neuroinflammation.Collectively,our results therefore indicate that:(1)5-7 d after stroke is a new time window critical for stroke functional recovery.(2)HDAC2 is the key mediator for the secondary functional loss after stroke.In the new time window,suppressing HDAC2 in the peri-infarct cortex of rodents by HD AC inhibitors,knockdown or knockout of HDAC2 promoted early and sustained stroke functional recovery.(3)Administration of MGCD0103 in the second functional loss phase overcome the functional impairment after stroke is probably achieved by epigenetically enhancing cells survival and neuroplasticity of surviving neurons as well as reducing neuroinflammation. |
PDF Full Text Request