The Effect Of NNOS-PSD-95 Coupling On Functional Reconstruction After Cerebral Ischaemia

Stroke greatly harms human health because of its high disability rate,high recurrence rate and high mortality rate.Sensorimotor and cognitive impairments caused by stroke lead survivors to cannot work or self-care.Currently,the tissue plasminogen activator?tPA?is mainly used for thrombolytic therapy.However,the use of tPA has a strict time window and it has bleeding risk.Due to the lack of other effective clinical drugs for the treatment of stroke,developing safe and valid drugs is especially important.Postsynaptic density protein-95?PSD-95?is a scaffolding protein that connects N-methyl-D-aspartate receptors?NMDARs?and neuronal nitric oxide synthase?nNOS?and forms ternary complex.Excessive activation of NMDARs caused by cerebralischaemiaincreasesnNOS-PSD-95interaction,leadingtoNO overproduction and neuronal injury.Disrupting nNOS-PSD-95 coupling reduces ischaemic damage at the acute stage after stroke.We developed an effective small-molecule compound,ZL006,which inhibits nNOS-PSD-95 interaction and has potent neuroprotective activity without obvious side-effects.Three to four hours after stroke is the prime time for the therapy.However,most of the patients have little knowledge about the disease,and other factors make them miss the best treatment time.Then,exploring the effective delayed-treatment drugs for stroke will be an important research direction.Although previous studies have shown that ZL006 is a potential drug for stroke,but it is limited in the acute phase.Whether it has therapeutic effect at the delayed stage after stroke?Inhibiting nNOS-PSD-95 coupling by ZL006 promotes functional recovery through increasing neuroprotection,neurogenesisandneuroplasticity.Fortheregulationof neuroplasticity,gamma-aminobutyric acid?GABA?is indispensable,because it can control the excitability of neurons.Evidence showed that the GABA-mediated tonic inhibition increases at the delayed stage after stroke,reducing excessive tonic inhibition is beneficial for recovery.NMDAR-PSD-95-nNOS is activated after ischemia,and it causes the release of NO which is used as a neuromodulator.NO can affect many transmitters release including GABA.Then,we suspected that delayed dissociation of nNOS from PSD-95 is likely to have treatment effect.Therefor,we investigated the following three questions:?1?whether delayed dissociation of nNOS from PSD-95 promotes functional recovery after cerebral ischaemia??2?Dose disrupting nNOS-PSD-95 coupling influence neuroplasticity??3?What are the potential molecule mechanisms responsible for the delayed treatment?Chapter 1.Delayed dissociation of nNOS from PSD-95 reduces tonic inhibition and promotes functional recovery after cerebral ischaemiaTo investigate the effect of nNOS-PSD-95 uncoupling on cerebral ischaemia,we used a membrane-permeable recombinant protein Tat-nNOS-N_1-133-133 and small molecule compound ZL006 to inhibit nNOS-PSD-95 association.Firstly,we detected the nNOS-PSD-95 interaction after photothrombotic stroke.The results showed that nNOS-PSD-95 coupling is increased after ischaemia and lasts at least 7 days.According to the results,we injected Tat-nNOS-N_1-133-133 into peri-infarct cortex from day 4 to day 10 after ischaemia.And we recorded the GABA-mediated tonic inhibition currents(I _tonic)in peri-infarct neurons by whole-cell voltage clamp between days 7 and 11 after ischaemia.We found that I _toniconic increased after ischaemia.Interestingly,Tat-nNOS-N_1-133-133 treatment reduced tonic inhibition in mice.However,the spontaneous inhibitory post-synaptic currents?sIPSC?was unchanged.And ZL006 incubation did not influence the evoked inhibitory post-synaptic currents?eIPSC?in normal hippocampus slices.The data confirmed that nNOS-PSD-95uncouplingattenuates GABA-mediated tonic inhibition rather than phasic inhibition.Reportedly,reducing tonic inhibition promotes functional recovery after stroke.So,we speculated that delayed dissociation of nNOS from PSD-95 could promote functional recovery after ischaemia.To address this notion,we injected Tat-nNOS-N_1-133-133 through microcannula or administrated ZL006 systemically from day 4 to day 10 after ischaemia,and conducted grid-walking task and cylinder task at day 11 to measure sensorimotor functions.The results proved that Tat-nNOS-N_1-133and ZL006 promoted functional recovery,because they rescued the increase of foot faults in the grid-walking task and forelimb asymmetry in the cylinder task induced by ischaemia.The improvement of motor functions due to delayed ZL006 treatment was abolished in nNOS^-/-mice,it indicated that the effect is dependent on nNOS.Moreover,we designed a recombinant virus AAV-nNOS-N_1-133-GFP which determines the long-term effect of nNOS-PSD-95 dissociation by expressing a region crucial for the nNOS-PSD-95 interaction,nNOS-N_1-133.AAV-nNOS-N_1-133-GFP was infused into the peri-infarct immediately after ischaemia,and we measured motor function at days 11,18,32,and 46.Compared with control virus AAV-GFP-infected mice,AAV-nNOS-N_1-133-GFP infected mice performed better in both the grid-walking task and the cylinder task.The results above have revealed that the tonic inhibition in peri-infarct increases significantly after ischaemia,delayed dissociation of nNOS from PSD-95 reduces tonic inhibition rather than phasic inhibition and promotes functional recovery.Chapter 2.Effect of delayed dissociation of nNOS-PSD-95 on neuroplasticityThe influence of reducing excessive tonic inhibition by nNOS-PSD-95dissociation on neuroplasticity is investigated in this chapter.Firstly,we used oxygen-glucose deprivation?OGD?to mimic the conditions of a peri-infarct cortex.Neurons and astrocytes were co-cultured for 9-10 days in vitro and exposed to OGD.Dendritic spine density,total dendrite length and terminal points significantly decreased after OGD.Incubation with Tat-nNOS-N_1-133-133 or?5-GABA_A receptors specific inverse agonist,L655,708 which can block tonic inhibition dramatically increased the spine density,the total dendrite length and terminal points did not change,compared with the OGD group.Tat-nNOS-N_1-133-133 and L655,708 incubation also reversed the decrease of Synapsin and PSD-95 expression.Thus,reducing excessive tonic inhibition improves dendrite spine density and synaptogenesis after ischaemia.To further confirm delayed dissociation of nNOS-PSD-95 regulates structural remodelling in the peri-infarct cortex,Tat-nNOS-N_1-133-133 was administrated from day 4to day 10 after after photothrombotic stroke,Golgi staining was performed at day 11.We found that ischaemia resulted a robust decrease of dendritic spine density,dendritic branch number and dendritic length,Tat-nNOS-N_1-133-133 treatment reversed the decline.Treatment with ZL006 had a similar effect to Tat-nNOS-N_1-133-133 for spine density.More importantly,ZL006 increased the frequency of miniature excitatory post-synaptic currents?mEPSCs?rather than amplitude,suggesting that synapse function is enhanced after nNOS-PSD-95 dissociation.However,inhibiting nNOS-PSD-95 coupling at a delayed stage had no effect on infarct volume,survival neurons and neorogenesis.These data suggested that delayed dissociation of nNOS from PSD-95 promotes functional recovery after cerebral ischaemia through blocking excessive tonic inhibition,improving structural remodelling and ameliorating synapse function.Chapter 3.Potential mechanisms underlying delayed nNOS-PSD-95 uncoupling-mediated functional recovery after ischaemiaIn this chapter,we studied the mechanisms responsible for delayed nNOS-PSD-95dissociation-inducedfunctionalrecoveryafterischaemia.Immunohistochemistry analysis revealed that ischaemia led to aberrant astrocytes activation in the peri-infarct cortex.The GABA level in the GFAP-labelled hypertrophic astrocytes increased remarkably,without a significant change in the neurons.Moreover,we used western blot to evaluate the expression of GABA transporters,including GAT-1 and GAT-3/4 respectively expressed in neurons and astrocytes.Our results showed that there was a down-regulation of GAT-1 but an up-regulation of GAT-3/4 from day 1 to day 7 after ischaemia.The above data indicated that GAT-3/4 existed in astrocytes might play an important part in regulating the tonic inhibition after ischaemia.To verify the opinion,the mice were treated with GAT-1-seletive antagonist NO-711 or GAT-3/4-selective antagonist SNAP-5114 from day 4 to day 11 after ischaemia.Only SNAP-5114treatment reduced foot faults and asymmetry index,further confirming the involvement of GAT-3/4 in tonic inhibition and functional recovery after ischaemia.we suspected that the function of GAT-3/4 was reversed,to release GABA,due to the high GABA level in the astrocytes and the effect of inhibiting GAT-3/4 on functional recovery.Accordingly,we recorded tonic inhibition mediated by ambient GABA.Blocking GAT-3/4 with SNAP-5114 increased I _toniconic in sham brain slices,suggesting that GAT-3/4 uptakes GABA in the normal cortex.However,I _toniconic decreased after application of SNAP-5114 to brain slices from vehicle-treated ischaemia mice,revealing that GAT-3/4 releases GABA after ischaemia.Thus,ischaemia can induce GAT-3/4 reversal.Interestingly,Tat-nNOS-N_1-133-133 treatment could rescue the decrease of I_toniconic caused bySNAP-5114.Meanwhile,theSNAP-5114canceledtheeffectof AAV-nNOS-N_1-133-GFP on functional recovery.These results shown that nNOS-PSD-95 uncoupling prevents the reversal of GAT-3/4 after ischaemia.However,the function was achieved by inhibiting astrocytes activation and GABA expression.Because Tat-nNOS-N_1-133-133 or ZL006 treatment declined the expression of GABA and glial fibrillary acidic protein?GFAP?,a specific marker for astrocytes activation.Western blot data shown that GABA synthetase including glutamic acid decarboxylase 67?GAD67?and monoamine oxidase B?MAOB?which exist in astrocytes up-regulated after ischaemia,both of these were down-regulated after Tat-nNOS-N_1-133-133 treatment.In the electrophysiological experiments,we found that similar to the effect of Tat-nNOS-N_1-133-133 treatment,AAV-nNOS-N_1-133-GFP-infected mice had increased I_toniconic currents in the peri-infarct cortex after SNAP-5114application.Lipopolysaccharide?LPS?,an astrocytes activator,abolished the effect of AAV-nNOS-N_1-133-GFP on GAT-3/4 reversal.Since nNOS-PSD-95 dissociation reduces the production of NO,we studied the regulation of NO on GABA level.Firstly,we found that nNOS-specific inhibitor vinyl-l-N-5-?1-imino-3-butenyl?-l-ornithine?L-VNIO?inhibited astrocytes activation,consistent with Tat-nNOS-N_1-133-133 treatment.But the combination of these two drugs did not enhance the inhibitory effect,suggesting that they have a common downstream signal?NO?.At the cellular level,we confirmed that NO level in neurons co-cultured with glia increased after OGD,while Tat-nNOS-N_1-133-133 rather than L655,708 could prevent the production of NO.Additionally,the NO donor led to high expression of GAD67,MAOB and GABA in cultured astrocytes.Finally,chromatin immunoprecipitation?ChIP?and biotin-switch assay indicated that ischaemia induced nNOS-PSD-95 interaction enhanced and NO overproduction.Increased NO resulted in significant S-nitrosylation of total proteins and total histone acetylation in cultured astrocytes.And it contributed to promoting histone acetylation in the Gad1 and Maob promoter regions and finally up-regulated the expression of GAD67 and MAOB in the peri-infarct tissue,but the effect was inhibited by Tat-nNOS-N_1-133.In summary,our results show that:?1?Delayed dissociation of nNOS from PSD-95 reduces tonic inhibition and promotes functional recovery.?2?Uncoupling nNOS-PSD-95 at the delayed stage improves structural remodelling and ameliorates synapse function.?3?Down-regulated of NO production induced by nNOS-PSD-95dissociation prevents astrocytes activation and GABA expression.Moreover,it rescues the GAT-3/4 reversal in pathological state,contributing to the functional recovery after cerebral ischaemia.