In the central nervous system,hyperpolarizationactivated cyclic nucleotide-gated(HCN)channels are essential to maintain normal neuronal function.Recent studies have shown that HCN channels may be involved in the patho...In the central nervous system,hyperpolarizationactivated cyclic nucleotide-gated(HCN)channels are essential to maintain normal neuronal function.Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury,but the mechanisms remain unclear.Autophagy is activated in cerebral ischemia,but its role in cell death/survival remains controversial.In this study,our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion(OGD/R)in hippocampal HT22 neurons.Furthermore,in the OGD/R group,p-mTOR,p-ULK1(Ser757),and p62 were significantly decreased,while p-ULK1(Ser317),atg5,and beclin1 were remarkably increased.ZD7288 did not change the expression of p-ULK1(Ser757),ULK1(Ser317),p62,Beclin1,and atg5,which are involved in regulating autophagosome formation.Besides,we found that OGD/R induced a significant increase in Cathepsin D expression,but not LAMP-1.Treatment with ZD7288 at 10μmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1.However,chloroquine(CQ),which decreases autophagosome-lysosome fusion,eliminated the correction of excessive autophagy and neuroprotection by ZD7288.Besides,shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-Ⅱand increased neuron survival in the OGD/R and transient global cerebral ischemia(TGCI)models,and CQ also eliminated the effects of HCN2-shRNA.Furthermore,we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNAtransfected HT22 neurons exposed to OGD/R or CQ.In HCN2-shRNA-transfected HT22 neurons,the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R;however,the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons.The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.展开更多
The thalamocortical(TC)circuit is closely asso-ciated with pain processing.The hyperpolarization-activated cyclic nucleotide-gated(HCN)2 channel is predominantly expressed in the ventral posterolateral thalamus(VPL)th...The thalamocortical(TC)circuit is closely asso-ciated with pain processing.The hyperpolarization-activated cyclic nucleotide-gated(HCN)2 channel is predominantly expressed in the ventral posterolateral thalamus(VPL)that has been shown to mediate neuropathic pain.However,the role of VPL HCN2 in modulating TC circuit activity is largely unknown.Here,by using optogenetics,neuronal trac-ing,electrophysiological recordings,and virus knockdown strategies,we showed that the activation of VPL TC neurons potentiates excitatory synaptic transmission to the hindlimb region of the primary somatosensory cortex(S1HL)as well as mechanical hypersensitivity following spared nerve injury(SNI)-induced neuropathic pain in mice.Either pharmaco-logical blockade or virus knockdown of HCN2(shRNA-Hcn2)in the VPL was sufficient to alleviate SNI-induced hyperalgesia.Moreover,shRNA-Hcn2 decreased the excitability of TC neurons and synaptic transmission of the VPL-S1HL circuit.Together,our studies provide a novel mechanism by which HCN2 enhances the excitability of the TC circuit to facilitate neuropathic pain.展开更多
基金supported by grants from the National Natural Science Foundation of China (85100929)the Natural Science Foundation of Hubei Province,China (2018CFB302 and 2019CFB446)the Youth Fund of Health and Family Planning Commission of Wuhan Municipality,Hubei Province,China (WX18Q13 and WX18Q22)。
文摘In the central nervous system,hyperpolarizationactivated cyclic nucleotide-gated(HCN)channels are essential to maintain normal neuronal function.Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury,but the mechanisms remain unclear.Autophagy is activated in cerebral ischemia,but its role in cell death/survival remains controversial.In this study,our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion(OGD/R)in hippocampal HT22 neurons.Furthermore,in the OGD/R group,p-mTOR,p-ULK1(Ser757),and p62 were significantly decreased,while p-ULK1(Ser317),atg5,and beclin1 were remarkably increased.ZD7288 did not change the expression of p-ULK1(Ser757),ULK1(Ser317),p62,Beclin1,and atg5,which are involved in regulating autophagosome formation.Besides,we found that OGD/R induced a significant increase in Cathepsin D expression,but not LAMP-1.Treatment with ZD7288 at 10μmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1.However,chloroquine(CQ),which decreases autophagosome-lysosome fusion,eliminated the correction of excessive autophagy and neuroprotection by ZD7288.Besides,shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-Ⅱand increased neuron survival in the OGD/R and transient global cerebral ischemia(TGCI)models,and CQ also eliminated the effects of HCN2-shRNA.Furthermore,we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNAtransfected HT22 neurons exposed to OGD/R or CQ.In HCN2-shRNA-transfected HT22 neurons,the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R;however,the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons.The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.
基金This work was supported by the National Natural Science Foundation of China(81960216,81903595,81860216,and 32060186)and the Natural Science Foundation of Jiangxi Province(20202BABL206049 and 20202BAB216043).
文摘The thalamocortical(TC)circuit is closely asso-ciated with pain processing.The hyperpolarization-activated cyclic nucleotide-gated(HCN)2 channel is predominantly expressed in the ventral posterolateral thalamus(VPL)that has been shown to mediate neuropathic pain.However,the role of VPL HCN2 in modulating TC circuit activity is largely unknown.Here,by using optogenetics,neuronal trac-ing,electrophysiological recordings,and virus knockdown strategies,we showed that the activation of VPL TC neurons potentiates excitatory synaptic transmission to the hindlimb region of the primary somatosensory cortex(S1HL)as well as mechanical hypersensitivity following spared nerve injury(SNI)-induced neuropathic pain in mice.Either pharmaco-logical blockade or virus knockdown of HCN2(shRNA-Hcn2)in the VPL was sufficient to alleviate SNI-induced hyperalgesia.Moreover,shRNA-Hcn2 decreased the excitability of TC neurons and synaptic transmission of the VPL-S1HL circuit.Together,our studies provide a novel mechanism by which HCN2 enhances the excitability of the TC circuit to facilitate neuropathic pain.
