Involvement Of Homer1 In Molecular Mechanism And Neuroprotection After Neuronal Injury

Background: Central nervous system injury is a common central nervous system disease induced by damage on brain or spinal cord tissues, which usually followed by neurological functional disorders. Neuronal damage and functional deficits of neurons is considered to be one of the most important mechanisms. Previous researches have studied many mechanisms of neuronal injury including glutamate induced excitotoxicity, calcium overload, oxidative stress and inflammation, among which glutamate, glutamate receptors and calcium metabolism related neuronal excitotoxicity has been deeply investigated. However, a lot of problems have not been determined, such as the exact role of m Glu R5 in neuronal injury, as well as mechanisms underlying the regulatory effects of calcium influx and intracellular calcium release on neuronal injury. Homer1 protein is a recently found scaffold protein of postsynaptic density, and has been shown to play important roles in neuronal function. Our previous studies found that the expression of Homer1 a protein significantly increased after traumatic neuronal injury, and could regulated neuronal injury through targeting m Glu Rs in the postsynaptic membrane. However, the effects of Homer1 a and Homer1b/c on neuronal injury and potential molecular mechanisms are not fully understood.Aims:(1) To investigate the potential protective effect of m Glu R5 on neuronal injury, and determine the possible molecular mechanism underlying glutamate induced neuronal injury, with focus on calcium metabolism and oxidative stress;(2) To investigate the potential protective effect of the store-operated calcium entry channels in glutamate induced neuronal injury, as well as the possible underlying mechanisms with focus on Homer1 protein;(3) To investigate the role of Homer1b/c in glutamate induced neuronal injury, and determine the relationship between Homer1b/c induced regulation of ER stress and mitochondrial dysfunction and its effect on neuronal injury;(4) To investigate the role of Homer1 a in glutamate induced neuronal injury, and determine the relationship between Homer1 a induced regulation of calcium influx and intracellular calcium release and its effect on neuronal injury.Methods:(1) Cell culture: cortical neurons cultured from mice;(2) In vitro neuronal injury model: glutamate induced neuronal injury in cortical neurons;(3) In vivo neuronal injury model: mouse closed head injury model induced by a weight-drop device;(4) Measurement of neuronal injury in vitro: cell viability, LDH release, Caspase-3 activity assay and neuronal apoptosis staining;(5) Measurement of neuronal injury in vivo: brain water content, brain tissue infarct volume, neurological severity score and TUNEL staining;(6) Regulation of Homer1 expressions: knockdown or overexpression of Homer1a/Homer1b/c by transfection with lentivirus vector;(7) Expression of related molecules: real-time fluorescent quantitative PCR, Western blot, immunofluorescence and immunohistochemistry.Results:(1) Glutamate induced neuronal damage in cultured cortical neurons was a calcium-dependent oxidative stress-mediated neuronal injury; m Glu R5 activation protected cultured cortical neurons against glutamate induced neuronal injury through regulating PKC-ERK pathways; m Glu R5 activation played a limited role in neuronal protection against traumatic brain injury;(2) SKF-96365 attenuated glutamate induced neuronal injury in a dose-dependent manner; SKF-96365 induced neuroprotection was partly dependent on the regulation of Homer1-Ca2+ signaling pathways;(3) Knockdown of Homer1b/c protected against glutamate induced neuronal injury; Knockdown of Homer1b/c reduced glutamate induced intracellular calcium release in cortical neurons; Knockdown of Homer1b/c inhibited glutamate induced ER stress; Knockdown of Homer1b/c preserved the mitochondrial function after excitotoxicity;(4) Overexpression of Homer1 a protected against glutamate induced neuronal injury; Overexpression of Homer1 a induced neuroprotection was partly dependent on the regulation of calcium influx and intracellular calcium release.Conclusion: The results presented in this study demonstrate that:(1) Activation of m Glu R5 exerts protective effects against glutamate induced neuronal injury, which is partly dependent on calcium-mediated oxidative stress;(2) The non-specific inhibitor of SOCE, SKF-96365, protects against glutamate induced intracellular calcium overload and neuronal injury, which is mediated by Homer1 pathway, but not dependent on the expression of TRPC1;(3) Knockdown of Homer1b/c reduces glutamate induced neuronal apoptosis through inhibition of ER stress and preservation of mitochondrial function;(4) Overexpression of Homer1 a attenuates glutamate induced ROS generation, oxidative stress and neuronal apoptosis through regulation of extracellular calcium influx and intracellular calcium release.