Neurological Function And Regulating Mechanism Of Homer1a In Traumatic Brain Injury

Background: Traumatic brain injury(TBI) is one of the significant causes of death or persistent disability. It has been estimated that about 2 million people suffer TBI in the United States each year. And almost one third of TBI victims are children and young population. In the world, the incidence of TBI is still increasing, partly because of widespread use of motor vehicle.TBI is a very complicated injury that could lead to varying degrees of contusion, diffuse axonal injury, hemorrhage, hypoxia, and neurodegeneration in the brain. The mechanisms of TBI have been defined as primary and secondary injury. Primary injury mechanisms result from the mechanical damage that occurs at the time of skull and brain impact, which is proved to be irreversible. The mechanisms of secondary injury include a large series of processes such as release of neurotransmitters(e.g. glutamate excitotoxicity), mitochondrial dysfunction, neuronal apoptosis, lipid degradation, initiation of inflammatory and immune responses, and so on. These mechanisms are proved to contribute to immediate cell death, early or late apoptotic programmed cell death and autophagy by TBI. Biomarkers are important for accurate diagnosis and therapy of complex disorders.Homer proteins are commonly known as scaffold proteins at postsynaptic density.Homer 1 is a widely studied member of the Homer protein family, comprising both synaptic structure and mediation of postsynaptic signaling transduction. Both an immediate-early gene encoding a Homer 1 variant and a constitutively expressed Homer 1 variant regulate receptor clustering and trafficking, intracellular calcium homeostasis, and intracellular molecule complex formation. Substantial preclinical investigations have implicated that each of these Homer 1 variants are associated with the etiology of many neurological diseases, such as pain, mental retardation syndromes, Alzheimer’s disease, schizophrenia, drug-induced addiction, and traumatic brain injury. Homer 1a(also called vesl-1s), as a short variant of Homer 1, was the first Homer protein to be isolated. The expression of Homer 1a encoded by an immediate-early gene(IEG), is very low under normal conditions and increases rapidly after neuronal activation.However, it is likely that a single biomarker will not reflect the full spectrum of the response of brain tissue to injury. It is still not well known that the key molecules, pathways and networks for a complex and multifaceted condition such as TBI.Purpose:(1) To establish a reliable traumatic brain injury model and observe the changes in pathology, morphology and behavioristics after TBI. Proteomics is used to systematically analyze differential protein expression in TBI conditions including, and thus, could provide insight into the potential mechanism;(2) to investigate the role of Homer1 a in TBI and characterize the protein expression profiles, pathways and networks related to Homer1 a. To verify the regulation and interactions among the proteins after TBI, including Homer1 a, MAPK pathway and Notch signaling pathway.Methods:(1) Animal model: The male C57BL/6 mice closed head injury model were using a head-attacked device;(2) brain injury assessment: water content of brain, neurological severity score;(3) pathology: HE staining, TUNEL staining, immunofluorescence staining and immunohistochemistry staining;(4) molecular biology: real-time fluorescent quantitative PCR, reverse transcription polymerase chain reaction(RT-PCR), western blot;(5) animal behavior: open field test(OFT), high plus maze, Morris water maze(MWM);(6) regulation of Homer1a: cortical injection with Homer1 a lentivirus vector, gene knockout mice: Homer1 a knockout mice;(6) Proteomics: one-dimensional gel electrophoresis in combination with label-free quantified nano-LC-MS/MS, Gene ontology(GO) and Ingenuity Pathway Analysis(IPA).Results: In closed head injury model, H&E staining was used to assess the lesion volumes and secondary tissue damage. H&E staining revealed that compare to the control, the loss of neuron was increased in cortex and hippocampus after TBI 24 h. And most of neurons were shunken with condensed cytoplasm.The number of TUNEL positive staining neurons was increased significantly after TBI 24 h compared to the control. Confirmed by western blot, the expression of Homer1 a was up-regulated but the expression of Homer1b/c was not changed after TBI. And the results of immunofluorescence and immunohistochemistry showed the number of Homer1 a positive staining neurons was elevated after TBI 24 h compared to control group. TBI also induced cerebral edema and impairment of neurological function. Using a variety of cognitive and behavioral tests(Morris water maze, elevated plus maze and open field test), the results indicated the learning and memory impairments, accompanied by a depressive behavior in mice after TBI. Proteomic and informatic tools were employed and the results showed that a series of proteins and pathways were involved in the TBI compared to the control. The differentially expressed proteins included the classic biomarkers(GFAP, S100 B, etc.) and Homer family(Homer1, Homer2 and Homer1a).In the further study, Homer1 a KO mice and Homer1 a lentivirus vector injected mice were used. Up-regulation of Homer1 a reduced the cerebral edema content and neurological functional impairment, while Homer1 a KO mice aggravate the cerebral edema and the impairment of neurological function. Cognitive and behavioral tests(Morris water maze, elevated plus maze and open field test) revealed that up-regulation of Homer1 a could partially reverse the depressive behavior, learning and memory impairments upon TBI. But Homer1 a KO mice worsen the abnormal behaviors after TBI. The analysis of proteomics to Homer1 a KO and control mice identified 6529 proteins. Among these proteins, 237 proteins were differentially expressed with fold ≥ 2.0 and informatic tools showed a number of pathways including protein ubiqiutination pathway, axonal guidance signaling, protein kinase A signaling, ERK/MAPK signaling and notch signaling were involved. The functionand disease analysis showed Homer1 a KO could affect the disorder of basal ganglia, synaptic depression, development of neurons, morphology of central nervous system, synaptic transmission, long-term depression and long-term potentiation. By western blot, the expression of cleaved caspase-3 in WT mice was signi?cantly elevated after TBI 24 h. In Homer1 a KO mice, the expression of cleaved caspase-3 was up-regulated on TBI 24 h compared to WT mice. In the mean time, compare to LV-con, Homer1 a overexpression by LV-H1 a could decrease the expression of cleaved caspase-3 after TBI 24 h. In accord with the result of proteomics, after TBI 24 h, Homer1 a KO could increase the expression of pERK, p-CREB and NICD, but decrease the expression of Glu R1 and Glu R2/3.Conclusion: The data in our investigation demostrates that Homer1 a, as a scaffold protein at postsynaptic density, contributes to a neuroprotective effect after traumatic brain injury. The analysis of proteomics to Homer1 a Regulation shows Homer1 a is a key molecule in multiple signaling pathway and network, especially in regulating ubiqiutination pathway, ERK/MAPK signaling and notch signaling.This study elucidates a potential biomarker and pharmacological target in a novel strategy of treatment against traumatic brain injury.