Proteome Research Of The Influences Of Vitamin A Deficiency On Neuropsychiatric Systems

Vitamin A is a micronutrient having multiple functions, including regulating early nervous system development and adult neurogenesis. Vitamin A deficiency (VAD) in adult rodents led to impairments of motor function and learning and memory abilities. And many clues suggest that Vitamin A cascade is associated with schizophrenia: Vitamin A pathway regulates the expression of some schizophrenia risk genes; the protein levels of Vitamin A transporters (TTR and APOE) are dysregulated in schizophrenia patients; the expression of Vitamin A transport and synthesis related molecules were found dysregulated. Furthermore, Vitamin A cascade is linked to Alzheimer disease. Vitamin A could regulate the gene expression of amyloid precursor protein (APP) and APP hydrolases. VAD animals displayed AD like molecular changes: APP accumulation, down-regulation of choline acetyltransferase and RARα.In the first part, we studied the motor ability, long term memory and thermal nociception in early VAD mouse, and detected the effect of combination of VAD and MK-801 (a NMDA receptor antagonist) on those behaviors. We found that early VAD mouse displayed higher locomotion and rearing abilities, and VAD induced pain sensitivity. Moreover, early VAD mouse showed hypersensitivity to MK-801 injection: VAD increased MK-801 induced stereotypy and ataxia, and amplified the suppression effect of MK-801 on rearing; the combination of VAD and MK-801 also lead to pain insensitivity. These abnormal behaviors reflect some faces of schizophrenia like symptoms: such as hyperactivity of locomotion and increased MK-801 induced stereotypy. However we didn't find the influence of VAD on long term horrible memory (step down test).After that, we took the proteomis technology to study the proteome changes in hippocampus, cortex and liver tissues of early VAD male mouse. We also studied the influences of retinoic acid (the active form of Vitamin A) synthesis inhibition upon proteome changes in U251 astrocytoma cells. We identified 23 VAD related protein spots in hippocampus, 46 altered protein spots in cortex, 38 altered protein spots in liver and 39 altered protein spots in U251 cells. Through bioinformatics analysis (Ingenuity pathway analysis, IPA), we found that VAD related proteins in cortex and U251 cells clustered to biological functions: oxidative stress response, neurotransmitters metabolism (serine and glutamate), mitochondrial function and energy metabolism (ATP synthesis); in cortex, VAD could influence NMDA 2B receptor signaling, cytoskeleton and transcript regulation factors, therefore altered the neuronal migration,growth,development and neuroplasticity. With western blot, up-regulation of PHGDH was also confirmed in U251 cells after inhibiting retinoic acid synthesis, and this up-regulation of PHGDH was also observed in cortex of VAD male mouse. The VAD altered proteins in hippocampus clustered to lipid metabolism, axonal guidance signaling and neuroplasticity. VAD could regulate the cytoskeleton and cell membrane related proteins to influence the neurotransmitters releasing, dopamine signaling, NMDA 2B receptor mediated signaling,neurogenesis and neuronal migration. In liver, VAD could induce abnormal carbohydrate metabolism and lipid metabolism, which were corresponding to our findings in cortex. Therefore all results suggest that early VAD could influence energy metabolism and cytoskeleton in cortex and hippocampus, influence the glutamate signaling and dopamine signaling, and finally lead to schizophrenia like behaviors in mouse: increased locomotion and increased MK-801 induced stereotypy. These findings further support the hypothesis that vitamin A pathway is associated with neuropsychiatric diseases.SynapsinⅡis a neuronal phosphoprotein, located in nervous synaptic vesicles. SynapinⅡis connected to cytoskeleton and participates in regulating the realease of neurotransmitters. Previous reports suggested that synapinⅡis associated to schizophrenia. The mRNA and protein level of synapinⅡwere found down-regulated in hippocampus of schizophrenia patients. There are significant association between some SNPs of synapinⅡand schizophrenia in Han, Korean and north European population. Furthermore, synapinⅡgene knockout mouse displayed some schizophrenia like behaviors: impairment of sensorimotor gating and social interaction. There are 2 kinds of splicing isoforms of synapinⅡ: synapinⅡa and synapinⅡb. There are several post-translational modification (PTM) variants of synapinⅡsplicing isoforms in 2-dimensonal gels. However, until now the knowledge of PTMs in synapinⅡisoforms is still limited.In the second part, we used multiple proteolytic enzymes and tandem mass spectrometric technologies (LC-ESI-QTOF, LC-ESI-TRAP) to study the protein sequence and PTMs of synapsin IIa and synapsin IIb in rat and mouse hippocampus. We identified 12 synapsin II protein spots in mouse hippocampus (6 IIa and 6 IIb) and 13 synapsin II proteins in rat hippocampus (6 IIa and 7 IIb).Among those, we got more than 90% sequence coverage (MS/MS) of 18 spots. We provided multiple PTM information of each PTM variants of synapsinII splicing isoforms, including phosphorylation, methylation, acetylation and deamidation. We found 7 new phosphorylation sites, 3 of them have been confirmed using phosphatase (T422 of rat synapsin IIa,S426 and T338 of rat synapsin IIb). Other 4 new phosphorylation sites were: S546 of rat synapsin IIa, T422 and S446 of rat synapsin IIb, S446 of mouse synapsin IIa. Furthermore, we found a new glycosylation site: N339 of rat synapsin IIb by MS/MS. These findings of PTMs provide important basis for annotating the molecular mechanisms of synapsin II in regulating neurotransmitters releasing in near future.