神經精神疾病致病因子的分子機轉

1. 神經精神疾病致病因子的分子機轉 • 牛痘病毒（vaccinia virus, VV）是一種痘病毒，整個病毒的生命週期是在宿主細胞的細胞質中完成。DNA 的基因體（genome）約有192Kb，可以表現出263個病毒蛋白。在多數的細胞中，病毒感染細胞之後會產生病毒後代;然而，在少數細胞中，病毒的生命週期被中止在post-entry的階段，顯示在特定的細胞株中，會有宿主限制的現象產生。對於牛痘病毒來說，在此病毒的其中一種會有宿主限制的細胞株CHO中，牛痘病毒要靠一個宿主範圍基因(host range gene) CP77來完成病毒的生命週期。我的論文主要著重於了解CP77在宿主限制的調控中所扮演的角色。我研究主題的第一部份是要探討CP77在結構及功能上的相關性。我們構築了能表現不同CP77截短片段的重組牛痘病毒，並且在HeLa及CHO細胞中監控這些截短蛋白的表現及測量其穩定性。實驗結果顯示，N端及C端具有保護作用，能在病毒感染的細胞中維持CP77蛋白的穩定性。第二部分是在研究CP77的後修飾轉譯作用。我們發現在病毒感染的HeLa及CHO細胞中，CP77都被ubiquitinated。並且CP77在細胞外也被sumoylated。最後，第三部份，根據之前我們實驗室的酵母菌雙雜交篩選實驗分析結果顯示，一個細胞內蛋白cytidine deaminase (CDA)會與CP77交互作用。在我研究報告中指出，CP77 以及CDA的確在GST-pull down實驗中有交互作用，顯示CDA可能參與調控牛痘病毒宿主限制。

2. Molecular Risk Factors in Neuropsychiatric Disorders • My researches are trying to identify the molecular risk factors in various neuropsychological disorders, including schizophrenia, bipolar mania patients, methamphetamine (MAP) addiction patients and Alzheimer’s disease (AD).The first part of my study was to deal the issue of psychosis in schizophrenia patients and MAP abusers. It is well known that dopaminergic pathway is the major neuronal substrate in the pathophysiology of psychosis development and in the rewarding process of addictive behaviors. The majority of released dopamine is reuptaked by the presynaptic dopamine transporter (DAT) to maintain the homeostasis of dopamine system. It is reasonabe to postulate that hyperdopaminergic state in psychosis might be in part due to the DAT dysfunction. Therefore, we first targeted the association of DAT and psychosis in MAP abusers and schizophrenia patients. One of the DAT gene polymorphism is 40-bp VNTR (variable number tandem repeats) which is located at the 3’-untranslated region of the DAT. However, we did not find the association of this DAT VNTR polymorphism with psychosis, neither in MAP psychosis nor schizophrenia patients. Dopaminergic rewarding system is the main pathway to be activated in addiction behavior. It has been also reported that DRD1A-48G polymorphism was associated with substance abuse. In addition to the dopaminergic rewarding, endocannabinoid system was noted to be a new target for addiction behavior through modulation of dopamine activity. Fatty acid amide hydrolase (FAAH) is the main catabolic enzyme in degradation of endocannabinoid and FAAH C385A polymorphism has been reported to be strongly associated with addiction behaviors. Therefore, we tested these two loci in our MAP addiction patients, but we could not replicate the positive association results in our patient samples. Our sample size is pretty large and the most important of all, our genetic distribution was no deviated from the Hardy-Weinberg equilibrium. We re-analyzed previous studies and showed that previous positive association study results might be false positive, because their genetic distribution was apparently deviated from the Hardy-Weinberg equilibrium.Compelling evidence suggests that neurodevelopment anomaly might be one of the major factors in the development of schizophrenia. Several molecules involved in the developmental process have been shown to the candidate genes in the pathogenesis of schizophrenia, such as Akt1, neuregulin, calcineurin and disrupted-in-schizophrenia 1(DISC1). Akt1 was shown to be lower in the peripheral lymphocytes and brain of schizophrenia patients. Akt1 deletion mice were shown to have impaired prepulse inhibition after amphetamine challenge. Therefore, lower Akt1 might be a risk factor to develop psychosis. Lower Akt1 might be also associated with neurodevelopment abnormalities which could be identified by clinical neurological soft sign evaluation. DISC1 is also shown to influence the cortical development. We also examine the DISC1 level in schizophrenia patients and MAP abusers simultaneously. The purpose to this study was trying to test if the lower Akt1 and DISC1 level was also a risk factor for development of MAP psychosis and the neurological abnormalities in schizophrenia patients. The results showed MAP would up-regulate Akt1 and DISC1 mRNA in the MAP abusers during acute phase. The Akt1 and DISC1 mRNA levels were of no difference between schizophrenia patients and controls. The expression level was also not associated with severity of neurological anomaly. However, we found a strong positive correlation between Akt1 and DISC1 level. The genetic association study showed an increase of G allele of SNP2 (rs1130214) in schizophrenia patients. The A allele of SNP4 (rs1130233) was shown to be associated with MAP abusers and schizophrenia. Further experiments are necessary to delineate the role of Akt1 and DISC1 in the development of psychosis.The second part of our study is to deal with the psychoneuroimmunology in bipolar mania patients. Psychological and physical stressors are known to induce immune alteration. The immunological cytokines may act as neurotransmitters in the CNS to modulate the hypothalamus-pituitary-adrenal (HPA) axis to control the stress hormone release and then modulate the immune response in a feedback mechanism. In addition to the well known HPA axis, the immune modulation in the hypothalamus might also induce circadian rhythm changes and affect the nearby limbic system, and then alter the neurobehavioral expression. Administration of cytokines would induce mood instability as depression in humans and experimental animals. Actually the interaction between nervous and immune system is complicated. Our study was trying to investigate the plasma levels of several immunologic variables in patients with bipolar mania. IL-1RA and sCD8 were found different in remitted bipolar patients as compared to normal controls. For TH1 cytokines, culture supernatant level of IFN-γ was found significantly lower in manic patients of both acute and remission stages as compared to normal controls. No significant difference was found in IL-2 level in pre-medicated acute manic patients compared to controls. For TH2 cytokines, no significant differences in IL-4 and IL-10 levels were observed. We showed that cell-mediated immune response was activated in patients with bipolar disorder during the pre-medication, medication, and the remission stages.The third part of our study was focusing upon AD patients. According the amyloid hypothesis of AD, the molecules involved in the amyloidogenic pathway would be the candidate genes associated with AD. BACE (beta-site APP cleaving enzyme) cleaves APP (amyloid precursor protein) is the rate-limiting step in the process of amyloidogenesis. Therefore, BACE polymorphisms would be the potential candidate markers associated with AD. We used SSCP (single strand conformational polymorphism) to find a 786C/G polymorphism in the BACE gene. However, this polymorphism was not associated with AD in our patients. In addition to the genetic marker association studies for AD patients, we were trying to survey other potential peripheral markers which could be of either diagnostic or prognostic value for clinical use. We used the proteomics method to screen the potential biomarkers in the sera of AD patients in comparison to the controls. Lower apolipoprotein A-I (ApoA-I) was noted in proteomics study and the lower ApoA-I was confirmed by direct assay of the serum of AD patients. The significance of lower ApoA-I in AD patients need further clarification. In summary, our research interest is to survey the molecular risk factors in neuropsychiatric disorders. The main topics were the etiology and psychosis and AD. In addition to the genetic polymorphism association study, we are trying to investigate the signaling molecules and other potential peripheral markers. The ultimate goal is to identify factors to be useful in diagnosis or prognosis prediction.