The Founction And Mechanisms Of Zdhhc15b And Bupropion During Neural System Development

Posttranslational modifications such as phosphorylation, glycosylation, lipidation markedly affect protein function. As a lipidation modification, palmitoylation is executed by a family of proteins that contain a unique cysteine-rich domain (cRD) with a conserved aspartate-histidine-histidine-cysteine (DHHC) signature motif. These kind of protein was called DHHC which was first found in yeast. Forward genetic screens have identified Erf2/Erf4 and ARK1 as DHHC proteins in yeast, and they are responsible for RAS2 and yeast casein kinase 2 modifications. In addition to the 7 DHHC proteins that have been identified in yeast,23 DHHC members classified into several subfamilies have been predicted in mammals.With the completion of identification of the 23 kinds of DHHC in mammals, gradually people got better understanding of the function this type of protein. Proteomics and family genetic analysis showed that, DHHC protein plays an important role in nervous system development related diseases and dysfunction of DHHC proteins contributes to a number of neurological disorders. For example, DHHC8 is associated with a higher risk to develop schizophrenia and other neuropsychiatric disorders. ZDHHC12 has been linked to regulation of amyloid precursor protein (APP) trafficking and metabolism. DHHC17 was originally identified as a huntingtin-interacting protein. A balanced reciprocal translocation between chromosomes X and the ZDHHC15 gene resulted in an absence ofZDHHC15 transcripts, which is associated with X-linked mental retardation (XLMR) and mutations in ZDHHC9 on the X-chromosome were also involved in XLMR. Moreover, increasing evidence shows that DHHC3 is involved in synapse function, while DHHC5 participates in neuronal differentiation in cultured cells, and DHHC17 deficiency is associated with a decreased striatal volume. The aforementioned studies clearly implicate DHHC proteins in neuronal development, likely through their ability to regulate gene expression in cell growth, fate, and function. But the mechanisms underline is not clear. Therefore, to clarify the role of DHHC protein and the molecular mechanisms in the nervous system become the focus of of neuroscience in recent years.With the accelerated pace of life, the incidence of depression increased year by year. While lots of efforts have been paid to studies of depression, but its pathogenesis is still mysterious. but the molecular and cellular mechanisms by which these agents exert their therapeutic effects are not well understood. Early hypotheses on the pathophysiology of major depression were based on aberrant intrasynaptic concentrations and the reduced release of mainly the neurotransmitters serotonin and norepinephrine. Study confirmed the content of neurotransmitters and receptors and functional changes are closely related with depression. It is expected, therefore, that serotonin and noradrenaline represent the major targets of current therapeutic interventions and drug development. But with the development of neurobiology, a simple neurotransmitter theory can not explain pathogenesis of depression. However, recent neuroimaging studies have demonstrated hippocampal volume is decreased in patients in the brains of depressed patients. It has been proposed that a decrease in adult hippocampal neurogenesis provides a biological and cellular basis for major depression. Considers that the reduced newborn hippocampal induced depression and antidepressants may increase cell proliferation reverse stress-induced decreases in hippocampal cell proliferation and neurogenesis. These findings support current theories proposing that major depressive disorders may be associated with impairment of structural plasticity and neural cellular resilience, and that antidepressants may act by correcting this dysfunction. But the mechanism of how it works is unclear.With the completion of genome sequencing project of zebrafish, we found that the gene in zebrafish and human genes conserved degree reached 85 percent, making zebrafish a model for the study of human disease. Meanwhile, the establishment of zebrafish developmental genetics technology provides a good foundation for the analysis of zebrafish models of human disease. Here, we investigate the role of Zdhhcl5b and bupropion in neurodevelopment in zebrafish.Chapter Ⅰ Zdhhcl5b regulates differentiation of diencephalic dopaminergic neurons in zebrafishBackgroundDHHC15, one of the characterized DHHC family members, encoding a 332-amino acids protein with four transmembrane domains and one DHHC-CRD feature domain. Compared with the other members, the current DHHC 15 reported still relatively small, and stay in vitro level. It is reported that DHHC15 is an candidate of x-linked mental retardation, and several neuronal protein can be modified by DHHC 15, suggesting DHHC15 play an important role in the nervous system. However, the precise role of DHHC 15 in the neural development remain largely elusive.Research contentTo detect the temporal and spatial expression of Zdhhcl5b during early embryonic development of zebrafish, whole-mount in situ hybridization (WISH) and RT-PCR was carried out. The result showed Zdhhcl5b transcript was maternally deposited and that ubiquitous zygotic expression was visible from the 1k-cell stage. From the stage of tailbud, the expression of Zdhhcl5b progressively concentrated in the anterior neural plate. From stage 18 hpf through stage 48 hpf, Zdhhcl5b was especially expressed in the diencephalon. The expression of Zdhhcl5b increased from 75% epiboly and endured the crest value from the stage 18 hpf to the stage 72 hpf. These foundings suggested a potential role of Zdhhc 15b in diencephalon development of zebrafish.To detect the function of Zdhhcl5b in diencephalons in developing zebrafish, we disrupted its translation with antisense morpholino oligonucleotides (MO) before the 8-cell period in zebrafish embryos. At 24hpf, the Zdhhcl5b morphants showed indistinguishable boundaries among different regions in the forebrain, with a slightly reduced size in comparison with controls. The forebrain, especially the diencephalon, is the cognitive learning center in zebrafish. The morphological defects in the diencephalon in Zdhhcl5b morphants led us to investigate learning behavior with a T-maze. We found that Zdhhcl5b mutation zebrafish resulted in learning and memory deficits. These foundings suggested Zdhhc15b played an important role in diencephalon development of zebrafish.DA neurons, as important components of the vertebrate diencephalon, play crucial roles in cognition, movement control, and endocrine modulation. The defects found in the diencephalon of Zdhhc15b morphants that were associated with poor learning behavior prompted us to investigate the potential role of Zdhhcl5b in DA neuronal development. Tyrosine hydroxylase (TH) and dopamine transporter (DAT) are special makers of DA neurons. With RT-PCR, we discovered that the expression of two reliable markers was significantly reduced at 24 and 48 h. This result suggests Zdhhcl5b may have a specific role in DA neurons. To confirm our findings, in situ hybridization of TH and DAT/Western blot (WB)/immunofluorescence of TH were carried out. We found that both TH/DAT-positive clusters were found in the diencephalon with fewer DA neurons in Taken together, we propose that Zdhhcl5b regulates the development of diencephalic DA neurons.During development, DA progenitor cells undergo an arrest in cell division and acquire differentiated features in response to differentiation signals to eventually mature into DA neurons. To investigate which stage of DA neurons was affected by Zdhhc15b, PCNA and BrdU analyses demonstrated were carried out. The result showed that Zdhhcl5b morphant have no obvious changes in the cell cycle as compared to controls. Therefore, Zdhhc15b was not necessary for DA progenitor proliferation and maintenance. Next, we found that ngnl, a DA progenitor marker, was comparably expressed in Zdhhcl5b morphants and controls, indicating that Zdhhcl5b was unlikely to be required for the specification of DA precursor cell identity. To test whether Zdhhc15b was involved in apoptosis of DA neurons, acridine orange apoptosis staining was performed at 24hpf, no increase in apoptotic cell death was found at this time point. To determine if Zdhhcl5b is required for the fate differentiation of DA neurons, WISH and RT-PCR were performed to examine the expression of DA neuronal differentiation factors. nurrl, which is expressed in immature DA neurons, decreased in Zdhhcl5b morphants. RT-PCR revealed that lmx1a and foxA, which sequentially regulate the specification and differentiation of neuroepithelial cells toward mature DA neurons, were reduced after Zdhhcl5b knockdown. The gene pitx3 that can induce the expression of immature DA neurons showed a similar decrease. In contrast, expression of enl/2 and Ebf2 showed no significant changes between control and Zdhhcl5b morphants. Futher research discovered that Shh and Wnt/β-catenin signaling pathway were involved in Zdhhcl5b regulated dopaminergic neurons differentiation. Our findings indicate that Zdhhcl5b acts as an intrinsic factor that specifically regulates DA neuron differentiation by recruiting the necessary series of transcription factors through Shh and Wnt/β-catenin signaling pathway.ConclusionIn the present study, we first used zebrafish to characterize the in vivo spatial and temporal expression pattern of Zdhhcl5b. Results of WISH showed that Zdhhcl5b was strongly expressed in the zebrafish diencephalon. Down-regulating Zdhhcl5b in developing zebrafish resulted in a fewer mature DA neurons, in turn yielding a smaller diencephalon with learning deficits.Further study revealed that Zdhhc15b was unnecessary for DA progenitor proliferation. Moreover, the same expression of ngnl in control and morphant zebrafish revealed that Zdhhcl5b was unlikely to be required for the specification of DA precursor cell identity. Therefore, we propose that Zdhhcl5b played a role in the differentiation of DA neurons. foxA2, lmx1a, and pitx3 were downregulated after Zdhhcl5b knockdown, leading us to conclude that Zdhhcl5b might affect DA neuronal differentiation by recruiting the required series of transcription factors through Shh and Wnt/β-catenin signaling pathway.Altogether, the paper provide new insight into the development of DA neurons and shed new light on the treatment of nervous system diseases caused by dysfunction of DA neurons like Alzheimer and Parkinson’s diseases.Chapter Ⅱ Effects of bupropion on protection and proliferation of zebrafish dopaminergic neuronsBackgroundDepression is a common mood disorder, with a high incidence、high morbidity and high recurrence rate. The incidence of depression has increased gradually in recent years.Depression has become a threat to people’s physical and mental health.Bupropion is a new antidepressant drug. With the in-depth study of bupropion, its roles in smoking cessation, improving alcohol dependence, and attention deficit hyperactivity disorder (attention deficit hyperactive disorder, ADHD) has been discovered. Despite these important advances, its mechanism is not fully understood. It is known to all that dopaminergic neurons is closely related to depression, whether bupropion has any effect on dopaminergic neurons and what the mechanism is both are problems to be solved in this research.In this study, we establish a damage model of dopaminergic neuron by MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) to study the effect of bupropion zebrafish dopaminergic neurons, and further discusses its mechanism.Research contentTo investigate whether bupropion plays a role in DA neurogenesis, we first performed in situ hybridization (WISH) of TH. The result showed that TH-positive clusters were found in diencephalon and MPTP caused a loss of diencephalic DA neurons significantly as compaired with control. But the situation in bupropion zebrafish was different. It improved the injury of MPTP on dopaminergic neurons, the number of DA neurons was increased with bupropion treatment and the phenotype was doze-depended. Subsequently, immunofluorescence of TH exhibited a similar pattern as shown by an increase in TH-positive DA neurons. WISH for DAT, another marker for mature DA neurons, also showed similar results. Taken together, the findings propose that bupropion maybe invoved in the development of diencephalic DA neurons.The forebrain, especially the diencephalon, is the cognitive learning center in zebrafish, and DA neurons were the executor. The founding above led us to investigate learning behavior with a T-maze. We found that MPTP treatment resulted in learning and memory deficits, but bupropion could improved the disorder of learning and memory ability caused by MPTP. Our results suggest bupropion may improve the learning impairment induced by MPTP through increasing the number of zebrafish dopaminergic neuron.Given the fact that the number of both TH+and DAT+neurons was increased in bupropion embryos, it would be intriguing to determine the protective effects of bupropion on DA neurons. In order to examine the role of bupropion in DA neurons apoptosis, acridine orange staining was performed at 24 hpf and 48hpf. The result showed the apoptotic cells in PT(posteriortuberculum, PT) area was increased dramatically when treated with MPTP as compared with control, but bupropion could reverse the situation. The results indicated that bupropion may protection DA neuron from apoptosis.In addition to the protective effect, several antidepressants are involved in neurogenesis. In order to further identify how bupropion effect DA neurogenesis, WISH of PCNA and BrdU analyses were performed. The results demonstrated that bupropion treated embryos got more number of neurons experienced proliferation in PT area where DA neurons existed. Therefore, bupropion may be necessary for DA progenitor proliferation and maintenance. WISH of ngnl was carried out to conform our findings, we found that ngnl, a DA progenitor marker, was expressed much stronger in bupropion treated embryos than MPTP embryos. These results imply that bupropion is critical for promoting the proliferation of DA progenitor.ConclusionHowever, the main mechanism of dopamine neurons regeneration and depression together with antidepressant treatment is unclear, moreover, the lack of clinical studies in humans, which will be a major issue to be resolved in the future. In short, we need to explore the function of dopamine neurons associated with depression in deep sight. Dopaminergic neurons regeneration may be the treatment of major depressive disorder and provide a new target for psychiatric disorders. A thorough understanding of the cellular and molecular mechanisms involved in the development of DA neuron will greatly facilitate the use of antidepressant therapy for depression treatment and other psychiatric diseases induced by DA neuron dysfunction.