| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 105 === The transport of neurotransmitters is essential for the regulation of nervous system. The abnormal level of neurotransmitters in brain often causes developmental problems and distinct behaviors. Therefore, understanding the mechanism of neurotransmitter transport is required for developing the treatment of neuron diseases. The limb region 1 domain containing 1 (LMBRD1) gene encodes two major proteins, named LMBD1 and NESI. The LMBD1 protein contains additional 73 amino acids residues at the N-terminus of NESI protein. Previous studies have shown that LMBD1 protein is a putative lysosomal vitamin B12 exporter. In addition, our studies further elucidated that LMBD1 protein serves as a specific adaptor protein and mediates the clathrin-mediated internalization of insulin receptor. Furthermore, our recent studies showed that Lmbrd1 heterozygous knockout (Lmbrd1+/-) mice exhibited distinct behaviors from normal mice such as fear, anxiety, pre-pulse inhibition, depression, sociability deficits, and insufficiency on learning memory. As the result of the abnormal behaviors of emotion and memory, our studies focus on striatum which involves in the regulation of emotion and memory reward. The data of positron emission tomography (PET) with 18F-DOPA showed an increase of dopamine level in Lmbrd1+/- mouse striatum. The specific aim of this study is to elucidate the mechanisms by which LMBD1 protein regulates dopamine transport. Results from the study of dopamine uptake in the aromatic L-amino acid decarboxylase (AADC) deficiency mouse model supported our hypothesis related to the function of LMBD1 protein in dopamine transport, indicating that the reduction of LMBD1 expression in the striatum of AADC deficiency mice with young age resulted in the dopamine accumulation to compensate the lack of dopamine biosynthesis. Dopamine autoreceptor D2 (D2autoR) and dopamine transporter (DAT) are responsible for dopamine transport. D2autoR regulates the dopamine exocytosis by negative feedback mechanism, whereas DAT directly transports extracellular dopamine into cells. In addition, D2autoR regulates the surface expression of DAT by PKA pathway, PKCβ-ERK pathway and by direct protein-protein interaction. In this study, dopamine uptake was increased when Lmbrd1 gene was knocked down in striatal STHdhQ7 cells. The phosphorylation of PKA was reduced in Lmbrd1+/- mouse striatum and Lmbrd1 knockdown STHdhQ7 cells, whereas the phosphorylation of ERK was increased. In addition, confocal image showed that LMBD1 partially colocalized with D2autoR. These data suggested that LMBD1 might play a role in regulating D2autoR and into downstream signal pathway. On the other hand, co-immunoprecipatation assay showed LMBD1 associated with DAT when co-expressed in HEK293T cells. GST pull down assay further demonstrated the interaction of the C terminus of LMBD1 with DAT present in STHdhQ7 cells. In addition, confocal image showed that DAT partially colocalized with LMBD1. These results suggested that LMBD1 might regulate D2autoR and DAT function involved in dopamine uptake.
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