LRRK2 regulates synaptic vesicle endocytosis.

The leucine-rich repeat kinase 2 (LRRK2) has been identified as the defective gene at the PARK8 locus causing the autosomal dominant form of Parkinson's disease (PD). Although several LRRK2 mutations were found in familial as well as sporadic PD patients, its physiological functions are not clearly defined. In this study, using yeast two-hybrid screening, we report the identification of Rab5b as an LRRK2-interacting protein. Indeed, our GST pull down and co-immunoprecipitation assays showed that it specifically interacts with LRRK2. In addition, subcellular fractionation and immunocytochemical analyses confirmed that a fraction of both proteins co-localize in synaptic vesicles. Interestingly, we found that alteration of LRRK2 expression by either overexpression or knockdown of endogenous LRRK2 in primary neuronal cells significantly impairs synaptic vesicle endocytosis. Furthermore, this endocytosis defect was rescued by co-expression of functional Rab5b protein, but not by its inactive form. Taken together, we propose that LRRK2, in conjunction with its interaction with Rab5b, plays an important role in synaptic function by modulating the endocytosis of synaptic vesicles.

Regulation of tyrosine hydroxylase gene expression by retinoic acid receptor.

Retinoic acid (RA), a derivative of vitamin A, critically controls brain patterning and neurogenesis during embryogenesis, and is known to regulate morphological differentiation of catecholaminergic neuronal cells. In this study, we investigated whether the retinoic acid receptor (RAR), a transcription factor specifically activated by all-trans-RA, could directly regulate transcription of tyrosine hydroxylase (TH), the first and rate-limiting step in the catecholamine biosynthesis pathway. First, treating TH-expressing human neuroblastoma SK-N-BE(2)C cells with all-trans RA resulted in an approximately 1.7-fold increase in endogenous TH mRNA expression, as determined by real-time PCR analysis. Second, when SK-N-BE(2)C cells were transiently co-transfected with the TH promoter-luciferase reporter construct, reporter gene expression was prominently activated by RAR in a ligand-dependent manner. Third, we identified a putative RAR responsive cis-regulatory element at - 1500 to - 1487 bp in the TH upstream promoter region by deletional and site-directed mutational analysis. Finally, we demonstrated that this putative motif directly interacts with RAR protein in a sequence-specific manner by means of an electrophoretic mobility shift assay. Taken together, our results indicate that the TH gene may be a direct downstream target of the RA signaling pathway and that RAR is able to activate TH transcription through interaction with an upstream sequence motif residing at - 1500 to - 1487 bp.

Regulation of the transcriptional activity of the tyrosine hydroxylase gene by androgen receptor.

Dopamine and the sex hormone testosterone are important factors regulating male sexual behavior. To investigate the possibility that these two factors are functionally interrelated, we investigated the potential role of the androgen receptor (AR) on transcriptional activity of the tyrosine hydroxylase (TH) gene that encodes the rate-limiting enzyme of the dopamine biosynthesis pathway. In this study, using transient co-transfection assays in TH-positive SK-N-BE(2)C and MN9D cells, we show that AR prominently transactivates TH promoter function in a ligand-dependent manner. Deletional and site-directed mutational analyses have mapped a putative androgen response element (ARE) in a region from -1562 to -1328 base pairs in the upstream TH promoter. We also found that DJ-1, one of recently identified genes whose mutations cause Parkinson's disease, down-regulated AR-dependent TH activation by approximately 50% in SK-N-BE(2)C cells. Based on these data, we propose that AR activates TH gene expression and that DJ-1 may modulate AR activity as a transcriptional co-repressor.