Parkinson's disease (PD) is the second most common neurodegenerative motor disorder, affecting approximately 1% of the population aged > or =60 years. Recent investigations have shown that in addition to motor symptoms such as bradykinesia, resting tremor, and gait instability, PD also causes non-motor symptoms such as insomnia, constipation, depression, and dementia. Most PD cases occurred sporadically, but 5-10% is inherited as familial PD, and several PD-causative genes have been identified and intensively studied. Autophagy is a self-degrading mechanism of balancing the energy source in response to nutrient shortage and various stresses, and is a tightly regulated and complicated process that generates double-membrane organelles. Autophagy failure has recently been observed in both animal PD models and human PD patients. The intention of this review is to introduce recent findings regarding the relationship between causative genetic mutations in PD and autophagy, from a clinical perspective.
Animals ; Autophagy* ; Constipation ; Dementia ; Depression ; Gait ; Humans ; Hypokinesia ; Intention ; Mitochondrial Degradation ; Organelles ; Parkinson Disease* ; Sleep Initiation and Maintenance Disorders ; Tremor

Extracellular vesicles (EVs) are small membranous vesicles that are secreted by various types of cells into biofluid or culture medium. EVs contain deoxyribonucleic acids, messenger ribonucleic acids (RNAs), microRNAs, lipids, and proteins derived from its cells of origin and can transfer those molecules to other targeted cells. Therefore, EVs can play important roles in intercellular communication. The findings of recent studies suggest that EVs can be used to spread protein aggregates in various neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In addition, it has been recognized that EVs can be used as a material for detecting biomarkers for such diseases or as a therapeutic tool.
Alzheimer Disease ; Biomarkers ; DNA ; MicroRNAs ; Neurodegenerative Diseases ; Parkinson Disease ; RNA

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.
Amino Acid Sequence ; Animals ; Exocytosis ; Female ; HEK293 Cells ; Humans ; Mice ; Molecular Sequence Data ; Mutant Proteins/metabolism ; Phosphorylation ; Phosphothreonine/metabolism ; Protein Binding ; Protein Interaction Mapping ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases/*metabolism ; Qa-SNARE Proteins/metabolism ; Rats ; Rats, Sprague-Dawley ; Synaptosomal-Associated Protein 25/*metabolism ; Synaptotagmins/metabolism ; Vesicle-Associated Membrane Protein 2/metabolism ; Vesicular Transport Proteins/chemistry/*metabolism

Mutations in the Leucine-rich repeat kinase 2 (LRRK2 ) gene are the most prevalent cause of familial Parkinson’s disease (PD). The increase in LRRK2 kinase activity observed in the pathogenic G2019S mutation is important for PD development. Several studies have reported that increased LRRK2 kinase activity and treatment with LRRK2 kinase inhibitors decreased and increased ciliogenesis, respectively, in mouse embryonic fibroblasts (MEFs) and retinal pigment epithelium (RPE) cells. In contrast, treatment of SH-SY5Y dopaminergic neuronal cells with PD-causing chemicals increased ciliogenesis. Because these reports were somewhat contradictory, we tested the effect of LRRK2 kinase activity on ciliogenesis in neurons. In SH-SY5Y cells, LRRK2 inhibitor treatment slightly increased ciliogenesis, but serum starvation showed no increase. In rat primary neurons, LRRK2 inhibitor treatment repeatedly showed no significant change. Little difference was observed between primary cortical neurons prepared from wild-type (WT) and G2019S +/- mice. However, a significant increase in ciliogenesis was observed in G2019S +/- compared to WT human fibroblasts, and this pattern was maintained in neural stem cells (NSCs) differentiated from the induced pluripotent stem cells (iPSCs) prepared from the same WT/G2019S fibroblast pair. NSCs differentiated from G2019S and its gene-corrected WT counterpart iPSCs were also used to test ciliogenesis in an isogenic background. The results showed no significant difference between WT and G2019S regardless of kinase inhibitor treatment and B27-deprivation-mimicking serum starvation. These results suggest that LRRK2 kinase activity may be not a direct regulator of ciliogenesis and ciliogenesis varies depending upon the cell type or genetic background.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2 ) gene are the most prevalent cause of familial Parkinson’s disease (PD). The increase in LRRK2 kinase activity observed in the pathogenic G2019S mutation is important for PD development. Several studies have reported that increased LRRK2 kinase activity and treatment with LRRK2 kinase inhibitors decreased and increased ciliogenesis, respectively, in mouse embryonic fibroblasts (MEFs) and retinal pigment epithelium (RPE) cells. In contrast, treatment of SH-SY5Y dopaminergic neuronal cells with PD-causing chemicals increased ciliogenesis. Because these reports were somewhat contradictory, we tested the effect of LRRK2 kinase activity on ciliogenesis in neurons. In SH-SY5Y cells, LRRK2 inhibitor treatment slightly increased ciliogenesis, but serum starvation showed no increase. In rat primary neurons, LRRK2 inhibitor treatment repeatedly showed no significant change. Little difference was observed between primary cortical neurons prepared from wild-type (WT) and G2019S +/- mice. However, a significant increase in ciliogenesis was observed in G2019S +/- compared to WT human fibroblasts, and this pattern was maintained in neural stem cells (NSCs) differentiated from the induced pluripotent stem cells (iPSCs) prepared from the same WT/G2019S fibroblast pair. NSCs differentiated from G2019S and its gene-corrected WT counterpart iPSCs were also used to test ciliogenesis in an isogenic background. The results showed no significant difference between WT and G2019S regardless of kinase inhibitor treatment and B27-deprivation-mimicking serum starvation. These results suggest that LRRK2 kinase activity may be not a direct regulator of ciliogenesis and ciliogenesis varies depending upon the cell type or genetic background.

For cancer gene therapy, cancer-specific over-expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.
Breast Neoplasms/*genetics/therapy ; Cell Cycle Proteins/*genetics/metabolism ; Cell Line, Tumor ; Cloning, Molecular ; Cytomegalovirus ; Dependovirus ; Female ; *Gene Targeting ; Gene Therapy ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Promoter Regions, Genetic/*genetics ; Ribonucleoside Diphosphate Reductase/*genetics/metabolism ; *Transcriptional Activation