Effects of HAP40 on clearance of mutant huntingtin and neurite outgrowth

• Main Authors: Yi-TingSu, 蘇意婷
• Other Authors: Lu-Shiun Her
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/93198244733074635519

碩士 === 國立成功大學 === 生命科學系碩博士班 === 101 === Huntington's disease (HD) is a dominant inherited neurodegenerative disorder caused by CAG repeats encoding polyglutamine (polyQ) expansion within the first exon of huntingtin (htt). The amino-terminal fragments of mutant huntingtin accumulate and form aggregates in neuronal nuclei and cytoplasm, primarily in the striatum and cortex of HD patients. It is still unclear whether aggregates are protective or detrimental for cell viability. Htt is enriched in the brain where it associates with microtubules and vesicles, such as Golgi apparatus, and mitochondria. HAP40 is a 40-kDa huntingtin-associated protein. Like huntingtin, HAP40 associates with microtubules and shuttles in axon. Previous studies have shown the huntingtin–HAP40 complex is an effector of Rab5 that regulates the dynamics of early endosomes through a switch from microtubules to F-actin. Endogenous HAP40 protein levels were increased in STHdhQ111 striatal cells and fibroblasts and brain tissue from human HD patients. Thus, up-regulation of HAP40 protein level might contribute to the selective neuropathology of HD. Previous studies had shown that aggregates of mutant N-Htt are enriched for components of the protein quality control machinery, such as ubiquitin, proteasome subunits, and chaperones. We found that overexpression of HAP40 promotes aggregation of mutant huntingtin in N2a cell. To investigate whether HAP40 affects ubiquitin proteasome system, we transfected N2a cell with HAP40, ubiquitin and N-Htt (WT or mutant). Overexpression of HAP40 enhances accumulation of polyubiquitinated protein. Depletion of Htt in HeLa cells results in Golgi disruption, similar to the effects by depletion of dynein and dynactin. In stable N2a cell lines expressing HAP40, the structures of Golgi were disrupted. During neurite outgrowth, actin and microtubules dynamics and membrane traffic play a role in initiation and elongation of neurites. In PC12 and N2a stable cell lines expressing HAP40, we found that HAP40 increase the percentage of cells with neurite.