Hsp27 In Peripheral Neuron Degenerative Diseases

• Main Authors: Pei-Yin Wen, 溫珮吟
• Other Authors: Mingli Hsieh
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/72229745683268384590

碩士 === 東海大學 === 生命科學系 === 96 === Charcot-Marie-Tooth (CMT) is a dominantly hereditary motor and sensory neurodegenerative disease. Previous studies showed that mutations of highly conserved α- crystallin domain in heat shock protein 27 (Hsp27) are responsible for axonal CMT type 2F. Hsp27, a member of small Hsps, has chaperone activity and anti-apoptotic function. Additionally, Hsp27 maintains the cell property by interacting with cytoskeleton. Therefore, we propose that because α-crystallin domain of Hsp27 is an important functional domain, missense mutation on the conserved residues of this region may disrupt normal functions of Hsp27, resulting in cytoplasmic aggregation and abnormal neuronal intermediate filament network . To look for novel mutation, we sequenced the α-crystallin domain of Hsp27 gene from CMT patients in Taiwan. Up to date, 14 patients with CMT disease have been examined but none of them contain mutations in α-crystallin domain of Hsp27. On the other hand, we successfully made Hsp27 constructs carrying various mutations on α- crystallin domain or other region by site-directed mutagenesis, including K112W, E119L, K123W, R127W, S135F, R140E, and P182L. The last four mutants were made according to pathogenic mutations and the others were changed from charged amino acids to non-charged ones. After transfecting different constructs into a mouse neuroblastoma cell line (N2a), similar expression levels of normal and mutant Hsp27 were confirmed by Western blot analysis. In addition, results from immunocytochemical staining demonstrated that only P182L led to severe cytoplasmic aggregates in N2a cells. Furthermore, DAPI staining was used to examine cell viability. Our results showed that pathogenic mutant R140E, but not R127W, significantly reduced cell viability, as compared with wild type Hsp27. However, other mutants do not have significant impacts on cell viability in our assay conditions. We conclude that different specific amino acid may be required for different functions of Hsp27. According to our results, we suggest that these charged residues on α-crystallin domain are not equally important for the functions we examined in the current study. However, we can not exclude the possibility that some amino acids may be involved in unidentified cellular functions of Hsp27. Currently, stable cell lines expressing normal or pathogenic mutant Hsp27, including R127W, S135F, R140E and P182L, are under construction. These cell models will be used to study the pathogenesis of CMT in the future.