Browsing by Author "Ahvenainen, Terhi"

Huntington's disease (HD) is a progressive neurodegenerative disorder that causes involuntary muscle movements, deteriorates muscle coordination and cognitive decline. Typical onset age of the disease is in mid age, although a juvenile form of HD is also known. The disease is inherited in an autosomal dominant manner via a mutation in the huntingtin gene (HTT). The characteristic mutation in HTT is an expansion of the glutamine stretch at the 5 end of the gene. Excessive amounts of glutamine residues alters the conformation and chemical features of the huntingtin protein (HTT) leading to accumulation of cellular aggregates. Although to date there are several known alterations in the cell that contribute to the disease, the pathogenesis of HD is not fully understood. Ubiquitin proteasome system (UPS) dismantles proteolytically unneeded or damaged proteins, which are targeted to proteolysis when ubiquitin tags are added to them. Deubiquitinating enzymes (DUB) recycle ubiquitin molecules by releasing them from proteasome substrates. Recycling of ubiquitin is critical to a cell as it maintains the free pool of the targeting molecule. Ubiquitin-specific protease 14 (USP14) is one of the DUB family enzymes and its distinctive function is to remove ubiquitin molecules from the tip of the ubiquitin chain and thus antagonize protein degradation. Although the specific function of the protein is unclear, it seems that USP14 operates as a fine regulator of protein turnover rate and in ER stress both in catalytic and non catalytic manner. The role of USP14 is especially emphasized in the nervous system, as it regulates synaptic transmission and neuronal development. Although it is suggested that dysfunction of UPS is involved in the pathogenesis of HD, the role of USP14 in the disease remains to be unknown. IU1 is a novel inhibitor of the catalytic domain of USP14. Studies with IU1 indicate that inhibition of USP14 enhances the clearance of aggregate prone proteins. The approach of this thesis was aimed to elucidate the routes of HD pathogenesis from diverse approaches. The general aim of the thesis was to investigate the role of USP14 in the wild-type PC6.3 cell model, and in the pathogenesis of HD by expressing HTT proteins with different lengths of glutamine stretches in PC6.3 cells. The specific aim of the study was to examine by western blot and microscopy analysis the pathogenic routes of HD that involve ER stress, oxidative stress, autophagy and mutant HTT aggregate dynamics. The function of USP14 was studied with overexpression of USP14, or by inhibiting its catalytic activity by IU1. The findings of this thesis show that overexpression of USP14 enhances the clearance of mutant HTT aggregates, and this effect is obtained in catalytic activity dependent manner. I show that upregulated USP14 is connected to improved clearance of mutant HTT and inhibition of autophagy, suggesting that the degradation is mediated via UPS. The catalytic activity of USP14 might also be important in ER stress regulation, as the results indicate that IU1 activates phosphorylation of both JNK and eIF2α. I was also able to establish a connection between USP14 and GADD34, as I show that GADD34 upregulates USP14. Finally, I show that catalytic inhibition of USP14 decreases the expression of antioxidant SOD2. The data in this thesis is lacking statistical significance, and it can be considered solely as a guideline. However, together these results indicate that the deubiquitinating activity of USP14 increases survival in PC6.3 cells in both a healthy and a HD model.