Huntington disease (HD) is the best know of the polyglutamine disorders which are caused by the excessive expansion of a CAG repeat in a transcribed gene. It is estimated that there are 1300 HD... Show moreHuntington disease (HD) is the best know of the polyglutamine disorders which are caused by the excessive expansion of a CAG repeat in a transcribed gene. It is estimated that there are 1300 HD patients in the Netherlands and even more people that are currently at risk. Translation of the CAG repeat results in proteins with an expanded polyglutamine repeat. Expansion of this repeat above a threshold of 36 results in an aggregation prone protein leading to disease onset around mid-life. In all polyglutamine diseases an inverse correlation is found between repeat expansion and age of disease onset. Neuronal intranuclear inclusions (NII) which contain at least part of the expanded protein are identified as hallmarks of the polyglutamine diseases. Inclusions have been suggested to function as protective storage sites implying that soluble misfolded proteins are the primary cause of toxicity. The aggregation prone properties of these proteins are causing the pathological gain of function due to interference with normal cellular function. The ubiquitin proteasome system (UPS) is responsible for the main protein degradation within the cell. The UPS contributes to cellular homeostasis by regulating the expression of essential proteins and degradation of excessive proteins. In addition, the UPS is involved in cellular protein quality control and responsible for the degradation of aberrant proteins that accumulate in neurodegeneration. Also expanded polyglutamine proteins can be degraded by the UPS in order to protect the cell from this aggregation prone protein. However, the expanded repeat is also difficult to degrade causing impairment of the proteasome and consequential accumulation. The general introduction in Chapter 1 gives an overview of several of the important aspects leading to neurodegeneration in HD and specifically the contribution of the UPS. Chapter 2 describes the discovery of molecular misreading which occurs on GAGAG motifs in different genes. The frameshift mutant of ubiquitin B (UBB+1) is formed by a dinucleotide deletion (_GU) in the mRNA. The mutant ubiquitin protein that is formed can no longer ubiquitinate substrate proteins but is a target for ubiquitination and subsequent proteasomal degradation. UBB+1 accumulates in several neurodegenerative diseases and is an in vivo marker for proteasomal inhibition. In Chapter 3, we investigated the accumulation of UBB+1 in HD and Spinocerebellar ataxia-3 (SCA3) and potential contribution to neurodegeneration. UBB+1 was found to accumulate in the cytoplasm and NII in the affected brain regions of HD and SCA3 demonstrating in vivo proteasome inhibition in these disorders. In a cellular model of HD we observed that UBB+1 results in inhibition of the proteasome which causes increased aggregate formation. In addition, a synergistic increase in polyglutamine induced cell death was found upon expression of UBB+1. These findings implicate UBB+1 as an aggravating factor in polyglutamine induced neurodegeneration and stresses the importance of the UPS for degradation of aberrant polyglutamine and UBB+1 proteins. UBB+1 transgenic mice show a mild inhibition of the proteasome. In Chapter 4 we tested the influence of this UPS inhibition by UBB+1 on Htt aggregation in vivo. Expression of expanded polyglutamine protein in the striatum of the UBB+1 transgenic mice showed a strong increase in NII formation compared to wildtype littermates. These results demonstrate in vivo that minor differences in UPS capacity can have major detrimental effects on the neuropathology of HD. The ubiquitin conjugating enzyme E2-25K has been shown to interact directly with Htt independent of polyglutamine repeat length. In Chapter 5, we investigated the localization of E2-25K in HD as well as the contribution to neurodegeneration. E2-25K co-localizes with a subset of NII in HD brain as well as with aggregates in apoptotic cells in vitro. Dominant negative E2-25K __ lacking the catalytic tail domain __ as well as an antisense construct decreased aggregate formation of expanded Htt. Additionally, mutant and antisense E2-25K reduced polyglutamine-induced cell death. These findings show that ubiquitination of E2-25K-targets contributes to aggregate formation as well as neuronal cell death in HD. Finally, in Chapter 6 the different findings of this thesis are discussed as well as further research and perspectives. In conclusion, the findings of this thesis illustrate the importance of the UPS for the cellular clearance of toxic proteins involved in neurodegeneration. The precise mechanism of specific neuronal dysfunction in HD is still unclear but is triggered by the gain of function of the polyglutamine repeat. Impairment of the UPS results in the further accumulation of aberrant proteins and subsequent neuronal dysfunction. Since HD is caused by protein expression from a mutant allele, more efficient degradation could protect the neurons from harmful polyglutamine proteins. Show less
