The ubiquitin proteasome system in Huntington disease : impairment of the proteolytic machinery aggravates huntingtin aggregation and toxicity

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 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.Prinses Beatrix Fonds, MAR 99-0113 Printing of thesis: Netherlands Institute for Neuroscience Stichting het Remmert Adriaan Laan fonds Vereniging van Huntington Leiden University Medical CenterUBL - phd migration 201

Computer aided synthesis: a game theoretic approach

In this invited contribution, we propose a comprehensive introduction to game theory applied in computer aided synthesis. In this context, we give some classical results on two-player zero-sum games and then on multi-player non zero-sum games. The simple case of one-player games is strongly related to automata theory on infinite words. All along the article, we focus on general approaches to solve the studied problems, and we provide several illustrative examples as well as intuitions on the proofs.Comment: Invitation contribution for conference "Developments in Language Theory" (DLT 2017

Proteasome Activator Enhances Survival of Huntington's Disease Neuronal Model Cells

In patients with Huntington's disease (HD), the proteolytic activity of the ubiquitin proteasome system (UPS) is reduced in the brain and other tissues. The pathological hallmark of HD is the intraneuronal nuclear protein aggregates of mutant huntingtin. We determined how to enhance UPS function and influence catalytic protein degradation and cell survival in HD. Proteasome activators involved in either the ubiquitinated or the non-ubiquitinated proteolysis were overexpressed in HD patients' skin fibroblasts or mutant huntingtin-expressing striatal neurons. Following compromise of the UPS, overexpression of the proteasome activator subunit PA28γ, but not subunit S5a, recovered proteasome function in the HD cells. PA28γ also improved cell viability in mutant huntingtin-expressing striatal neurons exposed to pathological stressors, such as the excitotoxin quinolinic acid and the reversible proteasome inhibitor MG132. These results demonstrate the specific functional enhancements of the UPS that can provide neuroprotection in HD cells

Disease-Associated Mutant Ubiquitin Causes Proteasomal Impairment and Enhances the Toxicity of Protein Aggregates

Protein homeostasis is critical for cellular survival and its dysregulation has been implicated in Alzheimer's disease (AD) and other neurodegenerative disorders. Despite the growing appreciation of the pathogenic mechanisms involved in familial forms of AD, much less is known about the sporadic cases. Aggregates found in both familial and sporadic AD often include proteins other than those typically associated with the disease. One such protein is a mutant form of ubiquitin, UBB+1, a frameshift product generated by molecular misreading of a wild-type ubiquitin gene. UBB+1 has been associated with multiple disorders. UBB+1 cannot function as a ubiquitin molecule, and it is itself a substrate for degradation by the ubiquitin/proteasome system (UPS). Accumulation of UBB+1 impairs the proteasome system and enhances toxic protein aggregation, ultimately resulting in cell death. Here, we describe a novel model system to investigate how UBB+1 impairs UPS function and whether it plays a causal role in protein aggregation. We expressed a protein analogous to UBB+1 in yeast (Ubext) and demonstrated that it caused UPS impairment. Blocking ubiquitination of Ubext or weakening its interactions with other ubiquitin-processing proteins reduced the UPS impairment. Expression of Ubext altered the conjugation of wild-type ubiquitin to a UPS substrate. The expression of Ubext markedly enhanced cellular susceptibility to toxic protein aggregates but, surprisingly, did not induce or alter nontoxic protein aggregates in yeast. Taken together, these results suggest that Ubext interacts with more than one protein to elicit impairment of the UPS and affect protein aggregate toxicity. Furthermore, we suggest a model whereby chronic UPS impairment could inflict deleterious consequences on proper protein aggregate sequestration

The additional value of patient-reported health status in predicting 1-year mortality after invasive coronary procedures: A report from the Euro Heart Survey on Coronary Revascularisation

Objective: Self-perceived health status may be helpful in identifying patients at high risk for adverse outcomes. The Euro Heart Survey on Coronary Revascularization (EHS-CR) provided an opportunity to explore whether impaired health status was a predictor of 1-year mortality in patients with coronary artery disease (CAD) undergoing angiographic procedures. Methods: Data from the EHS-CR that included 5619 patients from 31 member countries of the European Society of Cardiology were used. Inclusion criteria for the current study were completion of a self-report measure of health status, the EuroQol Questionnaire (EQ-5D) at discharge and information on 1-year follow-up, resulting in a study population of 3786 patients. Results: The 1-year mortality was 3.2% (n = 120). Survivors reported fewer problems on the five dimensions of the EQ-5D as compared with non-survivors. A broad range of potential confounders were adjusted for, which reached a p<0.10 in the unadjusted analyses. In the adjusted analyses, problems with self-care (OR 3.45; 95% CI 2.14 to 5.59) and a low rating (≤ 60) on health status (OR 2.41; 95% CI 1.47 to 3.94) were the most powerful independent predictors of mortality, among the 22 clinical variables included in the analysis. Furthermore, patients who reported no problems on all five dimensions had significantly lower 1-year mortality rates (OR 0.47; 95% CI 0.28 to 0.81). Conclusions: This analysis shows that impaired health status is associated with a 2-3-fold increased risk of all-cause mortality in patients with CAD, independent of other conventional risk factors. These results highlight the importance of including patients' subjective experience of their own health status in the evaluation strategy to optimise risk stratification and management in clinical practice