α-synuclein in Saccharomyces cerevisiae: model for aggregate clearance, cell survival and influence of autophagy

α-Synuclein ist ein neuronal exprimiertes Protein, das an neurodegenerativen Krankheiten wie Morbus Parkinson beteiligt ist. Fehlenhafte Faltung und Akkumulation in zytoplasmatische Einschlusskörper charakterisieren die Pathogenese dieser Krankheit. Diese sind auch durch Überexpression von α-synuclein in Hefezellen nachvollziehbar. In dieser Arbeit wurden das GAL1-Promotor gesteuerte Wildtyp (WT) α-Synuclein mit mehreren Varianten mit und ohne Fluoreszenzmarkierung im Modellsystem Saccharomyces cerevisiae untersucht. Die Überexpression des WT- und A53T-mutierten α-synucleins hemmte das Hefewachstum und resultierte in zytoplasmatischen Aggregaten. C-terminal fluoreszenzmarkierte Varianten zeigten die gleichen Effekte. Die Integration von drei WT- oder zwei A53T-Kopien in einen einzigen genomischen Lokus resultierten in einer signifikanten Wachstumshemmung sowie Akkumulation und wurden somit als Toxizitätsschwelle definiert. Die A30P Variante hatte nur einen gering hemmenden Effekt auf das Hefewachstum und bildete nur transiente Aggregate bei Überexpression. Im Gegensatz dazu zeigte die triple-Prolin-Variante A30P/A56P/A76P weder eine Wachstumshemmung noch nachweisbare Aggregate. Promoterabschaltungsexperimente zeigten, dass Hefezellen Aggregate auflösen können. Das 26S Proteasom spielt bei diesem α-Synuclein Abbau keine wichtige Rolle, da eine Proteasomhemmung durch das Medikament MG132 oder durch eine genetische cim3-1 Mutation die Auflösung der Aggregate wenig beeinflußt. Im Gegensatz dazu fuhrt die Hemmung durch den vakuolaren Proteaseninhibitor Phenylmethylsulfonylfluorid zu einer deutlichen Inhibition bei der Auflösung von α-Synuclein Aggregaten. Ebenso zeigte die Δpep4 Hefemutante eine verminderte Fähigkeit der Aggregatklärung. Die Autophagie-defiziente Hefemutante Δatg1 zeigte eine verzögerte Klärung, was auf eine Beteiligung von Autophagie an dem Prozess schließen lässt. Dies wurde dadurch bestätigt, dass α-synuclein-Aggregate auch reduziert wurden, wenn die Zellen mit dem Autophagie-indu zierenden Medikament Rapamycin behandelt wurden. Rapamycin vorbehandelte Zellen zeigten zusätzlich eine verminderte Aggregatbildung, was die Bedeutung von Autophagie in der α-synuclein Pathobiologie weiter validiert. Im Gegenzug hatte α-Synuclein auch einen Einfluss auf Autophagie. Während A30P α-Synuclein Autophagie transient hoch-regulieren konnte, hatte A53T einen hemmenden Effekt. WT und A53T α-synuclein zusätzlich verzögerten die Induktion von Autophagie. Eine cim3-1Δatg1 Doppelmutante klärte Aggregate nach Promotorabschaltung, was andeutet dass zusätzliche zellulare Mechanismen zur Klärung beitragen müssen. Diese Daten bieten neue Einsichten in die Prozesse, die Hefezellen benutzen um α-synuclein Aggregate zu entfernen und eröffnen neue Perspektiven für therapeutische Eingriffe

Inhibition of protein ubiquitination by paraquat and 1-methyl-4-phenylpyridinium impairs ubiquitin-dependent protein degradation pathways

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson’s disease (PD). Ubiquitin (Ub), alpha [α]-synuclein, p62/sequestosome 1 and oxidized proteins are major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effect of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP+, or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ–induced cell death. Inhibition of proteasomal activity by PQ was found to be a late event in cell death progression, and had no effect on either the toxicity of MPP+ or PQ, or the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins) and carbonylated proteins induced by PQ. PQ- and MPP+-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagic. We confirmed that PQ and MPP+ impaired autophagy flux, and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane associated foci in yeast cells. Our results demonstrate that inhibition of protein ubiquitination by PQ and MPP+ is involved in the dysfunction of Ub-dependent protein degradation pathways

The evidence for working memory training transfer effects on sentence comprehension and functional communication in persons with aphasia is inconclusive1

The primary aim of this study was to identify maintenance and generalization pattern in cognitive and language domains in persons with aphasia (PWA) following working memory (WM) training

A Cognitive Psychometric Investigation of Word Production and Phonological Error Rates in Logopenic Progressive Aphasia.

Purpose This study investigated the relationship between word production rates (WPRs) and phonological error rates (PERs) in generative and responsive tasks in logopenic progressive aphasia (lvPPA). We examined whether a portion of the reduced WPR during generative tasks related directly to phonological impairments affecting PER on all tasks, irrespective of other task differences that contributed to WPR. Method Two cognitive psychometric models were hypothesized and fit to the total number of words produced and the number of phonological errors produced by 22 participants on 10 tasks. Bayesian inference was used to construct posterior distributions of participant ability and task difficulty parameters. Model fit statistics were compared. Association strengths for average generative WPR and average responsive PER were also evaluated with linear least-squares regression. Results Average generative WPR and average responsive PER were significantly associated (r = -.77, p = .00002). A cognitive psychometric model that assumed reduced WPR on generative tasks reflects a portion of general phonological impairment yielded better fit than a model that ignored performance differences between generative and responsive tasks. Generative fluency tasks that elicited few phonological errors still reflected phonological impairment, via suppression. Individual participants were estimated to suppress between 62% and 93% of phonological errors on generative tasks that would have emerged on responsive tasks. Conclusions Suppression of phonological errors may present as decreased WPR on generative tasks in lvPPA. Failure to account for this suppression tendency may lead to overestimation of phonological ability. The findings indicate the need to account for task demands in assessing lvPPA

School of Science, Technology and Health : Research and Scholarship Handbook

The purpose of this handbook is to provide researchers —both faculty and students— with a summary of the best practices for conducting scientific research within the School of Science, Technology, and Health (SSTH). This handbook will address basic as well as advanced guidelines for the researchers, scientists, and students working in the laboratories of the SSTH. Use this as a ready reference for the most important institutional guidelines, regulations, policies, and procedures. All scientific research personnel, including research faculty, fellowship participants, graduate students, interns, laboratory technicians, undergraduate students under the direct or indirect supervision of SSTH faculty, or any other persons involved in any kind of scientific research are expected to be familiar with all relevant content herein. Faculty sponsors and/or the principal investigators will be responsible for providing access to this handbook to all scientific research personnel on their team, and for ensuring that the relevant information is conveyed effectively

Aggregate clearance of α-synuclein in Saccharomyces cerevisiae depends more on autophagosome and vacuole function than on the proteasome.

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention