Insights into alpha-synuclein and TorsinA biology

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.The yeast Saccharomyces cerevisiae has long been used to model complex cellular processes. As a eukaryote, much of its fundamental biology is conserved with higher organisms. As a single-celled, genetically tractable organism, it can easily be utilized for both high-throughput screening and hypothesis-driven analysis. Therefore, many groups use yeast to model disease-related proteins. One such model utilizes heterologous expression of [alpha]-synuclein ([alpha]-syn), a protein implicated in the progression of Parkinson's disease and other synucleinopathies. [alpha]-Syn expression in yeast is associated with many phenotypes that are recapitulated in higher organisms. Here, I used yeast to characterize two naturally occurring splice isoforms of [alpha]-syn, [alpha]-syn[delta]4 and [alpha]-syn[delta]6. Levels of these isoforms vary between synucleinopathies but little is known about their biology. I found that these splice isoforms display different localization patterns than full-length [alpha]-syn ([alpha]-synFL) and are less toxic in yeast. However, when expressed at a high level, both splice isoforms can exert toxicity and affect similar processes to [alpha]-synFL. Interestingly, the splice isoforms show differential responses to perturbations in sterol homeostasis. Studies concerning the relationship between sterol levels and synucleinopathy progression have been contradictory. Our findings reveal that [alpha]-syn[delta]4 is less sensitive to changes in sterol levels than [alpha]-synFL and [alpha]-syn[delta]6, suggesting that change in [alpha]-syn splice isoforms levels is a potential mechanism for these conflicting results. I also describe an attempt to model torsinA pathobiology in yeast. Mutations in torsinA cause early onset torsion dystonia, a devastating motor disorder. This protein has been described to function in regulating endoplasmic reticulum (ER) stress through the unfolded protein response (UPR). While I was unable to recapitulate a role for torsinA in the UPR in yeast, this model can serve as a platform for discovery of torsinA cofactors that enable it to act in this capacity, especially as more is uncovered concerning torsinA's role in the UPR. This thesis highlights both the strengths and limitations of modeling disease proteins in yeast. More specifically, my success with [alpha]-syn splice isoforms may provide insight into synucleinopathy etiology, while my inability to model torsinA-induced toxicity can inform subsequent attempts to study disease related proteins in yeast.by Julie S. Valastyan.Ph.D

TorsinA and the TorsinA-Interacting Protein Printor Have No Impact on Endoplasmic Reticulum Stress or Protein Trafficking in Yeast

Early-onset torsion dystonia is a severe, life-long disease that leads to loss of motor control and involuntary muscle contractions. While the molecular etiology of the disease is not fully understood, a mutation in an AAA+ ATPase, torsinA, has been linked to disease onset. Previous work on torsinA has shown that it localizes to the endoplasmic reticulum, where there is evidence that it plays roles in protein trafficking, and potentially also protein folding. Given the high level of evolutionary conservation among proteins involved in these processes, the ability of human such proteins to function effectively in yeast, as well as the previous successes achieved in examining other proteins involved in complex human diseases in yeast, we hypothesized that Saccharomyces cerevisiae might represent a useful model system for studying torsinA function and the effects of its mutants. Since torsinA is proposed to function in protein homeostasis, we tested cells for their ability to respond to various stressors, using a fluorescent reporter to measure the unfolded protein response, as well as their rate of protein secretion. TorsinA did not impact these processes, even after co-expression of its recently identified interacting partner, printor. In light of these findings, we propose that yeast may lack an additional cofactor necessary for torsinA function or proteins required for essential post-translational modifications of torsinA. Alternatively, torsinA may not function in endoplasmic reticulum protein homeostasis. The strains and assays we describe may provide useful tools for identifying and investigating these possibilities and are freely available.Howard Hughes Medical InstituteBachmann-Strauss Dystonia and Parkinson Foundatio

miRNA Expression in Colon Polyps Provides Evidence for a Multihit Model of Colon Cancer

Changes in miRNA expression are a common feature in colon cancer. Those changes occurring in the transition from normal to adenoma and from adenoma to carcinoma, however, have not been well defined. Additionally, miRNA changes among tumor subgroups of colon cancer have also not been adequately evaluated. In this study, we examined the global miRNA expression in 315 samples that included 52 normal colonic mucosa, 41 tubulovillous adenomas, 158 adenocarcinomas with proficient DNA mismatch repair (pMMR) selected for stage and age of onset, and 64 adenocarcinomas with defective DNA mismatch repair (dMMR) selected for sporadic (n = 53) and inherited colon cancer (n = 11). Sporadic dMMR tumors all had MLH1 inactivation due to promoter hypermethylation. Unsupervised PCA and cluster analysis demonstrated that normal colon tissue, adenomas, pMMR carcinomas and dMMR carcinomas were all clearly discernable. The majority of miRNAs that were differentially expressed between normal and polyp were also differentially expressed with a similar magnitude in the comparison of normal to both the pMMR and dMMR tumor groups, suggesting a stepwise progression for transformation from normal colon to carcinoma. Among the miRNAs demonstrating the largest fold up- or down-regulated changes (≥4), four novel (miR-31, miR-1, miR-9 and miR-99a) and two previously reported (miR-137 and miR-135b) miRNAs were identified in the normal/adenoma comparison. All but one of these (miR-99a) demonstrated similar expression differences in the two normal/carcinoma comparisons, suggesting that these early tumor changes are important in both the pMMR- and dMMR-derived cancers. The comparison between pMMR and dMMR tumors identified four miRNAs (miR-31, miR-552, miR-592 and miR-224) with statistically significant expression differences (≥2-fold change)

Coexpression of torsinA and printor does not uncover a phenotype stemming from torsinA expression.

<p>(A) The UPR of the indicated yeast strains was monitored by flow cytometry to detect expression from the UPRE-GFP construct upon stress with CPY* or 1.5 mM DTT. Coexpression of printor does not allow WT torsinA to reduce UPR-related stress. A 1-tailed Student's t-test was used to compare relative fluorescence of torsinA strains to the vector control. * = p<0.01. N = 6 independent trials per each sample. (B) The impact of torsinA with and without printor on trafficking of invertase. Cells were spotted on plates containing the pH sensitive dye, BCP. Simultaneous expression of torsinA and printor does not impact the rate of secretion.</p

TorsinA cannot rescue α-synuclein-induced toxicity in yeast.

<p>Assay showing the ability of yeast to grow in the presence or absence of α-synuclein (α-syn) with and without torsinA. Each row is a 5-fold dilution of the previous row. TorsinA cannot rescue α-syn-induced toxicity.</p

A Host-Produced Autoinducer-2 Mimic Activates Bacterial Quorum Sensing

Host-microbial symbioses are vital to health; nonetheless, little is known about the role cross-kingdom signaling plays in these relationships. In a process called quorum sensing, bacteria communicate with one another using extracellular signal molecules called autoinducers. One autoinducer, AI-2, is proposed to promote inter-species bacterial communication, including in the mammalian gut. We show that mammalian epithelia produce an AI-2 mimic activity in response to bacteria or tight-junction disruption. This AI-2 mimic is detected by the bacterial AI-2 receptor, LuxP/LsrB, and can activate quorum-sensing-controlled gene expression, including in the enteric pathogen Salmonella typhimurium. AI-2 mimic activity is induced when epithelia are directly or indirectly exposed to bacteria, suggesting that a secreted bacterial component(s) stimulates its production. Mutagenesis revealed genes required for bacteria to both detect and stimulate production of the AI-2 mimic. These findings uncover a potential role for the mammalian AI-2 mimic in fostering cross-kingdom signaling and host-bacterial symbioses

TorsinA can be expressed in the endoplasmic reticulum of yeast.

<p>(A) Diagram of constructs created for this work (upper panel) and summary of the different forms of torsinA used in these experiments (lower panel). TorsinA was localized to the endoplasmic reticulum (ER) using the signal sequence of the endogenous yeast protein KAR2 and an HDEL sequence. (B) Microscopy of yeast strains used. TorsinA was localized to the contiguous lumen of the nuclear envelope and ER. Some signal was also seen in the vacuole (arrow head), suggesting a portion of the protein was degraded. A representative frame is shown for each strain. Scale bar  =  2 µM. (C) Growth of torsinA-expressing yeast on plates. Each row is a 5-fold dilution of the previous row. Expression of wild type (WT) or mutant torsinA did not impact the growth rate. Uninduced plates included 1 mM methionine and induced plates lacked methionine.</p

TorsinA does not impact the unfolded protein response or trafficking in yeast.

<p>(A) A construct containing GFP driven by a unfolded protein response (UPR) sensitive promoter (UPRE-GFP) was used to monitor levels of the unfolded protein response (UPR) upon stress with 1.5 mM dithiothreitol (DTT) or mutant carboxypeptidase Y (CPY*). ERO1 served as a positive control. TorsinA is not able to reduce UPR levels caused by either stressor. Statistical analysis was conducted in comparison to the vector control strain with a 1-tailed Student's t-test. * = p<0.05, # = p<0.005, & = p<0.001. N = 6 independent trials per sample. (B)Growth of ero1-1 in the presence and absence of torsinA at 37°C. Each row is a 5-fold dilution of the previous row. TorsinA is not able to rescue the growth defect by the <i>ero1-1</i> mutation. Uninduced plates included 1 mM methionine and induced plates lacked methionine. (C) Trafficking of invertase, as monitored by halos produced by growth of torsinA-expressing strains on plates containing bromocresol purple (BCP). TorsinA does not impact the rate of secretion.</p