Role of Mechanisms of Efflux in Manganese Homeostasis and Detoxification

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Project Summary/Abstract Research Project: Mn is an essential element that becomes toxic at elevated cellular levels leading to the onset of an incurable neurotoxic syndrome. To date, research on Mn homeostasis and toxicosis in mammals has focused on the mechanisms of Mn influx but the identified influx transporters are neither specific for Mn nor regulated by cellular Mn levels. In contrast, the role of efflux of cytosolic Mn is understudied although efflux plays a significant role in maintaining homeostasis of other metals. We have now identified an important role for efflux of Mn via uptake into the Golgi apparatus followed by secretion in maintaining cellular Mn levels and protecting against excess Mn accumulation during elevated exposure. Based on this, our goal is to elucidate the regulatory mechanisms governing Mn efflux by the Golgi to so as to better understand the role of this fundamental process in Mn homeostasis and in the development of Mn-induced neurotoxicity. Our results indicate that uptake of Mn into the Golgi requires SPCA1, a Golgi-localized Ca/Mn pump and that SPCA1 rapidly traffics between the Golgi and endosomes. As subcellular trafficking of ion pumps regulate their activity, our first aim is to elucidate the role of SPCA1 trafficking in regulating Mn efflux. We also discovered that increased intra-Golgi Mn induces rapid degradation of the Golgi protein GPP130 and that GPP130 levels impact Mn efflux and toxicity. Therefore, our second aim is to elucidate the mechanism by which GPP130 regulates Mn efflux and toxicity. Finally, as additional factors regulating Mn efflux remain to be identified, our third aim is to perform a genome wide RNAi screen for proteins that alter control of Mn by the Golgi. In the screen, a modified version of GPP130 will serve as a novel sensor of Golgi lumenal Mn. The proposed studies will provide mechanistic understanding of a crucial but unexplored aspect of Mn homeostasis that is directly relevant to the pathobiology of Mn-induced neurotoxicity. Candidate/ Career Development Plan/ Environment: My goal is to be an independent investigator working broadly in the area of subcellular regulation of metal ion homeostasis as a means to understand the pathobiology of, and develop therapies for, diseases that occur due to metal toxicity. My academic path is well-suited to this goal. I embarked on a career in biomedical research after completing my medical training with the aim of making contributions to further our understanding of incurable human diseases. My graduate work, on the molecular mechanisms of pulmonary arterial hypertension, gave me the skills necessary to investigate clinically relevant cell biology questions. I have expanded these skills during my post-doctoral training. The K99 award is now crucial for my success because it will give me the protected time essential to complete my training under Dr. Adam Linstedt and help me initiate my independent career two years hence. Specific career development activities to aid my transition from trainee-to-faculty are as follows. I already perform my work independently and this will continue over the next two years. I am currently guiding a PHD student in her research and will continue to do so to gain experience in mentoring students. I will further improve my technical skills during this period and gain proficiency in yeast-2-hybrid, 2 D fluorescence gel electrophoresis and genome-wide RNAi screening. This will add to the skills I have already gained in the post- doctoral period (gene replacement after knockdown, structure function analyses of proteins and 3- and 4-D live and fixed cell microscopy). Due to the interdisciplinary nature of my work, we have established fruitful collaborations with Dr. Donald Smith, an expert in Mn toxicity; Dr. Mark Macbeth, a structural biology and Dr. Jonathan Minden, a biochemist. I will continue to interact regularly with these faculty over the next two years. For the award, they will serve as part of an advisory committee, chaired by Dr. Linstedt, which will meet every 3-4 months to ensure that I am making rapid progress. To further refine my scientific skills, I will also be attending several national and international conferences (e.g. GRC on Cell Biology of Metals in 2011; annual meeting of the American Society for Cell Biology) during the award period. Dr. Linstedt has an excellent track record as a mentor and working under his guidance for the next two years will provide me with the skills necessary to succeed in an independent setting. The innovative and interactive scientific atmosphere in CMU and the greater Pittsburgh area will also aid my training and research. Further, the work performed during the K99 period will help me generate more data, add to my publications and improve my credentials while applying for faculty positions two years hence. The work performed during the R00 period will raise multiple questions that will open exciting avenues for extended research in my career. Thus, the K99/R00 award will act directly aid my transition from post-doctoral trainee to junior faculty.