Jennifer R. Morgan - US grants

ABSTRACT The long term goal of this project is to identify the cellular and molecular mechanisms that give rise to the synaptic defects in patients with Parkinson's disease (PD) and other related neurological disorders. The pathological hallmark of these diseases includes abnormal levels of ¿-synuclein at synapses and throughout the neuron. While it is generally agreed that synucleins participate in synaptic vesicle trafficking, the exact steps of the vesicle trafficking pathway that are perturbed by altered levels of synuclein remain unclear. Thus, at present, it is not possible to design targeted strategies for improving synaptic function in PD. Experiments proposed here take the first steps toward this by identifying the precise synaptic vesicle trafficking defects caused excess synuclein at synapses, the mechanisms giving rise to these defects, and new strategies for reversing them. The experiments take advantage of two model synapses that are ideally suited for studies of synaptic vesicle trafficking, using both acute and genetic perturbations. The combination of highly quantitative biochemical assays to measure synuclein interactions, design of reagents to perturb these interactions, and detailed ultrastructural analyses provides the best opportunity to identify the cellular and molecular mechanisms leading to synuclein-induced synaptic vesicle trafficking defects. In initial studies, excess wild type synuclein causes a loss of synaptic vesicles, increased cisternae, and altered clathrin-coated profiles, consistent with inhibiting clathrin-mediated synaptic vesicle recycling. Going forward, proposed experiments are aimed at identifying the cellular mechanisms by which excess wild type ¿-synuclein and PD-related mutations (e.g. A30P, E46K, A53T) cause vesicle trafficking defects (Aims 1 and 2). Experiments will also investigate how synuclein interactions with specific synaptic binding partners (e.g. PI(4,5)P2 and the uncoating ATPase) contribute to the synaptic vesicle trafficking defects, and targeted strategies for disrupting these interactions will be assessed as a possible means for reversing synaptic defects (Aim 2 and 3). The proposed experiments are innovative because they are the first to use a combination of quantitative biochemical binding assays, acute perturbations, controlled stimulation conditions, and detailed ultrastructural analyses to identify the precise synaptic vesicle trafficking defects caused by excess synuclein or its mutations, which is ideally suited for the overall goal. The experiments are significant because they represent the first steps toward understanding the mechanisms giving rise to the synaptic defects, and they provide possible targeted, molecular strategies for improving synaptic function. Thus, these studies have direct implications for slowing or halting the neurodegeneration, cognitive deficits, and dementia in PD and other synucleinopathies.

The long term goal of this project is to identify the cellular and molecular mechanisms that give rise to the synaptic defects in Parkinson?s disease (PD), dementia with Lewy bodies (DLB), and variants of Alzheimer?s disease (AD) and to develop strategies for reversing them. A pathological hallmark of these diseases is aggregation of ?-synuclein throughout the neuron, including synapses. The synaptic aggregation of ?-synuclein is thought to be the cause of the cognitive deficits and dementia. While it is generally agreed that ?-synuclein accumulation at synapses impairs vesicle endocytosis, the underlying mechanisms remain unclear. Thus, at present, there are no known strategies for improving synaptic function in PD, DLB or AD because we don?t know the cellular or molecular targets. The proposed experiments take significant steps toward these goals by taking advantage of two classical vertebrate synapses that are ideally suited for studies on synaptic vesicle trafficking and by using both acute and genetic perturbations of ?-synuclein. The approach includes a combination of quantitative biochemical, electrophysiological, and imaging assays. One model for ?-synuclein toxicity suggests that it is initiated by formation of abnormal oligomers, while another proposes that build up of monomers is the trigger. Aim 1 will test predictions of both models at synapses by identifying how defined molecular species of ?-synuclein (monomers, dimers, higher molecular weight oligomers) affect vesicle trafficking and neurotransmission and the underlying mechanisms. Preliminary studies indicate that monomers and dimers produce distinct effects. Experiments in Aim 2 will determine the role for ?- synuclein self-association in producing synaptic defects by testing reagents that interfere with this process, including a drug with potential therapeutic value. Aim 3 is focused on reversing the synaptic defects caused by excess ?-synuclein by perturbing its association with Hsc70 chaperone protein, an idea that is supported by preliminary data. The experiments are innovative because they are the first to test the effects of defined molecular species of ?-synuclein at synapses, they continue the development of a new model synapse for these studies, and they will test several new reagents with potential for ameliorating the synaptic defects. The experiments are significant because they will elucidate the mechanisms by which excess??-synuclein causes synaptic deficits, and they will provide possible targeted, molecular strategies for improving synaptic function. Thus, these studies have direct implications for slowing or halting the neurodegeneration, cognitive deficits, and dementia in PD, DLB and other related diseases.

Project Summary: Pluripotent stem cell biology and regenerative medicine are rapidly growing fields with enormous biomedical implications across the entire spectrum of NIH?s clinical, translational, biomedical and behavioral portfolios. This R25 application seeks five years of sponsorship for our advanced training course entitled: Frontiers in Stem Cells and Regeneration (FrSCR) held annually at the MBL in Woods Hole under the Co-Directorship of Drs. Jennifer Morgan (MBL) and Ina Dobrinski (University of Calgary). FrSCR has been funded for the past five years as a new R25 award, and prior to that under U13 and T15 mechanisms. FrSCR continues to build on its demonstrated strengths and successes, and continues to expand to incorporate emerging, important biomedical concepts and sophisticated technological breakthroughs in regeneration and pluripotency. The FrSCR course introduces participating trainees to state-of-the-art research in the fields of pluripotent stem cell (PSC) biology and regenerative medicine, including biology and applications of multipotent adult stem cells (ASC). We provide trainees the necessary knowledge of laboratory techniques, career mentoring and instruction in the ethical, legal, and societal impact (ELSI) of PSC research to greatly enhance their successful entry into this field. In order to achieve this goal, we propose the following specific aims: 1) to provide in-depth instruction on the fundamental concepts in stem cell biology and regeneration; 2) to provide hands-on laboratory training in cutting-edge methods for experimentation with stem cells and regeneration; 3) to educate trainees on the open questions in the field, and how stem cells and regenerative biology can be used to solve problems related to human developmental, reproductive, aging, and neurological disorders, among others; 4) to provide opportunities for career planning and advancement, and to educate trainees on the legal, ethical, and regulatory landscape in which regenerative medicine research occurs, and 5) to foster a diverse, collegial environment, and to provide students with networking opportunities that lead to long-term interactions, mentorship, and collaborations. FrSCR is a dynamic and evolving entity that each year offers a fresh series of daily lectures on emerging concepts, followed by extended discussions, laboratory research, technologically intense workshops and informal seminars over a week-long period. The course is directed towards established investigators as well as advanced fellows and newly independent scientists who are committed to fundamental, translational or clinical research studies. The course addresses major current problems, followed by critical discussions and laboratory experiments in which advanced new techniques are presented to explore these problems. Thus, this course will provide a significant benefit to biomedical research through the training of new investigators in the field of stem cell biology and regeneration.