Talene A. Yacoubian, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Alpha-synuclein has a central role in the pathogenesis of Parkinson's disease, yet how it triggers dopaminergic neuron toxicity is poorly understood. Recent gene microarray studies in transgenic alpha-synuclein mice have shown that over-expression of alpha-synuclein results in decreased expression of a class of genes encoding the 14-3-3 proteins. Because the 14-3-3s regulate key signaling cascades, including apoptosis, the decrease in 14-3-3s may be an important step in alpha-synuclein-induced toxicity. The candidate has obtained preliminary data supporting the neuroprotective potential of the 14-3-3 proteins, particularly the theta isofom. Proposed experiments will determine the extent to which 14-3-3 isoforms can mitigate alpha-synuclein toxicity in both in vitro and in vivo models of Parkinson's disease and whether they can reduce apoptotic factor activity in these models. Potential mechanisms of how alpha-synuclein causes decreased 14-3-3 expression will be examined. Studies to evaluate the role of phosphorylation in the interaction between 14-3-3s and alpha-synuclein will also be performed. Results from these studies will look to validate the 14-3-3 proteins as potential targets for therapy in Parkinson's disease and related disorders. The research portion of the proposed career development program will further the candidate's understanding of Parkinson's disease pathophysiology and her training in experimental skills, such as in vivo animal work, epigenetic methods and the use of viral vectors for gene delivery. She will continue her subspecialty training and clinical practice in the diagnosis and treatment of Parkinson's disease and related disorders. In addition, the candidate will pursue formal studies in clinical research training to acquire knowledge on the major issues for converting a molecule neuroprotective in animal models into treatment for human patients. By the end of the five-year plan, the candidate expects to be fully equipped to direct research on Parkinson's disease mechanisms and the translation of such research into potential therapies. The 14-3-3 proteins appear promising, but whether or not they turn out to become practical neuroprotective agents, the experience gained in studying these proteins will have taught the candidate how to test whether candidate proteins are truly protective and, if so, how to begin developing them into clinical therapy. [unreadable]

DESCRIPTION (provided by applicant): The personal and societal costs of Parkinson's disease (PD) are expected to increase significantly in the next two decades. The mechanisms of neurodegeneration are not well understood, and no treatment clearly slows the neurodegenerative process in PD. Alpha-synuclein (?syn) is a protein that is central to PD pathogenesis, and recent studies show that the transmission of ?syn between different cell populations is key to its ability to cause toxicity. Release of ?syn is the first critical componen of transmission, and occurs through exosomal and non-exosomal mediated pathways. A second key feature of prion-like spread of ?syn is the uptake in target neurons, leading to consequent misfolding of endogenous ?syn. What mechanisms regulate the release and spread of ?syn pathology are not known. The 14-3-3 proteins are chaperone-like proteins that can reduce protein aggregation, regulate protein secretion, and promote cell survival. We have previously shown that 14-3-3s are protective in several models of PD and can regulate the exosomal release of LRRK2, a key protein implicated in PD. In this proposal, we present preliminary data that overexpression of the 14-3-3? isoform in ?syn-producing cells reduces the toxicity of released ?syn. Our central hypothesis is that 14-3-3 proteins can protect against ?syn toxicity by reducing the transmission of toxic ?syn species. In Aims 1 and 2, we will investigate whether 14-3-3s can regulate ?syn release through exosomes or alternative non-exosomal pathways and assess how any changes in release impacts paracrine ?syn toxicity. For these studies, we will use a paracrine inducible ?syn culture system in which released ?syn induced cell death in separately culture primary neurons. In Aim 1, we will use biochemical and imaging approaches to determine if 14-3-3s alter the amount and conformation of ?syn in exosomes. We will also assess how alterations in exosomal ?syn impact paracrine ?syn toxicity. In Aim 2, we will use similar techniques to test if 14-3-3s reduce ?syn release and toxicity through inhibition of the recycling endosomal pathway. In Aim 3, we will focus on the effects of 14-3-3s in target cells exposed to extracellular ?syn. Specifically, we will use in vitro and in vivo ?syn fibril models to test whether 14-3-3s can reduce ?syn uptake, aggregation, and toxicity in these models. If we can establish that 14-3-3s regulate the pathological transmission of ?syn, this would justify exploration of potential PD therapies targeting the 14-3-3s.

PROJECT SUMMARY Two critical proteins linked to Parkinson?s disease (PD) are alpha-synuclein (?syn) and LRRK2. Mutations in either gene cause autosomal dominant forms of PD, and GWAS studies have pointed to variants in both genes as risk factors for developing idiopathic PD. Although both proteins lead to a similar pathological outcome, how these two proteins cause neuronal injury and how they interact in the disease process are not well understood. Our key discovery is that 14-3-3? is a major regulator of both ?syn and LRRK2 and could be the missing link between ?syn and LRRK2. 14-3-3s are multifunctional, highly expressed brain proteins that act as chaperones, affect protein trafficking, and modulate enzymatic activity of their binding partners. Our research has highlighted the role of these critical proteins in PD and their interplay with both ?syn and LRRK2. We have observed that 14-3-3? acts as a chaperone to reduce ?syn aggregation and cell-to-cell transmission, and this same 14-3-3 isoform reduces the kinase activity and toxicity of mutant LRRK2. While our data clearly points to a potentially critical role for 14-3-3 dysfunction in Parkinson?s disease, a key question that remains is how 14-3-3??s endogenous functions could become impaired in PD. We propose that aberrant phosphorylation of 14-3-3? is the critical pathophysiologic event, and that increases in 14-3-3? phosphorylation promotes LRRK2 and ?syn effects in disease. In support of this hypothesis, we recently published data showing that 14-3-3? phosphorylation at S232 is dramatically elevated in the detergent-insoluble fractions from human PD and DLB brains. This increase in S232 phosphorylation correlates with cognitive decline and pathological severity measures, consistent with a role of S232 phosphorylation in the pathogenesis of neurodegeneration. In addition, we have observed that mitochondrial stress promotes 14-3-3? phosphorylation at S232 in culture. The S232D phosphomimetic mutant loses its protective effects in neurotoxin and ?syn culture models. Based on these data, we hypothesize that oxidative stress is a key upstream inducer of excessive 14-3-3? phosphorylation, leading to ?syn and LRRK2 toxicity. We recently created a conditional knock-in (KI) 14-3-3? S232D mouse line that will serve as the critical tool to understand the impact of 14-3-3 phosphorylation in PD. In Aim 1, we will test how 14-3-3? phosphorylation alters interactions with ?syn and modulates subsequent ?syn pathology. In Aim 2, we will test the impact of 14-3-3 phosphorylation on its interaction with LRRK2 and LRRK2 function. In Aim 3, we will examine whether 14-3-3? phosphorylation occurs early in sporadic PD and whether oxidative stress mediates toxicity via excessive 14-3-3 phosphorylation.

PROJECT SUMMARY: CLINICAL RESEARCH CORE Parkinson?s disease (PD) is the second most common neurodegenerative disorder. While current treatments mitigate the symptoms and disability associated with PD, no treatments slow or reverse the neurodegenerative process. Recent work points to a significant role for inflammation in PD pathogenesis. In work supported by the P20 Exploratory Project (NS09530), we found evidence for activation of innate immunity in animal models of PD, and obtained preliminary data implicating immunomodulation in human samples. Expanding this research to a more complete study of inflammatory changes in human subjects with PD is essential to define the role of inflammation in human disease and thus aid in the development of potential immunomodulatory disease modifying therapies. The primary goal of the Clinical Research Core is to establish a well-characterized cohort of early stage, idiopathic PD subjects for longitudinal study with regard to neuroinflammation. This cohort will provide clinical data, biospecimens, and imaging data that will directly support the three research projects proposed under the P50 grant. The Core will recruit, clinically assess, and follow annually 60 age- and sex- matched controls and 60 subjects with early stage, untreated, idiopathic PD at UAB. CSF, plasma, and peripheral blood mononuclear cells (PBMCs) will be collected at baseline, and plasma and PBMCs will be collected at annual follow-up visits. [18F]DPA-714 PET imaging will be performed on this cohort at baseline to assess central inflammation in early stage, idiopathic PD. All participant data will be submitted to the Data Management Resource (DMR) under the NINDS? Parkinson?s disease Biomarker Program (PDBP), and approved biospecimens will be sent to the NINDS repository BioSEND. In conjunction with Columbia University, the Core will also recruit LRRK2 G2019S carriers with PD (n=20) and without PD (n=20), and LRRK2 G2019S NON- carriers with PD (n=20) and without PD (n=20). Biospecimens and clinical data from patients previously genotyped for mutations in LRRK2 will feed directly into Project 3.

ABSTRACT The focus of the Medical Scientist (MD-PHD) Training Program (MSTP) at the University of Alabama at Birmingham (UAB) is to train outstanding young people to become successful physician scientists. Although the ultimate career pathway for individual trainees extends a broad spectrum from the conduct of basic biomedical research to clinical trials of novel therapeutic agents or procedures, the net effect of this cadre of investigators will be to increase the translation of basic biomedical understanding into clinical practice. Our Program focuses on the following goals: 1) to offer superb didactic training in both basic science education and the fundamentals of clinical education; 2) to train students in the tools necessary to become successful biomedical scientists, including grant and manuscript writing, as well as time and laboratory management; 3) to help students obtain the competencies to become excellent physicians, including clinical skills, medical knowledge, practice-based learning, interpersonal communication, professionalism, and evidence-based medicine; and 4) to graduate physician scientists who go on to become leaders in academic medical centers throughout the country. The UAB MSTP curriculum has three distinct phases: two years of basic science course work and summer research rotations (Pre-Clinical Phase), an extended research period culminating in the PhD degree (Research Phase), and final clinical training leading to the MD degree (Clinical Phase). Each year, our MSTP matriculates eight students into the first program year and accepts another one or two students through Advanced Transfer pathways (the NIH MD-PhD Partnership Training Program pathways, and MD or PhD to MSTP Advanced Transfer pathways). Our program currently has 72 students. Unique aspects and strengths of the UAB MSTP include the following: diverse student body (21% URM), extensive student involvement and ownership in the program, strong student representation in leadership positions in national organizations, vertical integration of students through program activities, exceptional core faculty preceptors across a broad array of specialties, an increasing number of students pursuing PhD training in programs outside of traditional biomedical sciences, a high funding rate of NRSA F30/31 or equivalent awards, an innovative continuing clinical education course that bridges the Pre-Clinical to Clinical phases, a very low attrition rate, low average time to completing the MSTP, and 100% residency match rate for MSTP graduates since the last renewal. Strong MSTP recruiting, leadership, and institutional support will ensure continued success of the UAB MSTP.

Recent evidence points to ?syn misfolding and cell-to-cell transmission as critical to neurodegeneration in Parkinson?s Disease (PD) and Dementia with Lewy Bodies (DLB). Key steps implicated in ?syn spread include release, uptake, misfolding, and impaired protein degradation, yet the key molecular mechanisms that regulate ?syn spread are poorly understood. Rab proteins are small GTPase proteins that control protein trafficking and degradation and have been implicated in ?syn pathogenesis. Among the Rab proteins, Rab27b is highly expressed at synaptic terminals in neurons in key brain areas affected in PD and DLB. Rab27b regulates synaptic vesicle (SV) exocytosis and recycling. In non-neuronal cells, Rab27b regulates the distal transport of lysosomes. We recently showed elevated Rab27b levels in ?syn models and in human PD and DLB. Furthermore, we observed that Rab27b KD increases ?syn toxicity by disrupting autophagic flux - pointing to a protective role for Rab27b in cells with high intracellular ?syn burden. Surprisingly, the effects of Rab27b were different in the ?syn fibril model, in which neurons were exposed to extracellular ?syn fibrils: Rab27 KO reduced fibrillary ?syn uptake to prevent ?syn inclusions and neuronal loss. Based on these data, we hypothesize that Rab27b plays two critical yet potentially opposing roles in ?syn handling: 1) Rab27b promotes ?syn autophagic-lysosomal degradation, yet 2) Rab27b can facilitate endocytosis of pathologic, extracellular ?syn as part of its role in SV recycling. Early in disease, we propose that Rab27b upregulation is neuroprotective: Rab27b aids intracellular ?syn clearance. Yet, as disease progresses and extracellular ?syn levels increase, Rab27b?s role in SV endocytosis becomes maladaptive, overcoming any protective function in protein degradation, and aids ?syn cell-to-cell transmission. Our proposed studies will examine the impact of Rab27b and its interactors on two critical biological processes implicated in ?syn pathogenesis. In Aim 1, we will test the mechanisms by which Rab27b promotes clearance of intracellular ?syn, and examine its interactions with other proteins involved in autophagic-lysosomal function. In Aim 2, we will elucidate the mechanisms by which Rab27b aids ?syn entry into neurons and determine its interaction with other synaptic trafficking proteins to mediate this effect. In Aim 3, we will test the overall consequences of Rab27b on ?syn pathology over time in two in vivo ?syn models. A greater understanding of the network of proteins that shape ?syn transmission will have significant impact on the development of future therapies for PD and DLB.

Summary/Abstract The Southeastern Medical Scientist Symposium (SEMSS) was established in 2010 by students from the University of Alabama at Birmingham (UAB), Emory University, and Vanderbilt University Medical Scientist Training Programs (MSTPs). The three programs have co-hosted the symposium for six years with the location rotating among Birmingham (2010, 2013), Atlanta (2011, 2014), and Nashville (2012, 2015). The objective of the symposium is to encourage a collaborative and interdisciplinary educational environment within the Southeast region of the United States. This fully student- organized symposium seeks to foster connections between the MD/PhD students at multiple institutions across the Southeast, exposing students to trends, challenges, and opportunities inherent in careers of academic physicians. Future SEMSS meetings will continue to rotate locations between Birmingham, Atlanta, and Nashville in order to optimize regional student participation. The program of each SEMSS has and will continue to include keynote speaker presentations, multiple topic-specific breakout sessions, MSTP student research oral and poster sessions, and social events. The breakout sessions are divided into sessions of interest to MD/PhD students, undergraduates, and residents/fellows. The meetings span two days, with content starting in the early afternoon on a Saturday and ending in the early afternoon on Sunday. The target audience for the SEMSS is MD/PhD students in training programs in the southeast and residents/fellows, MD students, and undergraduate students at southeastern institutions who have an interest in future careers as physician-scientists. An additional purpose of this symposium is to expose undergraduate students to physician-scientist trainees and faculty in order to foster excitement about careers in academic medicine and increase the pipeline of future physician-scientists. We think that this additional focus on undergraduate students from the region is critical to our purpose of enhancing the pipeline of students from backgrounds underrepresented in medicine (URIM), as according to US News and World Report, nine of the top twenty historically black colleges and universities (HBCUs) are located in Alabama, Georgia, Tennessee, Mississippi, or Louisiana.