Pallavi P. Gopal - US grants

? DESCRIPTION (provided by applicant): This proposal describes a 5 year career development plan for Dr. Pallavi Gopal to serve as a transition to successful independent physician-scientist. Dr. Gopal completed her clinical training in Anatomic Pathology and Neuropathology at the University of Pennsylvania (Penn), and is now developing an independent research and training program that will allow her to gain expertise in spatial and temporal dynamics of mRNA transport under physiological conditions and in disease. This proposal brings together diverse resources in RNA metabolism, molecular neuroscience and neuropathology and will provide superb training for Dr. Gopal to develop into an independent physician-scientist. Research will be performed under the mentorship of Dr. Erika Holzbaur, an internationally recognized expert in microtubule-based motors and real-time axon transport dynamics. This grant will provide protected time for Dr. Gopal to gain expertise in neuronal cytoskeletal, organelle, and RNA-protein dynamics through formal coursework, scientific seminars and meetings. The collaborative environment at Penn will foster utilization of novel techniques to conduct the proposed project and will provide Dr. Gopal with the training required to proceed towards a successful academic career. Amyotrophic lateral sclerosis (ALS) and front temporal lobar degeneration (FTLD) exist on two ends of a clinic pathologic spectrum but share clinical, genetic, and pathologic features. Ubiquitinated cytoplasmic inclusions composed of Tran's active response DNA-binding protein of 43 kDa (TDP-43) are a shared feature of sporadic ALS and the most common form of FTLD; there is concomitant loss of normal nuclear TDP-43 expression. Moreover, the discovery of disease-linked mutations in TDP-43 and other RNA processing proteins highlights altered RNA metabolism as a common pathogenic mechanism of neurodegeneration. However, our knowledge of how TDP-43 mislocalization disrupts its nuclear and cytoplasmic RNA processing functions and/or mediates toxicity in the cytoplasm is still incomplete. The research plan will utilize innovative approaches with real-time imaging techniques in primary neurons to test two main hypotheses: that loss of nuclear TDP-43 results in reduced dynamic flux of TDP-43 target mRNA and proteins in axons and that cytoplasmic redistribution of TDP-43 under pathological conditions results in mislocalization of TDP-43- associated mRNA. The specific aims are to: 1) Determine whether loss of nuclear TDP-43 function reduces axonal trafficking of synaptic proteins and organelle turn over and 2) Determine how (A) loss of cytoplasmic TDP-43 RNA binding function and (B) stress-induced cytoplasmic TDP-43 aggregation affect localization and trafficking of mRNA in axons and dendrites. These studies will provide temporal and spatial resolution of individual RNA transcripts in neurons in order to gain a clearer understanding of altered RNA metabolism in ALS/FTLD pathogenesis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are profoundly debilitating and fatal neurodegenerative diseases with overlapping clinical, pathologic and genetic features. Despite advances in our understanding of the pathology and genetic basis of ALS/FTD, the cellular mechanisms underlying neurodegeneration remain poorly understood, and current treatments extend life for only a few months. Almost all ALS patients and nearly half of FTD patients have pathologic aggregates composed of transactive response DNA-binding protein of 43 kDa (TDP-43), a DNA and RNA-binding protein with multiple roles in RNA stability, splicing and post-transcriptional RNA processing. Moreover, mutations in TDP-43 and other RNA-binding proteins cause familial and sporadic ALS/FTD, highlighting altered RNA metabolism as a common pathogenic mechanism of neurodegeneration. Recent studies have identified genetic interactions between TDP-43 and Ataxin-2, an RNA-binding protein that contains a polyglutamine (polyQ) tract normally 22-23 glutamines in length. Expansions of the Ataxin-2 polyQ tract (27-33 glutamines) increase risk for ALS and ALS-FTD overlap disease. However, the cellular and molecular mechanisms by which Ataxin-2 / TDP-43 interactions increase disease risk are unknown. The objective of this proposal is to determine the molecular basis of Ataxin-2 / TDP- 43 interactions and their impact on TDP-43 dependent RNA regulation, including RNA splicing and stability as well as spatiotemporal localization and translation of mRNA. In preliminary and published work, we and others find that TDP-43 and Ataxin-2 are components of neuronal ribonucleoprotein granules, RNA/protein-rich compartments that regulate mRNA stability, transport and translation. Our preliminary data show that Ataxin-2 polyQ expansions disrupt anterograde transport and fluorescence recovery after photobleaching of TDP-43 RNA granules that contain mutant Ataxin-2. Collectively, these data support our central hypothesis: Ataxin-2 polyQ expansions aberrantly scaffold TDP-43 / Ataxin-2 interactions and sequester TDP-43, disrupting nuclear and cytoplasmic functions of TDP-43. We will test this central hypothesis using complementary live-cell imaging and single-molecule imaging approaches, single-molecule FRET, translation assays, and RNA- sequencing/transcriptomics (i) to study the effect of Ataxin-2 polyQ expansions on TDP-43 transport and post- transcriptional regulation of TDP-43 target mRNAs in wild-type or Ataxin-2 mutant neurons; and (ii) to identify Ataxin-2 and TDP-43 domains required for aberrant interaction and to design small molecule inhibitors of TDP- 43 / Ataxin-2 polyQ interactions. The proposed research will provide new insights into (1) the molecular basis of Ataxin-2/TDP-43 interactions that confer increased ALS and ALS-FTD risk, (2) how Ataxin-2 polyQ expansions interact with TDP-43 to impact the transcriptome and spatiotemporal localization of mRNA in neurons, and (3) potential molecular targets for mechanism-based therapies.