Poly(ADP-ribose)-dependent TDP-43 pathology in oxidative stress (R21)

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Abstract Frontotemporal dementia (FTD) is an Alzheimer-related dementia disease (ADRD) and involves nuclear egress and cytoplasmic Transactivation response DNA binding protein 43 (TDP-43). The mechanisms for pathological export of TDP-43 or its accumulation in the cytoplasm is not clearly defined. Although, studies on disease-causing mutations have revealed that defects in nuclear import may be in part responsible for TDP-43 accumulation in the cytosol as these mutations change the properties of the protein. However, it remains largely unclear how TDP-43 accumulates in the cytosol, as the majority of the TDP-43 dependent pathologies are sporadic. TDP-43 contains two RNA recognition motifs (RRMs), a nuclear export signal (NES), and a nuclear localization signal (NLS) in its N-terminus, and binds poly (ADP-ribose) polymer (PAR) via a PAR-binding motif embedded in its NLS. Studies have shown that PAR inhibitors can inhibit TDP-43 pathology, and binding of PAR to TDP-43 can change its biophysical characteristics in solution. Exportin 1 (XPO1) mediates the NES-dependent export of proteins from the nucleus. TDP-43 is a 43kDa protein that may not require XPO1-dependent export under basal conditions but our data indicates that under neuronal stress conditions TDP-43 egresses the nucleus via XPO-1 dependent export. We hypothesize that in disease conditions like FTD type ADRD and related diseases; PAR binds TDP-43 in the nucleus and facilitates its interaction with XPO-1, which subsequently enhances the egress of TDP-43 from the nucleus to cytosol. This proposal is supported our preliminary data that both PARP inhibitor BMN673 and XPO-1 inhibitor KPT- 185 inhibit the cytosolic accumulation of TDP-43 in neurons and TDP-43 and XPO1 interact following PARP activation. In this proposal, we plan to study the alterations in TDP-43 in mouse cortical neurons exposed to oxidative stress. Oxidative stress is a common pathological process mediating protein mislocalization, aggregation, and cell death in virtually all neurological disorders including FTD. Oxidative stress via H2O2 in cortical neurons mediates a robust TDP-43 cytosolic localization. Therefore, it is likely that pathophysiological mechanisms identified in the oxidative stress model in cortical neurons can closely overlap/mimic pathological mechanisms in FTD and other ADRD involving changes/mislocalization of TDP-43. We will use biochemical, cell biological and imaging techniques in combination with proximity ligation assays, CryoEM, and Hydrogen/Deuterium Exchange mass spectrometry to assess the PAR-dependent alteration in TDP-43 and its interaction with XPO-1 that leads to an increase in the release of TDP-43 from the nucleus and subsequent accumulation/aggregation in the cytoplasm. These studies will help in understanding the pathophysiological mechanisms of TDP-43 accumulation in FTD, ALS, and other ADRD/dementia related diseases involving TDP-43