Targeting nuclear transport dysfunction in TDP-43 proteinopathies

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 DESCRIPTION (provided by applicant): TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative disorders that include FTDL and ALS. Unfortunately, the mechanisms underlying its pathogenesis are poorly understood. Interestingly, although normal TDP-43 is primarily in the nucleus, its cytosolic deposition and nuclear depletion characterize the disease state. This suggests that defects in nuclear import may play a pivotal role in the initiation of TDP-43 proteinopathies. In this regard, a positive correlation between formation of TDP-43 cytoplasmic inclusions and depletion of the nuclear transporter Karyopherin β (KPNB1) has been documented. Thus, the major goal of this application is to determine the disease-modifying ability of KPNB1 against TDP-43 using Drosophila as experimental model. Our central hypothesis is that KPNB1 will exert a protective activity against TDP-43 insults. This is supported by our preliminary data showing that Ketel, the Drosophila homologue of KPNB1, modifies human TDP-43 toxicity in flies. For instance, Ketel RNAi exacerbates the eye phenotypes of TDP-43WT and TDP-43M337V transgenes, while its overexpression robustly suppresses the neurotoxicity of TDP-43M337V. Interestingly, Ketel does not modify the eye phenotype of TDP-43NLS, which lacks a nuclear localization signal (NLS), neither the toxicity of FUS, a related DNA/RNA binding protein whose nuclear import is independent of KPNB1/Ketel. Strikingly, we also found that pathological TDP-43 induces cytoplasmic retention of NLS-tagged reporter proteins, which highlights a potential underlying cause of disease pathogenesis. Therefore, we plan to determine the molecular basis of this nuclear transport impairment and to define the therapeutic potential of targeting the nuclear transport machinery. Our specific aims are: (1) Characterize the interplay between Ketel/KPNB1 and TDP-43 toxicity. We will first co-express a group of wild type and mutant TDP-43 transgenes with various gain- and loss-of-function Ketel alleles. Then, we will assess the profiles of TDP-43 insolubility, cleavage, phosphorylation and subcellular distribution as well as the potential sequestration and/or down-regulation of Ketel. (2) Assess the ability of KPNB1 to prevent or reverse TDP-43 toxicity in the fly CNS. We will activate KPNB1 in the entire CNS starting at day 1 post-eclosion followed by independent activation of TDP-43WT and TDP-43M337V at later time points. Then, we will reverse the expression scheme, activating TDP-43 constructs first followed by KPNB1 to determine if KPNB1 can reverse or delay the course of the disease. The proposed research is innovative and highly significant because this will be the first systematic manipulation of KPNB1 and TDP-43 under different temporal patterns. Importantly, we expect to define whether KPNB1 has the ability to stop, delay, or prevent the toxicity associated with TDP-43 proteinopathies.