FLCN modifies the cytoplasmic translocation and aggregation of TDP-43

TDP-43 is a DNA/RNA binding protein whose cytoplasmic aggregation is associated with neuronal death in ALS and frontotemporal lobar degeneration (FTLD). TDP-43 has multiple cellular functions and shuttles between the nucleus and cytoplasm. However, in ALS and FTLD nuclear clearance of TDP-43 results in increased cytoplasmic localisation – a precursor to TDP-43 aggregation and stress granule formation. The mechanisms that regulate TDP-43 transport are not well understood but new research from Xia et al. (2015) has uncovered a role for FLCN in its nuclear export and the formation of stress granules.

TDP-43 is ubiquitously expressed and under normal conditions the majority of the protein is located within cell nucleus, only being sequestered to the cytoplasm under stress conditions. When TDP-43 is overexpressed in HEK293 the majority of cells show predominately nuclear localisation with a small percentage of cells showing cytoplasmic or diffuse localisation. However, concurrent overexpression of GFP-FLCN results in increased mislocalisation of TDP-43 to the cytoplasm. Xia et al. found this was due to enhancing FLCN-dependent nuclear export of TDP-43 rather than disruption of nuclear import. siRNA knockdown of FLCN results in reduced TDP-43 transport to the cytoplasm, even under stress conditions.

In the FLCN overexpression cells TDP-43 and GFP-FLCN colocalise in cytoplasmic punctate structures. Co-immunoprecipitation assays determined that FLCN and TDP-43, both wild type and mutant forms, directly interact in an RNA-independent manner. Xia et al. then used deletion mutation assays to map the interaction sites to FLCN residues 202-299 and TDP-43 RNA Recognition Motif 1 (RRM1).

The TDP-43 punctate structures are associated with the lysosomes and colocalise with ubiquitination and autophagy markers. TDP-43 colocalises with the stress granule marker G3BP1 only when GFP-FLCN is present to induce cytoplasmic TDP-43 localisation. In FLCN-depleted cells under stress TDP-43 dissociates from stress granules and then returns to the nucleus after conditions return to normal. The stress granules in these cells were smaller than those in control cells suggesting a role for TDP-43 in regulating the size of stress granules. As FLCN does not colocalise with G3BP1 it is most likely only indirectly impacting stress granule formation by mediating the cytoplasmic accumulation of TDP-43.

This research suggests a role for FLCN in enhancing the translocation of TDP-43 into the cytoplasm ahead of aggregation and stress granule production – although the exact mechanisms are unknown. Whilst interesting, these results are based on in vitro overexpression assays in a single, non-neuronal cell type. To understand the impact of this role for FLCN in neuronal pathology in ALS and FLTD further research is required to assess the interactions of endogenous TDP-43 and FLCN (Warren et al., 2004) in neuronal cell lines and ex vivo human samples. Additional research using kidney, lung and skin cells would be required to determine any pathological impacts in BHD patients. Studying endogenous proteins, using FLCN-specific antibodies, would also avoid the risk of the GFP tag affecting FLCN structure or function.

Increased AMPK signalling in motor neuron cell lines has also been shown to enhance nuclear clearing of TDP-43 (Liu et al., 2015). However, TDP-43 was found not to be a direct substrate of AMPK. As FLCN binds AMPK it is possible that FLCN is mediating the TDP-43 cytoplasmic localisation reported in response to changes in AMPK activity, and that depletion of FLCN could reduce this mislocalisation. TDP-43 was also recently linked to the alternative splicing of FNIP1 (De Conti et al., 2015) but further research is required to determine the impact of these interactions and any variations in isoform production on either ALS, FTLD or BHD pathology.

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