Abstract
TAR DNA-binding protein 43 (TDP-43) has emerged as a key player in many neurodegenerative pathologies, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Hallmarks of both FTLD and ALS are the toxic cytoplasmic inclusions of the prion-like C-terminal fragments of TDP-43 CTD (TDP-43 C-terminal domain), formed upon proteolytic cleavage of full-length TDP-43 in the nucleus and subsequent transport to the cytoplasm. Both full-length TDP-43 and its CTD are also known to form stress granules by coacervating with RNA in the cytoplasm during stress and may be involved in these pathologies. Furthermore, mutations in the PGRN gene, leading to haploinsufficiency and diminished function of progranulin (PGRN) protein, are strongly linked to FTLD and ALS. Recent reports have indicated that proteolytic processing of PGRN to smaller protein modules called granulins (GRNs) contributes to FTLD and ALS progression, with specific GRNs exacerbating TDP-43-induced cytotoxicity. Here we investigated the interactions between the proteolytic products of both TDP-43 and PGRN. Based on structural disorder and charge distributions, we hypothesized that GRN-3 and GRN-5 could interact with the TDP-43 CTD. We show that, under both reducing and oxidizing conditions, GRN-3 and GRN-5 interact with and differentially modulate TDP-43 CTD aggregation and/or liquid-liquid phase separation in vitro GRN-3 promoted insoluble aggregates of the TDP-43 CTD while GRN-5 mediated liquid-liquid phase separation. These results constitute the first observation of an interaction between GRNs and TDP-43, suggesting a mechanism by which attenuated PGRN function could lead to familial FTLD or ALS.
Highlights
TAR DNA-binding protein 43 (TDP-43) has emerged as a key player in many neurodegenerative pathologies, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS)
GRN-3 promoted insoluble aggregates of the TDP-43 CTD while GRN-5 mediated liquid–liquid phase separation. These results constitute the first observation of an interaction between GRNs and TDP-43, suggesting a mechanism by which attenuated PGRN function could lead to familial FTLD or ALS
Heritable FTLD can be classified into the following subtypes: the FTLD-TDP form, which is characterized by the presence of cytoplasmic inclusions of TAR DNA binding protein 43 (TDP-43) and constitutes about half of all cases of FTLD [7]; FTLD-Tau, in which cytoplasmic inclusions of hyperphosphorylated tau are observed; and FTLD-FUS, which is associated with inclusions of an RNA-binding protein called fused in sarcoma (FUS) (8 –12)
Summary
The 14.5-kDa construct of TDP-43 that constitutes residues 267– 414 in the C-terminal domain (TDP-43 CTD) is a major part of the C25 proteolysis fragment that forms aberrant inclusions within the cytosol in FTLD and ALS patients (Fig. 1a) [16]. Incubation of rGRN-5 with the TDP-43 CTD showed no discernable lag time but an instantaneous linear increase in ThT fluorescence for all stoichiometric incubations (Fig. 3d, insets) These data suggest that GRN-3 and GRN-5 interact differently with TDP-43 CTD to modulate the latter’s aggregation. RGRN-5 colocalized with the TDP-43 CTD within the LDs. To probe the internal dynamics of the LD droplets, FRAP was monitored temporally on the labeled TDP-43 CTD in the presence of RNA and GRNs. The fluorescence recovery rates for control LDs formed by the TDP-43 CTD and RNA showed nearly identical rates at 0 and 36 h, indicating preservation of internal mobility and dynamism over the incubation period (Fig. 6i). The cellular ramifications of this on stress response by stress granules remains to be seen
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