A “Two-hit” Hypothesis for Inclusion Formation by Carboxyl-terminal Fragments of TDP-43 Protein Linked to RNA Depletion and Impaired Microtubule-dependent Transport

Abstract

Carboxyl-terminal fragments (CTFs) of TDP-43 aggregate to form the diagnostic signature inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the biological significance of these CTFs and how they are generated remain enigmatic. To address these issues, we engineered mammalian cells with an inducible tobacco etch virus (TEV) protease that cleaves TDP-43 containing a TEV cleavage site. Regions of TDP-43 flanking the second RNA recognition motif (RRM2) are efficiently cleaved by TEV, whereas sites within this domain are more resistant to cleavage. CTFs containing RRM2 generated from de novo cleavage of nuclear TDP-43 are transported to the cytoplasm and efficiently cleared, indicating that cleavage alone is not sufficient to initiate CTF aggregation. However, CTFs rapidly aggregated into stable cytoplasmic inclusions following de novo cleavage when dynein-mediated microtubule transport was disrupted, RNA was depleted, or natively misfolded CTFs were introduced into these cells. Our data support a "two-hit" mechanism of CTF aggregation dependent on TDP-43 cleavage.The heterogeneous nuclear ribonucleoprotein (hnRNP) 2 TDP-43 (TAR DNA-binding protein of 43 kDa) forms pathological inclusions that are diagnostic hallmarks of amyotrophic lateral sclerosis (ALS) and the major form of frontotemporal lobar degeneration (FTLD-TDP) (1). Missense mutations in TDP-43 provide a genetic link between hnRNP functions and motor neuron disease (2, 3). TDP-43 is predominantly localized to the nucleus, where it regulates RNA splicing, mRNA stability, microRNA processing, and transcription (4 -6). However, in neurodegenerative TDP-43 proteinopathies like ALS and FTLD-TDP, a loss of nuclear TDP-43 coincides with cytoplasmic TDP-43 inclusions (1). These inclusions contain full-length TDP-43 and TDP-43 C-terminal fragments (CTFs) that are hyperphosphorylated and ubiquitinated. However, it is unclear how each of these species of TDP-43 contributes to inclusion formation or disease pathogenesis.TDP-43 is a typical 2XRRM-gly hnRNP composed of two tandem RNA recognition motifs (RRM1 and RRM2) followed by a glycine-rich carboxyl terminus ( Fig. 1) (7). Both RRMs retain nucleic acid binding properties, yet only RRM1 appears essential for RNA splicing (8). The glycine-rich domain is proposed to interact with other hnRNPs to synergistically affect alternative splicing of specific RNA transcripts (9, 10). A common feature of shuttling hnRNPs involved in RNA splicing is that their nuclear export is coupled to the export and maturation of mRNA in distinct ribonucleoprotein (RNP) complexes (11,12). In this respect, the nucleocytoplasmic shuttling of TDP-43 is dependent on its bipartite importin-␣ nuclear localization signal, a chromosome maintenance region 1 (CRM-1) nuclear export signal in RRM2, and undefined aspects of the carboxyl-terminal glycine-rich domain (13,14).CTFs are signatures of disease exclusively associated with TDP-43 pathology, and CTFs cleaved in the middle of RRM2 extending from amino acid 208 to t...

Metabolism and mis-metabolism of the neuropathological signature protein TDP-43

“…S1E, showing that enhanced cleavage of the pro-caspase 3 was observed in MG132-treated cells whereas Z-VAD treatment inhibited this enhancement. The data from cyclohexamide chase experiments showed that Z-VAD treatment increased the half-life of the TDP-43 protein from 31 h, a value similar to the findings of Pesiridis et al (Pesiridis et al, 2011), to over 75 h (Fig. 3A).…”

“…However, upon MG132 treatment, which causes accumulation of full-length TDP-43, owing to inhibition of UPS, and enhances the cleavage of the full-length TDP-43 to generate more TDP-35 and TDP-25 fragments, as the result of induction of caspase 3 Nonaka et al, 2009a), the amount of the cytosolic TDP-43-positive aggregates and/or insoluble TDP-43 species increases greatly. The above studies have suggested the importance of the TDP-35 and TDP-25 fragments and an elevated amount of the full-length TDP-43 in the formation of TDP-43-positive UBIs, a process in which preformed TDP-25 fibrils could act as the seed (Furukawa et al, 2011;Pesiridis et al, 2011) to trap the full-length TDP-43 in vitro in cultured cells and in vivo in diseased cells of patients with TDP-43 proteinopathies. However, data from other studies seem to indicate that the proteolytic processing of the full-length TDP-43 is not absolutely required for generation of the insoluble TDP-43, nor does the generation of insoluble TDP-43 species necessarily lead to the aggregate formation (Dormann et al, 2009;Kleinberger et al, 2010).…”

Targeted Depletion of TDP-43 Expression in the Spinal Cord Motor Neurons Leads to the Development of Amyotrophic Lateral Sclerosis-like Phenotypes in Mice

“…6. In the presence of TDP-43, these polyubiquitinated proteins then could form the TDP-43(ϩ) UBIs with TDP-43 (57,58). To what extent and how generally applicable this proposed scenario is to the ALS cases with TDP-43(ϩ) proteinopathies awaits to be validated in the future.…”

TDP‐35 sequesters TDP‐43 into cytoplasmic inclusions through binding with RNA

“…The role of the RRM domains in TDP-43 aggregation has been studied in detail [17][18][19], suggesting that RNA binding can inhibit TDP-43 aggregation in vitro [18]. The RRM1 moiety of TDP-35 plays a dominant role in binding with RNA/DNA and mediating association with TDP-43 [34], whereas RRM2 may collaborate with RRM1 to enhance the binding affinities with nucleic acids [30].…”

“…The RRM1 domain is essential for binding to single-strand RNA with six UG repeats [14], whereas the function of RRM2 is obscure since it is dispensable for binding to UG repeats [15]. The RNA binding ability is important for TDP-43 shuttling [16] and inclusion formation [17][18][19], but the mechanism by which the RNA exerts functions in formation of inclusions is largely unknown.…”