The extreme N-terminus of TDP-43 mediates the cytoplasmic aggregation of TDP-43 and associated toxicity in vivo

Abstract

Inclusions of Tar DNA- binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). Pathological TDP-43 exhibits the disease-specific biochemical signatures, which include its ubiquitination, phosphorylation and truncation. Recently, we demonstrated that the extreme N-terminus of TDP-43 regulates formation of abnormal cytoplasmic TDP-43 aggregation in cultured cells and primary neurons. However, it remained unclear whether this N-terminal domain mediates TDP-43 aggregation and the associated toxicity in vivo. To investigate this, we expressed a GFP-tagged TDP-43 with a nuclear localization signal mutation (GFP-TDP-43NLSm) and a truncated form without the extreme N-terminus (GFP-TDP-4310-414-NLSm) by adeno-associated viral (AAV) vectors in mouse primary cortical neurons and murine central nervous system. Compared to neurons containing GFP alone, expression of GFP-TDP-43NLSm resulted in the formation of ubiquitin-positive cytoplasmic inclusions and activation of caspase-3, an indicator of cell death. Moreover, mice expressing GFP-TDP-43NLSm proteins show reactive gliosis and develop neurological abnormalities. However, by deletion of TDP-43’s extreme N-terminus, these pathological alterations can be abrogated. Together, our study provides further evidence confirming the critical role of the extreme N-terminus of TDP-43 in regulating protein structure as well as mediating toxicity associated with its aggregation.

Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS

“…Although the aggregationprone 101 TDP43 C-terminus forms a core component of the cytoplasmic inclusions found in ALS patients [102][103][104][105][106][107][108][109] , emerging evidence suggests that N-terminal TDP43 fragments also contribute to ALS pathogenesis. N-terminal TDP43 fragments are observed in ALS patient spinal cord 110,111 , and in keeping with studies of RNA-binding proteins in yeast, the TDP43 RRMs misfold and aggregate in vitro without the C-terminal LCD to maintain solubility 65,91,[93][94][95][112][113][114] . Independent of the RRMs, the TDP43 N-terminus enhances TDP43 aggregation and toxicity 65,78,112,113 , potentially adding to sTDP43 insolubility and the impact of sTDP43 deposition in affected neurons.…”

“…N-terminal TDP43 fragments are observed in ALS patient spinal cord 110,111 , and in keeping with studies of RNA-binding proteins in yeast, the TDP43 RRMs misfold and aggregate in vitro without the C-terminal LCD to maintain solubility 65,91,[93][94][95][112][113][114] . Independent of the RRMs, the TDP43 N-terminus enhances TDP43 aggregation and toxicity 65,78,112,113 , potentially adding to sTDP43 insolubility and the impact of sTDP43 deposition in affected neurons.…”

Structural dissection of amyloid aggregates of TDP‐43 and its C‐terminal fragments TDP‐35 and TDP‐16

“…FTIR analysis reveals an extensive amount of non‐β‐sheet secondary structure in all constructs (~ 40–50%). Interestingly, the TDP‐43 NTD is involved in the sequestration of full‐length proteins into inclusions and is required for toxic aggregation in vitro and in vivo . Fawzi and coworkers recently identified a key amino acid position (S48) disrupting the liquid–liquid phase separation, reinforcing the idea of an NTD strongly involved in the assembly process .…”

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

“…Unlike the primarily disordered CTD, the TDP-43 NTD is highly conserved among animals (Appendix Fig S1; Ayala et al, 2005) and plays an essential role in TDP-43 splicing function (Zhang et al, 2013). Additionally, intact NTD is required for exogenous TDP-43 toxicity (Sasaguri et al, 2016) and for sequestration of endogenous TDP-43 in aggregates driven by expansion of an aggregation-prone region of the C-terminal domain (Romano et al, 2015). Together, these observations highlight the importance of NTD interactions with regard to cell function.…”