Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS
Kaitlin Weskamp, Elizabeth M. Tank, Roberto Miguez, Jonathon P. McBride, Nicolás B. Gómez, Matthew White, Ziqiang Lin, Carmen Moreno Gonzalez, Andrea Serio, Jemeen Sreedharan, Sami J. Barmada
Kaitlin Weskamp, Elizabeth M. Tank, Roberto Miguez, Jonathon P. McBride, Nicolás B. Gómez, Matthew White, Ziqiang Lin, Carmen Moreno Gonzalez, Andrea Serio, Jemeen Sreedharan, Sami J. Barmada
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Research Article Cell biology Neuroscience

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

Abstract

Cortical hyperexcitability and mislocalization of the RNA-binding protein TDP43 are highly conserved features in amyotrophic lateral sclerosis (ALS). Nevertheless, the relationship between these phenomena remains poorly defined. Here, we showed that hyperexcitability recapitulates TDP43 pathology by upregulating shortened TDP43 (sTDP43) splice isoforms. These truncated isoforms accumulated in the cytoplasm and formed insoluble inclusions that sequestered full-length TDP43 via preserved N-terminal interactions. Consistent with these findings, sTDP43 overexpression was toxic to mammalian neurons, suggesting neurodegeneration arising from complementary gain- and loss-of-function mechanisms. In humans and mice, sTDP43 transcripts were enriched in vulnerable motor neurons, and we observed a striking accumulation of sTDP43 within neurons and glia of ALS patients. Collectively, these studies uncover a pathogenic role for alternative TDP43 isoforms in ALS, and implicate sTDP43 as a key contributor to the susceptibility of motor neurons in this disorder.

Authors

Kaitlin Weskamp, Elizabeth M. Tank, Roberto Miguez, Jonathon P. McBride, Nicolás B. Gómez, Matthew White, Ziqiang Lin, Carmen Moreno Gonzalez, Andrea Serio, Jemeen Sreedharan, Sami J. Barmada

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Figure 6

sTDP43 overexpression leads to the cytoplasmic deposition and nuclear clearance of endogenous TDP43.

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sTDP43 overexpression leads to the cytoplasmic deposition and nuclear cl...
(A) HaloTag fusions of flTDP43 or sTDP43 were expressed in HEK293T cells and immunoprecipitated with HaloLink. Bound TDP43 was immunoblotted (IB) with a C-terminal TDP43 antibody. GAPDH served as a loading control. (B) Quantification of data shown in A, demonstrating the fraction of total TDP43 bound to flTDP43-Halo, sTDP43-Halo, or Halo alone. Data were combined from 3 replicates. *P < 0.05, **P < 0.01 by 1-way ANOVA with Dunnett’s post hoc test. (C) HEK293T cells were transfected with EGFP or EGFP-tagged sTDP43, and then immunostained using an antibody that recognizes the endogenous TDP43 C-terminus (Endo). Red, nuclear regions of interest (ROIs) determined by DAPI staining. (D) Nuclear, endogenous TDP43 is reduced by sTDP43 overexpression in HEK293T cells. EGFP n = 1537, sTDP43-EGFP n = 1997, 3 replicates. ****P < 0.0001 by 2-tailed t test. (E) Cytoplasmic endogenous TDP43 is elevated by sTDP43 overexpression in HEK293T cells. EGFP n = 129, sTDP43-EGFP n = 113, 3 replicates. ****P < 0.0001 by 2-tailed t test. (F) Primary mixed rodent cortical neurons were transfected with EGFP or EGFP-tagged sTDP43, and then immunostained using a C-terminal TDP43 antibody. Red, nuclear ROIs determined by DAPI staining. (G) sTDP43 overexpression resulted in a significant drop in nuclear, endogenous TDP43 in primary neurons (EGFP n = 395, EGFP-sTDP43 n = 323, 3 replicates; ****P < 0.0001 by 2-tailed t test), but this was not accompanied by increases in cytoplasmic, endogenous TDP43 (H) (EGFP n = 394, EGFP-sTDP43 n = 323, 3 replicates, NS by 2-tailed t test). Scale bars: 20 μm (C and F).