Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS
Kaitlin Weskamp, … , Jemeen Sreedharan, Sami J. Barmada
Kaitlin Weskamp, … , Jemeen Sreedharan, Sami J. Barmada
Published November 12, 2019
Citation Information: J Clin Invest. 2020;130(3):1139-1155. https://doi.org/10.1172/JCI130988.
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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 4

sTDP43 accumulates within the cytoplasm due to a putative NES.

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sTDP43 accumulates within the cytoplasm due to a putative NES. (A) Roden...
(A) Rodent primary mixed cortical neurons were transfected with mApple and EGFP-tagged TDP43 isoforms, and then imaged by fluorescence microscopy. (B) Amino acid sequence of the sTDP43 tail showing the putative NES identified using NetNES 1.1. Light blue, polar; purple, positively charged; green, hydrophobic residues. (C) sTDP43-EGFP was significantly more cytoplasmic compared with flTDP43-EGFP, while mutation of the putative NES (mNES) restores nuclear localization. N/C, nuclear/cytoplasmic. EGFP n = 481, flTDP43-EGFP n = 385, sTDP43-EGFP n = 456, sTDP43(mNES)-EGFP n = 490, stratified among 3 replicates. ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test. (D) Rodent primary mixed cortical neurons were transfected with 1 of 3 constructs: EGFP alone, EGFP fused to the sTDP43 C-terminus, or EGFP fused to the sTDP43 C-terminal tail harboring a mutated NES (mNES). (E) The sTDP43 C-terminus mislocalizes EGFP to the cytoplasm, and mislocalization depends on the putative NES. Shuttle-RFP, a construct with a strong NES, serves as a positive control for a cytoplasmic protein. EGFP n = 2490, Shuttle-RFP n = 2073, EGFP-tail n = 1956, EGFP-tail(mNES) n = 2482, combined from 3 replicates. ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test. Scale bars: 20 μm (A and D).