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 1

Hyperexcitability drives TDP43 accumulation in human iNeurons.

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Hyperexcitability drives TDP43 accumulation in human iNeurons. (A) Schem...
(A) Schematic of the cassette used to integrate Ngn1 and Ngn2 into the CLYBL safe harbor locus under a doxycycline-inducible (Tet-on) promoter. CLYBL, targeting sequence; Puro, puromycin resistance gene; pA, poly-A tail; P1, P2, promoters; RFP, mCherry; rtTA, reverse tetracycline-controlled transactivator; Ngn1 and -2, neurogenin 1 and 2; T2A, self-cleaving peptide; TRE, tetracycline response element. (B) Timeline depicting the differentiation of iPSCs into forebrain-like neurons within 2 weeks of doxycycline addition. (C) The resultant neurons are RFP positive and express the neuronal markers VGLUT1 and TUJ1. (D) Spontaneous neuronal activity visualized by the Ca2+ reporter gCaMP6f at 2 weeks. Activity was pharmacologically modulated with bath application of glutamate or TTX. Vehicle n = 257, glutamate n = 327, TTX n = 403, stratified among 3 replicates. ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test. (E) Treatment with TEA significantly increased neuronal activity. Vehicle n = 312, TEA n = 369, stratified among 3 replicates. ****P < 0.0001 by 2-tailed t test. (F) Example traces depicting changes in gCaMP6f fluorescence for each condition. (G) Heatmaps depicting global changes in activity. Each row represents 1 neuron, and each column represents a 20-second observation window. Thirty intervals were collected over a 12-hour period. Box color indicates the relative firing rate of each cell at each time point, ranging from low (blue) to high (red). (H) N-terminal TDP43 immunoreactivity was increased in TEA-treated iNeurons and decreased in TTX-treated iNeurons. (I) Density plot depicting the change in TDP43 immunoreactivity between conditions. Vehicle n = 110, TEA n = 113, TTX n = 96, 2 replicates; vertical lines indicate single neurons. *P < 0.05 by Kolmogorov-Smirnov test. (J) No change in TDP43 abundance was detected using an antibody directed against the C-terminus. (K) Density plot depicting the change in C-terminal TDP43 immunoreactivity between conditions. Vehicle n = 187, TEA n = 541, TTX n = 443, 2 replicates; vertical lines indicate single neurons. No significant differences as determined by the Kolmogorov-Smirnov test. Scale bars: 50 μm (top), 20 μm (bottom) (C); 20 μm (H and J).