Elicit: TDP-43 Biological Pathways (Public)

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TDP-43 Biological Pathways (Public)

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March 2, 2025

What biological pathways is TDP-43 known to be part of?

TDP-43 functions in seven major biological pathways: RNA metabolism, nucleocytoplasmic transport, stress response, mitochondrial function, synaptic function, inflammatory responses, and cell cycle regulation.

Abstract

Forty studies report that TDP-43 participates in a network of biological pathways. In several papers, TDP-43 regulates RNA metabolism by controlling alternative splicing, cryptic exon inclusion, and mRNA stability. Other studies document that TDP-43 affects nucleocytoplasmic transport by disrupting nuclear pore complex integrity and altering protein–RNA distribution. Reports also describe its role in stress response and protein quality control through modulation of stress granule dynamics, phase separation, and aggresome formation. In addition, TDP-43 has been linked to mitochondrial function via binding mitochondrial mRNAs and disrupting respiratory complex assembly, as well as to synaptic function and neurotransmitter regulation through effects on receptor recycling and synaptic protein expression. Some papers further implicate the protein in inflammatory and immune responses via activation of NF‑κB and inflammasome pathways, and in cell cycle regulation and DNA repair by modulating targets such as cyclin-dependent kinase expression and double-strand break repair.

Seven primary pathways emerge from these studies: 1. RNA metabolism and processing 2. Nucleocytoplasmic transport 3. Stress response and protein quality control 4. Mitochondrial function 5. Synaptic function and neurotransmitter regulation 6. Inflammatory and immune responses 7. Cell cycle regulation and DNA repair

Diverse experimental systems—including mouse models, cultured cells, and iPSC-derived neurons—support TDP-43’s multifaceted functions across these interrelated pathways.

Methods

We analyzed 40 sources from an initial pool of 493, using 6 screening criteria. Each paper was reviewed for 3 key aspects that mattered most to the research question. More on methods

Papers identified with Elicit search

n = 493

Papers screened using: Molecular Pathway Analysis, TDP-43 Function, Experimental Design, Study Type, Mechanistic Detail, Evidence Quality

n = 493

Papers screened out

n = 453

Papers included for extraction

n = 40

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Paper search

Using your research question “What biological pathways is TDP-43 known to be part of?”, we searched across over 126 million academic papers from the Semantic Scholar corpus. We retrieved the 493 papers most relevant to the query.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract all explicitly mentioned biological pathways or cellular processes that TDP-43 is involved in. Look in the methods, results, and discussion sections.

Specific areas to focus on:

If multiple pathways are mentioned, list them all. If a pathway is described but not explicitly named, summarize the description.

If no clear pathways are identified, write “No specific pathways explicitly described.”

Format examples:

Identify and list all molecular interactions and binding partners mentioned in the study.

Search in:

Extract:

If multiple interactions are found, list all. If no interactions are described, write “No molecular interactions reported.”

Format examples:

Extract specific functional outcomes or consequences resulting from TDP-43 interactions or modifications.

Look for:

Prioritize direct functional descriptions, not just descriptive observations.

If multiple consequences are found, list all. If no clear functional outcomes are described, write “No specific functional consequences reported.”

Format examples:

Results

Characteristics of Included Studies

Study

Study Design

Cellular/Model System

Pathways Investigated

Key Findings

Full text retrieved

“TDP‐43 Loss of Function,” 2016

In vitro study

Neuronal cell culture

Endosomal trafficking

The study reported that TDP-43 regulates endosomal trafficking and receptor recycling

No

Afroz et al., 2023

In vivo mouse model and in vitro studies

Mouse models, Amyotrophic Lateral Sclerosis (ALS) patient-derived microglia

Immune response, phagocytosis

The study reported that targeting TDP-43 C-terminal domain reduces pathology and enhances microglial function

No

Ayala et al., 2008

In vitro study

Human cells

Cell cycle regulation

The study reported that TDP-43 regulates Cyclin-dependent kinase 6 (Cdk6) expression and cell cycle progression

Yes

Chen, 2020

Review

Various

RNA metabolism, autophagy

The review reported that TDP-43 regulates cryptic exon splicing and autophagy pathways

No

Chou et al., 2017

In vitro and in vivo studies

Mouse primary neurons, human fibroblasts, induced pluripotent stem cell (iPSC)-derived neurons

Nucleocytoplasmic transport

The study reported that TDP-43 aggregates disrupt nuclear pore complexes and nucleocytoplasmic transport

Yes

Chou et al., 2018

In vitro and in vivo studies

Mouse primary neurons, human fibroblasts, iPSC-derived neurons

Nucleocytoplasmic transport

The study reported that TDP-43 pathology impairs nuclear protein import and RNA export

Yes

Deshaies et al., 2018

In vitro and in vivo studies

Human cells, mouse models

RNA splicing

The study reported that TDP-43 regulates alternative splicing of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1)

Yes

Fazal et al., 2021

iPSC-derived motor neuron study

Human iPSC-derived motor neurons

RNA metabolism, axonal transport

The study reported that TDP-43 mutations affect RNA processing and axonal transport

Yes

Feneberg et al., 2020

In vitro and in vivo studies

Mouse primary motor neurons

Protein-protein interactions, stress response

The study reported that TDP-43 interacts with proteins involved in stress granule formation and endosomal-extracellular transport

No

Feneberga et al., “Oxidative Stress in Motor Neurons”

In vitro and in vivo studies

Mouse primary motor neurons

Protein-protein interactions, stress response

The study reported that TDP-43 mutations affect stress granule formation and extracellular vesicle secretion

No

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Our analysis of the 40 studies on TDP-43 pathways reveals:

The diversity of cellular models and pathways investigated highlights the multifaceted nature of TDP-43’s functions and its potential involvement in various aspects of neurodegeneration.

Based on our analysis, we identified seven primary pathways implicated in the cellular mechanisms of TDP-43:

  1. RNA metabolism and processing
  2. Nucleocytoplasmic transport
  3. Stress response and protein quality control
  4. Mitochondrial function
  5. Synaptic function and neurotransmitter regulation
  6. Inflammatory and immune responses
  7. Cell cycle regulation and DNA repair

These diverse pathways highlight the multifaceted role of TDP-43 in cellular function and its potential impact on various aspects of neurodegeneration.

Thematic Analysis

RNA Processing and Regulation Pathways

Study

RNA Processing Function

Specific Targets/Mechanisms

Cellular Impact

Chen, 2020

Cryptic exon splicing regulation

Repression of cryptic exons

Maintenance of normal transcriptome

Deshaies et al., 2018

Alternative splicing regulation

hnRNP A1 pre-mRNA

Production of aggregation-prone hnRNP A1B isoform

Fazal et al., 2021

RNA processing, splicing regulation

Various mRNA targets

Altered gene expression in motor neurons

Gu et al., 2019

mRNA processing

Tau mRNA

Regulation of tau mRNA stability and exon 10 inclusion

Igaz et al., 2009

RNA splicing

No mention found

The study reported abnormal RNA splicing caused by TDP-43 C-terminal fragments

Kim et al., 2010

mRNA regulation

HDAC6 mRNA

Reduced expression of HDAC6

Koehler et al., 2022

Autoregulation, RNA binding

TDP-43 mRNA

Maintenance of TDP-43 protein levels

Lauranzano et al., 2015

RNA metabolism

TARDBP RNA targets

Regulation of TDP-43 target gene expression

Ling et al., 2010

RNA processing

No mention found

The study reported enhanced interaction with FUS/TLS affecting RNA processing

Long et al., 2024

miRNA biogenesis

miRNAs

Disruption of miRNA processing through Dicer interaction

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Our analysis of the included studies suggests that TDP-43 is involved in various RNA processing functions:

These findings suggest that TDP-43 plays a multifaceted role in RNA processing, potentially affecting various cellular processes through its interactions with different RNA targets.

Stress Response and Protein Quality Control Pathways

Study

Stress Response Mechanism

Protein Quality Control Function

Cellular Impact

Chen, 2020

Autophagy regulation

Regulation of ATG7 and other autophagy-related genes

Maintenance of cellular protein homeostasis

Chou et al., 2017

Stress granule dynamics

Interaction with nuclear pore complexes

Altered nucleocytoplasmic transport under stress

Feneberg et al., 2020

Stress granule formation

Interaction with stress granule proteins

Altered stress response under oxidative conditions

Feneberga et al., “Oxidative Stress in Motor Neurons”

Stress granule formation

Extracellular vesicle secretion

Impaired stress response and intercellular communication

Herzog et al., 2019

CREB signaling regulation

No mention found

Altered gene expression under stress conditions

Koehler et al., 2022

Phase separation

Autoregulation of TDP-43 levels

Maintenance of TDP-43 protein homeostasis

Luan et al., 2023

Integrated stress response activation

Regulation of stress-related gene expression

Enhanced TDP-43 aggregation and stress granule formation

Mann et al., 2019

Phase transitions

RNA binding-mediated regulation

Prevention of neurotoxic TDP-43 aggregation

McGurk et al., 2018

Stress granule localization

Interaction with poly(ADP-ribose)

Promotion of TDP-43 phase separation

Sidibé et al., 2020

Stress granule dynamics

G3BP1 mRNA stabilization

Regulation of stress granule assembly

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Our analysis of the table reveals several key findings regarding TDP-43’s role in cellular stress responses:

These findings highlight the complex and multifaceted role of TDP-43 in cellular stress responses, with a particular emphasis on stress granule dynamics and phase separation processes.

Synaptic Function and Neurotransmitter Pathways

Study

Synaptic Function

Neurotransmitter Regulation

Cellular Impact

“TDP‐43 Loss of Function,” 2016

Regulation of receptor recycling

Affects ErbB4 surface delivery

Impaired trophic signaling

Heyburn and Moussa, 2016

Regulation of synaptic protein expression

Affects vesicular glutamate levels

Altered neurotransmitter release

Sephton et al., 2010

Binding to RNAs involved in synaptic function

No mention found

Regulation of synaptic gene expression

Herzog et al., 2019

Regulation of dendritic complexity

No mention found

Altered neuronal morphology and potential synaptic changes

Fazal et al., 2021

Affects axonal transport

No mention found

Potential impact on synaptic function and neurotransmitter release

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Our analysis of the table reveals diverse effects of TDP-43 on synaptic function, neurotransmitter regulation, and cellular impact across the five studies:

The diversity of findings suggests that TDP-43 may have wide-ranging effects on neuronal function, affecting various aspects of synaptic activity, neurotransmitter regulation, and cellular processes. However, the lack of consistent findings across studies indicates a need for further research to establish more definitive patterns of TDP-43’s impact on neuronal function.

Pathway Interactions and Integration

Primary Pathway

Connected Pathways

Regulatory Points

Cellular Outcomes

RNA Processing

Stress Response, Synaptic Function

Splicing regulation, mRNA stability

Altered gene expression, protein homeostasis

Nucleocytoplasmic Transport

RNA Processing, Stress Response

Nuclear pore complex interactions

Disrupted RNA/protein localization, stress granule formation

Stress Response

RNA Processing, Protein Quality Control

Phase separation, stress granule dynamics

Altered stress granule assembly, protein aggregation

Mitochondrial Function

Stress Response, DNA Repair

Mitochondrial mRNA binding, unfolded protein response

Respiratory complex disruption, oxidative stress

Synaptic Function

RNA Processing, Protein Quality Control

Receptor recycling, synaptic protein expression

Altered neurotransmission, synaptic plasticity

Inflammatory Response

Stress Response, Mitochondrial Function

NF-κB and NLRP3 inflammasome activation

Microglial activation, neuroinflammation

DNA Repair

RNA Processing, Stress Response

Double-Strand Break (DSB) repair, R-loop metabolism

Genome stability, cellular survival

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Our analysis of the pathway interactions and integration reveals:

The studies we analyzed suggest interconnections between multiple pathways involving TDP-43, potentially indicating its multifaceted cellular functions. These interconnections may help explain the widespread cellular dysfunction observed in TDP-43-associated neurodegenerative diseases, as disruptions in one pathway could potentially have cascading effects on others.

References

Liam Chen\ (2020).The important functional role of TDP-43 plays in amyotrophic lateral sclerosis-frontotemporal dementia. Neural Regeneration Research

L. Heyburn, C. Moussa\ (2016).TDP-43 overexpression impairs presynaptic integrity. Neural Regeneration Research

Ching-Chieh Chou, Y. Zhang, Mfon E Umoh, Spencer Vaughan, Ileana Lorenzini, and 20 more\ (2017).TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Alzheimer's & Dementia

Wenzhang Wang, Luwen Wang, Junjie Lu, S. Siedlak, H. Fujioka, and 10 more\ (2016).The Inhibition of TDP-43 Mitochondrial Localization Blocks Its Neuronal Toxicity. Nature Network Boston

R. Mutihac, R. Mutihac, J. Alegre-Abarrategui, D. Gordon, Lucy Farrimond, and 3 more\ (2015).\ TARDBP pathogenic mutations increase cytoplasmic translocation of TDP-43 and cause reduction of endoplasmic reticulum Ca2+ signaling in motor neurons. Neurobiology of Disease

Weihua Zhao, D. Beers, S. Bell, Jinghong Wang, Shixiang Wen, and 2 more\ (2015).TDP-43 activates microglia through NF-κB and NLRP3 inflammasome. Experimental Neurology

L. Igaz, L. Kwong, A. Chen-Plotkin, M. Winton, T. Unger, and 4 more\ (2009).Expression of TDP-43 C-terminal Fragments in Vitro Recapitulates Pathological Features of TDP-43 Proteinopathies*. Journal of Biological Chemistry

Chien-Hsiung Yu, Sophia Davidson, C. Harapas, J. B. Hilton, Michael J. Mlodzianoski, and 21 more\ (2020).TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS. Cell

C. Sephton, Can Cenik, Alper Kucukural, E. Dammer, B. Cenik, and 7 more\ (2010).Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes. Journal of Biological Chemistry

J. Mann, A. Gleixner, J. Mauna, Edward Gomes, M. DeChellis-Marks, and 14 more\ (2019).RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43. Neuron

Shuo-Chien Ling, Claudio P. Albuquerque, Joo Seok Han, C. Lagier-Tourenne, Seiya Tokunaga, and 2 more\ (2010).ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proceedings of the National Academy of Sciences of the United States of America

No authors found (2016).TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons. EMBO Journal

Sang Hwa Kim, Naval P. Shanware, Michael J Bowler, R. Tibbetts\ (2010).Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA*. Journal of Biological Chemistry

Wei Luan, A. Wright, H. Brown-Wright, Sheng Le, Rebecca San Gil, and 5 more\ (2023).Early activation of cellular stress and death pathways caused by cytoplasmic TDP-43 in the rNLS8 mouse model of ALS and FTD. Molecular Psychiatry

Xing Sun, Yongjia Duan, Caixia Qin, Jian-Chiuan Li, Gang Duan, and 8 more\ (2018).Distinct multilevel misregulations of Parkin and PINK1 revealed in cell and animal models of TDP-43 proteinopathy. Cell Death and Disease

L. McGurk, Edward Gomes, Lin Guo, J. Mojsilovic-Petrovic, V. Tran, and 3 more\ (2018).Poly(ADP-Ribose) Prevents Pathological Phase Separation of TDP-43 by Promoting Liquid Demixing and Stress Granule Localization. Molecules and Cells

Josiah J. Herzog, Weijin Xu, Mugdha Deshpande, Reazur Rahman, H. Suib, and 3 more\ (2019).TDP-43 dysfunction restricts dendritic complexity by inhibiting CREB activation and altering gene expression. Proceedings of the National Academy of Sciences of the United States of America

Hadjara Sidibé, Yousra Khalfallah, S. Xiao, N. Gómez, Hana Fakim, and 15 more\ (2021).TDP-43 stabilizes G3BP1 mRNA: relevance to amyotrophic lateral sclerosis/frontotemporal dementia. Brain : a journal of neurology

E. Feneberg, D. Gordon, A. Thompson, M. Finelli, R. Dafinca, and 9 more\ (2020).An ALS-linked mutation in TDP-43 disrupts normal protein interactions in the motor neuron response to oxidative stress. Neurobiology of Disease

María M. Leal‐Lasarte, Jaime M Franco, A. Labrador-Garrido, D. Pozo, C. Roodveldt\ (2017).Extracellular TDP‐43 aggregates target MAPK/MAK/MRK overlapping kinase (MOK) and trigger caspase‐3/IL‐18 signaling in microglia. The FASEB Journal

Xiang Long, Mengni Jiang, Yongzhen Miao, Huanhuan Du, Ting Zhang, and 4 more\ (2024).TDP-43 mutations-induced defects in miRNA biogenesis and cytotoxicity by differentially obstructing Dicer activity in Drosophila and in vitro. bioRxiv

Y. Ayala, T. Misteli, F. Baralle\ (2008).TDP-43 regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent kinase 6 expression. Proceedings of the National Academy of Sciences of the United States of America

G. P. Ritson, Sara K Custer, Brian D. Freibaum, Jake B. Guinto, D. Geffel, and 8 more\ (2010).TDP-43 Mediates Degeneration in a Novel Drosophila Model of Disease Caused by Mutations in VCP/p97. Journal of Neuroscience

Zhipeng Yu, D. Fan, B. Gui, Lei Shi, C. Xuan, and 4 more\ (2012).Neurodegeneration-associated TDP-43 Interacts with Fragile X Mental Retardation Protein (FMRP)/Staufen (STAU1) and Regulates SIRT1 Expression in Neuronal Cells*. Journal of Biological Chemistry

Emily Feneberga, David Gordona, Alexander G. Thompsona, Mattéa J. Finellia, Ana Candalijaa, and 8 more\ (2020).Neurobiology of Disease An ALS-linked mutation in TDP-43 disrupts normal protein interactions in the motor neuron response to oxidative stress

Peng Wang, Jianwen Deng, Jie Dong, Jianghong Liu, E. Bigio, and 10 more\ (2019).TDP-43 induces mitochondrial damage and activates the mitochondrial unfolded protein response. PLoS Genetics

Kaitlin Weskamp, Elizabeth H M Tank, R. Miguez, Jonathon P. McBride, N. Gómez, and 6 more\ (2019).Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS. bioRxiv

Jade-Emmanuelle Deshaies, L. Shkreta, A. Moszczynski, Hadjara Sidibé, Sabrina Semmler, and 15 more\ (2018).TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis. Brain : a journal of neurology

Nicole F. Liachko, P. McMillan, T. Strovas, Elaine Loomis, Lynne Greenup, and 7 more\ (2014).The Tau Tubulin Kinases TTBK1/2 Promote Accumulation of Pathological TDP-43. PLoS Genetics

Hadjara Sidibé, Yousra Khalfallah, S. Xiao, N. Gómez, Elizabeth H M Tank, and 14 more\ (2020).TDP-43 stabilizes transcripts encoding stress granule protein G3BP1: potential relevance to ALS/FTD. bioRxiv

J. Mitra, Erika N. Guerrero, Erika N. Guerrero, Pavana M. Hegde, Nicole F. Liachko, and 15 more\ (2019).Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects. Proceedings of the National Academy of Sciences of the United States of America

Giulia Romano, Raffaella Klima, F. Feiguin\ (2020).TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity. BMC Biology

Y. Ke, Annika van Hummel, Carol Au, Gabriella Chan, W. S. Lee, and 16 more\ (2024).Targeting 14-3-3θ-mediated TDP-43 pathology in amyotrophic lateral sclerosis and frontotemporal dementia mice. Neuron

Jianlan Gu, Dandan Chu, Nana Jin, Feng Chen, Fei Liu\ (2019).Cyclic AMP-Dependent Protein Kinase Phosphorylates TDP-43 and Modulates Its Function in Tau mRNA Processing. Journal of Alzheimer's Disease

Seiji Watanabe, Hidekazu Inami, K. Oiwa, Yuri Murata, Shohei Sakai, and 5 more\ (2020).Aggresome formation and liquid–liquid phase separation independently induce cytoplasmic aggregation of TAR DNA-binding protein 43. Cell Death and Disease

Ching-Chieh Chou, Y. Zhang, Mfon E Umoh, Spencer Vaughan, Ileana Lorenzini, and 20 more\ (2018).TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nature Neuroscience

L. Koehler, Z. Grese, Alliny C S Bastos, Lohany D. Mamede, T. Heyduk, and 1 more\ (2022).TDP-43 Oligomerization and Phase Separation Properties Are Necessary for Autoregulation. Frontiers in Neuroscience

T. Afroz, Elodie Chevalier, M. Audrain, C. Dumayne, T. Ziehm, and 21 more\ (2023).Immunotherapy targeting the C-terminal domain of TDP-43 decreases neuropathology and confers neuroprotection in mouse models of ALS/FTD. Neurobiology of Disease

Raheem Fazal, Steven Boeynaems, A. Swijsen, Mathias De Decker, L. Fumagalli, and 14 more\ (2021).HDAC6 inhibition restores TDP‐43 pathology and axonal transport defects in human motor neurons with TARDBP mutations. EMBO Journal

E. Lauranzano, S. Pozzi, L. Pasetto, Riccardo Stucchi, Tania Massignan, and 8 more\ (2015).Peptidylprolyl isomerase A governs TARDBP function and assembly in heterogeneous nuclear ribonucleoprotein complexes. Brain : a journal of neurology

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Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects

J. Mitra, Erika N. Guerrero, Erika N. Guerrero, Pavana M. Hegde, Nicole F. Liachko, Haibo Wang, Velmarini Vasquez, Velmarini Vasquez, Junling Gao, Arvind Pandey, J. P. Taylor, B. Kraemer, Ping Wu, I. Boldogh, R. Garruto, Sankar Mitra, Sankar Mitra, K. S. Rao, M. Hegde, M. Hegde

Proceedings of the National Academy of Sciences of the United States of America·

2019·

216 citations

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Specific Biological Pathways Involving TDP-43

- mRNA processing and microRNA biogenesis - Stress granule formation - Regulation of neurite growth - Autophagy - Synaptic transmission - Inflammation and microglia infiltration - Nonhomologous end joining (NHEJ)-mediated DNA double-strand break repair - DNA damage response (DDR)

Molecular Interactions and Binding Partners of TDP-43

- Interacts with Ku70 - Interacts with p53 binding protein 1 (53BP1) - Acts as a scaffold for XRCC4-DNA ligase 4 complex - Interacts with DNA polymerase λ (Polλ) - Directly binds to DNA oligonucleotides mimicking DSBs

Functional Consequences of TDP-43 Interactions

Impaired DNA double-strand break repair, accumulation of DNA damage, sustained DDR signaling, neuronal apoptosis

Significance Amyotrophic lateral sclerosis (ALS) is a devastating, motor neuron degenerative disease without any cure to date. About 95% of ALS patients feature abnormalities in the RNA/DNA binding protein TDP-43, involving its nucleus-cytoplasmic mislocalization in spinal motor neurons. How TDP-43 pathology triggers neuronal apoptosis remains unclear. Here, we report that TDP-43 participates in the DNA damage response and its nuclear clearance in motor neurons causes DNA double-strand break repair defects in ALS. Our findings uncover a link between TDP-43 pathology and impaired DNA repair, and suggest potential avenues for DNA repair-targeted therapies for TDP-43–ALS. Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in Caenorhabditis elegans mutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in Caenorhabditis elegans mutant with TDP-1 loss-offunction. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

TDP-43 | DNA damage response | DNA double-strand break repair | amyotrophic lateral sclerosis | neurodegeneration A myotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal degenerative disease of motor neurons without an effective treatment. ALS affects neurons in the motor cortex, brainstem, and upper and lower spinal cord, gradually inducing muscle atrophy, denervation, and severe motor dysfunction. The ALS group of motor neuron diseases is highly complex, involving more than a dozen genes (reviewed in ref. 1). The transactivation response DNA-binding protein (TARDBP) of 43 kDa (TDP-43), has nuclear clearance, cytosolic sequestration/aggregation, and fragmentation in motor neurons characteristically observed in nearly 95% of sporadic ALS patients (2,3). ALS can develop both from familial (∼10% incidences) and sporadic causes (∼90% cases). Furthermore, a number of frontotemporal lobar degeneration (FTLD) patients develop ALS-like tau-negative, ubiquitin-positive inclusions of TDP-43 in cortical neurons, a subtype of motor neuron disease named FTLD-TDP (4).

TDP-43 protein, encoded by the TARDBP gene located on chromosome 1, is an RNA/DNA-binding protein of the hetero-geneous ribonucleoprotein family (5). Structurally, TDP-43 is composed of N-terminal domains comprising a bipartite nuclear localization sequence (NLS), two distinct RNA recognition motifs (RRM), namely RRM1 and RRM2, and a bipartite nuclear export sequence (NES). The C-terminal disordered domain comprising a prion-like motif is the primary contributor to its aggregation propensity (6). Since its first implication in ALS and FTLD (3,(7)(8)(9), the involvement of TDP-43 in mRNA processing and microRNA biogenesis has been well documented (reviewed in ref. 1). In addition, TDP-43 may act as a structural component in stress granule formation and in regulation of neurite growth (10). Studies have linked TDP-43 toxicity to other cellular pathways, including autophagy, loss of synaptic transmission, inflammation, and microglia infiltration, and their involvement in motor neuron death in both familial and sporadic forms of ALS (11,12). However, none of these processes exclusively drive the motor neurons to death, nor is their intervention sufficient to rescue degenerating neurons. Hence, further investigation is warranted to identify other functions of TDP-43 responsible for survival of motor neurons.

Significance

Amyotrophic lateral sclerosis (ALS) is a devastating, motor neuron degenerative disease without any cure to date. About 95% of ALS patients feature abnormalities in the RNA/DNA binding protein TDP-43, involving its nucleus-cytoplasmic mislocalization in spinal motor neurons. How TDP-43 pathology triggers neuronal apoptosis remains unclear. Here, we report that TDP-43 participates in the DNA damage response and its nuclear clearance in motor neurons causes DNA double-strand break repair defects in ALS. Our findings uncover a link between TDP-43 pathology and impaired DNA repair, and suggest potential avenues for DNA repair-targeted therapies for TDP-43-ALS.

In addition to its RNA-binding activity, TDP-43 also binds to DNA (13,14); however, its possible role in DNA transactions have not been investigated. Furthermore, significant accumulation of genomic damage is consistently observed in multiple neurodegenerative diseases and a previous proteomic study identified a key DNA repair protein "Ku" in the TDP-43 immunoprecipitation (IP) complex from human cells (15). This raised the unexplored possibility of TDP-43's involvement in DNA damage response (DDR).

Here, we have documented TDP-43's involvement in DDR as a key component of nonhomologous end joining (NHEJ), the major pathway for repair of DNA double-strand breaks (DSBs) in the postmitotic neurons. Our results showed significant DSB accumulation and reduced NHEJ levels in TDP-43-depleted human neural stem cell-derived motor neurons, as well as in sporadic ALS patients' spinal cord specimens with TDP-43 pathology. The NHEJ defects were due to reduced recruitment of X-ray repair crosscomplementing protein 4 (XRCC4)-like factor (XLF)-DNA ligase 4 (Lig4) complex at DSB sites, which is critical for break end ligation for NHEJ. Consistently, loss of TDP-43 correlated with reduced Lig4 activity. These observations, together with enhanced activation of the DDR factors, are consistent with the role of TDP-43 in DDR. Defective genome repair, the resulting persistent accumulation of unrepaired DSBs and sustained DDR activation, thus contribute to neuronal death in ALS and other TDP-43-associated neurodegenerative diseases. Our study thus provides a paradigm about the mechanism of TDP-43 toxicity, which may help develop DNA repair-targeted therapeutic approaches for ameliorating a broad range of motor neuron diseases involving TDP-43 pathology.

Results

TDP-43 Is a Component of the DDR Signaling for DSB Repair. Following up on a proteomic analysis by Taylor and coworkers (15), which showed the NHEJ-initiating DSB sensor protein Ku70 as an interacting partner of TDP-43 in mammalian cells, we confirmed the in cell association of TDP-43 with Ku70 by co-IP and in situ proximity ligation assay (PLA), using both endogenous TDP-43 and ectopic FLAG-TDP-43. These in cell studies were performed in three neuronal lines, namely differentiated neuroblastoma SH-SY5Y cell, human induced pluripotent stem cell (iPSC)-or fetal stem cell-derived neural progenitor (NP) cells (NPCs), as well as differentiated motor neurons. Human motor neurons were generated from a genetically unmodified human fetal neural stem cell (hNSC, K048) line (Fig. 1A and SI Appendix, Fig. S1B ) (16) and from an iPSC (KYOU-DXR0109B) line (SI Appendix, Fig. S1 C-F ) (17). Culture conditions and growth factors were optimized for generating up to ∼80% efficiency of differentiation into motor neurons. Motor neurons were used in most experiments that required <10,000 cells, including in cell studies by microscopy, comet, or PCR-based genome damage analyses. However, iPSC-derived NP cells (18) were used for co-IP studies, unless otherwise indicated. In view of Ku's high affinity for DNA, we eliminated the possibility of DNA-mediated protein-protein interactions by pretreating cell extracts with benzonase (DNase+ RNase) for 30 min at 37 °C, before performing co-IP assays. FLAG co-IP from total cell extracts of NP or differentiated SH-SY5Y cells (19,20) (SI Appendix, Fig. S1 A-E ) ectopically expressing FLAG-TDP-43, FLAG-Ku70, or empty FLAG-tag vector after treating with DSB-inducing topoisomerase II inhibitor etoposide or DMSO control, revealed strong interaction between TDP-43 and Ku70 (Fig. 1 B and C ). Induction of DNA damage significantly (>threefold) enhanced the interaction. The presence of phospho-(S139)-histone H2AX (γH2AX) in the co-IP complex of TDP-43 and Ku70 suggests the interaction of TDP-43 with Ku70 at the chromatin level. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) forms the DNA-PK holoenzyme after binding to Ku70/80 heterodimer, a key early response factor in NHEJ (21,22). PLA analysis in irradiated (IR, 3 Gy) NPCs, fixed at 30-min post-IR, showed strong interaction foci between TDP-43 and Ku70 or DNA-PKcs, compared with the control cells (SI Appendix, Fig. S2 A and B ). TDP-43 similarly showed enhanced interaction with p53 binding protein 1 (53BP1), via its DDR-linked phospho-(S1778)-53BP1 (23), in etoposide-treated cells as showed by endogenous 53BP1 co-IP using anti-53BP1 antibody (Ab) or normal rabbit IgG (SI Appendix, Fig. S2 C and D ). These data suggest the involvement of TDP-43 in early DSB response mechanisms.

We next examined the association of TDP-43 with key components of DDR signaling and the NHEJ pathway. Endogenous TDP-43 co-IP from nuclear extracts of the iPSC-derived NPC line (Fig. 1 D and E) treated with etoposide contained Ku70, p-(S1778)-53BP1, XRCC4/Lig4 complex, and DNA polymerase (Pol) λ, at ∼6to 10fold higher levels compared with that of control cells. Notably, the XRCC1/Lig3 complex, primarily involved in DNA single-strand break (SSB) repair (24), was not detected in the TDP-43 co-IP (Fig. 1F and SI Appendix, Fig. S2E ), suggesting specific involvement of TDP-43 in NHEJ-mediated DSB repair. Furthermore, NHEJ-associated X family DNA Pols (25,26), Polμ and Polλ, were present in the TDP-43 co-IP complex at a basal level; however, DNA damage-dependent enhanced association with TDP-43 was observed only for Polλ. Similar interaction patterns were observed in TDP-43 co-IP from chromatin extracts of differentiated SH-SY5Y cells (SI Appendix, Fig. S2 F and G ). In parallel, these interactions were confirmed by PLA in hNSC-derived motor neurons treated with etoposide or DMSO control (Fig. 1 G and H ).

To evaluate DNA damage dose-dependent enhancement of TDP-43's association with NHEJ and DDR factors, we isolated endogenous TDP-43 IP from nuclear extracts of NP cells after treatment with varying doses of etoposide (0.5-10 μM) (SI Appendix, Fig. S2 H and I ). The immunoblot (IB) analysis showed etoposide dose-dependent increase in the levels of repair factors Ku70, XRCC4, Lig4, and Polλ, together with DDR factors γH2AX, p53BP1, and p-(S1981)-ataxia-telangiectasia mutated (ATM). Moreover, another DSB-inducing radiomimetic drug bleomycin also induced TDP-43's association with γH2AX (SI Appendix, Fig. S5 ), indicating the ubiquitous nature of the interaction in response to DNA damage.

The kinetics of association and dissociation of TDP-43 with DDR factors further confirm its early recruitment and sustained retention/association as part of the DSB repair machinery, and its dissociation closely follows the time course of repair completion. SI Appendix, Fig. S3 H and I show the PLA of TDP-43 vs. Ku70 or p53BP1 at the indicated time intervals following etoposide treatment in NSC-derived motor neurons. The quantitation of average PLA signals showed that the association of TDP-43 with Ku70 and p53BP1 peaked at 5 min after etoposide treatment, before gradually dissociating by 1 h (SI Appendix, Fig. S3I ). Furthermore, IB analysis of endogenous TDP-43 co-IP from IR (6 Gy) SH-SY5Y cells showed similar association/dissociation patterns. IB of the input nuclear extracts confirmed activation of γH2AX and p53BP1 post-IR, peaking at 1-5 min before gradually disappearing with time (SI Appendix, Fig. S3A ). The level of Ku70 or TDP-43 was unaffected in the input, as expected. IB of co-IP eluates demonstrated a similar dynamic association of Ku70, p53BP1, and γH2AX with TDP-43 (SI Appendix, Fig. S3 B-D ). Notably, TDP-43's interaction with Ku70 and p53BP1 increased significantly, immediately (∼1 min) after DSB induction, followed by gradual dissociation to almost basal level of interaction at ∼2 h (SI Appendix, Fig. S3C ). The DDR and NHEJ factors identified in this study that interact with TDP-43 are schematically listed in SI Appendix, Fig. S3G .

TDP-43 Is Rapidly Recruited at DSB Sites and Retained Until Completion of Repair. We next evaluated binding of TDP-43 to DSB ends in chromatin by the damaged DNA IP assay (dDIP) (Fig. 2 A-C ). The bleomycin-treated cells were fixed with 4% paraformaldehyde and subjected to chromatin IP (ChIP) (25) with either anti-TDP-43 or anti-biotin Ab and control IgG. Binding of TDP-43 to chromatin was first confirmed by fold enrichment of two randomly selected housekeeping genes, GAPDH and β-actin, in control vs. treated TDP-43 ChIP eluates (Fig. 2B ). First, ChIP analysis using anti-biotin Ab, followed by a re-ChIP with anti-TDP-43 Ab showed enrichment of the same gene segments (Fig. 2C ), suggesting TDP-43's binding to the DSB ends. A similar dDIP analysis was performed in unstressed cells as control, where we did not observe a significant association of anti-biotin or anti-TDP-43 Ab with the selected gene segments in control cells (SI Appendix, Fig. S4A ).

To analyze the specificity and global nature of TDP-43's recruitment at the DSB sites generated either by genome-wide DSB induction with etoposide or at defined I-SceI endonuclease cleavage sites, we performed ChIP with anti-γH2AX Ab or mouse IgG, followed by re-ChIP with anti-TDP-43 Ab (Fig. 2 D-F ). For the ChIP assay, chromatin fragmentation was optimized to obtain 250-650 bp in sizes by sonication (SI Appendix, Fig. S4B ). For the first experiment, to test TDP-43's enrichment at etoposide-induced DSBs, cells were transfected with control or TDP-43 siRNA, 72 h before damage induction with etoposide (Fig. 2 D and E ). The first ChIP with anti-γH2AX Ab showed ∼threefold enrichment at a randomly selected HPRT gene segment, over corresponding IgG, which further increased by ∼twofold in TDP-43 knockdown (KD) cells (Fig. 2D ). The re-ChIP with anti-TDP-43 Ab showed a marked increase (∼15fold) in enrichment at the amplified HPRT gene segment, in-dicating TDP-43's association at etoposide-induced DSB sites. Significantly reduced amplification in TDP-43 KD cells confirmed the specificity of anti-TDP-43 Ab (Fig. 2E ). For the second experiment to test TDP-43's enrichment at defined DSBs, we used SH-SY5Y cells stably transfected with an I-SceI recognition sequence containing vector (pimEJ5GFP). ChIP was performed at 6 h posttransfection with or without the I-SceI expression vector (pCBASceI), with anti-TDP-43 Ab or control IgG as before. Amplification of DNA sequence adjacent to the I-SceI site demonstrated ∼10-fold enrichment of TDP-43 (Fig. 2F ). Together, these results suggest that TDP-43 is enriched in proximity of DNA DSBs following damage induction in the chromatin.

To gain further insight into the recruitment of TDP-43 to damaged chromatin, we analyzed repair kinetics after laser micro-IR (26) in NPCs transfected with either tdTomato reporter-tagged TDP-43 or EGFP reporter-tagged Ku70 vector constructs (Fig. 2 G and H). Given that Ku70, the key component of DNA-PK holoenzyme, is recruited at the DSB site very early, Ku70 recruitment at micro-IR-mediated chromatin damage served as a positive control (27). GFP-Ku70 was recruited at substantially high intensity within a minute at the laser track, followed by its gradual dissociation. Similarly, TDP-43-tdTomato was recruited at the laser track within 1 min (the earliest time point measured in this assay) with strong intensity, which was sustained for ∼15 min before a gradual decline. Furthermore, PLA of anti-TDP-43 vs. early DDR marker anti-γH2AX or anti-pATM Ab after IR (3 Gy) in iPSC-derived motor neurons was consistent with this scenario (Fig. 2 I and J ). The PLA foci analysis show the number of foci peaked within 1-5 min before gradually disappearing by 3 h (SI Appendix, Fig. S5B ).

To further evaluate the ability of TDP-43 to bind directly to the DSB ends, we performed an in vitro biotin-affinity pull-down assay (28) using purified recombinant TDP-43 (SI Appendix, Fig. S4C ) with three distinct DNA oligos mimicking a DSB, SSB, or intact duplex. The oligos were either 5′-biotinylated with one open blunt-end terminus (DSB) or both 5′-and 3′-biotinylated (intact duplex) or both ends biotinylated with an internal SSB nick. IB analysis of the biotin-affinity coeluated products against anti-TDP-43 Ab showed that TDP-43 binding required an unblocked DSB-like blunt terminus in DNA (SI Appendix, Fig. S4C ). Collectively, these results suggest TDP-43's direct binding to DSB ends, both in vitro and in chromatin. Moreover, the early recruitment of TDP-43 at DSB sites and its sustained presence until the repair completion suggest a vital role of TDP-43 in DSB repair/DDR signaling.

Loss of TDP-43 Causes Accumulation of DSBs in Neuronal Genome.

Based on TDP-43's cross-talk with DDR and NHEJ factors, we hypothesized that nuclear loss or functional inactivation of TDP-43 could cause unrepaired DSB accumulation. To test this, we first optimized TDP-43 KD in iPSC-derived NP cells using GFPtagged lentiviral shRNA transduction, which showed 80-90% uptake in neurons and ∼80% depletion of TDP-43 (SI Appendix, Fig. S6A ). Alkaline and neutral comet analyses of TDP-43 KD cells, 96 h post-shRNA transduction, showed ∼20-fold increase in DNA strand breaks compared with the control cells (SI Appendix, Fig. S6B ). Neutral comet analysis reflects DSBs exclusively, whereas alkaline comet analysis could be used to quantitate DSBs, SSBs, and other alkali-labile apurinic/apyrimidinic (AP) sites (29,30). Comparable increase in the mean alkaline vs. neutral comet tail moment in TDP-43 KD cells suggests that a majority of these breaks are DSBs. Furthermore, significant increase in the number of foci of γH2AX, p53BP1 and pATM at 96 h after TDP-43 KD, confirmed the accumulation of endogenous DSBs and DDR activation in neurons (SI Appendix, Fig. S6C ). The TUNEL assay at indicated time-points also suggested accumulation of DNA strand breaks (SI Appendix, Fig. S6D ). Consistent with our results in neurons, we observed similar accumulation of DSBs in TDP-43 siRNA-treated differentiated SH-SY5Y cells, as analyzed by the comet assay (SI Appendix, Fig. S6 E-G ), long-amplicon (LA) PCR-based amplification of isolated genomic DNA (SI Appendix, Fig. S6H ), and γH2AX foci accumulation (SI Appendix, Fig. S6I ).

DNA Damage and Activation of DDR Signaling After Conditional TDP-43 Knockout by Inducible CRISPR/Cas9 Technique. Given that TARDBP gene knockout (KO) is lethal to the neurons (31), we developed doxycycline (Dox)-inducible CRISPR/Cas9 (32) -based TDP-43 KO in an SH-SY5Y line (iCRISPR-TDP-43-KO-SH-SY5Y) to investigate the effect of dose-dependent depletion of TDP-43 in neurons. The iCRISPR/Cas9 strategy is schematically shown in Fig. 3A . iCRISPR-TDP-43-KO-SH-SY5Y cells were first differentiated with retinoic acid (10 μM) for 4 d (SI Appendix, Fig. S1A ) and then induced with Dox (5 μg/mL). The TDP-43 level was measured at 0, 2, 4, 6, and 8 d of induction. Complete TDP-43 KO was observed in 8 d (SI Appendix, Fig. S7 A and B ); however, the cells were detached from the petri dish and appeared nonviable by then. This was perhaps expected, because TDP-43 is essential for survival. On the other hand, ∼30%, 50%, and 75% depletion of TDP-43 was observed in viable cells after 2, 4, and 6 d of Dox induction, respectively (SI Appendix, Fig. S7 D and E ). IB of total extracts of iCRISPR-TDP-43-KO-SH-SY5Y cells showed activation of ATM as reflected in pATM level, as well as the formation of apoptosis markers cleaved poly-ADP ribose polymerase (PARP) 1 and cleaved caspase-3 (Fig. 3B ). Gradual increase in apoptotic cells after TDP-43 depletion was confirmed by FACS analysis using Annexin V/propidium iodide (PI) staining (SI Appendix, Fig. S7 D and E ) and by MTT assay (SI Appendix, Fig. S7F ). Neutral comet analysis showed presence of DSBs, the extent of which correlated with reduction in the TDP-43 level (Fig. 3C ). Similarly, LA-PCR analysis (33) showed a decrease in DNA integrity proportionate to TDP-43 level (Fig. 3D ). Notably, genome damage data on day 2 of Dox induction showed a marked increase in DSBs together with ATM activation (Fig. 3B and SI Appendix, Fig. S7C ), without a significant increase in apoptotic cell population, suggesting that DSB damage likely precedes cell death. Collectively, these studies demonstrate that conditional TDP-43 depletion is strongly correlated with unrepaired DSB accumulation, sustained DDR activation, and subsequently apoptotic cell death in neurons and, thus, underscores the essential role of TDP-43 in maintaining their genomic integrity.

TDP-43 Is Required for Optimal DSB Repair via NHEJ in Neurons. We next investigated whether TDP-43 depletion affected DSB repair via the NHEJ pathway in neurons. Comet analysis to monitor DSB repair kinetics showed significantly delayed repair in TDP-43 siRNA-transfected NP cells compared with control siRNAtreated cells, after exposure to bleomycin (Fig. 4 A and B ). While control cells showed nearly complete recovery from the exogenous genome damage by 6 h, ∼10-fold higher mean comet tail moment persisted in TDP-43 KD cells, indicating substantial reduction in DSB repair efficiency due to loss of TDP-43. Similar delay in DSB repair was reflected in the slower disappearance of 53BP1 foci in TDP-43 KD cells relative to control after bleomycin treatment (SI Appendix, Fig. S8A ). Finally, cell viability analysis by MTT or clonogenic survival assay indicated that TDP-43 depletion acted synergistically with IR or bleomycin to reduce cell viability (≥sixfold) (SI Appendix, Fig. S8 B and F ).

The delayed DSB repair of both intrinsic and induced DSBs in TDP-43 KD cells, together with the association of TDP-43 with NHEJ factors, suggest a function of TDP-43 in NHEJ, which we explored using an I-SceI plasmid-based NHEJ reporter assay (34). In this approach, neuronal cells were stably transfected with NHEJ reporter plasmid pimEJ5GFP harboring PGK-Puromycin cassette within the two I-SceI recognition sites. I-SceI sites are located between the promoter and GFP reporter coding sequence (schematically shown in Fig. 4C ) (35). Puromycin-resistant cells were sequentially treated with either control or TDP-43 siRNA for 72 h followed by pCBASceI transfection to introduce DSB (36). NHEJ-mediated error-free repair of I-SceI sites after break induction would result in expression of GFP, and therefore the percentage of GFP + cells would provide a relative measure of NHEJ (37). Notably, TDP-43 KD (∼90%) significantly reduced the percentage of GFP-expressing cells compared with control cells (Fig. 4 D and E ). A similar experiment in cycling HEK293 cells showed significantly reduced level of GFP expression in TDP-43 KD cells compared with that of control by FACS analysis as well as immunfluoresence (SI Appendix, Fig. S8 C and D ). DNA-PKcs inhibitor NU7441 served as positive control for NHEJ inhibition. Taken together, these results suggest that TDP-43 depletion causes reduction in NHEJ-mediated DSB repair proficiency. Furthermore, as described in SI Appendix, SI Results and Fig. S8 G-J, TDP-43 KD induced DSB repair defects were confirmed using a second shuttle vector-based approach (38).

Caenorhabditis elegans Mutant Lacking TDP-1 Is Sensitive to DNA Damage due to Defective NHEJ. To establish that involvement of TDP-43 in maintenance of genomic integrity is universal among all metazoans, we investigated whether TDP-1, the TDP-43 homolog in C. elegans, participated in DSB repair. Human TDP-43 and nematode TDP-1 are functional orthologs with similar RNA binding activities and have a conserved N terminal, NLS, and RRMs (Fig. 5A ) (39,40). We utilized standard laboratory strains N2 (Bristol) as WT and an endogenous knockin Tdp-1(ok803) loss-of-function strain (TDP-1ΔCTD; CK501) with deletion of C-terminal 299-aa residues lacking the NLS and the two RRMs (41). Sensitivities of WT or TDP-1ΔCTD C. elegans to genotoxic DSBs were assayed by evaluating numbers of viable embryos following ionizing radiation exposure. Stage-matched day 1 adult worms were irradiated for 10 min at 390 rad/min (total exposure of 40 Gy). IR C. elegans were allowed to lay eggs for 4 h, and the embryos were scored 24 h later for survival analysis by counting live worms vs. dead eggs. Data showed ∼25% increased lethality in CK501 compared with N2 after IR (Fig. 5B ). Total worm extracts (42,43) were then incubated with linearized plasmid substrate to analyze the plasmid recircularization efficiency via a colony formation assay (44). Mutant extract showed significantly reduced recircularization efficiency compared with N2 worms (Fig. 5C ), indicating impaired DSB repair. To further compare genome integrity in N2 vs. CK501 worms, genomic DNA was isolated from control or etoposide-treated worms and separated in 1% agarose gel electrophoresis and band intensity of intact DNA was quantified (SI Appendix, Fig. S9 A and B ). For this assay, etoposide treatment was continued for 24 h, and worms were either harvested immediately as 0-h recovery or allowed to recover for 24 h. Untreated mutant worms showed increased smear compared with WT, indicating the presence of higher basal level DNA damage. While the WT worms recovered from etoposide-induced DNA damage, CK501 worms showed persistent genome damage (indicated by smear and reduction in intact band intensity). Similarly, LA-PCR analysis from these C. elegans genomic DNA templates (45) consistently showed failure of mutant C. elegans to recover from etoposide-induced DNA damage, as measured by picogreen-based quantitation of PCR amplified products, suggesting defective DSB repair (Fig. 5D and SI Appendix, Fig. S9C ). Together, these studies establish the direct linkage between loss of TDP-1 function and the DSB repair defect in C. elegans. S1 and S2 . As shown in representative immunohistochemistry (IHC) images, the typical pathology of increased extranuclear TDP-43 was confirmed in spinal cord sections (cervical region) of both ALS-Guam and ALS-VA cases, and this was absent in appropriately matched controls (Fig. 6A , Left). Importantly, ALS spinal cord showed significantly higher staining for γH2AX and TUNEL compared with matched control spinal cord sections (Fig. 6A , Right). Fig. 6 B and C show the quantitation of γH2AX and TUNEL IHC signals per field from 10 sporadic ALS-VA and 5 ALS-Guam cases in comparison with matched controls. The ALS spinal cord also showed strong Thioflavin S + reactivity, indicating the presence of protein aggregates (SI Appendix, Fig. S10A ). Furthermore, genomic DNA isolated from both ALS-Guam and ALS-VA spinal cord tissue showed significantly reduced integrity due to the presence of strand breaks, as analyzed by LA-PCR (Fig. 6E and SI Appendix, Fig. S10 B-D ).

Given that sporadic ALS-TDP-43 proteinopathy may involve both TDP-43 aggregation and fragmentation, the status of the TDP-43 protein was analyzed by IB in the ALS-VA spinal cord (cervical) and matched controls. Notably, all of 10 ALS samples showed reduced TDP-43 monomer levels compared with controls (Fig. 6D and SI Appendix, Fig. S10H ) as well as significant increase in ubiquitinated proteins in sporadic ALS cases compared with the controls (SI Appendix, Fig. S11 ), consistent with the previous observation (3). In addition, the ALS samples also showed characteristic IB patterns representing TDP-43 aggregates/oligomeric forms and fragmentation, which were present at a negligible level in the controls. Furthermore, the presence of TDP-43 fragmentation and its aggregation in ALS samples were strongly correlated with higher levels of γH2AX and p53BP1 accumulation, together with the presence of cleaved PARP-1 and cleaved caspase-3, compared with their negligible level in controls (Fig. 6D ; quantitation of relative IB band intensity in SI Appendix, Fig. S10 E-G ), suggesting a link between accumulated DNA damage and the apoptotic death of affected spinal cord neurons.

To assess the linkage of TDP-43 pathology and genome damage accumulation in ALS spinal cord with DSB repair defects, we performed a plasmid recircularization assay, as an in vitro surrogate NHEJ assay with extracts from control and ALS spinal cord tissue. Four controls and 10 ALS-VA samples (SI Appendix, Table S1 ) were grouped into two sets each: control-I (containing control #1 and #2) and control-II (containing control #3 and #4); ALS-I (containing ALS#1-5) and ALS-II (containing ALS# 6-10). The grouped extracts were homogenously mixed with same amount of total protein from each sample. Both the ALS groups showed significant loss of monomeric TDP-43; as shown in Fig. 6D , the plasmid recircularization efficiency reduced about 50% in ALS tissue, suggesting defective DSB ligation (SI Appendix, Fig. S10I ). These data provide strong correlation of TDP-43 pathology, NHEJ defects, DSB damage, and neurodegeneration in ALS pathology.

TDP-43 Acts as a Scaffold in Recruiting XRCC4-Lig4 Complex at DSB for Efficient DNA Ligation in NHEJ. To gain molecular insights into role of TDP-43 in DSB repair, we examined recruitment of key NHEJ factors at damage sites, by testing their presence in γH2AX or 53BP1 co-IPs from nuclear extracts of NPCs first transfected with control or TDP-43 siRNA, following DSB induction with etoposide. IB of endogenous co-IPs showed substantially reduced association of both γH2AX (Fig. 7A and SI Appendix, Fig. S12A ) and 53BP1 (SI Appendix, Fig. S12 C and D ) with XRCC4, Lig4, as well as XLF, key factors in the NHEJ ligation complex after TDP-43 KD. It is important to note that unlike the XRCC4-Lig4-XLF complex, the association of Ku, pATM, and p53BP1 increased with γH2AX formation in absence of TDP-43, suggesting that TDP-43 primarily works downstream of 53BP1 in the NHEJ pathway. To further confirm reduced association of XRCC4 with DSB markers, we similarly performed FLAG-XRCC4 co-IP, which showed significantly reduced interactions with γH2AX and p53BP1 due to TDP-43 depletion (Fig. 7B and SI Appendix, Fig. S12B ). Levels of XLF and Lig4 in FLAG-XRCC4 co-IP remained unaffected, indicating TDP-43's role in recruitment of the NHEJ ligation complex, but may not on the complex formation itself. Defective in cell association of XRCC4 and Lig4 at DSB sites was further confirmed by a complementary PLA experiment between anti-53BP1 and anti-Lig4 or anti-XRCC4 Ab in NPCs treated with control or TDP-43 siRNA for 72 h (Fig. 7C and SI Appendix, Fig. S12G ), revealing ∼10-fold reduction in PLA signal due to TDP-43 down-regulation.

We next investigated whether reduced in cell interaction of DSB markers with the XRCC4/Lig4 complex is correlated in their actual recruitment at DSB sites, by ChIP/re-ChIP analyses.

A first ChIP with control IgG and anti-γH2AX Ab (as in Fig. 2D ) followed by re-ChIP with anti-XRCC4 Ab (SI Appendix, Fig. S12E ) or anti-53BP1 Ab (SI Appendix, Fig. S12F ) and respective control IgG, was performed in etoposide-treated NPC with or without TDP-43 KD. Quantitative PCR amplification of HPRT gene segment from ChIP products demonstrated a significantly reduced enrichment of XRCC4 after TDP-43 depletion. The enrichment of 53BP1 was increased after etoposide treatment, as expected, with an additional ∼fivefold increase after TDP-43 KD, consistent with co-IP results, suggesting enhanced accumulation of unrepaired DSBs. A similar re-ChIP analysis at defined I-SceI cleavage sites also confirmed defective recruitment of XRCC4 in the proximity of DSBs due to loss of TDP-43 (Fig. 7D ). Taken together, the ChIP data demonstrate that TDP-43 is required for optimal recruitment of the XRCC4-Lig4 complex at both genome-wide DSBs and I-SceI-induced defined break sites.

We next tested whether the reduced recruitment of XRCC4-Lig4 at DSBs in the absence of TDP-43 resulted in impaired DNA DSB ligation activity. For this experiment, endogenous XRCC4 IP complexes were isolated from etoposide-treated NPCs with or without TDP-43 KD by siRNA. IP eluates were used to test DNA ligation activity by two approaches: either using a 5′Cy3-labeled nicked duplex oligonucleotide substrate (Fig. 7E ) or a plasmid recircularization assay (SI Appendix, Fig. S12H ). Both assays showed significantly (∼four-to sixfold) reduced DNA ligation activity with the XRCC4 IP complex after TDP-43 KD, which was mostly rescued by the addition of recombinant TDP-43 to reaction mix. Absence of other DNA ligases, namely DNA Ligase1 (Lig1) and DNA Lig3, in XRCC4 co-IP complexes was confirmed by IB with respective Ab (SI Appendix, Fig. S13B ). Consistently, recombinant TDP-43 showed direct interaction with purified XRCC4-Lig4 complex in the absence of DNA (SI Appendix, Fig. S13A ). These data not only reveal a scaffolding activity of TDP-43 for recruiting the NHEJ ligation complex (schematically represented in SI Appendix, Fig. S12I ), but also provided a direct linkage of TDP-43 depletion and DSB ligation defects in the NHEJ pathway.

In summary, our data reveal that TDP-43 is a critical component of genomic DSB repair and the DDR signaling. Thus, TDP-43 pathology leading to its loss of functions in the ALS spinal cord neurons, impairs DSB repair by inhibiting NHEJ, leading to persistent accumulation of damage and sustained activation of DDR signaling, which contribute to neuronal cell death by apoptosis (Fig. 7F ).

Discussion

Since the discovery of TDP-43 toxicity in ALS in 2006 (3,7), research has mainly focused on the cytoplasmic aggregation of TDP-43 and disruption of its RNA-binding functions (46)(47)(48)(49)(50)(51). TDP-43, whose nuclear clearance is a hallmark of degenerating motor neurons in ALS, has also been shown to have the ability to bind to DNA (13,14). This makes TDP-43 a member of the distinct subgroup of RNA-binding proteins with functional DNA-binding activity, which we name RNA/DNA-binding proteins (RDBPs). However, the physiological functions/relevance of the DNA-binding activity of TDP-43, and the eventual loss of this function in disease-affected neurons, have not been previously investigated. Evidence for the presence of DNA damage in TDP-43 toxicity-linked neurodegenerative diseases (52,53), and identification of the DNA repair protein Ku in the TDP-43 interactome in human cells (15), provided the premise to investigate the role of TDP-43 in DDR in this study.

We have identified TDP-43 as an integral component of early DDR, which is activated for repair of DNA DSBs via NHEJ. TDP-43 showed strong in cell association with key DDR marker proteins γH2AX, pATM, and p53BP1 in a DNA damage dose-and timedependent manner. We induced DSBs using IR, radiomimetic bleomycin, or a DNA topoisomerase II inhibitor etoposide, all of which consistently showed TDP-43's association with DSB repair proteins and its recruitment at damage sites. Furthermore, laser ablation/live cell imaging revealed that TDP-43 was recruited at DSB sites as early as 1 min, together with Ku70, after damage induction and persisted at DSB until completion of repair. We confirmed recruitment of TDP-43 at DSBs using multiple complementary approaches including dDIP, ChIP/re-ChIP, and PLA with DSB markers, all of which consistently showed strong association of TDP-43 at DSBs. Importantly, TDP-43 was able to directly bind DNA oligonucleotide containing a DSB-mimicking blunt end in vitro. The DDR in mammalian cells is a complex and highly orchestrated signaling process. While there are minor variations in the proposed early events following DNA DSB damage, ATM together with the Mre11/Rad50/NBS1 complex is the first responder. Activated ATM (autophosphorylation at serine 1981) phosphorylates the H2AX bound to the DSB site, which then facilitates recruitment of other DDR proteins, including 53BP1, to the vicinity of the DNA break (54). Repair of DSBs can occur via one of three subpathways, two of which are homologous recombination in S/G2 cells, and NHEJ in all cells including postmitotic neurons. The third subpathway is via error-prone microhomology-dependent alternative end-joining and primarily detectable in immune cells for Ab diversification and in some drug-resistant tumors, and has not been shown to significantly contribute to DSB repair in neurons. NHEJ involves the DNA-PK holoenzyme (DNA-PKcs/Ku70/Ku80), a DNA polymerase (μ or λ), and XRCC4/DNA Lig4 (55,56).

Consistent with TDP-43's recruitment at DSBs and its stable in cell association with NHEJ factors, si/shRNA, or CRISPR/Cas9mediated loss of TDP-43 in multiple neuronal cell models, including human iPSC or NSC-derived motor neurons and differentiated SH-SY5Y and HEK293 cells, resulted in a marked increase in levels of unrepaired DSBs in the genome and activation of DDR signaling, even in unstressed conditions. It is important to mention that motor neuron differentiation from hNSCs was optimized to obtain up to ∼80% efficiency (SI Appendix, Fig. S1 C-F ) (17,18).

To gain molecular insight into TDP-43's role in specific DSB repair reactions, we tested the recruitment of key NHEJ factors by co-IP, ChIP, and PLA analyses. The data clearly demonstrated that loss of TDP-43 causes a strong inhibition in recruitment of XRCC4, Lig4, and XLF, three key components for DSB ligation in the NHEJ pathway. Because levels of these proteins were unchanged after TDP-43 KD, our findings suggested the involvement of defective protein-protein or protein-DNA interactions during NHEJ repair. The recruitment of Ku or 53BP1 was not inhibited in the absence of TDP-43, which suggested that TDP-43 might work as downstream factor of 53BP1 in NHEJ pathway, likely acting as a scaffold for recruiting/stabilizing the ligation complex. Furthermore, substantial impairment of DSB ligation activity in the XRCC4 immunocomplex isolated from neuronal cells lacking TDP-43, showed that reduced recruitment of the NHEJ ligation complex in the absence of TDP-43 indeed resulted in DNA endbreak ligation defect. As shown in SI Appendix, Fig. S13B , the XRCC4 co-IP contained Lig4 but not other nuclear DNA ligases (Lig3 or Lig1), thus reduced ligation activity of XRCC4 co-IP after TDP-43 KD could be directly attributed to Lig4 defect, the only DNA ligase involved in NHEJ. It is important to note that the level of XRCC4 and Lig4 were not affected in TDP-43-depleted cells, suggesting a direct role of TDP-43 in regulating the Lig4 function rather than via RNA processing. Our observation of TDP-43's pairwise interaction with the XRCC4/Lig4 complex in vitro supports this scenario. We recently reported the role of FUS in regulating recruitment and functions of the XRCC1/Lig3 complex in DNA SSB repair (57). Together these studies suggest a scaffolding role of RDBPs in DNA break sealing, likely critical for stabilizing the DNA termini at break sites.

The data in cultured motor neurons linking loss of TDP-43 to genomic DSB accumulation were further recapitulated in two in vivo model systems, including C. elegans lacking functional TDP-1 and human ALS patients' spinal cord tissue. Unlike in human neurons, though, hemizygous expression of functionally dead, truncated TDP-1 causes DNA damage accumulation but does not show motor phenotype in worms; however, it does make them highly sensitive to exogenously induced DSBs (γ-IR or etoposide treatment). Reduced plasmid recircularization in extracts of C. elegans lacking TDP-1, together with their increased lethality in response to DSB stress, provide strong in vivo evidence for the involvement of TDP-43 in DSB repair.

Here, we have provided evidence for the presence of unrepaired DSB damage and activation of DDR factors associated with clearance of nuclear TDP-43 in the spinal cord tissue of sporadic ALS patients. In addition, plasmid recircularization assay using spinal cord tissue extracts showed ∼70% reduction in DSB ligation in ALS compared with controls. Higher ALS predisposition has been reported in military veterans, perhaps due to potential exposure to lead metal complexes and other toxicants used in warfare combined with stress and other unknown factors; on the other hand, a significantly higher incidence of ALS in Guam residents was attributed to the consumption of unnatural amino acid β-methylamino-L-alanine through cycad products in the diet together with metal toxicity (58)(59)(60). TDP-43 pathology has also previously been reported in a majority of ALS-Guam patients (61,62), and this was confirmed in this study. Remarkably, the extent of genome damage in the ALS spinal cord also broadly correlated with the activation of apoptotic markers. Notably, we observed the presence of both truncated and aggregated TDP-43 species in the spinal cord of sporadic ALS patients (consistent with previous reports); however, both exhibited a comparable level of genome damage. This suggested the correlation of loss of monomeric TDP-43 to genomic instability in ALS patients.

Our studies thus report a key role of TDP-43 in DDR and DSB repair and suggest potential DNA repair based therapies for TDP-43-associated neurodegeneration.

Materials and Methods

For detailed and additional methods, see SI Appendix.

hNSC Culture and Differentiation. The hNSC (63) line (K048) was cultured as neurospheres in complete medium containing appropriate growth factors (16). The normal iPSC line (ATCC #KYOU-DXR0109B) was grown in CellMatrix basement membrane gel (Gibco) and Pluripotent Stem Cell SFM XF/FF media (Gibco) at 37 °C and 5% CO 2 . For differentiation into motor neurons, see details in SI Appendix.

C. elegans. C. elegans strains N2 (Bristol) and RB929 tdp-1 (ok803) were provided by the Caenorhabditis Genetic Center (Minneapolis, MN), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), and from the National Bioresource Project (Japan).

Human ALS Tissue Specimens. Human postmortem spinal cord tissue specimens from sporadic ALS patients and age-matched controls were obtained from two bio-repositories, namely the Department of Veterans Affairs Brain Biorepository (ALS-VA) and Binghampton Biorepository Archive (ALS-Guam) (SI Appendix, Tables S1 and S2 ).

CRISPR/Cas9-Mediated TDP-43 KO. Two single-guide RNAs were designed based on the exact DNA sequence of TDP-43 coding DNA region in SH-SY5Y cells. An inducible humanized Cas9 system was used for conditional knockout of TDP-43.

PLA. In situ protein-protein association was analyzed using PLA (Duolink; Sigma) as per the manufacturer's instructions (18,64).

Live-Cell Imaging of Micro-IR Based DSB Repair. For live cell imaging of DSB repair, cells were transfected with TDP-43-tdTOMATO or Ku70-EGFP reporter plasmids. DSBs were generated in a laser ablation track in the region of interest by a 405-nm diode laser, set to 100% transmission output for 150 iterations. Following IR, cells were maintained for indicated time periods scanning the fluorescence intensity of the laser-track for 30 min with 1 min interval. Data were collected from 5 to 15 cells and measured by ImageJ and normalized to the signal of the whole nucleus (26,65).

NHEJ Assay for the Repair of DSBs. We employed two plasmid based approaches to measure NHEJ-mediated DSB repair proficiency, namely the standard I-SceI based GFP reporter assay (35) and a shuttle plasmid containing unligatable DSB termini (38).

ACKNOWLEDGMENTS. The authors thank other members of M.L.H. laboratory, S. Rangaswamy, P. Basu, and V. Bora for assistance. Control and sporadic ALS spinal cord tissue specimens were provided by the Department of Veterans Affairs Biorepository. The Guamanian ALS specimens were obtained from Binghamton Biospecimen Archive. This research was primarily supported by National Institute of Neurological Disorders and Stroke-NIH Grant R01 NS088645; Muscular Dystrophy Association Grant MDA 294842; and the Houston Methodist Research Institute (M.L.H.).

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