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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TDP-43 induces mitochondrial damage and activates the mitochondrial unfolded protein response

Peng Wang, Jianwen Deng, Jie Dong, Jianghong Liu, E. Bigio, M. Mesulam, Tao Wang, Lei Sun, Li Wang, A. Lee, Warren A. McGee, Xiaoping Chen, K. Fushimi, Li Zhu, Jane Y. Wu

PLoS Genetics·

2019·

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Specific Biological Pathways Involving TDP-43

- RNA processing - Splicing regulation - mRNA trafficking - mRNA stability regulation - Mitochondrial complex I activity suppression - Mitochondrial ATP synthesis reduction - Mitochondrial ROS production increase - Activation of mitochondrial unfolded protein response (UPRmt)

Molecular Interactions and Binding Partners of TDP-43

Interacts with LonP1

Functional Consequences of TDP-43 Interactions

- Induces mitochondrial dysfunction - Decreases mitochondrial membrane potential - Increases production of reactive oxygen species (ROS) - Suppresses mitochondrial complex I activity - Reduces mitochondrial ATP synthesis - Activates the mitochondrial unfolded protein response (UPRmt) - Leads to early mitochondrial impairment preceding cell death - Exacerbates neurodegeneration when LonP1 is down-regulated

Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPRmt) in both cellular and animal models. Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases.

Introduction

TDP-43 proteinopathy is characterized by the presence of TDP-43 immunoreactive inclusion bodies in the affected tissues. Clinically, TDP-43 proteinopathy manifests as a spectrum of different neurodegenerative diseases, ranging from dementia (especially fronto-temporal lobar degeneration, FTLD) and motor neuron disease (MND) to traumatic brain injuries [1][2][3][4]. FTLD is a prevalent form of dementia with progressive atrophy of the frontal and/or temporal cortices [5][6][7]. Amyotrophic Lateral Sclerosis (ALS), a common form of MND, is characterized by a progressive loss of upper and lower motor neurons [8][9][10]. TDP-43 associated neurodegenerative diseases are clinically and genetically heterogeneous. A significant fraction of ALS patients exhibit cognitive impairment [11,12]; and ~15% of FTLD patients also show locomotor defects and meet the diagnostic criteria for ALS [12,13]. TDP-43-positive lesions are the most frequently identified pathology among FTLD and ALS cases and also present in ~50% AD samples [14][15][16]. However, the pathogenic mechanisms underlying TDP-43 proteinopathy remain unclear.

Mitochondrial damage is associated with a range of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and MNDs [17][18][19]. Mitochondrial changes have been detected in cellular and animal models for TDP-43 proteinopathy [16,[20][21][22][23][24][25][26][27]. It was recently reported that suppressing mitochondrial localization of TDP-43 blocked TDP-43 neurotoxicity [28]. However, mitochondrial morphological changes have not yet been characterized in patient samples, and the effects of TDP-43 on mitochondrial function remain controversial [27][28][29].

To maintain mitochondrial homeostasis, cells sense and respond to mitochondrial damage by activating a program known as the mitochondrial unfolded protein response (UPR mt ), which includes induction of mitochondrial chaperones assisting in proper protein folding, and of proteases promoting clearance of misfolded proteins [30][31][32]. Recent studies suggest a role of UPR mt in Alzheimer's disease, Parkinson's disease and ALS-SOD [33][34][35]. However, the role of UPR mt in TDP-43 proteinopathy has not been reported.

Here, we present a systematic study of TDP-43 proteinopathy combining cellular and animal models with patient samples. Analyses using electron microscopy (EM) reveal prominent mitochondrial damage in brain tissues from TDP-43 proteinopathy patients. These mitochondrial impairments include swollen and degenerated cristae or a complete loss of cristae. Similar mitochondrial cristae changes are detected in our cellular and animal models. Consistently, mitochondrial functional impairments are observed, including decreased mitochondrial membrane potential, reduced mitochondrial ATP synthesis and elevated mitochondrial ROS production. Our data show that mitochondrial impairment induced by TDP-43 is an early event, preceding cell death. Furthermore, induced TDP-43 expression leads to the activation of UPR mt in both cellular and fly models for TDP-43 proteinopathy. LonP1, one of the key mitochondrial proteases in UPR mt , plays an important role in the degradation of mitochondrial TDP-43. Consistent with the mRNA changes of LonP1 in cellular and fly models, LonP1 protein levels are increased in a fraction of the brain samples of patients affected by FTLD-TDP. Importantly, down-regulation of LonP1 in TDP-43 expressing flies not only induces more severe mitochondrial damage, but also advances disease onset and exacerbates the neurodegeneration phenotype in the animal model. These results suggest that LonP1 plays a protective role against TDP-43-induced neurotoxicity, especially at an early stage of the disease. Together, our data demonstrate that mitochondrial damage is a critical feature of TDP-43 proteinopathy and suggest that protecting mitochondria may have therapeutic potential.

Mitochondrial impairment in the brain samples of TDP-43 proteinopathy patients

To investigate the role of mitochondria in TDP-43 proteinopathy, we examined mitochondrial morphology in brain samples from patients using transmission electron microscopy (TEM) and immuno-electron microscopy (IEM). Following resin-embedding to obtain clear images of mitochondria, we analyzed brain samples from five patients with the pathological diagnosis of either FTLD-TDP or ALS-FTLD-TDP, together with the samples from three control subjects without any TDP-43 pathology (for details, see S1 Table ).

The majority of mitochondria in the control brain tissues showed normal morphology, with intact mitochondrial membrane and well-organized cristae (left panels in Fig 1A ). In contrast, more than 80% of mitochondria in the patient brains exhibited significant mitochondrial damage, especially abnormal cristae structure (Fig 1 ). Abnormal mitochondrial cristae presented as either a "vesicular" type with swollen cristae (marked by the arrows in the middle panels of IEM analyses of the brain tissues using a specific anti-TDP-43 antibody revealed that TDP-43 immunostaining signals were clearly detected inside mitochondria in the brain samples of both control and FTLD-TDP patients (marked by arrows in Fig 1C ; with enlarged views in insets), demonstrating that the endogenous TDP-43 protein is localized inside mitochondria, consistent with a recent report [28]. Interestingly, electron-dense TDP-43 positive protein aggregates were detected inside 1% of mitochondria in FTLD-TDP patient samples (arrowheads in Fig 1D ), but were not detected in any control samples. These EM analyses demonstrate that mitochondrial damage is a prominent feature in the pathology of brain tissues of TDP-43 proteinopathy patients.

Mitochondrial impairment in a cellular model of TDP-43 proteinopathy

To investigate the effects of TDP-43 on mitochondrial morphology and function in living cells, we established tetracycline (Tet) inducible HEK293 cell lines, expressing either wild type (Wt) or an ALS-associated TDP-43 mutant (A315T). Following Tet-induction for 24 hr, total cell lysates, cytoplasmic fractions and purified mitochondrial preparations were examined by Western blotting. The purity of the mitochondrial preparation was confirmed by the detection of mitochondrial protein TOM20 and the absence of the cytoplasmic GAPDH protein. Consistent with the IEM data from the human brain samples, the endogenous TDP-43 as well as the exogenously expressed Wt or ALS-mutant (A315T) TDP-43 were detected in purified mitochondria (Fig 2A ; for a longer exposure, see S1A Fig) , supporting the mitochondrial localization of the TDP-43 protein. Consistent with previous studies [36,37], expression of the exogenous TDP-43 suppressed expression of the endogenous TDP-43 (marked by "Endo" in Fig 2A ).

We next performed EM analyses of HEK293 cells expressing TDP-43 to characterize mitochondrial changes. In control cells, the vast majority of mitochondria exhibited normal morphology, with well-organized cristae (Fig 2B ). However, in cells expressing the A315T-mutant TDP-43, severe mitochondrial damage was detected, with significantly reduced mitochondrial sizes and impaired mitochondrial cristae 24 hr post-induction. When Wt TDP-43 was expressed, similar mitochondrial damage was also detected, although to a lesser extent (Fig 2B ;

TDP-43 induced mitochondrial dysfunction precedes cell death

To examine the temporal relationship between TDP-43-induced mitochondrial damage and cell death, we carried out a series of experiments using the Tet-inducible cells expressing Wtor A315T-mutant TDP-43 proteins at different time points (0, 24 or 36 hr) following induction of TDP-43 expression. We first measured mitochondrial membrane potential, ROS production and ATP synthesis (Fig 2C -2E To understand the mechanism by which increased TDP-43 expression suppressed mitochondrial ATP synthesis, we examined which mitochondrial complexes (complex I through V) in oxidative phosphorylation were affected. Interestingly, complex I activity was significantly reduced by 24 These data indicate that increased TDP-43 expression impairs mitochondrial ATP synthesis, possibly by suppression of mitochondrial complex I. TDP-43-induced reduction in the complex I activity was not likely the result of overall suppression of complex I genes by TDP-43, because quantitative PCR analyses of a number of complex I genes did not show a general reduction in the expression of these genes (see S2B Fig) . Future experiments are necessary to elucidate the mechanism by which TDP-43 suppresses the activity of complex I.

To examine cell death, cells were stained with an Annexin V-FITC/PI (propidium idodide) kit followed by flow cytometry analyses (Fig 3 ). Annexin V-positive/PI-negative, Annexin V- negative/PI-positive or Annexin V-positive/PI-positive staining indicates apoptosis, necroptosis or late apoptosis/necroptosis, respectively. Up to 36 hr post-induction, Annexin V-negative/PI-positive or PI/Annexin V double-postive cell populations did not show significant changes in TDP-43 expressing cells compared to the control group. Cells expressing A315Tmutant TDP-43 showed significantly increased cell death only after 36 hr post-induction of TDP-43 expression (2% cells showing Annexin V-positive/PI-negative staining; compared with ~0.5% in the control cells); whereas cells expressing wild type TDP-43 showed a less dramatic increase in cell death, also only after 36 hr post-induction (Fig 3A, Fig 3B) . It should be noted that at this time point only a small fraction (<5%; estimated by biochemical fractionation) of the total TDP-43 was detected in purified mitochondria (possibly due to the efficient degradation of mitochondrial TDP-43 before the disruption of the balanced mitochondrial proteostasis). Because mitochondrial dysfunction was observed at the 24 hr time point, these results demonstrate that TDP-43-induced mitochondrial dysfunction is an early event preceding cell death, suggesting that mitochondrial impairment may contribute to TDP-43 cytotoxicity.

TDP-43 induces mitochondrial damage and increases mitochondrial ROS production in a transgenic fly model of TDP-43 proteinopathy

To investigate TDP-43-induced mitochondrial damage in vivo, we examined transgenic flies expressing either Wt or A315T-mutant TDP-43 reported in our previous studies [40][41][42]. Transmission EM analyses of control fly eyes in 3-day old adult animals revealed intact ommatidial structures with seven rhabdomeres, whereas expression of either Wt or A315T-mutant TDP-43 in fly eyes led to severe ommatidial defects, often with a complete loss of rhabdomeres . Importantly, more than 85% of mitochondria in the photoreceptors expressing Wt or ALS-mutant TDP-43 exhibited swollen or vesicular cristae, whereas only ~5% of mitochondria in the control group showed damage (Fig 4A and Fig 4C) . In this setting, TDP-43 was expressed in photoreceptors under a strong GMR-Gal4 driver from an early stage, leading to rapid and severe mitochondrial damage. By the time of EM examination, >85% mitochondria showed damage in both Wt and A315T-mutant groups, not allowing us to detect differences between the two groups. It is remarkable that mitochondria in fly photoreceptors expressing either Wt or A315T-mutant TDP-43 showed similar mitochondrial cristae damage as those detected in the brain tissues of TDP-43 proteinopathy patients (see Fig 1A ). To examine whether the results observed were due to developmental defect(s), we used a system in which TDP-43 expression was induced only in adulthood using a temperature-sensitive tubulin-Gal80ts promoter with the GMR-Gal4 photoreceptor-specific driver or the Elav-Gal4 pan-neuronal driver (see S3 Fig The mitochondrion is a major source for the production of reactive oxygen species (ROS) [43]. Mitochondrial dysfunction can lead to the accumulation of ROS [44]. Furthermore, excessive ROS production affects neuronal survival and function [45,46]. We therefore examined whether TDP-43 expression affected mitochondrial ROS production in vivo using transgenic flies expressing TDP-43 in motor neurons. A fly line expressing mito-roGFP-Grx1, an in vivo mitochondrial ROS reporter [47], was crossed with either control RFP or TDP-43-RFP expressing flies. Ratiometric fluorescence confocal imaging was carried out to measure mitochondrial ROS levels in motor neurons expressing control (RFP) or TDP-43-RFP using a previously published protocol [47]. Significantly elevated mitochondrial ROS levels were detected in motor neurons expressing either Wt-or A315T-mutant TDP-43 as compared with the control group (see S4

Mitochondrial unfolded protein response is activated in cellular and animal models of TDP-43 proteinopathy

Our results presented above showed that increased TDP-43 expression led to mitochondrial cristae damage, reduced activities of mitochondrial OXPHOS complex I and IV, as well as decreased mitochondrial ATP synthesis. In addition, TDP-43 immuno-reactive aggregates were detected inside mitochondria of FTLD-TDP patient brain samples. These observations prompted us to examine if TDP-43 activated the mitochondrial unfolded protein response (UPR mt ).

Using our inducible HEK293 cells expressing Wt or A315T-mutant TDP-43, we examined mRNA levels of known genes critical for UPR mt , including ATF5, HSPA9 (mtHSP70), HSP60 and LonP1. Quantitative RT-PCR analyses revealed that by 48 hr post-induction of TDP-43 expression, mRNA levels of ATF5 and LonP1 were increased, and that by 72 hr post-induction, mRNA levels of ATF5, HSPA9, HSP60 and LonP1 were all increased in cells expressing either Wt-or A315T-mutant TDP-43 (Fig 5A). To investigate whether TDP-43 expression activated UPR mt in vivo, we induced TDP-43 expression in transgenic flies at the adult stage by heat shock using Elav-Gal4 pan-neuronal driver containing a temperature-sensitive tubulin-Gal80ts element, Elav-Gal4/tubulin-Gal80 ts driver [48] In female flies, by day 15 post-induction, HSP60A mRNA level was significantly increased in A315T-mutant expressing flies; and by day 30 post-induction, mRNA levels of HSP60A, Hsc-70-5, CG5045 (encoding ClpP) and two isoforms of Lon (the Drosophila ortholog of mammalian LonP1) were increased in TDP-43 expressing flies, especially those expressing A315Tmutant TDP-43. In male flies, the mRNA levels of all four genes were increased in flies expressing A315T-mutant TDP-43, and to a lesser extent in flies expressing Wt TDP-43, at 15 day post-induction. However, increased expression of only HSP60A, but not other three We next examined if protein levels of these UPR mt genes are altered in TDP-43 proteinopathy patient samples using a panel of brain samples characterized previously [40]. Western blotting analyses indicate that the average level of LonP1 protein in TDP-43 proteinopathy patient brains was higher than that in the control brains (Fig 5C, Fig 5D) . This is consistent with the possibility that UPR mt may be activated in a subset of TDP-43 proteinopathy patient brains. There was no significant difference between patient and control samples in the protein levels of either HSPA9 or HSP60. Together, these results support the notion that UPR mt is activated in cellular and animal models of TDP-43 proteinopathy as well as a subset of FTLD-TDP patient brains.

LonP1 interacts with TDP-43 and reduces the mitochondrial TDP-43 protein level

We further examined the relationship between LonP1 and TDP-43. LonP1 is a major mitochondrial matrix protease and a member of the evolutionarily conserved superfamily of AAA + ATPases. LonP1 plays a critical role in mitochondrial protein quality control by preferentially degrading misfolded or oxidized proteins [49]. We first tested whether TDP-43 interacted with LonP1 in a co-immunoprecipitation assay using an anti-Myc antibody in cells expressing Myc-tagged TDP-43. LonP1 was detected among immunoprecipitated proteins from cell lysates expressing either Wt or A315T-mutant TDP-43, but not the control lysates ( A number of studies suggest the roles of proteasome and autophagy in degradation of TDP-43 [50][51][52][53][54][55][56][57]. We then tested the effects of a proteasome inhibitor (MG132, MG) and an autophagy inhibitor (3-methyladenine, MA), and compared them with that of a LonP1 inhibitor [2-cyano-3,12-dioxooleana-1,9-dien-28-oicacid, CDDO (CD) [58] ] in the inducible TDP-43 expressing cells. Interestingly, neither the proteasome inhibitor (MG) nor the autophagy inhibitor (MA) had an effect on cell viability following induction of TDP-43 expression, whereas the LonP1 inhibitor (CD) specifically reduced the viability of cells expressing either (A) Expression levels of UPR mt related genes, including ATF5, HSPA9 (mtHSP70), HSP60 and LonP1, as detected by qPCR in TDP-43 inducible stable cells at different time points following induction of TDP-43 expression: 0, 36, 48 and 72-hr time points. HPRT1 was used as an internal control. (B) Expression levels of UPR mt related Drosophila genes in female and male flies, including HSP60, Hsc70-5 (mtHSP70), CG5045 (ClpP homolog) and Lon (Lon-RA and Lon-RC isoforms), as detected by qPCR in Elav-Gal4/Tub-Gal80 ts driven TDP-43 transgenic flies at day 15 and day 30 post-induction of TDP-43 expression. Actin 5C was used as an internal control. Fly genotypes: Ctr: Elav-Gal4/Tub-Gal80 ts /UAS-RFP; Wt: Elav-Gal4/Tub-Gal80 ts /UAS-Wt-TDP-43-RFP; A315T: Elav-Gal4/Tub-Gal80 ts /UAS-A315T-TDP-43-RFP. Data from 3 independent experiments (panels A and B) were analyzed using a two-way ANOVA with Bonferroni post hoc test ( � :P<0.05; �� :P<0.01; ��� : P<0.001). (C-D) Western blotting analyses using brains from control or FTLD-TDP patients show that LonP1 protein levels were higher in brain samples from patients affected by FTLD-TDP as compared with the control subjects. The levels of HSPA9 and HSP60 proteins were not changed in the patient brains. These brain samples have been reported previously [40]. Data were analyzed using StatPlus with a Student's t-test ( � :P<0.05; NS: not significant). We next examined whether increasing LonP1 expression suppressed TDP-43 cytotoxicity. Control (Ctr) or TDP-43 expressing cells were transfected with a vector control (-) or a Lon-P1-expressing plasmid (+) 24hr before Tet-induction; and cells were examined 36 hr postinduction. Increased LonP1 expression suppressed TDP-43 induced cytotoxicity (Fig 7B ). Quantification of Western blotting (WB) signals showed a ~2-fold increase in LonP1 expression, as normalized by actin levels. The total TDP-43 levels did not show significant changes (see S5C Fig), which is not unexpected because TDP-43 protein is predominantly nuclear, although it is the cytoplasmic/mitochondrial levels of TDP-43 that are correlated with neurotoxicity, as shown by published studies including ours [28,59].

We further tested whether down-regulating LonP1 altered TDP-43 induced cytotoxicity. TDP-43 inducible stable cells were transduced with a vector control virus (Ctr) or a lentivirus expressing shRNA specifically targeting LonP1 (KD) that reduced the LonP1 protein level by ~50%. LonP1 knockdown (KD) significantly reduced the viability in cells expressing TDP-43 (Fig 7C) . Fractionation experiments demonstrated that LonP1 down-regulation led to an increase in mitochondrial TDP-43 protein level in these cells although the cytosolic levels of TDP-43 were not dramatically affected (Fig 7D ), indicating that LonP1 decreases the mitochondrial TDP-43 protein level. To test whether TDP-43 could be directly degraded by LonP1, we established an in vitro protein degradation assay using purified recombinant LonP1 protein. Our data demonstrated that purified Wt or A315T TDP-43 protein was degraded by the purified recombinant LonP1 protein in a manner dependent on LonP1 concentrations (Fig

7E) and dependent on ATP (see S5D Fig).

A number of other mitochondrial proteases are involved in mitochondrial proteostasis. The mRNA level of CG5045, the Drosophila homolog of ClpP, was also increased in transgenic TDP-43 flies (see Fig 5B ). We thus examined if TDP-43 also interacted with ClpP. However, no detectable interaction between ClpP and TDP-43 was observed in a co-immunoprecipitation assay (supplemental S6A Fig) . Consistently, down-regulation of ClpP did not affect the mitochondrial TDP-43 level, as shown by WB analyses of purified mitochondria from cells

LonP1 protects against TDP-43-induced mitochondrial damage and neurodegeneration in vivo

To investigate whether altering Lon expression in vivo would modify neurodegeneration induced by TDP-43, we obtained fly lines over-expressing the Drosophila LonP1 ortholog, Lon, or expressing specific siRNA against Lon. Only one fly line overexpressing Lon was available, and it showed ~2-fold increase in Lon mRNA expression compared with control flies when the Elav-Gal4 driver was used (see S7A Fig) . However, over-expressing Lon by itself in control flies led to retinal degeneration. This prevented us from testing the effect of overexpressing Lon in TDP-43 flies.

On the other hand, two siLon fly lines were obtained, #1 and #2, which reduced Lon expression to ~30% and ~60%, respectively, of that in the control flies (see S7A Fig) . Down-regulating Lon expression by itself in control flies did not cause detectable phenotypes. The siLon#1 fly line showed more robust down-regulation efficiency and was thus used in subsequent experiments. We then crossed siLon flies with TDP-43 transgenic flies and examined retinal degeneration and locomotor function in adult flies expressing TDP-43 in photoreceptors or in all neurons respectively. Using the GMR-Gal4/tubulin-Gal80 ts driver, we monitored the progression of retinal degeneration during the adult stage following induction of TDP-43 expression by pulses of heat shock. Retinal degeneration was examined using TEM. By day 20 following TDP-43 induction, flies expressing TDP-43 exhibited profound retinal degeneration. The control flies showed normal photoreceptor organization, and heat shock per se did not affect photoreceptor development or maintenance as previously reported [60]. In contrast, retinae in flies expressing TDP-43 showed ommatidial disorganization with a clear reduction in rhabdomere numbers. The average number of rhabdomeres in flies expressing Wt or A315Tmutant TDP-43 was 6 or 5 respectively, as compared with 7 in the control flies (Fig 8A . These results show that Lon plays a protective role against TDP-43 induced neurodegeneration in these flies, especially during the early stage of the disease. Together, our data indicate that mitochondrial damage contributes to TDP-43-induced neurodegeneration.

Discussion

TDP-43 is a multi-functional RNA/DNA binding protein involved in multiple processes of gene regulation, from chromatin remodeling, DNA stability to RNA processing, including microRNA biogenesis, transcriptional and splicing regulation, mRNA trafficking as well as mRNA stability regulation [3,4,61]. Over a decade ago, TDP-43 was identified as a The locomotor index was measured in adult flies at different time points following induction of expression of Wt or A315T-mutant TDP-43 under the Elav-Gal4/Tub-Gal80 ts driver. Expression of TDP-43 in these flies led to progressive locomotor deficits, with A315T-mutant TDP-43 expressing flies showing a more severe phenotype. Down-regulation of Lon led to an earlier onset and more severe locomotor deficits in flies expressing TDP-43. The exacerbating effect of down-regulating Lon seemed to be more pronounced in male flies. More than 100 flies were analyzed in each group (precise fly numbers of each group in Supplementary Information). Data represent two independent experiments. Data in panels B, C, F and G were analyzed using a one-way ANOVA with Bonferroni post hoc test ( � :P<0.05; �� : P <0.01; ��� : P<0.001). Fly genotypes for panels A-D: Ctr: GMR-Gal4/Tub-Gal80 ts /UAS-RFP; Wt: GMR-Gal4/Tub-Gal80 ts /UAS-Wt-TDP43; Wt; siLon: GMR-Gal4/Tub-Gal80 ts /UAS-Wt-TDP-43/UAS-siLon; A315T: GMR-Gal4/Tub-Gal80 ts /UAS-A315T-TDP-43; A315T;siLon: GMR-Gal4/Tub-Gal80 ts /UAS-A315T-TDP-43/UAS-siLon. Fly genotypes for panels E-F:Wt: GMR-Gal4/UAS-Wt-TDP43; Wt;siLon: GMR-Gal4/UAS-Wt-TDP-43/UAS-siLon; A315T: GMR-Gal4/UAS-A315T-TDP-43; A315T;siLon: GMR-Gal4/UAS-A315T-TDP-43/ UAS-siLon. Fly genotypes for panel G: Ctr: Elav-Gal4/Tub-Gal80 ts /UAS-RFP; siLon: Elav-Gal4/Tub-Gal80 ts /UAS-RFP/UAS-siLon; Wt: Elav-Gal4/Tub-Gal80 ts /UAS-Wt-TDP-43; Wt; siLon: Elav-Gal4/Tub-Gal80 ts /UAS-Wt-TDP-43/UAS-siLon; A315T: Elav-Gal4/Tub-Gal80 ts /UAS-A315T-TDP-43; A315T; siLon: Elav-Gal4/Tub-Gal80 ts /UAS-A315T-TDP-43/UAS-siLon. https://doi.org/10.1371/journal.pgen.1007947.g008 characteristic protein in the inclusion bodies of tissues from patients affected by TDP-43 proteinopathy, including ALS-TDP and FTLD-TDP [1,62]. Since then, a large number of mutations in the TDP-43 gene have been identified in ALS patients, whereas dysregulation of TDP-43 gene expression or its function has been found in patients affected by FTLD and other neurodegenerative disorders [4,63,64].

Several groups have reported mitochondrial abnormalities in different models for TDP-43 proteinopathy, including abnormal mitochondrial clustering [24,26], and a shift in dynamics toward mitochondrial fragmentation [20,22,23,25]. A recent study reported the accumulation of TDP-43 in mitochondria in TDP-43 proteinopathy brain samples [28]. Of these studies, only one reported ultrastructural changes of mitochondria in mice expressing A315T-mutant TDP-43 [20]. However, it was not clear how widespread this damage was. There has not been, to our knowledge, a systematic morphological characterization of mitochondria in patient samples nor in TDP-43 proteinopathy model systems. Our study builds on these previous results by systematically and quantitatively examining TDP-43 induced mitochondrial damage using EM and other methods across different model systems and in patient samples. Our EM analyses clearly show that mitochondria frequently exhibited severe morphological impairment in TDP-43 proteinopathy patient samples and that such mitochondrial morphological changes are consistently detected across cellular and animal models of TDP- Recent studies indicate that cristae morphology determines the assembly and stability of respiratory chain super-complexes, and affects mitochondrial function [66,67]. It is not surprising that mitochondrial cristae are affected in a range of diseases, including neurodegenerative disorders. It has been reported that mitochondrial cristae are disrupted in Alzheimer's disease, showing concentric or parallel stacks [68,69]. A previous study from our group revealed that mitochondria in FTLD-FUS brain tissues showed a marked loss or disruption of cristae, with frequent detection of mitochondria in an "onion-like" deformed shape [70]. Data presented in this study demonstrate that vesicular or swollen mitochondrial cristae are a prominent feature not only in our cellular or animal models, but also in patient samples of TDP-43 proteinopathy (Fig 1 , Fig 2 and Fig 4) . Our results together with previous studies support the notion that mitochondrial impairment is a common pathogenic contributor to neurodegenerative diseases, and that distinct ultrastructural changes in mitochondria may reflect different mechanisms leading to mitochondrial damage.

Consistent with the morphological changes that we observed, mitochondrial membrane potential and mitochondrial ATP synthesis were reduced upon induction of TDP-43 expression (Fig 2 ). Interestingly, TDP-43 expression suppressed the activity of mitochondrial complex I, and to a lesser extent, complex IV, without affecting complexes II, III or V (Fig 2 ). The effect of TDP-43 on ATP synthesis and respiratory complexes has been examined in previous studies, but with discrepant results [23,[27][28][29]71]. Onesto and colleagues observed no change in the total ATP level and reduced mitochondrial membrane potential in fibroblasts from ALS-TDP patients (carrying the A382T mutation), consistent with our results; however, they observed no differences in mitochondrial complex activities. Kawamata and colleagues, on the other hand, reported that there were no mitochondrial bioenergetic defects in fibroblasts or transgenic mice expressing TDP-43 mutants, although mitochondrial calcium handling seemed to be affected [29]. In contrast, Wang and colleagues observed a decrease in ATP synthesis and a decrease in relative levels and activity in complex I from fibroblasts from ALS-TDP patients and HEK293 cells transiently overexpressing wild-type or three ALSmutants of TDP-43; however, they did not observe changes in the other complexes. Two groups provided evidence for mitochondrial dysfunction, including reduced mitochondrial respiration and ATP synthesis, in NSC-34 cells expressing ALS-mutant TDP-43 [27,71]. Further studies are necessary to resolve the discrepancy in these studies.

Our data presented here show that TDP-43 increases mitochondrial ROS production both in vitro and in vivo (Fig 2 ; S4 Fig) . Mitochondrion is a major site for ROS production, and excessive ROS accumulation can further damage mitochondria [43,72,73]. Although there were no detectable effects of TDP-43 on ROS production in cultured fibroblasts in the previous study [23], data from our cellular model show a clear increase in mitochondrial ROS production induced by TDP-43 (Fig 2 ). Furthermore, TDP-43 expression in fly motor neurons significantly increased mitochondrial ROS levels in vivo (S4 Fig) . It is interesting to note that the electron-dense TDP-43 positive aggregates detected inside mitochondria in TDP-43 proteinopathy patient brain samples (Fig 1D ) are reminiscent of the EM findings in lymphoblasts expressing LonP1 mutations of patients affected by cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome [74]. The mitochondrial abnormalities reported in these CODAS patients are similar to those detected in our TDP-43 proteinopathy patient samples, including swollen intra-or intercristal compartments, swollen or vesicular cristae and intra-mitochondrial aggregate-like structures (see Fig 1 ) [74]. Intriguingly, similar intra-mitochondrial aggregates were detected in flies expressing A315T-mutant TDP-43 only when Drosophila LonP1 homolog, Lon, was down-regulated (see S9 Fig) . Given that LonP1 is an ATP-dependent mitochondrial protease [49,74], and that mitochondrial ATP synthesis is suppressed by TDP-43, it is possible that reduced mitochondrial ATP synthesis might affect proteolytic activity of LonP1, resulting in further TDP-43 accumulation within mitochondria as the disease progresses and eventually leading to irreversible mitochondrial damage and the demise of affected neurons.

Our data from both mammalian cells and transgenic flies show that TDP-43 expression elicits UPR mt , a program that is evolutionarily conserved from nematodes to mammals. UPR mt induces expression of mitochondrial chaperones to assist in proper protein folding and proteases to promote clearance of misfolded proteins [30][31][32]75]. A variety of mitochondrial stresses induce UPR mt , including accumulation of misfolded proteins, depletion of mitochondrial DNA, ROS overload, perturbation of OXPHOS or mitochondrial translation, and disruption of the balance between mitochondrial-and nuclear-encoded proteins [30][31][32]76,77]. UPR mt has been reported in Parkinson's disease, Alzheimer's disease and ALS-SOD1 [33][34][35]. UPR mt activation detected in our cellular and animal models for TDP-43 proteinopathy could be the result of the combined effects of TDP-43, including mitochondrial accumulation of TDP-43 protein, increased ROS production, decreased membrane potential, impaired respiratory chain function and decreased mitochondrial ATP synthesis. To our knowledge, there were no previous reports of UPR mt in TDP-43 proteinopathy.

Consistent with qPCR results from cellular and fly models, the LonP1 protein level was upregulated in a fraction of patients affected by TDP-43 proteinopathy ( Recently, a new mechanism of mitochondria-mediated proteolysis, known as "mitochondria as guardian in cytosol (MAGIC)", was reported for degrading mis-foled proteins [78]. By MAGIC, cytosolic proteins prone to aggregation can be imported into mitochondria for degradation by mitochondria proteases in yeast and human cells, and PIM1 (encoding yeast Lon protease) is a major player in this process [78]. The complete machinery for MAGIC remains to be defined. Further studies are necessary to determine whether MAGIC is a major mechanism in mammalian proteostasis.

Together, our data led to a working model for the role of mitochondrial degradation of TDP-43 in the pathogenesis of TDP-43 proteinopathy (Fig 9 ). Under physiological conditions, TDP-43 is predominantly nuclear, although it shuttles between the nucleus and cytoplasm, with a small amount of TDP-43 transported into mitochondria. When TDP-43 mutations occur, or under certain cellular stresses, the mitochondrial TDP-43 level is increased. Excessive mitochondrial TDP-43 accumulation results in mitochondrial impairment, manifesting as mitochondrial membrane potential loss, mitochondrial ROS increase, and reduced mitochondrial ATP synthesis. Such TDP-43-induced mitochondrial damage triggers UPR mt , allowing the cell to initiate a series of responses to regain mitochondrial proteostasis by up-regulating mitochondrial proteases, including LonP1. It is likely at this early stage, before mitochondrial damage becomes irreparable, that mitochondrial stress responses enable the cell to reverse mitochondrial dysfunction. However, as the disease progresses, chronic cellular stresses lead to the excessive accumulation of TDP-43 in mitochondria, inducing irreversible mitochondrial damage. For example, persistent increase in the ROS level and severe reduction in ATP synthesis may result in a vicious cycle of suppression of LonP1 proteolytic activity and further accumulation of mitochondrial TDP-43 in spite of an increased protein level of LonP1, culminating in activation of cell death program(s). Data from our animal model and patient samples, together with our in vitro findings, support the notion that LonP1 may provide a protective mechanism against TDP-43 mediated neurotoxicity. It is noted that the time courses of TDP-43-induced UPR mt gene activation showed differences in male and female flies (Fig 5B ). Intriguingly, the exacerbation of locomotor deficits by Lon knockdown appeared to be more pronounced in male flies (Fig 8G ). This is consistent with a previous report that expression patterns of Lon protein isoforms were different between male and female flies and that Lon was required for gender-specific responses to oxidative stress [79]. The mechanisms underlying such gender-specific stress responses remain to be elucidated. Further work is necessary to determine whether the genderspecific response(s) play a significant role in humans against neurodegeneration.

Since the discovery of TDP-43-containing inclusion bodies in ALS and FTLD patient samples, intense efforts have been made to identify proteases capable of degrading TDP-43. A number of elegant studies have proposed possible involvement of different proteases in degrading TDP-43, including caspases, calpain and asparaginyl endopeptidase [56,[80][81][82][83][84][85][86]. None of the previously identified proteases have been shown to protect against TDP-43 induced neurotoxicity in vivo. Our biochemical experiments show that the endogenous TDP-43 and LonP1 interact with each other and that TDP-43 is degraded by the purified recombinant LonP1. Down-regulating LonP1 drosophila homolog, Lon, exacerbates TDP-43 induced mitochondrial damage and neurodegeneration. Together, these data provide previously unknown evidence that the mitochondrial protease LonP1 can protect against TDP-43 induced neurodegeneration in vivo. It will be interesting to investigate in the future whether genetic or epigenetic alterations that affect the expression or function of the human LonP1 gene may influence the onset or progression of TDP-43 proteinopathy. Our study suggests that improving mitochondrial function and reducing mitochondrial damage may provide therapeutic potential for patients affected by TDP-43 proteinopathy.

Ethics statement

De-identified postmortem human brain samples from autopsied tissues at the Neuropathology Core of the Cognitive Neurology & Alzheimer's Disease Center at Northwestern University were used following NIH and institutional guidelines. There was no research involving human subjects in this study. All animal studies were performed in accordance with national and institutional guidelines.

Cell cultures and transfection

HEK293 cells were cultured (37˚C, 5% CO2) in DMEM (Gibco), supplemented with 10% FBS (Gibco) and transfected as previously described [70]. HEK293-based T-Rex293 cells (Invitrogen) were transfected with pcDNA4 TO/myc-His plasmids (Invitrogen) expressing either Wt, or A315T-mutant TDP-43 following the manufacturer's manual. Control cells were transfected with an empty pcDNA4 vector. Individual clones of cells stably expressing TDP-43 were obtained following selection in zeocin (400 μg/mL). To induce TDP-43 expression, tetracycline (0.5μg/mL; unless specified otherwise) was added to the culture medium, and cells were cultured for different periods of time at 37˚C until harvesting. Western blotting was used to confirm induction of TDP-43 protein expression.

Fly strains and antibodies

Transgenic flies expressing the human TDP-43 (Wt or A315T-mutant) were described previously [40,41,87]. GMR-Gal4, OK371-Gal4, Elav-Gal4 and UAS-Lon-RNAi lines were obtained from the Bloomington Drosophila Stock Center (BDSC). Another UAS-Lon-RNAi fly line was obtained from the Vienna Drosophila Resource Center (VDRC). UAS-dLonOE was from the Kyoto Stock Center. The Tubulin-Gal80 ts (Tub-Gal80 ts ) line was kindly provided by Dr. A. Guo (IBP, CAS) [48]. The UAS-mito-roGFP2-Grx1 fly lines were kindly provided by Dr. T. Dick [47].

For flies under the Elav-Gal4/Tub-Gal80 ts -driver or GMR-Gal4/Tub-Gal80 ts -driver, parental flies were crossed and cultured at 18˚C, young flies after eclosion were transferred to 28˚C for 4 hr every day to induce TDP-43 expression. Other flies were all cultured at 25˚C. All flies were raised in standard fly food, 50% relative humidity, and 12hr-12hr light-dark cycles as described previously [41,70,87].

Antibodies used in this study include polyclonal rabbit-antibodies against TDP-43, ATP5A1, LonP1, HSPA9, ClpP, TOM20 and IMMT (ProteinTech Group Inc), as well as mouse monoclonal antibodies, anti-actin (ProteinTech Group Inc), anti-HSP60 (BD Biosciences) and anti-GAPDH (CWBIO). Rat-anti-dElav antibody is a kind gift from Dr. A. Guo.

Transmission electron microscopy and immuno-electron microscopy

Brain samples were evaluated for atrophy and for pathology by hematoxylin-eosin staining and immunostaining using corresponding antibodies, as previously described [40]. The brain tissue samples were fixed in 2.5% glutaraldehyde (GA, Electron Microscopy Sciences) for 2-3 hr at room temperature, after washing with PBS and fixation in 1% OsO4 buffer for 2 hr, the samples were dehydrated with graded ethanol solutions, and then embedded in Epon812 resin (SPI). Ultrathin sections (70 nm) were stained with 2% uranyl acetate for 30 minutes and then lead citrate for 10 minutes before imaging using an electron microscope (TecnaiTM Spirit, FEI).

For fly EM samples, fly heads were collected at day 3, fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) and 2.5% GA overnight at 4˚C. For HEK293 cells, cells were rinsed with PBS and then fixed in 2.5%GA overnight at 4˚C. TEM sections were prepared following protocols as described previously [88]. Fly heads and cells were then treated in the same manner as the brain tissues described above and sectioned on a Leica EM UC6/FC6 Ultramicrotome. After sections were transferred to copper grids, counter staining was performed with uranyl acetate and lead acetate before EM imaging.

Immuno-EM was carried out following our published protocol [70]. Briefly, samples were fixed in 2% PFA and 0.2% GA overnight. After rinsing with PBS, samples were embedded in 12% gelatin, dehydrated in 2.3M sucrose, subjected to ultrathin sectioning (70 nm) and then mounted on copper grids. After an additional rinse with PBS (with 1% BSA and 0.15% Glycine), samples were blocked in 5% goat serum (Electron Microscopy Sciences, EMS) for 30 minutes. Immunostaining was performed, incubating with primary antibodies for 2 hr followed by immunogold labeled secondary antibodies (EMS) for 1.5 hr. Following rinses with PBS, samples were re-fixed with 2.5% GA for 10 minutes and stained with 4% Uranyl acetate for 5 minutes, and imaged under a FEI TECNAI SPIRIT electron microscope.

Measurement of mitochondrial membrane potential by JC1

Mitochondrial membrane potential was measured in inducible TDP-43 cell lines using the mitochondrial dye JC1 (Invitrogen) following a published protocol [89]. Briefly, 48 hr before assay, inducible stable cells expressing the control vector or TDP-43 were seeded in 6-well plates. Tetracycline (1μg/mL) was added to induce TDP-43 expression for 0, 24 or 36 hr. Cells were detached using Trypsin-EDTA, rinsed in cold PBS and then stained using JC1 (5uM) for 20 minutes at 37˚C. Following staining, cells were measured using flow cytometry (BD FACS Calibur) and were analyzed by FlowJo software. Data were obtained from four independent experiments. More than 20,000 cells were measured per group in each experiment.

Measurement of mitochondrial ROS levels in fly motor neurons

Image acquisition and analyses of mitochondrial ROS of larval VNC motor neurons were performed according to published protocols with slight modifications [47]. Briefly, OK371-Gal4/ UAS-mito-roGFP2-Grx1 flies were crossed with female control or TDP-43 transgenic flies. Third instar wandering larvae were dissected in PBS containing 20mM N-ethyl maleimide (NEM) (Sigma-Aldrich), and incubated for 10 minutes. Larvae were then rinsed with PBS and then fixed with 4% PFA before mounting. Fixed larval ventral nerve chord (VNC) samples were imaged with a Leica SP8 confocal microscope equipped with a 40X oil immersion objective. Probe fluorescence was excited sequentially at 405 nm (reduced roGFP) and 488 nm (oxidized roGFP) (frame by frame) and detected at 500-530 nm. A ratio image was created by dividing a 405-nm image by the corresponding 488-nm image pixel-by-pixel, resulting in the ratio of reduced to oxidized roGFP. Images were processed and quantified using ImageJ.

Measurement of the total cellular ATP levels

The total cellular ATP level was measured using a CellTiter-Glo Luminescent Assay (Promega) according to the manufacturer's instruction. Briefly, 48 hr before assay, the control, Wt or ALS-mutant TDP-43 stale HEK293 cells were seeded in 96-well plates. One μg/mL tetracycline was added to induce TDP-43 expression for 0, 12, 24, or 36 hr. Following removal of the culture media and cell lysis, reaction mixtures were transferred to another opaque 96-well plate to measure luminescence. Luminescent signal values were normalized by the protein amount in each group to determine the total cellular ATP levels.

Mitochondrial purification

Mitochondrial isolation was performed according to published protocols with minor modifications [38,70]. Briefly, stable TDP-43-expressing HEK293 cells were suspended in isolation butter [0.22M mannitol, 0.07M sucrose, 20mM HEPES (pH 7.2), 1mM EGTA], homogenized with a Glass/Teflon Potter Elvehjem homogenizer (Bellco Glass Inc) and then fractionated by sequential centrifugation. Pellets (the mitochondrial fraction) were washed twice with wash buffer (0.25M sucrose, 50mM HEPES, 1mM EGTA, pH7.4) and were then resuspended in the same buffer. The protein amount was determined by the BCA protein assay (Pierce).

Fly mitochondrial purification was performed according to a published protocol with minor changes [90]. Sixty fly heads were collected under a microscope and were transferred into a Glass-Teflon Dounce homogenizer containing 500 μL of cold isolation buffer (225 mM Mannitol, 75 mM Sucrose, 10 mM MOPS and 1 mM EDTA, 2.5 mg/mL BSA) and homogenized on ice for 20 strokes. The homogenate was transferred to a 1.5 ml tube for centrifugation at 600 g for 10 min at 4˚C. The supernatant was centrifuged at 8,000 g for 10 min at 4˚C to enrich for mitochondria. Mitochondrial pellet was washed with 0.5 ml wash buffer (225 mM Mannitol, 75 mM Sucrose, 10 mM KCl, 10 mM Tris-HCl and 5 mM KH 2 PO4) and were then resuspended in the same buffer.

Mitochondrial ATP synthesis assay

Mitochondrial ATP synthesis was measured using a published protocol with minor modifications [39]. Briefly, equal amounts (30μg) of purified mitochondria were incubated with reaction substrates (0.15mM P1, P5-di (adenosine) pentaphosphate; 2mM malate; 2mM pyruvate; 0.1mM ADP) with or without oligomycin at 37˚C for 5 minutes. Reaction mixtures were stopped by adding boiling stop buffer (100mM Tris-HCl, 4mM EDTA, pH 7.4) and then an equal amount of CellTiter-Glo reagent (Promega) was added to measure ATP using a microplate reader. Mitochondrial ATP synthesis was quantified by subtracting the ATP content in the presence of oligomycin from the ATP content in the absence of oligomycin of the corresponding group.

Measuring activities of mitochondrial respiratory complexes

Stable inducible HEK293 cells expressing either the vector control or TDP-43 (Wt or A315Tmutant) were established as described above. Mitochondria were purified from these cells 24h following induction with tetracycline (1μg/mL) using a published protocol [70]. Briefly, mitochondria were collected from the boundary between 23% and 40% percoll of gradient centrifugation. Mitochondrial respiratory chain complex activities were measured following the published protocols [39,91]. Briefly, 10 μg of mitochondria were applied to a 100μl reaction mixture containing 30 mM KPO 4 pH7.2, 5mM MgCl 2 , 2.5 mg/mL BSA, 0.3 mM KCN, 0.13 mM NADH, 2 μg/mL antimycin A and 97.5 μM ubiquinone-1. The complex I specific activity was determined by the subtraction of the nonspecific activity in the presence of rotenone from the total NADH oxidase activity in the absence of rotenone. Complex II activity was measured in reaction mixture containing 30 mM KPO 4 (pH7.2), 5 mM MgCl 2 , 2.5 mg/mL BSA, 0.3 mM KCN, 50 μM DCPIP, 20mM succinate, 2 μg/mL antimycin A and 65 μM decylubiquinone. The complex II specific activity was determined by subtracting the nonspecific activity in the presence of malonate from the total ubiquinone reductase activity in the absence of malonate. Complex III and IV activities were measured by reduction and oxidation of cytochrome C, respectively, monitoring OD 550 respectively, as described previously [91]. Complex V activity was measured by subtracting non-specific activity in the presence of oligomycin following the published protocol [39].

Mitochondrial ROS detection assay

Mitochondrial ROS level was measured as described previously [70]. Briefly, 48 hr before the assay, inducible stable cells expressing the control or TDP-43 were seeded in 6-well plates. Tetracycline (1μg/mL) was added to induce TDP-43 expression for 0, 24, 36hr, respectively. Cells were detached using Trypsin-EDTA, rinsed in cold PBS and then stained with mitoSOX-Red for 20 min at 37˚C. After washes, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells were measured using flow cytometry (BD FACS ArialI) within 1 hr with analyses using the FlowJo software. Data were obtained from four independent experiments, with more than 20,000 cells were measured per group in each experiment.

Cell death detection assay

Cell death was measured using an Annexin V-FITC Apoptosis Detection Kit I (BD) according to the manufacturer's instructions. Briefly, 48 hr before the assay, inducible stable cells expressing the control or TDP-43 were seeded in 6-well plates. Tetracycline (1μg/mL) was added to induce TDP-43 expression for 0, 24 or 36 hr. Cells were detached by Trypsin-EDTA, rinsed in cold PBS and then stained with Annexin V-FITC and propidium iodide (PI) followed by immediate analyses (within 1 hr) using flow cytometry (BD FACS Calibur). Data were obtained from four independent experiments, and more than 20,000 cells were measured per group in each experiment.

Cell viability and cytotoxicity assays

Cell viability and cytotoxicity were determined using a CytoTox-ONE Homogeneous Membrane Integrity kit following the manufacturer's instructions (Promega). Briefly, the activity of lactate dehydrogenase (LDH) results in the generation of the fluorescent resorufin product, which was measured using a SPECTRAmax GEMINI XS (Molecular Device; excitation at 560 nm and emission at 590 nm). The cellular LDH activity quantifies the number of viable cells (cell viability); and the activity of LDH released in the culture media quantifies the number of non-viable cells that have lost membrane integrity (cytotoxicity).

Purification of LonP1 and in vitro protein degradation assay

A cDNA encoding the human LonP1 protein (amino acid residues 115-959) was cloned into vector pET32M3C [a modified version of the pET32a vector (Novagen, 69015-3)], expressed as an N-terminal thioredoxin and 6XHis-tagged protein and purified from E. coli (Rosetta strain, Novagen) following the published protocol [92]. Purified human LonP1 was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining and by immunoblotting using an anti-LonP1 antibody. Following Tet-induction (1μg/mL tetracycline) of the inducible HEK293 cells for 36hr, MycHis-tagged Wt or A315T-mutant TDP-43 protein was purified using Ni-Sepharose (GE Healthcare). Purified TDP-43 protein was incubated in a 30 μL in vitro degradation reaction system [20 mM Tris-HCl (pH8.0), 20 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 5 mM ATP] with different concentrations of purified LonP1 protein for 90 min at 37˚C. The reaction products were analyzed by Western blotting using the corresponding specific antibodies to detect TDP-43 and LonP1 proteins.

RNA Isolation and qRT-PCR

Total RNA was isolated from HEK293 cells or fly heads using TRizol reagent (Invitrogen) as described previously [70]. cDNA synthesis and qPCR were performed as described [30,70,79] using the corresponding primers (see S2 Table ). HPRT-1 and Actin5C were used as reference genes for mammalian cells and fly tissues, respectively.

Fly locomotor assays

The adult fly locomotor assay was carried out as described previously with minor modifications [41]. Briefly, flies were examined every 5 days with their locomotor index measured as the percentage of flies climbing above a 6-cm line in 15 seconds after they were tapped to the bottom of an empty vial. The experiment was repeated 10 times for each group.

Mitochondrial protein solubility assay

The protein solubility was examined as described previously with minor modifications [40]. Briefly, 100 fly heads were collected for mitochondrial purification. 100 μg of the mitochondrial fractions were resuspended in 200 μL RIPA lysis buffer containing 0.5% NP-40, extracted for 20 minutes on ice and then centrifuged at 12,000 g to collect the supernatant as the NP-40-soluble fraction and the pellet. The NP-40-insoluble pellet was then resuspended and extracted in 200 μL RIPA buffer containing 2% SDS for 20 minutes on ice. Following centrifugation at 12,000 g, the supernatant was collected as the SDS-soluble fraction. The SDS-insoluble pellet was then resuspended and extracted in 100 μL RIPA buffer containing 8 M urea for 20 minutes on ice. Following centrifugation at 12,000 g, the supernatant was collected as the urea-soluble fraction. All fractions were then subjected to Western blotting analysis.

Statistical analyses

Data were collected in Excel (Microsoft) and analyzed using GraphPad Prism 6 unless specified otherwise. Differences between two groups were analyzed using a Student's t-test. Multiple group comparisons were performed using a one-way or two-way analysis of variance (ANOVA) followed by post-hoc tests. The bar graphs with error bars represent mean ± standard error of the mean (SEM). Significance is indicated by asterisks: � , P < 0.05; �� , P< 0.01; ��� , P< 0.001.

availability

All relevant data are within the manuscript or supporting information.

Supporting information

S1 Table . Pathological and clinical diagnoses of subjects whose tissue samples were used in this study. All samples used were sequenced and confirmed that there were no mutations in known genes associated with ALS or FTLD, including TDP-43, FUS, C9orf72, GRN, SOD1 and MAPT, as reported previously [40]. Age, gender, Post-mortem interval (PMI; hours), together with pathological and clinical diagnoses, are included.

Acknowledgements

AcknowledgmentsWe are grateful to the anonymous reviewers whose constructive comments helped us in improve the paper and to Drs.Aaron Gitler and Jim Shorter for helpful suggestions.We thank members of the Wu lab for discussions.We thank the Center for Biological Imaging (CBI), Institute of Biophysics, Chinese Academy of Science for our electron microscopy work, and we are grateful to Shufeng Sun, Lei Sun, Can Peng and Li Wang for their help with preparing EM samples.We are grateful for Dr. T.P. Dick for generously providing mito-roGFP fly lines and providing valuable suggestions in the live imaging studies.We thank Drs. J. Chung and R. Bodmer for sharing fly lines.We thank David Kuo, and members of the Wu lab for discussions, suggestions and critical reading of the manuscript.

Funding

JD, PW, JL and LZ are supported by grants from the National Natural Science Foundation of China (31671174; 31501133; 31671452; 31701004) and from CAS Interdisciplinary Innovation Team.JD has been supported by a grant from the China Postdoctoral Science Foundation (2016M600137).PW is supported by a grant from the China Postdoctoral Science Foundation (2018M641498).TW is supported by the Chinese Ministry of Science and Technology (2014CB849700).JYW has been supported by ALS Therapy Alliance and NIH (RO1CA175360; RO1NS107396).EB and MM are supported by NIH (P30 AG13854; P30 AG016976; DC008552; AG045571).WAM is supported by NIH (F30 NS090893).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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