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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Aggresome formation and liquid–liquid phase separation independently induce cytoplasmic aggregation of TAR DNA-binding protein 43

Seiji Watanabe, Hidekazu Inami, K. Oiwa, Yuri Murata, Shohei Sakai, O. Komine, Akira Sobue, Y. Iguchi, M. Katsuno, K. Yamanaka

Cell Death and Disease·

2020·

38 citations

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Specific Biological Pathways Involving TDP-43

- RNA metabolism processes including pre-mRNA splicing, translational control, and mRNA stability - Liquid-liquid phase separation (LLPS) related to the formation of membrane-less organelles - Aggresome formation involving microtubules and HDAC6

Molecular Interactions and Binding Partners of TDP-43

Interacts with microtubule-related proteins (MRPs) such as PFN1, UBQLN2, TUBA4A, and CHMP2B; interacts with RNA-binding proteins (RBPs) such as FUS, TAF15, TIA1, and MATR3; involves histone deacetylase 6 (HDAC6) in aggresome formation.

Functional Consequences of TDP-43 Interactions

Disrupts RNA metabolism, including pre-mRNA splicing, translational control, and mRNA stability; Leads to cytoplasmic inclusion body formation; Involves aggresome formation and LLPS pathways; Contributes to ALS/FTLD pathology through distinct aggregation mechanisms

Abstract Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and a subtype of frontotemporal lobar degeneration (FTLD). Recent studies have suggested that the formation of cytoplasmic TDP-43 aggregates is dependent on a liquid–liquid phase separation (LLPS) mechanism. However, it is unclear whether TDP-43 pathology is induced through a single intracellular mechanism such as LLPS. To identify intracellular mechanisms responsible for TDP-43 aggregation, we established a TDP-43 aggregation screening system using a cultured neuronal cell line stably expressing EGFP-fused TDP-43 and a mammalian expression library of the inherited ALS/FTLD causative genes, and performed a screening. We found that microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) co-aggregated with TDP-43. MRPs and RBPs sequestered TDP-43 into the cytoplasmic aggregates through distinct mechanisms, such as microtubules and LLPS, respectively. The MRPs-induced TDP-43 aggregates were co-localized with aggresomal markers and dependent on histone deacetylase 6 (HDAC6), suggesting that aggresome formation induced the co-aggregation. However, the MRPs-induced aggregates were not affected by 1,6-hexanediol, an LLPS inhibitor. On the other hand, the RBPs-induced TDP-43 aggregates were sensitive to 1,6-hexanediol, but not dependent on microtubules or HDAC6. In sporadic ALS patients, approximately half of skein-like TDP-43 inclusions were co-localized with HDAC6, but round and granular type inclusion were not. Moreover, HDAC6-positive and HDAC6-negative inclusions were found in the same ALS patient, suggesting that the two distinct pathways are both involved in TDP-43 pathology. Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS) are involved in inducing the TDP-43 pathologies.

Introduction

Abnormal accumulation of TAR DNA-binding protein-43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by a selective loss of motor neurons, and a subtype of frontotemporal lobar degeneration (FTLD-TDP) 1 . TDP-43 is a DNA/RNA-binding protein that is predominantly localized in nuclei and plays multifunctional roles in RNA metabolism, including pre-mRNA splicing, translational control, and mRNA stability 2,3 . However, in most of the ALS and FTLD-TDP cases, TDP-43 is mislocalized into the cytoplasm and forms inclusion bodies 4,5 . In addition to this, more than 50 mutations in the TARDBP gene, encoding TDP-43, have been identified as a cause of inherited ALS 6 . These observations suggest that dysfunction of TDP-43 is a significant component of ALS pathogenesis. Thus, understanding the mechanism of TDP-43 aggregation will uncover the mechanistic basis for TDP-43 pathology and neurodegeneration in ALS/FTLD. Recently, a number of studies revealed that many kinds of RNA-binding proteins (RBPs), including TDP-43, spontaneously develop granule-like structures via a liquid-liquid phase separation (LLPS) mechanism 7,8 . LLPS is a process in which proteins and nucleotides abruptly segregate into two distinct phases, enabling the formation of intracellular membrane-less organelles 9 , such as p-bodies 10 and stress granules 11,12 . Under physiological conditions, LLPS enables to achieve high local concentrations for molecular interactions and rapid chemical reactions, and allow fast changes of molecules upon signaling for facilitating various intracellular biological processes, e.g., transcriptional regulation and signal transduction 13 . However, once excess amounts of proteins are accumulated together with dysregulation of LLPS, the complexes quickly transform into pathological inclusions that are often found in neurodegenerative diseases 14,15 . ALS causative gene products, including FUS, TIA-1, and, of course, TDP-43, are proposed to form aggregates via LLPS. Consistent with this hypothesis, recent studies have discovered that optical multimerization of cytoplasmic TDP-43 induces the aggregation and sequestration of endogenous nuclear TDP-43 into the cytoplasmic aggregates that are dependent on LLPS 16,17 .

However, whether LLPS is solely responsible for the cytoplasmic aggregation of TDP-43 remains unclear. Among the over 25 inherited ALS/FTLD causative genes, there is a considerable number of the genes encoding the proteins that have the functions different from RNA regulation 18,19 . For instance, SQSTM1 and TBK1 encode proteins involved in autophagy, while PFN1 and TUBA4A encode cytoskeletal proteins. Considering that TDP-43 pathology is also observed in the most of inherited ALS/ FTLD patients, there may be the intracellular mechanisms other than LLPS stimulating the formation of TDP-43 aggregates in ALS/FTLD lesions.

In this study, we aimed to determine the intracellular pathways involved in the formation of TDP-43 aggregates. Using a screening system of cytoplasmic TDP-43 aggregations induced by the expression of inherited ALS/FTLD causative genes, we established that, in addition to the RBPs, microtubule-related proteins (MRPs) also coaggregated with TDP-43 in the cytoplasm. The MRPs induced co-aggregation of TDP-43 via the formation of aggresomes, cytoplasmic aggregations that are dependent on microtubules. In contrast, the RBPs-induced aggregations were dependent on LLPS. Therefore, both the TDP-43 co-aggregation pathways were independent from each other. Moreover, we also showed that a subset of TDP-43 inclusions in spinal motor neurons of sporadic ALS cases were co-localized with histone deacetylase 6 (HDAC6), an aggresome marker. Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS) are involved in inducing the TDP-43 pathologies.

Antibodies

Antibodies used in this study are as follows; anti-HDAC6 (1:1000 for immunoblotting, 1:50 for immunohistochemistry, #7558, RRID: AB_10891804, Cell Signaling Technology, Danvers, MA, USA), anti-γ-tubulin (1:1000, #ab27074, RRID: AB_2211240, Abcam, Cambridge, UK), anti-vimentin (1:50, #3932, RRID: AB_2288553, Cell Signaling), anti-TDP-43 (1:1000, clone 3H8, #MABN45, EMD Millipore, Billerica, MA), antiβ-actin (1:5000, #5441, RRID: AB_476744, Sigma-Aldrich Co LLC, St. Louis, MO,USA), anti-RFP (1:1000, #PM005, RRID: AB_591279, Medical & Biological Laboratories (MBL) Co LTD, Nagoya, Japan).

Cell culture and small-interfering RNA (siRNA) transfection Mouse neuroblastoma Neuro2a (RRID: CVCL_0470) (N2a) cells were maintained and differentiated, as described elsewhere 20 . To establish the cell line stably expressing EGFP-fused human TDP-43 wild-type or nuclear localization signal (NLS)-deficient mutant 21 , we transfected linearized pcDNA3.1(+) (Thermo Fisher Scientific Inc, Waltham, MA, USA) inserted each TDP-43 complementary DNA (cDNA) with Lipofectamine 2000 (Thermo Fisher). The cells were cultured for two weeks in a growth medium containing 1.0 g/L G-418 (Nacalai Tesque, Kyoto, Japan). The colonies were isolated and confirmed the expression of EGFP-TDP-43 by fluorescent microscopy and immunoblotting. The established cell lines were maintained in a growth medium containing 1.0 g/L G-418. HeLa (CCL-2) and HEK293 (CRL-1573) cells, both obtained from ATCC, were maintained in Dulbecco's Modified Eagle's' Medium (DMEM) containing 4.5 g/L glucose supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Thermo Fisher) at 37 °C in a humidified chamber containing 5% CO 2 . Total RNAs of HeLa and HEK293 cells were isolated using the RNeasy-micro kit (QIAGEN, Hilden, Germany).

Stealth siRNA against the murine Hdac6 gene was obtained from Thermo Fisher. siRNA (20 nM) was transfected into N2a cells using Lipofectamine RNAi Max (Thermo Fisher) as described by the manufacturer.

TDP-43 aggregation screening using mammalian inherited ALS/FTLD causative gene expression library

Full-length wild-type cDNA of each familial ALS causative gene was amplified using the gene-specific primers from cDNA synthesized from total RNA of HeLa or HEK293 cells. The amplified cDNA was inserted into pmCherry-N1/C1 vectors (Clontech Laboratories Inc, Mountain View, CA, USA) using SLiCE reaction 22 . ALS/ FTLD-causing mutations were introduced according to the guidance of the Primestar Mutagenesis Basal Kit (Takara Bio, Shiga, Japan).

N2a cells stably expressing EGFP-fused TDP-43, which is described above, were seeded at 5.0 × 10 4 /well on a 4 well cover-slide chamber (AGC Techno Glass Inc, Shizuoka, Japan) coated with poly-D-lysine. On the next day, 0.25 µg/well plasmids to express inherited ALS/FTLD causative genes were transfected with Lipofectamine 2000 (Thermo Fisher) according to the manufacturer's guide. After 6 h of the transfection, the medium was changed into a differentiation medium (DMEM + 2%(v/v) fetal bovine serum and 2 mM N6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate (Nacalai Tesque, Kyoto, Japan)), and cultured for 48 h. Then, the non-fixed cells were observed using laser scanning confocal microscopy (LSM-700; Carl Zeiss AG, Oberkochen, Germany) and the equipped software (Zen; Carl Zeiss AG).

For the treatment with 1,6-hexanediol (1,6-HD) (Sigma), the medium was replaced with a fresh one with/ without 4%(v/v) 1,6-HD and incubated for 10 min at room temperature. Then, the non-fixed cells were observed using laser scanning confocal microscopy.

Immunoblotting and Immunofluorescence

Immunoblotting analyses were performed as described elsewhere 23 . For TDP-43 insolubility assay, the cells were first lysed in TBS supplemented with 1 mM EDTA, 1%(v/v) Triton X-100 and protease inhibitor cocktail by brief sonication on ice. After centrifugation at 15,000 × g, at 4 °C for 5 min, the supernatants were collected as soluble fraction. The pellets were resuspended in TBS supplemented with 1 mM EDTA and 2%(w/v) sodium-dodecyl sulfate (SDS) by brief sonication, then they were subjected to centrifugation at 15,000 × g, room temperature for 5 min. The supernatants were collected as insoluble fractions. Aliquots containing 15 µg/lane of soluble fractions and the same volume of corresponding insoluble fractions were analyzed by immunoblotting. For immunofluorescence analyses of γ-tubulin and vimentin, cultured N2a cells were transfected and cultured in a differentiation medium for 48 h. Then, the cells were fixed with ice-cold methanol at -30 °C for 20 min. After three times wash with TBS (50 mM Tris-HCl (pH 7.4), 150 mM NaCl), the cells were immunostained as described previously 23 . Briefly, the cells were incubated with primary antibodies at 4 °C for overnight, following incubation with secondary antibodies at room temperature for 2 h. Immunofluorescence images were obtained by a confocal laser scanning microscopy (LSM-700; Carl Zeiss AG, Oberkochen, Germany) and the equipped software (Zen; Carl Zeiss AG).

Postmortem human tissues

Specimens of spinal cords from six patients with sporadic ALS and two control patients with other neurological diseases were obtained by autopsy with informed consent (Supplementary Table S1 ). The diagnosis of ALS was confirmed by El Escorial diagnostic criteria as defined by the World Federation of Neurology. The ethics committee approved the collection of tissues and their use in this study of Nagoya University. We confirmed that informed consent of all the tested subjects were obtained. For immunofluorescence analyses, the sections were prepared from formalin-fixed and paraffinembedded tissues, deparaffinized, and incubated at 90 °C for 40 min in HistVT One (Nacalai Tesque).

Statistical analyses

All the data from immunofluorescence and semiquantitative immunoblotting were analyzed by an unpaired t-test for the comparison between two groups, or one-way ANOVA followed by the posthoc Tukey's multiple comparison t-test for the comparison among more than three groups, respectively. When the values were compared only with the control values, Dunnet's multiple comparison tests were used instead of Tukey's one. For all the ANOVA, we used Brown-Forsythe and Welch's correction not to assume normal distribution of the data. All the statistical analyzes were carried out using GraphPad Prism software (GraphPad Software, La Jolla, CA).

TDP-43 co-aggregation screening reveals the involvement of microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) in cytoplasmic TDP-43 aggregation

To identify intracellular pathways involved in cytoplasmic TDP-43 aggregation, we established a screening system for the co-aggregation of TDP-43 and ALS/FTLD causative proteins (Fig. 1A ). We generated N2a cells that stably expressed EGFP-fused wild-type or NLS-defective TDP-43 (TDP-43 WT or TDP-43 ΔNLS , respectively) as reporter cell lines to visualize TDP-43 aggregates (Fig. 1B ). When both the cells were cultured in normal conditions, no TDP-43 aggregates were observed. We also prepared an inherited ALS/FTLD causative gene expression library (19 genes in total, see Table 1 ). The library consisted of expression plasmids of mCherry-fused wildtype or two independent variants carrying ALS/FTLDlinked mutations for each of the genes. Then, we transfected each plasmid into the reporter cells and observed them after two days of incubation. We then screened the library to identify the ALS/FTLD genes that induce cytoplasmic TDP-43 aggregation in N2a reporter cells that stably express EGFP-TDP-43 ΔNLS . The screening results are summarized in Table 1 . Our screening identified that 14/19 (74%) gene products formed intracellular aggregates and 9 of them (9/19 in total, 47%) coaggregated with TDP-43 ΔNLS . Representative images for the cells expressing each of the inherited ALS/FTLD causative genes are shown in Supplementary Fig. S1 . We also found that the ALS/FTLD causative gene products are classified into three groups: gene products that induce co-aggregates with TDP-43 (Group 1) (Fig. 1C, F, G ), gene products that induce aggregates without TDP-43 (Group 2) (Fig. 1D, F, G ), and gene products that do not form aggregates (Group 3) (Fig. 1E-G ).

More importantly, all of the gene products in Group 1 induced cytoplasmic translocation and aggregation of TDP-43 WT (Fig. 2A and Supplementary Fig. S2 ). Interestingly, the Group 1 genes were involved in either microtubular dynamics or RNA regulation, suggesting that these two pathways are both involved in intracellular TDP-43 aggregation. Therefore, to determine the mechanisms of intracellular TDP-43 aggregation, we further classified Group 1 genes into microtubule-related proteins (MRPs; PFN1, UBQLN2, TUBA4A, and CHMP2B) and RNA-binding proteins (RBPs; FUS, TAF15, TIA1, and MART3) (Fig. 2B ). Notably, the genes encoding RBPs-induced TDP-43 co-aggregation only when their mutants were mislocalized into the cytoplasm, suggesting that leakage of RBPs from nuclei is closely associated with the cytoplasmic TDP-43 aggregation.

Furthermore, we also confirmed that the fluorescentbased screening results (Figs. 1 and 2 ) are well consistent with the results of TDP-43 insolubility assay (Fig. 3 ). The ALS/FTLD causative genes classified into Group 1 specifically decreased solubility of both TDP-43 ΔNLS -EGFP (Fig. 3B ) and endogenous TDP-43 (Fig. 3C ), supporting the notion that MRPs and RBPs are tightly involved in TDP-43 cytoplasmic aggregation.

Liquid-liquid phase separation (LLPS) is involved in RBPsinduced TDP-43 aggregation

Co-transfection of MRP (PFN1 C71G ) and RBP (FUS P525L ) in N2a reporter cells stably expressing EGFP-TDP-43 ΔNLS revealed that the aggregates induced by MRPs and RBPs were distinctly partitioned in cytoplasm (Supplementary Fig. S3 ). These results suggest that the mechanisms of MRPs-and RBPs-induced TDP-43 aggregation are independent of each other. Previous studies reported that FUS and TIA-1, representative RBPs classified into Group 1, form cytoplasmic aggregates through LLPS 24,25 . Thus, to investigate the mechanism of RBPs-induced aggregation, we treated the cells with 1,6-hexanediol (1,6-Hd), an inhibitor of LLPS, to assess the involvement of LLPS in the coaggregation of RBPs and TDP-43. As shown in Fig. 4A, B , 1,6-Hd drastically dissociated the cytoplasmic aggregates of RBPs, mutant FUS and TIA1. Consistent with the decreased number of RBPs-induced aggregates, sequestration of TDP-43 into the aggregates was also significantly prevented (Fig. 4C ). These observations suggest that LLPS drives the cytoplasmic co-aggregation of RBPs and TDP-43. In contrast, 1,6-Hd did not influence either the number of aggregates of MRPs (mutant PFN1 and UBQLN2) or coaggregates with TDP-43 (Fig. 4A-C ), suggesting that a mechanism independent of LLPS drives the co-aggregation of MRPs and TDP-43.

MRPs-induced TDP-43 co-aggregates are sensitive to nocodazole and co-localized with aggresomal markers

To examine whether the microtubules are essential for MRPs-induced TDP-43 aggregation, we then treated the cells with nocodazole, a microtubule destabilizing reagent. As expected, the nocodazole treatment substantially reduced the amount of co-aggregates of MRPs (PFN1 C71G ) and TDP-43 (Fig. 5A, B ). Intriguingly, the co-aggregates of MRPs and TDP-43 were co-localized with the aggresomal markers γ-tubulin (Fig. 5C ) and vimentin (Fig. 5D ). An aggresome is a peri-nuclear compartment where misfolded proteins accumulate 26,27 . Once the cellular protein degradation system is overwhelmed, the misfolded proteins are accumulated into aggresomes through microtubules for a cellular defense. Therefore, our findings suggest that TDP-43 is sequestered into aggregates of MRPs during aggresome formation. Meanwhile, RBPs-induced TDP-43 coaggregates (FUS P525L ) were not affected by nocodazole (Fig. 5A ) and did not co-localize with aggresomal markers (Fig. 5C, D ), suggesting that MRPs and RBPs sequester TDP-43 into their aggregates through distinct mechanisms.

Histone deacetylase 6 is associated with the coaggregation of MRPs and TDP-43

Histone deacetylase 6 (HDAC6) recognizes misfolded proteins and transports them to aggresomes through microtubules 28 . Previous reports showed that aggresomes Table 1 Results of TDP-43 co-aggregation screening with ALS/FTLD causative genes. are involved in the formation of inclusion bodies in the lesions of neurodegenerative diseases such as Huntington's disease 29,30 . Therefore, we next examined whether the elimination of Hdac6 by siRNA affected TDP-43 coaggregation with MRPs in N2a cells (Fig. 6A ). As shown in Fig. 6B-D , Hdac6 knock-down prevented the aggregation of MRPs, as well as TDP-43 co-aggregation with MRPs. Interestingly, relatively small cytoplasmic aggregates of MRPs were still observed in the cells with Hdac6 knock-down (Fig. 6B , asterisks); however, TDP-43 was not co-localized with them. Since HDAC6 is crucial for the transport of misfolded proteins to aggresomes 28,29 , this observation might suggest that TDP-43 is sequestered during compartmentalization. On the other hand, the Hdac6 knock-down did not affect RBPs-induced TDP-43 co-aggregation, indicating that Hdac6 was not involved in the TDP-43 sequestration into the LLPSinduced aggregates.

Different characteristics of TDP-43 inclusions in sporadic ALS suggest multiple pathways form TDP-43 pathology

The results of TDP-43 co-aggregation obtained by our screening system are based on the overexpression of the inherited ALS/FTLD causative genes. However, it is unclear whether the mechanisms for the formation of TDP-43 aggregation observed in our study are involved in the TDP-43 pathology of sporadic ALS. To examine the involvement of the aggresome pathway in TDP-43 pathology, we examined the co-localization of TDP-43 and HDAC6, an aggresomal marker, in spinal motor neurons of sporadic ALS patients. We found that approximately half of TDP-43 skein-like inclusions were co-localized with HDAC6 (75/160, 46.9% of total skeinlike inclusions) (Fig. 7A, B ). It should be noted that no colocalization of TDP-43 and HDAC6 was observed in round (0/52) or granular (0/33) TDP-43 inclusions. These observations support the hypothesis that TDP-43 skein-like inclusions are, at least partially, formed by a similar mechanism to aggresome formation. Importantly, both the HDAC6-positive and HDAC6-negative inclusions were found in every sporadic ALS patient, suggesting that the mechanisms of TDP-43 aggregation vary in each of the neurons. In conclusion, the different characteristics between skein-like and other types of TDP-43 inclusions suggest that TDP-43 pathology is developed via multiple complexed mechanisms, likely including both aggresome formation and LLPS, in sporadic ALS.

Discussion

In this study, we demonstrated that: (i) at least two distinct intracellular pathways were involved in TDP-43 cytoplasmic aggregation, including aggresome formation and LLPS; (ii) the two pathways were independent of each other; and (iii) in spinal motor neurons of sporadic ALS, both HDAC6-positive and HDAC6-negative aggregates were found in the same ALS patient. Therefore, to the best of our knowledge, we are the first to confirm that HDAC6-dependent aggresome formation is partially involved in skein-like TDP-43 inclusion of sporadic ALS.

Our study revealed that over 70% of the examined inherited ALS/FTLD causative 19 genes induced TDP-43 co-aggregation in cultured neuronal cells and the fluorescent-based observation of TDP-43 co-aggregates was well consistent with the TDP-43 insolubility examined by immunoblotting. Our screening system enabled us to assess whether any genes of interest induce cytoplasmic TDP-43 aggregation by a simple transfectionbased screening. Moreover, our screening system has been proved useful for investigating the mechanism of TDP-43 aggregation. Indeed, as described above, we identified two distinct mechanisms involved in TDP-43 aggregation by using this system (i.e., aggresome formation and LLPS). Our screening system will also be useful for identifying compounds that prevent cytoplasmic TDP-43 aggregation, which may contribute to a novel therapeutic strategy for ALS/FTLD. Although our screening system generally well recapitulated cytoplasmic TDP-43 aggregation in ALS/FTLD, our results were partially inconsistent with some previous reports on the pathology of inherited ALS 18,19 . Notably, we did not find any evidence of TDP-43 aggregation in the neuronal cells expressing the inherited ALS/FTLD causative genes related to protein degradation (e.g., VCP, UBQLN2, SQSTM1, and TBK1), although the previous studies reported TDP-43 pathology in inherited ALS patients with these mutations 18 . Moreover, the colocalization of TDP-43 and the inherited ALS/FTLD causative gene products are still controversial even though TDP-43 pathology is widely observed in the patients with ALS. For example, some studies have suggested the co-localization of TDP-43 with TIA-1 31 or FUS 32 but other studies do not 33,34 . One possible interpretation of this inconsistency is an experimental limitation of our screening system based on the overexpression of the inherited ALS/FTLD causative genes with a short incubation time before evaluation. Therefore, a careful interpretation is required to compare our results with the inherited ALS/FTLD neuropathology data.

The co-aggregation of TDP-43 and RBPs was sensitive to 1,6-Hd, suggesting that LLPS drives cytoplasmic RBPinduced aggregation. Nucleotides, enriched in nuclei, prevent aggregation driven by LLPS 35,36 . Indeed, the previous study demonstrated that an artificially designed RNA, which specifically binds to TDP-43, prevents intracellular aggregation of TDP-43 triggered by light-driven forced multimerization of TDP-43 in the cytoplasm 17 . This mechanism could explain our observations that RBPs only aggregated with TDP-43 when they leaked from nuclei into the cytoplasm. On the other hand, the MRPs-induced coaggregation of TDP-43 was dependent on aggresome formation. The role of aggresomes in the accumulation of misfolded proteins has been pointed out in various neurodegenerative diseases 37 . ALS-linked PFN1 mutant proteins that aggregated in the cytoplasm were dependent on microtubules, which was similar to our observations 38 . Intriguingly, these two pathways of TDP-43 aggregation are not complementary and are independent of each other. Inhibiting LLPS prevented RBPs-mediated TDP-43 aggregation, but did not affect MRPs-induced TDP-43 aggregation. Conversely, HDAC6 suppression and microtubular destabilization only affected MRPs-induced TDP-43 aggregation. All these observations indicate that targeting the intracellular mechanism specific to each pathway is key to preventing TDP-43 accumulation. Nonetheless, there is still a possibility that other independent pathways are also involved in cytoplasmic TDP-43 aggregation. Further investigation into the potential mechanisms of TDP-43 accumulation is required.

HDAC6 is a key molecule in aggresome formation. HDAC6 facilitates the transport of misfolded proteins by connecting microtubules and ubiquitinated proteins 28,37 . The involvement of HDAC6 has been reported in various neurodegenerative diseases such as Huntington's disease 29 , Parkinson's disease 28 , and Alzheimer's disease 30 . In ALS, it was reported that deletion of Hdac6 ameliorates the disease progression in SOD1-ALS mice 39 and TDP-43 deficiency drastically reduces HDAC6 expression in cultured cells 40 . Moreover, HDAC6 inhibition reversed axonal transports, which are defective in iPS-derived neurons from ALS patients with FUS mutation 41 . These observations suggest the possibility that HDAC6 is also involved in the ALS pathology similar to other neurodegenerative diseases and that it may be a therapeutic target for ALS through restoring axonal transport. In addition to this, our results strengthen the possibility that inhibition of HDAC6 is a potential therapeutic target for ALS by providing a novel mechanism for preventing microtubule-dependent TDP-43 pathology. HDAC6 inhibition is probably effective for MRPs-linked inherited ALS and partially to sporadic ALS.

Since most of the ALS cases are sporadic, it is important to identify the mechanism responsible for TDP-43 aggregation in the sporadic ALS. Recent reports showed that cytoplasmic TDP-43 was sequestered into aggresome dependent on microtubules in fibroblasts derived from sporadic ALS patients 42 . Consistent with the cited study, we found that TDP-43 was sequestered into aggregates of MRPs in N2a cells and approximately half of TDP-43 skein-like inclusions in sporadic ALS patients were co-localized with HDAC6, an aggresomal marker. Our results suggest that the HDAC6dependent aggresome pathway may be involved in the formation of skein-like inclusion in ALS/FTLD. However, we also found that both HDAC6-positive and HDAC6-negative skein-like inclusions were observed in the same sporadic ALS patient, as shown in Fig. 7B . In addition, the round and granular inclusions, which are not co-localized with HDAC6, were also found in the same sporadic ALS patient. These observations suggest that the mechanisms responsible for TDP-43 aggregation are different from each other among the remaining motor neurons in the one sporadic ALS patient and that the morphology of TDP-43 inclusions may reflect the corresponding intracellular mechanism. Further studies are needed to determine the mechanism responsible for each type of cytoplasmic TDP-43 aggregation in motor neurons. We found that the two distinct pathways, aggresome formation and LLPS, are independently involved in the formation of cytoplasmic TDP-43 inclusions in ALS. Although it is unclear whether each type of cytoplasmic TDP-43 aggregates provokes toxicities to neurons, our findings, together with the other studies, suggest the therapeutic potential of HDAC6 through the several lines of mechanisms in ALS/FTLD.

Conflict of interest

The authors declare that they have no conflict of interest.

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-020-03116-2).

Acknowledgements

AcknowledgementsThis work was supported by Grants-in-Aid for Scientific Research 18H02740, 18H04860, 19KK0214 (to K.Y.), and 17H04986 (to S.W.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan/Japan Society for the Promotion of Science (JSPS), AMED under Grant Number 20ek0109426h0001 (K.Y.), Uehara Memorial Foundation (K.Y. and S.W.), and the Public Foundation of Chubu Science and Technology Center (S.W.).

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