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Research article
Aggresome Formation and Liquid-liquid Phase Separation Independently Induce Cytoplasmic Aggregation of TAR DNA-binding protein 43
Koji Yamanaka
Nagoya University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4655-0035
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This work is licensed under a CC BY 4.0 License. Read Full License
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Background 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.
Methods 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. We performed a screening to identify the intracellular mechanisms responsible for TDP-43 aggregation. An immunofluorescence study was conducted using the sporadic ALS spinal cord sections.
Results 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.
Conclusion Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS ) are involved in inducing the TDP-43 pathologies.
Keywords
Amyotrophic lateral sclerosis (ALS), Frontotemporal lobar degeneration (FTLD), TAR DNA-binding protein 43 (TDP-43), aggresome, Histone deacetylase 6 (HDAC6)
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Koji Yamanaka
commented on 26 October, 2020
This work is published on Oct 23, 2020 as below. doi.org/10.1038/s41419-020-03116-2
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This is a preprint. It has not completed peer review.
Research article
Aggresome Formation and Liquid-liquid Phase Separation Independently Induce Cytoplasmic Aggregation of TAR DNA-binding protein 43
Koji Yamanaka
Nagoya University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4655-0035
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 16 Jun, 2020
Published
On 08 Oct, 2020
Community comments: 1
Metrics
Comments: 1
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version here.
Background 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.
Methods 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. We performed a screening to identify the intracellular mechanisms responsible for TDP-43 aggregation. An immunofluorescence study was conducted using the sporadic ALS spinal cord sections.
Results 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.
Conclusion Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS ) are involved in inducing the TDP-43 pathologies.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Koji Yamanaka
commented on 26 October, 2020
This work is published on Oct 23, 2020 as below. doi.org/10.1038/s41419-020-03116-2