Aggregation of the nucleic acid–binding protein TDP-43 occurs via distinct routes that are coordinated with stress granule formation

Todd J Cohen,Youjun Chen

doi:10.1074/jbc.ra118.006351

Todd J Cohen, Youjun Chen

Open Access

https://doi.org/10.1074/jbc.ra118.006351

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Abstract

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein, and its aggregation represents the defining pathology in amyotrophic lateral sclerosis (ALS) and related proteinopathies. Recent studies implicate cytoplasmic stress granules (SGs) as hubs that may facilitate TDP-43 aggregation. Here, using cellular fractionation, biochemical analyses, and histological assays, we show that TDP-43 targeted to the cytoplasm has multiple fates. Whereas a TDP-43 subpopulation is indeed recruited to SGs, mature aggregated TDP-43, produced with aggregate-prone TDP-43 variants or exposure to oxidative stress, generates distinct TDP-43 inclusions that are surprisingly devoid of SGs. Consistent with this observation, we found that SG components are predominantly excluded from TDP-43 pathology in motor neurons from individuals with ALS. We generated de novo SGs by expressing the fragile X protein (FMRP) and found that rather than directly engaging TDP-43 aggregates, SGs can sequester the proteostasis factor histone deacetylase 6 (HDAC6) and thereby impede TDP-43 clearance from cells. These findings indicate that SGs form distinct cytoplasmic structures that can indirectly enhance TDP-43 aggregation. Therapeutic approaches that inhibit SG formation may therefore be effective at suppressing TDP-43-mediated toxicity in patients with ALS and related TDP-43 proteinopathies.

Highlights

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid– binding protein, and its aggregation represents the defining pathology in amyotrophic lateral sclerosis (ALS) and related proteinopathies
We proposed that aberrantly acetylated TDP-43 triggers its aggregation in a similar manner to genetic TARDBP mutations, some of which reside within the RNA recognition motif domains and potentially modulate critical interactions between TDP-43 and target mRNA [11, 12]
To determine whether cytoplasmic TDP-43 is recruited to stress granules (SGs), we expressed TDP-43 lacking a nuclear localization sequence (TDP-43–⌬nuclear localization sequences (NLSs)) or a comparable variant containing aggregate-prone acetylation-mimic substitutions at residues Lys-145 and Lys-192 (TDP-43–⌬NLS– 2KQ), the latter of which generates very robust TDP-43 inclusions that more closely resemble ALS pathology [4]

Summary

Results

A small pool of nuclear TDP-43 relocalizes to SGs upon exposure to environmental stressors [17, 29]. To determine whether cytoplasmic TDP-43 is recruited to SGs, we expressed TDP-43 lacking a nuclear localization sequence (TDP-43–⌬NLS) or a comparable variant containing aggregate-prone acetylation-mimic substitutions at residues Lys-145 and Lys-192 (TDP-43–⌬NLS– 2KQ), the latter of which generates very robust TDP-43 inclusions that more closely resemble ALS pathology [4]. Cytoplasmic TDP-43 targeting alone did not cause significant accumulation within SGs (Fig. 1A), the more aggregate-prone TDP-43 mutant showed enhanced SG localization detected with multiple SG markers including FMRP and TIAR (Fig. 1, B and C, see cyan arrowheads marking SGs). An even more pronounced separation of these compartments was observed with TDP-43– ⌬NLS–2KQ (Fig. 1F) Quantification of these results showed a significant reduction in TDP-43 recruitment to SGs upon stress exposure (Fig. 1G). Cytoplasmic TDP-43 can integrate into SGs, it rapidly aggregates into a spectrum of SG-negative inclusions that are more reminiscent of the hallmark pathology seen in ALS patients

SG localization in ALS spinal cord

Discussion

Plasmids and cell culture

Fractionation and biochemical analysis

Immunocytochemistry and quantification

Histology of ALS spinal cord