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

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.

Novel reporters of mitochondria‐associated membranes (MAM), MAMtrackers, demonstrate MAM disruption as a common pathological feature in amyotrophic lateral sclerosis

“…Finally, we screened for MAM disruptions using MAMtracker‐Luc (N2a･MAMLuc stable cells) and a mammalian gene expression library of ALS‐causative genes 18 . The screening results are shown in Figure 5A‐D.…”

“…Although the amino acid sequence of split GFP was based on the previously reported one, 15 the cDNA sequence was modified using Codon Optimizer (IDT) to optimize its expression in mammalian cells. An ALS causative gene expression library was described elsewhere 18 . siRNA against murine Sigmar1 gene, an ortholog of human SIGMAR1 gene, was purchased from Thermo Fisher Scientific (siRNA ID: MSS237476).…”

Multi-phaseted problems of TDP-43 in selective neuronal vulnerability in ALS

“…The distinct histological traits of TDP-43 aggregate suggest multiple pathways for aggregation. Indeed, in cultured cells, cytoplasmic TDP-43 aggregation is driven by at least two distinct pathways upon expression of inherited ALS/FTLD causative genes: RNA-binding protein-mediated LLPS promoting granular-type aggregation and histone deacelylase 6 (HDAC6)-mediated aggresome formation promoting skein-like aggregation [ 97 ]. In the spinal cord of patients with ALS, most of the phosphorylated TDP-43 inclusions show significant skein-like immunoreactivity of lysine-145 acetylation in RRM1, which may be promoted by oxidative stress [ 50 ].…”

Liquid–liquid crystalline phase separation in biological filamentous colloids: nucleation, growth and order–order transitions of cholesteric tactoids

“…The most common form of liquid-liquid phase separation involves two biopolymers (e.g. a protein and RNA) 9 and is primarily regulated by enthalpic interactions. 7 The clas-ready been rationalized by the interaction between confinement, anisotropic surface energy, and Frank-Oseen elastic energy.…”