Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy

Abstract

Background Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Methods We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. Results We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. Conclusion Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration. Graphical Abstract

Nucleoporin Nsp1 surveils the phase state of FG-Nups

“…Since there is evidence that Nup62, the human orthologue of Nsp1, is linked to ALS and FTD related pathology [88][89][90] , we tested the effect of Nsp1FG expression on toxicity and localization of polyPR, a peptide linked to C9orf72 ALS. In yeast cells, as in many other models, mCerulean-tagged polyPR localizes in the nucleolus and nucleus and its expression is toxic 30,91 .…”

“…Our data contributes to a larger emerging theme of a tightly regulated interplay between FG-Nups, NTRs and the classical protein quality control system 54,55,85,100,101 . Interestingly, Nup62, the mammalian ortholog of Nsp1, has been found to participate in assemblies with disease-related IDPs, such as TDP43 89,90 and FUS 88,102 . In general, protein quality control of stably folded proteins is much better understood 100,101 than that of IDPs.…”

Nuclear pore complex and nucleocytoplasmic transport disruption in neurodegeneration

“…A number of studies have now documented the mislocalization and/or nuclear and cytoplasmic aggregation of a number of elements of the nuclear transport machinery including Nup62, RanGap1, and additional Nups and NTRs in familial and sporadic forms of ALS [35,42,[46][47][48]91,[98][99][100][101]. Interestingly, the cytoplasmic localization/mislocalization of Nups and NTRs has been linked to RNA-binding protein aggregation [42,48,102]. Specifically, the RNA-binding proteins TDP-43 and Fused in Sarcoma (FUS), which have been genetically linked to a small number of ALS cases [103,104], form characteristic insoluble aggregates in disease at least in part due to aberrant phase transitions [84,105].…”

“…Further, Nup62 and FUS directly interact and Nup62 impacts FUS droplet formation [48,106]. Moreover, nuclear import receptors have been shown to reverse aberrant phase separation and aggregation of TDP-43, FUS, and DPRs [102,[107][108][109][110] suggesting not only pathological but potentially therapeutic implications for NTR-RNA-binding protein interactions in neurodegeneration.…”

Proteomics elucidating physiological and pathological functions of TDP‐43

“…In addition, cytoplasmic aggregates of C-terminal truncated TDP-43 can recruit nuclear import elements such as importin-α and nuclear pore components such as the nuclear pore glycoprotein of 62 kDa (NUP62), disrupting general nuclear import and the nuclear lamina structure [48,125]. Interestingly, NUP62 co-aggregated with cytoplasmic TDP-43 can recruit karyopherin-β1, which in turn interacts with aggregated TDP-43 C-terminal fragment (CTF) to reduce aggregation [126]. This newly reported interaction hints to a complex network of interactions between TDP-43 and nuclear transport elements that is not limited to the importin system and opens new ways to reduce TDP-43 aggregation.…”