Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis

Roczniak-Ferguson, Agnes; Ferguson, Shawn M.

doi:10.1101/560144

Cited by 4 publications

(5 citation statements)

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“…Some TDP‐43 mutations compromise the protein's splicing ability for specific mRNA transcripts. For instance, alternative splicing of the POLDIP3 gene depends on RRM1, and P112H‐mutated TDP‐43 is deficient in restoring normal POLDIP3 splicing in TDP‐43 knockout cell lines 55,56 . A similar result has been reported for the ALS/FTD‐linked TDP‐43 K181E mutation, with that residue being located in close proximity to RRM1.…”

Section: Discussionsupporting

confidence: 62%

Frontotemporal dementia‐linked P112H mutation of TDP‐43 induces protein structural change and impairs its RNA binding function

et al. 2020

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TDP-43 forms the primary constituents of the cytoplasmic inclusions contributing to various neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Over 60 TDP-43 mutations have been identified in patients suffering from these two diseases, but most variations are located in the protein's disordered C-terminal glycine-rich region. P112H mutation of TDP-43 has been uniquely linked to FTD, and is located in the first RNA recognition motif (RRM1). This mutation is thought to be pathogenic, but its impact on TDP-43 at the protein level remains unclear. Here, we compare the biochemical and biophysical properties of TDP-43 truncated proteins with or without P112H mutation. We show that P112H-mutated TDP-43 proteins exhibit higher thermal stability, impaired RNA-binding activity, and a reduced tendency to aggregate relative to wild-type proteins. Near-UV CD, 2Dnuclear-magnetic resonance, and intrinsic fluorescence spectrometry further reveal that the P112H mutation in RRM1 generates local conformational changes surrounding the mutational site that disrupt the stacking interactions of the W113 side chain with nucleic acids. Together, these results support the notion that P112H mutation of TDP-43 contributes to FTD through functional impairment of RNA metabolism and/or structural changes that curtail protein clearance. K E Y W O R D S neurodegenerative disease, protein aggregation, RNA recognition motif, RNA-binding protein 1 | INTRODUCTION TDP-43 is a multifunctional protein that binds DNA and RNA, and it plays various roles in mRNA splicing, RNA transport, miRNA processing, and regulation of RNA stability and transcription. 1-3 TDP-43 contains an N-terminal domain (NTD) involved in protein dimerization, 4,5 two tandem RNA recognition motifs (RRM1 and RRM2) responsible for RNA binding, and an intrinsically disordered C-terminal glycine-rich region (G-rich) that exhibits prion-like properties involved in protein-protein interactions and aggregation (Figure 1a). 6-12 Cytoplasmic

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Section: Discussionsupporting

confidence: 62%

Frontotemporal dementia‐linked P112H mutation of TDP‐43 induces protein structural change and impairs its RNA binding function

et al. 2020

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“…To further assess the role of the RRM domains for TDP-43 nuclear localization and export without markedly increasing its molecular weight, we generated a series of constructs expressing V5-tagged wild-type and RRM mutants of TDP-43 ( Fig 6A ), including mutations of phenylalanine residues (5F◊L) and acetylation sites (2K◊Q) that are critical for RNA binding (Buratti & Baralle, 2001; Cohen et al , 2015), and a complete RRM deletion (ΔRRM1-2). Constructs were transiently transfected into a stable HeLa cell line largely depleted of TDP-43 by CRISPR ( Suppl fig 6A-B ) (generously provided by S. Ferguson (Roczniak-Ferguson & Ferguson, 2019)) to avoid potential confounding effects of hetero-oligomerization with endogenous TDP-43. High-content image analysis showed a significant drop in the steady-state N/C ratio of all three RRM mutants, presumably exaggerated for ΔRRM1-2 due to its small size (29 kD) in comparison to the point mutants (45 kD) ( Fig 6A-B ).…”

Section: Resultsmentioning

confidence: 99%

“…A single cell-derived clone of HeLa cells (ATCC) was maintained in OptiMEM (Gibco/ThermoFisher) with 4% FBS and penicillin-streptomycin. HEK293T cells (ATCC) and a monoclonal TDP-43 CRISPR-depleted HeLa cell line (a generous gift from Shawn Ferguson (Roczniak-Ferguson & Ferguson, 2019)), were maintained in DMEM (Gibco/ThermoFisher) with 10% FBS and penicillin-streptomycin. DLD1-wildtype cells (ATCC) and DLD1-NXF1-AID cells (Aksenova et al ., 2020) were maintained in DMEM (Gibco/ThermoFisher) with 10% FBS and penicillin-streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export

Duan

Zaepfel

Aksenova

et al. 2021

Preprint

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Nuclear clearance of the DNA/RNA-binding protein TDP-43 is a pathologic hallmark of amyotrophic lateral sclerosis and frontotemporal dementia that remains unexplained. Moreover, our current understanding of TDP-43 nucleocytoplasmic shuttling does not fully explain the predominantly nuclear localization of TDP-43 in healthy cells. Here, we used permeabilized and live-cell models to investigate TDP-43 nuclear export and the role of RNA in TDP-43 localization. We show that TDP-43 nuclear efflux occurs in low-ATP conditions and independent of active mRNA export, consistent with export by passive diffusion through nuclear pore channels. TDP-43 nuclear residence requires binding to GU-rich nuclear intronic pre-mRNAs, based on the induction of TDP-43 nuclear efflux by RNase and GU-rich oligomers and TDP-43 nuclear retention conferred by pre-mRNA splicing inhibitors. Mutation of TDP-43 RNA recognition motifs disrupts TDP-43 nuclear accumulation and abolishes transcriptional blockade-induced TDP-43 nuclear efflux, demonstrating strict dependence of TDP-43 nuclear localization on RNA binding. Thus, the nuclear abundance of GU-rich intronic pre-mRNAs, as dictated by the balance of transcription and pre-mRNA processing, regulates TDP-43 nuclear sequestration and availability for passive nuclear export.

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“…TDP-43 is a 414-amino acid nucleic-acid binding protein containing an N-terminal ubiquitin-like fold and two RNA recognition motifs, followed by a glycine-rich, low-sequence complexity prion-like domain at the C terminus. Although TDP-43 was originally described to harbor transcription repressor activity (12), the functions of TDP-43 has since been expanded to include a wide range of biological processes (7,(13)(14)(15), including biogenesis and processing of coding and noncoding RNAs (7,(13)(14)(15), mRNA transport (16), translation (17,18), mitochondrial function (19), autophagy (20,21), and organelle homeostasis (22). While TDP-43 is expressed in all major cell types within the central nervous system (CNS) (23), it is not known whether TDP-43 has cell-type-specific functions.…”

mentioning

confidence: 99%

Loss of TDP-43 in astrocytes leads to motor deficits by triggering A1-like reactive phenotype and triglial dysfunction

Peng

Agrawal

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Patients with amyotrophic lateral sclerosis (ALS) can have abnormal TDP-43 aggregates in the nucleus and cytosol of their surviving neurons and glia. Although accumulating evidence indicates that astroglial dysfunction contributes to motor neuron degeneration in ALS, the normal function of TDP-43 in astrocytes are largely unknown, and the role of astroglial TDP-43 loss to ALS pathobiology remains to be clarified. Herein, we show that TDP-43–deleted astrocytes exhibit a cell-autonomous increase in GFAP immunoreactivity without affecting astrocyte or microglia proliferation. At the transcriptomic level, TDP-43–deleted astrocytes resemble A1-reactive astrocytes and induce microglia to increase C1q expression. These astrocytic changes do not cause loss of motor neurons in the spinal cord or denervation at the neuromuscular junction. In contrast, there is a selective reduction of mature oligodendrocytes, but not oligodendrocyte precursor cells, suggesting triglial dysfunction mediated by TDP-43 loss in astrocytes. Moreover, mice with astroglial TDP-43 deletion develop motor, but not sensory, deficits. Taken together, our results demonstrate that TDP-43 is required to maintain the protective functions of astrocytes relevant to the development of motor deficits in mice.

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Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis

Cited by 4 publications

References 81 publications

Frontotemporal dementia‐linked P112H mutation of TDP‐43 induces protein structural change and impairs its RNA binding function

Frontotemporal dementia‐linked P112H mutation of TDP‐43 induces protein structural change and impairs its RNA binding function

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export

Loss of TDP-43 in astrocytes leads to motor deficits by triggering A1-like reactive phenotype and triglial dysfunction

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