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1.
Mol Neurodegener ; 15(1): 51, 2020 09 09.
Article in English | MEDLINE | ID: mdl-32907630

ABSTRACT

BACKGROUND: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response. METHODS: We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage. RESULTS: We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm. CONCLUSIONS: This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , DNA Damage/physiology , DNA End-Joining Repair/physiology , DNA-Binding Proteins/metabolism , Adult , Aged , Animals , Female , Humans , Male , Mice , Middle Aged , Motor Neurons/metabolism
2.
Front Immunol ; 9: 1017, 2018.
Article in English | MEDLINE | ID: mdl-29875767

ABSTRACT

Neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and glaucoma, affect millions of people worldwide. ALS is caused by the loss of motor neurons in the spinal cord, brainstem, and brain, and genetic mutations are responsible for 10% of all ALS cases. Glaucoma is characterized by the loss of retinal ganglion cells and is the most common cause of irreversible blindness. Interestingly, mutations in OPTN, encoding optineurin, are associated with both ALS and glaucoma. Optineurin is a highly abundant protein involved in a wide range of cellular processes, including the inflammatory response, autophagy, Golgi maintenance, and vesicular transport. In this review, we summarize the role of optineurin in cellular mechanisms implicated in neurodegenerative disorders, including neuroinflammation, autophagy, and vesicular trafficking, focusing in particular on the consequences of expression of mutations associated with ALS and glaucoma. This review, therefore showcases the impact of optineurin dysfunction in ALS and glaucoma.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Glaucoma/genetics , Mutation , Transcription Factor TFIIIA/genetics , Animals , Autophagy , Cell Cycle Proteins , Humans , Membrane Transport Proteins , Mice , Transcription Factor TFIIIA/physiology
3.
Hum Mol Genet ; 27(8): 1311-1331, 2018 04 15.
Article in English | MEDLINE | ID: mdl-29409023

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder and mutations in superoxide dismutase 1 (SOD1) account for 20% of familial ALS cases. The aetiology of ALS remains unclear, but protein misfolding, endoplasmic reticulum (ER) stress and neuronal apoptosis are implicated. We previously established that protein disulphide isomerase (PDIA1) is protective against ER stress and apoptosis in neuronal cells expressing mutant SOD1, and recently mutations in PDIA1 and related PDI family member endoplasmic reticulum protein 57 (ERp57/PDIA3), were associated with ALS. Here, we examined whether ERp57 is also protective against mutant SOD1 or whether distinct specificity exists amongst individual PDI family members. Neuronal cells co-expressing SOD1 and ERp57 were examined for inclusion formation, ER stress, ubiquitin proteasome system (UPS) dysfunction and apoptosis. Over-expression of ERp57 inhibited inclusion formation, ER stress, UPS dysfunction and apoptosis, whereas silencing of ERp57 expression enhanced mutant SOD1 inclusion formation, ER stress and toxicity, indicating a protective role for ERp57 against SOD1 misfolding. ERp57 also inhibited the formation of mutant SOD1 inclusions and apoptosis in primary cortical neurons, thus confirming results obtained from cell lines. ERp57 partially co-localized with TAR DNA-binding protein-43 (TDP-43)-positive inclusions in spinal cords from sporadic ALS patients, thus linking ERp57 to protein misfolding in human sporadic disease. Our results therefore imply that ERp57 has a protective role against pathological events induced by mutant SOD1 and they link ERp57 to the misfolding of TDP-43. This study therefore has implications for the design of novel therapeutics based on the activities of the PDI family of proteins.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , DNA-Binding Proteins/genetics , Endoplasmic Reticulum Stress/genetics , Neurons/metabolism , Protein Disulfide-Isomerases/genetics , Superoxide Dismutase-1/genetics , Aged , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Apoptosis , Cell Line , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Embryo, Mammalian , Female , Gene Expression Regulation , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Mutation , Neurons/pathology , Primary Cell Culture , Protein Disulfide-Isomerases/antagonists & inhibitors , Protein Disulfide-Isomerases/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Spinal Cord/metabolism , Spinal Cord/pathology , Superoxide Dismutase-1/metabolism
4.
Front Mol Neurosci ; 10: 119, 2017.
Article in English | MEDLINE | ID: mdl-28539871

ABSTRACT

Protein homeostasis, or proteostasis, has an important regulatory role in cellular function. Protein quality control mechanisms, including protein folding and protein degradation processes, have a crucial function in post-mitotic neurons. Cellular protein quality control relies on multiple strategies, including molecular chaperones, autophagy, the ubiquitin proteasome system, endoplasmic reticulum (ER)-associated degradation (ERAD) and the formation of stress granules (SGs), to regulate proteostasis. Neurodegenerative diseases are characterized by the presence of misfolded protein aggregates, implying that protein quality control mechanisms are dysfunctional in these conditions. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that are now recognized to overlap clinically and pathologically, forming a continuous disease spectrum. In this review article, we detail the evidence for dysregulation of protein quality control mechanisms across the whole ALS-FTD continuum, by discussing the major proteins implicated in ALS and/or FTD. We also discuss possible ways in which protein quality mechanisms could be targeted therapeutically in these disorders and highlight promising protein quality control-based therapeutics for clinical trials.

5.
Acta Neuropathol ; 130(5): 679-97, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26298469

ABSTRACT

Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , rab1 GTP-Binding Proteins/metabolism , Aged , Aged, 80 and over , Amyotrophic Lateral Sclerosis/pathology , Animals , Biological Transport/physiology , COP-Coated Vesicles/metabolism , COP-Coated Vesicles/pathology , Cell Line, Tumor , Cytoplasm/metabolism , Cytoplasm/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/pathology , Female , Golgi Apparatus/pathology , Humans , Male , Mice , Middle Aged , Mutation , Neurons/metabolism , Neurons/pathology , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Spinal Cord/metabolism , Spinal Cord/pathology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1
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