C9orf72-Associated Dipeptide Repeat Expansions Perturb ER-Golgi Vesicular Trafficking, Inducing Golgi Fragmentation and ER Stress, in ALS/FTD.

Hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are debilitating neurodegenerative conditions affecting either motor neurons (ALS) in the brain and spinal cord or neurons in the frontal and/or temporal cortical lobes (FTD). HREs undergo repeat-associated non-ATG (RAN) translation on both sense and anti-sense strands, generating five distinct dipeptide repeat proteins (DPRs), poly-GA, -GR, -GP, -PA and -PR. Perturbed proteostasis is well-recognised in ALS pathogenesis, including processes affecting the endoplasmic reticulum (ER) and Golgi compartments. However, these mechanisms have not been well characterised for C9orf72-mediated ALS/FTD. In this study we demonstrate that C9orf72 DPRs polyGA, polyGR and polyGP (× 40 repeats) disrupt secretory protein transport from the ER to the Golgi apparatus in neuronal cells. Consistent with this finding, these DPRs also induce fragmentation of the Golgi apparatus, activate ER stress, and inhibit the formation of the omegasome, the precursor of the autophagosome that originates from ER membranes. We also demonstrate Golgi fragmentation in cells undergoing RAN translation that express polyGP. Furthermore, dysregulated ER-Golgi transport was confirmed in C9orf72 patient dermal fibroblasts. Evidence of aberrant ER-derived vesicles in spinal cord motor neurons from C9orf72 ALS patients compared to controls was also obtained. These data thus confirm that ER proteostasis and ER-Golgi transport is perturbed in C9orf72-ALS in the absence of protein over-expression. Hence this study identifies novel molecular mechanisms associated with the ER and Golgi compartments induced by the C9orf72 HRE.

Molecular hallmarks of ageing in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal, severely debilitating and rapidly progressing disorder affecting motor neurons in the brain, brainstem, and spinal cord. Unfortunately, there are few effective treatments, thus there remains a critical need to find novel interventions that can mitigate against its effects. Whilst the aetiology of ALS remains unclear, ageing is the major risk factor. Ageing is a slowly progressive process marked by functional decline of an organism over its lifespan. However, it remains unclear how ageing promotes the risk of ALS. At the molecular and cellular level there are specific hallmarks characteristic of normal ageing. These hallmarks are highly inter-related and overlap significantly with each other. Moreover, whilst ageing is a normal process, there are striking similarities at the molecular level between these factors and neurodegeneration in ALS. Nine ageing hallmarks were originally proposed: genomic instability, loss of telomeres, senescence, epigenetic modifications, dysregulated nutrient sensing, loss of proteostasis, mitochondrial dysfunction, stem cell exhaustion, and altered inter-cellular communication. However, these were recently (2023) expanded to include dysregulation of autophagy, inflammation and dysbiosis. Hence, given the latest updates to these hallmarks, and their close association to disease processes in ALS, a new examination of their relationship to pathophysiology is warranted. In this review, we describe possible mechanisms by which normal ageing impacts on neurodegenerative mechanisms implicated in ALS, and new therapeutic interventions that may arise from this.

ALS/FTD-associated mutation in cyclin F inhibits ER-Golgi trafficking, inducing ER stress, ERAD and Golgi fragmentation.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.

Redox dysregulation as a driver for DNA damage and its relationship to neurodegenerative diseases.

Redox homeostasis refers to the balance between the production of reactive oxygen species (ROS) as well as reactive nitrogen species (RNS), and their elimination by antioxidants. It is linked to all important cellular activities and oxidative stress is a result of imbalance between pro-oxidants and antioxidant species. Oxidative stress perturbs many cellular activities, including processes that maintain the integrity of DNA. Nucleic acids are highly reactive and therefore particularly susceptible to damage. The DNA damage response detects and repairs these DNA lesions. Efficient DNA repair processes are therefore essential for maintaining cellular viability, but they decline considerably during aging. DNA damage and deficiencies in DNA repair are increasingly described in age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington's disease. Furthermore, oxidative stress has long been associated with these conditions. Moreover, both redox dysregulation and DNA damage increase significantly during aging, which is the biggest risk factor for neurodegenerative diseases. However, the links between redox dysfunction and DNA damage, and their joint contributions to pathophysiology in these conditions, are only just emerging. This review will discuss these associations and address the increasing evidence for redox dysregulation as an important and major source of DNA damage in neurodegenerative disorders. Understanding these connections may facilitate a better understanding of disease mechanisms, and ultimately lead to the design of better therapeutic strategies based on preventing both redox dysregulation and DNA damage.

The Role of DNA Damage in Neural Plasticity in Physiology and Neurodegeneration.

Damage to DNA is generally considered to be a harmful process associated with aging and aging-related disorders such as neurodegenerative diseases that involve the selective death of specific groups of neurons. However, recent studies have provided evidence that DNA damage and its subsequent repair are important processes in the physiology and normal function of neurons. Neurons are unique cells that form new neural connections throughout life by growth and re-organisation in response to various stimuli. This "plasticity" is essential for cognitive processes such as learning and memory as well as brain development, sensorial training, and recovery from brain lesions. Interestingly, recent evidence has suggested that the formation of double strand breaks (DSBs) in DNA, the most toxic form of damage, is a physiological process that modifies gene expression during normal brain activity. Together with subsequent DNA repair, this is thought to underlie neural plasticity and thus control neuronal function. Interestingly, neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, frontotemporal dementia, and Huntington's disease, manifest by a decline in cognitive functions, which are governed by plasticity. This suggests that DNA damage and DNA repair processes that normally function in neural plasticity may contribute to neurodegeneration. In this review, we summarize current understanding about the relationship between DNA damage and neural plasticity in physiological conditions, as well as in the pathophysiology of neurodegenerative diseases.

DNA Damage, Defective DNA Repair, and Neurodegeneration in Amyotrophic Lateral Sclerosis.

DNA is under constant attack from both endogenous and exogenous sources, and when damaged, specific cellular signalling pathways respond, collectively termed the "DNA damage response." Efficient DNA repair processes are essential for cellular viability, although they decline significantly during aging. Not surprisingly, DNA damage and defective DNA repair are now increasingly implicated in age-related neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). ALS affects both upper and lower motor neurons in the brain, brainstem and spinal cord, leading to muscle wasting due to denervation. DNA damage is increasingly implicated in the pathophysiology of ALS, and interestingly, the number of DNA damage or repair proteins linked to ALS is steadily growing. This includes TAR DNA binding protein 43 (TDP-43), a DNA/RNA binding protein that is present in a pathological form in almost all (97%) cases of ALS. Hence TDP-43 pathology is central to neurodegeneration in this condition. Fused in Sarcoma (FUS) bears structural and functional similarities to TDP-43 and it also functions in DNA repair. Chromosome 9 open reading frame 72 (C9orf72) is also fundamental to ALS because mutations in C9orf72 are the most frequent genetic cause of both ALS and related condition frontotemporal dementia, in European and North American populations. Genetic variants encoding other proteins involved in the DNA damage response (DDR) have also been described in ALS, including FUS, SOD1, SETX, VCP, CCNF, and NEK1. Here we review recent evidence highlighting DNA damage and defective DNA repair as an important mechanism linked to neurodegeneration in ALS.

The Complex Mechanisms by Which Neurons Die Following DNA Damage in Neurodegenerative Diseases.

Human cells are exposed to numerous exogenous and endogenous insults every day. Unlike other molecules, DNA cannot be replaced by resynthesis, hence damage to DNA can have major consequences for the cell. The DNA damage response contains overlapping signalling networks that repair DNA and hence maintain genomic integrity, and aberrant DNA damage responses are increasingly described in neurodegenerative diseases. Furthermore, DNA repair declines during aging, which is the biggest risk factor for these conditions. If unrepaired, the accumulation of DNA damage results in death to eliminate cells with defective genomes. This is particularly important for postmitotic neurons because they have a limited capacity to proliferate, thus they must be maintained for life. Neuronal death is thus an important process in neurodegenerative disorders. In addition, the inability of neurons to divide renders them susceptible to senescence or re-entry to the cell cycle. The field of cell death has expanded significantly in recent years, and many new mechanisms have been described in various cell types, including neurons. Several of these mechanisms are linked to DNA damage. In this review, we provide an overview of the cell death pathways induced by DNA damage that are relevant to neurons and discuss the possible involvement of these mechanisms in neurodegenerative conditions.

TDP-43 is a ubiquitylation substrate of the SCFcyclin F complex.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons in the brain and spinal cord. ALS and frontotemporal dementia (FTD) are overlapping diseases with shared pathological features. Affected neurons of people with ALS and FTD typically contain ubiquitin-immunoreactive inclusions, of which TDP-43 (Tar DNA-binding protein of 43 kDa) is a major component. However, what triggers the formation of these abnormal TDP-43 inclusions is unclear. Previously, we identified CCNF mutations in cohorts of familial and sporadic cases of ALS and FTD. CCNF encodes cyclin F, the substrate-binding component of a multiprotein E3 ubiquitin ligase complex that ubiquitylates and subsequently directs a set of protein substrates for proteasomal degradation. Here, we explored the relationship between cyclin F and TDP-43. METHODS: We used a series of complementary biochemical approaches including immunoprecipitations, in vitro ubiquitylation assays, immunofluorescence imaging and immunocytochemistry. Unpaired student t-tests were used to determine statistical significance of the results. RESULTS: In this study, we demonstrate that that the SCFcyclin F complex directly mediates the poly-ubiquitylation of TDP-43. Importantly, we demonstrate that cyclin F bearing the pathogenic ALS/FTD mutation, S621G, leads to aberrant ubiquitylation of TDP-43 as well as the accumulation of K48-ubiquitylated TDP-43 in neuron-like cells. Furthermore, we demonstrate that a patient carrying the ALS/FTD cyclin FS195R mutation displayed skein-like cytoplasmic TDP-43 aggregates, implying abnormal TDP-43 degradation in a CCNF mutation bearing patient. CONCLUSION: In summary, this study reports a direct ubiquitylation mechanism for TDP-43, revealing important insights into the regulation of cyclin F-mediated TDP-43 turnover and clues towards understanding the molecular origins of the ubiquitylated TDP-43 inclusions that are the hallmark pathological feature in ALS and FTD.

Riluzole does not ameliorate disease caused by cytoplasmic TDP-43 in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease commonly treated with riluzole, a small molecule that may act via modulation of glutamatergic neurotransmission. However, riluzole only modestly extends lifespan for people living with ALS, and its precise mechanisms of action remain unclear. Most ALS cases are characterised by accumulation of cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43), and understanding the effects of riluzole in models that closely recapitulate TDP-43 pathology may provide insights for development of improved therapeutics. We therefore investigated the effects of riluzole in female transgenic mice that inducibly express nuclear localisation sequence (NLS)-deficient human TDP-43 in neurons (NEFH-tTA/tetO-hTDP-43ΔNLS, 'rNLS8', mice). Riluzole treatment from the first day of hTDP-43ΔNLS expression did not alter disease onset, weight loss or performance on multiple motor behavioural tasks. Riluzole treatment also did not alter TDP-43 protein levels, solubility or phosphorylation. Although we identified a significant decrease in GluA2 and GluA3 proteins in the cortex of rNLS8 mice, riluzole did not ameliorate this disease-associated molecular phenotype. Likewise, riluzole did not alter the disease-associated atrophy of hindlimb muscle in rNLS8 mice. Finally, riluzole treatment beginning after disease onset in rNLS8 mice similarly had no effect on progression of late-stage disease or animal survival. Together, we demonstrate specific glutamatergic receptor alterations and muscle fibre-type changes reminiscent of ALS in female rNLS8 mice, but riluzole had no effect on these or any other disease phenotypes. Future targeting of pathways related to accumulation of TDP-43 pathology may be needed to develop better treatments for ALS.

The Mitochondrial-associated ER membrane (MAM) compartment and its dysregulation in Amyotrophic Lateral Sclerosis (ALS).

The endoplasmic reticulum (ER) and mitochondria connect at multiple contact sites to form a unique cellular compartment, termed the 'mitochondria-associated ER membranes' (MAMs). MAMs are hubs for signalling pathways that regulate cellular homeostasis and survival, metabolism, and sensitivity to apoptosis. MAMs are therefore involved in vital cellular functions, but they are dysregulated in several human diseases. Whilst MAM dysfunction is increasingly implicated in the pathogenesis of neurodegenerative diseases, its role in amyotrophic lateral sclerosis (ALS) is poorly understood. However, in ALS both ER and mitochondrial dysfunction are well documented pathophysiological events. Moreover, alterations to lipid metabolism in neurons regulate processes linked to neurodegenerative diseases, and a link between dysfunction of lipid metabolism and ALS has also been proposed. In this review we discuss the structural and functional relevance of MAMs in ALS and how targeting MAM could be therapeutically beneficial in this disorder.

Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations.

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.

Correction to: the Cysteine (Cys) Residues Cys-6 and Cys-111 in Mutant Superoxide Dismutase 1 (SOD1) A4V Are Required for Induction of Endoplasmic Reticulum Stress in Amyotrophic Lateral Sclerosis.

The original version of this article unfortunately contained an error in Fig. 3. The image shown for "C57S" was incorrect.

The Cysteine (Cys) Residues Cys-6 and Cys-111 in Mutant Superoxide Dismutase 1 (SOD1) A4V Are Required for Induction of Endoplasmic Reticulum Stress in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of motor neurons. Between 12 and 20% of inherited cases and approximately 1-2% of all cases are caused by mutations in the gene encoding dismutase 1 (SOD1). Mutant SOD1 A4V (alanine to valine) induces endoplasmic reticulum (ER) stress, which is increasingly implicated as a pathway to motor neuron degeneration and death in ALS. However, it remains unclear how ER stress is induced by mutant SOD1 A4V. Previous studies have established that it is induced early in pathophysiology and it precedes the formation of mutant SOD1 inclusions. SOD1 contains four cysteine residues, two of which form an intra-subunit disulphide bond involving Cys-57 and Cys-146. The remaining two cysteines, Cys-6 and Cys-111, remain unpaired and have been implicated in mutant SOD1 aggregation. In this study, we examined the relationship between the SOD1 A4V cysteine residues and aggregation, ER stress induction and toxicity. We report here that mutation of Cys-6 and Cys-111 in mutant SOD1 A4V, but not Cys-57 or Cys-146, ameliorates ER stress, inclusion formation and apoptosis in neuronal cell lines. These results imply that protein misfolding, induced by Cys-6 and Cys-111, is required for these pathological events in neuronal cells.

The Redox Activity of Protein Disulfide Isomerase Inhibits ALS Phenotypes in Cellular and Zebrafish Models.

Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in almost all cases of amyotrophic lateral sclerosis (ALS), and 20% of familial ALS cases are due to mutations in superoxide dismutase 1 (SOD1). Redox regulation is critical to maintain cellular homeostasis, although how this relates to ALS is unclear. Here, we demonstrate that the redox function of protein disulfide isomerase (PDI) is protective against protein misfolding, cytoplasmic mislocalization of TDP-43, ER stress, ER-Golgi transport dysfunction, and apoptosis in neuronal cells expressing mutant TDP-43 or SOD1, and motor impairment in zebrafish expressing mutant SOD1. Moreover, previously described PDI mutants present in patients with ALS (D292N, R300H) lack redox activity and were not protective against ALS phenotypes. Hence, these findings implicate the redox activity of PDI centrally in ALS, linking it to multiple cellular processes. They also imply that therapeutics based on PDI's redox activity will be beneficial in ALS.

CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis.

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

Emerging Evidence Highlighting the Importance of Redox Dysregulation in the Pathogenesis of Amyotrophic Lateral Sclerosis (ALS).

The cellular redox state, or balance between cellular oxidation and reduction reactions, serves as a vital antioxidant defence system that is linked to all important cellular activities. Redox regulation is therefore a fundamental cellular process for aerobic organisms. Whilst oxidative stress is well described in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), other aspects of redox dysfunction and their contributions to pathophysiology are only just emerging. ALS is a fatal neurodegenerative disease affecting motor neurons, with few useful treatments. Hence there is an urgent need to develop more effective therapeutics in the future. Here, we discuss the increasing evidence for redox dysregulation as an important and primary contributor to ALS pathogenesis, which is associated with multiple disease mechanisms. Understanding the connection between redox homeostasis, proteins that mediate redox regulation, and disease pathophysiology in ALS, may facilitate a better understanding of disease mechanisms, and lead to the design of better therapeutic strategies.

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins.

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as ß-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1ß secretion. Both caspase-1 and IL-1ß contribute to disease progression in the mouse SOD1G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1G93A mice microglia do not express NLRP3, and SOD1G93A protein generated IL-1ß in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1G93A mice. We show that both aggregated and soluble SOD1G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1ß cleavage, ASC speck formation, and the secretion of IL-1ß in a dose- and time-dependent manner. Importantly, SOD1G93A was unable to induce IL-1ß secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1ß secretion. Microglial NLRP3 upregulation was also observed in the TDP-43Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1G93A -mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.