scholarly journals The Impact of ALS-Associated Genes hnRNPA1, MATR3, VCP and UBQLN2 on the Severity of TDP-43 Aggregation

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1791
Author(s):  
Ana Bajc Česnik ◽  
Helena Motaln ◽  
Boris Rogelj
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Rna Binding ◽  
Neurodegenerative Disorder ◽  
Low Complexity ◽  
Wild Type ◽  
Disease Heterogeneity ◽  
Cytoplasmic Inclusions ◽  
The Impact ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder, characterized by cytoplasmic inclusions of RNA-binding protein TDP-43. Despite decades of research and identification of more than 50 genes associated with amyotrophic lateral sclerosis (ALS), the cause of TDP-43 translocation from the nucleus and its aggregation in the cytoplasm still remains unknown. Our study addressed the impact of selected ALS-associated genes on TDP-43 aggregation behavior in wild-type and aggregation prone TDP-43 in vitro cell models. These were developed by deleting TDP-43 nuclear localization signal and stepwise shortening its low-complexity region. The SH-SY5Y cells were co-transfected with the constructs of aggregation-prone TDP-43 and wild-type or mutant ALS-associated genes hnRNPA1, MATR3, VCP or UBQLN2. The investigated genes displayed a unique impact on TDP-43 aggregation, generating distinct types of cytoplasmic inclusions, similar to those already described as resembling prion strains, which could represent the basis for neurodegenerative disease heterogeneity.

scholarly journals RBM45 Modulates the Antioxidant Response in Amyotrophic Lateral Sclerosis through Interactions with KEAP1

2015 ◽  
Vol 35 (14) ◽  
pp. 2385-2399 ◽  
Author(s):  
Nadine Bakkar ◽  
Arianna Kousari ◽  
Tina Kovalik ◽  
Yang Li ◽  
Robert Bowser
Keyword(s):  
Oxidative Stress ◽  
Spinal Cord ◽  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Antioxidant Response ◽  
Cytoplasmic Inclusions ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective loss of motor neurons. Various factors contribute to the disease, including RNA binding protein dysregulation and oxidative stress, but their exact role in pathogenic mechanisms remains unclear. We have recently linked another RNA binding protein, RBM45, to ALS via increased levels of protein in the cerebrospinal fluid of ALS patients and its localization to cytoplasmic inclusions in ALS motor neurons. Here we show RBM45 nuclear exit in ALS spinal cord motor neurons compared to controls, a phenotype recapitulatedin vitroin motor neurons treated with oxidative stressors. We find that RBM45 binds and stabilizes KEAP1, the inhibitor of the antioxidant response transcription factor NRF2. ALS lumbar spinal cord lysates similarly show increased cytoplasmic binding of KEAP1 and RBM45. Binding of RBM45 to KEAP1 impedes the protective antioxidant response, thus contributing to oxidative stress-induced cellular toxicity. Our findings thus describe a novel link between a mislocalized RNA binding protein implicated in ALS (RBM45) and dysregulation of the neuroprotective antioxidant response seen in the disease.

scholarly journals TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009260
Author(s):  
Marta Giannini ◽  
Aleix Bayona-Feliu ◽  
Daisy Sproviero ◽  
Sonia I. Barroso ◽  
Cristina Cereda ◽  
...  
Keyword(s):  
Dna Damage ◽  
Amyotrophic Lateral Sclerosis ◽  
Rna Binding ◽  
Neurodegenerative Disorder ◽  
Genome Instability ◽  
Neuronal Model ◽  
Dna Breaks ◽  
Dna And Rna ◽  
Double Strand Dna Breaks ◽  
Lateral Sclerosis

TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. After showing with the non-neuronal model of HeLa cells that TDP-43 depletion increases R loops and associated genome instability, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, R loop-dependent increased DSBs and Fanconi Anemia repair centers. These results uncover a new role of TDP-43 in the control of co-transcriptional R loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.

Amyotrophic Lateral Sclerosis

2018 ◽  
Keyword(s):  
Quality Of Life ◽  
Amyotrophic Lateral Sclerosis ◽  
Health Care Professionals ◽  
New Technologies ◽  
Neurodegenerative Disorder ◽  
Well Being ◽  
Psychological Well Being ◽  
The Impact ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis: Understanding and optimizing quality of life and psychological well-being presents a comprehensive and up-to-date review of the enhancement of the lives of people with amyotrophic lateral sclerosis (ALS) and their caregivers. ALS is a progressive, fatal neurodegenerative disorder. No current medical therapy can reverse or stop its progression, and the promotion of quality of life and psychological well-being is a central component of ALS care. Health care professionals who work in this field should incorporate attention to psychological, emotional, and relational aspects of the disease into their approach to care. This book provides some of the knowledge and direction necessary for optimizing the quality of care for individuals with ALS and their caregivers. Topics discussed include an ALS-centred view of quality of life, depressive features, anxiety, resilience, cognitive impairment, complementary and alternative medicines, and psychological research. Specific elements of ALS, such as end-of-life concerns and bulbar dysfunction, are described through the lens of their psychological impact. There is extensive discussion of the development of new psychological treatments, as well as the impact and incorporation of new technologies, with the goal of fostering optimal quality of life and psychological well-being as key parts of a holistic approach to care for the patients and for those who are close to such individuals.

scholarly journals Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Yoshiaki Furukawa
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Superoxide Dismutase ◽  
Motor Neurons ◽  
Wild Type ◽  
Sporadic Als ◽  
Familial Form ◽  
Recent Developments ◽  
Lateral Sclerosis

Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS). While it remains controversial how SOD1 mutations lead to onset and progression of the disease, manyin vitroandin vivostudies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations insod1gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.

scholarly journals Positional motif analysis reveals the extent of specificity of protein-RNA interactions observed by CLIP

2021 ◽  
Author(s):  
Klara Kuret ◽  
Aram Gustav Amalietti ◽  
Jernej Ule
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Data Interpretation ◽  
Low Complexity ◽  
Motif Analysis ◽  
Link Type ◽  
Motif Enrichment ◽  
The Impact

AbstractBackgroundCrosslinking and immunoprecipitation (CLIP) is a method used to identify in vivo RNA– protein binding sites on a transcriptome-wide scale. With the increasing amounts of available data for RNA-binding proteins (RBPs), it is important to understand to what degree the enriched motifs specify the RNA binding profiles of RBPs in cells.ResultsWe develop positionally-enriched k-mer analysis (PEKA), a computational tool for efficient analysis of enriched motifs from individual CLIP datasets, which minimises the impact of technical and regional genomic biases by internal data normalisation. We cross-validate PEKA with mCross, and show that background correction by size-matched input doesn’t generally improve the specificity of detected motifs. We identify motif classes with common enrichment patterns across eCLIP datasets and across RNA regions, while also observing variations in the specificity and the extent of motif enrichment across eCLIP datasets, between variant CLIP protocols, and between CLIP and in vitro binding data. Thereby we gain insights into the contributions of technical and regional genomic biases to the enriched motifs, and find how motif enrichment features relate to the domain composition and low-complexity regions (LCRs) of the studied proteins.ConclusionsOur study provides insights into the overall contributions of regional binding preferences, protein domains and LCRs to the specificity of protein-RNA interactions, and shows the value of cross-motif and cross-RBP comparison for data interpretation. Our results are presented for exploratory analysis via an online platform in an RBP-centric and motif-centric manner (https://imaps.goodwright.com/apps/peka/). PEKA is available from https://github.com/ulelab/peka.

scholarly journals Characteristic Features of FUS Inclusions in Spinal Motor Neurons of Sporadic Amyotrophic Lateral Sclerosis

2020 ◽  
Vol 79 (4) ◽  
pp. 370-377 ◽  
Author(s):  
Kensuke Ikenaka ◽  
Shinsuke Ishigaki ◽  
Yohei Iguchi ◽  
Kaori Kawai ◽  
Yusuke Fujioka ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Rna Binding ◽  
Binding Protein ◽  
Specific Antigen ◽  
Antigen Retrieval ◽  
Technical Limitation ◽  
Cytoplasmic Inclusions ◽  
Sporadic Als ◽  
Lateral Sclerosis

Abstract Alterations of RNA metabolism caused by mutations in RNA-binding protein genes, such as transactivating DNA-binding protein-43 (TDP-43) and fused in sarcoma (FUS), have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Unlike the accumulation of TDP43, which is accepted as a pathological hall mark of sporadic ALS (sALS), FUS pathology in sALS is still under debate. Although immunoreactive inclusions of FUS have been detected in sALS patients previously, the technical limitation of signal detection, including the necessity of specific antigen retrieval, restricts our understanding of FUS-associated ALS pathology. In this study, we applied a novel detection method using a conventional antigen retrieval technique with Sudan Black B treatment to identify FUS-positive inclusions in sALS patients. We classified pathological motor neurons into 5 different categories according to the different aggregation characteristics of FUS and TDP-43. Although the granular type was more dominant for inclusions with TDP-43, the skein-like type was more often observed in FUS-positive inclusions, suggesting that these 2 proteins undergo independent aggregation processes. Moreover, neurons harboring FUS-positive inclusions demonstrated substantially reduced expression levels of dynactin-1, a retrograde motor protein, indicating that perturbation of nucleocytoplasmic transport is associated with the formation of cytoplasmic inclusions of FUS in sALS.

scholarly journals Combined Tissue-Fluid Proteomics to Unravel Phenotypic Variability in Amyotrophic Lateral Sclerosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Emanuela Leoni ◽  
Michael Bremang ◽  
Vikram Mitra ◽  
Irene Zubiri ◽  
Stephan Jung ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Proteomic Analysis ◽  
Rna Binding ◽  
Mononuclear Cells ◽  
Neurodegenerative Disorder ◽  
Phenotypic Variability ◽  
Tissue Fluid ◽  
Case Control Studies ◽  
Variable Site ◽  
Lateral Sclerosis

Abstract The lack of biomarkers for early diagnosis, clinical stratification and to monitor treatment response has hampered the development of new therapies for amyotrophic lateral sclerosis (ALS), a clinically heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. To identify new biomarkers and therapeutic targets, two separate proteomic workflows were applied to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS. Conventional multiplex (TMT) proteomic analysis of peripheral blood mononuclear cells (PBMCs) was performed alongside a recently introduced method to profile neuronal-derived proteins in plasma using brain tissue-enhanced isobaric tagging (TMTcalibrator). The combined proteomic analysis allowed the detection of regulated proteins linked to ALS pathogenesis (RNA-binding protein FUS, superoxide dismutase Cu-Zn and neurofilaments light polypeptide) alongside newly identified candidate biomarkers (myosin-9, fructose-bisphosphate aldolase and plectin). In line with the proteomic results, orthogonal immunodetection showed changes in neurofilaments and ApoE in bulbar versus limb onset fast progressing ALS. Functional analysis of significantly regulated features showed enrichment of pathways involved in regulation of the immune response, Rho family GTPases, semaphorin and integrin signalling. Our cross-phenotype investigation of PBMCs and plasma/brain proteins provides a more sensitive biomarker exploratory platform than conventional case-control studies in a single matrix. The reported regulated proteins may represent novel biomarker candidates and potentially druggable targets.

scholarly journals Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis

Brain ◽  
2019 ◽  
Vol 142 (9) ◽  
pp. 2572-2580 ◽  
Author(s):  
Giulia E Tyzack ◽  
Raphaelle Luisier ◽  
Doaa M Taha ◽  
Jacob Neeves ◽  
Miha Modic ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Nuclear Export ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Spinal Cord Tissue ◽  
Cytoplasmic Inclusions ◽  
Sporadic Als ◽  
Lateral Sclerosis ◽  
Fus Protein

Abstract Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

scholarly journals ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells

2020 ◽  
Author(s):  
Julia F. Riley ◽  
Heidi Hehnly ◽  
Carlos A. Castañeda
Keyword(s):  
Protein Quality ◽  
Wild Type ◽  
Disease Mutations ◽  
Ubiquitin Binding ◽  
Condensate Formation ◽  
The Impact ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation ◽  
In Cells

AbstractMutations in Ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells is unknown. Here, we employ live-cell imaging with photokinetic analysis to investigate how five patient-derived ALS-linked mutations in UBQLN2 impact stress-induced UBQLN2 condensate assembly and condensate material properties. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, cells transfected with mutant UBQLN2 contain fewer stress-induced UBQLN2 condensates than those with wild-type UBQLN2. Most strikingly, ectopically expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

scholarly journals The review of the methods to obtain non-neuronal cells to study glial influence on Amyotrophic Lateral Sclerosis pathophysiology at molecular level in vitro

2010 ◽  
Vol 25 (3) ◽  
pp. 281-289 ◽  
Author(s):  
Juliana Milani Scorisa ◽  
Tatiana Duobles ◽  
Gabriela Pintar de Oliveira ◽  
Jessica Ruivo Maximino ◽  
Gerson Chadi
Keyword(s):  
Spinal Cord ◽  
Amyotrophic Lateral Sclerosis ◽  
Transgenic Mice ◽  
Primary Cultures ◽  
Neuronal Cells ◽  
Disease Onset ◽  
Wild Type ◽  
Rt Pcr ◽  
Lateral Sclerosis

PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that displays a rapid evolution. Current treatments have failed to revert clinical symptoms because the mechanisms involved in the death of motoneuron are still unknown. Recent publications have put non-neuronal cells, particularly, astrocyte and microglia, in the scenario of pathophisiology of the disease. Animal models for ALS, particularly transgenic mice expressing the human SOD1 gene with a G93A mutation (hSOD1), are available and display the phenotype of the disease at cellular and clinical levels. However, it is a lack of detailed information regarding the methods to study the disease in vitro to better understand the contribution of non-neuronal cells in the onset and progression of the pathology. METHODS: Colonies of Swiss mice and transgenic mice expressing hSOD1 mutation as well as non-transgenic controls (wild-type) were amplified after a genotyping evaluation. Disease progression was followed behaviorally and mortality was registered. Highly purified primary cultures of astrocytes and microglia from mouse spinal cord were obtained. Cells were identified by means of GFAP and CD11B immunocytochemistry. The purity of astroglial and microglial cell cultures was also accompanied by means of Western blot and RT-PCR analyses employing a number of markers. RESULTS: The disease onset was about 105 days and the majority of transgenic mice displayed the disease symptoms by 125 days of age and reached the endpoint 20 days later. A substantial motor weakens was registered in the transgenic mice compared to wild-type at the end point. Immunocytochemical, biochemical and RT-PCR analyses demonstrated a highly purified primary cultures of spinal cord astrocytes and microglia. CONCLUSION: It is possible to achieve highly purified primary cultures of spinal cord astrocytes and microglia to be employed in cellular and molecular analyses of the influence of such non-neuronal cells in the pathophysiology of ALS.

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