Clinical and neuropathological features of ALS/FTD with TIA1 mutations

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent a disease continuum with common genetic causes and molecular pathology. We recently identified mutations in the T-cell restricted intracellular antigen-1 (TIA1) protein as a cause of ALS +/− FTD. TIA1 is an RNA-binding protein containing a low complexity domain (LCD) that promotes the assembly of membrane-less organelles, such as stress granules (SG). Whole exome sequencing of two family members with fALS/FTD revealed a novel missense mutation in the TIA1 LCD (P362L). Subsequent screening identified five more TIA1 mutations in six additional ALS patients, but none in controls. All mutation carriers presented with weakness, behavioral abnormalities or language impairments and had a final diagnosis of ALS +/− FTD. Autopsy on five TIA1 mutation carriers showed widespread neurodegeneration with TDP-43 pathology. Round eosinophilic inclusions in lower motor neurons were a consistent feature. Cellular assays revealed abnormal SG dynamics in the presence of TIA1 mutations. In summary, missense mutations in the LCD of TIA1 are a newly recognized cause of ALS/FTD with TDP-43 pathology and strengthen the role of RNA metabolism in the pathogenesis in this disease.

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Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

10.21203/rs.3.rs-933368/v1 ◽

2021 ◽

Author(s):

Shan Lu ◽

Jiaojiao Hu ◽

Bankhole Aladesuyi ◽

Alexander Goginashvili ◽

Sonia Vazquez-Sanchez ◽

...

Keyword(s):

Phase Separation ◽

Heat Shock ◽

Motor Neurons ◽

Rna Binding ◽

Isotope Labeling ◽

Small Heat Shock Protein ◽

Proteasome Inhibition ◽

Low Complexity ◽

Spinal Motor Neurons ◽

Hsp70 Family

Abstract While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70’s ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43’s RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

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Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

10.1101/2021.10.14.464447 ◽

2021 ◽

Author(s):

Shan Lu ◽

Jiaojiao Hu ◽

Olubankole Aladesuyi Arogundade ◽

Alexander Goginashvili ◽

Sonia Vazquez-Sanchez ◽

...

Keyword(s):

Phase Separation ◽

Heat Shock ◽

Motor Neurons ◽

Rna Binding ◽

Isotope Labeling ◽

Small Heat Shock Protein ◽

Proteasome Inhibition ◽

Low Complexity ◽

Spinal Motor Neurons ◽

Hsp70 Family

While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70's ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43's RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

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In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽

10.1093/genetics/159.4.1617 ◽

2001 ◽

Vol 159 (4) ◽

pp. 1617-1630

Author(s):

Suk-Won Jin ◽

Nancy Arno ◽

Adam Cohen ◽

Amy Shah ◽

Qijin Xu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Germ Cell ◽

Rna Binding ◽

Dominant Negative ◽

Missense Mutations ◽

Biochemical Tests ◽

Cell Fates ◽

Cytoplasmic Polyadenylation ◽

Binding Domains ◽

Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00087-15 ◽

2015 ◽

Vol 35 (14) ◽

pp. 2385-2399 ◽

Cited By ~ 12

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.

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RNA Binding Proteins and the Pathogenesis of Frontotemporal Lobar Degeneration

Annual Review of Pathology Mechanisms of Disease ◽

10.1146/annurev-pathmechdis-012418-012955 ◽

2019 ◽

Vol 14 (1) ◽

pp. 469-495 ◽

Cited By ~ 9

Author(s):

Jeffrey W. Hofmann ◽

William W. Seeley ◽

Eric J. Huang

Keyword(s):

Frontotemporal Lobar Degeneration ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Low Complexity ◽

Compelling Evidence ◽

Abnormal Protein ◽

Early Onset Dementia ◽

Cellular Pathways ◽

Liquid Liquid Phase Separation

Frontotemporal dementia is a group of early onset dementia syndromes linked to underlying frontotemporal lobar degeneration (FTLD) pathology that can be classified based on the formation of abnormal protein aggregates involving tau and two RNA binding proteins, TDP-43 and FUS. Although elucidation of the mechanisms leading to FTLD pathology is in progress, recent advances in genetics and neuropathology indicate that a majority of FTLD cases with proteinopathy involving RNA binding proteins show highly congruent genotype–phenotype correlations. Specifically, recent studies have uncovered the unique properties of the low-complexity domains in RNA binding proteins that can facilitate liquid–liquid phase separation in the formation of membraneless organelles. Furthermore, there is compelling evidence that mutations in FTLD genes lead to dysfunction in diverse cellular pathways that converge on the endolysosomal pathway, autophagy, and neuroinflammation. Together, these results provide key mechanistic insights into the pathogenesis and potential therapeutic targets of FTLD.

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Image-based deep learning reveals the responses of human motor neurons to stress and ALS

10.1101/2021.04.08.439054 ◽

2021 ◽

Author(s):

Colombine Verzat ◽

Jasmine Harley ◽

Rickie Patani ◽

Raphaëlle Luisier

Keyword(s):

Deep Learning ◽

Motor Neurons ◽

Rna Binding ◽

Relevant Information ◽

Morphological Attributes ◽

Fluorescent Markers ◽

Human Motor ◽

Microscopy Data ◽

Key Aspects ◽

Lateral Sclerosis

SUMMARYAlthough morphological attributes of cells and their substructures are recognized readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. In this study we integrate multichannel fluorescence high-content microscopy data with deep-learning imaging methods to reveal - directly from unsegmented images - novel neurite-associated morphological perturbations associated with (ALS-causing) VCP-mutant human motor neurons (MNs). Surprisingly, we reveal that previously unrecognized disease-relevant information is withheld in broadly used and often considered ‘generic’ biological markers of nuclei (DAPI) and neurons (βIII-tubulin). Additionally, we identify changes within the information content of ALS-related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP-mutant MN cultures. Furthermore, by analyzing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. Our study therefore reveals disease-relevant information contained in a range of both generic and more specific fluorescent markers, and establishes the use of image-based deep learning methods for rapid, automated and unbiased testing of biological hypotheses.

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RNA Is a Double-Edged Sword in ALS Pathogenesis

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.708181 ◽

2021 ◽

Vol 15 ◽

Author(s):

Benjamin L. Zaepfel ◽

Jeffrey D. Rothstein

Keyword(s):

Motor Neurons ◽

Cortical Neurons ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Metabolism ◽

Small Subset ◽

Toxic Rna ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that affects upper and lower motor neurons. Familial ALS accounts for a small subset of cases (<10–15%) and is caused by dominant mutations in one of more than 10 known genes. Multiple genes have been causally or pathologically linked to both ALS and frontotemporal dementia (FTD). Many of these genes encode RNA-binding proteins, so the role of dysregulated RNA metabolism in neurodegeneration is being actively investigated. In addition to defects in RNA metabolism, recent studies provide emerging evidence into how RNA itself can contribute to the degeneration of both motor and cortical neurons. In this review, we discuss the roles of altered RNA metabolism and RNA-mediated toxicity in the context of TARDBP, FUS, and C9ORF72 mutations. Specifically, we focus on recent studies that describe toxic RNA as the potential initiator of disease, disease-associated defects in specific RNA metabolism pathways, as well as how RNA-based approaches can be used as potential therapies. Altogether, we highlight the importance of RNA-based investigations into the molecular progression of ALS, as well as the need for RNA-dependent structural studies of disease-linked RNA-binding proteins to identify clear therapeutic targets.

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Cytoplasmic accumulation of FUS triggers early behavioral alterations linked to cortical neuronal hyperactivity and defects in inhibitory synapses

10.1101/2020.06.09.141556 ◽

2020 ◽

Author(s):

Jelena Scekic-Zahirovic ◽

Inmaculada Sanjuan-Ruiz ◽

Vanessa Kan ◽

Salim Megat ◽

Pierre De Rossi ◽

...

Keyword(s):

Neuronal Activity ◽

Rna Binding ◽

Gene Mutations ◽

Neuronal Loss ◽

Inhibitory Neurons ◽

Inhibitory Synapses ◽

Behavioral Alterations ◽

Behavioral Abnormalities ◽

Cytoplasmic Accumulation

AbstractGene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS, lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a profound increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo. Importantly, RNAseq analysis suggested involvement of defects in inhibitory neurons, that was confirmed by ultrastructural and morphological defects of inhibitory synapses and increased synaptosomal levels of mRNAs involved in inhibitory neurotransmission. Thus, cytoplasmic FUS triggers inhibitory synaptic deficits, leading to increased neuronal activity and behavioral phenotypes. FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, but also in other neurodegenerative diseases with FUS mislocalization.

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Tumour risks and genotype–phenotype correlations associated with germline variants in succinate dehydrogenase subunit genes SDHB, SDHC and SDHD

Journal of Medical Genetics ◽

10.1136/jmedgenet-2017-105127 ◽

2018 ◽

Vol 55 (6) ◽

pp. 384-394 ◽

Cited By ~ 56

Author(s):

Katrina A Andrews ◽

David B Ascher ◽

Douglas Eduardo Valente Pires ◽

Daniel R Barnes ◽

Lindsey Vialard ◽

...

Keyword(s):

Succinate Dehydrogenase ◽

Phenotypic Variability ◽

Cohort Analysis ◽

Cumulative Risk ◽

Clinical Information ◽

Missense Mutations ◽

Pathogenic Variants ◽

Mutation Carriers ◽

Kaplan Meier ◽

Age Related

BackgroundGermline pathogenic variants in SDHB/SDHC/SDHD are the most frequent causes of inherited phaeochromocytomas/paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. We estimate penetrance for symptomatic tumours and elucidate genotype–phenotype correlations in a large cohort of SDHB/SDHC/SDHD mutation carriers.MethodsA retrospective survey of 1832 individuals referred for genetic testing due to a personal or family history of phaeochromocytoma/paraganglioma. 876 patients (401 previously reported) had a germline mutation in SDHB/SDHC/SDHD (n=673/43/160). Tumour risks were correlated with in silico structural prediction analyses.ResultsTumour risks analysis provided novel penetrance estimates and genotype–phenotype correlations. In addition to tumour type susceptibility differences for individual genes, we confirmed that the SDHD:p.Pro81Leu mutation has a distinct phenotype and identified increased age-related tumour risks with highly destabilising SDHB missense mutations. By Kaplan-Meier analysis, the penetrance (cumulative risk of clinically apparent tumours) in SDHB and (paternally inherited) SDHD mutation-positive non-probands (n=371/67 with detailed clinical information) by age 60 years was 21.8% (95% CI 15.2% to 27.9%) and 43.2% (95% CI 25.4% to 56.7%), respectively. Risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4.2%(95% CI 1.1% to 7.2%). With retrospective cohort analysis to adjust for ascertainment, cumulative tumour risks for SDHB mutation carriers at ages 60 years and 80 years were 23.9% (95% CI 20.9% to 27.4%) and 30.6% (95% CI 26.8% to 34.7%).ConclusionsOverall risks of clinically apparent tumours for SDHB mutation carriers are substantially lower than initially estimated and will improve counselling of affected families. Specific genotype–tumour risk associations provides a basis for novel investigative strategies into succinate dehydrogenase-related mechanisms of tumourigenesis and the development of personalised management for SDHB/SDHC/SDHD mutation carriers.

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