RESUMEN
The assembly of membrane-less organelles such as stress granules (SGs) is emerging as central in helping cells rapidly respond and adapt to stress. Following stress sensing, the resulting global translational shutoff leads to the condensation of stalled mRNAs and proteins into SGs. By reorganizing cytoplasmic contents, SGs can modulate RNA translation, biochemical reactions, and signaling cascades to promote survival until the stress is resolved. While mechanisms for SG disassembly are not widely understood, the resolution of SGs is important for maintaining cell viability and protein homeostasis. Mutations that lead to persistent or aberrant SGs are increasingly associated with neuropathology and a hallmark of several neurodegenerative diseases. Mutations in CLN3 are causative of juvenile neuronal ceroid lipofuscinosis, a rare neurodegenerative disease affecting children also known as Batten disease. CLN3 encodes a transmembrane lysosomal protein implicated in autophagy, endosomal trafficking, metabolism, and response to oxidative stress. Using a HeLa cell model lacking CLN3, we now show that CLN3KO is associated with an altered metabolic profile, reduced global translation, and altered stress signaling. Furthermore, loss of CLN3 function results in perturbations in SG dynamics, resulting in assembly and disassembly defects, and altered expression of the key SG nucleating factor G3BP1. With a growing interest in SG-modulating drugs for the treatment of neurodegenerative diseases, novel insights into the molecular basis of CLN3 Batten disease may reveal avenues for disease-modifying treatments for this debilitating childhood disease.
Asunto(s)
Expresión Génica , Chaperonas Moleculares , Lipofuscinosis Ceroideas Neuronales , Gránulos de Estrés , Humanos , Células HeLa , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/fisiopatología , Gránulos de Estrés/genética , Gránulos de Estrés/patología , Estrés Fisiológico/genética , Transducción de Señal/genética , Expresión Génica/genética , Línea CelularRESUMEN
TDP-43 is an abundant and ubiquitously expressed nuclear protein that becomes dysfunctional in a spectrum of neurodegenerative diseases. TDP-43's ability to phase separate and form/enter biomolecular condensates of varying size and composition is critical for its functionality. Despite the high density of phase-separated assemblies in the nucleus and the nuclear abundance of TDP-43, our understanding of the condensate-TDP-43 relationship in this cellular compartment is only emerging. Recent studies have also suggested that misregulation of nuclear TDP-43 condensation is an early event in the neurodegenerative disease amyotrophic lateral sclerosis. This review aims to draw attention to the nuclear facet of functional and aberrant TDP-43 condensation. We will summarise the current knowledge on how TDP-43 containing nuclear condensates form and function and how their homeostasis is affected in disease.
RESUMEN
Paraspeckles are ribonucleoprotein granules assembled by NEAT1_2 lncRNA, an isoform of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1). Dysregulation of NEAT1_2/paraspeckles has been linked to multiple human diseases making them an attractive drug target. However currently NEAT1_2/paraspeckle-focused translational research and drug discovery are hindered by a limited toolkit. To fill this gap, we developed and validated a set of tools for the identification of NEAT1_2 binders and modulators comprised of biochemical and cell-based assays. The NEAT1_2 triple helix stability element was utilized as the target in the biochemical assays, and the cellular assay ('ParaQuant') was based on high-content imaging of NEAT1_2 in fixed cells. As a proof of principle, these assays were used to screen a 1,200-compound FDA-approved drug library and a 170-compound kinase inhibitor library and to confirm the screening hits. The assays are simple to establish, use only commercially-available reagents and are scalable for higher throughput. In particular, ParaQuant is a cost-efficient assay suitable for any cells growing in adherent culture and amenable to multiplexing. Using ParaQuant, we identified dual PI3K/mTOR inhibitors as potent negative modulators of paraspeckles. The tools we describe herein should boost paraspeckle studies and help guide the search, validation and optimization of NEAT1_2/paraspeckle-targeted small molecules.
Asunto(s)
Núcleo Celular , Paraspeckles , ARN Largo no Codificante , Humanos , Núcleo Celular/genética , Paraspeckles/efectos de los fármacos , Paraspeckles/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/química , Inhibidores de Proteínas Quinasas/farmacología , Descubrimiento de DrogasRESUMEN
Formation of cytoplasmic RNA-protein structures called stress granules (SGs) is a highly conserved cellular response to stress. Abnormal metabolism of SGs may contribute to the pathogenesis of (neuro)degenerative diseases such as amyotrophic lateral sclerosis (ALS). Many SG proteins are affected by mutations causative of these conditions, including fused in sarcoma (FUS). Mutant FUS variants have high affinity to SGs and also spontaneously form de novo cytoplasmic RNA granules. Mutant FUS-containing assemblies (mFAs), often called "pathological SGs", are proposed to play a role in ALS-FUS pathogenesis. However, structural differences between mFAs and physiological SGs remain largely unknown therefore it is unclear whether mFAs can functionally substitute for SGs and how they affect cellular stress responses. Here we used affinity purification to isolate mFAs and physiological SGs and compare their protein composition. We found that proteins within mFAs form significantly more physical interactions than those in SGs however mFAs fail to recruit many factors involved in signal transduction. Furthermore, we found that proteasome subunits and certain nucleocytoplasmic transport factors are depleted from mFAs, whereas translation elongation, mRNA surveillance and splicing factors as well as mitochondrial proteins are enriched in mFAs, as compared to SGs. Validation experiments for a mFA-specific protein, hnRNPA3, confirmed its RNA-dependent interaction with FUS and its sequestration into FUS inclusions in cultured cells and in a FUS transgenic mouse model. Silencing of the Drosophila hnRNPA3 ortholog was deleterious and potentiated human FUS toxicity in the retina of transgenic flies. In conclusion, we show that SG-like structures formed by mutant FUS are structurally distinct from SGs, prone to persistence, likely cannot functionally replace SGs, and affect a spectrum of cellular pathways in stressed cells. Results of our study support a pathogenic role for cytoplasmic FUS assemblies in ALS-FUS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Citoplasma/metabolismo , Cuerpos de Inclusión/metabolismo , Ratones , Mutación , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Gránulos de Estrés , Estrés FisiológicoRESUMEN
Proteins associated with familial neurodegenerative disease often aggregate in patients' neurons. Several such proteins, e.g. TDP-43, aggregate and are toxic when expressed in yeast. Deletion of the ATXN2 ortholog, PBP1, reduces yeast TDP-43 toxicity, which led to identification of ATXN2 as an amyotrophic lateral sclerosis (ALS) risk factor and therapeutic target. Likewise, new yeast neurodegenerative disease models could facilitate identification of other risk factors and targets. Mutations in SS18L1, encoding the calcium-responsive transactivator (CREST) chromatin-remodeling protein, are associated with ALS. We show that CREST is toxic in yeast and forms nuclear and occasionally cytoplasmic foci that stain with Thioflavin-T, a dye indicative of amyloid-like protein. Like the yeast chromatin-remodeling factor SWI1, CREST inhibits silencing of FLO genes. Toxicity of CREST is enhanced by the [PIN+] prion and reduced by deletion of the HSP104 chaperone required for the propagation of many yeast prions. Likewise, deletion of PBP1 reduced CREST toxicity and aggregation. In accord with the yeast data, we show that the Drosophila ortholog of human ATXN2, dAtx2, is a potent enhancer of CREST toxicity. Downregulation of dAtx2 in flies overexpressing CREST in retinal ganglion cells was sufficient to largely rescue the severe degenerative phenotype induced by human CREST. Overexpression caused considerable co-localization of CREST and PBP1/ATXN2 in cytoplasmic foci in both yeast and mammalian cells. Thus, co-aggregation of CREST and PBP1/ATXN2 may serve as one of the mechanisms of PBP1/ATXN2-mediated toxicity. These results extend the spectrum of ALS associated proteins whose toxicity is regulated by PBP1/ATXN2, suggesting that therapies targeting ATXN2 may be effective for a wide range of neurodegenerative diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Ataxina-2/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Animales Modificados Genéticamente , Ataxina-2/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Priones/metabolismo , Células Ganglionares de la Retina/patología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transactivadores/genéticaRESUMEN
Topoisomerases are essential enzymes that recognize and modify the topology of DNA to allow DNA replication and transcription to take place. Topoisomerases are divided into type I topoisomerases, that cleave one DNA strand to modify DNA topology, and type II, that cleave both DNA strands. Topoisomerases normally rapidly religate cleaved-DNA once the topology has been modified. Topoisomerases do not recognize specific DNA sequences, but actively cleave positively supercoiled DNA ahead of transcription bubbles or replication forks, and negative supercoils (or precatenanes) behind, thus allowing the unwinding of the DNA-helix to proceed (during both transcription and replication). Drugs that stabilize DNA-cleavage complexes with topoisomerases produce cytotoxic DNA damage and kill fast-dividing cells; they are widely used in cancer chemotherapy. Oligonucleotide-recognizing topoisomerase inhibitors (OTIs) have given drugs that stabilize DNA-cleavage complexes specificity by linking them to either: (i) DNA duplex recognizing triplex forming oligonucleotide (TFO-OTIs) or DNA duplex recognizing pyrrole-imidazole-polyamides (PIP-OTIs) (ii) or by conventional Watson-Crick base pairing (WC-OTIs). This converts compounds from indiscriminate DNA-damaging drugs to highly specific targeted DNA-cleaving OTIs. Herein we propose simple strategies to enable DNA-duplex strand invasion of WC-OTIs giving strand-invading SI-OTIs. This will make SI-OTIs similar to the guide RNAs of CRISPR/Cas9 nuclease bacterial immune systems. However, an important difference between OTIs and CRISPR/Cas9, is that OTIs do not require the introduction of foreign proteins into cells. Recent successful oligonucleotide therapeutics for neurodegenerative diseases suggest that OTIs can be developed to be highly specific gene editing agents for DNA lesions that cause neurodegenerative diseases.
Asunto(s)
Enfermedades Neurodegenerativas , Oligonucleótidos , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Superhelicoidal , Humanos , Imidazoles , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/genética , Nylons , Oligonucleótidos/química , Pirroles , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II , Inhibidores de Topoisomerasa/farmacología , Inhibidores de Topoisomerasa/uso terapéuticoRESUMEN
Pathological changes involving TDP-43 protein ('TDP-43 proteinopathy') are typical for several neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). FTLD-TDP cases are characterized by increased binding of TDP-43 to an abundant lncRNA, NEAT1, in the cortex. However it is unclear whether enhanced TDP-43-NEAT1 interaction represents a protective mechanism. We show that accumulation of human TDP-43 leads to upregulation of the constitutive NEAT1 isoform, NEAT1_1, in cultured cells and in the brains of transgenic mice. Further, we demonstrate that overexpression of NEAT1_1 ameliorates TDP-43 toxicity in Drosophila and yeast models of TDP-43 proteinopathy. Thus, NEAT1_1 upregulation may be protective in TDP-43 proteinopathies affecting the brain. Approaches to boost NEAT1_1 expression in the CNS may prove useful in the treatment of these conditions.
Asunto(s)
Esclerosis Amiotrófica Lateral/prevención & control , Encéfalo/metabolismo , Proteínas de Unión al ADN/toxicidad , Demencia Frontotemporal/prevención & control , Neuroblastoma/prevención & control , ARN Largo no Codificante/genética , Proteinopatías TDP-43/prevención & control , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Drosophila melanogaster , Demencia Frontotemporal/etiología , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroblastoma/etiología , Neuroblastoma/metabolismo , Neuroblastoma/patología , ARN Largo no Codificante/administración & dosificación , Saccharomyces cerevisiae , Proteinopatías TDP-43/etiología , Proteinopatías TDP-43/metabolismo , Proteinopatías TDP-43/patologíaRESUMEN
FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.
Asunto(s)
Neuronas Motoras/metabolismo , Proteína FUS de Unión a ARN/genética , Animales , Encéfalo/metabolismo , Citoplasma/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Proteína FUS de Unión a ARN/metabolismo , Médula Espinal/metabolismoRESUMEN
The accurate diagnosis and clinical management of the growth restriction disorder Silver Russell Syndrome (SRS) has confounded researchers and clinicians for many years due to the myriad of genetic and epigenetic alterations reported in these patients and the lack of suitable animal models to test the contribution of specific gene alterations. Some genetic alterations suggest a role for increased dosage of the imprinted CYCLIN DEPENDENT KINASE INHIBITOR 1C (CDKN1C) gene, often mutated in IMAGe Syndrome and Beckwith-Wiedemann Syndrome (BWS). Cdkn1c encodes a potent negative regulator of fetal growth that also regulates placental development, consistent with a proposed role for CDKN1C in these complex childhood growth disorders. Here, we report that a mouse modelling the rare microduplications present in some SRS patients exhibited phenotypes including low birth weight with relative head sparing, neonatal hypoglycemia, absence of catch-up growth and significantly reduced adiposity as adults, all defining features of SRS. Further investigation revealed the presence of substantially more brown adipose tissue in very young mice, of both the classical or canonical type exemplified by interscapular-type brown fat depot in mice (iBAT) and a second type of non-classic BAT that develops postnatally within white adipose tissue (WAT), genetically attributable to a double dose of Cdkn1c in vivo and ex-vivo. Conversely, loss-of-function of Cdkn1c resulted in the complete developmental failure of the brown adipocyte lineage with a loss of markers of both brown adipose fate and function. We further show that Cdkn1c is required for post-transcriptional accumulation of the brown fat determinant PR domain containing 16 (PRDM16) and that CDKN1C and PRDM16 co-localise to the nucleus of rare label-retaining cell within iBAT. This study reveals a key requirement for Cdkn1c in the early development of the brown adipose lineages. Importantly, active BAT consumes high amounts of energy to generate body heat, providing a valid explanation for the persistence of thinness in our model and supporting a major role for elevated CDKN1C in SRS.
Asunto(s)
Tejido Adiposo Pardo/crecimiento & desarrollo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Impresión Genómica , Síndrome de Silver-Russell/genética , Factores de Transcripción/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Adulto , Animales , Temperatura Corporal , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Mutación , Fenotipo , Síndrome de Silver-Russell/metabolismo , Síndrome de Silver-Russell/patología , Factores de Transcripción/genéticaRESUMEN
Fused in sarcoma (FUS) is an RNA-binding protein involved in pathogenesis of several neurodegenerative diseases. Aggregation of mislocalized FUS into non-amyloid inclusions is believed to be pivotal in the development of cell dysfunction, but the mechanism of their formation is unclear. Using transient expression of a panel of deletion and chimeric FUS variants in various cultured cells, we demonstrated that FUS accumulating in the cytoplasm nucleates a novel type of RNA granules, FUS granules (FGs), that are structurally similar but not identical to physiological RNA transport granules. Formation of FGs requires FUS N-terminal prion-like domain and the ability to bind specific RNAs. Clustering of FGs coupled with further recruitment of RNA and proteins produce larger structures, FUS aggregates (FAs), that resemble but are clearly distinct from stress granules. In conditions of attenuated transcription, FAs lose RNA and dissociate into RNA-free FUS complexes that become precursors of large aggresome-like structures. We propose a model of multistep FUS aggregation involving RNA-dependent and RNA-independent stages. This model can be extrapolated to formation of pathological inclusions in human FUSopathies.
Asunto(s)
Citoplasma/metabolismo , Proteína FUS de Unión a ARN/metabolismo , ARN/genética , ARN/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Humanos , Ratones , Modelos Biológicos , Mutación , Agregación Patológica de Proteínas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Transcripción GenéticaRESUMEN
Paraspeckles are nuclear bodies formed by a set of specialized proteins assembled on the long non-coding RNA NEAT1; they have a role in nuclear retention of hyperedited transcripts and are associated with response to cellular stress. Fused in sarcoma (FUS) protein, linked to a number of neurodegenerative disorders, is an essential paraspeckle component. We have shown that its recruitment to these nuclear structures is mediated by the N-terminal region and requires prion-like activity. FUS interacts with p54nrb/NONO, a major constituent of paraspeckles, in an RNA-dependent manner and responds in the same way as other paraspeckle proteins to alterations in cellular homeostasis such as changes in transcription rates or levels of protein methylation. FUS also regulates NEAT1 levels and paraspeckle formation in cultured cells, and FUS deficiency leads to loss of paraspeckles. Pathological gain-of-function FUS mutations might be expected to affect paraspeckle function in human diseases because mislocalized amyotrophic lateral sclerosis (ALS)-linked FUS variants sequester other paraspeckle proteins into aggregates formed in cultured cells and into neuronal inclusions in a transgenic mouse model of FUSopathy. Furthermore, we detected abundant p54nrb/NONO-positive inclusions in motor neurons of patients with familial forms of ALS caused by FUS mutations, but not in other ALS cases. Our results suggest that both loss and gain of FUS function can trigger disruption of paraspeckle assembly, which may impair protective responses in neurons and thereby contribute to the pathogenesis of FUSopathies.
Asunto(s)
Proteína FUS de Unión a ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Línea Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Cuerpos de Inclusión Intranucleares/metabolismo , Ratones , Ratones Transgénicos , ARN Largo no Codificante/metabolismoRESUMEN
Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1-359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1-359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5-4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1-359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.
Asunto(s)
Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Axones/metabolismo , Neuronas Motoras/metabolismo , Señales de Localización Nuclear , Proteína FUS de Unión a ARN/biosíntesis , Eliminación de Secuencia , Secuencias de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Axones/patología , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/patología , Humanos , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Fenotipo , ARN , Proteína FUS de Unión a ARN/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases are characterised by dysfunction of a host of RNA-binding proteins (RBPs) and a severely disrupted RNA metabolism. Recently, RBP-harbouring phase-separated complexes, ribonucleoprotein (RNP) granules, have come into the limelight as "crucibles" of neuronal pathology in ALS. RNP granules are indispensable for the multitude of regulatory processes underlying cellular RNA metabolism and serve as critical organisers of cellular biochemistry. Neurons, highly specialised cells, heavily rely on RNP granules for efficient trafficking, signalling and stress responses. Multiple RNP granule components, primarily RBPs such as TDP-43 and FUS, are affected by ALS mutations. However, even in the absence of mutations, RBP proteinopathies represent pathophysiological hallmarks of ALS. Given the high local concentrations of RBPs and RNAs, their weakened or enhanced interactions within RNP granules disrupt their homeostasis. Thus, the physiological process of phase separation and RNP granule formation, vital for maintaining the high-functioning state of neuronal cells, becomes their Achilles heel. Here, we will review the recent literature on the causes and consequences of abnormal RNP granule functioning in ALS and related disorders. In particular, we will summarise the evidence for the network-level dysfunction of RNP granules in these conditions and discuss considerations for therapeutic interventions to target RBPs, RNP granules and their network as a whole.
Asunto(s)
Esclerosis Amiotrófica Lateral , Gránulos Citoplasmáticos , Ribonucleoproteínas , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Ribonucleoproteínas/metabolismo , Animales , Gránulos Citoplasmáticos/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Orgánulos/metabolismoRESUMEN
TDP-43 protein is dysregulated in several neurodegenerative diseases, which often have a multifactorial nature and may have extrinsic stressors as a "second hit." TDP-43 undergoes reversible nuclear condensation in stressed cells including neurons. Here, we demonstrate that stress-inducible nuclear TDP-43 condensates are RNA-depleted, non-liquid assemblies distinct from the known nuclear bodies. Their formation requires TDP-43 oligomerization and ATP and is inhibited by RNA. Using a confocal nanoscanning assay, we find that amyotrophic lateral sclerosis (ALS)-linked mutations alter stress-induced TDP-43 condensation by changing its affinity to liquid-like ribonucleoprotein assemblies. Stress-induced nuclear condensation transiently inactivates TDP-43, leading to loss of interaction with its protein binding partners and loss of function in splicing. Splicing changes are especially prominent and persisting for STMN2 RNA, and STMN2 protein becomes rapidly depleted early during stress. Our results point to early pathological changes to TDP-43 in the nucleus and support therapeutic modulation of stress response in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Núcleo Celular , Proteínas de Unión al ADN , Empalme del ARN , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Empalme del ARN/genética , Núcleo Celular/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Estrés Fisiológico , Animales , RatonesRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs). Understanding the genetic and molecular factors influencing ALS survival is crucial for disease management and therapeutics. In this study, we introduce a deep learning-powered genetic analysis framework to link rare noncoding genetic variants to ALS survival. Using data from human induced pluripotent stem cell (iPSC)-derived MNs, this method prioritizes functional noncoding variants using deep learning, links cis-regulatory elements (CREs) to target genes using epigenomics data, and integrates these data through gene-level burden tests to identify survival-modifying variants, CREs, and genes. We apply this approach to analyze 6,715 ALS genomes, and pinpoint four novel rare noncoding variants associated with survival, including chr7:76,009,472:C>T linked to CCDC146. CRISPR-Cas9 editing of this variant increases CCDC146 expression in iPSC-derived MNs and exacerbates ALS-specific phenotypes, including TDP-43 mislocalization. Suppressing CCDC146 with an antisense oligonucleotide (ASO), showing no toxicity, completely rescues ALS-associated survival defects in neurons derived from sporadic ALS patients and from carriers of the ALS-associated G4C2-repeat expansion within C9ORF72. ASO targeting of CCDC146 may be a broadly effective therapeutic approach for ALS. Our framework provides a generic and powerful approach for studying noncoding genetics of complex human diseases.
RESUMEN
The discovery of a causative link between dysfunction of a number of RNA-binding proteins with prion-like domains and the development of certain (neuro)degenerative diseases has completely changed our perception of molecular mechanisms instigating pathological process in these disorders. Irreversible aggregation of these proteins is a crucial pathogenic event delineating a type of proteinopathy. FUS (fused in sarcoma) is a prototypical member of the class, and studies into the causes and consequences of FUSopathies have been instrumental in characterizing the processes leading to deregulation of RNA metabolism in neurodegeneration. In vivo models of FUSopathy have provided critical insights into the mechanisms of FUS toxicity and clues on the role of non-amyloid aggregates, which are hallmarks of these diseases. The present review summarizes the data on FUS aggregation signatures in available model organisms on the basis of overexpression of FUS variants.
Asunto(s)
Modelos Biológicos , Unión Proteica , Proteína FUS de Unión a ARN/metabolismo , Animales , Humanos , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genéticaRESUMEN
We describe an autosomal dominant, multi-generational, amyotrophic lateral sclerosis (ALS) pedigree in which disease co-segregates with a heterozygous p.Y374X nonsense mutation within TDP-43. Mislocalization of TDP-43 and formation of insoluble TDP-43-positive neuronal cytoplasmic inclusions is the hallmark pathology in >95% of ALS patients. Neuropathological examination of the single case for which CNS tissue was available indicated typical TDP-43 pathology within lower motor neurons, but classical TDP-43-positive inclusions were absent from motor cortex. The mutated allele is transcribed and translated in patient fibroblasts and motor cortex tissue, but overall TDP-43 protein expression is reduced compared to wild-type controls. Despite absence of TDP-43-positive inclusions we confirmed deficient TDP-43 splicing function within motor cortex tissue. Furthermore, urea fractionation and mass spectrometry of motor cortex tissue carrying the mutation revealed atypical TDP-43 protein species but not typical C-terminal fragments. We conclude that the p.Y374X mutation underpins a monogenic, fully penetrant form of ALS. Reduced expression of TDP-43 combined with atypical TDP-43 protein species and absent C-terminal fragments extends the molecular phenotypes associated with TDP-43 mutations and with ALS more broadly. Future work will need to include the findings from this pedigree in dissecting the mechanisms of TDP-43-mediated toxicity.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mutación , LinajeRESUMEN
PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.
Asunto(s)
Transporte de ARN , ARN , Humanos , Transporte Activo de Núcleo Celular , Expresión Génica , HomeostasisRESUMEN
Amyotrophic lateral sclerosis (ALS) is characterised by substantial loss of both upper and lower motor neuron function, with sensory and cognitive systems less affected. Though heritable forms of the disease have been described, the vast majority of cases are sporadic with poorly defined underlying pathogenic mechanisms. Here we demonstrate that the neurological pathology induced in transgenic mice by overexpression of γ-synuclein, a protein not previously associated with ALS, recapitulates key features of the disease, namely selective damage and loss of discrete populations of upper and lower motor neurons and their axons, contrasted by limited effects upon the sensory system.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Axones/patología , Neuronas Motoras/patología , Médula Espinal/patología , gamma-Sinucleína/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Percepción del Tacto/fisiología , gamma-Sinucleína/metabolismoRESUMEN
BACKGROUND: Recent clinical studies have demonstrated that dimebon, a drug originally designed and used as a non-selective antihistamine, ameliorates symptoms and delays progress of mild to moderate forms of Alzheimer's and Huntington's diseases. Although the mechanism of dimebon action on pathological processes in degenerating brain is elusive, results of studies carried out in cell cultures and animal models suggested that this drug might affect the process of pathological accumulation and aggregation of various proteins involved in the pathogenesis of proteinopathies. However, the effect of this drug on the pathology caused by overexpression and aggregation of alpha-synuclein, including Parkinson's disease (PD), has not been assessed. OBJECTIVE: To test if dimebon affected alpha-synuclein-induced pathology using a transgenic animal model. METHODS: We studied the effects of chronic dimebon treatment on transgenic mice expressing the C-terminally truncated (1-120) form of human alpha-synuclein in dopaminergic neurons, a mouse model that recapitulates several biochemical, histopathological and behavioral characteristics of the early stage of PD. RESULTS: Dimebon did not improve balance and coordination of aging transgenic animals or increase the level of striatal dopamine, nor did it prevent accumulation of alpha-synuclein in cell bodies of dopaminergic neurons. CONCLUSION: Our observations suggest that in the studied model of alpha-synucleinopathy dimebon has very limited effect on certain pathological alterations typical of PD and related diseases.