RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disorder characterized by progressive muscular weakness due to the selective loss of motor neurons. Mutations in the gene Fused in Sarcoma (FUS) were identified as one cause of ALS. Here, we report that mutations in FUS lead to upregulation of synaptic proteins, increasing synaptic activity and abnormal release of vesicles at the synaptic cleft. Consequently, FUS-ALS neurons showed greater vulnerability to glutamate excitotoxicity, which raised neuronal swellings (varicose neurites) and led to neuronal death. Fragile X mental retardation protein (FMRP) is an RNA-binding protein known to regulate synaptic protein translation, and its expression is reduced in the FUS-ALS lines. Collectively, our data suggest that a reduction of FMRP levels alters the synaptic protein dynamics, leading to synaptic dysfunction and glutamate excitotoxicity. Here, we present a mechanistic hypothesis linking dysregulation of peripheral translation with synaptic vulnerability in the pathogenesis of FUS-ALS.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Adulto , Humanos , Esclerose Lateral Amiotrófica/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Mutação , Glutamatos/metabolismo , Proteína FUS de Ligação a RNA/genéticaRESUMO
Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.
Assuntos
Quadruplex G , Hepatite B Crônica , Humanos , Vírus da Hepatite B/genética , DNA Circular/genética , DNA Circular/metabolismo , Separação de Fases , DNA Viral/genética , DNA Viral/metabolismo , Hepatite B Crônica/genética , Hepatite B Crônica/metabolismo , Hepatócitos/metabolismo , Replicação Viral/genéticaRESUMO
The hypomethylation of fused in sarcoma (FUS) in frontotemporal lobar degeneration promotes the formation of irreversible condensates of FUS. However, the mechanisms by which these hypomethylated FUS condensates cause neuronal dysfunction are unknown. Here we report that expression of FUS constructs mimicking hypomethylated FUS causes aberrant dendritic FUS condensates in CA1 neurons. These hypomethylated FUS condensates exhibit spontaneous, and activity induced movement within the dendrite. They impair excitatory synaptic transmission, postsynaptic density-95 expression, and dendritic spine plasticity. These neurophysiological defects are dependent upon both the dendritic localisation of the condensates, and their ability to undergo liquid-liquid phase separation. These results indicate that the irreversible liquid-liquid phase separation is a key component of hypomethylated FUS pathophysiology in sporadic FTLD, and this can cause synapse dysfunction in sporadic FTLD.
Assuntos
Demência Frontotemporal , Degeneração Lobar Frontotemporal , Humanos , Separação de Fases , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Degeneração Lobar Frontotemporal/genética , Metilação de DNARESUMO
Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations.
Assuntos
Doença de Charcot-Marie-Tooth , Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Interferência de RNA , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/terapia , Doença de Charcot-Marie-Tooth/metabolismo , Mutação , Hidrolases/genética , Camundongos TransgênicosRESUMO
Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy but requires high levels of the adaptor protein DCAF15. Consequently, novel approaches to deplete RBM39 in an DCAF15-independent manner are required. Here, we uncover that RBM39 autoregulates via the inclusion of a poison exon into its own pre-mRNA and identify the cis-acting elements that govern this regulation. We also determine the NMR solution structures of RBM39's tandem RNA recognition motifs (RRM1 and RRM2) bound to their respective RNA targets, revealing how RRM1 recognises RNA stem loops whereas RRM2 binds specifically to single-stranded N(G/U)NUUUG. Our results support a model where RRM2 selects the 3'-splice site of a poison exon and the RRM3 and RS domain stabilise the U2 snRNP at the branchpoint. Our work provides molecular insights into RBM39-dependent 3'-splice site selection and constitutes a solid basis to design alternative anti-cancer therapies.
Assuntos
Neoplasias , Splicing de RNA , Splicing de RNA/genética , Motivos de Ligação ao RNA , Sítios de Splice de RNA , Homeostase , Fatores de Processamento de RNA/genética , Neoplasias/genéticaRESUMO
The purinoceptor P2X7R is a promising therapeutic target for tauopathies, including Alzheimer's disease (AD). Pharmacological inhibition or genetic knockdown of P2X7R ameliorates cognitive deficits and reduces pathological tau burden in mice that model aspects of tauopathy, including mice expressing mutant human frontotemporal dementia (FTD)-causing forms of tau. However, disagreements remain over which glial cell types express P2X7R and therefore the mechanism of action is unresolved. Here, we show that P2X7R protein levels increase in human AD post-mortem brain, in agreement with an upregulation of P2RX7 mRNA observed in transcriptome profiles from the AMP-AD consortium. P2X7R protein increases mirror advancing Braak stage and coincide with synapse loss. Using RNAScope we detect P2RX7 mRNA in microglia and astrocytes in human AD brain, including in the vicinity of senile plaques. In cultured microglia, P2X7R activation modulates the NLRP3 inflammasome pathway by promoting the formation of active complexes and release of IL-1ß. In astrocytes, P2X7R activates NFκB signalling and increases production of the cytokines CCL2, CXCL1 and IL-6 together with the acute phase protein Lcn2. To further explore the role of P2X7R in a disease-relevant context, we expressed wild-type or FTD-causing mutant forms of tau in mouse organotypic brain slice cultures. Inhibition of P2X7R reduces insoluble tau levels without altering soluble tau phosphorylation or synaptic localisation, suggesting a non-cell autonomous role of glial P2X7R on pathological tau aggregation. These findings support further investigations into the cell-type specific effects of P2X7R-targeting therapies in tauopathies.
Assuntos
Doença de Alzheimer , Demência Frontotemporal , Tauopatias , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Microglia/metabolismo , RNA Mensageiro/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatias/metabolismoRESUMO
Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.
Assuntos
Venenos , Humanos , Proteínas e Peptídeos de Choque Frio/metabolismo , Temperatura Baixa , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: A rare coding variant, P522R, in the phospholipase C gamma 2 (PLCG2) gene has been identified as protective against late-onset Alzheimer's disease (AD), but the mechanism is unknown. PLCG2 is exclusively expressed in microglia within the central nervous system, and altered microglial function has been implicated in the progression of AD. METHODS: Healthy control hiPSCs were CRISPR edited to generate cells heterozygous and homozygous for the PLCG2P522R variant. Microglia derived from these hiPSC's were used to investigate the impact of PLCγ2P522R on disease relevant processes, specifically microglial capacity to take up amyloid beta (Aß) and synapses. Targeted qPCR assessment was conducted to explore expression changes in core AD linked and microglial genes, and mitochondrial function was assessed using an Agilent Seahorse assay. RESULTS: Heterozygous expression of the P522R variant resulted in increased microglial clearance of Aß, while preserving synapses. This was associated with the upregulation of a number of genes, including the anti-inflammatory cytokine Il-10, and the synapse-linked CX3CR1, as well as alterations in mitochondrial function, and increased cellular motility. The protective capacity of PLCγ2P522R appeared crucially dependent on (gene) 'dose', as cells homozygous for the variant showed reduced synapse preservation, and a differential gene expression profile relative to heterozygous cells. CONCLUSION: These findings suggest that PLCγ2P522R may result in increased surveillance by microglia, and prime them towards an anti-inflammatory state, with an increased capacity to respond to increasing energy demands, but highlights the delicate balance of this system, with increasing PLCγ2P522R 'dose' resulting in reduced beneficial impacts.
Assuntos
Doença de Alzheimer , Fosfolipase C gama , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Humanos , Microglia/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Sinapses/metabolismoRESUMO
Alternative pre-mRNA splicing allows for the production of multiple mRNAs from an individual gene, which not only expands the protein-coding potential of the genome but also enables complex mechanisms for the post-transcriptional control of gene expression. Regulation of alternative splicing entails a combinatorial interplay between an abundance of trans-acting splicing factors, cis-acting regulatory sequence elements and their concerted effects on the core splicing machinery. Given the extent and biological significance of alternative splicing in humans, it is not surprising that aberrant splicing patterns can cause or contribute to a wide range of diseases. In this introductory chapter, we outline the mechanisms that govern alternative pre-mRNA splicing and its regulation and discuss how dysregulated splicing contributes to human diseases affecting the motor system and the brain.
Assuntos
Processamento Alternativo , Precursores de RNA , Doença/genética , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transativadores/metabolismoRESUMO
Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.
Assuntos
Proteína FUS de Ligação a RNA/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Células HEK293 , Células HeLa , Humanos , Mapeamento de Interação de Proteínas , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/isolamento & purificaçãoRESUMO
Genes encoding replication-dependent histones lack introns, and the mRNAs produced are a unique class of RNA polymerase II transcripts in eukaryotic cells that do not end in a polyadenylated tail. Mature mRNAs are thus formed by a single endonucleolytic cleavage that releases the pre-mRNA from the DNA and is the only processing event necessary. U7 snRNP is one of the key factors that determines the cleavage site within the 3'UTR of replication-dependent histone pre-mRNAs. We have previously showed that the FUS protein interacts with U7 snRNA/snRNP and regulates the expression of histone genes by stimulating transcription and 3' end maturation. Mutations in the FUS gene first identified in patients with amyotrophic lateral sclerosis (ALS) lead to the accumulation of the FUS protein in cytoplasmic inclusions. Here, we report that mutations in FUS lead to disruption of the transcriptional activity of FUS and mislocalization of U7 snRNA/snRNP in cytoplasmic aggregates in cellular models and primary neurons. As a consequence, decreased transcriptional efficiency and aberrant 3' end processing of histone pre-mRNAs were observed. This study highlights for the first time the deregulation of replication-dependent histone gene expression and its involvement in ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Regulação da Expressão Gênica , Histonas/metabolismo , Mutação , Proteína FUS de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U7/genética , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Perfilação da Expressão Gênica , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Neurociências , Plasmídeos/metabolismo , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genéticaRESUMO
RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.
Assuntos
Reparo do DNA/genética , Proteína FUS de Ligação a RNA/genética , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , Células HEK293 , Células HeLa , Humanos , Ligação Proteica/genética , Domínios Proteicos/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Erythropoietic protoporphyria (EPP) is a rare disease in which patients experience severe light sensitivity. It is caused by a deficiency of ferrochelatase (FECH), the last enzyme in heme biosynthesis (HBS). The lack of FECH causes accumulation of its photoreactive substrate protoporphyrin IX (PPIX) in patients' erythrocytes. Here, we explored an approach for the treatment of EPP by decreasing PPIX synthesis using small-molecule inhibitors directed to factors in the HBS pathway. We generated a FECH-knockout clone from K562 erythroleukemia cells, which accumulates PPIX and undergoes oxidative stress upon light exposure. We used these matched cell lines to screen a set of publicly available inhibitors of factors in the HBS pathway. Inhibitors of the glycine transporters GlyT1 and GlyT2 lowered levels of PPIX and markers of oxidative stress selectively in K56211B4 cells, and in primary erythroid cultures from an EPP patient. Our findings open the door to investigation of glycine transport inhibitors for HBS disorders.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Protoporfiria Eritropoética/tratamento farmacológico , Protoporfirinas/farmacologia , Células Cultivadas , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Humanos , Células K562 , Estrutura Molecular , Protoporfiria Eritropoética/metabolismoRESUMO
Mutations in the RNA-binding protein Fused in Sarcoma (FUS) cause early-onset amyotrophic lateral sclerosis (ALS). However, a detailed understanding of central RNA targets of FUS and their implications for disease remain elusive. Here, we use a unique blend of crosslinking and immunoprecipitation (CLIP) and NMR spectroscopy to identify and characterise physiological and pathological RNA targets of FUS. We find that U1 snRNA is the primary RNA target of FUS via its interaction with stem-loop 3 and provide atomic details of this RNA-mediated mode of interaction with the U1 snRNP. Furthermore, we show that ALS-associated FUS aberrantly contacts U1 snRNA at the Sm site with its zinc finger and traps snRNP biogenesis intermediates in human and murine motor neurons. Altogether, we present molecular insights into a FUS toxic gain-of-function involving direct and aberrant RNA-binding and strengthen the link between two motor neuron diseases, ALS and spinal muscular atrophy (SMA).
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Linhagem Celular , Predisposição Genética para Doença/genética , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Neurônios Motores/metabolismo , Mutação , Domínios e Motivos de Interação entre Proteínas , RNA Nuclear Pequeno/química , Proteína FUS de Ligação a RNA/química , Ribonucleoproteína Nuclear Pequena U1/químicaRESUMO
Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.
Assuntos
Esclerose Lateral Amiotrófica/genética , Homeostase/genética , Proteína FUS de Ligação a RNA/genética , Animais , Citoplasma/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Íntrons/genética , Mutação com Perda de Função , Camundongos , Camundongos Knockout , Mutação/genética , Splicing de RNA/genética , Superóxido Dismutase-1/genética , Proteína com Valosina/genéticaRESUMO
Regulatory non-protein coding RNAs perform a remarkable variety of complex biological functions. Previously, we demonstrated a role of the human non-coding vault RNA1-1 (vtRNA1-1) in inhibiting intrinsic and extrinsic apoptosis in several cancer cell lines. Yet on the molecular level, the function of the vtRNA1-1 is still not fully clear. Here, we created HeLa knock-out cell lines revealing that prolonged starvation triggers elevated levels of apoptosis in the absence of vtRNA1-1 but not in vtRNA1-3 knock-out cells. Next-generation deep sequencing of the mRNome identified the PI3K/Akt pathway and the ERK1/2 MAPK cascade, two prominent signaling axes, to be misregulated in the absence of vtRNA1-1 during starvation-mediated cell death conditions. Expression of vtRNA1-1 mutants identified a short stretch of 24 nucleotides of the vtRNA1-1 central domain as being essential for successful maintenance of apoptosis resistance. This study describes a cell signaling-dependent contribution of the human vtRNA1-1 to starvation-induced programmed cell death.
Assuntos
Apoptose/genética , RNA não Traduzido/metabolismo , Transdução de Sinais/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sistema de Sinalização das MAP Quinases , Nucleotídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , InaniçãoRESUMO
Amyotrophic lateral sclerosis (ALS) is a relentless and fatal neurological disease characterized by the selective degeneration of motor neurons. No effective therapy is available for this disease. Several lines of evidence indicate that alteration of RNA metabolism, including microRNA (miRNA) processing, is a relevant pathogenetic factor and a possible therapeutic target for ALS. Here, we showed that the abundance of components in the miRNA processing machinery is altered in a SOD1-linked cellular model, suggesting consequent dysregulation of miRNA biogenesis. Indeed, high-throughput sequencing of the small RNA fraction showed that among the altered miRNAs, miR-129-5p was increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients. We demonstrated that miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD. ELAVL4/HuD is predominantly expressed in neurons, where it controls several key neuronal mRNAs. Overexpression of pre-miR-129-1 inhibited neurite outgrowth and differentiation via HuD silencing in vitro, while its inhibition with an antagomir rescued the phenotype. Remarkably, we showed that administration of an antisense oligonucleotide (ASO) inhibitor of miR-129-5p to an ALS animal model, SOD1 (G93A) mice, result in a significant increase in survival and improved the neuromuscular phenotype in treated mice. These results identify miR-129-5p as a therapeutic target that is amenable to ASO modulation for the treatment of ALS patients.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo , Proteína Semelhante a ELAV 4 , Humanos , Camundongos , MicroRNAs/genética , Oligonucleotídeos Antissenso/farmacologia , Superóxido Dismutase-1 , Regulação para CimaRESUMO
Mutations in fused in sarcoma (FUS) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. Here we show the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. Our data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas do Complexo SMN/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Drosophila/genética , Drosophila/metabolismo , Feminino , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurônios Motores/metabolismo , Mutação , Fenótipo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/toxicidade , Proteínas do Complexo SMN/genética , Fatores de Transcrição/metabolismoRESUMO
Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers, with a median survival of only three to six months. Standard treatment options and even targeted therapies have so far failed to improve long-term overall survival. Thus, novel treatment modalities for ATC, such as immunotherapy, are urgently needed. CD47 is a "don't eat me" signal, which prevents cancer cells from phagocytosis by binding to signal regulatory protein alpha on macrophages. So far, the role of macrophages and the CD47-signal regulatory protein alpha signaling axis in ATC is not well understood. Methods: This study analyzed 19 primary human ATCs for macrophage markers, CD47 expression, and immune checkpoints by immunohistochemistry. ATC cell lines and a fresh ATC sample were assessed by flow cytometry for CD47 expression and macrophage infiltration, respectively. CD47 was blocked in phagocytosis assays of co-cultured macrophages and ATC cell lines. Anti-CD47 antibody treatment was administered to ATC cell line xenotransplanted immunocompromised mice, as well as to tamoxifen-induced ATC double-transgenic mice. Results: Human ATC samples were heavily infiltrated by CD68- and CD163-expressing tumor-associated macrophages (TAMs), and expressed CD47 and calreticulin, the dominant pro-phagocytic molecule. In addition, ATC tissues expressed the immune checkpoint molecules programmed cell death 1 and programmed death ligand 1. Blocking CD47 promoted the phagocytosis of ATC cell lines by macrophages in vitro. Anti-CD47 antibody treatment of ATC xenotransplanted mice increased the frequency of TAMs, enhanced the expression of macrophage activation markers, augmented tumor cell phagocytosis, and suppressed tumor growth. In double-transgenic ATC mice, CD47 was expressed on tumor cells, and blocking CD47 increased TAM frequencies. Conclusions: Targeting CD47 or CD47 in combination with programmed cell death 1 may potentially improve the outcomes of ATC patients and may represent a valuable addition to the current standard of care.
Assuntos
Antígenos de Diferenciação/imunologia , Antígeno CD47/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Receptores Imunológicos/imunologia , Carcinoma Anaplásico da Tireoide/imunologia , Neoplasias da Glândula Tireoide/imunologia , Evasão Tumoral/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos de Diferenciação/metabolismo , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Imunoterapia , Técnicas In Vitro , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Transplante de Neoplasias , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores Imunológicos/metabolismo , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fused in sarcoma (FUS) is an RNA binding protein involved in regulating many aspects of RNA processing and linked to several neurodegenerative diseases. Transcriptomics studies indicate that FUS binds a large variety of RNA motifs, suggesting that FUS RNA binding might be quite complex. Here, we present solution structures of FUS zinc finger (ZnF) and RNA recognition motif (RRM) domains bound to RNA. These structures show a bipartite binding mode of FUS comprising of sequence-specific recognition of a NGGU motif via the ZnF and an unusual shape recognition of a stem-loop RNA via the RRM. In addition, sequence-independent interactions via the RGG repeats significantly increase binding affinity and promote destabilization of structured RNA conformation, enabling additional binding. We further show that disruption of the RRM and ZnF domains abolishes FUS function in splicing. Altogether, our results rationalize why deciphering the RNA binding mode of FUS has been so challenging.