RESUMEN
Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-ß (Aß) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aß in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aß co-aggregates account for ~50% of the mass of diffusible Aß aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aß tune disease-related functions of Aß aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aß. Selectively removing non-lipidated apoE4-Aß co-aggregates enhances clearance of toxic Aß by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Apolipoproteína E4 , Apolipoproteínas E , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Humanos , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Animales , Apolipoproteína E4/metabolismo , Apolipoproteína E4/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Ratones , Femenino , Agregado de Proteínas , Masculino , Agregación Patológica de Proteínas/metabolismo , Ratones Transgénicos , Neuroglía/metabolismoRESUMEN
Mitochondrial dysfunction is a common feature of C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD); however, it remains unclear whether this is a cause or consequence of the pathogenic process. Analysing multiple aspects of mitochondrial biology across several Drosophila models of C9orf72-ALS/FTD, we found morphology, oxidative stress, and mitophagy are commonly affected, which correlated with progressive loss of locomotor performance. Notably, only genetic manipulations that reversed the oxidative stress levels were also able to rescue C9orf72 locomotor deficits, supporting a causative link between mitochondrial dysfunction, oxidative stress, and behavioural phenotypes. Targeting the key antioxidant Keap1/Nrf2 pathway, we found that genetic reduction of Keap1 or pharmacological inhibition by dimethyl fumarate significantly rescued the C9orf72-related oxidative stress and motor deficits. Finally, mitochondrial ROS levels were also elevated in C9orf72 patient-derived iNeurons and were effectively suppressed by dimethyl fumarate treatment. These results indicate that mitochondrial oxidative stress is an important mechanistic contributor to C9orf72 pathogenesis, affecting multiple aspects of mitochondrial function and turnover. Targeting the Keap1/Nrf2 signalling pathway to combat oxidative stress represents a therapeutic strategy for C9orf72-related ALS/FTD.
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Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Modelos Animales de Enfermedad , Demencia Frontotemporal , Proteína 1 Asociada A ECH Tipo Kelch , Mitocondrias , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Fenotipo , Transducción de Señal , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Mitocondrias/metabolismo , Animales , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Humanos , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Especies Reactivas de Oxígeno/metabolismo , Mitofagia/genética , Dimetilfumarato/farmacología , MasculinoRESUMEN
The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream, playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS); however, the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression, compromised barrier integrity, and increased P-glycoprotein transporter activity. In addition, mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover, our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally, C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.
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Esclerosis Amiotrófica Lateral , Barrera Hematoencefálica , Células Endoteliales , Humanos , Esclerosis Amiotrófica Lateral/genética , Barrera Hematoencefálica/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Células Endoteliales/metabolismoRESUMEN
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.
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to â¼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS.
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Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Animales , Ratones , Dipéptidos , Proteína C9orf72 , Transporte Activo de Núcleo Celular , Células HEK293 , Péptidos , Neuronas Motoras , ARN , Factores de Empalme Serina-ArgininaRESUMEN
New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.
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COVID-19 , SARS-CoV-2 , COVID-19/genética , Cromatografía Liquida , Codón sin Sentido , ARN Polimerasas Dirigidas por ADN/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Estudio de Asociación del Genoma Completo , Humanos , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , SARS-CoV-2/genética , Espectrometría de Masas en Tándem , Proteinas del Complejo de Replicasa Viral , Replicación Viral/genéticaRESUMEN
The blood-brain barrier (BBB) is a highly specialized and dynamic compartment which regulates the uptake of molecules and solutes from the blood. The relevance of the maintenance of a healthy BBB underpinning disease prevention as well as the main pathomechanisms affecting BBB function will be detailed in this review. Barrier disruption is a common aspect in both neurodegenerative diseases, such as amyotrophic lateral sclerosis, and neurodevelopmental diseases, including autism spectrum disorders. Throughout this review, conditions altering the BBB during the earliest and latest stages of life will be discussed, revealing common factors involved. Due to the barrier's role in protecting the brain from exogenous components and xenobiotics, drug delivery across the BBB is challenging. Potential therapies based on the BBB properties as molecular Trojan horses, among others, will be reviewed, as well as innovative treatments such as stem cell therapies. Additionally, due to the microbiome influence on the normal function of the brain, microflora modulation strategies will be discussed. Finally, future research directions are highlighted to address the current gaps in the literature, emphasizing the idea that common therapies for both neurodevelopmental and neurodegenerative pathologies exist.
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Barrera Hematoencefálica , Enfermedades Neurodegenerativas , Humanos , Barrera Hematoencefálica/patología , Enfermedades Neurodegenerativas/patología , Transporte Biológico , Encéfalo/patología , Sistemas de Liberación de MedicamentosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with no effective cure. Astrocytes display a toxic phenotype in ALS and contribute to motoneuron (MN) degeneration. Modulating astrocytes' neurotoxicity can reduce MN death. Our previous studies showed the beneficial effect of mesenchymal stem cell (MSC) administration in SOD1G93A ALS mice, but the mechanisms are still unclear. We postulated that the effects could be mediated by extracellular vesicles (EVs) secreted by MSCs. We investigated, by immunohistochemical, molecular, and in vitro functional analyses, the activity of MSC-derived EVs on the pathological phenotype and neurotoxicity of astrocytes isolated from the spinal cord of symptomatic SOD1G93A mice and human astrocytes (iAstrocytes) differentiated from inducible neural progenitor cells (iNPCs) of ALS patients. In vitro EV exposure rescued mouse and human ALS astrocytes' neurotoxicity towards MNs. EVs significantly dampened the pathological phenotype and neuroinflammation in SOD1G93A astrocytes. In iAstrocytes, exposure to EVs increased the antioxidant factor Nrf2 and reduced reactive oxygen species. We previously found nine miRNAs upregulated in MSC-derived EVs. Here, the transfection of SOD1G93A astrocytes with single miRNA mimics reduced astrocytes' activation and the expression of neuroinflammatory factors. Moreover, miR-466q and miR-467f mimics downregulate Mapk11, while miR-466m-5p and miR-466i-3p mimics promote the nuclear translocation of Nrf2. In iAstrocytes, transfection with miR-29b-3p mimic upregulated NQO1 antioxidant activity and reduced neurotoxicity towards MNs. MSC-derived EVs modulate astrocytes' reactive phenotype and neurotoxicity through anti-inflammatory and antioxidant-shuttled miRNAs, thus representing a therapeutic strategy in ALS.
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Esclerosis Amiotrófica Lateral , Vesículas Extracelulares , Células Madre Mesenquimatosas , MicroARNs , Enfermedades Neurodegenerativas , Humanos , Ratones , Animales , Esclerosis Amiotrófica Lateral/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Ratones Transgénicos , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismoRESUMEN
Dipeptide repeat (DPR) proteins are aggregation-prone polypeptides encoded by the pathogenic GGGGCC repeat expansion in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic ß-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and -independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to-motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly-GA cellular uptake and propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.
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Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Demencia Frontotemporal , Esclerosis Amiotrófica Lateral/patología , Proteína C9orf72/genética , Dipéptidos , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Humanos , Neuronas Motoras/metabolismoRESUMEN
Three-dimensional (3D) cultures, so-called organoids, have emerged as an attractive tool for disease modeling and therapeutic innovations. Here, we aim to determine if boundary cap neural crest stem cells (BC) can survive and differentiate in gelatin-based 3D bioprinted bioink scaffolds in order to establish an enabling technology for the fabrication of spinal cord organoids on a chip. BC previously demonstrated the ability to support survival and differentiation of co-implanted or co-cultured cells and supported motor neuron survival in excitotoxically challenged spinal cord slice cultures. We tested different combinations of bioink and cross-linked material, analyzed the survival of BC on the surface and inside the scaffolds, and then tested if human iPSC-derived neural cells (motor neuron precursors and astrocytes) can be printed with the same protocol, which was developed for BC. We showed that this protocol is applicable for human cells. Neural differentiation was more prominent in the peripheral compared to central parts of the printed construct, presumably because of easier access to differentiation-promoting factors in the medium. These findings show that the gelatin-based and enzymatically cross-linked hydrogel is a suitable bioink for building a multicellular, bioprinted spinal cord organoid, but that further measures are still required to achieve uniform neural differentiation.
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Células-Madre Neurales , Organoides , Gelatina , Humanos , Cresta Neural , Médula EspinalRESUMEN
New therapeutic targets are a valuable resource in the struggle to reduce the morbidity and mortality associated with the COVID-19 pandemic, caused by the SARS-CoV-2 virus. Genome-wide association studies (GWAS) have identified risk loci, but some loci are associated with co-morbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins; EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. Lung-specific eQTLs were identified from GTEx (v7) for each of the 332 host proteins. Aggregating COVID-19 GWAS statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19 which survived stringent multiple testing correction. EXOSC2 is a component of the RNA exosome and indeed, LC-MS/MS analysis of protein pulldowns demonstrated an interaction between the SARS-CoV-2 RNA polymerase and the majority of human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression, impeded SARS-CoV-2 replication and upregulated oligoadenylate synthase ( OAS) genes, which have been linked to a successful immune response against SARS-CoV-2. Reduced EXOSC2 expression did not reduce cellular viability. OAS gene expression changes occurred independent of infection and in the absence of significant upregulation of other interferon-stimulated genes (ISGs). Targeted depletion or functional inhibition of EXOSC2 may be a safe and effective strategy to protect at-risk individuals against clinical COVID-19.
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Topoisomerase1 (TOP1)-mediated chromosomal breaks are endogenous sources of DNA damage that affect neuronal genome stability. Whether TOP1 DNA breaks are sources of genomic instability in Huntington's disease (HD) is unknown. Here, we report defective 53BP1 recruitment in multiple HD cell models, including striatal neurons derived from HD patients. Defective 53BP1 recruitment is due to reduced H2A ubiquitination caused by the limited RNF168 activity. The reduced availability of RNF168 is caused by an increased interaction with p62, a protein involved in selective autophagy. Depletion of p62 or disruption of the interaction between RNAF168 and p62 was sufficient to restore 53BP1 enrichment and subsequent DNA repair in HD models, providing new opportunities for therapeutic interventions. These findings are reminiscent to what was described for p62 accumulation caused by C9orf72 expansion in ALS/FTD and suggest a common mechanism by which protein aggregation perturb DNA repair signaling.
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Roturas del ADN , Reparación del ADN , Enfermedad de Huntington/metabolismo , Proteína Sequestosoma-1/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Enfermedad de Huntington/genética , Neuronas/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
Amyotrophic lateral sclerosis (ALS) is a complex disease that leads to motor neuron death. Despite heritability estimates of 52%, genome-wide association studies (GWASs) have discovered relatively few loci. We developed a machine learning approach called RefMap, which integrates functional genomics with GWAS summary statistics for gene discovery. With transcriptomic and epigenetic profiling of motor neurons derived from induced pluripotent stem cells (iPSCs), RefMap identified 690 ALS-associated genes that represent a 5-fold increase in recovered heritability. Extensive conservation, transcriptome, network, and rare variant analyses demonstrated the functional significance of candidate genes in healthy and diseased motor neurons and brain tissues. Genetic convergence between common and rare variation highlighted KANK1 as a new ALS gene. Reproducing KANK1 patient mutations in human neurons led to neurotoxicity and demonstrated that TDP-43 mislocalization, a hallmark pathology of ALS, is downstream of axonal dysfunction. RefMap can be readily applied to other complex diseases.
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Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Proteínas Adaptadoras Transductoras de Señales/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Muerte Celular/genética , Proteínas del Citoesqueleto/genética , Estudio de Asociación del Genoma Completo , Humanos , Células Madre Pluripotentes Inducidas/patología , Neuronas Motoras/patologíaRESUMEN
Although controversial, the amyloid cascade hypothesis remains central to the Alzheimer's disease (AD) field and posits amyloid-beta (Aß) as the central factor initiating disease onset. In recent years, there has been an increase in emphasis on studying the role of low molecular weight aggregates, such as oligomers, which are suggested to be more neurotoxic than fibrillary Aß. Other Aß isoforms, such as truncated Aß, have also been implicated in disease. However, developing a clear understanding of AD pathogenesis has been hampered by the complexity of Aß biochemistry in vitro and in vivo. This review explores factors contributing to the lack of consistency in experimental approaches taken to model Aß aggregation and toxicity and provides an overview of the different techniques available to analyse Aß, such as electron and atomic force microscopy, nuclear magnetic resonance spectroscopy, dye-based assays, size exclusion chromatography, mass spectrometry and SDS-PAGE. The review also explores how different types of Aß can influence Aß aggregation and toxicity, leading to variation in experimental outcomes, further highlighting the need for standardisation in Aß preparations and methods used in current research.
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Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Animales , HumanosRESUMEN
Disruption to protein homeostasis caused by lysosomal dysfunction and associated impairment of autophagy is a prominent pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). The most common genetic cause of ALS/FTD is a G4C2 hexanucleotide repeat expansion in C9orf72 (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 repeat transcripts gives rise to dipeptide repeat (DPR) proteins that have been shown to be toxic and may contribute to disease etiology. Genetic variants in TMEM106B have been associated with frontotemporal lobar degeneration with TDP-43 pathology and disease progression in C9ALS/FTD. TMEM106B encodes a lysosomal transmembrane protein of unknown function that is involved in various aspects of lysosomal biology. How TMEM106B variants affect C9ALS/FTD is not well understood but has been linked to changes in TMEM106B protein levels. Here, we investigated TMEM106B function in the context of C9ALS/FTD DPR pathology. We report that knockdown of TMEM106B expression exacerbates the accumulation of C9ALS/FTD-associated cytotoxic DPR proteins in cell models expressing RAN-translated or AUG-driven DPRs as well as in C9ALS/FTD-derived iAstrocytes with an endogenous G4C2 expansion by impairing autophagy. Loss of TMEM106B caused a block late in autophagy by disrupting autophagosome to autolysosome maturation which coincided with impaired lysosomal acidification, reduced cathepsin activity, and juxtanuclear clustering of lysosomes. Lysosomal clustering required Rab7A and coincided with reduced Arl8b-mediated anterograde transport of lysosomes to the cell periphery. Increasing Arl8b activity in TMEM106B-deficient cells not only restored the distribution of lysosomes, but also fully rescued autophagy and DPR protein accumulation. Thus, we identified a novel function of TMEM106B in autophagosome maturation via Arl8b. Our findings indicate that TMEM106B variants may modify C9ALS/FTD by regulating autophagic clearance of DPR proteins. Caution should therefore be taken when considering modifying TMEM106B expression levels as a therapeutic approach in ALS/FTD.
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BACKGROUND: Pathological interactions between ß-amyloid (Aß) and tau drive synapse loss and cognitive decline in Alzheimer's disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aß-induced synaptotoxicity in AD is not well understood. METHODS: We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human Aß that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of Aß before being added to naïve rodent or human neuron cultures. A cytokine, identified in unbiased screens of stimulated astrocyte media and in postmortem human AD brain lysates was also applied to neurons, including those pre-treated with a chemokine receptor antagonist. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures. RESULTS: We found that conditioned medium from stimulated astrocytes induces exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C-X-C motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal C-X-C motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and Aß-stimulated astrocyte secretions. CONCLUSIONS: Our data indicate that astrocytes exacerbate the synaptotoxic effects of Aß via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine-receptor pair as a novel target for therapeutic intervention in AD.
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Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/toxicidad , Astrocitos/patología , Quimiocina CXCL1/antagonistas & inhibidores , Quimiocina CXCL1/química , Sinapsis/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Espinas Dendríticas/patología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Receptores de Interleucina-8B/antagonistas & inhibidores , Proteínas tau/química , Proteínas tau/toxicidadRESUMEN
BACKGROUND: Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. METHODS: Here, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. RESULTS: Our study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1-3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. CONCLUSIONS: Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.
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Transporte Activo de Núcleo Celular/fisiología , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , Neuronas/metabolismo , ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Drosophila , Humanos , Neuronas/patología , Neuroprotección/fisiologíaRESUMEN
Increasing evidence supports the involvement of DNA damage in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Elevated levels of DNA damage are consistently observed in both sporadic and familial forms of ALS and may also play a role in Western Pacific ALS, which is thought to have an environmental cause. The cause of DNA damage in ALS remains unclear but likely differs between genetic subgroups. Repeat expansion in the C9ORF72 gene is the most common genetic cause of familial ALS and responsible for about 10% of sporadic cases. These genetic mutations are known to cause R-loops, thus increasing genomic instability and DNA damage, and generate dipeptide repeat proteins, which have been shown to lead to DNA damage and impairment of the DNA damage response. Similarly, several genes associated with ALS including TARDBP, FUS, NEK1, SQSTM1 and SETX are known to play a role in DNA repair and the DNA damage response, and thus may contribute to neuronal death via these pathways. Another consistent feature present in both sporadic and familial ALS is the ability of astrocytes to induce motor neuron death, although the factors causing this toxicity remain largely unknown. In this review, we summarise the evidence for DNA damage playing a causative or secondary role in the pathogenesis of ALS as well as discuss the possible mechanisms involved in different genetic subtypes with particular focus on the role of astrocytes initiating or perpetuating DNA damage in neurons.