RESUMEN
Expansion of intronic GGGGCC repeats in the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Transcription of the expanded repeats results in the formation of RNA-containing nuclear foci and altered RNA metabolism. In addition, repeat-associated non-AUG (RAN) translation of the expanded GGGGCC-repeat sequence results in the production of highly toxic dipeptide-repeat (DPR) proteins. GGGGCC repeat-containing transcripts form G-quadruplexes, which are associated with formation of RNA foci and RAN translation. Zfp106, an RNA-binding protein essential for motor neuron survival in mice, suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Here, we show that Zfp106 inhibits formation of RNA foci and significantly reduces RAN translation caused by GGGGCC repeats in cultured mammalian cells, and we demonstrate that Zfp106 coexpression reduces the levels of DPRs in C9orf72 patient-derived cells. Further, we show that Zfp106 binds to RNA G-quadruplexes and causes a conformational change in the G-quadruplex structure formed by GGGGCC repeats. Together, these data demonstrate that Zfp106 suppresses the formation of RNA foci and DPRs caused by GGGGCC repeats and suggest that the G-quadruplex RNA-binding function of Zfp106 contributes to its suppression of GGGGCC repeat-mediated cytotoxicity.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína C9orf72 , G-Cuádruplex , Proteínas de Unión al ARN , ARN , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Unión Proteica , Biosíntesis de Proteínas , ARN/metabolismo , ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.
Asunto(s)
Proteína C9orf72/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Dipéptidos/metabolismo , Proteolisis , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular , Codón Iniciador/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Expansión de las Repeticiones de ADN/genética , Modelos Animales de Enfermedad , Drosophila/efectos de los fármacos , Demencia Frontotemporal/patología , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/patología , Isoquinolinas/farmacología , Longevidad/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Biosíntesis de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Interferencia de ARN , Sulfonamidas/farmacologíaRESUMEN
In addition to motor neurone degeneration, up to 50% of amyotrophic lateral sclerosis (ALS) patients present with cognitive decline. Understanding the neurobiological changes underlying these cognitive deficits is critical, as cognitively impaired patients exhibit a shorter survival time from symptom onset. Given the pathogenic role of synapse loss in other neurodegenerative diseases in which cognitive decline is apparent, such as Alzheimer's disease, we aimed to assess synaptic integrity in the ALS brain. Here, we have applied a unique combination of high-resolution imaging of post-mortem tissue with neuropathology, genetic screening and cognitive profiling of ALS cases. Analyses of more than 1 million synapses using two complimentary high-resolution techniques (electron microscopy and array tomography) revealed a loss of synapses from the prefrontal cortex of ALS patients. Importantly, synapse loss was significantly greater in cognitively impaired cases and was not due to cortical atrophy, nor associated with dementia-associated neuropathology. Interestingly, we found a trend between pTDP-43 pathology and synapse loss in the frontal cortex and discovered pTDP-43 puncta at a subset of synapses in the ALS brains. From these data, we postulate that synapse loss in the prefrontal cortex represents an underlying neurobiological substrate of cognitive decline in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/psicología , Disfunción Cognitiva/patología , Corteza Prefrontal/patología , Sinapsis/patología , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Atrofia , Disfunción Cognitiva/etiología , Disfunción Cognitiva/genética , Disfunción Cognitiva/metabolismo , Estudios de Cohortes , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosforilación , Corteza Prefrontal/metabolismo , Corteza Prefrontal/ultraestructura , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Importance: Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) holds the promise to arrest or cure monogenic disease if it can be determined which genetic change to create without inducing unintended cellular dysfunction and how to deliver this technology to the target organ reliably and safely. Clinical trials for blood and liver disorders, for which delivery of CRISPR is not limiting, show promise, yet no trials have begun for central nervous system (CNS) indications. Observations: The CNS is arguably the most challenging target given its innate exclusion of large molecules and its defenses against bacterial invasion (from which CRISPR originates). Herein, the types of CRISPR editing (DNA cutting, base editing, and templated repair) and how these are applied to different genetic variants are summarized. The challenges of delivering genome editors to the CNS, including the viral and nonviral delivery vehicles that may ultimately circumvent these challenges, are discussed. Also, ways to minimize the potential in vivo genotoxic effects of genome editors through delivery vehicle design and preclinical off-target testing are considered. The ethical considerations of germline editing, a potential off-target outcome of any gene editing therapy, are explored. The unique regulatory challenges of a human-specific therapy that cannot be derisked solely in animal models are also discussed. Conclusions and Relevance: An understanding of both the potential benefits and challenges of CRISPR gene therapy better informs the scientific, clinical, regulatory, and timeline considerations of developing CRISPR gene therapy for neurologic diseases.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Animales , Humanos , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Terapia Genética , Edición Génica , Sistema Nervioso CentralRESUMEN
Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.