RESUMEN
A hexanucleotide repeat expansion in the C9orf72 gene has been identified as the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. The expanded hexanucleotide repeat is translated by an unconventional mechanism to produce five species of dipeptide repeat (DPR) proteins, glycine-proline (GP), glycine-alanine (GA), glycine-arginine (GR), proline-alanine (PA) and proline-arginine (PR). Of these, the arginine-rich ones, PR and GR, are highly toxic in a variety of model systems, ranging from human cells, to Drosophila, to even the budding yeast, Saccharomyces cerevisiae. We recently performed a genetic screen in yeast for modifiers of PR toxicity and identified suppressors and enhancers, many of which function in nucleocytoplasmic transport. Whether or not GR toxicity involves similar mechanisms to PR is unresolved. Therefore, we performed a genetic screen in yeast to identify modifiers of GR toxicity and compared the results of the GR screen to results from our previous PR screen. Surprisingly, there was only a small degree of overlap between the two screens, suggesting potential for distinct toxicity mechanisms between PR and GR.
Asunto(s)
Proteína C9orf72/toxicidad , Proteínas Recombinantes/toxicidad , Secuencias Repetitivas de Aminoácido , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteína C9orf72/genética , Pruebas Genéticas , Humanos , Técnicas Microbiológicas/métodos , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Mutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Inactivación de Genes/métodos , Pruebas Genéticas , Humanos , RNA-Seq , Mutaciones Letales Sintéticas/genética , Células U937RESUMEN
C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Núcleo Celular/metabolismo , Expansión de las Repeticiones de ADN/fisiología , Dipéptidos/metabolismo , Demencia Frontotemporal/metabolismo , Proteínas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Esclerosis Amiotrófica Lateral/genética , Animales , Proteína C9orf72 , Núcleo Celular/genética , Células Cultivadas , Dipéptidos/genética , Demencia Frontotemporal/genética , Eliminación de Gen , Humanos , Ratones , Proteínas/genética , LevadurasRESUMEN
We hypothesized that microglia in the ventral posterolateral (VPL) nucleus of the thalamus are reactive following peripheral nerve injury, and that inhibition of microglia by minocycline injection in the VPL attenuates thermal hyperalgesia. Our results show increased expression of OX-42 co-localized with phosphorylated p38MAPK (P-p38) in the VPL seven days after chronic constriction injury (CCI) of the sciatic nerve. However, astrocytic GFAP expression in the VPL is unchanged 7 and 14 days after CCI. Microinjection of minocycline into the VPL contralateral to CCI reverses thermal hyperalgesia, whereas vehicle injection has no effect on paw withdrawal latency. Minocycline abrogates the increased expression of OX-42 in the VPL after CCI. Therefore, peripheral nerve injury favors a hyperactive microglial phenotype in the VPL, suggesting remote neuroimmune signaling from the damaged nerve to the brain, concomitant with neuropathic behavior that is reversed by local intervention in the VPL to inhibit microglia.