RESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative condition characterized by the progressive deterioration of motor neurons in the cortex and spinal cord. Using an automated robotic microscope platform that enables the longitudinal tracking of thousands of single neurons, we examine the effects a large library of compounds on modulating the survival of primary neurons expressing a mutation known to cause ALS. The goal of our analysis is to identify the few potentially beneficial compounds among the many assayed, the vast majority of which do not extend neuronal survival. This resembles the large-scale simultaneous inference scenario familiar from microarray analysis, but transferred to the survival analysis setting due to the novel experimental setup. We apply a three-component mixture model to censored survival times of thousands of individual neurons subjected to hundreds of different compounds. The shrinkage induced by our model significantly improves performance in simulations relative to performing treatment-wise survival analysis and subsequent multiple testing adjustment. Our analysis identified compounds that provide insight into potential novel therapeutic strategies for ALS.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Modelos Estadísticos , Análisis de Supervivencia , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/mortalidad , Simulación por Computador , Humanos , Neuronas Motoras/efectos de los fármacosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2α phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2α phosphorylation in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.
Asunto(s)
Adenina/análogos & derivados , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Indoles/farmacología , Adenina/farmacología , Análisis de Varianza , Animales , Ataxinas , Proteínas de Unión al ADN/genética , Drosophila melanogaster , Ontología de Genes , Ensayos Analíticos de Alto Rendimiento , Humanos , Immunoblotting , Inmunohistoquímica , Proteínas Luminiscentes , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Proteínas de Unión a Poli(A)/metabolismo , Interferencia de ARN , Retina/metabolismo , Retina/ultraestructura , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequeñas , Médula Espinal/citología , Médula Espinal/metabolismo , Proteína Fluorescente RojaRESUMEN
In Parkinson's disease (PD), dopamine depletion alters neuronal activity in the direct and indirect pathways and leads to increased synchrony in the basal ganglia network. However, the origins of these changes remain elusive. Because GABAergic interneurons regulate activity of projection neurons and promote neuronal synchrony, we recorded from pairs of striatal fast-spiking (FS) interneurons and direct- or indirect-pathway MSNs after dopamine depletion with 6-OHDA. Synaptic properties of FS-MSN connections remained similar, yet within 3 days of dopamine depletion, individual FS cells doubled their connectivity to indirect-pathway MSNs, whereas connections to direct-pathway MSNs remained unchanged. A model of the striatal microcircuit revealed that such increases in FS innervation were effective at enhancing synchrony within targeted cell populations. These data suggest that after dopamine depletion, rapid target-specific microcircuit organization in the striatum may lead to increased synchrony of indirect-pathway MSNs that contributes to pathological network oscillations and motor symptoms of PD.