RESUMO
The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.
Assuntos
Membrana Celular/química , Proteínas PrPC/análise , Príons/antagonistas & inibidores , Animais , Caseína Quinase II/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HEK293 , Harmalina/análogos & derivados , Harmalina/farmacologia , Hematoxilina/análogos & derivados , Hematoxilina/farmacologia , Humanos , Camundongos , Neuroblastoma , Proteínas PrPC/genética , Príons/biossíntese , Príons/toxicidade , Quinacrina/farmacologia , Tacrolimo/farmacologiaRESUMO
Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Astrócitos/enzimologia , Exossomos/enzimologia , Neurônios Motores/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Superóxido Dismutase/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Morte Celular/genética , Exossomos/genética , Exossomos/patologia , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Mutação , Proteínas do Tecido Nervoso/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Proteína com ValosinaRESUMO
In prion diseases, the infectious isoform of the prion protein (PrP(Sc)) may subvert a normal, physiological activity of the cellular isoform (PrP(C)). A deletion mutant of the prion protein (Delta105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP(C) and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Delta105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Delta105-125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Delta105-125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.