RESUMO
In Huntington's disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon 1 reporter, which showed remarkably high sensitivity and specificity in detecting mHTT seeding species in HD patient biosamples. We showed that the seeding-competent mHTT species in HD CSF are significantly elevated upon disease onset and with the progression of neuropathological grades. Mechanistically, we showed that mHTT seeding activities in patient CSF could be ameliorated by the overexpression of chaperone DNAJB6 and by antibodies against the polyproline domain of mHTT. Together, our study developed a selective and scalable cell-based tool to investigate mHTT seeding activities in HD CSF, and demonstrated that the CSF mHTT seeding species are significantly associated with certain disease states. This seeding activity can be ameliorated by targeting specific domain or proteostatic pathway of mHTT, providing novel insights into such pathological activities.
Assuntos
Líquido Cefalorraquidiano/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteína Huntingtina/metabolismo , Doença de Huntington/patologia , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Agregação Patológica de Proteínas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Linhagem Celular , Éxons/genética , Feminino , Genes Reporter/genética , Proteínas de Choque Térmico HSP40/genética , Humanos , Proteína Huntingtina/líquido cefalorraquidiano , Proteína Huntingtina/genética , Doença de Huntington/líquido cefalorraquidiano , Doença de Huntington/genética , Microscopia Intravital , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/genética , Mutação , Proteínas do Tecido Nervoso/genética , Agregação Patológica de Proteínas/líquido cefalorraquidiano , Agregação Patológica de Proteínas/genética , Domínios Proteicos/genética , Engenharia de Proteínas , Dobramento de ProteínaRESUMO
Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT.
Assuntos
Doença de Huntington/líquido cefalorraquidiano , Doença de Huntington/genética , Mutação , Proteínas do Tecido Nervoso/genética , Peptídeos/líquido cefalorraquidiano , Agregação Patológica de Proteínas/líquido cefalorraquidiano , Animais , Células Cultivadas , Feminino , Humanos , Proteína Huntingtina , Masculino , Microscopia Eletrônica , Agregação Patológica de Proteínas/patologia , Ratos , Ratos Transgênicos , TransfecçãoRESUMO
Toxic metals accumulation in brain has a significant role in the pathogenesis of Alzheimer's disease (AD) by accelerating amyloid ß (Aß) peptide aggregation. Aß has high affinity for iron and copper resulting in the generation of neurotoxic hydrogen peroxide, oxidative stress and free radical formation. Water-soluble conjugated polyfluorene derivative poly(9,9-bis(6-sulphate hexyl) fluorene-alt-1,4-phenylene) sodium salt (P1) binds Fe(3+) heme proteins selectively in cerebrospinal fluid (CSF), including ferritin in the Aß fibrils and diminishes their accumulation. Hence, therapeutic strategies involving clearance of Aß from brain plaques, metal removal, structurally modifying the aggregates, and preventing them from aggregating again into toxic polypeptides are vital strategies to control AD pathogenesis.