RESUMEN
Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca(2+) signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca(2+) increases. NAADP-evoked Ca(2+) signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca(2+)-dependent trafficking in Parkinson disease.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/genética , Enfermedad de Parkinson/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Lisosomas/metabolismo , Lisosomas/patología , NADP/análogos & derivados , NADP/genética , NADP/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca2+-dependent hubs for signaling.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Membranas/metabolismo , NADP/análogos & derivados , Señalización del Calcio/fisiología , Línea Celular Tumoral , Células Cultivadas , Células HeLa , Humanos , Lisosomas/metabolismo , NADP/metabolismo , Fosforilación/fisiologíaRESUMEN
Lysosomes are abundant organelles best known for their crucial role in macromolecule turnover. Lysosome dysfunction features in several diseases exemplified by the lysosomal storage disorders and is often associated with marked changes in lysosome structure. Lysosomal morphology may therefore serve as a sensitive readout of endocytic well-being. Here we describe methods for monitoring lysosome morphology in fixed and live cells using fluorescent probes and electron microscopy.
Asunto(s)
Lisosomas/ultraestructura , Células Cultivadas , Microscopía por Crioelectrón , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes/química , Humanos , Microscopía Electrónica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Forma de los Orgánulos , Análisis de la Célula Individual , Coloración y Etiquetado , Fijación del TejidoRESUMEN
BACKGROUND: Cognitive biases may contribute to delusion persistence. We tested this in a longitudinal study of first episode psychosis (FEP). METHODS: 34 FEP patients completed assessments of delusions and Jumping to Conclusions (JTC) at baseline and 12-month follow-up. RESULTS: JTC was associated with baseline delusion severity (t(32)=2.7, p=0.01). Baseline delusions persisted at follow-up for 8/20 participants (40%), who all jumped to conclusions (8/8, 100%), compared to half of those with no or changeable delusions (14/26, 54%; χ(2) (df=1)=5.7, p=0.03; Phi=0.4). CONCLUSION: Findings implicate cognitive biases in delusion persistence, and support the potential to reduce delusions through reasoning-focused interventions.