RESUMEN
The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis-mass spectrometry (LiP-MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP-MS detects well-characterized PPIs, including antibody-target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson's disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.
Asunto(s)
alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , Mapeo de Interacción de Proteínas , Espectrometría de Masas , Unión Proteica , Proteolisis , Enfermedad de Parkinson/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Mapas de Interacción de Proteínas , Conformación Proteica , Amiloide/metabolismo , Amiloide/química , Proteoma/metabolismoRESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative disorder and a central role for α-synuclein (αSyn; SNCA) in disease aetiology has been proposed based on genetics and neuropathology. To better understand the pathological mechanisms of αSyn, we generated induced pluripotent stem cells (iPSCs) from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of the SNCA locus and differentiated them into dopaminergic neurons (DAns). iPSC-derived DAn from PD patients carrying either mutation showed increased intracellular αSyn accumulation, and DAns from patients carrying the SNCA triplication displayed oligomeric αSyn pathology and elevated αSyn extracellular release. Transcriptomic analysis of purified DAns revealed perturbations in expression of genes linked to mitochondrial function, consistent with observed reduction in mitochondrial respiration, impairment in mitochondrial membrane potential, aberrant mitochondrial morphology and decreased levels of phosphorylated DRP1Ser616. Parkinson's iPSC-derived DAns showed increased endoplasmic reticulum stress and impairments in cholesterol and lipid homeostasis. Together, these data show a correlation between αSyn cellular pathology and deficits in metabolic and cellular bioenergetics in the pathology of PD.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Diferenciación Celular , Dinaminas/metabolismo , Estrés del Retículo Endoplásmico/genética , Metabolismo Energético/genética , Humanos , Metabolismo de los Lípidos/genética , Potencial de la Membrana Mitocondrial , Mitocondrias/ultraestructura , Mutación , Enfermedad de Parkinson/metabolismo , RNA-Seq , Sinucleinopatías/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Inflammation of the brain and the consequential immunological responses play pivotal roles in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia (FTD). Microglia, the resident macrophage cells of the brain, have also emerged as key players in neuroinflammation. As primary human microglia from living subjects are normally not accessible to researchers, there is a pressing need for an alternative source of authentic human microglia which allows modeling of neurodegeneration in vitro. Several protocols for induced pluripotent stem cell (iPSC)-derived microglia have recently been developed and provide unlimited access to patient-derived material. In this present study, we give an overview of iPSC-derived microglia models in monoculture and coculture systems, their advantages and limitations, and how they have already been used for disease phenotyping. Furthermore, we outline some of the gene engineering tools to generate isogenic controls, the creation of gene knockout iPSC lines, as well as covering reporter cell lines, which could help to elucidate complex cell interaction mechanisms in the microglia/neuron coculture system, for example, microglia-induced synapse loss. Finally, we deliberate on how said cocultures could aid in personalized drug screening to identify patient-specific therapies against neurodegeneration. Stem Cells 2019;37:724-730.
Asunto(s)
Enfermedad de Alzheimer/terapia , Esclerosis Amiotrófica Lateral/terapia , Demencia Frontotemporal/terapia , Microglía/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/terapia , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Técnicas de Cocultivo , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Técnicas de Inactivación de Genes , Ingeniería Genética/métodos , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Inflamación , Microglía/efectos de los fármacos , Microglía/patología , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/patología , Fármacos Neuroprotectores/uso terapéutico , Nootrópicos/uso terapéutico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patologíaRESUMEN
The Parkinson's disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease and is expressed in immune subsets. Here, we characterize a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes using human induced pluripotent stem cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-γ in these cells, including a 187-kDa cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles but is recruited to LAMP1+/RAB9+ "maturing" phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Macrófagos/metabolismo , Fagosomas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Diferenciación Celular , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/citología , Interferón gamma/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Macrófagos/citología , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismoRESUMEN
A recent gamma-retroviral clinical Chronic Granulomatous Disease (CGD) gene therapy (GT) trial achieved proof-of-concept but was accompanied by activation of oncogenes and transgene silencing. The ubiquitous chromatin opening element (UCOE) comprises the sequences of two divergently oriented house-keeping gene promoters and is known to have anti-silencing properties. In a screen we identified two novel UCOE constructs that prevent adjacent promoter methylation in P19 cells. Experiments were continued with the shorter UCOE constructs in induced pluripotent stem cells (iPSC) derived from a p47phox-deficient CGD patient. The iPSC line was transduced with the lentiviral GT vectors expressing P47 under the constitutively active SFFV promoter with UCOE element (UCOE_SF) and without UCOE element (SF) adjacent to the SFFV promoter. The iPSC were expanded before propagation towards neutrophils. 20 days after transduction the UCOE_SF vector was protected from methylation in iPSC as previously shown in P19 cells, whereas the SF vector was heavily methylated in iPSC. The UCOE_SF vector maintained stable transgene expression in iPSC, macrophages and neutrophils, whereas the SF vector was strongly silenced. The UCOE_SF vector stably restored ROS production in neutrophils, whereas for the SF vector the count of ROS producing cells was marginal. To conclude, we have shown that the prevention of transgene silencing by UCOE is functionally and mechanistically preserved upon terminal neutrophil differentiation.
RESUMEN
To examine the pathogenic role of α-synuclein (αS) in Parkinson's Disease, we have generated induced Pluripotent Stem Cell lines from early onset Parkinson's Disease patients with SNCA A53T and SNCA Triplication mutations, and in this study have differentiated them to PSC-macrophages (pMac), which recapitulate many features of their brain-resident cousins, microglia. We show that SNCA Triplication pMac, but not A53T pMac, have significantly increased intracellular αS versus controls and release significantly more αS to the medium. SNCA Triplication pMac, but not A53T pMac, show significantly reduced phagocytosis capability and this can be phenocopied by adding monomeric αS to the cell culture medium of control pMac. Fibrillar αS is taken up by pMac by actin-rearrangement-dependent pathways, and monomeric αS by actin-independent pathways. Finally, pMac degrade αS and this can be arrested by blocking lysosomal and proteasomal pathways. Together, these results show that macrophages are capable of clearing αS, but that high levels of exogenous or endogenous αS compromise this ability, likely a vicious cycle scenario faced by microglia in Parkinson's disease.
Asunto(s)
Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Fagocitosis/efectos de los fármacos , alfa-Sinucleína/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Diferenciación Celular , Femenino , Dosificación de Gen , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Enfermedad de Parkinson/patología , Células Madre Pluripotentes , alfa-Sinucleína/genéticaRESUMEN
Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology.