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1.
Cell ; 187(14): 3671-3689.e23, 2024 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-38866017

RESUMEN

Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.


Asunto(s)
Supervivencia Celular , Neuronas Dopaminérgicas , FN-kappa B , Factor de Necrosis Tumoral alfa , Proteína p53 Supresora de Tumor , Neuronas Dopaminérgicas/metabolismo , Animales , Humanos , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ratones , Supervivencia Celular/efectos de los fármacos , Transducción de Señal , Enfermedad de Parkinson/metabolismo , Células Madre Pluripotentes/metabolismo , Apoptosis , Modelos Animales de Enfermedad , Sistemas CRISPR-Cas
2.
Annu Rev Biochem ; 92: 435-464, 2023 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-37018845

RESUMEN

The polyamines putrescine, spermidine, and spermine are abundant polycations of vital importance in mammalian cells. Their cellular levels are tightly regulated by degradation and synthesis, as well as by uptake and export. Here, we discuss the delicate balance between the neuroprotective and neurotoxic effects of polyamines in the context of Parkinson's disease (PD). Polyamine levels decline with aging and are altered in patients with PD, whereas recent mechanistic studies on ATP13A2 (PARK9) demonstrated a driving role of a disturbed polyamine homeostasis in PD. Polyamines affect pathways in PD pathogenesis, such as α-synuclein aggregation, and influence PD-related processes like autophagy, heavy metal toxicity, oxidative stress, neuroinflammation, and lysosomal/mitochondrial dysfunction. We formulate outstanding research questions regarding the role of polyamines in PD, their potential as PD biomarkers, and possible therapeutic strategies for PD targeting polyamine homeostasis.


Asunto(s)
Enfermedad de Parkinson , Trastornos Parkinsonianos , Animales , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Poliaminas/metabolismo , Neuroprotección , Espermidina/metabolismo , Mamíferos/metabolismo
3.
Cell ; 186(24): 5394-5410.e18, 2023 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-37922901

RESUMEN

Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.


Asunto(s)
Terapia Genética , Enfermedad de Parkinson , Animales , Humanos , Ratones , Cuerpo Estriado/metabolismo , Levodopa/uso terapéutico , Levodopa/genética , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/terapia , Primates , Receptores de Dopamina D1/metabolismo , Modelos Animales de Enfermedad
4.
Cell ; 186(22): 4868-4884.e12, 2023 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-37863056

RESUMEN

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.


Asunto(s)
Envejecimiento , Humor Acuoso , Inteligencia Artificial , Biopsia Líquida , Proteómica , Humanos , Envejecimiento/metabolismo , Humor Acuoso/química , Biopsia , Enfermedad de Parkinson/diagnóstico
5.
Cell ; 186(18): 3845-3861.e24, 2023 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-37591240

RESUMEN

Dopaminergic projections regulate various brain functions and are implicated in many neuropsychiatric disorders. There are two anatomically and functionally distinct dopaminergic projections connecting the midbrain to striatum: nigrostriatal, which controls movement, and mesolimbic, which regulates motivation. However, how these discrete dopaminergic synaptic connections are established is unknown. Through an unbiased search, we identify that two groups of antagonistic TGF-ß family members, bone morphogenetic protein (BMP)6/BMP2 and transforming growth factor (TGF)-ß2, regulate dopaminergic synapse development of nigrostriatal and mesolimbic neurons, respectively. Projection-preferential expression of their receptors contributes to specific synapse development. Downstream, Smad1 and Smad2 are specifically activated and required for dopaminergic synapse development and function in nigrostriatal vs. mesolimbic projections. Remarkably, Smad1 mutant mice show motor defects, whereas Smad2 mutant mice show lack of motivation. These results uncover the molecular logic underlying the proper establishment of functionally segregated dopaminergic synapses and may provide strategies to treat relevant, projection-specific disease symptoms by targeting specific BMPs/TGF-ß and/or Smads.


Asunto(s)
Cuerpo Estriado , Dopamina , Animales , Ratones , Mesencéfalo , Motivación , Movimiento , Sinapsis
6.
Cell ; 185(11): 1943-1959.e21, 2022 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-35545089

RESUMEN

Parthanatos-associated apoptosis-inducing factor (AIF) nuclease (PAAN), also known as macrophage migration inhibitor factor (MIF), is a member of the PD-D/E(X)K nucleases that acts as a final executioner in parthanatos. PAAN's role in Parkinson's disease (PD) and whether it is amenable to chemical inhibition is not known. Here, we show that neurodegeneration induced by pathologic α-synuclein (α-syn) occurs via PAAN/MIF nuclease activity. Genetic depletion of PAAN/MIF and a mutant lacking nuclease activity prevent the loss of dopaminergic neurons and behavioral deficits in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Compound screening led to the identification of PAANIB-1, a brain-penetrant PAAN/MIF nuclease inhibitor that prevents neurodegeneration induced by α-syn PFF, AAV-α-syn overexpression, or MPTP intoxication in vivo. Our findings could have broad relevance in human pathologies where parthanatos plays a role in the development of cell death inhibitors targeting the druggable PAAN/MIF nuclease.


Asunto(s)
Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Enfermedad de Parkinson , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Endonucleasas/metabolismo , Ratones , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo
7.
Cell ; 185(12): 2035-2056.e33, 2022 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-35688132

RESUMEN

Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.


Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Humanos , Enfermedad de Parkinson/metabolismo , Cuerpos de Procesamiento , Estabilidad del ARN , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
8.
Cell ; 185(17): 3214-3231.e23, 2022 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-35907404

RESUMEN

Although mutations in mitochondrial-associated genes are linked to inflammation and susceptibility to infection, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) perturbs mitochondrial homeostasis and reprograms cell death pathways in macrophages. When the inflammasome is activated in Lrrk2G2019S macrophages, elevated mitochondrial ROS (mtROS) directs association of the pore-forming protein gasdermin D (GSDMD) to mitochondrial membranes. Mitochondrial GSDMD pore formation then releases mtROS, promoting a switch to RIPK1/RIPK3/MLKL-dependent necroptosis. Consistent with enhanced necroptosis, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and severe immunopathology. Our findings suggest a pivotal role for GSDMD as an executer of multiple cell death pathways and demonstrate that mitochondrial dysfunction can direct immune outcomes via cell death modality switching. This work provides insights into how LRRK2 mutations manifest or exacerbate human diseases and identifies GSDMD-dependent necroptosis as a potential target to limit Lrrk2G2019S-mediated immunopathology.


Asunto(s)
Mitocondrias , Necroptosis , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animales , Humanos , Inflamasomas , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Macrófagos , Ratones , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo
9.
Cell ; 184(13): 3519-3527.e10, 2021 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-34107286

RESUMEN

Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.


Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Secuencia de Aminoácidos , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/ultraestructura , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína
10.
Cell ; 184(4): 931-942.e18, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33571431

RESUMEN

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.


Asunto(s)
Receptores de Dopamina D1/química , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Transducción de Señal , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/análogos & derivados , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Secuencia de Aminoácidos , Secuencia Conservada , Microscopía por Crioelectrón , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Dopamina D1/ultraestructura , Receptores de Dopamina D2/ultraestructura , Homología Estructural de Proteína
11.
Cell ; 181(3): 590-603.e16, 2020 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-32272060

RESUMEN

Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.


Asunto(s)
Neurogénesis/fisiología , Neuroglía/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Sistemas CRISPR-Cas/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Modelos Animales de Enfermedad , Dopamina/metabolismo , Regulación de la Expresión Génica/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades del Sistema Nervioso/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Células Ganglionares de la Retina/fisiología
12.
Cell ; 182(6): 1508-1518.e16, 2020 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-32783917

RESUMEN

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.


Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Microtúbulos/metabolismo , Enfermedad de Parkinson/metabolismo , Citoplasma/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Humanos , Microscopía Electrónica de Transmisión , Microtúbulos/química , Modelos Químicos , Mutación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fosfotransferasas/química , Fosfotransferasas/metabolismo , Dominios Proteicos , Repeticiones WD40
13.
Cell ; 182(2): 270-296, 2020 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-32707093

RESUMEN

Mammals have two specialized vascular circulatory systems: the blood vasculature and the lymphatic vasculature. The lymphatic vasculature is a unidirectional conduit that returns filtered interstitial arterial fluid and tissue metabolites to the blood circulation. It also plays major roles in immune cell trafficking and lipid absorption. As we discuss in this review, the molecular characterization of lymphatic vascular development and our understanding of this vasculature's role in pathophysiological conditions has greatly improved in recent years, changing conventional views about the roles of the lymphatic vasculature in health and disease. Morphological or functional defects in the lymphatic vasculature have now been uncovered in several pathological conditions. We propose that subtle asymptomatic alterations in lymphatic vascular function could underlie the variability seen in the body's response to a wide range of human diseases.


Asunto(s)
Vasos Linfáticos/metabolismo , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Historia del Siglo XXI , Humanos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Linfangiogénesis , Enfermedades Linfáticas/genética , Enfermedades Linfáticas/historia , Enfermedades Linfáticas/patología , Metástasis Linfática , Vasos Linfáticos/anatomía & histología , Vasos Linfáticos/citología , Neoplasias/metabolismo , Neoplasias/patología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética
14.
Annu Rev Cell Dev Biol ; 36: 237-264, 2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-32749865

RESUMEN

Parkinson's disease (PD) is a leading cause of neurodegeneration that is defined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transforming insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has revealed central roles for protein aggregation, mitochondrial damage, and defects in endolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein-sorting-associated protein 35 (VPS35) and LRRK2 being implicated in a common signaling pathway. A greater understanding of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets.


Asunto(s)
Enfermedad de Parkinson/metabolismo , Animales , Autofagia , Vesículas Citoplasmáticas/metabolismo , Humanos , Mitocondrias/metabolismo , Enfermedad de Parkinson/genética , Agregado de Proteínas , Transporte de Proteínas
15.
Cell ; 172(5): 897-909.e21, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29474918

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.


Asunto(s)
Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Genoma Humano , Transcriptoma/genética , Empalme Alternativo/genética , Elementos Alu/genética , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Estudios de Cohortes , Familia , Femenino , Sitios Genéticos , Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Intrones/genética , Masculino , Repeticiones de Minisatélite/genética , Modelos Genéticos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Nucleótido Esparcido Corto , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo
16.
Cell ; 167(6): 1469-1480.e12, 2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27912057

RESUMEN

The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein α-synuclein (αSyn), often resulting in motor dysfunction as exemplified by Parkinson's disease (PD). Using mice that overexpress αSyn, we report herein that gut microbiota are required for motor deficits, microglia activation, and αSyn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of αSyn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.


Asunto(s)
Enfermedad de Parkinson/microbiología , Enfermedad de Parkinson/patología , Animales , Encéfalo/patología , Disbiosis/patología , Ácidos Grasos/metabolismo , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/fisiopatología , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Ratones , Microglía/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatología , alfa-Sinucleína/metabolismo
17.
Mol Cell ; 83(18): 3314-3332.e9, 2023 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-37625404

RESUMEN

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.


Asunto(s)
Enfermedades Neurodegenerativas , Proteínas de Saccharomyces cerevisiae , Animales , Humanos , alfa-Sinucleína/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo
18.
Mol Cell ; 83(14): 2524-2539.e7, 2023 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-37390818

RESUMEN

Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.


Asunto(s)
Enfermedad de Parkinson , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Enfermedad de Parkinson/metabolismo , Canales de Potasio/metabolismo
19.
Mol Cell ; 83(17): 3188-3204.e7, 2023 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-37683611

RESUMEN

Failure to clear damaged mitochondria via mitophagy disrupts physiological function and may initiate damage signaling via inflammatory cascades, although how these pathways intersect remains unclear. We discovered that nuclear factor kappa B (NF-κB) essential regulator NF-κB effector molecule (NEMO) is recruited to damaged mitochondria in a Parkin-dependent manner in a time course similar to recruitment of the structurally related mitophagy adaptor, optineurin (OPTN). Upon recruitment, NEMO partitions into phase-separated condensates distinct from OPTN but colocalizing with p62/SQSTM1. NEMO recruitment, in turn, recruits the active catalytic inhibitor of kappa B kinase (IKK) component phospho-IKKß, initiating NF-κB signaling and the upregulation of inflammatory cytokines. Consistent with a potential neuroinflammatory role, NEMO is recruited to mitochondria in primary astrocytes upon oxidative stress. These findings suggest that damaged, ubiquitinated mitochondria serve as an intracellular platform to initiate innate immune signaling, promoting the formation of activated IKK complexes sufficient to activate NF-κB signaling. We propose that mitophagy and NF-κB signaling are initiated as parallel pathways in response to mitochondrial stress.


Asunto(s)
FN-kappa B , Transducción de Señal , FN-kappa B/genética , Quinasa I-kappa B/genética , Proteínas Serina-Treonina Quinasas/genética , Mitocondrias/genética
20.
Mol Cell ; 82(1): 44-59.e6, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34875213

RESUMEN

Mutations in PINK1 cause autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the translocase of the outer mitochondrial membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein, we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.


Asunto(s)
Proteínas de Insectos/metabolismo , Mitocondrias/enzimología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Proteínas Quinasas/metabolismo , Tribolium/enzimología , Animales , Línea Celular Tumoral , Activación Enzimática , Estabilidad de Enzimas , Humanos , Proteínas de Insectos/genética , Cinética , Mitocondrias/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/genética , Simulación del Acoplamiento Molecular , Mutación , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/genética , Relación Estructura-Actividad , Tribolium/genética
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