RESUMO
Vacuolar protein sorting 35 (VPS35) is a core component of the retromer complex, crucial to endosomal protein sorting and intracellular trafficking. We recently linked a mutation in VPS35 (p.D620N) to familial parkinsonism. Here, we characterize human VPS35 and retromer function in mature murine neuronal cultures and investigate neuron-specific consequences of the p.D620N mutation. We find VPS35 localizes to dendritic spines and is involved in the trafficking of excitatory AMPA-type glutamate receptors (AMPARs). Fundamental neuronal processes, including excitatory synaptic transmission, AMPAR surface expression and synaptic recycling are altered by VPS35 overexpression. VPS35 p.D620N acts as a loss-of-function mutation with respect to VPS35 activity regulating synaptic transmission and AMPAR recycling in mouse cortical neurons and dopamine neuron-like cells produced from induced pluripotent stem cells of human p.D620N carriers. Such perturbations to synaptic function likely produce chronic pathophysiological stress upon neuronal circuits that may contribute to neurodegeneration in this, and other, forms of parkinsonism.
Assuntos
Mutação de Sentido Incorreto , Neurônios/metabolismo , Doença de Parkinson/genética , Receptores de Glutamato/metabolismo , Proteínas de Transporte Vesicular/genética , Animais , Espinhas Dendríticas/metabolismo , Humanos , Camundongos , Transporte Proteico , Sinapses/metabolismoRESUMO
X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder endemic to Panay Island (Philippines). Patients present with generalizing dystonia and parkinsonism. Genetic changes surrounding the TAF1 (TATA-box binding protein associated factor 1) gene have been associated with XDP inducing a degeneration of striatal spiny projection neurons. There is little knowledge about the pathophysiology of this disorder. Our objective was to generate and analyze an in-vitro model of XDP based on striatal neurons differentiated from induced pluripotent stem cells (iPSC). We generated iPSC from patient and healthy control fibroblasts (3 affected, 3 controls), followed by directed differentiation of the cultures towards striatal neurons. Cells underwent characterization of immunophenotype as well as neuronal function, glutamate receptor properties and calcium dynamics by whole-cell patch-clamp recordings and calcium imaging. Furthermore, we evaluated expression levels of AMPA receptor subunits and voltage-gated calcium channels by quantitative real-time PCR. We observed no differences in basic electrophysiological properties. Application of the AMPA antagonist NBQX led to a more pronounced reduction of postsynaptic currents in XDP neurons. There was a higher expression of AMPA receptor subunits in patient-derived neurons. Basal calcium levels were lower in neurons derived from XDP patients and cells with spontaneous calcium transients were more frequent. Our data suggest altered glutamate response and calcium dynamics in striatal XDP neurons.
Assuntos
Cálcio/metabolismo , Distúrbios Distônicos/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Adulto , Canais de Cálcio/metabolismo , Corpo Estriado/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de AMPA/metabolismoRESUMO
OBJECTIVES: To study the frequency of different gene mutations in patients with early-onset parkinsonism and bilateral subthalamic nucleus deep brain stimulation (STN-DBS) and the short- and long-term surgical outcome in mutation-positive (MUT+) and -negative (MUT-) patients. METHODS: Eighty patients with disease onset at age Assuntos
Terapia por Estimulação Elétrica/estatística & dados numéricos
, Doença de Parkinson/genética
, Doença de Parkinson/terapia
, Proteínas Quinases/genética
, Núcleo Subtalâmico/cirurgia
, Ubiquitina-Proteína Ligases/genética
, Adulto
, Idoso
, Análise Mutacional de DNA
, Terapia por Estimulação Elétrica/métodos
, Feminino
, Seguimentos
, Lateralidade Funcional/genética
, Frequência do Gene
, Predisposição Genética para Doença/genética
, Testes Genéticos
, Genótipo
, Heterozigoto
, Humanos
, Masculino
, Pessoa de Meia-Idade
, Mutação/genética
, Doença de Parkinson/fisiopatologia
, Estudos Prospectivos
, Recuperação de Função Fisiológica/genética
, Núcleo Subtalâmico/fisiopatologia
, Resultado do Tratamento