RESUMO
Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson's disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found that LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell-type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilize and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Humanos , Ratos , Animais , Idoso , Doença de Parkinson/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mitofagia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Mitocôndrias/metabolismoRESUMO
Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson's disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chambered microfluidic platform and established a cortico-striato-nigral microcircuit partially recapitulating the striatal presynaptic landscape in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, cortical glutamatergic projections facilitated MSN synaptic activity, and dopaminergic transmission enhanced maturation of MSNs in vitro. Replacement of wild-type iPSC-derived dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation led to a depolarisation of resting membrane potential and an increase in rheobase in iPSC-MSNs, as well as a reduction in both voltage-gated sodium and potassium currents. Such deficits were resolved in late microcircuit cultures, and could be reversed in younger cultures with antagonism of protein kinase A activity in iPSC-MSNs. Taken together, our results highlight the unique utility of modelling striatal neurons in a modular physiological circuit to reveal mechanistic insights into GBA1 mutations in PD.
RESUMO
The brain is spatially organized and contains unique cell types, each performing diverse functions and exhibiting differential susceptibility to neurodegeneration. This is exemplified in Parkinson's disease with the preferential loss of dopaminergic neurons of the substantia nigra pars compacta. Using a Parkinson's transgenic model, we conducted a single-cell spatial transcriptomic and dopaminergic neuron translatomic analysis of young and old mouse brains. Through the high resolving capacity of single-cell spatial transcriptomics, we provide a deep characterization of the expression features of dopaminergic neurons and 27 other cell types within their spatial context, identifying markers of healthy and aging cells, spanning Parkinson's relevant pathways. We integrate gene enrichment and genome-wide association study data to prioritize putative causative genes for disease investigation, identifying CASR as a regulator of dopaminergic calcium handling. These datasets represent the largest public resource for the investigation of spatial gene expression in brain cells in health, aging, and disease.
Assuntos
Neurônios Dopaminérgicos , Doença de Parkinson , Camundongos , Animais , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Transcriptoma/genética , Substância Negra/metabolismo , Estudo de Associação Genômica Ampla , Envelhecimento/genética , Perfilação da Expressão GênicaRESUMO
During AD pathology, Tau protein levels progressively increase from early pathological stages. Tau altered expression causes an unbalance of Tau subcellular localization in the cytosol and in the nuclear compartment leading to synaptic dysfunction, neuronal cell death and neurodegeneration as a consequence. Due to the relevant role of epigenetic remodellers in synaptic activity in physiology and in neurodegeneration, in particular of TRIM28 and HDAC1, we investigated the relationship between Tau and these epigenetic factors. By molecular, imaging and biochemical approaches, here we demonstrate that Tau altered expression in the neuronal cell line SH-SY5y does not alter TRIM28 and HDAC1 expression but it induces a subcellular reduction of HDAC1 in the nuclear compartment. Remarkably, HDAC1 reduced activity modulates the expression of synaptic genes in a way comparable to that observed by Tau increased levels. These results support a competitive relationship between Tau levels and HDAC1 subcellular localization and nuclear activity, indicating a possible mechanism mediating the alternative role of Tau in the pathological alteration of synaptic genes expression.
RESUMO
Parkinson's disease (PD) is characterized by a progressive deterioration of motor and cognitive functions. Although death of dopamine neurons is the hallmark pathology of PD, this is a late-stage disease process preceded by neuronal dysfunction. Here we describe early physiological perturbations in patient-derived induced pluripotent stem cell (iPSC)-dopamine neurons carrying the GBA-N370S mutation, a strong genetic risk factor for PD. GBA-N370S iPSC-dopamine neurons show an early and persistent calcium dysregulation notably at the mitochondria, followed by reduced mitochondrial membrane potential and oxygen consumption rate, indicating mitochondrial failure. With increased neuronal maturity, we observed decreased synaptic function in PD iPSC-dopamine neurons, consistent with the requirement for ATP and calcium to support the increase in electrophysiological activity over time. Our work demonstrates that calcium dyshomeostasis and mitochondrial failure impair the higher electrophysiological activity of mature neurons and may underlie the vulnerability of dopamine neurons in PD.
RESUMO
Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models.
Assuntos
Doença de Parkinson , Humanos , Neurônios Dopaminérgicos/metabolismo , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Mutação , Crescimento Neuronal , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Processamento de Proteína Pós-Traducional , ProteômicaRESUMO
Parkinson's disease (PD) is an age-related neurodegenerative disorder with no known cure. In order to better understand the pathological mechanisms which lead to neuronal cell death and to accelerate the process of drug discovery, a reliable in vitro model is required. Unfortunately, research into PD and neurodegeneration in general has long suffered from a lack of adequate in vitro models, mainly due to the inaccessibility of live neurons from vulnerable areas of the human brain. Recent reprogramming technologies have recently made it possible to reliably derive human induced pluripotent stem cells (iPSCs) from patients and healthy subjects to generate specific, difficult to obtain, cellular sub-types. These iPSC-derived cells can be employed to model disease to better understand pathological mechanisms and underlying cellular vulnerability. Therefore, in this chapter, we will discuss the techniques involved in the reprogramming of somatic cells into iPSCs, the evolution of iPSC differentiation methods and their application in neurodegenerative disease modeling.
Assuntos
Neurônios Dopaminérgicos , Células-Tronco Pluripotentes Induzidas , Modelos Biológicos , Doença de Parkinson , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/metabolismoRESUMO
Formation of Tau aggregates is a common pathological feature of tauopathies and their accumulation directly correlates with cytotoxicity and neuronal degeneration. Great efforts have been made to understand Tau aggregation and to find therapeutics halting or reversing the process, however, progress has been slowed due to the lack of a suitable method for monitoring Tau aggregation. We developed a cell-based assay allowing to detect and quantify Tau aggregation in living cells. The system is based on the FRET biosensor CST able to monitor the molecular dynamic of Tau aggregation in different cellular conditions. We probed candidate compounds that could block Tau hyperphosphorylation. In particular, to foster the drug discovery process, we tested kinase inhibitors approved for the treatment of other diseases. We identified the ERK inhibitor PD-901 as a promising therapeutic molecule since it reduces and prevents Tau aggregation. This evidence establishes the CST cell-based aggregation assay as a reliable tool for drug discovery and suggests that PD-901 might be a promising compound to be tested for further preclinical studies on AD.
RESUMO
Tau displacement from microtubules is the first step in the onset of tauopathies and is followed by toxic protein aggregation. However, other non-canonical functions of Tau might have a role in these pathologies. Here, we demonstrate that a small amount of Tau localizes in the nuclear compartment and accumulates in both the soluble and chromatin-bound fractions. We show that favoring Tau nuclear translocation and accumulation, by Tau overexpression or detachment from MTs, increases the expression of VGluT1, a disease-relevant gene directly involved in glutamatergic synaptic transmission. Remarkably, the P301L mutation, related to frontotemporal dementia FTDP-17, impairs this mechanism leading to a loss of function. Altogether, our results provide the demonstration of a direct physiological role of Tau on gene expression. Alterations of this mechanism may be at the basis of the onset of neurodegeneration.
Assuntos
Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteínas tau/genética , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/genética , Expressão Gênica/genética , Células HeLa , Humanos , Microtúbulos/genética , Mutação/genética , Tauopatias/genéticaRESUMO
Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer's disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.