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Parkinson's disease is a common, incurable neurodegenerative disorder that is clinically heterogeneous: it is likely that different cellular mechanisms drive the pathology in different individuals. So far it has not been possible to define the cellular mechanism underlying the neurodegenerative disease in life. We generated a machine learning-based model that can simultaneously predict the presence of disease and its primary mechanistic subtype in human neurons. We used stem cell technology to derive control or patient-derived neurons, and generated different disease subtypes through chemical induction or the presence of mutation. Multidimensional fluorescent labelling of organelles was performed in healthy control neurons and in four different disease subtypes, and both the quantitative single-cell fluorescence features and the images were used to independently train a series of classifiers to build deep neural networks. Quantitative cellular profile-based classifiers achieve an accuracy of 82%, whereas image-based deep neural networks predict control and four distinct disease subtypes with an accuracy of 95%. The machine learning-trained classifiers achieve their accuracy across all subtypes, using the organellar features of the mitochondria with the additional contribution of the lysosomes, confirming the biological importance of these pathways in Parkinson's. Altogether, we show that machine learning approaches applied to patient-derived cells are highly accurate at predicting disease subtypes, providing proof of concept that this approach may enable mechanistic stratification and precision medicine approaches in the future.
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Mutations in the SNCA gene cause autosomal dominant Parkinson's disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small ß-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34-41 post differentiation. Once midbrain identity fully developed, at day 48-62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.
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Aggregation of alpha-synuclein (α-Syn) drives Parkinson's disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)-derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Cardiolipinas/metabolismo , Humanos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Type 2 diabetes (T2DM) is an established risk factor for developing Parkinson's disease (PD), but its effect on disease progression is not well understood. OBJECTIVE: The aim of this study was to investigate the influence of T2DM on aspects of disease progression in PD. METHODS: We analyzed data from the Tracking Parkinson's study to examine the effects of comorbid T2DM on PD progression and quality of life by comparing symptom severity scores assessing a range of motor and nonmotor symptoms. RESULTS: We identified 167 (8.7%) patients with PD and T2DM (PD + T2DM) and 1763 (91.3%) patients with PD without T2DM (PD). After controlling for confounders, patients with T2DM had more severe motor symptoms, as assessed by Movement Disorder Society Unified Parkinson's Disease Rating Scale, Part III (25.8 [0.9] vs. 22.5 [0.3] P = 0.002), and nonmotor symptoms, as assessed by Non-Motor Symptoms Scale total (38.4 [2.5] vs. 31.8 [0.7] P < 0.001), and were significantly more likely to report loss of independence (odds ratio, 2.08; 95% confidence interval [CI]: 1.34-3.25; P = 0.001) and depression (odds ratio, 1.62; CI: 1.10-2.39; P = 0.015). Furthermore, over time, patients with T2DM had significantly faster motor symptom progression (P = 0.012), developed worse mood symptoms (P = 0.041), and were more likely to develop substantial gait impairment (hazard ratio, 1.55; CI: 1.07-2.23; P = 0.020) and mild cognitive impairment (hazard ratio, 1.7; CI: 1.24-2.51; P = 0.002) compared with the PD group. CONCLUSIONS: In the largest study to date, T2DM is associated with faster disease progression in Parkinson's, highlighting an interaction between these two diseases. Because it is a potentially modifiable metabolic state, with multiple peripheral and central targets for intervention, it may represent a target for alleviating parkinsonian symptoms and slowing progression to disability and dementia. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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Disfunción Cognitiva , Diabetes Mellitus Tipo 2 , Enfermedad de Parkinson , Disfunción Cognitiva/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/epidemiología , Progresión de la Enfermedad , Humanos , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/epidemiología , Calidad de Vida/psicologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of ß-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson's disease, and highlights a new mechanism by which lipid peroxidation causes cell death.
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Calcio/metabolismo , Ferroptosis , Hierro/metabolismo , Peroxidación de Lípido , Enfermedad de Parkinson , alfa-Sinucleína/metabolismo , Células Cultivadas , Células Madre Embrionarias Humanas , Humanos , Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patologíaRESUMEN
The molecular events causing memory loss and neuronal cell death in Alzheimer's disease (AD) over time are still unknown. Here we found that picomolar concentrations of soluble oligomers of synthetic beta amyloid (Aß42) aggregates incubated with BV2 cells or rat astrocytes caused a sensitised response of Toll-like receptor 4 (TLR4) with time, leading to increased production of TNF-α. Aß aggregates caused long term potentiation (LTP) deficit in hippocampal slices and predominantly neuronal cell death in co-cultures of astrocytes and neurons, which was blocked by TLR4 antagonists. Soluble Aß aggregates cause LTP deficit and neuronal death via an autocrine/paracrine mechanism due to TLR4 signalling. These findings suggest that the TLR4-mediated inflammatory response may be a key pathophysiological process in AD.
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Péptidos beta-Amiloides/fisiología , Neuronas/fisiología , Agregado de Proteínas/fisiología , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Embrión de Mamíferos , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Neuronas/efectos de los fármacos , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/fisiopatología , Agregación Patológica de Proteínas/psicología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
In the original publication of this article, the author Magarida Rodrigues was written incorrectly.
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Despite the wealth of genomic and transcriptomic data in Parkinson's disease (PD), the initial molecular events are unknown. Using LD score regression analysis, we show significant enrichment in PD heritability within regulatory sites for LPS-activated monocytes and that TLR4 expression is highest within human substantia nigra, the most affected brain region, suggesting a role for TLR4 inflammatory responses. We then performed extended incubation of cells with physiological concentrations of small alpha-synuclein oligomers observing the development of a TLR4-dependent sensitized inflammatory response with time, including TNF-α production. ROS and cell death in primary neuronal cultures were significantly reduced by TLR4 antagonists revealing that an indirect inflammatory mechanism involving cytokines produced by glial cells makes a major contribution to neuronal death. Prolonged exposure to low levels of alpha-synuclein oligomers sensitizes TLR4 responsiveness in astrocytes and microglial, explaining how they become pro-inflammatory, and may be an early causative event in PD.
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Astrocitos/metabolismo , Microglía/metabolismo , Enfermedad de Parkinson/metabolismo , Receptor Toll-Like 4/metabolismo , alfa-Sinucleína/metabolismo , Animales , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Muerte Celular , Citocinas/metabolismo , Humanos , Inflamación/patología , Microglía/patología , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/patología , Sustancia Negra/patologíaRESUMEN
The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250â nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-ß. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.
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Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Neuronas/patología , Enfermedad de Parkinson/patología , Agregado de Proteínas , alfa-Sinucleína/química , Péptidos beta-Amiloides/metabolismo , Aptámeros de Péptidos/química , Humanos , Agregación Patológica de Proteínas , alfa-Sinucleína/metabolismoRESUMEN
Misfolding and aggregation of the proteins amyloid-ß, tau and alpha-synuclein is the predominant pathology underlying the neurodegenerative disorders, Alzheimer's and Parkinson's disease. While end stage insoluble products of aggregation have been well characterised in human and animal models of disease, accumulating evidence from biophysical, cellular and in vivo studies has shown that soluble intermediates of aggregation, or oligomers, may be the key species that mediate toxicity and underlie seeding and spreading in disease. Here, we review the process of protein misfolding, and the intrinsic and extrinsic processes that cause the native states of the key aggregating proteins to undergo conformational change to form oligomers and ultimately fibrils. We discuss the structural features of the key toxic intermediate, and describe the putative mechanisms by which oligomers may cause cell toxicity. Finally, we explore the potential therapeutic approaches raised by the oligomer hypothesis in neurodegenerative disease.
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Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Enfermedad de Parkinson/fisiopatología , Agregado de Proteínas , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Péptidos beta-Amiloides/química , Animales , Humanos , Conformación Proteica , alfa-Sinucleína/química , Proteínas tau/químicaRESUMEN
Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.
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Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Animales , Técnicas de Cocultivo , Células Madre Embrionarias/metabolismo , Humanos , Peroxidación de Lípido , Poro de Transición de la Permeabilidad Mitocondrial , Oxidación-Reducción , Técnicas de Placa-Clamp , Permeabilidad , Proteómica , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: The aggregation of the protein É-synuclein (ÉS) underlies a range of increasingly common neurodegenerative disorders including Parkinson's disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ÉS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ÉS aggregation in vitro in the presence of two ÉS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ÉS. RESULTS: We show that both nanobodies inhibit the formation of ÉS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ÉS, leading to a dramatic reduction in oligomer-induced cellular toxicity. CONCLUSIONS: The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.
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Anticuerpos de Dominio Único/metabolismo , alfa-Sinucleína/metabolismo , Humanos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Unión ProteicaRESUMEN
Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.
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Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Astrocitos/patología , Modelos Biológicos , Neuronas Motoras/patología , Proteína que Contiene Valosina/metabolismo , Supervivencia Celular , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Estrés del Retículo Endoplásmico , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mutación/genética , Degeneración Nerviosa/patología , Neurogénesis , Estrés Oxidativo , Fenotipo , Sinapsis/patologíaRESUMEN
The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson's disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule FÓ§rster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.
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The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5-140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 10(4), are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.
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Modelos Biológicos , Priones/metabolismo , alfa-Sinucleína/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Adult hippocampal neurogenesis is a highly dynamic process in which new cells are born, but only some of which survive. Of late it has become clear that these surviving newborn neurons have functional roles, most notably in certain forms of memory. Conventional methods to look at adult neurogenesis are based on the quantification of the number of newly born neurons using a simple cell counting methodology. However, this type of approach fails to capture the dynamic aspects of the neurogenic process, where neural proliferation, death and differentiation take place continuously and simultaneously. In this paper, we propose a simple mathematical approach to better understand the adult neurogenic process in the hippocampus which in turn will allow for a better analysis of this process in disease states and following drug therapies.
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Hipocampo/fisiología , Modelos Neurológicos , Neurogénesis/fisiología , Neuronas/fisiología , Animales , Área Bajo la Curva , Bromodesoxiuridina , Recuento de Células/métodos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Fármacos del Sistema Nervioso Central/farmacología , Proteínas de Dominio Doblecortina , Fluoxetina/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Antígeno Ki-67/metabolismo , Masculino , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuropéptidos/metabolismo , Factores de TiempoRESUMEN
The present study was undertaken to characterize neuronal activity-dependent expression and release of vascular endothelial growth factor (VEGF) from rat hippocampal neurons and its contribution to neuronal functions. Increased levels of VEGF164 mRNA were evident both in cultured neurons and slices, but not astrocytes, following membrane depolarization with KCl. Activity-dependent expression of VEGF, as well as its release, was dependent on the activation of the N-methyl-d-aspartate receptors or L-type voltage-activated calcium channels. A brief (10 min) application of recombinant VEGF165 to neurons elicited a slow rise in cytosolic Ca2+ in a VEGFR2 dependent manner. The VEGF-induced Ca2+ responses required Ca2+ influx, phospholipase Cgamma and Ca2+ stores. An inhibitor of transient receptor potential canonical channels reduced the VEGF-induced Ca2+ responses by 50%, suggesting the involvement of transient receptor potential canonical channels in the VEGF-mediated responses. The same brief stimulus with VEGF led to long-term synaptic enhancement dependent on protein synthesis. VEGF had prominent effects on the activation calcium/calmodulin protein kinase II and cAMP responsive element binding protein as well as extracellular signal-regulated protein kinase and mammalian target of rapamycin-all in a VEGFR2 dependent manner. Our findings suggest that VEGF released from neuronal cells plays a local role in Ca2+ influx and synaptic transmission that may influence the generation of long-term changes in synaptic efficacy.