RESUMO
α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson's disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology.
Assuntos
Proteína 2 Associada à Membrana da Vesícula , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Humanos , Ligação Proteica , Animais , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Células HEK293 , Eletricidade Estática , Separação de FasesRESUMO
Fundamental discoveries have shaped our molecular understanding of presynaptic processes, such as neurotransmitter release, active zone organization and mechanisms of synaptic vesicle (SV) recycling. However, certain regulatory steps still remain incompletely understood. Protein liquid-liquid phase separation (LLPS) and its role in SV clustering and active zone regulation now introduce a new perception of how the presynapse and its different compartments are organized. This article highlights the newly emerging concept of LLPS at the synapse, providing a systematic overview on LLPS tendencies of over 500 presynaptic proteins, spotlighting individual proteins and discussing recent progress in the field. Newly discovered LLPS systems like ELKS/liprin-alpha and Eps15/FCho are put into context, and further LLPS candidate proteins, including epsin1, dynamin, synaptojanin, complexin and rabphilin-3A, are highlighted.
Assuntos
Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Sinapses/fisiologia , Transmissão Sináptica , Endocitose , Exocitose , Monoéster Fosfórico Hidrolases/isolamento & purificação , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-ß 1-42 (Aß42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteostase , alfa-Sinucleína/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Linhagem Celular Tumoral , Feminino , Serina Peptidase 2 de Requerimento de Alta Temperatura A/genética , Serina Peptidase 2 de Requerimento de Alta Temperatura A/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fragmentos de Peptídeos/genética , Ratos , Ratos Sprague-Dawley , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , alfa-Sinucleína/genéticaRESUMO
Postnatally derived cultures of ventral mesencephalic neurons offer several crucial advantages over embryonic ventral mesencephalic cultures, including a higher content of TH-positive cells and the ability to derive cells from the substantia nigra, which contains the neurons most vulnerable to Parkinson's disease. On the other hand, these cultures are more challenging to produce consistently. Here, we provide an easy-to-implement protocol for culturing postnatal ventral mesencephalic cells from the substantia nigra (SN) and the ventral tegmental area using commercially available media, dishes, and general lab equipment, avoiding extensive material and equipment purchases. The protocol can be completed in about 5 h and provides ventral midbrain neuron cultures on cortex glia feeder layers in three weeks' time. The protocol uses an optimized protease digestion, tissue storage in Hibernate A during dissection and purification of neurons on an OptiPrep density gradient.
RESUMO
Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.
Assuntos
Cálcio/metabolismo , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Técnicas In Vitro , Metabolismo dos Lipídeos , Microscopia Eletrônica de Transmissão , Ressonância Magnética Nuclear Biomolecular , Terminações Pré-Sinápticas/metabolismo , Agregados Proteicos , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Sinaptossomos/metabolismo , alfa-Sinucleína/ultraestruturaRESUMO
α-Synuclein is known as a presynaptic protein that binds to small synaptic vesicles. Recent studies suggest that α-synuclein is not only attracted to these tiny and therewith highly curved membranes, but that in fact the sensing and regulation of membrane curvature is part of its physiological function. Moreover, recent studies have suggested that α-synuclein plays a role in the endocytosis of synaptic vesicles, and have provided support for a function of α-synuclein during exo- and endocytosis in which curvature sensing and membrane stabilization are crucial steps. This review aims to highlight recent research in the field and adds a new picture on the function of α-synuclein in maintaining synaptic homeostasis upon intense and repetitive neuronal activity.
Assuntos
Endocitose , Exocitose , alfa-Sinucleína/metabolismo , Humanos , Neurônios/metabolismoRESUMO
The function of intact organelles, whether mitochondria, Golgi apparatus or endoplasmic reticulum (ER), relies on their proper morphological organization. It is recognized that disturbances of organelle morphology are early events in disease manifestation, but reliable and quantitative detection of organelle morphology is difficult and time-consuming. Here we present a novel computer vision algorithm for the assessment of organelle morphology in whole cell 3D images. The algorithm allows the numerical and quantitative description of organelle structures, including total number and length of segments, cell and nucleus area/volume as well as novel texture parameters like lacunarity and fractal dimension. Applying the algorithm we performed a pilot study in cultured motor neurons from transgenic G93A hSOD1 mice, a model of human familial amyotrophic lateral sclerosis. In the presence of the mutated SOD1 and upon excitotoxic treatment with kainate we demonstrate a clear fragmentation of the mitochondrial network, with an increase in the number of mitochondrial segments and a reduction in the length of mitochondria. Histogram analyses show a reduced number of tubular mitochondria and an increased number of small mitochondrial segments. The computer vision algorithm for the evaluation of organelle morphology allows an objective assessment of disease-related organelle phenotypes with greatly reduced examiner bias and will aid the evaluation of novel therapeutic strategies on a cellular level.
Assuntos
Algoritmos , Esclerose Lateral Amiotrófica/patologia , Biometria/métodos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Mitocôndrias/patologia , Animais , Modelos Animais de Doenças , Estudos de Avaliação como Assunto , Camundongos Transgênicos , Neurônios/patologia , Projetos PilotoRESUMO
Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by a progressive loss of motor neurons. Although the etiology remains unclear, disturbances in Ca2+ homoeostasis and protein folding are essential features of neurodegeneration. The correct folding of proteins is managed by folding proteins, which are regulated by Ca2+ levels. Therefore, Ca(2+)-sensitive folding proteins represent an important link between disturbed Ca2+ handling and protein misfolding in amyotrophic lateral sclerosis. In the first part of this review, we focus on Ca2+ handling in the endoplasmic reticulum and mitochondria in terms of their roles in protein misfolding. In the second part, we draw attention to the main Ca(2+)-sensitive folding proteins that play a role in motor neuron degeneration such as calreticulin and calnexin, which are involved in the folding of glycosylated proteins. In addition, calmodulin and the Ca2+/calmodulin-dependent protein kinase are discussed as one correlation to oxidative stress. The heat shock protein endoplasmin is associated with the anti-apoptotic insulin-like growth factor pathway that is altered in amyotrophic lateral sclerosis. Grp78, which influences Ca2+ homeostasis in the intraluminal endoplasmic reticulum is upregulated in mice models and amyotrophic lateral sclerosis patients and constitutes a core component of the unfolded protein response. Lastly, the protein disulfide isomerase family is responsible for mediating oxidative protein folding in the endoplasmic reticulum.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Cálcio/fisiologia , Dobramento de Proteína , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/fisiologia , Chaperona BiP do Retículo Endoplasmático , Homeostase/fisiologia , Humanos , CamundongosRESUMO
Motor neurons vulnerable to the rapidly progressive deadly neurodegenerative disease amyotrophic lateral sclerosis (ALS) inherently express low amounts of calcium binding proteins (CaBP), likely to allow physiological motor neuron firing frequency modulation. At the same time motor neurons are susceptible to AMPA receptor mediated excitotoxicity and internal calcium deregulation which is not fully understood. We analysed ER mitochondria calcium cycle (ERMCC) dynamics with subsecond resolution in G93A hSOD1 overexpressing motor neurons as a model of ALS using fluorescent calcium imaging. When comparing vulnerable motor neurons and non-motor neurons from G93A hSOD1 mice and their non-transgenic littermates, we found a decelerated cytosolic calcium clearance in the presence of G93A hSOD1. While both non-transgenic as well as G93A hSOD1 motor neurons displayed large mitochondrial calcium uptake by the mitochondrial uniporter (mUP), the mitochondrial calcium extrusion system was altered in the presence of G93A hSOD1. In addition, ER calcium uptake by the sarco-/endoplasmic reticulum ATPase (SERCA) was increased in G93A hSOD1 motor neurons. In survival assays, blocking the mitochondrial sodium calcium exchanger (mNCE) by CGP37157 as well as inhibiting SERCA by cyclopiazonic acid showed protective effects against kainate induced excitotoxicity. Thus, our study shows for the first time that the functional consequence of G93A hSOD1 overexpression in intact motor neurons is indeed a disturbance of the ER mitochondria calcium cycle, and identified two promising targets for therapeutic intervention in the pathology of ALS.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Neurônios Motores/ultraestrutura , Superóxido Dismutase/genética , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Clonazepam/análogos & derivados , Clonazepam/farmacologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/genética , Compostos de Rutênio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Medula Espinal/citologia , Tetrodotoxina/farmacologia , Tiazepinas/farmacologia , Resposta a Proteínas não Dobradas/genética , Verapamil/farmacologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder of motor neurons leading to death of the patients, mostly within 2-5 years after disease onset. The pathomechanism of motor neuron degeneration is only partially understood and therapeutic strategies based on mechanistic insights are largely ineffective. The discovery of reliable biomarkers of disease diagnosis and progression is the sine qua non of both the revelation of insights into the ALS pathomechanism and the assessment of treatment efficacies. Proteomic approaches are an important pillar in ALS biomarker discovery. Cerebrospinal fluid is the most promising body fluid for differential proteome analyses, followed by blood (serum, plasma), and even urine and saliva. The present study provides an overview about reported peptide/protein biomarker candidates that showed significantly altered levels in certain body fluids of ALS patients. These findings have to be discussed according to proposed pathomechanisms to identify modifiers of disease progression and to pave the way for the development of potential therapeutic strategies. Furthermore, limitations and advantages of proteomic approaches for ALS biomarker discovery in different body fluids and reliable validation of biomarker candidates have been addressed.