RESUMO
α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson's disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Our results show that α-Syn colocalizes with PIP2 and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P2 levels on the plasma membrane. In accord with their effects on PI(4,5)P2 levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P2 levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn-mediated membrane trafficking.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitose , Fosfatidilinositol 4,5-Difosfato/metabolismo , alfa-Sinucleína/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , alfa-Sinucleína/genéticaRESUMO
It has been suggested that extracellular alpha synuclein (αSyn) can mediate neuroinflammation in Parkinson's disease, and that αSyn affects B-cell maturation. However, the function of αSyn in T cells is poorly understood. We hypothesized that αSyn can affect CD4+ T-cell proliferation and activity. We found that αSyn deficiency exacerbates disease progression in 8 weeks old C57BL6/J EAE-induced mice, and that αSyn-deficient CD4+ T cells have increased pro-inflammatory response to myelin antigen relative to wild-type cells, as measured by cytokine secretion of interleukin IL-17 and interferon gamma. Furthermore, expression of αSyn on a background of αSyn knockout mitigates the inflammatory responses in CD4+ T cells. We discovered that elevated levels of Nurr1, a transcription factor belonging to the orphan nuclear receptor family, are associated with the pro-inflammatory profile of αSyn-deficient CD4+ T cells. In addition, we demonstrated that silencing of Nurr1 expression using an siRNA reduces IL-17 levels and increases the levels of IL-10, an anti-inflammatory cytokine. Study of αSyn-mediated cellular pathways in CD4+ T cells may provide useful insights into the development of pro-inflammatory responses in immunity, providing future avenues for therapeutic intervention.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Ativação Linfocitária/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , alfa-Sinucleína/deficiência , Animais , Proliferação de Células , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Inflamação/imunologia , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Células Th1/imunologia , alfa-Sinucleína/genética , alfa-Sinucleína/fisiologiaRESUMO
α-Synuclein overexpression (ASOX) drives the formation of toxic aggregates in neurons vulnerable in Parkinson's disease (PD), including dopaminergic neurons of the substantia nigra (SN) and cholinergic neurons of the dorsal motor nucleus of the vagus (DMV). Just as these populations differ in when they exhibit α-synucleinopathies during PD pathogenesis, they could also differ in their physiological responses to ASOX. An ASOX-mediated hyperactivity of SN dopamine neurons, which was caused by oxidative dysfunction of Kv4.3 potassium channels, was recently identified in transgenic (A53T-SNCA) mice overexpressing mutated human α-synuclein. Noting that DMV neurons display extensive α-synucleinopathies earlier than SN dopamine neurons while exhibiting milder cell loss in PD, we aimed to define the electrophysiological properties of DMV neurons in A53T-SNCA mice. We found that DMV neurons maintain normal firing rates in response to ASOX. Moreover, Kv4.3 channels in DMV neurons exhibit no oxidative dysfunction in the A53T-SNCA mice, which could only be recapitulated in wild-type mice by glutathione dialysis. Two-photon imaging of redox-sensitive GFP corroborated the finding that mitochondrial oxidative stress was diminished in DMV neurons in the A53T-SNCA mice. This reduction in oxidative stress resulted from a transcriptional downregulation of voltage-activated (Cav) calcium channels in DMV neurons, which led to a reduction in activity-dependent calcium influx via Cav channels. Thus, ASOX induces a homeostatic remodeling with improved redox signaling in DMV neurons, which could explain the differential vulnerability of SN dopamine and DMV neurons in PD and could promote neuroprotective strategies that emulate endogenous homeostatic responses to ASOX (e.g., stressless pacemaking) in DMV neurons. SIGNIFICANCE STATEMENT: Overexpression of mutant α-synuclein causes Parkinson's disease, presumably by driving neurodegeneration in vulnerable neuronal target populations. However, the extent of α-synuclein pathology (e.g., Lewy bodies) is not directly related to the degree of neurodegeneration across various vulnerable neuronal populations. Here, we show that, in contrast to dopamine neurons in the substantia nigra, vagal motoneurons do not enhance their excitability and oxidative load in response to chronic mutant α-synuclein overexpression. Rather, by downregulating their voltage-activated calcium channels, vagal motoneurons acquire a stressless form of pacemaking that diminishes mitochondrial and cytosolic oxidative stress. Emulating this endogenous adaptive response to α-synuclein overexpression could lead to novel strategies to protect dopamine neurons and perhaps delay the onset of Parkinson's disease.
Assuntos
Relógios Biológicos , Neurônios Motores , Doença de Parkinson/fisiopatologia , Nervo Vago/fisiologia , alfa-Sinucleína/biossíntese , alfa-Sinucleína/genética , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/genética , Neurônios Dopaminérgicos/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Estresse Oxidativo , Canais de Potássio Shal/metabolismo , Transdução de Sinais/genética , Substância Negra/citologia , Substância Negra/fisiologia , Nervo Vago/citologiaRESUMO
α-Synuclein (aS) is a protein abundant in presynaptic nerve terminals in Parkinson disease (PD) and is a major component of intracellular Lewy bodies, the pathological hallmark of neurodegenerative disorders such as PD. Accordingly, the relationships between aS structure, its interaction with lipids, and its involvement in neurodegeneration have attracted great interest. Previously, we reported on the interaction of aS with brain polyunsaturated fatty acids, in particular docosahexaenoic acid (DHA). aS acquires an α-helical secondary structure in the presence of DHA and, in turn, affects DHA structural and aggregative properties. Moreover, aS forms a covalent adduct with DHA. Here, we provide evidence that His-50 is the main site of this covalent modification. To better understand the role of His-50, we analyzed the effect of DHA on aS-derived species: a naturally occurring variant, H50Q; an oxidized aS in which all methionines are sulfoxides (aS4ox); a fully lysine-alkylated aS (acetyl-aS); and aS fibrils, testing their ability to be chemically modified by DHA. We show, by mass spectrometry and spectroscopic techniques, that H50Q and aS4ox are modified by DHA, whereas acetyl-aS is not. We correlated this modification with aS structural features, and we suggest a possible functional role of aS in sequestering the early peroxidation products of fatty acids, thereby reducing the level of highly reactive lipid species. Finally, we show that fibrillar aS loses almost 80% of its scavenging activity, thus lacking a potentially protective function. Our findings linking aS scavenging activity with brain lipid composition suggest a possible etiological mechanism in some neurodegenerative disorders.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Neuroproteção , alfa-Sinucleína/metabolismo , Ácido Araquidônico/metabolismo , Sítios de Ligação , Encéfalo/metabolismo , Dicroísmo Circular , Ácidos Docosa-Hexaenoicos/metabolismo , Humanos , Metabolismo dos Lipídeos , Lisina/química , Espectrometria de Massas , Metionina/química , Oxigênio/química , Doença de Parkinson/metabolismo , Estrutura Secundária de Proteína , Tripsina/químicaRESUMO
The validity of α-synuclein (α-Syn) as a biomarker for Parkinson's disease (PD) is still under investigation. Conventional methods for capture and quantitation of α-Syn protein in human samples are primarily based on anti-α-Syn antibodies. Specific and competent antibodies were raised against α-Syn. However, capture by anti-α-Syn antibodies may be limited to specific epitope recognition, attributed to protein structure or post-translational modifications. Hence, antibody-based methods for α-Syn capture raise a concern regarding their efficacy to detect the intracellular, unfolded α-Syn pool. An alternative is α-Syn capture by membrane lipids, i.e., to utilize the biochemical property of α-Syn to specifically bind membrane lipids and acquire a characteristic structure following binding. We determined α-Syn levels in human samples using immobilized lipids for α-Syn capture. The lipids used for α-Syn capture consist of phosphatidyl inositol (PI), phosphatidyl serine (PS), and phosphatidyl ethanolamine (PE). Addition of mono-sialoganglioside, GM1 ganglioside, to the immobilized lipids significantly improved α-Syn detection. Following capture, the lipid-bound α-Syn was detected using an anti-α-Syn antibody. Total α-Syn levels in whole blood cells (WBC), cerebrospinal fluid (CSF), and saliva were determined by the lipid-ELISA method.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Doença de Parkinson/sangue , Doença de Parkinson/líquido cefalorraquidiano , Fosfolipídeos/metabolismo , alfa-Sinucleína , Adulto , Anticorpos/química , Células Sanguíneas/química , Feminino , Gangliosídeo G(M1)/metabolismo , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/diagnóstico , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Saliva/química , alfa-Sinucleína/sangue , alfa-Sinucleína/líquido cefalorraquidianoRESUMO
Calcium influx elevates mitochondrial oxidant stress (mOS) in dorsal motor nucleus of the vagus (DMV) neurons that are prone to Lewy body pathologies in presymptomatic Parkinson's disease (PD) patients. In experimental PD models, treatment with isradipine, the dihydropyridine with the highest affinity to Cav1.3 channels, prevents subthreshold calcium influx via Cav1.3 channels into midbrain dopamine neurons and protects them from mOS. In DMV neurons, isradipine is also effective in reducing mOS despite overwhelming evidence that subthreshold calcium influx is negligible compared with spike-triggered influx. To solve this conundrum we combined slice electrophysiology, two-photon laser scanning microscopy, mRNA profiling, and computational modeling. We find that the unusually depolarized subthreshold voltage trajectory of DMV neurons is positioned between the relatively hyperpolarized activation curve of Cav1.3 channels and that of other high-voltage activated (HVA) calcium channels, thus creating a functional segregation between Cav1.3 and HVA calcium channels. The HVA channels flux the bulk of calcium during spikes but can only influence pacemaking through their coupling to calcium-activated potassium currents. In contrast, Cav1.3 currents, which we show to be more than an order-of-magnitude smaller than the HVA calcium currents, are able to introduce sufficient inward current to speed up firing. However, Kv4 channels that are constitutively open in the subthreshold range guarantee slow pacemaking, despite the depolarizing action of Cav1.3 and other pacemaking currents. We propose that the efficacy of isradipine in preventing mOS in DMV neurons arises from its mixed effect on Cav1.3 channels and on HVA Cav1.2 channels.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Neurônios Motores/metabolismo , Nervo Vago/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio Tipo L/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/fisiologia , Canais de Potássio Cálcio-Ativados/metabolismo , Nervo Vago/citologia , Nervo Vago/fisiologiaRESUMO
While α-Synuclein (α-Syn) is mainly detected as a cytosolic protein, a portion of it is recovered bound to membranes. It is suggested that binding to membrane phospholipids controls α-Syn structure, physiology and pathogenesis. We aimed at investigating the role, of the positive charged lysine residues at the KTKEGV repeat motif, in mediating α-Syn associations with membrane phospholipids and in α-Syn oligomerization and aggregation. Specifically, two positive lysine (K) residues were replaced with two negative glutamic acid (E) residues at either the first or second KTKEGV repeat motifs. The effect of these mutations on membrane binding was determined by a quantitative phospholipid ELISA assay and compared to wild-type α-Syn and to the Parkinson's disease-causing mutations, A30P, E46K and A53T. We found that the K to E substitutions affected α-Syn binding to phospholipids. In addition, K to E substitutions resulted in a dramatically lower level of soluble α-Syn oligomers and larger intracellular inclusions. Together, our results suggest a critical role for lysine residues at the N-terminal repeat domain in the pathophysiology of α-Syn.
Assuntos
Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Doença de Parkinson/genéticaRESUMO
Ultrastructural, neurochemical, and molecular alterations within the striatum are associated with the onset and progression of Parkinson's disease (PD). In PD, the dopamine-containing neurons in the substantia nigra pars compacta (SNc) degenerate and reduce dopamine-containing innervations to the striatum. The loss of striatal dopamine is associated with enhanced corticostriatal glutamatergic plasticity at the early stages of PD. However, with disease progression, the glutamatergic corticostriatal white matter tracts (WMTs) also degenerate. We analyzed the levels of Mu opioid receptors (MORs) in the corticostriatal WMTs, as a function of α-Synuclein (α-Syn) toxicity in transgenic mouse brains. Our data show an age-dependent loss of MOR expression levels in the striatum and specifically, within the caudal striatal WMTs in α-Syn tg mouse brains. The loss of MOR expression is associated with degeneration of the myelinated axons that are localized within the corticostriatal WMTs. In brains affected with late stages of PD, we detect evidence confirming the degeneration of myelinated axons within the corticostriatal WMTs. We conclude that loss of corticostriatal MOR expression is associated with degeneration of corticostriatal WMT in α-Syn tg mice, modeling PD.
RESUMO
Alpha-synuclein (alphaS) is an abundant neuronal cytoplasmic protein implicated in Parkinson's disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, alphaS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in alphaS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that alphaS acts with PUFAs to enhance the internalization of the membrane-binding dye, FM 1-43. Specifically, alphaS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin-mediated endocytosis in neuronal and non-neuronal cultured cells. Moreover, alphaS expression and PUFA-enhanced basal and -evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted alphaS mouse brains. We suggest that alphaS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.
Assuntos
Clatrina/metabolismo , Endocitose , Ácidos Graxos Insaturados/metabolismo , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Multimerização Proteica , Transporte Proteico , Receptores da Transferrina/metabolismo , Solubilidade , Técnicas de Cultura de Tecidos , Transferrina/metabolismo , alfa-Sinucleína/deficiência , alfa-Sinucleína/genéticaRESUMO
Alpha-synuclein (alphaS) is a protein involved in the cytopathology and genetics of Parkinson disease and is thought to affect mitochondrial complex I activity. Previous studies have shown that mitochondrial toxins and specifically inhibitors of complex I activity enhance alphaS pathogenesis. Here we show that alphaS overexpression specifically inhibits complex I activity in dopaminergic cells and in A53T alphaS transgenic mouse brains. Importantly, our results indicate that the inhibitory effect on complex I activity is not associated with alphaS-related pathology. Specifically, complex I activity measured in purified mitochondria from A53T alphaS transgenic mouse brains was not affected by mouse age; Parkinson disease-like symptoms; levels of alphaS soluble oligomers; levels of insoluble, lipid-associated alphaS; or alphaS intraneuronal depositions in vivo. Likewise, no correlation was found between complex I activity and polyunsaturated fatty acid-induced alphaS depositions in Lewy body-like inclusions in cultured dopaminergic cells. We further show that the effect of alphaS on complex I activity is not due to altered mitochondrial protein levels or affected complex I assembly. Based on the results herein, we suggest that alphaS expression negatively regulates complex I activity as part of its normal, physiological role.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Transgenes , alfa-Sinucleína/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Dopamina/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , alfa-Sinucleína/genéticaRESUMO
Recent data support an involvement of defects in homeostasis of phosphoinositides (PIPs) in the pathophysiology of Parkinson's disease (PD). Genetic mutations have been identified in genes encoding for PIP-regulating and PIP-interacting proteins, that are associated with familial and sporadic PD. Many of these proteins are implicated in vesicular membrane trafficking, mechanisms that were recently highlighted for their close associations with PD. PIPs are phosphorylated forms of the membrane phospholipid, phosphatidylinositol. Their composition in the vesicle's membrane of origin, as well as membrane of destination, controls vesicular membrane trafficking. We review the converging evidence that points to the involvement of PIPs in PD. The review describes PD- and PIP-associated proteins implicated in clathrin-mediated endocytosis and autophagy, and highlights the involvement of α-synuclein in these mechanisms.
Assuntos
Doença de Parkinson , Autofagia , Endocitose , Humanos , Doença de Parkinson/genética , Fosfatidilinositóis/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Mannitol, a natural alcoholic-sugar, was recently suggested as a potential disease-modifying agent in Parkinson's disease. In animal models of the disease, mannitol interferes with the formation of α-synuclein fibrils, inhibits the formation of α-synuclein oligomers and leads to phenotypic recovery of impaired motor functions. Parkinson's patients who consume mannitol report improvements of both motor and non-motor symptoms. Safety of long-term use of oral mannitol, tolerable dose and possible benefit, however, were never clinically studied. We studied the safety of oral mannitol in Parkinson's disease and assessed the maximal tolerable oral dose by conducting a phase IIa, randomized, double-blind, placebo-controlled, single-center, dose-escalating study (ClinicalTrials.gov Identifier: NCT03823638). The study lasted 36 weeks and included four dose escalations of oral mannitol or dextrose to a maximal dose of 18 g per day. The primary outcome was the safety of oral mannitol, as assessed by the number of adverse events and abnormal laboratory results. Clinical and biochemical efficacy measures were collected but were not statistically-powered. Fourteen patients receiving mannitol completed the trial (in addition to eight patients on placebo). Mannitol-related severe adverse events were not observed. Gastrointestinal symptoms limited dose escalation in 6/14 participants on mannitol. None of the clinical or biochemical efficacy secondary outcome measures significantly differed between groups. We concluded that long-term use of 18 g per day of oral mannitol is safe in Parkinson's disease patients but only two third of patients tolerate this maximal dose. These findings should be considered in the design of future efficacy trials.
RESUMO
alpha-Synuclein (alphaS) is a presynaptic protein implicated in Parkinson's disease (PD). Growing evidence implicates mitochondrial dysfunction, oxidative stress, and alphaS-lipid interactions in the gradual accumulation of alphaS in pathogenic forms and its deposition in Lewy bodies, the pathological hallmark of PD and related synucleinopathies. The peroxisomal biogenesis disorders (PBD), with Zellweger syndrome serving as the prototype of this group, are characterized by malformed and functionally impaired peroxisomes. Here we utilized the PBD mouse models Pex2-/-, Pex5-/-, and Pex13-/- to study the potential effects of peroxisomal dysfunction on alphaS-related pathogenesis. We found increased alphaS oligomerization and phosphorylation and its increased deposition in cytoplasmic inclusions in these PBD mouse models. Furthermore, we show that alphaS abnormalities correlate with the altered lipid metabolism and, specifically, with accumulation of long chain, n-6 polyunsaturated fatty acids that occurs in the PBD models.
Assuntos
Transtornos Peroxissômicos/metabolismo , Peroxissomos/metabolismo , alfa-Sinucleína/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/deficiência , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Sobrevivência Celular/genética , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Biogênese de Organelas , Estresse Oxidativo/fisiologia , Transtornos Peroxissômicos/genética , Transtornos Peroxissômicos/patologia , FosforilaçãoRESUMO
α-Synuclein (α-Syn) protein is implicated in the pathogenesis of Parkinson disease (PD). It is primarily cytosolic and interacts with cell membranes. α-Syn also occurs in the nucleus. Here we investigated the mechanisms involved in nuclear translocation of α-Syn. We analyzed alterations in gene expression following induced α-Syn expression in SH-SY5Y cells. Analysis of upstream regulators pointed at alterations in transcription activity of retinoic acid receptors (RARs) and additional nuclear receptors. We show that α-Syn binds RA and translocates to the nucleus to selectively enhance gene transcription. Nuclear translocation of α-Syn is regulated by calreticulin and is leptomycin-B independent. Importantly, nuclear translocation of α-Syn following RA treatment enhances its toxicity in cultured neurons and the expression levels of PD-associated genes, including ATPase cation transporting 13A2 (ATP13A2) and PTEN-induced kinase1 (PINK1). The results link a physiological role for α-Syn in the regulation of RA-mediated gene transcription and its toxicity in the synucleinopathies.
RESUMO
BACKGROUND: α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson's disease (PD). α-Syn has been shown to associate with membranes and bind acidic phospholipids. However, the physiological importance of these associations to the integrity of axons is not fully clear. METHODS: Biochemical, immunohistochemical and ultrastructural analyses in cultured neurons, transgenic mouse brains, PD and control human brains. RESULTS: We analyzed the ultrastructure of cross-sectioned axons localized to white matter tracts (WMTs), within the dorsal striatum of old and symptomatic α-Syn transgenic mouse brains. The analysis indicated a higher density of axons of thinner diameter. Our findings in cultured cortical neurons indicate a role for α-Syn in elongation of the main axon and its collaterals, resulting in enhanced axonal arborization. We show that α-Syn effect to enhance axonal outgrowth is mediated through its activity to regulate membrane levels of the acidic phosphatidylinositol 4,5-bisphosphate (PI4,5P2). Moreover, our findings link α-Syn- enhanced axonal growth with evidence for axonal injury. In relevance to disease mechanisms, we detect in human brains evidence for a higher degree of corticostriatal glutamatergic plasticity within WMTs at early stages of PD. However, at later PD stages, the respective WMTs in the caudate are degenerated with accumulation of Lewy pathology. CONCLUSIONS: Our results show that through regulating PI4,5P2 levels, α-Syn acts to elongate the main axon and collaterals, resulting in a higher density of axons in the striatal WMTs. Based on these results we suggest a role for α-Syn in compensating mechanisms, involving corticostriatal glutamatergic plasticity, taking place early in PD.
Assuntos
Axônios/ultraestrutura , Encéfalo/metabolismo , Plasticidade Neuronal/fisiologia , Doença de Parkinson , alfa-Sinucleína/metabolismo , Animais , Axônios/metabolismo , Encéfalo/ultraestrutura , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Vias Neurais/metabolismo , Vias Neurais/fisiopatologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Substância Branca/metabolismo , Substância Branca/ultraestruturaRESUMO
Alpha-synuclein (alphaS) and beta-synuclein (betaS) are homologous proteins implicated in Parkinson's disease and related synucleinopathies. While alphaS is neurotoxic and its aggregation and deposition in Lewy bodies is related to neurodegeneration, betaS is considered as a potent inhibitor of alphaS aggregation and toxicity. No mechanism for the neuroprotective role of betaS has been described before. Here, we report that similar to alphaS, betaS normally occurs in lipid-associated, soluble oligomers in wild-type (WT) mouse brains. We partially purified betaS and alphaS proteins from whole mouse brain by size exclusion followed by ion exchange chromatography and found highly similar elution profiles. Using this technique, we were able to partially separate betaS from alphaS and further separate betaS monomer from its own oligomers. Importantly, we show that although alphaS and betaS share high degree of similarities, betaS oligomerization is not affected by increasing cellular levels of polyunsaturated fatty acids (PUFAs), while alphaS oligomerization is dramatically enhanced by PUFA. We show the in vivo occurrence of hetero-oligomers of alphaS and betaS and suggest that betaS expression inhibits PUFA-enhanced alphaS oligomerization by forming hetero-oligomers up to a quatramer that do not further propagate.
Assuntos
Lipídeos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , beta-Sinucleína/química , beta-Sinucleína/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Transformada , Cromatografia por Troca Iônica/métodos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Ácidos Graxos Insaturados/farmacologia , Humanos , Imunoprecipitação/métodos , Substâncias Macromoleculares , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , alfa-Sinucleína/deficiênciaRESUMO
OBJECTIVE: To determine whether blood cells expressed α-Syn can differentiate Parkinson's disease (PD) from healthy controls (HC). METHODS: The concentrations of α-Syn were determined in samples of blood cell pellets using a quantitative Lipid-ELISA assay. In addition, the levels of total protein, hemoglobin, iron and H-ferritin were determined. The study includes samples from the Biofind cohort (n = 46 PD and 45 HC) and results were validated with an additional cohort (n = 35 PD and 28 HC). RESULTS: A composite biomarker consisting of the concentrations of total α-Syn, proteinase-K resistant (PKres ) α-Syn and phospho-Serine 129 α-Syn (PSer 129), is designed based on the analysis of the discovery BioFIND cohort. This composite biomarker differentiates a PD subgroup, presenting motor symptoms without dementia from a HC group, with a convincing accuracy, represented by an AUC = 0.81 (95% CI, 0.71 to 0.92). Closely similar results were obtained for the validation cohort, that is, AUC = 0.81, (95% CI, 0.70 to 0.94). INTERPRETATION: Our results demonstrate the potential usefulness of blood cells expressed α-Syn as a biomarker for PD.
Assuntos
Células Sanguíneas/metabolismo , Doença de Parkinson/sangue , Doença de Parkinson/diagnóstico , alfa-Sinucleína/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Accumulation of misfolded proteins as insoluble aggregates occurs in several neurodegenerative diseases. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), alpha-synuclein (alpha S) accumulates in insoluble inclusions. To identify soluble alpha S oligomers that precede insoluble aggregates, we probed the cytosols of mesencephalic neuronal (MES) cells, normal and alpha S-transgenic mouse brains, and normal, PD, and DLB human brains. All contained highly soluble oligomers of alpha S whose detection was enhanced by delipidation. Exposure of living MES neurons to polyunsaturated fatty acids (PUFAs) increased alpha S oligomer levels, whereas saturated FAs decreased them. PUFAs directly promoted oligomerization of recombinant alphaS. Transgenic mice accumulated soluble oligomers with age. PD and DLB brains had elevated amounts of the soluble, lipid-dependent oligomers. We conclude that alpha S interacts with PUFAs in vivo to promote the formation of highly soluble oligomers that precede the insoluble alpha S aggregates associated with neurodegeneration.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismo , Doença por Corpos de Lewy/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/metabolismo , Fatores Etários , Substituição de Aminoácidos , Animais , Encéfalo/metabolismo , Química Encefálica , Linhagem Celular , Citosol/química , Ácidos Graxos/farmacologia , Ácidos Graxos Insaturados/farmacologia , Humanos , Substâncias Macromoleculares , Mesencéfalo/citologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Sinucleínas , alfa-SinucleínaRESUMO
α-Synuclein is a protein involved in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We investigated the role of neuronal α-Syn in myelin composition and abnormalities. The phospholipid content of purified myelin was determined by 31P NMR in two mouse lines modeling PD, PrP-A53T α-Syn and Thy-1 wt-α-Syn. Significantly higher levels of phospholipids were detected in myelin purified from brains of these α-Syn transgenic mouse models than in control mice. Nevertheless, myelin ultrastructure appeared intact. To further investigate the effect of α-Syn on myelin abnormalities, we systematically analyzed the striatum, a brain region associated with neurodegeneration in PD. An age and disease-dependent loss of myelin basic protein (MBP) signal was detected by immunohistochemistry in striatal striosomes (patches). The age-dependent loss of MBP signal was associated with lower P25α levels in oligodendrocytes. In addition, we found that α-Syn inhibited oligodendrocyte maturation and the formation of membranous sheets in vitro. Based on these results we concluded that neuronal α-Syn is involved in the regulation and/or maintenance of myelin phospholipid. However, axonal hypomyelination in the PD models is evident only in progressive stages of the disease and associated with α-Syn toxicity.
Assuntos
Encéfalo/metabolismo , Bainha de Mielina/metabolismo , Neurônios/metabolismo , Fosfolipídeos/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/patologia , Encéfalo/ultraestrutura , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Neurônios/patologia , Neurônios/ultraestrutura , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Presenilina-1/genética , Presenilina-1/metabolismo , alfa-Sinucleína/deficiência , alfa-Sinucleína/genéticaRESUMO
A marker for diagnosis of Parkinson's disease (PD), which reflects on the occurrence of peripheral pathogenic mechanisms, would potentially improve therapy. The significance of α-Synuclein (α-Syn) expression in red blood cells (RBC) is currently unclear. Here we investigated whether RBC's-expressed α-Syn may associate with PD. To this aim, we determined the levels of total and proteinase K-resistant α-Syn in samples of packed red blood cells (PRBCs). Twenty-one individuals with PD at various disease stages and 15 healthy controls, with similar demographic features, were recruited to this study. α-Syn levels were determined by their biochemical property to bind phospholipids, using a phospholipid-ELISA assay. A significantly lower ratio of total-to-proteinase K-resistant α-Syn levels was detected in PD patients than in the healthy control group. However, there was considerable overlap between the two groups. Suggesting a need for additional markers to be tested in combination with α-Syn levels. To the best of our knowledge, this is the first evidence for an association between RBCs-expressed α-Syn and pathogenic mechanisms involved in PD.