RESUMO
Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.
Assuntos
Cálcio , alfa-Sinucleína , Cálcio/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Membrana Celular/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de LigaçãoRESUMO
α-Synuclein (α-syn) phosphorylation at serine 129 (pS129α-syn) is substantially increased in Lewy body disease, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129α-syn (WTα-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129α-syn as a measure of efficacy in clinical trials.
Assuntos
Amiloide , Doença por Corpos de Lewy , Doença de Parkinson , Agregação Patológica de Proteínas , alfa-Sinucleína , Amiloide/metabolismo , Humanos , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Fosforilação , Agregados Proteicos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Serina/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Collapsin response mediator protein 2 (CRMP2) is a member of a protein family, which is highly involved in neurodevelopment, but most of its members become heavily downregulated in adulthood. CRMP2 is an important factor in neuronal polarization, axonal formation and growth cone collapse. The protein remains expressed in adulthood, but is more region specific. CRMP2 is present in adult corpus callosum (CC) and in plastic areas like prefrontal cortex and hippocampus. CRMP2 has been implicated as one of the risk-genes for Schizophrenia (SZ). Here, a CRMP2 conditional knockout (CRMP2-cKO) mouse was used as a model of SZ to investigate how it could affect the white matter and therefore brain connectivity. Multielectrode electrophysiology (MEA) was used to study the function of corpus callosum showing an increase in conduction velocity (CV) measured as Compound Action Potentials (CAPs) in acute brain slices. Light- and electron-microscopy, specifically Serial Block-face Scanning Electron Microscopy (SBF-SEM), methods were used to study the structure of CC in CRMP2-cKO mice. A decrease in CC volume of CRMP2-cKO mice as compared to controls was observed. No differences were found in numbers nor in the size of CC oligodendrocytes (OLs). Similarly, no differences were found in myelin thickness or in node of Ranvier (NR) structure. In contrast, abnormally smaller axons were measured in the CRMP2-cKO mice. Using these state-of-the-art methods it was possible to shed light on specific parts of the dysconnectivity aspect of deletion of CRMP2 related to SZ and add details to previous findings helping further understanding the disease. This paper substantiates the white matter changes in the absence of CRMP2 and ties it to the role it plays in this complex disorder.
Assuntos
Axônios , Corpo Caloso , Animais , Camundongos , Axônios/fisiologia , Encéfalo , Camundongos Knockout , Bainha de Mielina , Neurônios/metabolismoRESUMO
Two recent, high-profile manuscripts reported negative results with two parallel approaches of passive immunization targeting α-synuclein in a population of patients with early Parkinson's disease (PD). These phase II studies failed to show a bona fide disease-modifying neuroprotective effect on PD progression, despite the evidence that these antibodies effectively bind native α-synuclein in human serum. Here, we discuss the possible reasons that could help explain the lack of clinical efficacy. In particular, we highlight (1) the wealth of evidence supporting the notion of α-synuclein as a valid therapeutic target; (2) the lack of evidence of target engagement in the aforementioned studies, especially of the elusive oligomeric species, the likely culprits in disease pathogenesis and/or its propagation; (3) the limitations, especially in terms of timing passive immunization, of preclinical models, where the same α-synuclein antibodies succeeded in mitigating disease manifestations; (4) the consideration of possibly intervening at an even earlier stage of disease in future trials; and (5) the multitude of strategies beyond passive immunization that could be used to combat α-synuclein-mediated neurodegeneration, if in the end the current approach is not fruitful. Overall, our perception is that converging developments in the field, among them novel bioassays and biomarkers, improved cellular and animal models and objective measurements of motor activities integrated into clinical trials, if further optimized, will gradually move the momentum of the field forward. This, to better test the concept of whether α-synuclein-targeting therapies can indeed deliver the "holy grail" of neuroprotection to the benefit of the PD community. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Humanos , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/imunologia , Anticorpos/imunologia , Anticorpos/uso terapêutico , Biomarcadores , Doença de Parkinson/tratamento farmacológico , Resultado do TratamentoRESUMO
Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.
Assuntos
Integrina alfaXbeta2 , Macrófagos , Doença de Parkinson , Fagossomos , Agregação Patológica de Proteínas , alfa-Sinucleína , Humanos , Integrina alfaXbeta2/química , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Doença de Parkinson/genética , Doença de Parkinson/imunologia , Doença de Parkinson/patologia , Fagossomos/química , Fagossomos/genética , Fagossomos/imunologia , Fagossomos/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/imunologia , Agregação Patológica de Proteínas/patologia , Estrutura Quaternária de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/imunologiaRESUMO
Neuronal aggregates of misfolded alpha-synuclein protein are found in the brain and periphery of patients with Parkinson's disease. Braak and colleagues have hypothesized that the initial formation of misfolded alpha-synuclein may start in the gut, and then spread to the brain via peripheral autonomic nerves hereby affecting several organs, including the heart and intestine. Age is considered the greatest risk factor for Parkinson's disease, but the effect of age on the formation of pathology and its propagation has not been studied in detail. We aimed to investigate whether propagation of alpha-synuclein pathology from the gut to the brain is more efficient in old versus young wild-type rats, upon gastrointestinal injection of aggregated alpha-synuclein. Our results demonstrate a robust age-dependent gut-to-brain and brain-to-gut spread of alpha-synuclein pathology along the sympathetic and parasympathetic nerves, resulting in age-dependent dysfunction of the heart and stomach, as observed in patients with Parkinson's disease. Moreover, alpha-synuclein pathology is more densely packed and resistant to enzymatic digestion in old rats, indicating an age-dependent maturation of alpha-synuclein aggregates. Our study is the first to provide a detailed investigation of alpha-synuclein pathology in several organs within one animal model, including the brain, skin, heart, intestine, spinal cord and autonomic ganglia. Taken together, our findings suggest that age is a crucial factor for alpha-synuclein aggregation and complete propagation to heart, stomach and skin, similar to patients. Given that age is the greatest risk factor for human Parkinson's disease, it seems likely that older experimental animals will yield the most relevant and reliable findings. These results have important implications for future research to optimize diagnostics and therapeutics in Parkinson's disease and other age-associated synucleinopathies. Increased emphasis should be placed on using aged animals in preclinical studies and to elucidate the nature of age-dependent interactions.
Assuntos
Envelhecimento/patologia , Disautonomias Primárias/etiologia , alfa-Sinucleína/toxicidade , Envelhecimento/metabolismo , Animais , Sistema Nervoso Autônomo/efeitos dos fármacos , Sistema Nervoso Autônomo/metabolismo , Sistema Nervoso Autônomo/patologia , Encéfalo/patologia , Duodeno/efeitos dos fármacos , Duodeno/patologia , Rim/patologia , Músculo Esquelético/patologia , Miocárdio/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Disautonomias Primárias/metabolismo , Disautonomias Primárias/patologia , Agregação Patológica de Proteínas/patologia , Ratos Endogâmicos F344 , Pele/patologia , Medula Espinal/patologia , Estômago/efeitos dos fármacos , Estômago/patologiaRESUMO
Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25α to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25α. Similar to earlier studies, p25α increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25α transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25α did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 µM KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells.
Assuntos
Atrofia de Múltiplos Sistemas/metabolismo , Atrofia de Múltiplos Sistemas/patologia , Prolil Oligopeptidases/antagonistas & inibidores , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Linhagem Celular , Sobrevivência Celular , Humanos , Atrofia de Múltiplos Sistemas/tratamento farmacológico , Atrofia de Múltiplos Sistemas/etiologia , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Fosforilação , Agregação Patológica de Proteínas/tratamento farmacológicoRESUMO
In Parkinson's disease, dopamine-containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build-up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH-over-expressing mice (TH-HI) using a BAC-transgenic approach that results in over-expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH-HI mice had a 3-fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH-HI mice showed increased striatal production of H2 O2 and reduced glutathione levels. In addition, TH-HI mice had elevated striatal levels of the neurotoxic dopamine metabolites 3,4-dihydroxyphenylacetaldehyde and 5-S-cysteinyl-dopamine and were more susceptible than wild-type mice to the effects of amphetamine and methamphetamine. These results demonstrate that increased TH alone is sufficient to produce oxidative stress in vivo, build up autotoxic dopamine metabolites, and augment toxicity.
Assuntos
Anfetamina/farmacologia , Catecolaminas/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Estresse Oxidativo , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Dopamina/análogos & derivados , Dopamina/metabolismo , Feminino , Dosagem de Genes , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
BACKGROUND: Synucleinopathies are characterized by neurodegeneration and deposition of the presynaptic protein α-synuclein in pathological protein inclusions. Growing evidence suggests the complement system not only has physiological functions in the central nervous system, but also is involved in mediating the pathological loss of synapses in Alzheimer's disease. However, it is not established whether the complement system has a similar role in the diseases Parkinson's disease, Dementia with Lewy bodies, and multiple system atrophy (MSA) that are associated with α-synuclein aggregate pathology. METHODS: To investigate if the complement system has a pathological role in synucleinopathies, we assessed the effect of the complement system on the viability of an α-synuclein expressing cell model and examined direct activation of the complement system by α-synuclein in a plate-based activation assay. Finally, we investigated the levels of the initiator of the classical pathway, C1q, in postmortem brain samples from MSA patients. RESULTS: We demonstrate that α-synuclein activates the classical complement pathway and mediates complement-dependent toxicity in α-synuclein expressing SH-SY5Y cells. The α-synuclein-dependent cellular toxicity was rescued by the complement inhibitors RaCI (inhibiting C5) and Cp20 (inhibiting C3). Furthermore, we observed a trend for higher levels of C1q in the putamen of MSA subjects than that of controls. CONCLUSION: α-Synuclein can activate the classical complement pathway, and the complement system is involved in α-synuclein-dependent cellular cytotoxicity suggesting the system could play a prodegenerative role in synucleinopathies.
Assuntos
Via Clássica do Complemento/fisiologia , Corpos de Inclusão/metabolismo , Córtex Visual/metabolismo , alfa-Sinucleína/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Humanos , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Córtex Visual/patologiaRESUMO
Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.
Assuntos
Atrofia de Múltiplos Sistemas/genética , Doenças Neurodegenerativas/genética , Sinucleinopatias/patologia , alfa-Sinucleína/genética , Animais , Linhagem Celular , Humanos , Corpos de Inclusão/patologia , Camundongos , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas/patologia , Proteínas do Tecido Nervoso/genética , Oligodendroglia/metabolismo , Conformação Proteica , Deficiências na Proteostase/genética , Substância Negra/patologia , alfa-Sinucleína/toxicidadeRESUMO
The pathological aggregation of the presynaptic protein α-synuclein (α-syn) and propagation through synaptically coupled neuroanatomical tracts is increasingly thought to underlie the pathophysiological progression of Parkinson's disease (PD) and related synucleinopathies. Although the precise molecular mechanisms responsible for the spreading of pathological α-syn accumulation in the CNS are not fully understood, growing evidence suggests that de novo α-syn misfolding and/or neuronal internalization of aggregated α-syn facilitates conformational templating of endogenous α-syn monomers in a mechanism reminiscent of prions. A refined understanding of the biochemical and cellular factors mediating the pathological neuron-to-neuron propagation of misfolded α-syn will potentially elucidate the etiology of PD and unravel novel targets for therapeutic intervention. Here, we discuss recent developments on the hypothesis regarding trans-synaptic propagation of α-syn pathology in the context of neuronal vulnerability and highlight the potential utility of novel experimental models of synucleinopathies.
Assuntos
Doença de Parkinson/patologia , Príons/metabolismo , Agregação Patológica de Proteínas , alfa-Sinucleína/metabolismo , Animais , Humanos , Doença de Parkinson/metabolismoRESUMO
Parkinson's disease (PD) is a currently incurable disease and the number of patients is expected to increase due to the extended human lifespan. α-Synuclein is a pathological hallmark of PD and variations and triplications of the gene encoding α-synuclein are strongly correlated with the risk of developing PD. Decreasing α-synuclein is therefore a promising therapeutic strategy for the treatment of PD. We have previously demonstrated that Polo-like kinase 2 (PLK-2) regulates α-synuclein protein levels by modulating the expression of α-synuclein mRNA. In this study, we further expand the knowledge on this pathway and show that it depends on down-stream modulation of Glycogen-synthase kinase 3 ß (GSK-3ß). We show that PLK-2 inhibition only increases α-synuclein levels in the presence of active GSK-3ß in both cell lines and primary neuronal cultures. Furthermore, direct inhibition of GSK-3ß decreases α-synuclein protein and mRNA levels in our cell model and overexpression of Leucine-rich repeat kinase 2, known to activate GSK-3ß, increases α-synuclein levels. Finally, we show an increase in endogenous α-synuclein in primary neurons when increasing GSK-3ß activity. Our findings demonstrate a not previously described role of endogenous GSK-3ß activity in the PLK-2 mediated regulation of α-synuclein levels. This finding opens up the possibility of GSK-3ß as a novel target for decreasing α-synuclein levels by the use of small molecule compounds, hereby serving as a disease modulating strategy.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , alfa-Sinucleína/metabolismo , Animais , Glicogênio Sintase Quinase 3 beta/genética , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , alfa-Sinucleína/genéticaRESUMO
Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies.
Assuntos
Cálcio/química , Cálcio/metabolismo , Citosol/química , Agregados Proteicos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , alfa-Sinucleína/metabolismo , Animais , Encéfalo/patologia , Caenorhabditis elegans , Linhagem Celular , Células Cultivadas , Retículo Endoplasmático/metabolismo , Humanos , Indóis/farmacologia , Corpos de Lewy , Masculino , Camundongos , Doença de Parkinson/patologia , Ratos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidoresRESUMO
Multiple system atrophy (MSA) is characterized by the presence of distinctive glial cytoplasmic inclusions (GCIs) within oligodendrocytes that contain the neuronal protein alpha-synuclein (aSyn) and the oligodendroglia-specific phosphoprotein TPPP/p25α. However, the role of oligodendroglial aSyn and p25α in the formation of aSyn-rich GCIs remains unclear. To address this conundrum, we have applied human aSyn (haSyn) pre-formed fibrils (PFFs) to rat wild-type (WT)-, haSyn-, or p25α-overexpressing oligodendroglial cells and to primary differentiated oligodendrocytes derived from WT, knockout (KO)-aSyn, and PLP-haSyn-transgenic mice. HaSyn PFFs are readily taken up by oligodendroglial cells and can recruit minute amounts of endogenous aSyn into the formation of insoluble, highly aggregated, pathological assemblies. The overexpression of haSyn or p25α accelerates the recruitment of endogenous protein and the generation of such aberrant species. In haSyn PFF-treated primary oligodendrocytes, the microtubule and myelin networks are disrupted, thus recapitulating a pathological hallmark of MSA, in a manner totally dependent upon the seeding of endogenous aSyn. Furthermore, using oligodendroglial and primary cortical cultures, we demonstrated that pathology-related S129 aSyn phosphorylation depends on aSyn and p25α protein load and may involve different aSyn "strains" present in oligodendroglial and neuronal synucleinopathies. Importantly, this hypothesis was further supported by data obtained from human post-mortem brain material derived from patients with MSA and dementia with Lewy bodies. Finally, delivery of haSyn PFFs into the mouse brain led to the formation of aberrant aSyn forms, including the endogenous protein, within oligodendroglia and evoked myelin decompaction in WT mice, but not in KO-aSyn mice. This line of research highlights the role of endogenous aSyn and p25α in the formation of pathological aSyn assemblies in oligodendrocytes and provides in vivo evidence of the contribution of oligodendroglial aSyn in the establishment of aSyn pathology in MSA.
Assuntos
Atrofia de Múltiplos Sistemas/patologia , Proteínas do Tecido Nervoso/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Ratos , Sinucleinopatias/metabolismoRESUMO
he original version of this article.
RESUMO
The conversion of endogenous alpha-synuclein (asyn) to pathological asyn-enriched aggregates is a hallmark of Parkinson's disease (PD). These inclusions can be detected in the central and enteric nervous system (ENS). Moreover, gastrointestinal symptoms can appear up to 20 years before the diagnosis of PD. The dual-hit hypothesis posits that pathological asyn aggregation starts in the ENS, and retrogradely spreads to the brain. In this study, we tested this hypothesis by directly injecting preformed asyn fibrils into the duodenum wall of wild-type rats and transgenic rats with excess levels of human asyn. We provide a meticulous characterization of the bacterial artificial chromosome (BAC) transgenic rat model with respect to initial propagation of pathological asyn along the parasympathetic and sympathetic pathways to the brainstem, by performing immunohistochemistry at early time points post-injection. Induced pathology was observed in all key structures along the sympathetic and parasympathetic pathways (ENS, autonomic ganglia, intermediolateral nucleus of the spinal cord (IML), heart, dorsal motor nucleus of the vagus, and locus coeruleus (LC)) and persisted for at least 4 months post-injection. In contrast, asyn propagation was not detected in wild-type rats, nor in vehicle-injected BAC rats. The presence of pathology in the IML, LC, and heart indicate trans-synaptic spread of the pathology. Additionally, the observed asyn inclusions in the stomach and heart may indicate secondary anterograde propagation after initial retrograde spreading. In summary, trans-synaptic propagation of asyn in the BAC rat model is fully compatible with the "body-first hypothesis" of PD etiopathogenesis. To our knowledge, this is the first animal model evidence of asyn propagation to the heart, and the first indication of bidirectional asyn propagation via the vagus nerve, i.e., duodenum-to-brainstem-to-stomach. The BAC rat model could be very valuable for detailed mechanistic studies of the dual-hit hypothesis, and for studies of disease modifying therapies targeting early pathology in the gastrointestinal tract.
Assuntos
Encéfalo/metabolismo , Neurônios/metabolismo , Doença de Parkinson/patologia , Sinapses/metabolismo , alfa-Sinucleína/metabolismo , Animais , Sistema Nervoso Autônomo/metabolismo , Sistema Nervoso Autônomo/patologia , Encéfalo/patologia , Modelos Animais de Doenças , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologia , Neurônios/patologia , Doença de Parkinson/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Medula Espinal/patologia , Sinapses/patologia , alfa-Sinucleína/administração & dosagemRESUMO
Parkinson's disease (PD) affects motor function through degenerative processes and synaptic transmission impairments in the basal ganglia. None of the treatments available delays or stops the progression of the disease. While α-synuclein pathological accumulation represents a hallmark of the disease in its idiopathic form, leucine rich repeat kinase 2 (LRRK2) is genetically associated with familial and sporadic forms of PD. The genetic information suggests that LRRK2 kinase activity plays a role in the pathogenesis of the disease. To support a potential link between LRRK2 and α-synuclein in the pathophysiological mechanisms underlying PD, the effect of LRRK2 ablation or LRRK2 kinase pharmacological inhibition were studied in rats with adeno-associated virus-induced (AAV) α-synuclein overexpression in the nigrostriatal pathway. We first report that viral overexpression of α-synuclein induced increased burst firing in subthalamic neurons. Aberrant firing pattern of subthalamic neurons has also been reported in PD patients and neurotoxin-based animal models, and is hypothesized to play a key role in the appearance of motor dysfunction. We further report that genetic LRRK2 ablation, as well as pharmacological inhibition of LRRK2 kinase activity with PFE-360, reversed the aberrant firing pattern of subthalamic neurons induced by AAV-α-synuclein overexpression. This effect of LRRK2 modulation was not associated with any neuroprotective effect or motor improvement. Nonetheless, our findings may indicate a potential therapeutic benefit of LRRK2 kinase inhibition by normalizing the aberrant neuronal activity of subthalamic neurons induced by AAV-α-synuclein, a neurophysiological trait recapitulating observations in PD.
Assuntos
Potenciais de Ação/fisiologia , Dependovirus/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/deficiência , Transtornos Parkinsonianos/metabolismo , Núcleo Subtalâmico/metabolismo , alfa-Sinucleína/biossíntese , Potenciais de Ação/efeitos dos fármacos , Animais , Dependovirus/genética , Feminino , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Transtornos Parkinsonianos/genética , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Ratos Transgênicos , Núcleo Subtalâmico/efeitos dos fármacos , alfa-Sinucleína/genéticaRESUMO
Plasma total and nervous system derived exosomal (NDE) α-synuclein have been determined as potential biomarkers of Parkinson's disease (PD). To explore the utility of plasma α-synuclein in the prodromal phase of PD, plasma total and NDE α-synuclein were evaluated in baseline and 2-year follow-up samples from 256 individuals recruited as part of the Parkinson's Associated Risk Syndrome (PARS) study. The results demonstrated that baseline and longitudinal increases in total α-synuclein predicted progression of cognitive decline in hyposmic individuals with dopamine transporter (DAT) binding reduction. On the other hand, a longitudinal decrease in NDE α-synuclein predicted worsening cognitive scores in hyposmic individuals with DAT binding reduction. Finally, in individuals with faster DAT progression, decreasing NDE/total α-synuclein ratio was associated with a larger reduction in DAT from baseline to follow-up. These results suggest that, though underlying mechanisms remain to be defined, alterations in plasma total and NDE α-synuclein concentrations are likely associated with PD progression, especially in the aspect of cognitive impairment, at early stages of the disease.
Assuntos
Disfunção Cognitiva/sangue , Disfunção Cognitiva/diagnóstico , Doença de Parkinson/sangue , Doença de Parkinson/diagnóstico , alfa-Sinucleína/sangue , Idoso , Biomarcadores/sangue , Disfunção Cognitiva/psicologia , Estudos Transversais , Feminino , Seguimentos , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/psicologia , Projetos Piloto , Fatores de RiscoRESUMO
Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy comprise a group of neurodegenerative diseases termed synucleinopathies. Synucleinopathie are, characterized by presence of inclusion bodies in degenerating brain cells which contain aggregated α-synuclein phosphorylated on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease cellular degeneration. In vitro phosphorylation demonstrates that PKR can directly bind and phosphorylate monomeric and filamenteous α-synuclein on Ser129. Inhibition and knockdown of PKR reduce Ser129 phosphorylation in different models (SH-SY5Y ASYN cells, OLN-AS7 cells, primary mouse hippocampal neurons, and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-ß-1b, lead to increased levels of phosphorylated Ser129 α-synuclein that is completely blocked by simultaneous PKR inhibition. These results reveal a direct link between PKR and the phosphorylation and toxicity of α-synuclein, and they support that neuroinflammatory processes play a role in modulating the pathogenicity of α-synuclein.
Assuntos
Hipocampo/metabolismo , alfa-Sinucleína/metabolismo , eIF-2 Quinase/metabolismo , Animais , Animais Recém-Nascidos , Morte Celular/fisiologia , Linhagem Celular Transformada , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , eIF-2 Quinase/antagonistas & inibidoresRESUMO
Oligomeric species of various proteins are linked to the pathogenesis of different neurodegenerative disorders. Consequently, there is intense focus on the discovery of novel inhibitors, e.g. small molecules and antibodies, to inhibit the formation and block the toxicity of oligomers. In Parkinson disease, the protein α-synuclein (αSN) forms cytotoxic oligomers. The flavonoid epigallocatechin gallate (EGCG) has previously been shown to redirect the aggregation of αSN monomers and remodel αSN amyloid fibrils into disordered oligomers. Here, we dissect EGCG's mechanism of action. EGCG inhibits the ability of preformed oligomers to permeabilize vesicles and induce cytotoxicity in a rat brain cell line. However, EGCG does not affect oligomer size distribution or secondary structure. Rather, EGCG immobilizes the C-terminal region and moderately reduces the degree of binding of oligomers to membranes. We interpret our data to mean that the oligomer acts by destabilizing the membrane rather than by direct pore formation. This suggests that reduction (but not complete abolition) of the membrane affinity of the oligomer is sufficient to prevent cytotoxicity.