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1.
Nat Commun ; 11(1): 4727, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948752

RESUMO

The apolipoprotein E (APOE) gene contains both the major common risk variant for late onset Alzheimer's disease (AD), e4, and the major neuroprotective variant, e2. Here we examine the association of APOE e2 with multiple neurodegenerative pathologies, leveraging the NACC v. 10 database of 1557 brains that included 130 e2 carriers and 679 e4 carriers in order to examine potential neuroprotective effects. For AD-related pathologies of amyloid plaques and Braak stage, e2 had large and highly significant protective effects contrasted with e3/e3 and e4 carriers with odds ratios of about 0.50 for e3 contrasts and 0.10 for e4 contrasts. When we separately examined e2/e4 carriers, risk for AD pathologies was similar to that of e4 carriers, not e2 carriers. For multiple fronto-temporal lobar pathologies and tauopathies, e2 was not significantly associated with pathology. In sum, we found that e2 was associated with large but circumscribed protective effects.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Apolipoproteína E2/genética , Apolipoproteína E2/metabolismo , Genótipo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Feminino , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Modelos Logísticos , Masculino , Placa Amiloide/patologia , Tauopatias/metabolismo , alfa-Sinucleína/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(33): 20305-20315, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32737160

RESUMO

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson's disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.


Assuntos
Doença de Parkinson , alfa-Sinucleína/química , Amiloide/química , Amiloide/metabolismo , Animais , Células Cultivadas , Microscopia Crioeletrônica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Conformação Proteica , Ratos , Ratos Sprague-Dawley , alfa-Sinucleína/síntese química , alfa-Sinucleína/metabolismo
3.
PLoS One ; 15(8): e0238075, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833982

RESUMO

Parkinson disease is the most common neurodegenerative movement disorder, estimated to affect one in twenty-five individuals over the age of 80. Mutations in glucocerebrosidase 1 (GBA1) represent the most common genetic risk factor for Parkinson disease. The link between GBA1 mutations and α-synuclein accumulation, a hallmark of Parkinson disease, is not fully understood. Following our recent finding that Gba1 mutations lead to increased α-synuclein accumulation in mice, we have studied the effects of a single injection of mouse α-synuclein pre-formed fibrils into the striatum of Gba1 mice that carry a L444P knock-in mutation. We found significantly greater formation and spread of α-synuclein inclusions in Gba1-transgenic mice compared to wild-type controls. This indicates that the Gba1 L444P mutation accelerates α-synuclein pathology and spread.


Assuntos
Técnicas de Introdução de Genes , Glucosilceramidase/genética , Mutação , Agregados Proteicos/genética , alfa-Sinucleína/química , Animais , Humanos , Injeções , Camundongos , Neostriado/metabolismo , alfa-Sinucleína/metabolismo
4.
PLoS One ; 15(8): e0237328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790707

RESUMO

α-Synuclein (αSyn) fibrils spread from one neuronal cell to another. This prion-like phenomenon is believed to contribute to the progression of the pathology in Parkinson's disease and other synucleinopathies. The binding of αSyn fibrils originating from affected cells to the plasma membrane of naïve cells is key in their prion-like propagation propensity. To interfere with this process, we designed polypeptides derived from proteins we previously showed to interact with αSyn fibrils, namely the molecular chaperone Hsc70 and the sodium/potassium pump NaK-ATPase and assessed their capacity to bind αSyn fibrils and/or interfere with their take-up by cells of neuronal origin. We demonstrate here that polypeptides that coat αSyn fibrils surfaces in such a way that they are changed affect αSyn fibrils binding to the plasma membrane components and/or their take-up by cells. Altogether our observations suggest that the rationale design of αSyn fibrils polypeptide binders that interfere with their propagation between neuronal cells holds therapeutic potential.


Assuntos
Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Animais , Linhagem Celular , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSC70/farmacologia , Humanos , Camundongos , Modelos Moleculares , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Peptídeos/química , Príons/antagonistas & inibidores , Príons/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/farmacologia
6.
Toxicol Lett ; 332: 164-170, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32659473

RESUMO

Manganese (Mn) is an environmental pollutant having a toxic effect on Parkinson's disease, with significant damage seen in the neurons of basal ganglia. Hence, Mn pollution is a public health concern. A Sprague-Dawley rat model was used to determine the damage to basal nuclei, and the effect of Mn intake was detected using the Morris water maze test and transmission electron microscopy. The SH-SY5Y cell line was exposed to Mn, and downstream signaling was assessed to determine the mechanism of toxicity. Mn exposure injured neurons, repressing GABAAR receptors and inducing GABABR receptors. The synergistic effect of the GABABR receptor and Kir6.1-SUR1 or Kir6.2-SUR1 was found to be one of the potential factors for the secretion of α-synuclein. The accumulation of α-synuclein regulated downstream factors calmodulin (CAM) cAMP response element-binding protein (CREB), thereby impairing learning and memory. Other genes downstream of CREB, rather than the feedback regulation of CREB, and brain-derived neurotrophic factor might also be involved.


Assuntos
Canais KATP/efeitos dos fármacos , Intoxicação por Manganês/metabolismo , Receptores de GABA/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Animais , Gânglios da Base/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Masculino , Intoxicação por Manganês/psicologia , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/psicologia , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-B/efeitos dos fármacos
7.
Life Sci ; 257: 118019, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32629002

RESUMO

Parkinson's disease (PD) is a disease of the human nervous system with an onset, in the sixth and seventh decades of the human life. Chiefly perceived as progressive degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) with the ensued loss of dopamine in the striatum and the presence of Lewy bodies, consisting of α-synuclein agglomeration. In which the neuronal bridge between substantia nigra and striatum plays an advent role in the motor system. Dilapidation of these neurons results in dopamine depletion which in-turn makes hay to PD. Eventually, the etiology and pathogenesis of PD were still on a hike of dilemma. Traditional Chinese medicine (TCM), including Chinese herbal remedies, acupuncture, and manipulative therapies, is commonly used as an adjunctive therapy in different diseases, particularly neurological diseases, in Asian countries. Additionally, TCM might improve the prognoses and the quality of life of patients with PD because it induces less adverse drug reactions. The present review describes research on the various neuroprotective components and herbal extracts from herbal medicines in the context of addressing the effects of PD.


Assuntos
Medicina Tradicional Chinesa/métodos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/terapia , Animais , Encéfalo/metabolismo , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina , Neurônios Dopaminérgicos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Parte Compacta da Substância Negra/metabolismo , Substância Negra/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(32): 18977-18983, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719116

RESUMO

Parkinson's disease is associated with α-synuclein (α-syn), a cytosolic protein enriched in presynaptic terminals. The biological function of α-syn remains elusive; however, increasing evidence suggests that the protein is involved in the regulation of synaptic vesicle fusion, signifying the importance of α-syn-lipid interactions. We show that α-syn preferentially binds to GM1-rich, liquid-ordered lipid domains on cytoplasmic membranes by using unroofed cells, which encapsulates lipid complexity and cellular topology. Moreover, proteins (Rab3a, syntaxin-1A, and VAMP2) involved in exocytosis also localize with α-syn, supporting its proposed functional role in exocytosis. To investigate how these lipid/protein interactions influence α-syn at the residue level, α-syn was derivatized with an environmentally sensitive fluorophore (7-nitrobenz-2-oxa-1,3-diazol-4-yl [NBD]) at different N- and C-terminal sites. Measurements of NBD fluorescence lifetime distributions reveal that α-syn adopts a multitude of membrane-bound conformations, which were not recapitulated in simple micelle or vesicle models, indicating an exquisite sensitivity of the protein to the complex lipid environment. Interestingly, these data also suggest the participation of the C terminus in membrane localization, which is generally overlooked and thus emphasize the need to use cellularly derived and biologically relevant membranes for biophysical characterization. Collectively, our results demonstrate that α-syn is more conformationally dynamic at the membrane interface than previously appreciated, which may be important for both its physiological and pathological functions.


Assuntos
Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , alfa-Sinucleína/química , Gangliosídeo G(M1)/metabolismo , Humanos , Cinética , Lipídeos de Membrana/química , Microdomínios da Membrana/química , Microdomínios da Membrana/genética , Ligação Proteica , Transporte Proteico , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
9.
Toxicol Lett ; 331: 188-199, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32569805

RESUMO

Methamphetamine (METH) is a highly addictive psychostimulant drug whose abuse can cause many health complications. Our previous studies have shown that METH exposure increases α-synuclein (α-syn) expression. Recently, it was shown that α-syn could be transferred from neurons to astrocytes via exosomes. However, the specific role of astrocytes in α-syn pathology involved in METH neurotoxicity remains unclear. The objective of this study was to determine whether exosomes derived from METH-treated neurons contain pathological α-syn and test the hypothesis that exosomes can transfer pathological α-syn from neurons to astrocytes. To this end, using animal and cell line coculture models, we show that exosomes isolated from METH-treated SH-SY5Y cells contained pathological α-syn. Furthermore, the addition of METH exosomes to the medium of primary cultured astrocytes induced α-syn aggregation and inflammatory responses in astrocytes. Then, we evaluated changes in nuclear receptor related 1 protein (Nurr1) expression and the levels of inflammatory cytokines in primary cultured astrocytes exposed to METH or α-syn. We found that METH or α-syn exposure decreased Nurr1 expression and increased proinflammatory cytokine expression in astrocytes. Our results indicate that α-syn can be transferred from neuronal cells to astrocytes through exosomes. When internalized α-syn accumulated in astrocytes, the cells produced inflammatory responses. Nurr1 may play a crucial role in this process and could be a therapeutic target for inflammatory damage caused by METH.


Assuntos
Astrócitos/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/toxicidade , Exossomos/metabolismo , Metanfetamina/toxicidade , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Astrócitos/imunologia , Astrócitos/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/metabolismo , Hipocampo/citologia , Humanos , Injeções Intraperitoneais , Masculino , Camundongos Endogâmicos C57BL , Neurônios/imunologia , Neurônios/metabolismo , Síndromes Neurotóxicas/imunologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Cultura Primária de Células , Sinucleinopatias/imunologia , Sinucleinopatias/metabolismo
10.
Nat Commun ; 11(1): 2820, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499486

RESUMO

As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn's aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.


Assuntos
Agregados Proteicos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Benzotiazóis/metabolismo , Cálcio/metabolismo , Humanos , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Fosforilação , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Relação Estrutura-Atividade
11.
Nat Commun ; 11(1): 2729, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483166

RESUMO

Aggregation and spreading of α-Synuclein (αSyn) are hallmarks of several neurodegenerative diseases, thus monitoring human αSyn (hαSyn) in animal models or cell cultures is vital for the field. However, the detection of native hαSyn in such systems is challenging. We show that the nanobody NbSyn87, previously-described to bind hαSyn, also shows cross-reactivity for the proteasomal subunit Rpn10. As such, when the NbSyn87 is expressed in the absence of hαSyn, it is continuously degraded by the proteasome, while it is stabilized when it binds to hαSyn. Here, we exploit this feature to design a new Fluorescent Reporter for hαSyn (FluoReSyn) by fusing NbSyn87 to fluorescent proteins, which results in fluorescence signal fluctuations depending on the presence and amounts of intracellular hαSyn. We characterize this biosensor in cells and tissues to finally reveal the presence of transmittable αSyn in human cerebrospinal fluid, demonstrating the potential of FluoReSyn for clinical research and diagnostics.


Assuntos
Citosol/metabolismo , Proteínas Luminescentes/metabolismo , Anticorpos de Domínio Único/metabolismo , alfa-Sinucleína/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Citosol/química , Feminino , Fluorescência , Células HEK293 , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Masculino , Microscopia de Fluorescência por Excitação Multifotônica , Pessoa de Meia-Idade , Neurônios/citologia , Neurônios/metabolismo , Ratos Wistar , Anticorpos de Domínio Único/genética , alfa-Sinucleína/líquido cefalorraquidiano , alfa-Sinucleína/genética
12.
Proc Natl Acad Sci U S A ; 117(25): 14178-14186, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513706

RESUMO

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).


Assuntos
Lipídeos de Membrana/metabolismo , Membranas/metabolismo , alfa-Sinucleína/metabolismo , Fluoresceínas , Humanos , Cinética , Bicamadas Lipídicas/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Fosfatidilgliceróis/química , Ligação Proteica , alfa-Sinucleína/genética
13.
PLoS One ; 15(6): e0235198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32598365

RESUMO

Parkinson´s disease is characterized by the accumulation of proteinaceous aggregates in Lewy bodies and Lewy Neurites. The main component found in such aggregates is α-synuclein. Here, we investigate how bovine eye lens crystallin proteins influence the aggregation kinetics of α-synuclein at mildly acidic pH (5.5) where the underlying aggregation mechanism of this protein is dominated by secondary nucleation of monomers on fibril surface providing an autocatalytic amyloid amplification process. Bovine α-, ßH- and γB-crystallins were found to display chaperone-like activity inhibiting α-synuclein aggregation. This effect was shown to be time-dependent, with early additions of α-crystallin capable of retarding and even inhibiting aggregation during the time frame of the experiment. The inhibitory nature of crystallins was further investigated using trap and seed kinetic experiments. We propose crystallins interact with mature α-synuclein fibrils, possibly binding along the surfaces and at fibril free ends, inhibiting both elongation and monomer-dependent secondary nucleation processes in a mechanism that may be generic to some chaperones that prevent the onset of protein misfolding related pathologies.


Assuntos
Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas , alfa-Cristalinas/metabolismo , alfa-Sinucleína/metabolismo , beta-Cristalinas/metabolismo , gama-Cristalinas/metabolismo , Amiloide/metabolismo , Animais , Bovinos , Clonagem Molecular , Escherichia coli/genética , Humanos , Cinética , Cristalino/metabolismo , Ligação Proteica
14.
Neurology ; 95(2): e155-e165, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32561678

RESUMO

OBJECTIVE: To determine whether Lewy body disease subgroups have different clinical profiles. METHODS: Participants had dementia, autopsy-confirmed transitional or diffuse Lewy body disease (TLBD or DLBD) (n = 244), or Alzheimer disease (AD) (n = 210), and were seen at least twice (mean follow-up 6.2 ± 3.8 years). TLBD and DLBD groups were partitioned based on the presence or absence of neocortical neurofibrillary tangles using Braak staging. Four Lewy body disease subgroups and AD were compared on clinical features, dementia trajectory, and onset latency of probable dementia with Lewy bodies (DLB) or a DLB syndrome defined as probable DLB or dementia with one core feature of parkinsonism or probable REM sleep behavior disorder. RESULTS: In TLBD and DLBD without neocortical tangles, diagnostic sensitivity was strong for probable DLB (87% TLBD, 96% DLBD) and the DLB syndrome (97% TLBD, 98% DLBD) with median latencies <1 year from cognitive onset, and worse baseline attention-visual processing but better memory-naming scores than AD. In DLBD with neocortical tangles, diagnostic sensitivity was 70% for probable DLB and 77% for the DLB syndrome with respective median latencies of 3.7 years and 2.7 years from cognitive onset, each associated with tangle distribution. This group had worse baseline attention-visual processing than AD, but comparable memory-naming impairment. TLBD with neocortical tangles had 48% diagnostic sensitivity for probable DLB and 52% for the DLB syndrome, with median latencies >6 years from cognitive onset, and were cognitively similar to AD. Dementia trajectory was slowest for TLBD without neocortical tangles, and fastest for DLBD with neocortical tangles. CONCLUSIONS: The phenotypic expression of DLB was associated with the distribution of α-synuclein and tau pathology.


Assuntos
Doença por Corpos de Lewy/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Atenção , Cognição , Progressão da Doença , Feminino , Humanos , Doença por Corpos de Lewy/classificação , Doença por Corpos de Lewy/psicologia , Masculino , Memória , Pessoa de Meia-Idade , Neocórtex/patologia , Emaranhados Neurofibrilares/patologia , Desempenho Psicomotor , Sensibilidade e Especificidade
15.
Proc Natl Acad Sci U S A ; 117(26): 15209-15220, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541058

RESUMO

Preclinical assessment of the therapeutic potential of dopamine (DA) neuron replacement in Parkinson's disease (PD) has primarily been performed in the 6-hydroxydopamine toxin model. While this is a good model to assess graft function, it does not reflect the pathological features or progressive nature of the disease. In this study, we establish a humanized transplantation model of PD that better recapitulates the main disease features, obtained by coinjection of preformed human α-synuclein (α-syn) fibrils and adeno-associated virus (AAV) expressing human wild-type α-syn unilaterally into the rat substantia nigra (SN). This model gives rise to DA neuron dysfunction and progressive loss of DA neurons from the SN and terminals in the striatum, accompanied by extensive α-syn pathology and a prominent inflammatory response, making it an interesting and relevant model in which to examine long-term function and integrity of transplanted neurons in a PD-like brain. We transplanted DA neurons derived from human embryonic stem cells (hESCs) into the striatum and assessed their survival, growth, and function over 6 to 18 wk. We show that the transplanted cells, even in the presence of ongoing pathology, are capable of innervating the DA-depleted striatum. However, on closer examination of the grafts, we found evidence of α-syn pathology in the form of inclusions of phosphorylated α-syn in a small fraction of the grafted DA neurons, indicating host-to-graft transfer of α-syn pathology, a phenomenon that has previously been observed in PD patients receiving fetal tissue grafts but has not been possible to demonstrate and study in toxin-based animal models.


Assuntos
Células-Tronco Embrionárias/fisiologia , Transplante de Células-Tronco , Sinucleinopatias , alfa-Sinucleína/metabolismo , Animais , Sobrevivência Celular , Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo , Feminino , Humanos , Inflamação , Degeneração Neural , Ratos , Ratos Sprague-Dawley , Substância Negra/citologia
16.
Proc Natl Acad Sci U S A ; 117(20): 10865-10875, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32366666

RESUMO

Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.


Assuntos
Endocitose/fisiologia , Miosinas/química , Miosinas/metabolismo , Polieletrólitos/metabolismo , alfa-Sinucleína/metabolismo , Linhagem Celular , Clatrina/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Modelos Moleculares , Doença de Parkinson/metabolismo , Conformação Proteica , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
17.
Adv Exp Med Biol ; 1233: 223-236, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274759

RESUMO

The proteostasis network controls the balance between protein synthesis, folding, function, and degradation, and ensures proteins are recycled when they are no longer needed or become damaged, avoiding unwanted aggregation and accumulation. In various neurological disorders, such as Parkinson's disease (PD) and other synucleinopathies, the accumulation of misfolded and aggregated alpha-synuclein (aSyn) is considered a central event in the onset and progression of disease. During aging, there is a decline in the activity of various degradation machineries, and the overall buffering capacity of the proteostasis network starts to decrease. Such decline is thought to play a pivotal role in PD, causing aSyn to build-up due to compromised clearance, which in turn contributes to further disease progression.In this chapter, we summarize central findings related to aSyn accumulation and degradation, as well as to the consequences of the toxic effects caused by aSyn on proteostasis. We also highlight some of the factors and pathways that may be used as potential targets for therapeutic interventions in PD.


Assuntos
Doença de Parkinson/metabolismo , Proteostase , Humanos , Doença de Parkinson/tratamento farmacológico , alfa-Sinucleína/metabolismo
18.
PLoS One ; 15(4): e0232019, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343709

RESUMO

Parkinson's disease (PD) is a common neurodegenerative disorder which is mostly sporadic but familial-linked PD (FPD) cases have also been found. The first reported gene mutation that linked to PD is α-synuclein (α-syn). Studies have shown that mutations, increased expression or abnormal processing of α-syn can contribute to PD, but it is believed that multiple mechanisms are involved. One of the contributing factors is post-translational modification (PTM), such as phosphorylation of α-syn at serine 129 by G-protein-coupled receptor kinases (GRKs) and casein kinase 2α (CK2α). Another known important contributing factor to PD pathogenesis is oxidative and nitrosative stress. In this study, we found that GRK6 and CK2α can be S-nitrosylated by nitric oxide (NO) both in vitro and in vivo. S-nitrosylation of GRK6 and CK2α enhanced their kinase activity towards the phosphorylation of α-syn at S129. In an A53T α-syn transgenic mouse model of PD, we found that increased GRK6 and CK2α S-nitrosylation were observed in an age dependent manner and it was associated with an increased level of pSer129 α-syn. Treatment of A53T α-syn transgenic mice with Nω-Nitro-L-arginine (L-NNA) significantly reduced the S-nitrosylation of GRK6 and CK2α in the brain. Finally, deletion of neuronal nitric oxide synthase (nNOS) in A53T α-syn transgenic mice reduced the levels of pSer129 α-syn and α-syn in an age dependent manner. Our results provide a novel mechanism of how NO through S-nitrosylation of GRK6 and CK2α can enhance the phosphorylation of pSer129 α-syn in an animal model of PD.


Assuntos
Caseína Quinase II/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Óxido Nítrico/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Fatores Etários , Animais , Caseína Quinase II/química , Modelos Animais de Doenças , Quinases de Receptores Acoplados a Proteína G/química , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Óxido Nítrico Sintase Tipo I/genética , Nitroarginina/administração & dosagem , Nitroarginina/farmacologia , Estresse Nitrosativo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Fosforilação , Serina/metabolismo , alfa-Sinucleína/química
19.
Artigo em Chinês | MEDLINE | ID: mdl-32306690

RESUMO

Objective: To investigate the effects of Paraquat on autophagy level in SH-SY5Y cell and the mechanism of abnormal aggregation of α-synuclein. Methods: Human neuroblastoma cell (SH-SY5Y cell) was used as model of dopaminergic neurons in vitro. The cells were treated with different concentrations of PQ (0, 18.75, 37.5, 75, 150, 300, 600 µmol/L) for 24 hours, and induced by 150 µmol/L PQ for 0, 12, 24, 36, 48, 60, 72, 96 hours to detect the relative survival rate of cells and determine dose/time-effect relationship. The cells were treated with concentration of 0, 75, 150, 300, 600 µmol/L PQ for 24 hours, and induced by 150 µmol/L PQ for different hours to detect intracellular LDH activity. The expression levels of autophagy-related proteins(LC3I, LC3II, Beclin1 , Vps34 and p62) and α-synuclein were detected by Western blot. The gene expression level of α-synuclein was assayed by Real-time quantitative PCR. The expression level of α-synuclein was also evaluated by immunofluorescence. The cells were pretreated with 100 nmol/L autophagy inducer rapamycin (RAPA) for 6 hours. The expression levels of autophagy-related proteins and α-synuclein were detected by Western blot. Results: CCK8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner; Compared with control group, the activity of LDH in the cell supernatant increased significantly after PQ exposure (P<0.05) ; Western blot analysis showed the ratio of autophagy-related protein LC3II/LC3I, Beclin1 and Vps34 protein expression were significantly lower after PQ treatment while the expression of p62 protein was higher (P<0.05) ; The protein and gene expression of α-synuclein were increased significantly after PQ treatment (P <0.05) ; Immunofluorescence showed the fluorescence intensity of α- synuclein in cells was significantly enhanced (P <0.05) . Compared with PQ group, the expression levels of autophagy-related proteins LC3II/LC3I and Beclin1 were significantly increased whlie α-synuclein protein level was decreased after RAPA induction (P<0.05) . Similarly, the IF result showed the fluorescence signal of α- synuclein significantly decreased after RAPA induction (P<0.05) . Conclusion: Paraquat induced autophagy dysfunction in SH-SY5Y cells, which leads to an abnormal aggregation of α-synuclein.


Assuntos
Autofagia , Neurônios Dopaminérgicos/efeitos dos fármacos , Paraquat/toxicidade , Agregação Patológica de Proteínas , alfa-Sinucleína/metabolismo , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/citologia , Humanos
20.
Nat Commun ; 11(1): 1943, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327648

RESUMO

Kidney fibrosis is a highly deleterious process and a final manifestation of chronic kidney disease. Alpha-(α)-synuclein (SNCA) is an actin-binding neuronal protein with various functions within the brain; however, its role in other tissues is unknown. Here, we describe the expression of SNCA in renal epithelial cells and demonstrate its decrease in renal tubules of murine and human fibrotic kidneys, as well as its downregulation in renal proximal tubular epithelial cells (RPTECs) after TGF-ß1 treatment. shRNA-mediated knockdown of SNCA in RPTECs results in de novo expression of vimentin and α-SMA, while SNCA overexpression represses TGF-ß1-induced mesenchymal markers. Conditional gene silencing of SNCA in RPTECs leads to an exacerbated tubulointerstitial fibrosis (TIF) in two unrelated in vivo fibrotic models, which is associated with an increased activation of MAPK-p38 and PI3K-Akt pathways. Our study provides an evidence that disruption of SNCA signaling in RPTECs contributes to the pathogenesis of renal TIF by facilitating partial epithelial-to-mesenchymal transition and extracellular matrix accumulation.


Assuntos
Nefropatias/patologia , Rim/patologia , alfa-Sinucleína/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/genética , Vimentina/metabolismo , alfa-Sinucleína/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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