RESUMO
Astrocyte reactivity is disease- and stimulus-dependent, adopting either a proinflammatory A1 phenotype or a protective, anti-inflammatory A2 phenotype. Recently, we demonstrated, using cell culture, animal models and human brain samples, that dopaminergic neurons produce and secrete higher levels of the chemokine-like signaling protein Prokineticin-2 (PK2) as a compensatory protective response against neurotoxic stress. As astrocytes express a high level of PK2 receptors, herein, we systematically characterize the role of PK2 in astrocyte structural and functional properties. PK2 treatment greatly induced astrocyte migration, which was accompanied by a shift in mitochondrial energy metabolism, a reduction in proinflammatory factors, and an increase in the antioxidant genes Arginase-1 and Nrf2. Overexpression of PK2 in primary astrocytes or in the in vivo mouse brain induced the A2 astrocytic phenotype with upregulation of key protective genes and A2 reactivity markers including Arginase-1 and Nrf2, PTX3, SPHK1, and TM4SF1. A small-molecule PK2 agonist, IS20, not only mimicked the protective effect of PK2 in primary cultures, but also increased glutamate uptake by upregulating GLAST. Notably, IS20 blocked not only MPTP-induced reductions in the A2 phenotypic markers SPHK1 and SCL10a6 but also elevation of the of A1 marker GBP2. Collectively, our results reveal that PK2 regulates a novel neuron-astrocyte signaling mechanism by promoting an alternative A2 protective phenotype in astrocytes, which could be exploited for development of novel therapeutic strategies for PD and other related chronic neurodegenerative diseases. PK2 signals through its receptors on astrocytes and promotes directed chemotaxis. PK2-induced astrocyte reactivity leads to an increase in antioxidant and anti-inflammatory proteins while increasing glutamate uptake, along with decreased inflammatory factors. © 2018 Wiley Periodicals, Inc.
Assuntos
Astrócitos/metabolismo , Quimiotaxia/fisiologia , Hormônios Gastrointestinais/metabolismo , Neuropeptídeos/metabolismo , Animais , Arginase/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Hormônios Gastrointestinais/administração & dosagem , Regulação da Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Neuropeptídeos/administração & dosagem , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/administração & dosagem , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
Sustained neuroinflammation mediated by resident microglia is recognized as a key pathophysiological contributor to many neurodegenerative diseases, including Parkinson's disease (PD), but the key molecular signaling events regulating persistent microglial activation have yet to be clearly defined. In the present study, we examined the role of Fyn, a non-receptor tyrosine kinase, in microglial activation and neuroinflammatory mechanisms in cell culture and animal models of PD. The well-characterized inflammogens LPS and TNFα rapidly activated Fyn kinase in microglia. Immunocytochemical studies revealed that activated Fyn preferentially localized to the microglial plasma membrane periphery and the nucleus. Furthermore, activated Fyn phosphorylated PKCδ at tyrosine residue 311, contributing to an inflammogen-induced increase in its kinase activity. Notably, the Fyn-PKCδ signaling axis further activated the LPS- and TNFα-induced MAP kinase phosphorylation and activation of the NFκB pathway, implying that Fyn is a major upstream regulator of proinflammatory signaling. Functional studies in microglia isolated from wild-type (Fyn(+/+)) and Fyn knock-out (Fyn(-/-)) mice revealed that Fyn is required for proinflammatory responses, including cytokine release as well as iNOS activation. Interestingly, a prolonged inflammatory insult induced Fyn transcript and protein expression, indicating that Fyn is upregulated during chronic inflammatory conditions. Importantly, in vivo studies using MPTP, LPS, or 6-OHDA models revealed a greater attenuation of neuroinflammatory responses in Fyn(-/-) and PKCδ (-/-) mice compared with wild-type mice. Collectively, our data demonstrate that Fyn is a major upstream signaling mediator of microglial neuroinflammatory processes in PD. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) is a complex multifactorial disease characterized by the progressive loss of midbrain dopamine neurons. Sustained microglia-mediated neuroinflammation has been recognized as a major pathophysiological contributor to chronic degenerative processes in PD; however, the key molecular signaling mechanisms underlying microglial activation are not entirely clear. Herein, we identified a novel role for the non-receptor tyrosine kinase Fyn in regulating neuroinflammatory responses in microglia. Our data clearly suggest that the Fyn-PKCδ signaling axis acts as a major upstream signaling mediator of the sustained neuroinflammatory processes in cell culture and animal models of PD. Our finding has important clinical significance to PD because it identifies Fyn as a potential translational target for intervention of progressive neurodegenerative processes in PD.
Assuntos
Encefalite/etiologia , Microglia/metabolismo , Doença de Parkinson/complicações , Proteína Quinase C-delta/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Animais , Fracionamento Celular/métodos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/ultraestrutura , Óxido Nítrico/metabolismo , Oxidopamina/toxicidade , Doença de Parkinson/etiologia , Fosforilação , Proteína Quinase C-delta/genética , Proteínas Proto-Oncogênicas c-fyn/genética , Fator de Necrose Tumoral alfa/farmacologia , Tirosina , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Chronic microglial activation has been linked to the progressive degeneration of the nigrostriatal dopaminergic neurons evidenced in Parkinson's disease (PD) pathogenesis. The exact etiology of PD remains poorly understood. Although both oxidative stress and neuroinflammation are identified as co-contributors in PD pathogenesis, signaling mechanisms underlying neurodegenerative processes have yet to be defined. Indeed, we recently identified that protein kinase C delta (PKCδ) activation is critical for induction of dopaminergic neuronal loss in response to neurotoxic stressors. However, it remains to be defined whether PKCδ activation contributes to immune signaling events driving microglial neurotoxicity. In the present study, we systematically investigated whether PKCδ contributes to the heightened microglial activation response following exposure to major proinflammatory stressors, including α-synuclein, tumor necrosis factor α (TNFα), and lipopolysaccharide (LPS). We report that exposure to the aforementioned inflammatory stressors dramatically upregulated PKCδ with a concomitant increase in its kinase activity and nuclear translocation in both BV-2 microglial cells and primary microglia. Importantly, we also observed a marked upregulation of PKCδ in the microglia of the ventral midbrain region of PD patients when compared to age-matched controls, suggesting a role for microglial PKCδ in neurodegenerative processes. Further, shRNA-mediated knockdown and genetic ablation of PKCδ in primary microglia blunted the microglial proinflammatory response elicited by the inflammogens, including ROS generation, nitric oxide production, and proinflammatory cytokine and chemokine release. Importantly, we found that PKCδ activated NFκB, a key mediator of inflammatory signaling events, after challenge with inflammatory stressors, and that transactivation of NFκB led to translocation of the p65 subunit to the nucleus, IκBα degradation and phosphorylation of p65 at Ser536. Furthermore, both genetic ablation and siRNA-mediated knockdown of PKCδ attenuated NFκB activation, suggesting that PKCδ regulates NFκB activation subsequent to microglial exposure to inflammatory stimuli. To further investigate the pivotal role of PKCδ in microglial activation in vivo, we utilized pre-clinical models of PD. We found that PKCδ deficiency attenuated the proinflammatory response in the mouse substantia nigra, reduced locomotor deficits and recovered mice from sickness behavior in an LPS-induced neuroinflammation model of PD. Likewise, we found that PKCδ knockout mice treated with MPTP displayed a dampened microglial inflammatory response. Moreover, PKCδ knockout mice exhibited reduced susceptibility to the neurotoxin-induced dopaminergic neurodegeneration and associated motor impairments. Taken together, our studies propose a pivotal role for PKCδ in PD pathology, whereby sustained PKCδ activation drives sustained microglial inflammatory responses and concomitant dopaminergic neurotoxicity consequently leading to neurobehavioral deficits. We conclude that inhibiting PKCδ activation may represent a novel therapeutic strategy in PD treatment.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Microglia/metabolismo , Doença de Parkinson/metabolismo , Proteína Quinase C-delta/metabolismo , Animais , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Mesencéfalo/citologia , Camundongos Knockout , Microglia/efeitos dos fármacos , Modelos Teóricos , Degeneração Neural/patologia , Doença de Parkinson/patologia , Transdução de Sinais/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The oxidative stress-sensitive protein kinase Cδ (PKCδ) has been implicated in dopaminergic neuronal cell death. However, little is known about the epigenetic mechanisms regulating PKCδ expression in neurons. Here, we report a novel mechanism by which the PKCδ gene can be regulated by histone acetylation. Treatment with histone deacetylase (HDAC) inhibitor sodium butyrate (NaBu) induced PKCδ expression in cultured neurons, brain slices, and animal models. Several other HDAC inhibitors also mimicked NaBu. The chromatin immunoprecipitation analysis revealed that hyperacetylation of histone H4 by NaBu is associated with the PKCδ promoter. Deletion analysis of the PKCδ promoter mapped the NaBu-responsive element to an 81-bp minimal promoter region. Detailed mutagenesis studies within this region revealed that four GC boxes conferred hyperacetylation-induced PKCδ promoter activation. Cotransfection experiments and Sp inhibitor studies demonstrated that Sp1, Sp3, and Sp4 regulated NaBu-induced PKCδ up-regulation. However, NaBu did not alter the DNA binding activities of Sp proteins or their expression. Interestingly, a one-hybrid analysis revealed that NaBu enhanced transcriptional activity of Sp1/Sp3. Overexpression of the p300/cAMP-response element-binding protein-binding protein (CBP) potentiated the NaBu-mediated transactivation potential of Sp1/Sp3, but expressing several HDACs attenuated this effect, suggesting that p300/CBP and HDACs act as coactivators or corepressors in histone acetylation-induced PKCδ up-regulation. Finally, using genetic and pharmacological approaches, we showed that NaBu up-regulation of PKCδ sensitizes neurons to cell death in a human dopaminergic cell model and brain slice cultures. Together, these results indicate that histone acetylation regulates PKCδ expression to augment nigrostriatal dopaminergic cell death, which could contribute to the progressive neuropathogenesis of Parkinson disease.
Assuntos
Neurônios Dopaminérgicos/patologia , Epigênese Genética , Histonas/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteína Quinase C-delta/metabolismo , Regulação para Cima , Acetilação/efeitos dos fármacos , Animais , Encéfalo/patologia , Ácido Butírico/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Epigênese Genética/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteína Quinase C-delta/genética , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição Sp/química , Fatores de Transcrição Sp/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
We recently identified a compensatory survival role for protein kinase D1 (PKD1) in protecting dopaminergic neurons from oxidative insult. To investigate the molecular mechanism of Prkd1 gene expression, we cloned the 5'-flanking region (1620-bp) of the mouse Prkd1 gene. Deletion analyses revealed that the -250/+113 promoter region contains full promoter activity in MN9D dopaminergic neuronal cells. In silico analysis of the Prkd1 promoter uncovered binding sites for key redox transcription factors including Sp1 and NF-κB. Over-expression of Sp1, Sp3, and NF-κB-p65 proteins stimulated Prkd1 promoter activity. Binding of Sp3 and NF-κB-p65 to the Prkd1 promoter was confirmed using chromatin immunoprecipitation. Treatment with the Sp inhibitor mithramycin A significantly attenuated Prkd1 promoter activity and PKD1 mRNA and protein expression. Further mechanistic studies revealed that inhibition of histone deacetylation and DNA methylation up-regulated PKD1 mRNA expression. Importantly, negative modulation of PKD1 signaling by pharmacological inhibition or shRNA knockdown increased dopaminergic neuronal sensitivity to oxidative damage in a human mesencephalic neuronal cell model. Collectively, our findings demonstrate that Sp1, Sp3, and NF-κB-p65 can transactivate the mouse Prkd1 promoter and that epigenetic mechanisms, such as DNA methylation and histone modification, are key regulatory events controlling the expression of pro-survival kinase PKD1 in dopaminergic neuronal cells. Previously, we demonstrated that protein kinase D1 (PKD1) plays a survival role during the early stage of oxidative stress in dopaminergic neuronal cells.
Assuntos
Neurônios Dopaminérgicos/patologia , Epigênese Genética/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteína Quinase C/genética , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Células Cultivadas , Imunoprecipitação da Cromatina , Clonagem Molecular , Decitabina , Deleção de Genes , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Dados de Sequência Molecular , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117-4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP(+)) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP(+)-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP(+)-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.
Assuntos
Apoptose/fisiologia , Neurônios Dopaminérgicos/metabolismo , Peptidilprolil Isomerase/metabolismo , Regulação para Cima , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , 1-Metil-4-fenilpiridínio/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Peptidilprolil Isomerase de Interação com NIMA , Naftoquinonas/farmacologia , Neurotoxinas/farmacologia , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/genética , Doença de Parkinson Secundária/metabolismo , Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/genética , Interferência de RNA , DNA Polimerase Dirigida por RNA , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismoRESUMO
Aggregation of tau into filamentous inclusions underlies Alzheimer's disease (AD) and numerous other neurodegenerative tauopathies. The pathogenesis of tauopathies remains unclear, which impedes the development of disease-modifying treatments. Here, by systematically analyzing human tripartite motif (TRIM) proteins, we identified a few TRIMs that could potently inhibit tau aggregation. Among them, TRIM11 was markedly down-regulated in AD brains. TRIM11 promoted the proteasomal degradation of mutant tau as well as superfluous normal tau. It also enhanced tau solubility by acting as both a molecular chaperone to prevent tau misfolding and a disaggregase to dissolve preformed tau fibrils. TRIM11 maintained the connectivity and viability of neurons. Intracranial delivery of TRIM11 through adeno-associated viruses ameliorated pathology, neuroinflammation, and cognitive impairments in multiple animal models of tauopathies. These results suggest that TRIM11 down-regulation contributes to the pathogenesis of tauopathies and that restoring TRIM11 expression may represent an effective therapeutic strategy.
Assuntos
Agregação Patológica de Proteínas , Tauopatias , Animais , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatias/genética , Tauopatias/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Chronic environmental exposure to manganese (Mn) can cause debilitating damage to the central nervous system. However, its potential toxic effects on the enteric nervous system (ENS) have yet to be assessed. OBJECTIVE: We examined the effect of Mn on the ENS using both cell and animal models. METHOD: Rat enteric glial cells (EGCs) and mouse primary enteric cultures were exposed to increasing concentrations of Mn and cell viability and mitochondrial health were assessed using various morphological and functional assays. C57BL/6 mice were exposed daily to a sublethal dose of Mn (15mg/kg/d) for 30 d. Gut peristalsis, enteric inflammation, gut microbiome profile, and fecal metabolite composition were assessed at the end of exposure. RESULTS: EGC mitochondria were highly susceptible to Mn neurotoxicity, as evidenced by lower mitochondrial mass, adenosine triphosphate-linked respiration, and aconitase activity as well as higher mitochondrial superoxide, upon Mn exposure. Minor differences were seen in the mouse model: specifically, longer intestinal transit times and higher levels of colonic inflammation. CONCLUSION: Based on our findings from this study, Mn preferentially induced mitochondrial dysfunction in a rat EGC line and in vivo resulted in inflammation in the ENS. https://doi.org/10.1289/EHP7877.
Assuntos
Sistema Nervoso Entérico , Microbioma Gastrointestinal , Animais , Manganês/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , RatosRESUMO
Human-derived neuronal cell lines are progressively being utilized in understanding neurobiology and preclinical translational research as they are biologically more relevant than rodent-derived cells lines. The Lund human mesencephalic (LUHMES) cell line comprises human neuronal cells that can be differentiated to post-mitotic neurons and is increasingly being used as an in vitro model for various neurodegenerative diseases. A previously published 2-step differentiation procedure leads to the generation of post-mitotic neurons within 5-days, but only a small proportion (10%) of the total cell population tests positive for tyrosine hydroxylase (TH). Here we report on a novel differentiation protocol that we optimized by using a cocktail of neurotrophic factors, pleiotropic cytokines, and antioxidants to effectively generate proportionately more dopaminergic neurons within the same time period. Visualization and quantification of TH-positive cells revealed that under our new protocol, 25% of the total cell population expressed markers of dopaminergic neurons with the TH-positive neuron count peaking on day 5. These neurons showed spontaneous electrical activity and responded to known Parkinsonian toxins as expected by showing decreased cell viability and dopamine uptake and a concomitant increase in apoptotic cell death. Together, our results outline an improved method for generating a higher proportion of dopaminergic neurons, thus making these cells an ideal neuronal culture model of Parkinson's disease (PD) for translational research.
Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/metabolismo , Modelos Neurológicos , Células-Tronco Neurais/metabolismo , Doença de Parkinson/metabolismo , Pesquisa Translacional Biomédica , Antígenos de Diferenciação/biossíntese , Linhagem Celular , Neurônios Dopaminérgicos/patologia , Humanos , Células-Tronco Neurais/patologia , Doença de Parkinson/patologia , Doença de Parkinson/terapiaRESUMO
Neurodegenerative diseases are characterized by the formation and propagation of protein aggregates, especially amyloid fibrils. However, what normally suppresses protein misfolding and aggregation in metazoan cells remains incompletely understood. Here, we show that TRIM11, a member of the metazoan tripartite motif (TRIM) family, both prevents the formation of protein aggregates and dissolves pre-existing protein deposits, including amyloid fibrils. These molecular chaperone and disaggregase activities are ATP independent. They enhance folding and solubility of normal proteins and cooperate with TRIM11 SUMO ligase activity to degrade aberrant proteins. TRIM11 abrogates α-synuclein fibrillization and restores viability in cell models of Parkinson's disease (PD). Intracranial adeno-associated viral delivery of TRIM11 mitigates α-synuclein-mediated pathology, neurodegeneration, and motor impairments in a PD mouse model. Other TRIMs can also function as ATP-independent molecular chaperones and disaggregases. Thus, we define TRIMs as a potent and multifunctional protein quality-control system in metazoa, which might be applied to treat neurodegenerative diseases.
Assuntos
Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Doença de Parkinson/patologia , Agregados ProteicosRESUMO
Occupational or environmental exposure to manganese (Mn) can lead to the development of "Manganism," a neurological condition showing certain motor symptoms similar to Parkinson's disease (PD). Like PD, Mn toxicity is seen in the central nervous system mainly affecting nigrostriatal neuronal circuitry and subsequent behavioral and motor impairments. Since the first report of Mn-induced toxicity in 1837, various experimental and epidemiological studies have been conducted to understand this disorder. While early investigations focused on the impact of high concentrations of Mn on the mitochondria and subsequent oxidative stress, current studies have attempted to elucidate the cellular and molecular pathways involved in Mn toxicity. In fact, recent reports suggest the involvement of Mn in the misfolding of proteins such as α-synuclein and amyloid, thus providing credence to the theory that environmental exposure to toxicants can either initiate or propagate neurodegenerative processes by interfering with disease-specific proteins. Besides manganism and PD, Mn has also been implicated in other neurological diseases such as Huntington's and prion diseases. While many reviews have focused on Mn homeostasis, the aim of this review is to concisely synthesize what we know about its effect primarily on the nervous system with respect to its role in protein misfolding, mitochondrial dysfunction, and consequently, neuroinflammation and neurodegeneration. Based on the current evidence, we propose a 'Mn Mechanistic Neurotoxic Triad' comprising (1) mitochondrial dysfunction and oxidative stress, (2) protein trafficking and misfolding, and (3) neuroinflammation.
RESUMO
The aggregation of α-synuclein (αSyn) is considered a key pathophysiological feature of certain neurodegenerative disorders, collectively termed synucleinopathies. Given that a prion-like, cell-to-cell transfer of misfolded αSyn has been recognized in the spreading of αSyn pathology in synucleinopathies, we investigated the biological mechanisms underlying the propagation of the disease with respect to environmental neurotoxic stress. Considering the potential role of the divalent metal manganese (Mn2+) in protein aggregation, we characterized its effect on αSyn misfolding and transmission in experimental models of Parkinson's disease. In cultured dopaminergic neuronal cells stably expressing wild-type human αSyn, misfolded αSyn was secreted through exosomes into the extracellular medium upon Mn2+ exposure. These exosomes were endocytosed through caveolae into primary microglial cells, thereby mounting neuroinflammatory responses. Furthermore, Mn2+-elicited exosomes exerted a neurotoxic effect in a human dopaminergic neuronal model (LUHMES cells). Moreover, bimolecular fluorescence complementation (BiFC) analysis revealed that Mn2+ accelerated the cell-to-cell transmission of αSyn, resulting in dopaminergic neurotoxicity in a mouse model of Mn2+ exposure. Welders exposed to Mn2+ had increased misfolded αSyn content in their serum exosomes. Stereotaxically delivering αSyn-containing exosomes, isolated from Mn2+-treated αSyn-expressing cells, into the striatum initiated Parkinsonian-like pathological features in mice. Together, these results indicate that Mn2+ exposure promotes αSyn secretion in exosomal vesicles, which subsequently evokes proinflammatory and neurodegenerative responses in both cell culture and animal models.
Assuntos
Corpo Estriado/metabolismo , Neurônios Dopaminérgicos/metabolismo , Exossomos/metabolismo , Manganês/toxicidade , Doença de Parkinson Secundária/metabolismo , Agregação Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Exossomos/patologia , Manganês/farmacologia , Camundongos , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/patologia , Príons/metabolismo , Agregação Patológica de Proteínas/induzido quimicamente , Agregação Patológica de Proteínas/patologiaRESUMO
Chronic, sustained inflammation underlies many pathological conditions, including neurodegenerative diseases. Divalent manganese (Mn2+) exposure can stimulate neurotoxicity by increasing inflammation. In this study, we examined whether Mn2+ activates the multiprotein NLRP3 inflammasome complex to promote neuroinflammation. Exposing activated mouse microglial cells to Mn2+ substantially augmented NLRP3 abundance, caspase-1 cleavage, and maturation of the inflammatory cytokine interleukin-1ß (IL-1ß). Exposure of mice to Mn2+ had similar effects in brain microglial cells. Furthermore, Mn2+ impaired mitochondrial ATP generation, basal respiratory rate, and spare capacity in microglial cells. These data suggest that Mn-induced mitochondrial defects drove the inflammasome signal amplification. We found that Mn induced cell-to-cell transfer of the inflammasome adaptor protein ASC in exosomes. Furthermore, primed microglial cells exposed to exosomes from Mn-treated mice released more IL-1ß than did cells exposed to exosomes from control-treated animals. We also observed that welders exposed to manganese-containing fumes had plasma exosomes that contained more ASC than did those from a matched control group. Together, these results suggest that the divalent metal manganese acts as a key amplifier of NLRP3 inflammasome signaling and exosomal ASC release.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Exossomos/metabolismo , Inflamassomos/metabolismo , Manganês/farmacologia , Microglia/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Caspase 1/metabolismo , Células Cultivadas , Interleucina-1beta/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microglia/metabolismo , SoldagemRESUMO
Persistent microglia-mediated neuroinflammation is a major pathophysiological contributor to the progression of Parkinson's disease (PD), but the cell-signaling mechanisms governing chronic neuroinflammation are not well understood. Here, we show that Fyn kinase, in conjunction with the class B scavenger receptor CD36, regulates the microglial uptake of aggregated human α-synuclein (αSyn), which is the major component of PD-associated Lewy bodies. αSyn can effectively mediate LPS-independent priming and activation of the microglial NLRP3 inflammasome. Fyn kinase regulates both of these processes; it mediates PKCδ-dependent NF-κB-p65 nuclear translocation, leading to inflammasome priming, and facilitates αSyn import into microglia, contributing to the generation of mitochondrial reactive oxygen species and consequently to inflammasome activation. In vivo experiments using A53T and viral-αSyn overexpression mouse models as well as human PD neuropathological results further confirm the role of Fyn in NLRP3 inflammasome activation. Collectively, our study identifies a novel Fyn-mediated signaling mechanism that amplifies neuroinflammation in PD.
Assuntos
Inflamassomos/metabolismo , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-fyn/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animais , Antígenos CD36/metabolismo , Dependovirus/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Gliose/metabolismo , Gliose/patologia , Humanos , Interleucina-1beta/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregados Proteicos , Proteína Quinase C-delta/metabolismo , Proteínas Proto-Oncogênicas c-fyn/deficiência , Espécies Reativas de Oxigênio/metabolismoRESUMO
Many chronic neurodegenerative disorders share a common pathogenic mechanism involving the aggregation and deposition of misfolded proteins. Recently, it was shown that these aggregated proteins could be transferred from one cell to another via extracellular nanovesicles called exosomes. Initially thought to be a means of cellular waste removal, exosomes have since been discovered to actively participate in cell-to-cell communication. Importantly, various inflammatory and signaling molecules, as well as small RNAs are selectively packaged in these vesicles. Considering the important role of environmental manganese (Mn) in Parkinson's disease (PD)-like neurological disorders, we characterized the effect of Mn on exosome content and release using an MN9D dopaminergic cell model of PD, which was generated to stably express wild-type human α-synuclein (αSyn). Mn exposure (300µM MnCl2) for 24h induced the release of exosomes into the extracellular media prior to cytotoxicity, as determined by NanoSight particle analysis and electron microscopy. Strikingly, Western blot analysis revealed that Mn treatment in αSyn-expressing cells increases the protein Rab27a, which regulates the release of exosomes from cells. Moreover, next-generation sequencing showed more small RNAs in exosomes isolated from Mn-exposed cells than from control exosomes. Our miRNA profiling analysis led to the discovery of increased expression of certain miRNAs previously shown to regulate key biological pathways, including protein aggregation, autophagy, inflammation and hypoxia. Collectively, our results provide a glimpse of Mn's role in modulating extracellular miRNA content through exosomal release from dopaminergic neuronal cells and thus potentially contributing to progressive neurodegeneration. Further characterization of extracellular miRNAs and their targets will have major impacts on biomarker discovery and translational strategies for environmentally linked neurodegenerative diseases including PD.
Assuntos
Exossomos/metabolismo , Manganês/toxicidade , MicroRNAs/metabolismo , Doença de Parkinson Secundária/metabolismo , alfa-Sinucleína/metabolismo , Células Cultivadas , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Humanos , Deficiências na Proteostase/induzido quimicamenteRESUMO
Chronic manganese (Mn) exposure induces neurotoxicity, which is characterized by Parkinsonian symptoms resulting from impairment in the extrapyramidal motor system of the basal ganglia. Mitochondrial dysfunction and oxidative stress are considered key pathophysiological features of Mn neurotoxicity. Recent evidence suggests astrocytes as a major target of Mn neurotoxicity since Mn accumulates predominantly in astrocytes. However, the primary mechanisms underlying Mn-induced astroglial dysfunction and its role in metal neurotoxicity are not completely understood. In this study, we examined the interrelationship between mitochondrial dysfunction and astrocytic inflammation in Mn neurotoxicity. We first evaluated whether Mn exposure alters mitochondrial bioenergetics in cultured astrocytes. Metabolic activity assessed by MTS assay revealed an IC50 of 92.68µM Mn at 24h in primary mouse astrocytes (PMAs) and 50.46µM in the human astrocytic U373 cell line. Mn treatment reduced mitochondrial mass, indicative of impaired mitochondrial function and biogenesis, which was substantiated by the significant reduction in mRNA of mitofusin-2, a protein that serves as a ubiquitination target for mitophagy. Furthermore, Mn increased mitochondrial circularity indicating augmented mitochondrial fission. Seahorse analysis of bioenergetics status in Mn-treated astrocytes revealed that Mn significantly impaired the basal mitochondrial oxygen consumption rate as well as the ATP-linked respiration rate. The effect of Mn on mitochondrial energy deficits was further supported by a reduction in ATP production. Mn-exposed primary astrocytes also exhibited a severely quiescent energy phenotype, which was substantiated by the inability of oligomycin to increase the extracellular acidification rate. Since astrocytes regulate immune functions in the CNS, we also evaluated whether Mn modulates astrocytic inflammation. Mn exposure in astrocytes not only stimulated the release of proinflammatory cytokines, but also exacerbated the inflammatory response induced by aggregated α-synuclein. The novel mitochondria-targeted antioxidant, mito-apocynin, significantly attenuated Mn-induced inflammatory gene expression, further supporting the role of mitochondria dysfunction and oxidative stress in mediating astrogliosis. Lastly, intranasal delivery of Mn in vivo elevated GFAP and depressed TH levels in the olfactory bulbs, clearly supporting the involvement of astrocytes in Mn-induced dopaminergic neurotoxicity. Collectively, our study demonstrates that Mn drives proinflammatory events in astrocytes by impairing mitochondrial bioenergetics.
Assuntos
Astrócitos/efeitos dos fármacos , Encefalite/induzido quimicamente , Manganês/toxicidade , Mitocôndrias/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Encefalite/metabolismo , Concentração Inibidora 50 , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismoRESUMO
Chronic exposure to environmental toxins has been known to initiate or aggravate various neurological disorders, carcinomas and other adverse health effects. Uptake by naïve cells of pathogenic factors such as danger-associated molecules, mRNAs, miRNAs or aggregated proteins leads to disruption in cellular homeostasis further resulting in inflammation and disease propagation. Although early research tended to focus solely on exosomal removal of unwanted cellular contents, more recent reports indicate that these nano-vesicles play an active role in intercellular signaling. Not only is the exosomal cargo cell type-specific, but it also differs between healthy and dying cells. Moreover, following exosome uptake by naïve cells, the contents from these vesicles can alter the fate of recipient cells. Since exosomes can traverse long distances, they can influence distantly located cells and tissues. This review briefly explores the role played by environmental toxins in stimulating exosome release in the context of progressive neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's, as well as certain cancers such as lung, liver, ovarian, and tracheal carcinomas.
Assuntos
Exposição Ambiental , Poluentes Ambientais/toxicidade , Exossomos/efeitos dos fármacos , Doenças Neurodegenerativas/induzido quimicamente , Comunicação Celular , Humanos , Doenças Neurodegenerativas/patologiaRESUMO
Protein misfolding and aggregation are key pathological features of many neurodegenerative diseases including Parkinson's disease (PD) and other forms of human Parkinsonism. PD is a complex and multifaceted disorder whose etiology is not fully understood. However, several lines of evidence support the multiple hit hypothesis that genetic vulnerability and environmental toxicants converge to trigger PD pathology. Alpha-synuclein (α-Syn) aggregation in the brain is an important pathophysiological characteristic of synucleinopathies including PD. Epidemiological and experimental studies have shown that metals and pesticides play a crucial role in α-Syn aggregation leading to the onset of various neurodegenerative diseases including PD. In this review, we will emphasize key findings of several epidemiological as well as experimental studies of metal- and pesticide-induced α-Syn aggregation and neurodegeneration. We will also discuss other factors such as traumatic brain injury and oxidative insult in the context of α-Syn-related neurodegenerative processes.
Assuntos
Agregados Proteicos/fisiologia , alfa-Sinucleína/genética , alfa-Sinucleína/fisiologia , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , Doença de Parkinson/metabolismo , Praguicidas/efeitos adversos , Deficiências na Proteostase/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Prion protein (PrP) misfolding and oligomerization are key pathogenic events in prion disease. Copper exposure has been linked to prion pathogenesis; however, its mechanistic basis is unknown. We resolve, with single-molecule precision, the molecular mechanism of Cu(2+)-induced misfolding of PrP under physiological conditions. We also demonstrate that misfolded PrPs serve as seeds for templated formation of aggregates, which mediate inflammation and degeneration of neuronal tissue. Using a single-molecule fluorescence assay, we demonstrate that Cu(2+) induces PrP monomers to misfold before oligomer assembly; the disordered amino-terminal region mediates this structural change. Single-molecule force spectroscopy measurements show that the misfolded monomers have a 900-fold higher binding affinity compared to the native isoform, which promotes their oligomerization. Real-time quaking-induced conversion demonstrates that misfolded PrPs serve as seeds that template amyloid formation. Finally, organotypic slice cultures show that misfolded PrPs mediate inflammation and degeneration of neuronal tissue. Our study establishes a direct link, at the molecular level, between copper exposure and PrP neurotoxicity.
Assuntos
Cobre/química , Proteínas Priônicas/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Proteínas Priônicas/química , Proteínas Priônicas/genética , Proteínas Priônicas/toxicidade , Agregados Proteicos/fisiologia , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteína Quinase C-delta/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidadeRESUMO
Mitochondrial dysfunction, oxidative stress and neuroinflammation have been implicated as key mediators contributing to the progressive degeneration of dopaminergic neurons in Parkinson's disease (PD). Currently, we lack a pharmacological agent that can intervene in all key pathological mechanisms, which would offer better neuroprotective efficacy than a compound that targets a single degenerative mechanism. Herein, we investigated whether mito-apocynin (Mito-Apo), a newly-synthesized and orally available derivative of apocynin that targets mitochondria, protects against oxidative damage, glial-mediated inflammation and nigrostriatal neurodegeneration in cellular and animal models of PD. Mito-Apo treatment in primary mesencephalic cultures significantly attenuated the 1-methyl-4-phenylpyridinium (MPP(+))-induced loss of tyrosine hydroxylase (TH)-positive neuronal cells and neurites. Mito-Apo also diminished MPP(+)-induced increases in glial cell activation and inducible nitric oxide synthase (iNOS) expression. Additionally, Mito-Apo decreased nitrotyrosine (3-NT) and 4-hydroxynonenol (4-HNE) levels in primary mesencephalic cultures. Importantly, we assessed the neuroprotective property of Mito-Apo in the MPTP mouse model of PD, wherein it restored the behavioral performance of MPTP-treated mice. Immunohistological analysis of nigral dopaminergic neurons and monoamine measurement further confirmed the neuroprotective effect of Mito-Apo against MPTP-induced nigrostriatal dopaminergic neuronal loss. Mito-Apo showed excellent brain bioavailability and also markedly attenuated MPTP-induced oxidative markers in the substantia nigra (SN). Furthermore, oral administration of Mito-Apo significantly suppressed MPTP-induced glial cell activation, upregulation of proinflammatory cytokines, iNOS and gp91phox in IBA1-positive cells of SN. Collectively, these results demonstrate that the novel mitochondria-targeted compound Mito-Apo exhibits profound neuroprotective effects in cellular and pre-clinical animal models of PD by attenuating oxidative damage and neuroinflammatory processes.