RESUMO
α-Synuclein is a key pathogenic protein that aggregates in hallmark lesions in Parkinson's disease and other α-synucleinopathies. Prior in vitro studies demonstrated that it is a substrate for cross-linking by transglutaminase 2 (TG2) into higher-order species. Here we investigated whether this increased aggregation occurs in vivo and whether TG2 exacerbates α-synuclein toxicity in Mus musculus and Saccharomyces cerevisiae. Compared with α-synuclein transgenic (Syn(Tg)) mice, animals double transgenic for human α-synuclein and TG2 (TG2(Tg)/Syn(Tg)) manifested greater high-molecular-weight insoluble species of α-synuclein in brain lysates and developed α-synuclein aggregates in the synaptic vesicle fraction. In addition, larger proteinase K-resistant aggregates developed, along with increased thioflavin-S-positive amyloid fibrils. This correlated with an exaggerated neuroinflammatory response, as seen with more astrocytes and microglia. Further neuronal damage was suggested by greater morphological disruption of nerve fibers and a trend toward decreased c-Fos immunoreactive neurons. Finally, the performance of TG2(Tg)/Syn(Tg) animals on motor behavioral tasks was worse relative to Syn(Tg) mice. Greater toxicity of α-synuclein was also demonstrated in yeast cells coexpressing TG2. Our findings demonstrate that TG2 promotes the aggregation of α-synuclein in vivo and that this is associated with aggravated toxicity of α-synuclein and its downstream neuropathologic consequences.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Agregação Patológica de Proteínas , Saccharomyces cerevisiae/metabolismo , Transglutaminases/metabolismo , alfa-Sinucleína/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/fisiologia , Humanos , Locomoção , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Agregados Proteicos , Proteína 2 Glutamina gama-Glutamiltransferase , Vesículas Sinápticas/metabolismoRESUMO
α-Synuclein (α-Syn) is a key protein that accumulates as hyperphosphorylated aggregates in pathologic hallmark features of Parkinson's disease (PD) and other neurodegenerative disorders. Phosphorylation of this protein at serine 129 is believed to promote its aggregation and neurotoxicity, suggesting that this post-translational modification could be a therapeutic target. Here, we demonstrate that phosphoprotein phosphatase 2A (PP2A) dephosphorylates α-Syn at serine 129 and that this activity is greatly enhanced by carboxyl methylation of the catalytic C subunit of PP2A. α-Syn-transgenic mice raised on a diet supplemented with eicosanoyl-5-hydroxytryptamide, an agent that enhances PP2A methylation, dramatically reduced both α-Syn phosphorylation at Serine 129 and α-Syn aggregation in the brain. These biochemical changes were associated with enhanced neuronal activity, increased dendritic arborizations, and reduced astroglial and microglial activation, as well as improved motor performance. These findings support the notion that serine 129 phosphorylation of α-Syn is of pathogenetic significance and that promoting PP2A activity is a viable disease-modifying therapeutic strategy for α-synucleinopathies such as PD.
Assuntos
Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Serotonina/análogos & derivados , alfa-Sinucleína/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Células Cultivadas , Dendritos/genética , Dendritos/metabolismo , Dendritos/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Metilação , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Fosforilação/fisiologia , Serotonina/metabolismo , alfa-Sinucleína/genéticaRESUMO
Consumption of coffee is associated with reduced risk of Parkinson's disease (PD), an effect that has largely been attributed to caffeine. However, coffee contains numerous components that may also be neuroprotective. One of these compounds is eicosanoyl-5-hydroxytryptamide (EHT), which ameliorates the phenotype of α-synuclein transgenic mice associated with decreased protein aggregation and phosphorylation, improved neuronal integrity and reduced neuroinflammation. Here, we sought to investigate if EHT has an effect in the MPTP model of PD. Mice fed a diet containing EHT for four weeks exhibited dose-dependent preservation of nigral dopaminergic neurons following MPTP challenge compared to animals given control feed. Reductions in striatal dopamine and tyrosine hydroxylase content were also less pronounced with EHT treatment. The neuroinflammatory response to MPTP was markedly attenuated, and indices of oxidative stress and JNK activation were significantly prevented with EHT. In cultured primary microglia and astrocytes, EHT had a direct anti-inflammatory effect demonstrated by repression of lipopolysaccharide-induced NFκB activation, iNOS induction, and nitric oxide production. EHT also exhibited a robust anti-oxidant activity in vitro. Additionally, in SH-SY5Y cells, MPP(+)-induced demethylation of phosphoprotein phosphatase 2A (PP2A), the master regulator of the cellular phosphoregulatory network, and cytotoxicity were ameliorated by EHT. These findings indicate that the neuroprotective effect of EHT against MPTP is through several mechanisms including its anti-inflammatory and antioxidant activities as well as its ability to modulate the methylation and hence activity of PP2A. Our data, therefore, reveal a strong beneficial effect of a novel component of coffee in multiple endpoints relevant to PD.
Assuntos
Anti-Inflamatórios/farmacologia , Café/química , Intoxicação por MPTP/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Intoxicação por MPTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Neurodegenerative disorders are characterized by progressive neuronal loss and the aggregation of disease-specific pathogenic proteins in hallmark neuropathologic lesions. Many of these proteins, including amyloid Αß, tau, α-synuclein and huntingtin, are cross-linked by the enzymatic activity of transglutaminase 2 (TG2). Additionally, the expression and activity of TG2 is increased in affected brain regions in these disorders. These observations along with experimental evidence in cellular and mouse models suggest that TG2 can contribute to the abnormal aggregation of disease causing proteins and consequently to neuronal damage. This accumulating evidence has provided the impetus to develop inhibitors of TG2 as possible neuroprotective agents. However, TG2 has other enzymatic activities in addition to its cross-linking function and can modulate multiple cellular processes including apoptosis, autophagy, energy production, synaptic function, signal transduction and transcription regulation. These diverse properties must be taken into consideration in designing TG2 inhibitors. In this review, we discuss the biochemistry of TG2, its various physiologic functions and our current understanding about its role in degenerative diseases of the brain. We also describe the different approaches to designing TG2 inhibitors that could be developed as potential disease-modifying therapies.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Doenças Neurodegenerativas/metabolismo , Transglutaminases/fisiologia , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/química , Humanos , Camundongos , Camundongos Transgênicos , Doenças Neurodegenerativas/tratamento farmacológico , Conformação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/antagonistas & inibidores , Transglutaminases/químicaRESUMO
Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase 3 family, is activated by oxidative stress. The death-signaling pathway mediated by ASK1 is inhibited by DJ-1, which is linked to recessively inherited Parkinson's disease (PD). Considering that DJ-1 deficiency exacerbates the toxicity of the mitochondrial complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we sought to investigate the direct role and mechanism of ASK1 in MPTP-induced dopamine neuron toxicity. In the present study, we found that MPTP administration to wild-type mice activates ASK1 in the midbrain. In ASK1 null mice, MPTP-induced motor impairment was less profound, and striatal dopamine content and nigral dopamine neuron counts were relatively preserved compared to wild-type littermates. Further, microglia and astrocyte activation seen in wild-type mice challenged with MPTP was markedly attenuated in ASKâ»/â» mice. These data suggest that ASK1 is a key player in MPTP-induced glial activation linking oxidative stress with neuroinflammation, two well recognized pathogenetic factors in PD. These findings demonstrate that ASK1 is an important effector of MPTP-induced toxicity and suggest that inhibiting this kinase is a plausible therapeutic strategy for protecting dopamine neurons in PD.