RESUMEN
Aging and exposure to environmental toxins including MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) are strong risk factors for developing Parkinson's disease (PD), a common neurologic disorder characterized by selective degeneration of midbrain dopaminergic (DAergic) neurons and astrogliosis. Aging and PD impair the subventricular zone (SVZ), one of the most important brain regions for adult neurogenesis. Because inflammation and oxidative stress are the hallmarks of aging and PD, we investigated the nature, timing, and signaling mechanisms contributing to aging-induced SVZ stem/neuroprogenitor cell (NPC) inhibition in aging male mice and attempted to determine to what extent manipulation of these pathways produces a functional response in the outcome of MPTP-induced DAergic toxicity. We herein reveal an imbalance of Nrf2-driven antioxidant/anti-inflammatory genes, such as Heme oxygenase1 in the SVZ niche, starting by middle age, amplified upon neurotoxin treatment and associated with an exacerbated proinflammatory SVZ microenvironment converging to dysregulate the Wingless-type MMTV integration site (Wnt)/ß-catenin signaling, a key regulatory pathway for adult NPCs. In vitro experiments using coculture paradigms uncovered aged microglial proinflammatory mediators as critical inhibitors of NPC proliferative potential. We also found that interruption of PI3K (phosphatidylinositol3-kinase)/Akt and the Wnt/Fzd/ß-catenin signaling cascades, which switch glycogen synthase kinase 3ß (GSK-3ß) activation on and off, were causally related to the impairment of SVZ-NPCs. Moreover, a synergy between dysfunctional microglia of aging mice and MPTP exposure further inhibited astrocyte proneurogenic properties, including the expression of key Wnts components. Last, pharmacological activation/antagonism studies in vivo and in vitro suggest the potential that aged SVZ manipulation is associated with DAergic functional recovery.
Asunto(s)
Envejecimiento/metabolismo , Mesencéfalo/metabolismo , Células-Madre Neurales/metabolismo , Trastornos Parkinsonianos/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Células Cultivadas , Técnicas de Cocultivo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , Neuroglía/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismoRESUMEN
In Parkinson's disease (PD), neurogenesis is impaired in the subventricular zone (SVZ) of postmortem human PD brains, in primate nonhuman and rodent models of PD. The vital role of Wingless-type MMTV integration site (Wnt)/ß-catenin signaling in the modulation of neurogenesis, neuroprotection, and synaptic plasticity coupled to our recent findings uncovering an active role for inflammation and Wnt/ß-catenin signaling in MPTP-induced loss and repair of nigrostriatal dopaminergic (DAergic) neurons prompted us to study the impact of neuroinflammation and the Wnt/ß-catenin pathway in the response of SVZ neuroprogenitors (NPCs) in MPTP-treated mice. In vivo experiments, using bromodeoxyuridine and cell-specific markers, and ex vivo time course analyses documented an inverse correlation between the reduced proliferation of NPCs and the generation of new neuroblasts with the phase of maximal exacerbation of microglia reaction, whereas a shift in the microglia proinflammatory phenotype correlated with a progressive NPC recovery. Ex vivo and in vitro experiments using microglia-NPC coculture paradigms pointed to NADPH-oxidase (gpPHOX(91)), a major source of microglial ROS, and reactive nitrogen species as candidate inhibitors of NPC neurogenic potential via the activation of glycogen synthase 3 (pGSK-3ß(Tyr216)), leading to loss of ß-catenin, a chief downstream transcriptional effector. Accordingly, MPTP/MPP(+) (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) caused ß-catenin downregulation and pGSK-3ß(Tyr216) overexpression, whereas manipulation of Wnt/ß-catenin signaling with RNA interference-mediated GSK-3ß knockdown or GSK-3ß antagonism reversed MPTP-induced neurogenic impairment ex vivo/in vitro or in vivo. Reciprocally, pharmacological modulation of inflammation prevented ß-catenin downregulation and restored neurogenesis, suggesting the possibility to modulate this endogenous system with potential consequences for DAergic neuroprotection and self-repair.
Asunto(s)
Mediadores de Inflamación/administración & dosificación , Intoxicación por MPTP/metabolismo , Plasticidad Neuronal/fisiología , Enfermedad de Parkinson/metabolismo , Receptor Cross-Talk/fisiología , Células Madre/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/antagonistas & inhibidores , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/uso terapéutico , Animales , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen/métodos , Mediadores de Inflamación/fisiología , Intoxicación por MPTP/tratamiento farmacológico , Intoxicación por MPTP/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Receptor Cross-Talk/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/patología , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
BACKGROUND: Dopamine-synthesizing (dopaminergic, DA) neurons in the ventral midbrain (VM) constitute a pivotal neuronal population controlling motor behaviors, cognitive and affective brain functions, which generation critically relies on the activation of Wingless-type MMTV integration site (Wnt)/ß-catenin pathway in their progenitors. In Parkinson's disease, DA cell bodies within the substantia nigra pars compacta (SNpc) progressively degenerate, with causes and mechanisms poorly understood. Emerging evidence suggests that Wnt signaling via Frizzled (Fzd) receptors may play a role in different degenerative states, but little is known about Wnt signaling in the adult midbrain. Using in vitro and in vivo model systems of DA degeneration, along with functional studies in both intact and SN lesioned mice, we herein highlight an intrinsic Wnt1/Fzd-1/ß-catenin tone critically contributing to the survival and protection of adult midbrain DA neurons. RESULTS: In vitro experiments identifie Fzd-1 receptor expression at a mRNA and protein levels in dopamine transporter (DAT) expressing neurons, and demonstrate the ability of exogenous Wnt1 to exert robust neuroprotective effects against Caspase-3 activation, the loss of tyrosine hydroxylase-positive (TH+) neurons and [3H] dopamine uptake induced by different DA-specific insults, including serum and growth factor deprivation, 6-hydroxydopamine and MPTP/MPP+. Co-culture of DA neurons with midbrain astrocytes phenocopies Wnt1 neuroprotective effects, whereas RNA interference-mediated knockdown of Wnt1 in midbrain astrocytes markedly reduces astrocyte-induced TH+ neuroprotection. Likewise, silencing ß-catenin mRNA or knocking down Fzd-1 receptor expression in mesencephalic neurons counteract astrocyte-induced TH+ neuroprotection. In vivo experiments document Fzd-1 co-localization with TH+ neurons within the intact SNpc and blockade of Fzd/ß-catenin signaling by unilateral infusion of a Fzd/ß-catenin antagonist within the SN induces reactive astrocytosis and acutely inhibits TH+ neuron survival in ipsilateral SNpc, an effect efficiently prevented by pharmacological activation of ß-catenin signaling within the SNpc. CONCLUSION: These results defining a novel Wnt1/Fzd-1/ß-catenin astrocyte-DA autoprotective loop provide a new mechanistic inside into the regulation of pro-survival processes, with potentially relevant consequences for drug design or drug action in Parkinson's disease.
RESUMEN
BACKGROUND: Current evidence suggests a role of neuroinflammation in the pathogenesis of Parkinson's disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of basal ganglia injury. Reportedly, nonsteroidal anti-inflammatory drugs (NSAIDs) mitigate DAergic neurotoxicity in rodent models of PD. Consistent with these findings, epidemiological analysis indicated that certain NSAIDs may prevent or delay the progression of PD. However, a serious impediment of chronic NSAID therapy, particularly in the elderly, is gastric, renal and cardiac toxicity. Nitric oxide (NO)-donating NSAIDs, have a safer profile while maintaining anti-inflammatory activity of parent compounds. We have investigated the oral activity of the NO-donating derivative of flurbiprofen, [2-fluoro-α-methyl (1,1'-biphenyl)-4-acetic-4-(nitrooxy)butyl ester], HCT1026 (30 mg kg(-1) daily in rodent chow) in mice exposed to the parkinsonian neurotoxin MPTP. METHODS: Ageing mice were fed with a control, flurbiprofen, or HCT1026 diet starting ten days before MPTP administration and continuing for all the experimental period. Striatal high affinity synaptosomal dopamine up-take, motor coordination assessed with the rotarod, tyrosine hydroxylase (TH)- and dopamine transporter (DAT) fiber staining, stereological cell counts, immunoblotting and gene expression analyses were used to assess MPTP-induced nigrostriatal DAergic toxicity and glial activation 1-40 days post-MPTP. RESULTS: HCT1026 was well tolerated and did not cause any measurable toxic effect, whereas flurbiprofen fed mice showed severe gastrointestinal side-effects. HCT1026 efficiently counteracted motor impairment and reversed MPTP-induced decreased synaptosomal [3H]dopamine uptake, TH- and DAT-stained fibers in striatum and TH+ neuron loss in substantia nigra pars compacta (SNpc), as opposed to age-matched mice fed with a control diet. These effects were associated to a significant decrease in reactive macrophage antigen-1 (Mac-1)-positive microglial cells within the striatum and ventral midbrain, decreased expression of iNOS, Mac-1 and NADPH oxidase (PHOX), and downregulation of 3-Nitrotyrosine, a peroxynitrite finger print, in SNpc DAergic neurons. CONCLUSIONS: Oral treatment with HCT1026 has a safe profile and a significant efficacy in counteracting MPTP-induced dopaminergic (DAergic) neurotoxicity, motor impairment and microglia activation in ageing mice. HCT1026 provides a novel promising approach towards the development of effective pharmacological neuroprotective strategies against PD.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Antiinflamatorios no Esteroideos/inmunología , Antiinflamatorios no Esteroideos/uso terapéutico , Cuerpo Estriado/patología , Actividad Motora/efectos de los fármacos , Óxido Nítrico/metabolismo , Enfermedad de Parkinson/fisiopatología , Sustancia Negra/patología , Animales , Antiinflamatorios no Esteroideos/farmacología , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Dopaminérgicos , Flurbiprofeno/administración & dosificación , Flurbiprofeno/análogos & derivados , Flurbiprofeno/inmunología , Flurbiprofeno/farmacología , Flurbiprofeno/uso terapéutico , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Intoxicación por MPTP , Antígeno de Macrófago-1/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Oxidorreductasas/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Prueba de Desempeño de Rotación con Aceleración Constante , Sustancia Negra/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Oxidative stress is implicated in the death of dopaminergic neurons in sporadic forms of Parkinson's disease. Because oxidative stress can be modulated endogenously by uncoupling proteins (UCPs), we hypothesized that specific neuronal expression of UCP2, one member of the UCP family that is rapidly induced in the CNS following insults, could confer neuroprotection in a mouse model of Parkinson's disease. We generated transgenic mice overexpressing UCP2 in catecholaminergic neurons under the control of the tyrosine hydroxylase promoter (TH-UCP2). In these mice, dopaminergic neurons of the substantia nigra showed a twofold elevation in UCP2 expression, elevated uncoupling of their mitochondria, and a marked reduction in indicators of oxidative stress, an effect also observed in the striatum. Upon acute exposure to 1,2,3,6-methyl-phenyl-tetrahydropyridine, TH-UCP2 mice showed neuroprotection and retention of locomotor functions. Our data suggest that UCP2 may represent a drug target for slowing the progression of Parkinson's disease.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Dopamina/metabolismo , Proteínas de Transporte de Membrana/biosíntesis , Proteínas Mitocondriales/biosíntesis , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Canales Iónicos , Masculino , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Transgénicos , Proteínas Mitocondriales/genética , Tirosina 3-Monooxigenasa/biosíntesis , Tirosina 3-Monooxigenasa/genética , Proteína Desacopladora 2 , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismoRESUMEN
The G to A single nucleotide polymorphisms (SNPs), at position -376, -308 and -238 in the promoter of the tumor necrosis factor alpha (TNF) gene, have been independently correlated with numerous diseases. Alleles TNF(-376A) and TNF(-238A) are normally found throughout the world with very low frequencies. We investigated the frequency of these SNPs in Sicilian subjects hospitalized after traumatic brain injury and in three groups of subjects from northern Sardinia: healthy subjects and individuals with multiple sclerosis or ischemic stroke. While no significant difference was found between healthy and disease subjects, the frequency of TNF(-376A) and TNF(-238A) was elevated up to 10 times in Sardinia compared to Sicily and other populations throughout the world. These elevated frequencies may be the result of genetic drift or of selective pressure on TNF itself or on neighboring genes, including the HLA. Malaria, endemic to Sardinia until the end of the 1940s, and the bubonic plague, are among the possible causes of selection. These findings indicate that Sardinia is an ideal location to further elucidate the correlation between TNF or HLA polymorphisms and diseases, including multiple sclerosis and type-I diabetes, present with an unusually high frequency and co-morbidity in Sardinia.
Asunto(s)
Polimorfismo de Nucleótido Simple , Factor de Necrosis Tumoral alfa/genética , Diabetes Mellitus Tipo 1/genética , Frecuencia de los Genes , Humanos , Italia , Esclerosis Múltiple/genética , Accidente Cerebrovascular/genéticaRESUMEN
Glucocorticoid (GC) hormones play a central role in the bidirectional communication between the neuroendocrine and the immune systems and exert, via GC receptors (GR), potent immunosuppressive and anti-inflammatory effects. In this study, we report that GR deficiency of transgenic mice expressing GR antisense RNA from early embryonic life has a dramatic impact in programming the susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. GR deficiency renders mice resistant to myelin oligodendrocyte glycoprotein-induced EAE, and such mice do not develop clinical or histological signs of disease compared with EAE-susceptible wild-type mice. Resistance to EAE in GR-deficient mice is associated not with endogenous GC levels, but with a significant reduction in spleen and lymph node cell proliferation. The use of NO inhibitors in vitro indicates that NO is the candidate immunosuppressor molecule. GR-deficient mice develop 3- to 6-fold higher nitrite levels in the periphery and are resistant to NO inhibition by GCs. Specific inhibition of NO production in vivo by treatment with the inducible NO synthase inhibitor, L-N(6)-(1-iminoethyl)-lysine, suppressed circulating nitrites, increased myelin oligodendrocyte glycoprotein-specific cell proliferation, and rendered GR-deficient mice susceptible to EAE. Thus, life-long GR deficiency triggers inducible NO synthase induction and NO generation with consequent down-regulation of effector cell proliferation. These findings identify a novel link among GR, NO, and EAE susceptibility and highlight NO as critical signaling molecule in bidirectional communication between the hypothalamic-pituitary-adrenocortical axis and the immune system.