RESUMEN
Currently there is no neuroprotective or neurorestorative therapy for Parkinson's disease. Here we report that transient receptor potential vanilloid 1 (TRPV1) on astrocytes mediates endogenous production of ciliary neurotrophic factor (CNTF), which prevents the active degeneration of dopamine neurons and leads to behavioural recovery through CNTF receptor alpha (CNTFRα) on nigral dopamine neurons in both the MPP(+)-lesioned or adeno-associated virus α-synuclein rat models of Parkinson's disease. Western blot and immunohistochemical analysis of human post-mortem substantia nigra from Parkinson's disease suggests that this endogenous neuroprotective system (TRPV1 and CNTF on astrocytes, and CNTFRα on dopamine neurons) might have relevance to human Parkinson's disease. Our results suggest that activation of astrocytic TRPV1 activates endogenous neuroprotective machinery in vivo and that it is a novel therapeutic target for the treatment of Parkinson's disease.
Asunto(s)
Astrocitos/metabolismo , Factor Neurotrófico Ciliar/metabolismo , Neuronas Dopaminérgicas/metabolismo , Neuroprotección , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Sustancia Negra/metabolismo , Animales , Subunidad alfa del Receptor del Factor Neurotrófico Ciliar/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Femenino , Humanos , Regeneración Nerviosa , Enfermedad de Parkinson/fisiopatología , Ratas , Sustancia Negra/citología , Sustancia Negra/patología , Canales Catiónicos TRPV/metabolismoRESUMEN
Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.
Asunto(s)
Calpaína/metabolismo , Neuronas Dopaminérgicas/metabolismo , Proteoma/metabolismo , 1-Metil-4-fenilpiridinio/farmacología , Animales , ATPasas Transportadoras de Arsenitos/genética , ATPasas Transportadoras de Arsenitos/metabolismo , Calpaína/antagonistas & inhibidores , Muerte Celular , Línea Celular , Dipéptidos/farmacología , Dipéptidos/uso terapéutico , Neuronas Dopaminérgicas/efectos de los fármacos , Electroforesis en Gel Bidimensional/métodos , Glicina/análogos & derivados , Glicina/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Ionomicina/farmacología , Periferinas/genética , Periferinas/metabolismo , Proteómica/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
This study examined whether ethyl pyruvate (EP) promotes the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. MPTP induced degeneration of nigrostriatal DA neurons and glial activation as visualized by tyrosine hydroxylase, macrophage Ag complex-1, and/or glial fibrillary acidic protein immunoreactivity. Western blotting and immunohistochemistry showed activation of microglial NADPH oxidase and astroglial myeloperoxidase (MPO) and subsequent reactive oxygen species/reactive nitrogen species production and oxidative DNA damage in the MPTP-treated substantia nigra. Treatment with EP prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This neuroprotection afforded by EP was associated with the suppression of astroglial MPO expression, NADPH oxidase-, and/or inducible NO synthase-derived reactive oxygen species/reactive nitrogen species production by activated microglia. Interestingly, EP was found to protect DA neurons from 1-methyl-4-phenyl-pyridinium neurotoxicity in cocultures of mesencephalic neurons and microglia but not in neuron-enriched mesencephalic cultures devoid of microglia. The present findings show that EP may inhibit glial-mediated oxidative stress, suggesting that EP may have therapeutic value in the treatment of aspects of Parkinson's disease related to glia-derived oxidative damage.
Asunto(s)
Dopamina/fisiología , Neuroglía/inmunología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/inmunología , Piruvatos/uso terapéutico , Sustancia Negra/inmunología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/antagonistas & inhibidores , Animales , Células Cultivadas , Técnicas de Cocultivo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/inmunología , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Dopamina/efectos adversos , Antagonistas de Dopamina/administración & dosificación , Antagonistas de Dopamina/uso terapéutico , Mediadores de Inflamación/administración & dosificación , Mediadores de Inflamación/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Neuroglía/efectos de los fármacos , Neuroglía/patología , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/inmunología , Enfermedad de Parkinson/patología , Piruvatos/administración & dosificación , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Sustancia Negra/patologíaRESUMEN
This study examined whether the cannabinoid receptor type 1 (CB(1)) receptor contributes to the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. MPTP induced significant loss of nigrostriatal DA neurons and microglial activation in the substantia nigra (SN), visualized with tyrosine hydroxylase or macrophage Ag complex-1 immunohistochemistry. Real-time PCR, ELISA, Western blotting, and immunohistochemistry disclosed upregulation of proinflammatory cytokines, activation of microglial NADPH oxidase, and subsequent reactive oxygen species production and oxidative damage of DNA and proteins in MPTP-treated SN, resulting in degeneration of DA neurons. Conversely, treatment with nonselective cannabinoid receptor agonists (WIN55,212-2 and HU210) led to increased survival of DA neurons in the SN, their fibers and dopamine levels in the striatum, and improved motor function. This neuroprotection by cannabinoids was accompanied by suppression of NADPH oxidase reactive oxygen species production and reduced expression of proinflammatory cytokines from activated microglia. Interestingly, cannabinoids protected DA neurons against 1-methyl-4-phenyl-pyridinium neurotoxicity in cocultures of mesencephalic neurons and microglia, but not in neuron-enriched mesencephalic cultures devoid of microglia. The observed neuroprotection and inhibition of microglial activation were reversed upon treatment with CB(1) receptor selective antagonists AM251 and/or SR14,716A, confirming the involvement of the CB(1) receptor. The present in vivo and in vitro findings clearly indicate that the CB(1) receptor possesses anti-inflammatory properties and inhibits microglia-mediated oxidative stress. Our results collectively suggest that the cannabinoid system is beneficial for the treatment of Parkinson's disease and other disorders associated with neuroinflammation and microglia-derived oxidative damage.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/efectos adversos , Cuerpo Estriado/inmunología , Inhibidores de Crecimiento/fisiología , Microglía/efectos de los fármacos , Microglía/inmunología , Neurotoxinas/efectos adversos , Receptor Cannabinoide CB1/fisiología , Sustancia Negra/inmunología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/antagonistas & inhibidores , Animales , Benzoxazinas/farmacología , Células Cultivadas , Técnicas de Cocultivo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Dopaminérgicos/administración & dosificación , Dopaminérgicos/efectos adversos , Dronabinol/análogos & derivados , Dronabinol/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/patología , Morfolinas/farmacología , Naftalenos/farmacología , Neuronas/efectos de los fármacos , Neuronas/inmunología , Neuronas/patología , Neurotoxinas/administración & dosificación , Trastornos Parkinsonianos/inmunología , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/prevención & control , Receptor Cannabinoide CB1/metabolismo , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismoRESUMEN
The present study examined whether the antidepressant paroxetine promotes the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. MPTP induced degeneration of nigrostriatal DA neurons and glial activation as visualized by tyrosine hydroxylase, macrophage Ag complex-1, and/or glial fibrillary acidic protein immunoreactivity. Real-time PCR, Western blotting, and immunohistochemistry showed upregulation of proinflammatory cytokines, activation of microglial NADPH oxidase and astroglial myeloperoxidase, and subsequent reactive oxygen species production and oxidative DNA damage in the MPTP-treated substantia nigra. Treatment with paroxetine prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This neuroprotection afforded by paroxetine was associated with the suppression of astroglial myeloperoxidase expression and/or NADPH oxidase-derived reactive oxygen species production and reduced expression of proinflammatory cytokines, including IL-1beta, TNF-alpha, and inducible NO synthase, by activated microglia. The present findings show that paroxetine may possess anti-inflammatory properties and inhibit glial activation-mediated oxidative stress, suggesting that paroxetine and its analogues may have therapeutic value in the treatment of aspects of Parkinson's disease related to neuroinflammation.
Asunto(s)
Encéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Enfermedad de Parkinson Secundaria/prevención & control , Paroxetina/farmacología , Sustancia Negra/efectos de los fármacos , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Antidepresivos de Segunda Generación/farmacología , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Dopamina/metabolismo , Activación Enzimática/efectos de los fármacos , Inmunohistoquímica , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/fisiopatología , Peroxidasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sustancia Negra/citología , Sustancia Negra/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In the present study, we investigated the effects of IL-13, a well-known anti-inflammatory cytokine, on the thrombin-treated hippocampus in vivo. NeuN immunohistochemistry and Nissl staining revealed significant loss of hippocampal CA1 neurons upon intrahippocampal injection of thrombin. This neurotoxicity was accompanied by substantial microglial activation, as evident from OX-42 immunohistochemistry results. In parallel, Western blot analysis and hydroethidine histochemistry disclosed activation of NADPH oxidase, generation of reactive oxygen species, and oxidative damage in the hippocampal CA1 area showing hippocampal neuron degeneration. Interestingly, immunohistochemical and biochemical experiments showed that intrahippocampal injection of thrombin increased IL-13 immunoreactivity and IL-13 levels as early as 8 h after thrombin, reaching a peak at 7 days, which was maintained up to 14 days. Moreover, double-label immunohistochemistry revealed IL-13 immunoreactivity exclusively in activated microglia. IL-13-neutralizing Abs significantly rescued CA1 hippocampal neurons from thrombin neurotoxicity. In parallel, neutralization of IL-13 inhibited activation of NADPH oxidase, reactive oxygen species production, and oxidative damage. Additionally, IL-13 neutralization suppressed the expression of inducible NO synthase and several proinflammatory cytokines. To our knowledge, the present study is the first to show that IL-13 triggers microglial NADPH oxidase-derived oxidative stress, leading to the degeneration of hippocampal neurons in vivo, as occurs in cases of Alzheimer's disease.
Asunto(s)
Apoptosis/inmunología , Hipocampo/inmunología , Interleucina-13/fisiología , Microglía/enzimología , Microglía/inmunología , NADPH Oxidasas/metabolismo , Neuronas/inmunología , Estrés Oxidativo/inmunología , Animales , Activación Enzimática/inmunología , Femenino , Regulación Enzimológica de la Expresión Génica/inmunología , Hipocampo/enzimología , Hipocampo/patología , Interleucina-13/biosíntesis , Interleucina-13/genética , Microglía/patología , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/fisiología , Neuronas/enzimología , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Trombina/administración & dosificación , Trombina/toxicidad , Regulación hacia Arriba/inmunologíaRESUMEN
BACKGROUND: Gangliosides, sialic acid-containing glycosphingolipids exist in mammalian cell membranes particularly neuronal membranes. The trisialoganglioside (GT1b) is one of the major brain gangliosides and acts as an endogenous regulator in the brain. We previously showed GT1b induces mesencephalic dopaminergic (DA) neuronal death, both in vivo and in vitro. We further investigate the underlying mechanisms of GT1b neurotoxicity. RESULTS: Consistent with earlier findings, GT1b attenuated the DA neuron number and dopamine uptake level in mesencephalic cultures. Morphological evidence revealed GT1b-induced chromatin condensation and nuclear fragmentation as well as an increased number of TUNEL-positive cells, compared to control cultures. Interestingly, while GT1b enhanced caspase-3 activity, DEVD, a caspase-3 inhibitor, failed to rescue DA neuronal death. Immunoblot analysis revealed that GT1b inactivates Akt through dephosphorylation at both Ser473 and Thr308, subsequent dephosphorylation of GSK-3beta, a substrate of Akt, and hyperphosphorylation of tau, downstream of GSK-3beta. Moreover, a GSK-3beta specific inhibitor, L803-mt, attenuated tau phosphorylation and rescued DA neurons from cell death in mesencephalic cultures. CONCLUSION: Our data provide novel evidence that a Akt/GSK-3beta/tau-dependent, but not caspase-3 signaling pathway plays a pivotal role in GT1b-mediated neurotoxic actions on mesencephalic DA neurons.
Asunto(s)
Dopamina/metabolismo , Gangliósidos/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Mesencéfalo/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas tau/metabolismo , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Caspasa 3/metabolismo , Recuento de Células , Células Cultivadas , Femenino , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Microscopía Inmunoelectrónica , Neuronas/citología , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Current pharmacological treatments for Parkinson's disease (PD) are focused on symptomatic relief, but not on disease modification, based on the strong belief that PD is caused by irreversible dopaminergic neuronal death. Thus, the concept of the presence of dormant dopaminergic neurons and its possibility as the disease-modifying therapeutic target against PD have not been explored. Here we show that optogenetic activation of substantia nigra pars compacta (SNpc) neurons alleviates parkinsonism in acute PD animal models by recovering tyrosine hydroxylase (TH) from the TH-negative dormant dopaminergic neurons, some of which still express DOPA decarboxylase (DDC). The TH loss depends on reduced dopaminergic neuronal firing under aberrant tonic inhibition, which is attributed to excessive astrocytic GABA. Blocking the astrocytic GABA synthesis recapitulates the therapeutic effect of optogenetic activation. Consistently, SNpc of postmortem PD patients shows a significant population of TH-negative/DDC-positive dormant neurons surrounded by numerous GABA-positive astrocytes. We propose that disinhibiting dormant dopaminergic neurons by blocking excessive astrocytic GABA could be an effective therapeutic strategy against PD.
Asunto(s)
Astrocitos/metabolismo , Neuronas Dopaminérgicas/fisiología , Degeneración Nerviosa/fisiopatología , Enfermedad de Parkinson/fisiopatología , Tirosina 3-Monooxigenasa/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Pérdida de Tono Postural/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Persona de Mediana Edad , Enfermedad de Parkinson/terapia , Ratas , Ratas Wistar , Tirosina 3-Monooxigenasa/antagonistas & inhibidores , Ácido gamma-Aminobutírico/biosíntesisRESUMEN
The present study investigated whether thrombin can induce the production of reactive oxygen species (ROS) through activation of neuronal NADPH oxidase and whether this contributes to oxidative damage and consequently to neurodegeneration. Immunocytochemical and biochemical evidence demonstrated that, in neuron-enriched hippocampal cultures, thrombin induces neurodegeneration in a dose-dependent manner. In parallel, ROS production was evident as assessed by analyzing DCF and hydroethidine. Real-time PCR analysis, at various time points after thrombin treatment, also demonstrated that expression of NADPH oxidase subunits (p47(phox) and p67(phox)) occurs. In addition, Western blot analysis and double-label immunocytochemistry showed an up-regulation in the expression of cytosolic components (Rac 1 and p67(phox)), the translocation of cytosolic proteins (p47(phox) and p67(phox)) to the membrane, and the localization of gp91(phox) or p47(phox) expression in hippocampal neurons of cultures and CA1 layer. The thrombin-induced ROS production, protein oxidation, and loss of cultured hippocampal neurons were partially attenuated by an NADPH oxidase inhibitor and/or by several antioxidants. Collectively, the present study is the first to demonstrate that, in cultured hippocampal neurons, thrombin-induced neurotoxicity is, at least in part, caused by neuronal NADPH oxidase-mediated oxidative stress. This strongly suggests that thrombin can act as an endogenous neurotoxin, and inhibitors of thrombin and/or antioxidants can be useful agents for treating oxidative stress-mediated hippocampal neurodegenerative diseases, such as Alzheimer's disease.
Asunto(s)
Hipocampo/metabolismo , NADPH Oxidasas/metabolismo , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Estrés Oxidativo/fisiología , Trombina/toxicidad , Animales , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Trastornos Heredodegenerativos del Sistema Nervioso/tratamiento farmacológico , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Trastornos Heredodegenerativos del Sistema Nervioso/fisiopatología , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , NADPH Oxidasas/efectos de los fármacos , NADPH Oxidasas/genética , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/fisiopatología , Neuronas/efectos de los fármacos , Neurotoxinas/antagonistas & inhibidores , Neurotoxinas/metabolismo , Neurotoxinas/toxicidad , Estrés Oxidativo/efectos de los fármacos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Trombina/antagonistas & inhibidores , Trombina/metabolismoRESUMEN
Transient receptor potential vanilloid subtype 1 (TRPV1), also known as vanilloid receptor 1 (VR1), is a nonselective cation channel that is activated by a variety of ligands, such as exogenous capsaicin (CAP) or endogenous anandamide (AEA), as well as products of lipoxygenases. Cannabinoid type 1 (CB1) receptor belongs to the G protein-coupled receptor superfamily and is activated by cannabinoids such as AEA and exogenous Delta-9-tetrahydrocannabinol (THC). TRPV1 and CB1 receptors are widely expressed in the brain and play many significant roles in various brain regions; however, the issue of whether TRPV1 or CB1 receptors mediate neuroprotection or neurotoxicity remains controversial. Furthermore, functional crosstalk between these two receptors has been recently reported. It is therefore timely to review current knowledge regarding the functions of these two receptors and to consider new directions of investigation on their roles in the brain.
Asunto(s)
Encéfalo/metabolismo , Receptor Cannabinoide CB1/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Encéfalo/anatomía & histología , Moduladores de Receptores de Cannabinoides/metabolismo , Capsaicina/química , Capsaicina/metabolismo , Endocannabinoides , Fármacos Neuroprotectores/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Fármacos del Sistema Sensorial/química , Fármacos del Sistema Sensorial/metabolismoRESUMEN
The effects of capsaicin (CAP), a transient receptor potential vanilloid subtype 1 (TRPV1) agonist, were determined on nigrostriatal dopamine (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). The results showed that TRPV1 activation by CAP rescued nigrostriatal DA neurons, enhanced striatal DA functions and improved behavioral recovery in MPTP-treated mice. CAP neuroprotection was associated with reduced expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1ß) and reactive oxygen species/reactive nitrogen species from activated microglia-derived NADPH oxidase, inducible nitric oxide synthase or reactive astrocyte-derived myeloidperoxidase. These beneficial effects of CAP were reversed by treatment with the TRPV1 antagonists capsazepine and iodo-resiniferatoxin, indicating TRPV1 involvement. This study demonstrates that TRPV1 activation by CAP protects nigrostriatal DA neurons via inhibition of glial activation-mediated oxidative stress and neuroinflammation in the MPTP mouse model of PD. These results suggest that CAP and its analogs may be beneficial therapeutic agents for the treatment of PD and other neurodegenerative disorders that are associated with neuroinflammation and glial activation-derived oxidative damage.
Asunto(s)
Antioxidantes/farmacología , Capsaicina/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Intoxicación por MPTP/metabolismo , Neuroglía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo , Animales , Antioxidantes/uso terapéutico , Capsaicina/uso terapéutico , Neuronas Dopaminérgicas/metabolismo , Intoxicación por MPTP/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/metabolismo , Neuroglía/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Óxido Nítrico Sintasa de Tipo II/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
Intranigral injection of the transient receptor potential vanilloid subtype 1 (TRPV1; also known as VR1) agonist capsaicin (CAP) into the rat brain, or treatment of rat mesencephalic cultures with CAP, resulted in cell death of dopaminergic (DA) neurons, as visualized by immunocytochemistry. This in vivo and in vitro effect was ameliorated by the TRPV1 antagonist capsazepine (CZP) or iodo-resiniferatoxin, suggesting the direct involvement of TRPV1 in neurotoxicity. In cultures, both CAP and anandamide (AEA), an endogenous ligand for both TRPV1 and cannabinoid type 1 (CB1) receptors, induced degeneration of DA neurons, increases in intracellular Ca2+ ([Ca2+]i), and mitochondrial damage, which were inhibited by CZP, the CB1 antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) or the intracellular Ca2+ chelator BAPTA/AM. We also found that CAP or AEA increased mitochondrial cytochrome c release as well as immunoreactivity to cleaved caspase-3 and that the caspase-3 inhibitor z-Asp-Glu-Val-Asp-fmk protected DA neurons from CAP- or AEA-induced neurotoxicity. Additional studies demonstrated that treatment of mesencephalic cultures with CB1 receptor agonist (6aR)-trans 3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d] pyran-9-methanol (HU210) also produced degeneration of DA neurons and increases in [Ca2+]i, which were inhibited by AM251 and BAPTA/AM. The CAP-, AEA-, or HU210-induced increases in [Ca2+]i were dependent on extracellular Ca2+, with significantly different patterns of Ca2+ influx. Surprisingly, CZP and AM251 reversed HU210- or CAP-induced neurotoxicity by inhibiting Ca2+ influx, respectively, suggesting the existence of functional cross talk between TRPV1 and CB1 receptors. To our knowledge, this study is the first to demonstrate that the activation of TRPV1 and/or CB1 receptors mediates cell death of DA neurons. Our findings suggest that these two types of receptors, TRPV1 and CB1, may contribute to neurodegeneration in response to endogenous ligands such as AEA.
Asunto(s)
Muerte Celular/fisiología , Dopamina/fisiología , Canales Iónicos/fisiología , Mesencéfalo/citología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Animales , Ácidos Araquidónicos/farmacología , Calcio/metabolismo , Capsaicina/farmacología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Endocannabinoides , Inmunohistoquímica , Canales Iónicos/efectos de los fármacos , Ligandos , Mitocondrias/fisiología , Alcamidas Poliinsaturadas , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB1/fisiología , Sustancia Negra/citología , Canales Catiónicos TRPVRESUMEN
The cannabinoid (CB2) receptor type 2 has been proposed to prevent the degeneration of dopamine neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. However, the mechanisms underlying CB2 receptor-mediated neuroprotection in MPTP mice have not been elucidated. The mechanisms underlying CB2 receptor-mediated neuroprotection of dopamine neurons in the substantia nigra (SN) were evaluated in the MPTP mouse model of Parkinson's disease (PD) by immunohistochemical staining (tyrosine hydroxylase, macrophage Ag complex-1, glial fibrillary acidic protein, myeloperoxidase (MPO), and CD3 and CD68), real-time PCR and a fluorescein isothiocyanate-labeled albumin assay. Treatment with the selective CB2 receptor agonist JWH-133 (10 µg kg(-1), intraperitoneal (i.p.)) prevented MPTP-induced degeneration of dopamine neurons in the SN and of their fibers in the striatum. This JWH-133-mediated neuroprotection was associated with the suppression of blood-brain barrier (BBB) damage, astroglial MPO expression, infiltration of peripheral immune cells and production of inducible nitric oxide synthase, proinflammatory cytokines and chemokines by activated microglia. The effects of JWH-133 were mimicked by the non-selective cannabinoid receptor WIN55,212 (10 µg kg(-1), i.p.). The observed neuroprotection and inhibition of glial-mediated neurotoxic events were reversed upon treatment with the selective CB2 receptor antagonist AM630, confirming the involvement of the CB2 receptor. Our results suggest that targeting the cannabinoid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with glial activation, BBB disruption and peripheral immune cell infiltration.
Asunto(s)
Barrera Hematoencefálica/patología , Neuronas Dopaminérgicas/patología , Enfermedad de Parkinson Secundaria/patología , Receptor Cannabinoide CB2/inmunología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Barrera Hematoencefálica/inmunología , Citocinas/análisis , Citocinas/inmunología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/inmunología , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/inmunología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Neuroprotección , Enfermedad de Parkinson Secundaria/inmunología , Sustancia Negra/inmunología , Sustancia Negra/patologíaRESUMEN
The present study examined whether thrombin-induced microglial activation could contribute to death of dopaminergic neurons in the rat substantia nigra (SN) in vivo. Seven days after thrombin injection into the SN, tyrosine hydroxylase immunohistochemistry showed a significant loss of nigral dopaminergic neurons. In parallel, thrombin-activated microglia, visualized by immunohistochemical staining using antibodies against the complement receptor type 3 (OX-42) and the major histocompatibility complex class II antigens were also observed in the SN, where degeneration of nigral neurons was found. Reverse transcription PCR at various time points demonstrated that activated microglia in vivo exhibited an early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and several proinflammatory cytokines, including interleukin 1beta (IL-1beta), IL-6, and tumor necrosis factor alpha. Western blot analysis and double-label immunohistochemistry showed an increase in the expression of iNOS and COX-2 and the colocalization of these proteins within microglia. The thrombin-induced loss of SN dopaminergic neurons was partially inhibited by NG-nitro-L-arginine methyl ester hydrochloride, an NOS inhibitor, and by DuP-697, a COX-2 inhibitor. Additional studies demonstrated that extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) were activated in the SN as early as 30 min after thrombin injection, and that these kinases were localized within microglia. Inhibition of ERK1/2 and p38 MAPK reduced iNOS and COX-2 mRNA expression and rescued dopaminergic neurons in the SN. The present results strongly suggest that microglial activation triggered by endogenous compound(s) such as thrombin may be involved in the neuropathological processes of dopaminergic neuronal cell death that occur in Parkinson's disease.
Asunto(s)
Microglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Trastornos Parkinsonianos/inducido químicamente , Sustancia Negra/efectos de los fármacos , Trombina/farmacología , Animales , Recuento de Células , Ciclooxigenasa 2 , Citocinas/metabolismo , Progresión de la Enfermedad , Dopamina/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Inmunohistoquímica , Isoenzimas/biosíntesis , Microglía/metabolismo , Microglía/patología , Microinyecciones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Neuronas/patología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Ratas , Ratas Sprague-Dawley , Técnicas Estereotáxicas , Sustancia Negra/patología , Tirosina 3-Monooxigenasa/biosíntesisRESUMEN
The present study examined the effects of ATP on the striatum of Sprague-Dawley rats. Intrastriatal administration of ATP produced dose-dependent striatal lesions as confirmed by cresyl violet staining. Additional immunostaining using neuronal nuclear protein (NeuN), OX-42 and GFAP antibodies revealed that ATP caused death of both neurons and glial cells. The nonmetabolizable ATP analogue ATPgammaS and P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP) mimicked ATP effects, whereas either P2Y receptor agonist ADP or P1 receptor agonist adenosine did not. The P2 receptor antagonist reactive blue 2, but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) attenuated ATP-induced striatal injury. These results suggest that intrastriatal administration of ATP causes P2X receptor-mediated cell death in the striatum and support the hypothesis that extracellular ATP can be an important mediator of neuropathological events of brain injuries.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antígenos CD , Antígenos de Neoplasias , Antígenos de Superficie , Proteínas Aviares , Proteínas Sanguíneas , Hipoxia-Isquemia Encefálica/metabolismo , Neostriado/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Neurotoxinas/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Basigina , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipoxia-Isquemia Encefálica/fisiopatología , Glicoproteínas de Membrana/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Neostriado/efectos de los fármacos , Neostriado/fisiopatología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Neuroglía/efectos de los fármacos , Neuroglía/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Neurotoxinas/toxicidad , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2XRESUMEN
The present study examined whether Interleukin-13 (IL-13) or IL-4, an anti-inflammatory cytokine, could induce cell death of activated microglia by prothrombin kringle-2 (pKr-2) which is a domain of prothrombin distinct from thrombin. Microglia cell death was detected at eight days after co-treatment of pKr-2 with IL-13/IL-4 in vitro. This cell death was assessed by live assay, dead assay, TUNEL and MTT assay. In parallel, reactive oxygen species (ROS) production was evident as assessed by superoxide assay, WST-1 and analyzing DCF in combination of pKr-2 and IL-13 or IL-4 treated microglia. The IL-13/IL-4-enhanced ROS production and cell death in pKr-2 activated microglia was partially inhibited by an NADPH oxidase inhibitor, apocynin and/or by several antioxidants. Moreover, Western blot analysis showed a significant increase in cyclooxygenase-2 (COX-2) expression in combination of pKr-2 and IL-13 or IL-4 treated microglia, which was partially inhibited by apocynin and an antioxidant, trolox. Additional studies demonstrated that microglia cell death was reversed by treatment with COX-2 inhibitor, NS398. Our data strongly suggest that oxidative stress and COX-2 activation through NADPH oxidase may contribute to IL-13/IL-4 induced cell death of pKr-2 activated microglia.
Asunto(s)
Interleucina-13/farmacología , Interleucina-4/farmacología , Kringles , Microglía/efectos de los fármacos , Microglía/metabolismo , Estrés Oxidativo/efectos de los fármacos , Protrombina/química , Acetofenonas/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Ciclooxigenasa 2/metabolismo , Inhibidores Enzimáticos/farmacología , Etiquetado Corte-Fin in Situ , NADPH Oxidasas/metabolismo , Nitrobencenos/farmacología , Protrombina/farmacología , Ratas , Sulfonamidas/farmacología , Sales de Tetrazolio , TiazolesRESUMEN
The present study examined whether capsaicin (CAP), an agonist of transient receptor potential vanilloid subtype 1 (TRPV1) can prevent 1-methyl-4-phenylpyridinium (MPP(+))-induced dopaminergic (DA) neuronal death in the substantia nigra (SN). Unilateral injection of MPP(+) into the median forebrain bundle of rat brain resulted in a significant loss of nigral DA neurons, assessed by tyrosine hydroxylase (TH) immunostaining. In parallel, activation of microglia, visualized by OX-42 and OX-6 immunostaining were also observed in the SN, where degeneration of nigral neurons was found. By contrast, MPP(+) neurotoxicity was partially inhibited by co-treatment with MPP(+) and CAP. Interestingly, CAP significantly decreased not only immunoreactivity of OX-42 and OX-6 but also production of microglia-derived reactive oxygen species (ROS) in the SN of MPP(+)-treated rats. In experiments designed to further verify effectiveness of CAP against microglia-derived neurotoxicity, CAP inhibited ROS production and blocked MPP(+)-induced death of DA neurons in co-cultures of mesencephalic neurons and microglia, but not in microglia-free, neuron-enriched mesencephalic cultures. This beneficial effect was reversed by capsazepine, an antagonist of TRPV1, expressed in microglia, indicating TRPV1 involvement. Our data demonstrate for the first time that CAP may inhibit microglial activation-mediated oxidative stress via TRPV1, suggesting that CAP and its analogs may have therapeutic value by inhibiting microglial activation and/or ROS generation that occurs in Parkinson's disease.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Mesencéfalo/citología , Microglía/fisiología , Degeneración Nerviosa/metabolismo , Estrés Oxidativo/fisiología , Canales Catiónicos TRPV/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Análisis de Varianza , Animales , Antígenos CD/metabolismo , Capsaicina/uso terapéutico , Recuento de Células , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Interacciones Farmacológicas , Femenino , Lateralidad Funcional , Microglía/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/prevención & control , Neurotoxinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Fármacos del Sistema Sensorial/uso terapéutico , Sustancia Negra/efectos de los fármacos , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Parkinson's disease (PD) is characterized by degeneration of nigrostriatal dopaminergic (DA) neurons. Mice treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) exhibit microglial activation-induced oxidative stress and inflammation, and nigrostriatal DA neuronal damage, and thus serve as an experimental model of PD. Here, we report that fluoxetine, one of the most commonly prescribed antidepressants, prevents MPTP-induced degeneration of nigrostriatal DA neurons and increases striatal dopamine levels with the partial motor recovery. This was accompanied by inhibiting transient expression of proinflammatory cytokines and inducible nitric oxide synthase; and attenuating microglial NADPH oxidase activation, reactive oxygen species/reactive nitrogen species production, and consequent oxidative damage. Interestingly, fluoxetine was found to protect DA neuronal damage from 1-methyl-4-phenyl-pyridinium (MPP(+)) neurotoxicity in co-cultures of mesencephalic neurons and microglia but not in neuron-enriched mesencephalic cultures devoid of microglia. The present in vivo and in vitro findings show that fluoxetine may possess anti-inflammatory properties and inhibit glial activation-mediated oxidative stress. Therefore, we carefully propose that neuroprotection of fluoxetine might be associated with its anti-inflammatory properties and could be employed as novel therapeutic agents for PD and other disorders associated with neuroinflammation and microglia-derived oxidative damage.
Asunto(s)
Dopamina/metabolismo , Fluoxetina/uso terapéutico , Intoxicación por MPTP/prevención & control , Microglía/metabolismo , Degeneración Nerviosa/prevención & control , Neuronas/patología , Recuperación de la Función/efectos de los fármacos , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacocinética , 1-Metil-4-fenilpiridinio/antagonistas & inhibidores , 1-Metil-4-fenilpiridinio/farmacología , Animales , Recuento de Células/métodos , Técnicas de Cocultivo/métodos , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Citocinas/metabolismo , Fluoxetina/farmacología , Intoxicación por MPTP/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/inmunología , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Degeneración Nerviosa/inducido químicamente , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/efectos de los fármacos , Prueba de Desempeño de Rotación con Aceleración Constante/métodos , Sustancia NegraRESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative disorder caused by selective degeneration of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Although mitochondrial abnormality, oxidative stress and proteasomal dysfunction are recognized as major contributors to the progression of PD, there is a limited understanding of the key molecular events that provoke degeneration of DA neurons. Using a proteomic approach, we attempted to identify profiles of proteins with altered expression levels in rats following unilateral stereotaxic injection of 6-hydroxydopamine into the SNc. Protein expression profiles of these proteins in the substantia nigra and the striatum were made using two-dimensional gel electrophoresis in conjunction with a mass spectrometry. More than 70 identified proteins displayed significant differences in their temporal and spatial expression pattern between experimental and vehicle-operated control groups. Based on the identity of the proteins, we further searched for potential binding partners using biological databases available on the web and constructed a protein interaction network. Among several interconnected proteins in the network, we verified the interaction between prohibitin and the NADH-ubiquinone oxidoreductase 30kDa subunit (NDUFS3 subunit; a mitochondrial complex I subunit) by co-immunoprecipitation. We also confirmed, using immunohistochemical localization, that both prohibitin and the NDUFS3 subunit were increased in the dying DA neurons, suggesting its potential role in regulating mitochondrial function in dying DA neurons. Furthermore, knockdown of prohibitin accelerated 6-hydroxydopamine-induced cell death in SH-SY5Y cells. Our results raise the possibility that interconnected proteins in the network may positively or negatively impact the progression of DA neuronal death.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/patología , Modelos Animales de Enfermedad , Oxidopamina/toxicidad , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodos , Animales , Línea Celular Tumoral , Redes Reguladoras de Genes/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Masculino , Neuroblastoma/metabolismo , Neuroblastoma/patología , Trastornos Parkinsonianos/genética , Prohibitinas , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas Represoras/deficiencia , Proteínas Represoras/genéticaRESUMEN
Lipopolysaccharide (LPS)-induced microglial activation causes degeneration of nigral dopaminergic (DA) neurons. Here, we examined whether fluoxetine prevents LPS-induced degeneration of DA in the rat substantia nigra (SN) in vivo. Seven days after LPS injection into the SN, immunostaining for tyrosine hydroxylase (TH) revealed a significant loss of nigral DA neurons. Parallel activation of microglia (visualized by OX-42 and ED1 immunohistochemistry), production of reactive oxygen species (ROS) (assessed by hydroethidine histochemistry), and degeneration of nigral DA neurons were also observed in the SN. Western blot analyses and double-label immunohistochemistry showed an increase in the expression of inducible nitric oxide synthase (iNOS) within activated microglia. LPS also induced translocation of p67(phox), the cytosolic component of NADPH oxidase, to the membrane of SN microglia, indicating activation of NADPH oxidase. The LPS-induced loss of nigral DA neurons was partially inhibited by fluoxetine, and the observed neuroprotective effects were associated with fluoxetine-mediated suppression of microglial NADPH oxidase activation and iNOS upregulation, and decreased ROS generation and oxidative stress. These results suggest that fluoxetine and analogs thereof may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglia-derived oxidative damage.