RESUMEN
Tyrosine hydroxylase (TH) catalyzes hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine, the initial and rate-limiting step in the synthesis of dopamine, noradrenaline, and adrenaline. Mutations in the human TH gene are associated with hereditary motor disorders. The common C886T mutation identified in the mouse Th gene results in the R278H substitution in the enzyme molecule. We investigated the impact of this mutation on the TH activity in the mouse midbrain. The TH activity in the midbrain of Mus musculus castaneus (CAST) mice homozygous for the 886C allele was higher compared to C57BL/6 and DBA/2 mice homozygous for the 886T allele. Notably, this difference in the enzyme activity was not associated with changes in the Th gene mRNA levels and TH protein content. Analysis of the TH activity in the midbrain in mice from the F2 population obtained by crossbreeding of C57BL/6 and CAST mice revealed that the 886C allele is associated with a high TH activity. Moreover, this allele showed complete dominance over the 886T allele. However, the C886T mutation did not affect the levels of TH protein in the midbrain. These findings demonstrate that the C886T mutation is a major genetic factor determining the activity of TH in the midbrain of common laboratory mouse strains. Moreover, it represents the first common spontaneous mutation in the mouse Th gene whose influence on the enzyme activity has been demonstrated. These results will help to understand the role of TH in the development of adaptive and pathological behavior, elucidate molecular mechanisms regulating the activity of TH, and explore pharmacological agents for modulating its function.
Asunto(s)
Ratones Endogámicos C57BL , Tirosina 3-Monooxigenasa , Animales , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Ratones , Mutación , Encéfalo/metabolismo , Ratones Endogámicos DBA , Mesencéfalo/metabolismo , Mesencéfalo/enzimología , Masculino , AlelosRESUMEN
We present a robust, fresh-frozen approach to immunohistochemistry (IHC), without committing the tissue to IHC via fixation and cryopreservation while maintaining long-term storage, using LiCor-based infrared (IR) quantification for sensitive assessment of TH in immunoreacted midbrain sections for quantitative comparison across studies. In fresh-frozen tissue stored up to 1 year prior to IHC reaction, we found our method to be highly sensitive to rotenone treatment in 3-month-old Sprague-Dawley rats, and correlated with a significant decline in rotarod latency-to-fall measurement by approximately 2.5 fold. The measured midbrain region revealed a 31 % lower TH signal when compared to control (p < 0.01 by t test, n = 5). Bivariate analysis of integrated TH counts versus rotarod latency-to-fall indicates a positive slope and modest but significant correlation of R2 = 0.68 (p < 0.05, n = 10). These results indicate this rapid, instrument-based quantification method by IR detection successfully quantifies TH levels in rat brain tissue, while taking only 5 days from euthanasia to data output. This approach also allows for the identification of multiple targets by IHC with the simultaneous performance of downstream molecular analysis within the same animal tissue, allowing for the use of fewer animals per study.
Asunto(s)
Mesencéfalo/química , Mesencéfalo/enzimología , Desempeño Psicomotor/fisiología , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/metabolismo , Animales , Insecticidas/toxicidad , Masculino , Mesencéfalo/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Rotenona/toxicidad , Espectroscopía Infrarroja Corta/métodos , Factores de TiempoRESUMEN
Parkinson's disease is characterized by a loss of dopaminergic neurons in the ventral midbrain. This disease is diagnosed when around 50% of these neurons have already died; consequently, therapeutic treatments start too late. Therefore, an urgent need exists to find new targets involved in the onset and progression of the disease. Phosphodiesterase 7 (PDE7) is a key enzyme involved in the degradation of intracellular levels of cyclic adenosine 3', 5'-monophosphate in different cell types; however, little is known regarding its role in neurodegenerative diseases, and specifically in Parkinson's disease. We have previously shown that chemical as well as genetic inhibition of this enzyme results in neuroprotection and anti-inflammatory activity in different models of neurodegenerative disorders, including Parkinson's disease. Here, we have used in vitro and in vivo models of Parkinson's disease to study the regulation of PDE7 protein levels. Our results show that PDE7 is upregulated after an injury both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures and after lipopolysaccharide or 6-hidroxydopamine injection in the Substantia nigra pars compacta of adult mice. PDE7 increase takes place mainly in degenerating dopaminergic neurons and in microglia cells. This enhanced expression appears to be direct since 6-hydroxydopamine and lipopolysaccharide increase the expression of a 962-bp fragment of its promoter. Taking together, these results reveal an essential function for PDE7 in the pathways leading to neurodegeneration and inflammatory-mediated brain damage and suggest novel roles for PDE7 in neurodegenerative diseases, specifically in PD, opening the door for new therapeutic interventions.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/metabolismo , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/patología , Animales , Apoptosis , Línea Celular , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/genética , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/enzimología , Neuronas Dopaminérgicas/patología , Embrión de Mamíferos/enzimología , Humanos , Masculino , Mesencéfalo/enzimología , Mesencéfalo/patología , Neuroglía/enzimología , Neuroglía/patología , Oxidopamina , Regiones Promotoras Genéticas/genética , Ratas Wistar , Sustancia Negra/enzimología , Sustancia Negra/patologíaRESUMEN
Parkinson's disease (PD) is a prevalent neurodegenerative movement disorder that is characterized pathologically by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Despite intensive research, the etiology of PD remains poorly understood. Interestingly, recent studies have implicated neuronal energy dysregulation as one of the key perpetrators of the disease. Supporting this, we have recently demonstrated that pharmacological or genetic activation of AMP kinase (AMPK), a master regulator of cellular energy homeostasis, rescues the pathological phenotypes of Drosophila models of PD. However, little is known about the role of AMPK in the mammalian brain. As an initial attempt to clarify this, we examined the expression of AMPK in rodent brains and found that phospho-AMPK (pAMPK) is disproportionately distributed in the adult mouse brain, being high in the ventral midbrain where the SN resides and relatively lower in regions such as the cortex-reflecting perhaps the unique energy demands of midbrain DA neurons. Importantly, the physiologically higher level of midbrain pAMPK is significantly reduced in aged mice and also in Parkin-deficient mice; the loss of function of which in humans causes recessive Parkinsonism. Not surprisingly, the expression of PGC-1α, a downstream target of AMPK activity, and a key regulator of mitochondrial biogenesis, mirrors the expression pattern of pAMPK. Similar observations were made with PINK1-deficient mice. Finally, we showed that metformin administration restores the level of midbrain pAMPK and PGC-1α expression in Parkin-deficient mice. Taken together, our results suggest that the disruption of AMPK-PGC-1α axis in the brains of individuals with Parkin or PINK1 mutations may be a precipitating factor of PD, and that pharmacological AMPK activation may represent a neuroprotective strategy for the disease.
Asunto(s)
Adenilato Quinasa/metabolismo , Mesencéfalo/enzimología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Asociadas a la Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/deficiencia , Ubiquitina-Proteína Ligasas/deficiencia , Envejecimiento/metabolismo , Animales , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Evaluación Preclínica de Medicamentos , Metabolismo Energético , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Metformina/farmacología , Metformina/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Especificidad de Órganos , Proteínas Asociadas a la Enfermedad de Parkinson/deficiencia , Proteínas Asociadas a la Enfermedad de Parkinson/genética , Porción Compacta de la Sustancia Negra/enzimología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/biosíntesis , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Fosforilación , Proteínas Quinasas/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Common disorders, including diabetes and Parkinson's disease, are caused by a combination of environmental factors and genetic susceptibility. However, defining the mechanisms underlying gene-environment interactions has been challenging due to the lack of a suitable experimental platform. Using pancreatic ß-like cells derived from human pluripotent stem cells (hPSCs), we discovered that a commonly used pesticide, propargite, induces pancreatic ß-cell death, a pathological hallmark of diabetes. Screening a panel of diverse hPSC-derived cell types we extended this observation to a similar susceptibility in midbrain dopamine neurons, a cell type affected in Parkinson's disease. We assessed gene-environment interactions using isogenic hPSC lines for genetic variants associated with diabetes and Parkinson's disease. We found GSTT1-/- pancreatic ß-like cells and dopamine neurons were both hypersensitive to propargite-induced cell death. Our study identifies an environmental chemical that contributes to human ß-cell and dopamine neuron loss and validates a novel hPSC-based platform for determining gene-environment interactions.
Asunto(s)
Ciclohexanos/toxicidad , Diabetes Mellitus/inducido químicamente , Neuronas Dopaminérgicas/efectos de los fármacos , Interacción Gen-Ambiente , Células Secretoras de Insulina/efectos de los fármacos , Plaguicidas/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Diferenciación Celular , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/enzimología , Glutatión Transferasa/deficiencia , Glutatión Transferasa/genética , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/enzimología , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Mesencéfalo/enzimología , Ratones , Modelos Biológicos , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/enzimologíaRESUMEN
Kyotorphin (KTP; L-tyrosyl-l-arginine), an opioid-like analgesic discovered in the bovine brain, is potentially a neuromodulator because of its localization in synaptosomes, the existence of a specific KTP receptor, and the presence of its biosynthetic enzyme in the brain. KTP is formed in the brain from its constituent amino acids, L-tyrosine and L-arginine, by an enzyme termed KTP synthetase. However, the latter has never been identified. We aimed to test the hypothesis that tyrosyl-tRNA synthetase (TyrRS) is also KTP synthetase. We found that recombinant hTyrRS synthesizes KTP from tyrosine, arginine, and ATP, with Kmâ¯=â¯1400⯵M and 200⯵M for arginine and tyrosine, respectively. TyrRS knockdown of PC12 cells with a small interfering RNA (siRNA) in the presence of 1.6â¯mM tyrosine, arginine, proline, or tryptophan significantly reduced the level of KTP, but not those of tyrosine-tyrosine, tyrosine-proline, or tyrosine-tryptophan. siRNA treatment did not affect cell survival or proliferation. In mice, TyrRS levels were found to be greater in the midbrain and medulla oblongata than in other brain regions. When arginine was administered 2â¯h prior to brain dissection, the KTP levels in these regions plus olfactory bulb significantly increased, although basal brain KTP levels remained relatively even. Our conclusion is further supported by a positive correlation across brain regions between TyrRS expression and arginine-accelerated KTP production.
Asunto(s)
Endorfinas/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Bulbo Raquídeo/enzimología , Mesencéfalo/enzimología , Péptido Sintasas/biosíntesis , Tirosina-ARNt Ligasa/biosíntesis , Animales , Endorfinas/genética , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Bulbo Raquídeo/citología , Mesencéfalo/citología , Ratones , Células PC12 , Péptido Sintasas/genética , Ratas , Tirosina-ARNt Ligasa/genéticaRESUMEN
Mitochondrial and lysosomal dysfunction have been implicated in substantia nigra dopaminergic neurodegeneration in Parkinson's disease (PD), but how these pathways are linked in human neurons remains unclear. Here we studied dopaminergic neurons derived from patients with idiopathic and familial PD. We identified a time-dependent pathological cascade beginning with mitochondrial oxidant stress leading to oxidized dopamine accumulation and ultimately resulting in reduced glucocerebrosidase enzymatic activity, lysosomal dysfunction, and α-synuclein accumulation. This toxic cascade was observed in human, but not in mouse, PD neurons at least in part because of species-specific differences in dopamine metabolism. Increasing dopamine synthesis or α-synuclein amounts in mouse midbrain neurons recapitulated pathological phenotypes observed in human neurons. Thus, dopamine oxidation represents an important link between mitochondrial and lysosomal dysfunction in PD pathogenesis.
Asunto(s)
Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Lisosomas/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Enfermedad de Parkinson/metabolismo , Animales , Antioxidantes/farmacología , Inhibidores de la Calcineurina/farmacología , Línea Celular , Modelos Animales de Enfermedad , Glucosilceramidasa/deficiencia , Humanos , Melaninas/metabolismo , Mesencéfalo/enzimología , Mesencéfalo/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Proteína Desglicasa DJ-1/genética , Sustancia Negra/enzimología , Sustancia Negra/metabolismo , Tacrolimus/farmacología , alfa-Sinucleína/metabolismoRESUMEN
The identity of higher-order neurons and circuits playing an associative role to control renal function is not well understood. We identified specific neural populations of rostral elements of brain regions that project multisynaptically to the kidneys in 3-6 days after injecting a retrograde tracer pseudorabies virus (PRV)-614 into kidney of 13 adult male C57BL/6J strain mice. PRV-614 infected neurons were detected in a number of mesencephalic (e.g. central amygdala nucleus), telencephalic regions and motor cortex. These divisions included the preoptic area (POA), dorsomedial hypothalamus (DMH), lateral hypothalamus, arcuate nucleus (Arc), suprachiasmatic nucleus (SCN), periventricular hypothalamus (PeH), and rostral and caudal subdivision of the paraventricular nucleus of the hypothalamus (PVN). PRV-614/Tyrosine hydroxylase (TH) double-labeled cells were found within DMH, Arc, SCN, PeH, PVN, the anterodorsal and medial POA. A subset of neurons in PVN that participated in regulating sympathetic outflow to kidney was catecholaminergic or serotonergic. PRV-614 infected neurons within the PVN also contained arginine vasopressin or oxytocin. These data demonstrate the rostral elements of brain innervate the kidney by the neuroanatomical circuitry.
Asunto(s)
Encéfalo/virología , Herpesvirus Suido 1/fisiología , Riñón/inervación , Vías Nerviosas , Animales , Encéfalo/enzimología , Masculino , Mesencéfalo/enzimología , Mesencéfalo/virología , Ratones , Ratones Endogámicos C57BL , Vías Nerviosas/anatomía & histología , Vías Nerviosas/virología , Núcleo Hipotalámico Paraventricular/enzimología , Núcleo Hipotalámico Paraventricular/virología , Telencéfalo/enzimología , Telencéfalo/virología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
In this study, to study the effect of rhynchophylline on TH in midbrain of methamphetamine-induced conditioned place preference (CPP) adult zebrafish, place preference adult zebrafish models were established by methamphetamine (40µg/g) and the expression of TH was observed by immunohistochemistry technique and Western blot. Ketamine (150µg/g), high dose of rhynchophylline (100µg/g) group can significantly reduce the place preference; immunohistochemistry results showed that the number of TH-positive neurons in midbrain was increased in the methamphetamine model group, whereas less TH-positive neurons were found in the ketamine group and high dosage rhynchophylline group. Western blot results showed that the expression of TH protein was significantly increased in the model group, whereas less expression was found in the ketamine group, high dosage rhynchophylline group. Our data pointed out that TH plays an important role in the formation of methamphetamine-induced place preference in adult zebrafish. Rhynchophylline reversed the expression of TH in the midbrain demonstrates the potential effect of mediates methamphetamine induced rewarding effect.
Asunto(s)
Condicionamiento Operante/efectos de los fármacos , Alcaloides Indólicos/farmacología , Metanfetamina/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Pez Cebra , Trastornos Relacionados con Anfetaminas , Animales , Conducta Animal/efectos de los fármacos , Ketamina/farmacología , Masculino , Mesencéfalo/citología , Mesencéfalo/enzimología , Neuronas/enzimología , OxindolesRESUMEN
Disruption of the cyclic feature of the day-night environment can cause negative effects on daily activity and advanced brain functions such as learning, memory and decision-making behaviour. These functions in songbirds, including corvids, involve the hippocampus, pallium and midbrain, as revealed by ZENK (a neuronal activation marker) and tyrosine hydroxylase (TH) expressions. TH is rate-limiting marker enzyme of the biosynthesis of dopamine, widely implicated in learning and memory. Here, we measured ZENK and TH immunoreactivity in the hippocampal, pallial and midbrain regions in response to cognitive performance (learning-memory retrieval) tests in Indian house crows (Corvus splendens) exposed to constant light environment (LL) with controls on 12h light:12h darkness. Along with the decay of circadian rhythm in activity behaviour, LL caused a significant decline in the cognitive performance. There was also a decrease under LL in the activity of neurons in the hippocampus, medial and central caudal nidopallium, and hyperpallium apicale, which are widely distributed with TH-immunoreactive fibres. Further, under LL, TH- immunoreactive neurons were reduced in number in midbrain dopamine synthesis sites, the venteral tegmental area (VTA) and substantia nigra (SN), with a negative correlation of co-localized ZENK/TH- immunoreactive cells on errors during the association tasks. These results show decreased activity of learning and memory neural systems, and underscore the role of dopamine in reduced cognitive performance of diurnal corvids with disrupted circadian rhythms under an abrupt light environment.
Asunto(s)
Hipocampo/enzimología , Aprendizaje/fisiología , Memoria/fisiología , Mesencéfalo/enzimología , Tirosina 3-Monooxigenasa/metabolismo , Animales , Cuervos , Dopamina/metabolismo , Luz , Neuronas/metabolismo , Pájaros Cantores , Sustancia Negra/enzimologíaRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder that leads to destruction of the midbrain dopaminergic (DA) neurons. This phenomenon is related to apoptosis and its activation can be blocked by the pituitary adenylate cyclase-activating polypeptide (PACAP). Growing evidence indicates that autophagy, a self-degradation activity that cleans up the cell, is induced during the course of neurodegenerative diseases. However, the role of autophagy in the pathogenesis of neuronal disorders is yet poorly understood and the potential ability of PACAP to modulate the related autophagic activation has never been significantly investigated. Hence, we explored the putative autophagy-modulating properties of PACAP in in vitro and in vivo models of PD, using the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively, to trigger alterations of DA neurons. In both models, following the toxin exposure, PACAP reduced the autophagic activity as evaluated by the production of LC3 II, the modulation of the p62 protein levels, and the formation of autophagic vacuoles. The ability of PACAP to inhibit autophagy was also observed in an in vitro cell assay by the blocking of the p62-sequestration activity produced with the autophagy inducer rapamycin. Thus, the results demonstrated that autophagy is induced in PD experimental models and that PACAP exhibits not only anti-apoptotic but also anti-autophagic properties.
Asunto(s)
Neuronas Dopaminérgicas/enzimología , Intoxicación por MPTP/enzimología , Mesencéfalo/enzimología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Animales , Línea Celular Tumoral , Neuronas Dopaminérgicas/patología , Inducción Enzimática , Humanos , Intoxicación por MPTP/genética , Intoxicación por MPTP/patología , Masculino , Mesencéfalo/patología , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genéticaRESUMEN
The experiment was conducted to investigate the effect of high dietary copper on catecholamine concentration and dopamine-ß-hydroxylase (DßH) activity in hypothalami and midbrains of growing pigs. Forty-five crossbred weanling pigs with an average body weight of 7.5 kg were randomly assigned to three groups of 15 each to receive a control diet containing 10 mg/kg Cu (diet A) and diets containing 125 (diet B) or 250 (diet C) mg Cu/kg DM for 45 days. Compared to the control, Cu supplementation at both 125 and 250 mg Cu/kg DM increased average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency. High dietary copper increased midbrain and hypothalami dopamine (DA) and norepinephrine (NE) concentrations and midbrain dopamine-ß-hydroxylase activity. However, increasing dietary Cu had no effect on hypothalami dopamine-ß-hydroxylase activity.
Asunto(s)
Catecolaminas/metabolismo , Cobre/administración & dosificación , Dieta , Hipotálamo/metabolismo , Mesencéfalo/metabolismo , Animales , Dopamina beta-Hidroxilasa/metabolismo , Hipotálamo/enzimología , Mesencéfalo/enzimología , PorcinosRESUMEN
Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here, we show that GABA co-release in dopamine neurons does not use the conventional GABA-synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol (EtOH) at concentrations seen in blood alcohol after binge drinking, and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Consumo Excesivo de Bebidas Alcohólicas , Neuronas Dopaminérgicas/metabolismo , Mesencéfalo/metabolismo , Recompensa , Ácido gamma-Aminobutírico/biosíntesis , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Animales , Consumo Excesivo de Bebidas Alcohólicas/sangre , Consumo Excesivo de Bebidas Alcohólicas/enzimología , Consumo Excesivo de Bebidas Alcohólicas/genética , Neuronas Dopaminérgicas/enzimología , Etanol/sangre , Etanol/farmacología , Evolución Molecular , Femenino , Técnicas de Silenciamiento del Gen , Masculino , Mesencéfalo/citología , Mesencéfalo/enzimología , Redes y Vías Metabólicas , Ratones , Retinal-Deshidrogenasa , Eliminación de SecuenciaRESUMEN
Diminished lysosomal function can lead to abnormal cellular accumulation of specific proteins, including α-synuclein, contributing to disease pathogenesis of vulnerable neurons in Parkinson's disease (PD) and related α-synucleinopathies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase (GCase), and mutations in GBA1 are a prominent genetic risk factor for PD. Previous studies showed that in sporadic PD, and in normal aging, GCase brain activity is reduced and levels of corresponding glycolipid substrates are increased. The present study tested whether increasing GCase through AAV-GBA1 intra-cerebral gene delivery in two PD rodent models would reduce the accumulation of α-synuclein and protect midbrain dopamine neurons from α-synuclein-mediated neuronal damage. In the first model, transgenic mice overexpressing wildtype α-synuclein throughout the brain (ASO mice) were used, and in the second model, a rat model of selective dopamine neuron degeneration was induced by AAV-A53T mutant α-synuclein. In ASO mice, intra-cerebral AAV-GBA1 injections into several brain regions increased GCase activity and reduced the accumulation of α-synuclein in the substantia nigra and striatum. In rats, co-injection of AAV-GBA1 with AAV-A53T α-synuclein into the substantia nigra prevented α-synuclein-mediated degeneration of nigrostriatal dopamine neurons by 6 months. These neuroprotective effects were associated with altered protein expression of markers of autophagy. These experiments demonstrate, for the first time, the neuroprotective effects of increasing GCase against dopaminergic neuron degeneration, and support the development of therapeutics targeting GCase or other lysosomal genes to improve neuronal handling of α-synuclein.
Asunto(s)
Neuronas Dopaminérgicas/enzimología , Terapia Genética/métodos , Glucosilceramidasa/genética , Mesencéfalo/enzimología , Enfermedades Neurodegenerativas/terapia , alfa-Sinucleína/metabolismo , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Vectores Genéticos , Glucosilceramidasa/metabolismo , Humanos , Masculino , Mesencéfalo/patología , Ratones Transgénicos , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/patología , Ratas Sprague-Dawley , alfa-Sinucleína/genéticaRESUMEN
Rotenone induced neuronal toxicity in ventral mesencephalic (VM) dopaminergic (DA) neurons in culture is widely accepted as an important model for the investigation of Parkinson's disease (PD). However, little is known about developmental stage dependent toxic effects of rotenone on VM neurons in vitro. The objective of present study is to investigate the effect of rotenone on developing VM neurons at immature versus mature stages. Primary VM neurons were cultured in the absence of glial cells. Exposure of VM neurons to rotenone for 2 days induced cell death in both immature and mature neurons in a concentration-dependent manner, but to a greater extent in mature neurons. While rotenone-treated mature VM neurons showed α-synuclein aggregation and sensitivity to DA neurons, immature VM neurons exhibited only DA neuronal sensitivity but not α-synuclein aggregation. In addition, on rotenone treatment, enhancement of caspase-3 activity and reactive oxygen species (ROS) production were higher in mature VM neurons than in immature neurons. These results suggest that even though both mature and immature VM neurons are sensitive to rotenone, their manifestations differ from each other, with only mature VM neurons exhibiting Parkinsonian conditions.
Asunto(s)
Mesencéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Rotenona/toxicidad , Animales , Caspasa 3/metabolismo , Células Cultivadas , Curcumina/farmacología , Femenino , Mesencéfalo/enzimología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Inflammatory events involving activated microglia have been recognized to play an important role in pathogenesis of various neurodegenerative disorders including Parkinson disease. Compounds regulating activation profiles of microglia may provide therapeutic benefits for Parkinson disease characterized by degeneration of midbrain dopaminergic neurons. Here we examined the effect of macelignan, a compound derived from nutmeg, on inflammatory degeneration of midbrain dopaminergic neurons. Treatment of midbrain slice cultures with interferon (IFN)-γ and lipopolysaccharide (LPS) caused a substantial decrease in viable dopaminergic neurons and an increase in nitric oxide (NO) production indicated by extracellular nitrite accumulation. Application of macelignan (10 µM) concomitantly with LPS prevented the loss of dopaminergic neurons. Besides nitrite accumulation, up-regulation of inducible NO synthase protein expression in response to IFN-γ/LPS was confirmed by Western blotting, and immunohistochemical examination revealed expression of inducible NO synthase in a subpopulation of Iba-1-poitive microglia. However, macelignan did not affect any of these NO-related parameters. On the other hand, macelignan promoted expression of arginase-1 in midbrain slice cultures irrespective of the presence or the absence of IFN-γ/LPS treatment. Arginase-1 expression was mainly localized in a subpopulation of Iba-1-positive cells. Importantly, the neuroprotective effect of macelignan was antagonized by N(ω)-hydroxy-nor-L-arginine, a specific arginase inhibitor. The neuroprotective effect of macelignan was also prevented by GW9662, a peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Overall, these results indicate that macelignan, a compound with PPARγ agonist activity, can provide neuroprotective effect on dopaminergic neurons in an arginase-dependent but NO-independent manner.
Asunto(s)
Arginasa/biosíntesis , Neuronas Dopaminérgicas/enzimología , Lignanos/farmacología , Mesencéfalo/enzimología , Microglía/enzimología , Degeneración Nerviosa/enzimología , Animales , Animales Recién Nacidos , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Inflamación/patología , Lignanos/uso terapéutico , Mesencéfalo/efectos de los fármacos , Microglía/efectos de los fármacos , Degeneración Nerviosa/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Técnicas de Cultivo de Órganos , Ratas , Ratas WistarRESUMEN
Perinatal asphyxia (PA) is a leading cause of neuronal damage in newborns, resulting in long-term neurological and cognitive deficits, in part due to impairment of mesostriatal and mesolimbic neurocircuitries. The insult can be as severe as to menace the integrity of the genome, triggering the overactivation of sentinel proteins, including poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 overactivation implies increased energy demands, worsening the metabolic failure and depleting further NAD(+) availability. Using a global PA rat model, we report here evidence that hypoxia increases PARP-1 activity, triggering a signalling cascade leading to nuclear translocation of the NF-κB subunit p65, modulating the expression of IL-1ß and TNF-α, pro-inflammatory molecules, increasing apoptotic-like cell death in mesencephalon of neonate rats, monitored with Western blots, qPCR, TUNEL and ELISA. PARP-1 activity increased immediately after PA, reaching a maximum 1-8 h after the insult, while activation of the NF-κB signalling pathway was observed 8 h after the insult, with a >twofold increase of p65 nuclear translocation. IL-1ß and TNF-α mRNA levels were increased 24 h after the insult, together with a >twofold increase in apoptotic-like cell death. A single dose of the PARP-1 inhibitor nicotinamide (0.8 mmol/kg, i.p.), 1 h post delivery, prevented the effect of PA on PARP-1 activity, p65 translocation, pro-inflammatory cytokine expression and apoptotic-like cell death. The present study demonstrates that PA leads to PARP-1 overactivation, increasing the expression of pro-inflammatory cytokines and cell death in mesencephalon, effects prevented by systemic neonatal nicotinamide administration, supporting the idea that PARP-1 inhibition represents a therapeutic target against the effects of PA.
Asunto(s)
Asfixia Neonatal/metabolismo , Asfixia/metabolismo , Mesencéfalo/metabolismo , Niacinamida/administración & dosificación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Asfixia/enzimología , Asfixia Neonatal/enzimología , Humanos , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Mesencéfalo/efectos de los fármacos , Mesencéfalo/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Nitrative stress is a key component of the pathogenic process in Parkinson's disease (PD), but the relative roles of constitutive neuronal nitric oxide synthase (n-NOS) and inducible nitric oxide synthase (i-NOS) in glial cells remain unresolved. We have investigated the effects of a range of concentrations of the selective n-NOS inhibitor ARR17477, and the selective i-NOS inhibitor 1400W, on MPP(+)-induced cell death in foetal ventral mesencephalic (VM) dopaminergic cultures. MPP(+) induced a loss of TH-positive neurones accompanied by an increase in immunoreactivity for GFAP and OX-6 as markers of astrocytes and activated microglia, respectively, and induced i-NOS immunoreactivity. Unexpectedly, MPP(+) treatment did not induce 3-NT immunoreactivity in the cultures. ARR17477 and 1400W alone had no effect on the number of TH-positive cells or on the number of GFAP or OX-6 positive cells. ARR17477 did not prevent the MPP(+)-induced decrease in TH-positive neurones and had no effect on the increased number of GFAP- and OX-6-positive cells. By contrast, 1400W caused a concentration-dependent preservation of TH-positive neurones in the presence of MPP(+). It also significantly reduced the number of OX-6-immunoreactive cells and there was a small reduction in GFAP immunoreactivity. The results suggest a major role for i-NOS-mediated nitrative stress in microglia in MPP(+)-induced dopaminergic cell death and this may have important implications for developing neuroprotective strategies for PD.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Picolinas/toxicidad , Amidinas/farmacología , Animales , Anticuerpos Monoclonales/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Bencilaminas/farmacología , Recuento de Células , Células Cultivadas , Neuronas Dopaminérgicas/enzimología , Neuronas Dopaminérgicas/patología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Mesencéfalo/efectos de los fármacos , Mesencéfalo/enzimología , Mesencéfalo/patología , Microglía/efectos de los fármacos , Microglía/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/enzimología , Cultivo Primario de Células , Ratas Wistar , Tiofenos/farmacología , Tirosina/análogos & derivados , Tirosina/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
OBJECTIVE: Lesch-Nyhan disease (LND) is caused by congenital deficiency of the purine recycling enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Affected patients have a peculiar neurobehavioral syndrome linked with reductions of dopamine in the basal ganglia. The purpose of the current studies was to determine the anatomical basis for the reduced dopamine in human brain specimens collected at autopsy. METHODS: Histopathological studies were conducted using autopsy tissue from 5 LND cases and 6 controls. Specific findings were replicated in brain tissue from an HGprt-deficient knockout mouse using immunoblots, and in a cell model of HGprt deficiency by flow-activated cell sorting (FACS). RESULTS: Extensive histological studies of the LND brains revealed no signs suggestive of a degenerative process or other consistent abnormalities in any brain region. However, neurons of the substantia nigra from the LND cases showed reduced melanization and reduced immunoreactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. In the HGprt-deficient mouse model, immunohistochemical stains for TH revealed no obvious loss of midbrain dopamine neurons, but quantitative immunoblots revealed reduced TH expression in the striatum. Finally, 10 independent HGprt-deficient mouse MN9D neuroblastoma lines showed no signs of impaired viability, but FACS revealed significantly reduced TH immunoreactivity compared to the control parent line. INTERPRETATION: These results reveal an unusual phenomenon in which the neurochemical phenotype of dopaminergic neurons is not linked with a degenerative process. They suggest an important relationship between purine recycling pathways and the neurochemical integrity of the dopaminergic phenotype.
Asunto(s)
Dopamina/deficiencia , Neuronas Dopaminérgicas/patología , Síndrome de Lesch-Nyhan/genética , Síndrome de Lesch-Nyhan/patología , Mesencéfalo/enzimología , Mesencéfalo/patología , Fenotipo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Niño , Preescolar , Cuerpo Estriado/enzimología , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Dopamina/genética , Neuronas Dopaminérgicas/enzimología , Humanos , Hipoxantina Fosforribosiltransferasa/deficiencia , Hipoxantina Fosforribosiltransferasa/genética , Síndrome de Lesch-Nyhan/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Sustancia Negra/enzimología , Sustancia Negra/patología , Tirosina 3-Monooxigenasa/deficiencia , Tirosina 3-Monooxigenasa/genética , Adulto JovenRESUMEN
Parkinson's disease is a neurodegenerative disease whose hallmark pathological features include a selective loss of dopaminergic neurons in the midbrain. Ciclooxygenase-2 activity induction and oxidative stress have been implicated in the aetiology of Parkinson's disease and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of Parkinson disease. Upon administration of fish oil, melatonin and vitamin E, neuroprotective effects on MPTP-induced neurotoxicity have been indicated. The aim of this study was to investigate the time course and compare the potency of these agents alone, on several parameters such as COX-2 and lipid peroxides (LPO) products associated with MPTP neurotoxicity in midbrain homogenates of C57BL/6 mice. Using fish oil (0.0368 g EPA and 0.0184 g DHA, per day), melatonin (10 mg/kg/day), and vitamin E (50 mg/Kg/day) we have now shown that COX-2 activity, LPO and nitrite/nitrate levels were significantly increased in MPTP treated mice (p < 0.001) while fish oil, melatonin and vitamin E treatment were capable of decreasing significantly the outcome of all above noted parameters (p < 0.05). The effect of fish oil on COX-2 activity and nitrite/nitrate levels was more profound than that of vitamin E or melatonin while the latter was more effective on reducing the LPO levels compared to fish oil and vitamin E. In conclusion, the outcome of the neuroprotective effects of these agents is long lasting and of variable potency indicating a different anti-inflammatory mode of action.