RESUMO
Inhibition of glucosylceramide synthase (GCS) has been proposed as a therapeutic strategy for the treatment of Parkinson's Disease (PD), particularly in patients where glycosphingolipid accumulation and lysosomal impairment are thought to be contributing to disease progression. Herein, we report the late-stage optimization of an orally bioavailable and CNS penetrant isoindolinone class of GCS inhibitors. Starting from advanced lead 1, we describe efforts to identify an improved compound with a lower human dose projection, minimal P-glycoprotein (P-gp) efflux, and acceptable pregnane X receptor (PXR) profile through fluorine substitution. Our strategy involved the use of predicted volume ligand efficiency to advance compounds with greater potential for low human doses down our screening funnel. We also applied minimized electrostatic potentials (Vmin) calculations for hydrogen bond acceptor sites to rationalize P-gp SAR. Together, our strategies enabled the alignment of a lower human dose with reduced P-gp efflux, and favorable PXR selectivity for the discovery of compound 12.
RESUMO
Glutamate plays a key role in cognition and mood, and it has been shown that inhibiting ionotropic glutamate receptors disrupts cognition, while enhancing ionotropic receptor activity is pro-cognitive. One approach to elevating glutamatergic tone has been to antagonize presynaptic metabotropic glutamate receptor 2 (mGluR2). A desire for selectivity over the largely homologous mGluR3 motivated a strategy to achieve selectivity through the identification of mGluR2 negative allosteric modulators (NAMs). Extensive screening and optimization efforts led to the identification of a novel series of 4-arylquinoline-2-carboxamides. This series was optimized for mGluR2 NAM potency, clean off-target activity, and desirable physical properties, which resulted in the identification of improved C4 and C7 substituents. The initial lead compound from this series was Ames-positive in a single strain with metabolic activation, indicating that a reactive metabolite was likely responsible for the genetic toxicity. Metabolic profiling and Ames assessment across multiple analogs identified key structure-activity relationships associated with Ames positivity. Further optimization led to the Ames-negative mGluR2 negative allosteric modulator MK-8768.
RESUMO
Parkinson's disease is the second most prevalent progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations in GBA, the gene that encodes for the lysosomal enzyme glucosylcerebrosidase, are a major genetic risk factor for the development of Parkinson's disease potentially through the accumulation of glucosylceramide and glucosylsphingosine in the CNS. A therapeutic strategy to reduce glycosphingolipid accumulation in the CNS would entail inhibition of the enzyme responsible for their synthesis, glucosylceramide synthase (GCS). Herein, we report the optimization of a bicyclic pyrazole amide GCS inhibitor discovered through HTS to low dose, oral, CNS penetrant, bicyclic pyrazole urea GCSi's with in vivo activity in mouse models and ex vivo activity in iPSC neuronal models of synucleinopathy and lysosomal dysfunction. This was accomplished through the judicious use of parallel medicinal chemistry, direct-to-biology screening, physics-based rationalization of transporter profiles, pharmacophore modeling, and use a novel metric: volume ligand efficiency.
RESUMO
Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.
Assuntos
Benzoxazóis/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Glucosilceramidase/genética , Glucosiltransferases/antagonistas & inibidores , Glicoesfingolipídeos/metabolismo , Lisossomos/efeitos dos fármacos , Sinucleinopatias/metabolismo , alfa-Sinucleína/efeitos dos fármacos , Animais , Neurônios Dopaminérgicos/metabolismo , Técnicas In Vitro , Lisossomos/metabolismo , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Cultura Primária de Células , Agregados Proteicos , Ratos , Sinucleinopatias/genética , alfa-Sinucleína/metabolismoRESUMO
Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher's disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson's disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD. The importance of GCase in PD necessitates preclinical models in which to study GCase-related mechanisms and novel therapeutic approaches, as well as to elucidate the molecular mechanisms leading to enhanced PD risk in GBA1 mutation carriers. The aim of this study was to develop and characterize a novel GBA1 mouse model and to facilitate wide accessibility of the model with phenotypic data. Herein we describe the results of molecular, biochemical, histological, and behavioral phenotyping analyses in a GBA1 D409V knock-in (KI) mouse. This mouse model exhibited significantly decreased GCase activity in liver and brain, with substantial increases in glycosphingolipid substrates in the liver. While no changes in the number of dopamine neurons in the substantia nigra were noted, subtle changes in striatal neurotransmitters were observed in GBA1 D409V KI mice. Alpha-synuclein pathology and inflammation were not observed in the nigrostriatal system of this model. In summary, the GBA1 D409V KI mouse model provides an ideal model for studies aimed at pharmacodynamic assessments of potential therapies aiming to restore GCase.
Assuntos
Glucosilceramidase/metabolismo , Glicoesfingolipídeos/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Técnicas de Introdução de Genes , Glucosilceramidase/genética , Immunoblotting , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Parkinsonianos/enzimologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Mutação Puntual/genéticaRESUMO
Multiple genome-wide association studies (GWAS) in Parkinson disease (PD) have identified a signal at chromosome 4p16.3; however, the causal variant has not been established for this locus. Deep investigation of the region resulted in one identified variant, the rs34311866 missense SNP (p.M393T) in TMEM175, which is 20 orders of magnitude more significant than any other SNP in the region. Because TMEM175 is a lysosomal gene that has been shown to influence α-synuclein phosphorylation and autophagy, the p.M393T variant is an attractive candidate, and we have examined its effect on TMEM175 protein and PD-related biology. After knocking down each of the genes located under the GWAS peak via multiple shRNAs, only TMEM175 was found to consistently influence accumulation of phosphorylated α-synuclein (p-α-syn). Examination of the p.M393T variant showed effects on TMEM175 function that were intermediate between the wild-type (WT) and knockout phenotypes, with reduced regulation of lysosomal pH in response to starvation and minor changes in clearance of autophagy substrates, reduced lysosomal localization, and increased accumulation of p-α-syn. Finally, overexpression of WT TMEM175 protein reduced p-α-syn, while overexpression of the p.M393T variant resulted in no change in α-synuclein phosphorylation. These results suggest that the main signal in the chromosome 4p16.3 PD risk locus is driven by the TMEM175 p.M393T variant. Modulation of TMEM175 may impact α-synuclein biology and therefore may be a rational therapeutic strategy for PD.
Assuntos
Doença de Parkinson/genética , Polimorfismo de Nucleotídeo Único , Canais de Potássio/genética , alfa-Sinucleína/metabolismo , Linhagem Celular , Cromossomos Humanos Par 4/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Fosforilação , Canais de Potássio/metabolismoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinson's disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis. A high throughput screen of our compound collection afforded a number of promising indazole leads which were truncated in order to identify a minimum pharmacophore. Further optimization of these indazoles led to the development of MLi-2 (1): a potent, highly selective, orally available, brain-penetrant inhibitor of LRRK2.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indazóis/química , Indazóis/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Indazóis/administração & dosagem , Indazóis/farmacocinética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Simulação de Acoplamento Molecular , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/enzimologia , Ratos , Ratos WistarRESUMO
Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD.
Assuntos
Autofagossomos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Canais de Potássio/genética , alfa-Sinucleína/química , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/patologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Lisossomos/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Modelos Biológicos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Canais de Potássio/deficiência , Cultura Primária de Células , Agregados Proteicos/efeitos dos fármacos , Ratos , alfa-Sinucleína/farmacologiaRESUMO
Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiofenos/farmacologia , Administração Oral , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
Neuronal inclusions of hyperphosphorylated and aggregated tau protein are a pathological hallmark of several neurodegenerative tauopathies, including Alzheimer's disease (AD). The hypothesis of tau transmission in AD has emerged from histopathological studies of the spatial and temporal progression of tau pathology in postmortem patient brains. Increasing evidence in cellular and animal models supports the phenomenon of intercellular spreading of tau. However, the molecular and cellular mechanisms of pathogenic tau transmission remain unknown. The studies described herein investigate tau pathology propagation using human neurons derived from induced pluripotent stem cells. Neurons were seeded with full-length human tau monomers and oligomers and chronic effects on neuronal viability and function were examined over time. Tau oligomer-treated neurons exhibited an increase in aggregated and phosphorylated pathological tau. These effects were associated with neurite retraction, loss of synapses, aberrant calcium homeostasis, and imbalanced neurotransmitter release. In contrast, tau monomer treatment did not produce any measureable changes. This work supports the hypothesis that tau oligomers are toxic species that can drive the spread of tau pathology and neurodegeneration. SIGNIFICANCE STATEMENT: Several independent studies have implicated tau protein as central to Alzheimer's disease progression and cell-to-cell pathology propagation. In this study, we investigated the ability of different tau species to propagate pathology in human neurons derived from induced pluripotent stem cells, which to date has not been shown. We demonstrated that tau oligomers, but not monomers, induce accumulation of pathological, hyperphosphorylated tau. This effect was accompanied with neurite degeneration, loss of synapses, aberrant calcium homeostasis, imbalanced neurotransmitter release, and ultimately with neuronal death. This study bridges various tau pathological phenotypes into a single and relevant induced pluripotent stem cell neuronal model of human disease that can be applied to the discovery of the mechanisms of tau-induced neurodegeneration.
Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Proteínas tau/metabolismo , Proteínas tau/toxicidade , Análise de Variância , Cálcio/metabolismo , Sobrevivência Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Masculino , Microfluídica , Microscopia de Força Atômica , Neurotransmissores/metabolismo , Fosforilação , Transporte Proteico/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas tau/químicaRESUMO
Aging, the main risk factor for Parkinson's disease (PD), is associated with increased α-synuclein levels in substantia nigra pars compacta (SNc). Excess α-synuclein spurs Lewy-like pathology and dysregulates the activity of protein phosphatase 2A (PP2A). PP2A dephosphorylates many neuroproteins, including the catecholamine rate-limiting enzyme, tyrosine hydroxylase (TH). A loss of nigral dopaminergic neurons induces PD movement problems, but before those abnormalities occur, behaviors such as olfactory loss, anxiety, and constipation often manifest. Identifying mouse models with early PD behavioral changes could provide a model in which to test emerging therapeutic compounds. To this end, we evaluated mice expressing A53T mutant human (A53T) α-synuclein for behavior and α-synuclein pathology in olfactory bulb, adrenal gland, and gut. Aging A53T mice exhibited olfactory loss and anxiety that paralleled olfactory and adrenal α-synuclein aggregation. PP2A activity was also diminished in olfactory and adrenal tissues harboring insoluble α-synuclein. Low adrenal PP2A activity co-occurred with TH hyperactivity, making this the first study to link adrenal synucleinopathy to anxiety and catecholamine dysregulation. Aggregated A53T α-synuclein recombinant protein also had impaired stimulatory effects on soluble recombinant PP2A. Collectively, the data identify an excellent model in which to screen compounds for their ability to block the spread of α-synuclein pathology associated with pre-motor stages of PD.
Assuntos
Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , alfa-Sinucleína/genética , Glândulas Suprarrenais/enzimologia , Glândulas Suprarrenais/metabolismo , Envelhecimento/patologia , Animais , Ansiedade/genética , Ansiedade/psicologia , Western Blotting , Química Encefálica/fisiologia , Progressão da Doença , Alimentos , Trato Gastrointestinal/enzimologia , Trato Gastrointestinal/metabolismo , Genótipo , Hipercinese/genética , Hipercinese/psicologia , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Atividade Motora/fisiologia , Neurônios/fisiologia , Transtornos Parkinsonianos/enzimologia , Proteína Fosfatase 2/metabolismo , Olfato/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common forms of inheritable Parkinson's disease and likely play a role in sporadic disease as well. LRRK2 is a large multidomain protein containing two key groups, a Ras-like GTP binding domain and a serine, threonine kinase domain. Mutations in the LRRK2 gene that associate with Parkinson's disease reside primarily within the two functional domains of the protein, suggesting that LRRK2 function is critical to the pathogenesis of the disease. The most common LRRK2 mutation increases kinase activity, making LRRK2 kinase inhibition an attractive target for small molecule drug development. However, the physiological function of LRRK2 kinase as well as its endogenous protein substrates remains poorly understood and has hindered drug development efforts. Recent advances in LRRK2 biology have revealed several potential cellular roles, interacting proteins, and putative physiological substrates. Together, a picture emerges of a complex multifunctional protein that exists in multiple cellular compartments. Through unclear mechanisms, LRRK2 kinase regulates cytoskeleton architecture through control of protein translation, phosphorylation of cytoskeletal proteins, and response to cellular stressors. This article will briefly cover some interesting recent studies in LRRK2 cellular biology and highlight emerging cellular models of LRRK2 kinase function.
Assuntos
Neurônios/fisiologia , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Proteínas do Citoesqueleto/ultraestrutura , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Neurônios/enzimologia , Doença de Parkinson/patologia , Biossíntese de Proteínas/genética , Modificação Traducional de Proteínas/genética , Proteínas Serina-Treonina Quinases/química , Especificidade por Substrato/genética , Especificidade por Substrato/fisiologiaRESUMO
Gastrointestinal disorders, particularly severe constipation and delayed gastric emptying, are core symptoms of Parkinson's disease that affect most patients. However, the neuropathological substrate and physiological basis for this dysfunction are poorly defined. To begin to explore these phenomena in laboratory models of PD, rats were treated with either vehicle or rotenone (2.0 mg/kg, i.p.; 5 days/week) for 6-weeks. Myenteric plexus alpha-synuclein aggregate pathology and neuron loss were assessed 3-days and 6-months after the last rotenone injection. Gastrointestinal motility was assessed at 3-days, 1-month and 6-months after the last rotenone injection. Rotenone treatment caused an acute reduction in alpha-synuclein-immunoreactivity, but this was followed 6 months later by a robust increase in aggregate pathology and cytoplasmic inclusions that were similar in appearance to enteric Lewy-bodies in idiopathic PD. Rotenone-treated rats also had a moderate but permanent loss of small intestine myenteric neurons and an associated modest slowing of gastrointestinal motility 6-months after treatment. Our results suggest that a circumscribed exposure to an environmental toxicant can cause the delayed appearance of parkinsonian alpha-synuclein pathology in the enteric nervous system and an associated functional deficit in gastrointestinal motility. The rotenone model may therefore, provide a means to investigate pathogenic mechanisms and to test new therapeutic interventions into gastrointestinal dysfunction in PD.
Assuntos
Gastroenteropatias/induzido quimicamente , Gastroenteropatias/patologia , Inseticidas/farmacologia , Rotenona/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 3 , Ingestão de Alimentos/efeitos dos fármacos , Formiatos/farmacologia , Gastroenteropatias/etiologia , Gastroenteropatias/metabolismo , Motilidade Gastrointestinal/efeitos dos fármacos , Masculino , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/etiologia , Doença de Parkinson/patologia , Ratos , Ratos Endogâmicos Lew , Estatísticas não Paramétricas , alfa-Sinucleína/efeitos dos fármacos , alfa-Sinucleína/metabolismoRESUMO
The systemic rotenone model of Parkinson's disease (PD) accurately replicates many aspects of the pathology of human PD and has provided insights into the pathogenesis of PD. The major limitation of the rotenone model has been its variability, both in terms of the percentage of animals that develop a clear-cut nigrostriatal lesion and the extent of that lesion. The goal here was to develop an improved and highly reproducible rotenone model of PD. In these studies, male Lewis rats in three age groups (3, 7 or 12-14 months) were administered rotenone (2.75 or 3.0 mg/kg/day) in a specialized vehicle by daily intraperitoneal injection. All rotenone-treated animals developed bradykinesia, postural instability, and/or rigidity, which were reversed by apomorphine, consistent with a lesion of the nigrostriatal dopamine system. Animals were sacrificed when the PD phenotype became debilitating. Rotenone treatment caused a 45% loss of tyrosine hydroxylase-positive substantia nigra neurons and a commensurate loss of striatal dopamine. Additionally, in rotenone-treated animals, alpha-synuclein and poly-ubiquitin positive aggregates were observed in dopamine neurons of the substantia nigra. In summary, this version of the rotenone model is highly reproducible and may provide an excellent tool to test new neuroprotective strategies.
Assuntos
Discinesia Induzida por Medicamentos/fisiopatologia , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/fisiopatologia , Rotenona/toxicidade , Substância Negra/efeitos dos fármacos , Substância Negra/fisiopatologia , Animais , Modelos Animais de Doenças , Dopamina/deficiência , Discinesia Induzida por Medicamentos/patologia , Hipocinesia/induzido quimicamente , Injeções Intraperitoneais , Masculino , Rigidez Muscular/induzido quimicamente , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurotoxinas/toxicidade , Transtornos Parkinsonianos/patologia , Ratos , Ratos Endogâmicos Lew , Reprodutibilidade dos Testes , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Ubiquitinas/efeitos dos fármacos , Ubiquitinas/metabolismo , Desacopladores/toxicidade , alfa-Sinucleína/efeitos dos fármacos , alfa-Sinucleína/metabolismoRESUMO
The objective of this study was to determine if the phosphodiesterase 5 (PDE-5) inhibitor, sildenafil, could be used as a neuroprotective agent in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) murine model of Parkinson's disease. The underlying hypothesis of these studies is that blockade of PDE-5 catabolism of cGMP will attenuate the loss of nigrostriatal dopamine (NSDA) neurons following chronic neurotoxin exposure. Chronic MPTP-treated mice were administered sildenafil using three different regimens. Animals were: 1) treated with sildenafil and then exposed to chronic MPTP; 2) treated concurrently with sildenafil and MPTP; and 3) first exposed to MPTP and subsequently treated with sildenafil. End points of neurotoxicity included dopamine (DA) and tyrosine hydroxylase (TH) concentrations in NSDA axon terminals in the striatum, and stereological cell counts of TH immunoreactive neurons in the substantia nigra. Results reveal that sildenafil did not prevent neurotoxicity produced by chronic MPTP exposure regardless of the treatment paradigms employed. On the other hand, sildenafil did not produce any deleterious effect on NSDA neuron function nor did it potentiate the neurotoxic effects of MPTP. These results suggest that sildenafil would not accelerate DA cell loss when used as a treatment for erectile dysfunction in men diagnosed with Parkinson's disease.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transtornos Parkinsonianos/tratamento farmacológico , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Substância Negra/efeitos dos fármacos , Sulfonas/farmacologia , Sulfonas/uso terapêutico , Animais , Axônios/metabolismo , Western Blotting , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Fosfodiesterase 5 , Purinas/farmacologia , Purinas/uso terapêutico , Citrato de Sildenafila , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Many neurodegenerative diseases associated with functional Tau dysregulation, including Alzheimer's disease (AD) and other tauopathies, also show alpha-synuclein (alpha-Syn) pathology, a protein associated with Parkinson's disease (PD) pathology. Here we show that treatment of primary mesencephalic neurons (48 h) or subchronic treatment of wild-type (WT) mice with the Parkinsonism-inducing neurotoxin MPP+/MPTP, results in selective dose-dependent hyperphosphorylation of Tau at Ser396/404 (PHF-1-reactive Tau, p-Tau), with no changes in pSer202 but with nonspecific increases in pSer262 levels. The presence of alpha-Syn was absolutely mandatory to observe MPP+/MPTP-induced increases in p-Tau levels, since no alterations in p-Tau were seen in transfected cells not expressing alpha-Syn or in alpha-Syn-/- mice. MPP+/MPTP also induced a significant accumulation of alpha-Syn in both mesencephalic neurons and in WT mice striatum. MPTP/MPP+ lead to differential alterations in p-Tau and alpha-Syn levels in a cytoskeleton-bound, vs. a soluble, cytoskeleton-free fraction, inducing their coimmunoprecipitation in the cytoskeleton-free fraction and neuronal soma. Subchronic MPTP exposure increased sarkosyl-insoluble p-Tau in striatum of WT but not alpha-Syn-/- mice. These studies describe a novel mechanism for MPTP neurotoxicity, namely a MPTP-inducible, strictly alpha-Syn-dependent, increased formation of PHF-1-reactive Tau, suggesting convergent overlapping pathways in the genesis of clinically divergent diseases such as AD and PD.
Assuntos
Intoxicação por MPTP/fisiopatologia , alfa-Sinucleína/genética , Proteínas tau/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Modelos Animais de Doenças , Humanos , Intoxicação por MPTP/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Transfecção , alfa-Sinucleína/deficiência , alfa-Sinucleína/metabolismoRESUMO
Tyrosine hydroxylase (TH) protein, phosphorylated at serine-40, serine-31 and serine-19, and enzyme catalytic activity were compared under basal conditions and in activated nigrostriatal dopamine (NSDA) neurons of wild-type and homozygous alpha-synuclein knockout mice. Mice were injected with the D2 antagonist raclopride to stimulate NSDA neuronal activity in the presence or absence of supplemental l-tyrosine. There was no difference in phosphorylated TH levels or TH catalytic activity between wild-type and alpha-synuclein knockout mice under basal conditions or following raclopride-induced acceleration of NSDA activity. In wild-type animals, tyrosine administration potentiated the raclopride-induced increase in phosphorylated TH and enzyme activity. However, tyrosine administration did not enhance phosphorylated TH levels or enzyme catalytic activity in raclopride-stimulated NSDA neurons in alpha-synuclein knockout mice. These findings suggest that alpha-synuclein plays a role in the ability of tyrosine to either enhance TH phosphorylation or hinder TH inactivation during accelerated neuronal activity. The present study supports the hypothesis that alpha-synuclein functions as a molecular chaperone protein that regulates the phosphorylation state of TH in a substrate and activity-dependent manner.
Assuntos
Corpo Estriado/fisiologia , Neurônios/fisiologia , Substância Negra/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/fisiologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Ácido Homovanílico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Fosforilação , Reação em Cadeia da Polimerase , Racloprida/farmacologia , Substância Negra/efeitos dos fármacos , alfa-Sinucleína/deficiência , alfa-Sinucleína/genéticaRESUMO
The functional role of alpha-synuclein in the pathogenesis of Parkinson's disease (PD) is not fully understood. Systemic exposure of alpha-synuclein-deficient mice to neurotoxins provides a direct approach to evaluate how alpha-synuclein may mediate cell death in a common murine model of PD. To this end, wild-type and homozygous alpha-synuclein knock-out mice were treated with sub-chronic and prolonged, chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In the sub-chronic model, wild-type and alpha-synuclein knock-out mice were treated for five consecutive days with MPTP (1-25 mg/kg, s.c.) or vehicle, and sacrificed 3 days following the last injection. The prolonged, chronic model consisted of two injections of MPTP (1-20 mg/kg, s.c.) per week for 5 weeks, with co-administration of probenecid (250 mg/kg, i.p.), and animals were sacrificed 3 weeks following the last injection. Sub-chronic administration of MPTP caused a dramatic, dose-dependent decrease in striatal dopamine (DA) concentrations, while an attenuated response was observed in alpha-synuclein knock-out mice. Similarly, prolonged, chronic administration of MPTP produced a dose-dependent decrease in striatal DA concentrations, and a corresponding loss of striatal vesicular monoamine transporter (VMAT-2) protein in wild-type mice. However, mice lacking alpha-synuclein had an attenuated loss of striatal DA concentrations, while no loss of striatal VMAT-2 protein was observed. Both sub-chronic and prolonged, chronic administration of MPTP caused an increase in the 3,4-dihydroxyphenylacetic acid (DOPAC) to DA ratio in wild-type mice, but not in mice lacking alpha-synuclein. Despite attenuated toxicity, elevated lactate concentrations were observed in alpha-synuclein knock-out mice following prolonged, chronic MPTP administration. The results of this study provide evidence that alpha-synuclein null mice have an attenuated response to the toxic effects of MPTP exposure, even over prolonged periods of time and that the biochemical sequela of a protracted insult to nigrostriatal DA neurons are distinct between mice with and without alpha-synuclein expression.