RESUMO
The accumulation of α-synuclein inclusions in vulnerable neuronal populations pathologically defines Lewy body diseases including Parkinson's disease. Recent pre-clinical studies suggest poly(ADP-ribose) polymerase-1 activation and the subsequent generation of poly(ADP-ribose) polymer represent key steps in the formation of toxic α-synuclein aggregates and neurodegeneration. Several studies suggest that the inhibition of poly(ADP-ribose) polymerase-1 activity via the poly(ADP-ribose) polymerase-1/2 small molecule inhibitor ABT-888 (Veliparib), a drug in clinical trials for different cancers, may prevent or ameliorate α-synuclein fibril-induced aggregation, inclusion formation and dopaminergic neurodegeneration. Herein, we evaluated the effects of poly(ADP-ribose) polymer on α-synuclein fibrillization in vitro, the effects of ABT-888 on the formation of fibril-seeded α-synuclein inclusions in primary mouse cortical neurons and the effects of an in-diet ABT-888 dosage regimen with the intracranial injection of α-synuclein fibrils into the mouse dorsal striatum. We found that poly(ADP-ribose) polymer minimally but significantly increased the rate of spontaneously formed α-synuclein fibrils in vitro. Machine-learning algorithms that quantitatively assessed α-synuclein inclusion counts in neurons, both in primary cultures and in the brains of fibril-injected mice, did not reveal differences between ABT-888- and vehicle-treated groups. The in-diet administered ABT-888 molecule demonstrated outstanding brain penetration in mice; however, dopaminergic cell loss in the substantia nigra caused by α-synuclein fibril injections in the striatum was similar between ABT-888- and vehicle-treated groups. α-Synuclein fibril-induced loss of dopaminergic fibres in the dorsal striatum was also similar between ABT-888- and vehicle-treated groups. We conclude that additional pre-clinical evaluation of ABT-888 may be warranted to justify further exploration of ABT-888 for disease modification in Lewy body diseases.
RESUMO
Alpha-synuclein (αSyn) pathology is a hallmark of Parkinson's disease. Here we show that lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a regulator of αSyn pathology and cytotoxicity. LPCAT1 is upregulated by αSyn E35K E46K E61K (3K) in human M17 neuroblastoma cells and primary rat cortical neurons, and in postmortem brain tissue from PD patients with confirmed αSyn aggregate pathology. Suppression of LPCAT1 reduces αSyn accumulations and toxicity in our neuroblastoma αSyn 3K overexpression model. Further overexpression of LPCAT1 promotes pS129 αSyn positive aggregation in primary neurons in the αSyn pre-formed fibril (PFF) model. A phospholipid product of LPCAT1 enzymatic activity, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, similarly promotes neuronal PFF seeded aggregation. Using a pH sensitive PFF model we provide evidence that αSyn fibrils have altered endo-lysosomal processing under LPCAT1 enhancement, suggesting less aggregate degradation. Our data demonstrates that LPCAT1 and associated phospholipids can regulate αSyn pathology.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Doença de Parkinson/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/patologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fosforilcolina/metabolismo , Ratos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Here, we report the independent discovery and validation of stearoyl-CoA desaturase (SCD) as a modulator of α-synuclein (αSyn)-induced pathology and toxicity in cell-based Parkinson's disease (PD) models. We identified SCD as top altered gene from transcriptional profiling in primary neurons exogenously expressing αSyn with the amplified familial PD mutation 3K. Thus, we sought to further explore SCD as a therapeutic target in neurodegeneration. We report that SCD inhibitors are toxic to early human and rat neuron cultures while displaying minimal toxicity to late cultures. The fatty acid product of SCD, oleic acid (OLA), fully rescues this toxicity in early cultures, suggesting on-target toxicity. Furthermore, SCD inhibition rescues αSyn 3K-induced toxicity in late primary neurons. We also confirm that SCD inhibitors reduce formation of αSyn accumulations, while OLA increases these accumulations in an αSyn 3K neuroblastoma model. However, we identify a caveat with this model where αSyn 3K levels can be suppressed by high SCD inhibitor concentrations, obscuring true effect size. Further, we show that both SCD1 or SCD5 knock-down reduce αSyn 3K accumulations and toxicity, making both a putative drug target. Overall, we confirm key findings of published data on SCD inhibition and its benefits in αSyn accumulation and stress models. The differential neurotoxicity induced by SCD inhibition based on neuron culture age must be accounted for when researching SCD in neuron models and has potential clinical implications. Lastly, our gene profiling studies also revealed novel putative genes connected to αSyn neurotoxicity that are worth further study.
Assuntos
Neuroblastoma , Doença de Parkinson , Animais , Humanos , Neurônios , Ratos , Estearoil-CoA Dessaturase/genética , alfa-Sinucleína/genéticaRESUMO
Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson's disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient-derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD-causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)-human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Glucosilceramidase/metabolismo , alfa-Sinucleína/metabolismo , Animais , Animais Recém-Nascidos , Glucosilceramidase/genética , Metabolismo dos Lipídeos , Lipídeos/química , Aprendizagem em Labirinto , Camundongos , Atividade Motora , Proteínas Recombinantes , alfa-Sinucleína/químicaRESUMO
Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD.
Assuntos
Adenosina Trifosfatases/metabolismo , Lisossomos/enzimologia , Proteínas de Membrana/metabolismo , Mutação , Doença de Parkinson/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Astrócitos/enzimologia , Encéfalo/metabolismo , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , ATPases Translocadoras de Prótons , Regulação para CimaRESUMO
Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression.
Assuntos
Antiparkinsonianos/farmacologia , Doença de Parkinson/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , alfa-Sinucleína/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Injeções Intraventriculares , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/metabolismoRESUMO
Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2-3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.
Assuntos
Proteínas Mitocondriais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Feminino , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/genética , Lisina/metabolismo , Masculino , Microscopia Confocal , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Serina/genética , Serina/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Aggregate-prone mutant proteins, such as α-synuclein and huntingtin, play a prominent role in the pathogenesis of various neurodegenerative disorders; thus, it has been hypothesized that reducing the aggregate-prone proteins may be a beneficial therapeutic strategy for these neurodegenerative disorders. Here, we identified two previously described glucosylceramide (GlcCer) synthase inhibitors, DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol and Genz-123346(Genz), as enhancers of autophagy flux. We also demonstrate that GlcCer synthase inhibitors exert their effects on autophagy by inhibiting AKT-mammalian target of rapamycin (mTOR) signaling. More importantly, siRNA knock down of GlcCer synthase had the similar effect as pharmacological inhibition, confirming the on-target effect. In addition, we discovered that inhibition of GlcCer synthase increased the number and size of lysosomal/late endosomal structures. Although inhibition of GlcCer synthase decreases levels of mutant α-synuclein in neurons, it does so, according to our data, through autophagy-independent mechanisms. Our findings demonstrate a direct link between glycosphingolipid biosynthesis and autophagy in primary neurons, which may represent a novel pathway with potential therapeutic value for the treatment of Parkinson's disease. Inhibition of GlcCer synthase enhances autophagy by inhibiting AKT-mTOR signaling, and increases the number and size of lysosomal/late endosomal structures. Furthermore, inhibition of GlcCer synthase decreased levels of mutant α-synuclein in neurons, which may represent a potential therapeutic target for Parkinson's disease.
Assuntos
Autofagia/fisiologia , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Neurônios/fisiologia , Animais , Western Blotting , Células Cultivadas , Dioxanos/farmacologia , Feminino , Glicoesfingolipídeos/biossíntese , Células HEK293 , Humanos , Masculino , Meperidina/análogos & derivados , Meperidina/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína Oncogênica v-akt/metabolismo , Doença de Parkinson/genética , Fosforilação , Cultura Primária de Células , Pirrolidinas/farmacologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Genetic mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to autosomal dominant Parkinson's disease. The most prevalent mutation, G2019S, results in enhanced LRRK2 kinase activity that potentially contributes to the etiology of Parkinson's disease. Consequently, disease progression is potentially mediated by poorly characterized phosphorylation-dependent LRRK2 substrate pathways. To address this gap in knowledge, we transduced SH-SY5Y neuroblastoma cells with LRRK2 G2019S via adenovirus, then determined quantitative changes in the phosphoproteome upon LRRK2 kinase inhibition (LRRK2-IN-1 treatment) using stable isotope labeling of amino acids in culture combined with phosphopeptide enrichment and LC-MS/MS analysis. We identified 776 phosphorylation sites that were increased or decreased at least 50% in response to LRRK2-IN-1 treatment, including sites on proteins previously known to associate with LRRK2. Bioinformatic analysis of those phosphoproteins suggested a potential role for LRRK2 kinase activity in regulating pro-inflammatory responses and neurite morphology, among other pathways. In follow-up experiments, LRRK2-IN-1 inhibited lipopolysaccharide-induced tumor necrosis factor alpha (TNFα) and C-X-C motif chemokine 10 (CXCL10) levels in astrocytes and also enhanced multiple neurite characteristics in primary neuronal cultures. However, LRRK2-IN-1 had almost identical effects in primary glial and neuronal cultures from LRRK2 knockout mice. These data suggest LRRK2-IN-1 may inhibit pathways of perceived LRRK2 pathophysiological function independently of LRRK2 highlighting the need to use multiple pharmacological tools and genetic approaches in studies determining LRRK2 function.
Assuntos
Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteômica , Adenoviridae/genética , Animais , Astrócitos/metabolismo , Células Cultivadas , Quimiocina CXCL10/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Lipopolissacarídeos/farmacologia , Espectrometria de Massas , Camundongos , Camundongos Knockout , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Fosforilação , Plasmídeos/genética , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Titânio/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
While there is now substantial evidence that 5-HT(6) antagonism leads to significantly improved cognitive ability, the mechanism(s) and/or pathway(s) involved are poorly understood. We have evaluated the consequence of chronic administration of the 5-HT(6) receptor antagonists SB-271046 and SB-399885 on neural cell adhesion molecule polysialylation state (NCAM PSA), a neuroplastic mechanism necessary for memory consolidation. Quantitative analysis of NCAM PSA immunopositive neurons in the dentate gyrus of drug-treated animals revealed a dose-dependent increase in polysialylated cell frequency following treatment with both SB-271046 and SB-399885. These effects could not be attributed to increased neurogenesis, as no difference in the rate of bromodeoxyuridine incorporation was apparent between the control and drug-treated groups. A substantial increase in the frequency of polysialylated cells in layer II of the entorhinal and perirhinal cortices was also observed, brain regions not previously associated with neurogenesis. Chronic treatment with SB-271046 or SB-399885 also significantly increased the activation of dentate polysialylation that is specific to learning. This effect does not occur with other cognition-enhancing drugs, such as tacrine, and this action potentially differentiates 5-HT(6) receptor antagonism as an unique neuroplastic mechanism for cognitive processes which may slow or reverse age/neurodegenerative related memory deficits.
Assuntos
Giro Denteado/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Moléculas de Adesão de Célula Nervosa/farmacologia , Neurônios/metabolismo , Piperazinas/farmacologia , Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Ácidos Siálicos/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Antimetabólitos , Bromodesoxiuridina , Proliferação de Células/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Giro Denteado/citologia , Relação Dose-Resposta a Droga , Córtex Entorrinal/citologia , Córtex Entorrinal/efeitos dos fármacos , Hipocampo/citologia , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Estrogens have long been implicated in influencing cognitive processes, yet the molecular mechanisms underlying these effects and the roles of the estrogen receptors alpha (ERalpha) and beta (ERbeta) remain unclear. Using pharmacological, biochemical and behavioral techniques, we demonstrate that the effects of estrogen on hippocampal synaptic plasticity and memory are mediated through ERbeta. Selective ERbeta agonists increased key synaptic proteins in vivo, including PSD-95, synaptophysin and the AMPA-receptor subunit GluR1. These effects were absent in ERbeta knockout mice. In hippocampal slices, ERbeta activation enhanced long-term potentiation, an effect that was absent in slices from ERbeta knockout mice. ERbeta activation induced morphological changes in hippocampal neurons in vivo, including increased dendritic branching and increased density of mushroom-type spines. An ERbeta agonist, but not an ERalpha agonist, also improved performance in hippocampus-dependent memory tasks. Our data suggest that activation of ERbeta can regulate hippocampal synaptic plasticity and improve hippocampus-dependent cognition.
Assuntos
Receptor beta de Estrogênio/metabolismo , Estrogênios/metabolismo , Hipocampo/metabolismo , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Estrogênios/agonistas , Estrogênios/farmacologia , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ovariectomia , Fosforilação/efeitos dos fármacos , Ratos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Deficits in attention and response inhibition are apparent across several neurodegenerative and neuropsychiatric disorders for which current pharmacotherapy is inadequate. While it is difficult to model such executive processes in animals, the 5-choice serial reaction time test (5-CSRTT), which originated from the continuous performance test (CPT) in humans, may serve as a useful translational assay for efficacy in these key behavioral domains. At Wyeth and Abbott, we recently investigated the utility of employing the 5-CSRTT in adult rats. This involved training and testing groups of rats over an extended period of several months and required the animals to learn to nose-poke into one of five apertures following presentation of a brief visual stimulus in that aperture in order to obtain a food reward. When the stimulus duration was short, the rat had to pay close attention to make a correct choice--a nose-poke into the aperture with the brief visual stimulus. We evaluated nicotine and the histamine H(3) receptor antagonist, ciproxifan, since compounds targeting both nicotinic and histaminergic neurotransmission are currently under investigation for treating cognitive dysfunction in ADHD, AD and schizophrenia. After approximately 12 weeks of training, rats were tested with drug when they had achieved stable performance. Nicotine (0.2, 0.4 mg/kg s.c.) significantly improved accuracy and reduced errors of omission (reflecting improved attention and vigilance) when baseline performance was <90% correct. In contrast, nicotine tended to worsen accuracy when baseline performance was >90% correct. Using the same test paradigm, ciproxifan (3mg/kg i.p.) reduced premature responding, a measure of impulsivity. Under conditions of variable stimulus duration, ciproxifan also improved accuracy and decreased impulsivity. In summary, we have replicated previous findings by others of positive effects of nicotine on attention, but also showed that this is dependent on baseline performance. We also expanded on previous positive findings by others with ciproxifan on attention and both Wyeth and Abbott demonstrate for the first time decreased impulsivity with this mechanism.
Assuntos
Atenção/efeitos dos fármacos , Comportamento de Escolha/efeitos dos fármacos , Imidazóis/farmacologia , Comportamento Impulsivo/fisiopatologia , Nicotina/farmacologia , Tempo de Reação/efeitos dos fármacos , Animais , Atenção/fisiologia , Comportamento de Escolha/fisiologia , Interações Medicamentosas , Antagonistas dos Receptores Histamínicos/farmacologia , Masculino , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Ratos Long-Evans , Tempo de Reação/fisiologiaRESUMO
SB-399885 (N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide) has high affinity for human recombinant and native 5-HT(6) receptors, with pK(i) values 9.11+/-0.03 and 9.02+/-0.05, respectively and is a potent competitive antagonist (pA(2) 7.85+/-0.04). It displays over 200-fold selectivity for the 5-HT(6) receptor over all other receptors, ion channels and enzymes tested to date. SB-399885 inhibited ex vivo [(125)I]SB-258585 (4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide) binding with an ED(50) of 2.0+/-0.24 mg/kg p.o. in rats. It had a minimum effective dose of 1 mg/kg p.o. in a rat maximal electroshock seizure threshold test and a long duration of action, overall demonstrating an excellent pharmacokinetic-pharmacodynamic correlation. Repeated administration of this agent (10 mg/kg p.o., b.i.d. for 7 days) significantly reversed a scopolamine-induced deficit (0.5 mg/kg i.p.) in a rat novel object recognition paradigm. Moreover, in aged rats (22 months old) SB-399885 (10 mg/kg p.o., b.i.d. for 7 days) fully reversed the age-dependent deficit in water maze spatial learning compared to vehicle-treated age-matched controls and significantly improved recall of the task measured by increases in the searching of the target quadrant on post-training days 1, 3 and 7. In vivo microdialysis in the rat medial prefrontal cortex demonstrated that acute SB-399885 (10 mg/kg p.o.) significantly increased extracellular acetylcholine levels. These data demonstrate that SB-399885 is a potent, selective, brain penetrant, orally active 5-HT(6) receptor antagonist with cognitive enhancing properties that are likely to be mediated by enhancements of cholinergic function. These studies provide further support for the potential therapeutic utility of 5-HT(6) receptor antagonists in disorders characterised by cognitive deficits such as Alzheimer's disease and schizophrenia.
Assuntos
Envelhecimento/psicologia , Cognição/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Piperazinas/farmacologia , Receptores de Serotonina/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Sulfonamidas/farmacologia , Acetilcolina/metabolismo , Animais , Química Encefálica/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Eletrochoque , Células HeLa , Humanos , Masculino , Microdiálise , Piperazinas/farmacocinética , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Convulsões/prevenção & controle , Estimulação Química , Sulfonamidas/farmacocinéticaRESUMO
Phosphodiesterase-4 (PDE4) belongs to an important family of proteins that regulates the intracellular level of cyclic adenosine monophosphate (cAMP). Several lines of evidence indicate that targeting PDE4 with selective inhibitors may offer novel strategies in the treatment of age-related memory impairment and Alzheimer's disease. The rationale for such an approach stems from preclinical studies indicating that PDE4 inhibitors can counteract deficits in long-term memory caused by pharmacological agents, aging or overexpression of mutant forms of human amyloid precursor proteins. In addition to their pro-cognitive and pro-synaptic plasticity properties, PDE4 inhibitors are potent neuroprotective, neuroregenerative and anti-inflammatory agents. Based on the fact that Alzheimer's disease is a progressive neurodegenerative disorder that is characterised by cognitive impairment, and that neuroinflammation is now recognised as a prominent feature in Alzheimer's pathology, we have concluded that targeting PDE4 with selective inhibitors may offer a novel therapy aimed at slowing progression, prevention and, eventually, therapy of Alzheimer's disease.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Inibidores de Fosfodiesterase/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Cognição/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Modelos Animais de Doenças , Humanos , Transtornos da Memória/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Inibidores de Fosfodiesterase/efeitos adversos , Inibidores de Fosfodiesterase/uso terapêutico , Transmissão Sináptica/efeitos dos fármacosRESUMO
The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.
Assuntos
Receptores de GABA-B/metabolismo , Receptores de GABA/metabolismo , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetinae , AMP Cíclico/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Ensaio Radioligante , Receptores de GABA/efeitos dos fármacos , Receptores de GABA-B/efeitos dos fármacos , Radioisótopos de EnxofreRESUMO
N-(2,5-Dibromo-3-fluorophenyl)-4-methoxy-3-piperazin-1-ylbenzenesulfonamide (SB-357134) potently inhibited [125I]SB-258585 and [3H]LSD binding in a HeLa cell line expressing human 5-HT(6) receptors (pK(i)=8.6 and 8.54, respectively). Furthermore, SB-357134 inhibited [125I]SB-258585 binding in human caudate--putamen and in rat and pig striatum membranes (pK(i)=8.82, 8.44, and 8.61, respectively). SB-357134 displayed over 200-fold selectivity for the 5-HT(6) receptor versus 72 other receptors and enzymes. 5-HT-stimulated cyclic AMP (cAMP) accumulation in human 5-HT(6) receptors was competitively antagonised by SB-357134 (pA(2)=7.63). SB-357134 inhibited ex vivo [125I]SB-258585 binding in the rat with an ED(50) of 4.9 +/- 1.3 mg/kg po, 4 h postdose. In the rat maximal electroshock seizure threshold (MEST) test, SB-357134 produced a potent and dose-dependent increase in seizure threshold, with a minimum effective dose of 0.1 mg/kg po. At 10 mg/kg po, maximum activity occurred between 4 and 6 h postdose. Good exposure was observed with SB-357134 at 10 mg/kg po, reaching maximal blood and brain concentrations of 4.3 +/- 0.2 and 1.3 +/- 0.06 microM, respectively, 1 h postdose. In addition, SB-357134 (10 mg/kg po) enhanced memory and learning following chronic administration (twice a day for 7 days) in the rat water maze. Overall, these studies demonstrate that SB-357134 is a potent, selective, brain penetrant, and orally active 5-HT(6) receptor antagonist.