RESUMEN
In multiple neurodevelopmental and neurodegenerative disorders, endosomal changes correlate with changes in exosomes. We examined this linkage in the brain of mice that received cocaine injections for two weeks starting at 2.5 months of age. Cocaine caused a decrease in the number of both neuronal early and late endosomes and exosomes in the brains of male but not female mice. The response to cocaine in ovariectomized females mirrored male, demonstrating that these sex-differences in response to cocaine are driven by hormonal differences. Moreover, cocaine increased the amount of α-synuclein per exosome in the brain of females but did not affect exosomal α-synuclein content in the brain of males, a sex-difference eliminated by ovariectomy. Enhanced packaging of α-synuclein into female brain exosomes with the potential for propagation of pathology throughout the brain suggests a mechanism for the different response of females to chronic cocaine exposure as compared to males.
Asunto(s)
Cocaína , Exosomas , Animales , Cocaína/farmacología , Endosomas , Femenino , Masculino , Ratones , Neuronas/patología , alfa-SinucleínaRESUMEN
Cocaine is a highly addictive stimulant with diverse effects on physiology. Recent studies indicate the involvement of extracellular vesicles (EVs) secreted by neural cells in the cocaine addiction process. It is hypothesized that cocaine affects secretion levels of EVs and their cargos, resulting in modulation of synaptic transmission and plasticity related to addiction physiology and pathology. Lipids present in EVs are important for EV formation and for intercellular lipid exchange that may trigger physiological and pathological responses, including neuroplasticity, neurotoxicity, and neuroinflammation. Specific lipids are highly enriched in EVs compared to parent cells, and recent studies suggest the involvement of various lipids in drug-induced synaptic plasticity during the development and maintenance of addiction processes. Therefore, we examined interstitial small EVs isolated from the brain of mice treated with either saline or cocaine, focusing on the effects of cocaine on the lipid composition of EVs. We demonstrate that 12 days of noncontingent repeated cocaine (10 mg/kg) injections to mice, which induce locomotor sensitization, cause lipid composition changes in brain EVs of male mice as compared with saline-injected controls. The most prominent change is the elevation of GD1a ganglioside in brain EVs of males. However, cocaine does not affect the EV lipid profiles of the brain in female mice. Understanding the relationship between lipid composition in EVs and vulnerability to cocaine addiction may provide insight into novel targets for therapies for addiction.
Asunto(s)
Encéfalo/efectos de los fármacos , Cocaína/toxicidad , Inhibidores de Captación de Dopamina/toxicidad , Vesículas Extracelulares/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Caracteres Sexuales , Animales , Encéfalo/metabolismo , Encéfalo/patología , Cocaína/administración & dosificación , Inhibidores de Captación de Dopamina/administración & dosificación , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patología , Femenino , Inyecciones Intraperitoneales , Metabolismo de los Lípidos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Deficits in N-methyl-D-aspartate receptor (NMDAR) function are increasingly linked to persistent negative symptoms and cognitive deficits in schizophrenia. Accordingly, clinical studies have been targeting the modulatory site of the NMDA receptor, based on the decreased function of NMDA receptor, to see whether increasing NMDA function can potentially help treat the negative and cognitive deficits seen in the disease. Glycine and D-serine are endogenous ligands to the NMDA modulatory site, but since high doses are needed to affect brain levels, related compounds are being developed, for example glycine transport (GlyT) inhibitors to potentially elevate brain glycine or targeting enzymes, such as D-amino acid oxidase (DAAO) to slow the breakdown and increase the brain level of D-serine. In the present study we further evaluated the effect of DAAO inhibitors 5-chloro-benzo[d]isoxazol-3-ol (CBIO) and sodium benzoate (NaB) in a phencyclidine (PCP) rodent mouse model to see if the inhibitors affect PCP-induced locomotor activity, alter brain D-serine level, and thereby potentially enhance D-serine responses. D-Serine dose-dependently reduced the PCP-induced locomotor activity at doses above 1000 mg/kg. Acute CBIO (30 mg/kg) did not affect PCP-induced locomotor activity, but appeared to reduce locomotor activity when given with D-serine (600 mg/kg); a dose that by itself did not have an effect. However, the effect was also present when the vehicle (Trappsol(®)) was tested with D-serine, suggesting that the reduction in locomotor activity was not related to DAAO inhibition, but possibly reflected enhanced bioavailability of D-serine across the blood brain barrier related to the vehicle. With this acute dose of CBIO, D-serine level in brain and plasma were not increased. Another weaker DAAO inhibitor NaB (400 mg/kg), and NaB plus D-serine also significantly reduced PCP-induced locomotor activity, but without affecting plasma or brain D-serine level, arguing against a DAAO-mediated effect. However, NaB reduced plasma L-serine and based on reports that NaB also elevates various plasma metabolites, for example aminoisobutyric acid (AIB), a potential effect via the System A amino acid carrier may be involved in the regulation of synaptic glycine level to modulate NMDAR function needs to be investigated. Acute ascorbic acid (300 mg/kg) also inhibited PCP-induced locomotor activity, which was further attenuated in the presence of D-serine (600 mg/kg). Ascorbic acid may have an action at the dopamine membrane carrier and/or altering redox mechanisms that modulate NMDARs, but this needs to be further investigated. The findings support an effect of D-serine on PCP-induced hyperactivity. They also offer suggestions on an interaction of NaB via an unknown mechanism, other than DAAO inhibition, perhaps through metabolomic changes, and find unexpected synergy between D-serine and ascorbic acid that supports combined NMDA glycine- and redox-site intervention. Although mechanisms of these specific agents need to be determined, overall it supports continued glutamatergic drug development.
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D-Aminoácido Oxidasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Modelos Animales , Fenciclidina/administración & dosificación , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Cuerpo Estriado/metabolismo , Locomoción/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Serina/sangre , Serina/metabolismo , Serina/farmacologíaRESUMEN
Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states.
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Conducta Animal/efectos de los fármacos , Proteínas de Unión al Calcio/administración & dosificación , Calpaína/antagonistas & inhibidores , Longevidad/efectos de los fármacos , Tauopatías/prevención & control , Tauopatías/fisiopatología , Animales , Calpaína/metabolismo , Inhibidores de Cisteína Proteinasa/administración & dosificación , Femenino , Masculino , Ratones , Ratones Transgénicos , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/prevención & control , Tasa de Supervivencia , Tauopatías/patología , Resultado del Tratamiento , Proteínas tau/efectos de los fármacos , Proteínas tau/genéticaRESUMEN
Cocaine, an addictive psychostimulant, has a broad mechanism of action, including the induction of a wide range of alterations in brain metabolism and mitochondrial homeostasis. Our group recently identified a subpopulation of non-microvesicular, non-exosomal extracellular vesicles of mitochondrial origin (mitovesicles) and developed a method to isolate mitovesicles from brain parenchyma. We hypothesised that the generation and secretion of mitovesicles is affected by mitochondrial abnormalities induced by chronic cocaine exposure. Mitovesicles from the brain extracellular space of cocaine-administered mice were enlarged and more numerous when compared to controls, supporting a model in which mitovesicle biogenesis is enhanced in the presence of mitochondrial alterations. This interrelationship was confirmed in vitro. Moreover, cocaine affected mitovesicle protein composition, causing a functional alteration in mitovesicle ATP production capacity. These data suggest that mitovesicles are previously unidentified players in the biology of cocaine addiction and that target therapies to fine-tune brain mitovesicle functionality may be beneficial to mitigate the effects of chronic cocaine exposure.
Asunto(s)
Cocaína , Vesículas Extracelulares , Ratones , Animales , Cocaína/metabolismo , Cocaína/farmacología , Vesículas Extracelulares/metabolismo , Encéfalo/metabolismo , Mitocondrias/metabolismo , BiologíaRESUMEN
Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(-)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3- and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na+ channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.
Asunto(s)
Benzotropina/farmacología , Cocaína/análogos & derivados , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Condicionamiento Operante/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Femenino , Indicadores y Reactivos , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Neocórtex/citología , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Embarazo , Unión Proteica , Conformación Proteica , Ensayo de Unión Radioligante , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Sodio/metabolismo , Canales de Sodio/metabolismo , Relación Estructura-Actividad , Natación/psicología , Veratridina/farmacologíaRESUMEN
Neurofilament (NFL) proteins have recently been found to play unique roles in synapses. NFL is known to interact with the GluN1 subunit of N-methyl-D-aspartic acid (NMDAR) and be reduced in schizophrenia though functional consequences are unknown. Here we investigated whether the interaction of NFL with GluN1 modulates synaptic transmission and schizophrenia-associated behaviors. The interaction of NFL with GluN1 was assessed by means of molecular, pharmacological, electrophysiological, magnetic resonance spectroscopy (MRS), and schizophrenia-associated behavior analyses. NFL deficits cause an NMDAR hypofunction phenotype including abnormal hippocampal function, as seen in schizophrenia. NFL-/- deletion in mice reduces dendritic spines and GluN1 protein levels, elevates ubiquitin-dependent turnover of GluN1 and hippocampal glutamate measured by MRS, and depresses hippocampal long-term potentiation. NMDAR-related behaviors are also impaired, including pup retrieval, spatial and social memory, prepulse inhibition, night-time activity, and response to NMDAR antagonist, whereas motor deficits are minimal. Importantly, partially lowering NFL in NFL+/- mice to levels seen regionally in schizophrenia, induced similar but milder NMDAR-related synaptic and behavioral deficits. Our findings support an emerging view that central nervous system neurofilament subunits including NFL in the present report, serve distinctive, critical roles in synapses relevant to neuropsychiatric diseases.
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Espinas Dendríticas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteínas de Neurofilamentos/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Conducta Animal , Femenino , Ácido Glutámico/metabolismo , Hipocampo/patología , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones NoqueadosRESUMEN
Traditional models of schizophrenia have focused primarily upon dopaminergic (DA) dysregulation. In contrast, more recent models focus on dysfunction of glutamatergic systems, acting particularly through N-methyl-D-aspartate (NMDA) receptors. NMDA receptors in brain are regulated by glycine, acting via a strychnine-insensitive regulatory site, and by glycine (GlyT1) transporters that maintain low glycine levels in the immediate vicinity of the NMDA receptor complex. The present study investigates the role of NMDA receptors in the modulation of striatal dopamine release in vitro, and of glycine transport inhibitors (GTIs) as potential psychotherapeutic agents in schizophrenia. In striatum, NMDA receptors exert dual excitatory/inhibitory effects, with inhibition reflecting activity of local GABAergic feedback regulation. We have previously demonstrated effectiveness of glycine in regulating [3H]DA release both in vivo and in vitro, consistent with its beneficial clinical effects. In the present study, similar effects were observed for the high-affinity GTI (+)N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy-)propyl]sarcosine (NFPS), and for a range of high-affinity GTIs with appropriate rank order of potency. In addition, (+)NFPS significantly stimulated NMDA-induced [3H]GABA release. Effects, of GTIs, were blocked by the glycine-site antagonists L689,560 and HA-966, and the GABA(B) antagonists phaclofen and CGP 52432, confirming the roles of both the NMDA-associated glycine-site and presynaptic GABA(B) receptors in NMDA receptor-mediated regulation of striatal DA release in vitro. Endogenous DA hyperactivity is associated with prominent positive symptoms in schizophrenia. The present results are consistent with recent clinical studies showing significant effectiveness of glycine-site agonists and GTIs in reduction of persistent positive, as well as negative, symptoms in schizophrenia.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Compuestos de Bifenilo/farmacología , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Glicina/metabolismo , Terminales Presinápticos/metabolismo , Sarcosina/análogos & derivados , Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inhibidores , Animales , Cuerpo Estriado/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Antagonistas de Receptores de GABA-B , Proteínas de Transporte de Glicina en la Membrana Plasmática , Ratones , Ratones Endogámicos BALB C , N-Metilaspartato/farmacología , Técnicas de Cultivo de Órganos , Terminales Presinápticos/efectos de los fármacos , Receptores de GABA-B/metabolismo , Receptores de Glicina/antagonistas & inhibidores , Receptores de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Sarcosina/farmacología , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
This study was designed to understand molecular and cellular mechanisms underlying aggressive behaviors in mice exposed to repeated interactions in their homecage with conspecifics. A resident-intruder procedure was employed whereby two males were allowed to interact for 10 min trials, and aggressive and/or submissive behaviors (e.g., degree of attacking, biting, chasing, grooming, rearing, or upright posture) were assessed. Following 10 days of behavioral trials, brains were removed and dissected into specific regions including the cerebellum, frontal cortex, hippocampus, midbrain, pons, and striatum. Gene expression analysis was performed using real-time quantitative polymerase-chain reaction (qPCR) for catechol-O-methyltransferase (COMT) and tyrosine hydroxylase (TH). Compared to naive control mice, significant up regulation of COMT expression of residents was observed in the cerebellum, frontal cortex, hippocampus, midbrain, and striatum; in all of these brain regions the COMT expression of residents was also significantly higher than that of intruders. The intruders also had a significant down regulation (compared to naive control mice) within the hippocampus, indicating a selective decrease in COMT expression in the hippocampus of submissive subjects. Immunoblot analysis confirmed COMT up regulation in the midbrain and hippocampus of residents and down regulation in intruders. qPCR analysis of TH expression indicated significant up regulation in the midbrain of residents and concomitant down regulation in intruders. These findings implicate regionally- and behaviorally-specific regulation of COMT and TH expression in aggressive and submissive behaviors. Additional molecular and cellular characterization of COMT, TH, and other potential targets is warranted within this animal model of aggression.
Asunto(s)
Agresión/fisiología , Encéfalo/metabolismo , Catecol O-Metiltransferasa/metabolismo , Regulación de la Expresión Génica/fisiología , Predominio Social , Tirosina 3-Monooxigenasa/metabolismo , Análisis de Varianza , Animales , Encéfalo/anatomía & histología , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Familial Parkinson's disease (PD) has been linked to point mutations and duplication of the alpha-synuclein gene and mutant alpha-synuclein expression increases the vulnerability of neurons to exogenous insults. In this study, we analyzed the levels of dopamine and its metabolites in the olfactory bulb (OB), and nigrostriatal regions of transgenic mice expressing human, mutant A53T alpha-synuclein (alpha-syn tg) and their non-transgenic (ntg) littermates using a sub-toxic, moderate dose of MPTP to determine if mutant human alpha-synuclein sensitizes the central dopaminergic systems to oxidative stress. We observed that after a single, sub-lethal MPTP injection, dopamine levels were reduced in striatum and SN in both the alpha-syn tg and ntg mice. In the olfactory bulb, a region usually resistant to MPTP toxicity, levels were reduced only in the alpha-syn tg mice. In addition, we identified a significant increase in dopamine metabolism in the alpha-syn transgenic, but not ntg mice. Finally, MPTP treatment of alpha-syn tg mice was associated with a marked elevation in the oxidative product, 3-nitrotyrosine that co-migrated with alpha-synuclein. Cumulatively, the data support the hypothesis that mutant alpha-synuclein sensitizes dopaminergic neurons to neurotoxic insults and is associated with greater oxidative stress. The alpha-syn tg line is therefore useful to study the genetic and environmental inter-relationship in PD.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Dopamina/metabolismo , Neuronas/efectos de los fármacos , alfa-Sinucleína/metabolismo , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Ratones , Ratones Transgénicos , Mutación , Neuronas/metabolismo , alfa-Sinucleína/genéticaRESUMEN
Nicotine or cocaine, when administered intravenously, induces an increase of extracellular dopamine in the nucleus accumbens. The nicotine-mediated increase was shown to occur at least in part through increase of the activity of dopamine neurons in the ventral tegmental area. As part of our continuing studies of the mechanisms of nicotine effects in the brain, in particular, effects on reward and cognitive mechanisms, in the present study we examined the role of various receptors in the ventral tegmental area in nicotine and cocaine reward. We assayed inhibition of the increase of dopamine in the nucleus accumbens induced by intravenous nicotine or cocaine administration by antagonists administered into the ventral tegmental area. Nicotine-induced increase of accumbal dopamine release was inhibited by intrategmental nicotinic (mecamylamine), muscarinic (atropine), dopaminergic (D1: SCH 23390, D2: eticlopride), and NMDA glutamatergic (MK 801) and GABAB (saclofen) antagonists, but not by AMPA-kainate (CNQX, GYKI52466) antagonists under our experimental circumstances. The intravenous cocaine-induced increase of dopamine in the nucleus accumbens was inhibited by muscarinic (atropine), dopamine 2 (eticlopride), and GABAB (saclofen) antagonists but not by antagonists to nicotinic (mecamylamine), dopamine D1 (SCH 23390), glutamate (MK 801), or AMPA-kainate (CNQX, GYKI52466) receptors. Antagonists administered in the ventral tegmental area in the present study had somewhat different effects when they were previously administered intravenously. When administered intravenously atropine did not inhibit cocaine effects. The inhibition by atropine may be indirect, since this compound, when administered intrategmentally, decreased basal dopamine levels in the accumbens. The findings indicate that a number of receptors in the ventral tegmental area mediate nicotine-induced dopamine changes in the nucleus accumbens, a major component of the nicotine reward mechanism. Some, but not all, of these receptors in the ventral tegmental area also seem to participate in the reward mechanism of cocaine. The importance of local receptors in the ventral tegmental area was further indicated by the increase in accumbal dopamine levels after intrategmental administration of nicotine or also cocaine.
Asunto(s)
Dopamina/metabolismo , Nicotina/farmacología , Núcleo Accumbens/fisiología , Receptores de Neurotransmisores/fisiología , Área Tegmental Ventral/fisiología , Animales , Atropina/farmacología , Benzazepinas/farmacología , Cocaína/farmacología , Maleato de Dizocilpina/farmacología , Cinética , Masculino , Núcleo Accumbens/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Dopaminérgicos/efectos de los fármacos , Receptores Dopaminérgicos/fisiología , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/fisiología , Receptores de Neurotransmisores/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiología , Área Tegmental Ventral/efectos de los fármacosRESUMEN
The effects of acute lorazepam challenges on plasma (p) HVA, MHPG, and 5-HIAA, and their relationship to drug-induced cognitive and motor deficits and the apolipoprotein (APOE)-epsilon4 allele were examined. Eighteen healthy elderly (8 epsilon4 carriers) received placebo or acute oral lorazepam doses (0.5 mg or 1 mg) in random sequence, 1-week apart. Cognitive assessment and plasma levels of pHVA, pMHPG, and p5-HIAA were determined at baseline and at 1, 2.5, and 5 h postchallenge. There was no drug-to-placebo difference in monoamine levels and no consistent relationship between changes in monoamine levels and cognitive performance, regardless of epsilon4 status. However, the 1.0 mg dose increased p5-HIAA in epsilon4 carriers, whereas it caused a reduction in noncarriers. Higher baseline pMHPG and p5-HIAA levels were associated with better baseline memory. The epsilon4 allele may modulate the effect of lorazepam on p5-HIAA, but further studies are needed to confirm this finding and elucidate its possible significance.