RESUMO
Activation of AMPA receptors (AMPARs) in the nucleus accumbens is necessary for the reinstatement of cocaine-seeking behavior, an animal model of drug craving and relapse. AMPARs are tetrameric protein complexes that consist of GluA1-4 subunits, of which GluA2 imparts calcium permeability. Adenosine deaminase acting on RNA 2 (ADAR2) is a nuclear enzyme that is essential for editing GluA2 pre-mRNA at Q/R site 607. Unedited GluA2(Q) subunits form calcium-permeable AMPARs (CP-AMPARs), whereas edited GluA2(R) subunits form calcium-impermeable channels (CI-AMPARs). Emerging evidence suggests that the reinstatement of cocaine seeking is associated with increased synaptic expression of CP-AMPARs in the nucleus accumbens. However, the role of GluA2 Q/R site editing and ADAR2 in cocaine seeking is unclear. In the present study, we investigated the effects of forced cocaine abstinence on GluA2 Q/R site editing and ADAR2 expression in the nucleus accumbens. Our results demonstrate that 7 days of cocaine abstinence is associated with decreased GluA2 Q/R site editing and reduced ADAR2 expression in the accumbens shell, but not core, of cocaine-experienced rats compared with yoked saline controls. To examine the functional significance of ADAR2 and GluA2 Q/R site editing in cocaine seeking, we used viral-mediated gene delivery to overexpress ADAR2b in the accumbens shell. Increased ADAR2b expression in the shell attenuated cocaine priming-induced reinstatement of drug seeking and was associated with increased GluA2 Q/R site editing and surface expression of GluA2-containing AMPARs. Taken together, these findings support the novel hypothesis that an increased contribution of accumbens shell CP-AMPARs containing unedited GluA2(Q) promotes cocaine seeking. Therefore, CP-AMPARs containing unedited GluA2(Q) represent a novel target for cocaine addiction pharmacotherapies.
Assuntos
Adenosina Desaminase/metabolismo , Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Comportamento de Procura de Droga/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de AMPA/metabolismo , Adenosina Desaminase/genética , Animais , Cálcio/metabolismo , Condicionamento Operante/efeitos dos fármacos , Desoxirribonucleases de Sítio Específico do Tipo II/administração & dosagem , Regulação da Expressão Gênica/fisiologia , Masculino , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Edição de RNA/efeitos dos fármacos , Edição de RNA/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Autoadministração , Transdução GenéticaRESUMO
A yeast artificial chromosome (YAC) mouse model of Huntington's disease (YAC128) develops motor abnormalities, age-dependent striatal atrophy and neuronal loss. Alteration of neurotransmitter receptors, particularly glutamate and dopamine receptors, is a pathological hallmark of Huntington's disease. We therefore analyzed neurotransmitter receptors in symptomatic YAC128 Huntington's disease mice. We found significant increases in N-methyl-d-aspartate, AMPA and metabotropic glutamate receptor binding, which were not due to increases in receptor subunit mRNA expression levels. Subcellular fractionation analysis revealed increased levels of glutamate receptor subunits in synaptic membrane fractions from YAC128 mice. We found no changes in dopamine, GABA or adenosine receptor binding, nor did we see alterations in dopamine D1, D2 or adenosine A2a receptor mRNA levels. The receptor abnormalities in YAC128 transgenic mice thus appear limited to glutamate receptors. We also found a significant decrease in preproenkephalin mRNA in the striatum of YAC128 mice, which contrasts with the lack of change in levels of mRNA encoding neurotransmitter receptors. Taken together, the abnormal and selective increases in glutamate receptor subunit expression and binding are not due to increases in receptor subunit expression and may exert detrimental effects. The decrease in preproenkephalin mRNA suggests a selective transcriptional deficit, as opposed to neuronal loss, and could additionally contribute to the abnormal motor symptoms in YAC128 mice.
Assuntos
Cromossomos Artificiais de Levedura/genética , Doença de Huntington/genética , Doença de Huntington/fisiopatologia , Receptores de Glutamato/fisiologia , Animais , Autorradiografia , Western Blotting , Encefalinas/metabolismo , Humanos , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Precursores de Proteínas/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de AMPA/metabolismo , Receptores Dopaminérgicos/metabolismo , Receptores de GABA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Purinérgicos P1/metabolismo , Frações Subcelulares/metabolismoRESUMO
Transcriptional dysregulation is a central pathogenic mechanism in Huntington's disease (HD); HD and transgenic mouse models of HD demonstrate down-regulation of specific genes at the level of mRNA expression. Furthermore, neuronal intranuclear inclusions (NIIs) have been identified in the brains of R6/2 mice and HD patients. One possibility is that NIIs contribute to transcriptional dysregulation by sequestering transcription factors. We therefore assessed the relationship between NIIs and transcriptional dysregulation in the R6/2 mouse, using double-label in situ hybridization combined with immunohistochemistry, and laser capture microdissection combined with quantitative real-time PCR. There was no difference in transcript levels of specific genes between NII-positive and NII-negative neurons. These results demonstrate that NIIs do not cause decreases in D2, PPE and PSS mRNA levels in R6/2 striatum and therefore are not involved in the down-regulation of these specific genes in this HD model. In addition, these observations argue against the notion that NIIs protect against transcriptional dysregulation in HD.