Characterization of a novel JNK-mediated mechanism of cannabinoid tolerance

描述（由申请人提供）：本研究将调查导致 9-THC 耐受的细胞、分子和生理机制。我们已经培育出表达大麻素受体 1 (CB) 脱敏抗性形式的突变小鼠 (S426A/S430A)，其对 9-THC 表现出延迟耐受性。 1 然而，S426A/S430A 突变体最终对 ?9-THC 完全耐受。用c-Jun N-末端激酶(JNK)抑制剂治疗S426A/S430A突变体消除了对α9-THC镇痛作用的耐受性，表明该信号传导途径可能是在S426A/S430A突变体小鼠中观察到的残留耐受性的原因。将通过测量野生型、S426A/S430A x JNK1 敲除 (KO) 和 S426A/S430A x JNK2 KO 双突变小鼠对 9-THC 镇痛、低温和强直作用的耐受性来确定参与大麻素耐受性的特定 JNK 亚型。将在 S426A/S430A 单突变小鼠中检查 SP600125（JNK 抑制剂）对低温、强直和镇痛耐受的预防作用的剂量反应曲线，以确定该抑制剂的最佳剂量。将进行微阵列分析，检查载体和 SP600125 处理的 S426A/S430A 突变体之间以及 S426A/S430A 单突变体和 S426A/S430A x JNK1 和 S426A/S430A x JNK2 KO 双突变体之间的基因表达差异，以确定负责 JNK 介导的大麻素耐受性的分子靶标。通过微阵列分析确定的所有假定 JNK 靶点都将使用定量实时 PCR 进行验证。本研究的目的是确定负责介导对 9-THC 镇痛作用耐受的 JNK 形式，并确定负责大麻素耐受的 JNK 分子和生化靶点。

DESCRIPTION (provided by applicant): This study will investigate the cellular, molecular, and physiological mechanisms responsible for tolerance to ?9-THC. We have produced mutant mice (S426A/S430A) expressing a desensitization- resistant form of the cannabinoid receptor 1 (CB) that exhibit delayed tolerance for ?9-THC. 1 However S426A/S430A mutants eventually become completely tolerant to ?9-THC. Treatment of S426A/S430A mutant with an inhibitor of c-Jun N-terminal kinase (JNK) eliminates tolerance to the analgesic effects of ?9-THC suggesting that this signaling pathway might be responsible for the residual tolerance observed in S426A/S430A mutant mice. The specific JNK isoform involved in cannabinoid tolerance will be determined by measuring tolerance for the analgesic, hypothermic, and cataleptic effects of ?9-THC in wild-type, S426A/S430A x JNK1 knockout (KO), and S426A/S430A x JNK2 KO double mutant mice. A dose response curve for the preventative effects of SP600125 (JNK inhibitor) on hypothermic, cataleptic, and analgesic tolerance will be examined in S426A/S430A single mutant mice to determine an optimal dosage for this inhibitor. Microarray analyses examining differences in gene expression between vehicle and SP600125-treated S426A/S430A mutants as well between S426A/S430A single mutants and S426A/S430A x JNK1 and S426A/S430A x JNK2 KO double mutants will be done to determine the molecular targets responsible for JNK-mediated cannabinoid tolerance. All putative JNK targets identified by microarray analysis will be validated using quantitative real-time PCR. The goal of this study is to determine the form of JNK responsible for mediating tolerance to the analgesic effects of ?9-THC and also to identify the molecular and biochemical targets of JNK that are responsible for cannabinoid tolerance.