Characterization of a novel JNK-mediated mechanism of cannabinoid tolerance

描述（由申请人提供）：本研究将调查细胞，分子和生理机制负责耐受？9-四氢大麻酚我们已经产生了突变小鼠（S426 A/S430 A）表达的脱敏抗性形式的大麻素受体1（CB）表现出延迟耐受？9-四氢大麻酚1然而，S426 A/S430 A突变体最终变得完全耐受？9-四氢大麻酚用c-Jun N-末端激酶（JNK）抑制剂治疗S426 A/S430 A突变体消除了对？9-THC表明该信号通路可能负责在S426 A/S430 A突变小鼠中观察到的残留耐受性。具体的JNK亚型参与大麻素耐受性将通过测量耐受性的镇痛，体温过低，和僵硬的影响？9-野生型、S426 A/S430 A x JNK 1敲除（KO）和S426 A/S430 A x JNK 2 KO双突变小鼠中的THC。将在S426 A/S430 A单突变小鼠中检查SP 600125（JNK抑制剂）对低温、僵硬和镇痛耐受性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，检查溶媒和SP 600125处理的S426 A/S430 A突变体之间以及S426 A/S430 A单突变体与S426 A/S430 A x JNK 1和S426 A/S430 A x JNK 2 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。将使用定量实时PCR验证通过微阵列分析鉴定的所有推定JNK靶标。本研究的目的是确定JNK的形式负责介导耐受的镇痛效果？9-THC，并确定负责大麻素耐受性的JNK的分子和生物化学靶点。

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.