Characterization of a novel JNK-mediated mechanism of cannabinoid tolerance

描述（由申请人提供）：本研究将探讨对- 9-THC耐受的细胞、分子和生理机制。我们已经产生了表达脱敏抵抗形式的大麻素受体1 （CB）的突变小鼠（S426A/S430A），表现出对- 9-THC的延迟耐受性。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突变体可消除对？9-THC提示该信号通路可能是S426A/S430A突变小鼠残留耐受的原因。参与大麻素耐受性的特定JNK异构体将通过测量大麻素的镇痛、低温和催化作用的耐受性来确定。9-THC在野生型、S426A/S430A × JNK1敲除（KO）和S426A/S430A × JNK2 KO双突变小鼠中的表达。我们将在S426A/S430A单突变小鼠中研究SP600125 （JNK抑制剂）对低温、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。微阵列分析将检测载药和sp600125处理的S426A/S430A突变体之间的基因表达差异，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jnk介导的大麻素耐受的分子靶点。所有通过微阵列分析确定的JNK靶点将使用实时荧光定量PCR进行验证。本研究的目的是确定JNK的形式负责介导对镇痛作用的耐受？9-THC，并确定JNK的分子和生化靶标，这些靶标负责大麻素耐受性。

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.