第IIa類組蛋白去乙醯酶在古柯鹼成癮之角色

Abstract

藥物濫用是一個尚未見有效治療方式並且值得注意的社會經濟議題。長時間的藥物濫用會導致不正常的神經適應(neural adaptation)發生，目前非正常的神經適應被認為是藥物成癮的病因之一，可能導致終身行為上的異常。組蛋白乙醯化（Histone acetylation）是目前被研究最多的表觀遺傳學（epigenetics）機制之一，其受到組蛋白乙醯基轉移酶（histone acetyltransferases, HATs）和組蛋白去乙醯酶（histone deacetylases, HDACs）兩種酵素的調控，組蛋白乙醯化可藉由改變基因的表現進而影響神經和行為學上的可塑性(plasticity)和適應性，而神經和行為學上的可塑性被認為和藥物成癮相關。目前多數的研究是透過非選擇性的組蛋白去乙醯酶抑制劑或HDAC基因轉殖鼠來研究HDAC在古柯鹼成癮的動物模型中扮演的角色。 然而，不同種類的組蛋白去乙醯酶在大腦正回饋迴路（brain reward circuitry）中可能扮演著不同的角色。例如，抑制HDAC3被證明可以幫助古柯鹼的戒斷（extinction），但抑制HDAC1則被認為會有反效果。因此，各別種類的組蛋白去乙醯酶在成癮性疾病中扮演的角色是值得被研究的。在我們的研究中，我們檢視第IIa類組蛋白去乙醯酶選擇性抑制劑，包含WJ26220（選擇性HDAC4, 5, 7抑制劑）和LMK235（選擇性HDAC4, 5抑制劑），在古柯鹼之場域偏好性試驗（cocaine-induced place preference test, CPP）中對古柯鹼成癮表現的影響，藉此來闡明第IIa類組蛋白去乙醯酶（包含組蛋白去乙醯酶4, 5和7）在古柯鹼成癮中扮演著什麼樣的角色。 我們利用古柯鹼訓練8到10週大的C57BL/6雄性小鼠產生場域偏好性。小鼠待在愛好箱（preferred chamber）內和非愛好箱（non-preferred chamber）內的時間差異被定義為CPP score。此外，我們利用免疫螢光染色計算被DAPI標記並於組蛋白H3-lysine14（H3K14）位置有乙醯化的細胞數量，來檢視小鼠伏隔核核心部位(nucleus accumbens core, NAc core)組蛋白乙醯化的程度。我們也量測伏隔核核心部位中具有HDAC7免疫螢光標記的面積來評估HDAC7蛋白的表現量。 在三天場域偏好性訓練期中，於給予古柯鹼注射之前三十分鐘先給予腹腔注射賦形劑、WJ26220（選擇性HDAC4, 5, 7抑制劑，1 mg/kg）或LMK235（選擇性HDAC4, 5抑制劑，0.05 mg/kg），在沒有影響小鼠活動力的前提下，WJ26220顯著性抑制了古柯鹼誘發之場域偏好程度（CPP scores: 118±11 sec vs. 206±15 sec, n=10, p<0.001），然而LMK235以及vehicle則不影響CPP結果。另外，給予小鼠連續三天每天經腹腔注射WJ26220（1 mg/kg）或LMK235（0.05 mg/kg），我們發現小鼠伏隔核核心之組蛋白乙醯化的程度顯著性的增加了，由此結果我們可證實WJ26220和LMK235確可分布至腦中並有效抑制該部位之HDAC。綜合上述結果可推論WJ26220抑制古柯鹼誘發之場域偏好程度的效果可能主要是來自於抑制HDAC7。而透過免疫螢光染色我們觀察到，經過三天每日注射一劑古柯鹼的場域偏好性訓練期後，小鼠伏隔核核心部位之HDAC7蛋白表現量顯著性地增加，因此我們也推測古柯鹼導致之HDAC7表現量的增加可能為古柯鹼誘發場域偏好性產生的成因之一。 總結，我們的場域偏好性實驗結果首度顯示，第IIa類組蛋白去乙醯酶對於組蛋白的修飾於古柯鹼成癮中扮有關鍵性的角色。因此，第IIa類組蛋白去乙醯酶選擇性抑制劑(例如: WJ26220)對古柯鹼成癮應具有臨床治療的潛力。

Drug abuse is an unmet medical need and a noteworthy social and economic issue. A long-term drug abuse may result in abnormal neural adaptations thought to be involved in the pathogenesis of addictive disorders and drive life-long behavioral abnormalities. Histone acetylation, one of most studied epigenetic mechanisms, regulated by enzymes of histone acetyltransferases (HATs) and histone deacetylases (HDACs), has been recently reported to influence the neural and behavioral plasticity associated with drug addiction by altering gene expressions. Most of these studies investigated the role of HDACs, enzymes that repress histone acetylation, in animal models of cocaine acquisition by using non-selective HDAC inhibitors (also called pan-HDAC inhibitors), and some by using HDAC subtype-transgenic mice. However, different subtypes of HDACs play opposite roles in brain reward circuitry. For example, HDAC3 inhibition was shown to improve cocaine extinction while HDAC1 inhibition inhibited extinction. Therefore, it is worthwhile to investigate the role of specific subtype of HDACs in addictive disorders. In this study, we revealed the role of class IIa HDACs including HDAC4, 5 and 7 in cocaine-induced addiction by examining the effects of WJ26220, a selective HDAC4, 5, 7 inhibitor, and LMK235, a selective HDAC4, 5 inhibitor, on the acquisition of cocaine-induced place preference. C57BL/6 mice (male, 8-10 weeks) were trained to addict to cocaine by a 5 day-bias paradigm of cocaine-paired conditioned place preference (CPP) test. The time spent difference between cocaine-preferred and non-preferred chambers was defined as the CPP score. Besides, we accessed histone acetylation in the nucleus accumbens (NAc) core, measured by the number of cells with co-immunoreactivity to both acetylated-histone H3 lysine 14 (Ac-H3K14) and DAPI by immunofluorescence. HDAC7 expression was also accessed in NAc core by immunofluorescence which measured the area of immunoreactivity to HDAC7. WJ26220 (1 mg/kg, i.p.), a selective HDAC4, 5, 7 inhibitor, or LMK235 (0.05 mg/kg, i.p.), a selective HDAC4, 5 inhibitor, was pretreated 30 minutes before cocaine-injection in the 3 days’ cocaine-pairing period. Daily pretreatment with WJ26220 30 minutes before cocaine-injection significantly attenuated cocaine-induced CPP (CPP scores: 118±11 sec vs. 206±15 sec, n=10, p<0.001) without affecting spontaneous motor activity. However, daily pretreatment with LMK235 or the vehicle, did not affect cocaine CPP. Nevertheless, histone acetylation in the NAc core was significantly increased after daily i.p. treatment of WJ26220 (1 mg/kg) or LMK235 (0.05 mg/kg) for 3 days. The latter results support the effectiveness of WJ26220 and LMK235 in terms of HDAC inhibition and brain permeability. The effectiveness of WJ26220 (a HDAC4, 5, 7 inhibitor) but not LMK235 (a selective HDAC4, 5 inhibitor), in attenuating cocaine-induced CPP suggests that HDAC7 inhibition contributes to WJ26220-induced attenuation of cocaine CPP. In parallel, the immunofluorescence showed an enhancement of HDAC7 expression in NAc core after 3 days’ cocaine conditioning, suggesting the involvement of HDAC7 activation in the formation of cocaine CPP. Taken together, our results for the first time suggest that histone modification by class IIa histone deacetylases may plays a pathogentic role in the acquisition phase of cocaine CPP, and that selective class IIa HDAC inhibitors (e.g. WJ26220) may be are potential therapeutic agents for cocaine addiction.