多巴胺亢奮之神經精神疾患的全新治療標的:小腦中含a6次單元之γ-氨基丁酸-A受體

Abstract

我們在一則臨床案例報告發現，一位患有頑固性抽動的小女孩，發現在其服用苦藍盤汁液的萃取物一小時後，她的抽動症狀獲得有效緩解。因此，我們從苦藍盤葉的乙醇萃取物中，鑑定並分離出活性成分的純化合物hispidulin。hispidulin是γ-氨基丁酸-A(GABAA)受體的正向異位調控者，有著微莫耳濃度（μM）的親和力，其中包含α6次單元(α6 GABAARs)，這種次單元特異性地表現在小腦的顆粒細胞中。我們的研究團隊首先發現hispidulin能顯著地改善由甲基安非他命（methamphetamine）及NMDA 通道阻斷劑所引起的前脈衝抑制不良（PPI），此動物模型可模擬某些神經精神疾病患者的感覺門控系統(sensorimotor gating)功能下降。由在動物上模擬思覺失調症的負向症狀模式，發現也能改善在小鼠上慢性給予天使塵（phencyclindine）來誘導社交行行為退縮的現象。 然而hispidulin並非選擇性地只作用於GABAARs受體，而我們的維也納合作研究團隊(Varagic et al., 2013)在該時間點合成Compound 6。Compound 6 是第一個對α6 GABAARs有選擇性的化合物。經我們的團隊研究後發現，Compound 6和hispidulin一樣可以改善由甲基安非他命所引起的前脈衝抑制不良。接下來我們以動物模型對Compound 6 進行一系列的研究，其中包括甲基安非他命（促多巴胺釋放激素）所引起的活動力增加現象，以及阿樸嗎啡（多巴胺促進劑）所誘使的刻版化攀爬行行為，這兩種動物疾病模型皆可模擬因多巴胺活性過高所致的神經精神疾病，例如思覺失調症(舊稱精神分裂症)。同時一個非競爭α6GABAAR 的拮抗劑furosemide 也以小腦局部注射方式被使用在甲基安非他命所引起的活動力增加模型，用以拮抗Compound 6的作用。 第一部分的動物行行為模式實驗，我們發現腹腔注射給予特定濃度的Compound 6，會修復甲基安非他命所造成活動力增加的現象，在該濃度下Compound 6 不會改變及阿樸嗎啡所誘使的刻版化攀爬行行為。此濃度也不會造成肌肉功能減弱、自主活動力下降、焦慮且小腦內注射furosemide可有效地抑制Compound 6 所造成修復甲基安非他命活動力力增加的效果。這些結果顯示Compound 6 能修復甲基安非他命所造成活動力增加的現象。藉由這些藥理學試驗我們釐清三個重點，分別是小腦顆粒細胞中的確實為Compound 6的作用目標，且Compound 6的有效濃度不會造成BDZ藥物的類似副作用，且Compound 6能夠穿過血腦屏障進而作用在小腦。 除此之外，我們經由在背側紋狀體注射Slitrk-1的小分子核糖核酸，形成引發動物的刻版化行為模型進一步模擬妥瑞氏症（Tourette syndrome, TS），來檢測Compound 6是否有治療TS的潛力；另外也使用Compound 6的氘衍生物RV-I-29來驗證此動物模型。在成人的紋狀體中，Slitrk-1僅表現在膽鹼能的中間神經元，且死後解剖研究顯示TS病患的膽鹼能的中間神經元所含Slitrk-1相對於一般人更少，這些研究暗示Slitrk-1的含量降低可能是造成TS的病因之一，而Compound 6與 RV-I-29能夠顯著降低此模型中的刻版化行為。 經由以上結果，我們推測Compound 6能藉由正向調控小腦顆粒細胞中的α6 GABAARs，以加強小腦抑制紋狀體中的多巴胺活性，而非直接抑制多巴胺的受體。也因此我們期待α6 GABAARs的異位正向調控機制可以成為新的TS治療方向。

In a case study, we found the intractable motor tics in a pediatric patient were subsided within 1 hour after taking the leaf extract of a local herb, Clerodendron inerme Gaertn (CI) (Fan et al., 2009). Then, we have identified an active constituent, hispidulin, from the ethanol extract of CI leaves, which is a positive allosteric modulator (PAM) of GABA-A receptors, including the α6 subunit-containing GABAARs (α6 GABAARs).α6 GABAARs are exclusively expressed in cerebellar granule cells, the trigeminal ganglia and cochlear nucleus. Since Hispidulin is not selective to α6 GABAAR, we obtained the first selective α6 GABAAR PAM, Compound 6, from our Vienna cooperative team (Varagic et al., 2013). Compound 6, like Hispidulin, effectively rescued the impairment of prepulse inhibition of the startle response (PPI) induced by methamphetamine, a measurement of the sensorimotor gating function that is deficit in several neuropsychiatric disorders, including tic disorders and schizophrenia. Here, we further conducted a comprehensive study examining effects of Compound 6 in various behavioral models mimicking tic disorders, including the hyperlocomotion induced by methamphetamine (a dopamine releaser), stereotypy climbing behaviors induced by apomorphine (a dopamine receptor agonist) as well as the stereotypy behaviors induced by intra-dorsal striatum injection of the siRNA of Slitrk-1, an animal model mimicking Tourette syndrome (TS). We also test the Slitrk-1 KD mice model by RV-I-29, which is a –OCD3 (deuterated) derivative of Compound 6. In the adult striatum, Slitrk-1 is only expressed in cholinergic interneurons that were reduced in postmortem brains of TS subjects. We also validated the α6 GABAAR in the cerebellum is the action target of Compound 6 using anatomical and pharmacological approaches. Intraperitoneal (i.p.) injection of Compound 6 significantly rescued methamphetamine-induced hyperlocomotion. This effect was significantly antagonized by intra-cerebellum microinjection of furosemide, a selective α6GABAAR antagonist. On the other hand, Compound 6 (i.p.) did not affect stereotypy climbing behaviors induced by apomorphine, a dopamine receptor agonist. Compound 6 and RV-I-29 also significantly reduced the stereotypy behaviors in mice induced by knocking down striatal Slitrk. The Slitrk KD mice may be an efficacy animal model to screen TS treating compound, so we further test the model by RV-I-29, which is another PAM of GABAA receptors. However, Compound 6 did not affect the spontaneous motor activity, motor coordination in the rotarod test or the grip strength, neither displayed any significant sedation or anxiolytic activity in sedation assessment and elevated plus maze test. These results suggest that Compound 6 can successfully pass through the blood brain barrier to rescue methamphetamine-induced hyperlocomotion possibly via enhancing cerebellar inhibitory control on the striatal dopaminergic activity through positively modulating α6 GABAARs in cerebellar granule cells, but not affect dopamine receptor response directly. It is also suggested that the α6 GABAAR in the cerebellum is a new target for the treatment of TS or tic disorders. The PAM selective to α6 GABAARs may be a novel class of anti-tic therapy.