紋狀體Slitrk1基因在小鼠刻板行為之角色：涉及乙醯膽鹼與多巴胺神經傳遞

Abstract

妥瑞症是一個好發在孩童時期的神經發育障礙症，且男性的平均發生率較女性高。妥瑞症的主要症狀為運動抽動及聲音抽動，特徵為不自主且重複性的快速動作如搖頭晃腦或者擤鼻子、清喉嚨，現今妥瑞症的療法多半只能改善症狀而無法完全根治或者會造成強烈副作用，因此，妥瑞症至今仍被美國食品藥品監督管理局列為孤兒病。許多研究指出妥瑞症的病因與皮質-基底核-丘腦迴路功能異常有關，但其詳細的機制仍未臻了解。妥瑞症的基因研究已辨識出數個相關的突變基因，其中包含SLITRK1基因，SLITRK1/Slitrk1蛋白質在哺乳類大腦發育時期會表現在皮質-基底核-丘腦迴路，至成年時期則只存於紋狀體膽鹼能中間神經元內，SLITRK1/Slitrk1蛋白質的功能為調控神經突的生長以及突觸形成。此外，一篇屍驗研究發現妥瑞症病人的紋狀體膽鹼能中間神經元相較於正常對照組明顯減少。上述的發現皆指出紋狀體膽鹼能中間神經元的SLITRK1/Slitrk1在妥瑞症的病理成因中扮演重要角色。我們先前的實驗已發現被注射針對Slitrk1信使核糖核酸的小分子干擾核糖核酸到紋狀體中靜默掉Slitrk1基因的成鼠（Slitrk1基因敲落鼠）會明顯展現較多且較持久的妥瑞症相似異常行為──刻板行為，相較於被注射無同源性的小分子干擾核糖核酸的對照組成鼠（scramble鼠）。 本篇研究旨在探討Slitrk1基因敲落誘發之刻板行為背後的相關機制。透過微透析實驗收集小鼠紋狀體的透析液，接著以高效液相層析儀量測數種神經傳遞物的含量。為了探究哪些神經傳遞物之受體參與在Slitrk1基因敲落所誘發之刻板行為，我們給予Slitrk1基因敲落鼠預期能減少刻板行為的特定受體之活化劑、拮抗劑或正向異位調節劑；另一方面，我們給予scramble鼠預期能增加刻板行為的特定受體之拮抗劑，進而分析這些藥物對刻板行為的影響。Slitrk1基因敲落鼠的紋狀體神經傳遞物含量分析結果顯示：（1） 誘發性而非持續性乙醯膽鹼含量下降；（2） 誘發性多巴胺含量下降，而持續性多巴胺含量增加；（3）持續性γ-氨基丁酸、穀氨酸及血清素含量並未顯著改變。刻板行為結果顯示多巴胺受體D2拮抗劑（haloperidol, 0.3 mg/kg, i.p.）及γ-氨基丁酸A型受體活化劑（muscimol, 1 μg/0.5 μl, i.str.）顯著減少Slitrk1基因敲落誘發之刻板行為；而α4β2菸鹼型乙醯膽鹼受體拮抗劑（DHβE, 18 μg/0.5 μl, i.str.）顯著增加正常老鼠的刻板行為。然而我們亦觀察到α4β2菸鹼型乙醯膽鹼受體活化劑（sazetidine-A, 10 pmol/0.5 μl, i.str.）、乙醯膽鹼酯酶抑制劑（donepezil, 100 ng/0.5 μl, i.str.）、蕈毒鹼型乙醯膽鹼M4受體的正向異位調節劑（VU0152100, 12.5 ng/0.5 μl, i.str.）以及多巴胺受體D1拮抗劑（SCH23390, 1 μg/0.5 μl, i.str.）無法顯著減弱Slitrk1基因敲落誘發之刻板行為；而蕈毒鹼型乙醯膽鹼M4受體拮抗劑（PD102807, 39 ng/0.5 μl, i.str.）無法顯著增加正常老鼠的刻板行為。由上述的實驗結果，我們認為α4β2菸鹼型乙醯膽鹼受體而非蕈毒鹼型乙醯膽鹼M4受體、多巴胺受體D2而非D1、以及γ-氨基丁酸A型受體在刻板行為中扮演重要之角色。最後，我們也發現Slitrk1基因敲落所造成的影響是可逆的，包含誘發的刻板行為與減少的紋狀體誘發性多巴胺含量，此可逆效果亦與減少的Slitrk1蛋白質表現量回復有所呼應。 綜上所述，本篇研究發現Slitrk1基因敲落所誘發之刻板行為可歸因為紋狀體乙醯膽鹼及多巴胺系統失衡，亦可能涉及γ-氨基丁酸系統。紋狀體膽鹼能中間神經元Slitrk1缺乏可能造成Slitrk1調控興奮性突觸形成的正常功能受損，進而導致誘發性乙醯膽鹼含量下降。紋狀體誘發性多巴胺含量下降可能是因為誘發性乙醯膽鹼含量下降所直接導致，而持續性多巴胺含量增加則可能是間接的補償作用造成，或者是紋狀體誘發性γ-氨基丁酸含量下降所導致。藥理學研究指出α4β2菸鹼型乙醯膽鹼受體、多巴胺受體D2與γ-氨基丁酸A型受體在刻板行為中扮演關鍵性的角色，這些神經傳遞系統的失能最終致使中型多棘神經元的功能受損，因而產生過多刻板行為。最後，臨床妥瑞症所用藥物haloperidol顯著減少Slitrk1基因敲落所誘發之刻板行為的效果，支持了Slitrk1基因敲落鼠作為未來開發妥瑞症新穎藥物或療法之模式動物的潛能。

Tourette syndrome (TS) is a neurodevelopmental disease prevalent among children, with a predominance in male. The symptoms of TS are motor and vocal tics, which are involuntary and stereotypic movements and phonations. Current treatments for TS are mostly symptomatic, addressing an unmet medical need for curing TS. Thus, new drugs for TS are defined by FDA as orphan drugs. The pathogenesis of TS remains elusive, while most studies support the involvement of impaired cortico-basal ganglia-thalamo-cortical circuit. Genetic studies of TS patients have identified several mutated genes, including the SLITRK1 gene. The SLITRK1/Slitrk1 protein expression is delicately regulated in the cortical-striatal-thalamo-cortical circuit during brain development, and confined in the striatal cholinergic interneurons (ChIs) in the adult striatum of human, monkey, and rodents. The function of SLITRK1 is related to neurite outgrowth and synapse formation. A post-mortem study has shown that the striatal ChIs in TS patients were significantly reduced, compared to normal controls. These findings suggest that SLITRK1/Slitrk1 in striatal ChIs plays an important role in the pathogenesis of TS. Our previous study indeed revealed that mice with the striatal Slitrk1 knocked down (Slitrk1-KD mice), induced by intra-striatal microinjection of Slitrk1 siRNA, manifested significantly more and longer stereotypic behaviors compared to the control group of mice injected with a scramble siRNA (scramble mice). In this study, we aim to elucidate the underlying mechanisms of Slitrk1 KD-elicited stereotypic behaviors. Slitrk1-KD mice were induced via intra-striatal (i.str.) microinjection of Slitrk1 siRNA into dorsomedial striatum bilaterally, and the control mice were i.str. injected with a scramble siRNA. Various striatal neurotransmitter levels were assessed by microdialysis-HPLC. To investigate the receptor subtypes involved in Slitrk1 KD-induced stereotypic behaviors, the antagonists, agonists, or positive allosteric modulator (PAM) of targeting receptor subtypes expected to decrease stereotypic behaviors were given to Slitrk1-KD mice, and the antagonists targeting the receptor subtypes expected to induce stereotypic behaviors were given to normal mice, and their effects on stereotypic behaviors were examined. From microdialysis-HPLC measurements of striatal neurotransmitters, we found that (1) evoked but not basal acetylcholine (ACh) levels were declined; (2) evoked dopamine (DA) levels were decreased, while tonic DA levels were increased; and (3) basal GABA, glutamate (Glu) and serotonin (5-HT) levels did not alter in the striatum of Slitrk1-KD mice in comparison to scramble mice. Stereotypic behavior tests showed that D2R antagonist (haloperidol, 0.3 mg/kg, i.p.), and GABAAR agonist (muscimol, 1 μg/0.5 μl, i.str.) decreased stereotypic behaviors in Slitrk1-KD mice, respectively; and an α4β2 nicotinic acetylcholine receptor (nAChR) antagonist (DHβE, 18 μg/0.5 μl, i.str.), increased stereotypic behaviors in normal mice. However, we did not observe significant reducing effects of an α4β2 nAChR agonist (sazetidine-A, 10 pmol/0.5 μl, i.str.); anti-cholinesterase (donepezil, 100 ng/0.5 μl, i.str.); muscarinic receptor type 4 (M4R) PAM (VU0152100, 12.5 ng/0.5 μl, i.str.); and D1R antagonist (SCH23390, 1 μg/0.5 μl, i.str.) in Slitrk1-KD mice. Besides, M4R antagonist (PD102807, 39 ng/0.5 μl, i.str.), did not markedly increase stereotypic behaviors in normal mice. These results suggest that α4β2 nicotinic, instead of M4 muscarinic, ACh receptors, D2, instead of D¬1, dopamine receptors, and GABAA receptors play pivotal roles in stereotypic behaviors. In the end, we demonstrated the reversibility of Slitrk1 KD-induced effects, including increased stereotypic behaviors and reduced striatal evoked DA levels. This reversible effect is associated with the reversed decreased striatal Slitrk1 protein levels. Collectively, it is suggested that Slitrk1 KD-induced stereotypic behaviors are attributed to dysfunctional striatal ACh and DA systems, and perhaps the GABA system. Striatal Slitrk1 deficiency in ChIs may lead to decreased evoked ACh, possibly due to the impaired function of Slitrk1 to form excitatory synapses. Diminished striatal evoked DA levels may be resulted from decreased striatal evoked ACh levels, while increased striatal tonic DA levels may be due to indirect compensatory effects or declined striatal evoked GABA levels. Pharmacologic studies suggest the important roles of α4β2 nAChR, D2R and GABAAR in stereotypic behaviors. The dysfunction of these neurotransmitter systems ultimately leads to disrupted function of medium spiny neurons (MSNs), contributing to stereotypic behaviors. Finally, the effectiveness of haloperidol in reducing Slitrk1 KD-induced stereotypic behaviors supports the predictive validity of this mouse model for further investigation of novel treatments for TS.