Slitrk1在成年小鼠紋狀體膽鹼能神經元中的關鍵作用: 對妥瑞症的啟示

Abstract

妥瑞症（Tourette Syndrome, TS），是一種常發生於兒童時期以非自主的運動和聲音抽動為特徵的神經發展疾患。根據精神疾病診斷與統計手冊第五版修訂版(DSM-5-TR)的診斷標準，妥瑞症的診斷符合下列幾項症狀: (1)需要出現多種運動型抽動症狀和至少一個聲語型抽動症狀, (2)這些抽動症狀需要反覆出現持續超過一年, (3) 抽動症狀須於18歲之前就出現, (4)這些症狀不能歸因於藥物或其他病毒感染的因素。妥瑞症患者所表現的抽動症狀可區分為簡單型抽動症狀, 如快速、孤立的動作，例如眨眼或清喉嚨; 和複雜型抽動症狀, 像是涉及一連串的動作或聲音，例如模仿他人的行為或是咒罵等等。妥瑞症患者常見的共病症包括注意力不足過動症（ADHD）和強迫症（OCD）。從病因學角度看，妥瑞症的成因涉及遺傳、環境和免疫因素的共同影響，尤其是許多研究都指出妥瑞症具有高度的遺傳性。從神經生物學角度看，妥瑞症與基底核功能失調有關，造成那些抑制不必要動作的腦內迴路受到影響，例如許多功能性影像學研究已指出連接大腦皮質-紋狀體-視丘-大腦皮質的迴路功能失常是妥瑞症患者主要的致病原因。 最近的研究則聚焦於紋狀體內膽鹼能中間神經元(Cholinergic interneurons, ChIs)的作用，這些膽鹼能中間神經元能調節多巴胺的釋放，並在運動控制和行為調節中發揮關鍵作用。紋狀體內的膽鹼能中間神經元的數量只佔所有紋狀體神經元的1-2％左右，但它們在腦中是最豐富的乙醯膽鹼區域，而且在紋狀體中投射出密集的纖維樹突，以調控下游的中型多棘神經元(Medium spiny neurons, MSNs)及其他γ-氨基丁酸中間神經元(GABAergic interneurons) 的功能，近一步影響個體的動作或行為。紋狀體膽鹼能中間神經元特殊的電生理特性使其不需要接受外來刺激便可以自行產生動作電位，所以又稱為紋狀體節律器。病理研究指出妥瑞症患者紋狀體中的膽鹼能中間神經元數量比一般人減少了將近50~60%左右，這是首次發現紋狀體膽鹼能中間神經元在妥瑞症病理成因的重要性。 臨床研究發現位於13q31.1染色體上的SLIT和NTRK-like 1（SLITRK1）基因變異與妥瑞症可能有相關性。SLITRK1/Slitrk1蛋白質對於神經樹突發育與突觸形成至關重要，這對神經連接和功能的發展是必需的。SLITRK1/Slitrk1在紋狀體神經元中的表現受到發育性的調控，以小鼠為例，在胚胎和青春期早期階段達到高峰，不過在成年期幾乎消失。但值得注意的是在成年階段Slitrk1的表現僅限於特定的膽鹼能中間神經元，這代表Slitrk1與維持成年階段ChIs的正常功能可能有關，因此我們提出的假設是降低成年小鼠紋狀體內Slitrk1的表現會讓小鼠的紋狀體膽鹼能中間神經元功能失常，進一步導致出現類似抽動的行為。 本研究將Slitrk1或Scramble小干擾RNA（small interfere RNA, siRNA）注射於成年的C57BL/6J雄性小鼠的背側紋狀體內，產生了Slitrk1基因敲落（Slitrk1-KD）小鼠與Scramble對照組，並分別比較兩者的刻板動作及類似抽動症狀的行為、感覺動作門閾過濾反應、Slitrk1 蛋白質表現量、紋狀體膽鹼中間神經元的數量與活性及電生理特性、神經傳導物質的濃度、與小鼠刻板行為對藥物的反應等等。我們發現Slitrk1-KD小鼠表現出更頻繁且持續時間更長的簡單和複雜的刻板行為，高峰期為注射後3天，但10天後逐漸恢復至基線。這些小鼠還出現顯著的前脈衝抑制缺陷和去除左前掌膠帶的延遲期增加，代表它們的感覺運動門控功能受損。電生理研究顯示，相較於Slitrk1陽性的膽鹼能中間神經元，Slitrk1陰性的膽鹼能中間神經元的膜電阻較高且神經興奮性較低。Slitrk1-KD小鼠表現出誘發性乙醯膽鹼和誘發性多巴胺釋放明顯降低的情況，但是基礎性多巴胺釋放量則被增強，顯示出神經傳導物質會有動態性的變化。儘管自發性活動量相近，但Slitrk1-KD小鼠對甲基安非他命誘導的高運動性反應較低，這指向多巴胺受體功能改變，尤其是多巴胺D2受體，進一步由對小鼠施與多巴胺D2拮抗劑以及對阿朴吗啡(apomorphine)的增強反應證實成年小鼠的多巴胺D2受體反應性增加了。 本研究發現Slitrk1對於維持紋狀體膽鹼能中間神經元的活動和隨後的多巴胺傳遞至關重要，Slitrk1-KD小鼠在某些方面模擬了妥瑞症的特徵，特別是在神經傳導系統失調和受體敏感性變化方面，提供了對該疾病神經生物學基礎的洞察。與其他需要壓力才能表現抽動行為的妥瑞症小鼠模型不同的是，Slitrk1-KD小鼠可自發性地出現這些症狀，突顯出潛在的紋狀體功能障礙主要涉及膽鹼能中間神經元功能改變與乙醯膽鹼和多巴胺等神經傳導物質的濃度變化有關。本研究的結論顯示出Slitrk1對於維持小鼠成年期紋狀體膽鹼中間神經元的功能具有其關鍵角色，對正常的神經遞質動態至關重要，且紋狀體膽鹼能中間神經元可能成為發展治療妥瑞症抽動症狀的潛在目標，尤其是對傳統治療反應不佳的病人。

Tourette Syndrome (TS), also known as Gilles de la Tourette Syndrome, is a common neurodevelopmental disorder in children, characterized by involuntary motor and vocal tics. According to DSM-5-TR criteria, the diagnosis of TS requires multiple motor tics and at least one vocal tic that persist for over a year, start before age 18, and are not attributable to medications or other medical conditions. Tics in TS are classified as either simple—such as quick, isolated movements like eye blinking or throat clearing—or complex, involving sequences of movements or sounds such as echopraxia and coprolalia, respectively. Common comorbidities in TS patients include ADHD and OCD. From an etiological perspective, TS involves genetic, environmental, and immunological factors, with recent research highlighting genetic predispositions. Neurobiologically, TS is associated with dysfunction in the basal ganglia, which affects brain circuits that suppress unwanted actions. Research also focuses on the role of striatal cholinergic interneurons (ChIs), which modulate dopamine and play crucial roles in motor control and behavior regulation. ChIs, constituting only 1-2% of total neurons, project dense arbors of fibers across the stratum, making it the most acetylcholine-rich brain region. Disruptions in ChI function are linked with TS, underlining the importance of these neurons in the disorder's pathology. The SLIT and NTRK-like 1 (SLITRK1) gene, located on chromosome 13q31.1, has been implicated in TS, though its association varies. SLITRK1 is critical for neurite development and synaptic formation, essential for neuronal connectivity and function. Its expression in striatal neurons is developmentally regulated, peaking during embryonic and adolescent stages but nearly vanishing in adulthood. Notably, in adult stages, its expression is restricted to specific cholinergic interneurons (ChIs), indicating its role in maintaining ChI functions and potentially influencing cortico-striatal circuits involved in TS. The study investigated Slitrk1 knockdown (Slitrk1-KD) mice in comparison to Scramble control groups, treated respectively with Slitrk1 and Scramble siRNA. The Slitrk1-KD group exhibited significantly more frequent and prolonged bouts of both simple and complex stereotypic behaviors, peaking 3 days post-siRNA injection and returning to baseline after 10 days. These mice also displayed significant deficits in prepulse inhibition (PPI) and increased latency in removing adhesive tape, suggesting impaired sensorimotor gating and function. A reduction in the number of active striatal ChIs, identified by decreased phospho-S6 ribosomal protein (pS6RP) staining, correlated with reduced Slitrk1 expression. Electrophysiological studies showed that Slitrk1-negative (Slitrk1(-)) ChIs had higher membrane resistance and lower excitability than Slitrk1-positive (Slitrk1(+))ChIs. Slitrk1-KD mice exhibited reduced evoked acetylcholine (ACh) and dopamine release, but increased tonic dopamine release, indicating altered neurotransmitter dynamics. Despite similar levels of spontaneous activity, these mice were less responsive to methamphetamine-induced hyperlocomotion, pointing to altered dopamine receptor function, particularly in D2 receptors, further evidenced by their increased response to apomorphine. These findings suggest that Slitrk1 is essential for maintaining the activity of striatal ChIs and subsequent dopaminergic transmission for normal motor function. The study concludes that Slitrk1-KD mice model certain aspects of TS, particularly regarding neurotransmitter system dysregulation and receptor sensitivity changes, offering insights into the neurobiological underpinnings of the disorder. Unlike other TS models that require stress to manifest tic behaviors, Slitrk1-KD mice spontaneously exhibited these symptoms, highlighting potential intrinsic striatal dysfunction, primarily involving ChIs and dopamine signaling. These conclusions emphasize the crucial role of Slitrk1 in striatal ChIs for normal neurotransmitter dynamics and its potential as a therapeutic target in TS, especially for those TS patients with symptoms resistant to standard treatments.