以典型瑞特氏症模式小鼠研究運動障礙之療癒

Abstract

瑞特氏症 (Rett syndrome, RTT)是由於第二型甲基CpG結合蛋白 (methyl - CpG binding protein 2, MECP2) 基因發生突變所造成的一種神經發育疾病。當小鼠的MeCP2缺失時，會產生許多類似瑞特氏症患者的運動障礙，其中包含運動活力低下、運動協調與運動學習能力缺損，此運動障礙可能由於γ-胺基丁酸(GABA)與多巴胺神經訊號傳遞功能失調所造成。先前研究發現Mecp2基因剔除小鼠的運動障礙伴隨大腦皮質與紋狀體中GABA合成的減少，我們因此嘗試利用藥理的方式增加GABA傳訊，測試其是否可改善Mecp2基因剔除小鼠的運動障礙。我們發現Mecp2基因剔除以及條件缺失公鼠，經由管餵方式給予100 mg/kg厚朴生藥 (cortex Magnoliae)連續七天後，對於平衡桿行走測試以及加速滾輪測試有改善的趨勢，但是對於Mecp2T158A點突變公鼠以及條件缺失母鼠卻沒有效果。我們進而檢測和厚朴酚合成純化物 (以下簡稱MH101)對運動障礙的改善效果。在行為測試前九十分鐘腹腔給予 1 mg/kg的MH101並觀察待測鼠在加速滾輪上的掉落延宕時間， 發現Mecp2條件缺失母鼠在投藥後，與控制組母鼠在同樣藥物處理下相近，顯示MH101有改善運動障礙的效果。以免疫染色法觀察c-Fos蛋白表現量檢測神經細胞的活性，發現在投予MH101後，控制組母鼠的c-Fos蛋白表現量在紋狀體之內背側區有顯著的增加，且Mecp2條件缺失母鼠相對應的腦區也有增加的趨勢，而其他皮質腦區卻皆無明顯改變，其結果顯示內背側紋狀體的活性增加可能與Mecp2條件缺失母鼠的運動協調能力增加有所關聯。另一方面，先前研究指出瑞特氏症模式小鼠的前端紋狀體中，有μ型類鴉片受體表現量減少以及第二型多巴胺受體的過度表現，所以我們嘗試在野生型小鼠的前端紋狀體活化其μ型類鴉片受體或是減少第二型多巴胺受體的神經傳導，檢測是否能夠增加其運動能力。實驗結果顯示，不論是給予μ型類鴉片受體促效劑或是第二型多巴胺受體拮抗劑皆可有效防止手術後之運動活力表現的缺損，因此，於前端紋狀體活化μ型類鴉片受體或阻斷第二型多巴胺受體可能改善小鼠之運動障礙。綜上所述，本研究藉由改善神經傳導的缺失緩解瑞特氏症模式小鼠部份的運動障礙，提供典型瑞特氏症可能有效的治療方式。

Rett Syndrome (RTT) is a neurodevelopmental disorder caused by mutations of the methyl-CpG-binding protein 2 (MECP2). Mice with deficient MeCP2 recapitulate many RTT-like motor symptoms, including hypoactivity, deficits in motor coordination, and motor learning, those are associated with hypofunction of GABAergic and dopaminergic neurotransmission. Previous study found that decreased GABA synthesis in the striatum is accompanied by motor deficits in Mecp2-null mice, here we attempt to examine whether pharmacological interventions by increasing GABA transmission could ameliorate motor deficits in Mecp2 mutant mice. We found that Mecp2-null mice and conditional knockout (cKO) male mice, but not mice carrying Mecp2T158A point mutation and cKO old female mice, administered with the cortex Magnoliae for 7 days via oral gavage at the dose of 100 mg/kg, showed a trend of improvement on open field test, beam walking task or rotarod task. We next tested the therapeutic effects of MH101 (a synthetic analog of honokiol),which is a GABAA receptor agonist derived from the bark of the plant Magnolia officinalis. We found that female cKO mice showed improvement on the rotarod performance after administration of 1 mg/kg MH101 90 mins prior to behavior tests. By examining expression of c-Fos protein, an indicator of neuronal activity, in different cortical and striatal regions with immunohistochemistry, we found that the MH101 treatment increased c-Fos expression in dorsomedial part of the rostral striatum in both control and cKO mice, but without significant alteration in the cortical neuron activity, suggesting that neuronal activity in the dorsomedial part of the rostral striatum may be related to increased motor learning in Mecp2 cKO female mice. On the other hand, the reduced expression of mu opioid receptor 1 and increased dopamine D2 receptor in the rostral striatum of RTT-like mice had been demonstrated in our previous study, we thus try to test whether activation of MOR1 or blockade of DRD2 in the rostral striatum could enhance motor function in wild-type mice. The results indicated that both treatments prevented post-surgery hypoactivity, suggesting that opioid and dopaminergic drugs could be alternative choices for improving motor deficits in RTT-like animal models. Taken together, our findings provide a proof-of-principle for novel pharmacological therapeutics to ameliorate motor deficits in mouse models of typical Rett syndrome.