摘要:隨著人口結構日益老化,罹患神經退化性疾病患者與日俱增,對於社會與經濟是一大負擔與挑戰。釐清誘發疾病的致病機轉,研發減緩病程甚至是保護神經系統的治療策略,是目前的重要課題。巴金森氏症(Parkinson’s disease)為常見的神經退化性疾病,病患除表現出動作遲緩的症狀之外,亦會有非運動方面的症狀,其中以腸胃蠕動遲緩、便祕為最常見,這些腸胃道症狀也被認為是巴金森氏症的前驅症狀,甚至早在動作出現緩慢之前十幾年即已存在。現階段的證據顯示引起少數遺傳性巴金森氏症的致病基因變異與環境危險因子的交互作用,是導致大腦黑質中多巴胺神經細胞內α-synuclein 堆積(即路易士體Lewy body)的堆積與神經元漸進性缺失的原因。最近的研究發現,腸道中的微生物菌種為環境危險因子之一,藉由特殊腸道細菌的聚生與細菌代謝物短鍊脂肪酸,活化腸胃道中發炎反應,加速大腦中多巴胺神經的退化與路易士體的累積。臨床上,有10%的病患帶有家族遺傳史,目前已發現十幾個導致遺傳性巴金森氏症的致病基因,其中以Leucine-rich repeat kinase (LRRK2)最常見,LRRK2 G2019S 不僅是常見的突變為點,LRRK2 基因多型性也會增加偶發性巴金森氏症的致病風險。然而,引起巴金森氏症的基因變異背景與腸道菌群引致發炎之間的交互作用機轉目前仍未明。為探討此議題,本研究第一部分將募集巴金森氏症患者與健康成年人,研究兩者之間的腸道微生物體(microbiota)與發炎指標(糞便中鈣衛蛋白)的差異,並在巴金森氏症患者中探討這些指標與調控腸道免疫發炎反應的類鐸受體(toll-like receptors,TLRs)與LRRK2 基因變異間的交互作用對於疾病病程與症狀嚴重程度的影響; 我們也將長期追蹤巴金森患者,並探討合併腸道微生物體資訊、糞便與血液中短鍊脂肪酸與發炎相關係細胞激素濃度及TLRs 與LRRK2 基因變異之整合指標是否可以當作疾病進展的生物指標。第二部分,我們將以LRRK2 G2019S 基因轉殖鼠為動物模式。在餵食2.5% 低濃度的dextran sulphate sodium (DSS)給予小鼠腸道輕微慢性刺激促進發炎反應後,探討腸道神經細胞是否藉由調控腸道發炎反應的第二(TLR2)與第四類鐸受體(TLR4)促進下游激酶活性,基於glycogen synthase 3β (GSK3β)為TLR2,TLR4 與Lrrk2 激酶的共同下游,我們假設在此基因與腸道發炎的環境刺激下,將導致α-synuclein磷酸化增加,加速腸道神經細胞中α-synuclein 的累積。第三部分,我們將以穩定表現帶有LRRK2 G2019S 的SH-SY5Y 人類多巴胺神經細胞株與microglial 細胞株共同培養為細胞模式,探討腸道神經中的α-synuclein 是否會藉由誘發發炎細胞激素通過血腦障壁的非神經之體液路徑,藉由刺激大腦中的神經膠質細胞上的類鐸受體,以釋放發炎物質導致神經細胞凋亡,並同時刺激多巴胺神經細胞上的類鐸受體引發下游訊息反應,藉由和Lrrk2 變異的激酶活性間的交互作用,促使多巴胺神經細胞中的α-synuclein 磷酸化與累積,與多巴胺神經細胞凋亡。第四部份,我們將以TLR2與TLR4 抑制劑探究其對神經細胞退化與α-synuclein 的累積的保護效果。本研究之結果預計將對巴金森氏症的致病機轉有更深入之瞭解,同時也提供了一個對於未來以抑制腸道發炎及TLRs 相關神經發炎反應為機轉治療的策略方向。
Abstract: As society ages, the increasing burden for care of neurodegenerative disorders has become animportant issue. Research for elucidating disease pathophysiology and development of mechanism targeted therapy are current unmet needs.Parkinson disease (PD) is a common neurodegenerative disorder and is presented with progressivemotor dysfunction. Non-motor symptoms, such as gastrointestinal problems (especially constipations)may precede motor symptoms by decades and are regarded as prodrome symptoms of PD. The etiology ofPD comes from the interplay between genetic and environmental risk factors, which contribute tointraneuronal α-synuclein aggregations, namely Lewy bodies, and dopaminergic neuron degeneration. Inthe past decade, several PD-causative genes have been identified to cause familial forms of PD. Among these genes, LRRK2 gene mutation is most prevalent. LRRK2 carrying the G2019S mutation is the most common mutation in autosomal-dominant form of PD and genetic polymorphisms of LRRK2 also increase risk of sporadic PD. Recently, gut microbiota is recognized as a player in aggravating dopaminergic neuronal degeneration through microglia activation in a PD mouse model, reinforcing the crucial role of gut-brain axis dysregulation in neurodegenerative disorders. However, how gut microbiota and related gut inflammation interact with genetic mutation background to cause α-synuclein aggregations and neuronal degeneration remains largely unknown.To address these issues, the current research will recruit patients with PD and healthy adults to compare the fecal microbiota, its metabolite short chain fatty acid (SCFA), gut and plasma inflammatory markers between these two groups. As toll-like receptors (TLRs) mediate gut innate immune responses, we also investigate the interaction between aforementioned fecal and plasma inflammatory markers and LTRs genetic variants in the symptoms severity and disease progression of PD by longitudinally follow up the recruited PD patients. We believe that the results of this first part of the study will identify an integrated biomarker panel, incorporating fecal, plasma and genetic markers, for risk and diseaseprogression of PD. Next, by applying LRRK2 G2019S transgenic mice model, we will examine the intraneuronal α-synuclein aggregations in enteric nervous plexus (ENS) neurons of LRRK2 G2019S micefollowing feeding with dextran sulphate sodium (DSS) to induce inflammatory colitis condition. We postulate that TLRs signaling pathway, especially TLR2 and TLR4, and its interaction with aberrant Lrrk2 kinase activity would synergistically promote α-synuclein phosphorylation, aggregations and decrease its intra-cellular degradation by activating the common downstream GSK3β activity and inhibit lysomsome and proteasome degradation pathways. In the third part of this proposal, we will use the coculture system of stably expressed LRRK2 G2019S SH-SY5Y human dopaminergic neuronal cell line andhuman microglia cell lines to investigate the mechanisms how gut inflammation or gut microbiotametabolite, SCFA, contribute to central dopaminergic neuron degeneration. We hypothesize that these gutrelated inflammatory markers or secreted α-synuclein from ENS neurons would stimulate both microgliaand neurons, that activates neuroinflammation and provoke dopaminergic neuronal TLRs signaling andinteracting with Lrrk2 kinase activity to propagate α-synucleins aggregations and aggravateddopaminergic neuronal degeneration. Finally, we will examine the potential neuroprotective effects andinhibiting the α-synuclein aggregation abilities of antagonists of TLR2 and TLR4 in the chronic colitismice model carrying LRRK2 G2019S mutations.Our results will have a better understanding of PD pathophysiology and will open a new avenue forTLRs-targeted therapy in PD.