腸道菌相和腸道發炎相關基因變異於巴金森病致病機轉之角色

Abstract

巴金森病是第二大神經退化性疾病，由基因與環境的交互作用所形成的。根據病理學家Braak的解剖發現，路易氏體最早可能於腸神經叢中出現，經由迷走神經由下而上的傳至大腦。研究指出，巴金森病患的腸道，不僅是致炎性腸內菌較一般人來得多，不論是發炎生物標記，促炎細胞因子也都較正常人得高，代表巴金森病患腸道內有發炎的現象。而許多證據顯示，腸道感染或發炎，會導致α-synuclein(路易氏體的主成份)的產生。所以我們認為，人體內第一線抵抗這些致炎腸內菌的先天免疫系統中的Toll-like receptor (TLR)可能在巴金森病的形成過程，佔有一定的角色。有趣的是，不只是發炎性腸道疾病在多個流行病學研究被發現是巴金森病的風險因子之一，全基因定序研究更發現，巴金森病和發炎性腸道疾病有共同的LRRK2風險基因的變異，顯示此基因可能與巴金森病腸道的致病機轉有關。而就我們所知，LRRK2蛋白具有調控TLR所傳遞的發炎反應功能的角色。 整體而言，巴金森病的致病機轉與腸道菌叢不良(dysbiosis)和腸道發炎相關但詳細機轉不明，因此，本研究想要探討巴金森症患者中腸道內TRL2, TLR4, LRRK2與phospho-α-synuclein(p-α-syn)的表現與正常健康對照組之差異，同時亦釐清疾病嚴重度和這些相關蛋白質的表現是否有相關性。 材料與方法 本研究收案曾於臺灣大學附設醫院曾接受過常規大腸鏡檢查且符合UK PD Brain Bank Criteria的巴金森病患以及健康受試者。取得的腸道切片檢體經由石蠟包埋保存並使用BioGenex detection system進行LRRK2, TRL2, TLR4, 與p-α-syn, 等的免疫組織化學染色。染色完成後掃描成電子檔，並使用影像分析軟體，StrataQuest，進行表現量分析。統計方面，Mann-Whitney U test會用來比較巴金森病患與控制組。Kruskals-Wallis test則用來比較控制組，早期巴金森(H Y stage I, II)與後期巴金森(H Y stage III-V)患者。另使用Linear mixed-effects model來計算同一病患在不同時間點切片的分析結果。 結果 本研究共收集19位巴金森病患(42個腸道粘膜切片)，與年齡相符之7位健康控制組受試者(8個腸道粘膜切片)，以供免疫組織化學染色。染色後將玻片掃描成電子檔加以進行分析。而在所有巴金森病患中，共有6位病人在發病前與發病後接受兩次以上腸鏡檢查並有黏膜切片可供分析。我們發現，LRRK2在巴金森病患的腸道粘膜的表現相較於控制組顯著增加 (LRRK2陽性的細胞數與總細胞數的比例為2.63% vs. 0.13%, P=0.016)。而TLR2, TLR4，和p-α-syn則無統計上顯著差別。當比較控制組，初期巴金森症患者(H Y stage I II)，與後期巴金森患者(H Y stage III-V)腸道粘膜的蛋白質表現時，LRRK2的表現和疾病嚴重程度則有正相關的趨勢(P=0.06)。發病後的腸道LRRK2與TLR2表現，則較發病前有增加的趨勢，雖然沒有達到統計上的顯著。 結論 巴金森病患腸道粘膜的LRRK2表現相較於控制組是增加的，並與疾病嚴重程度呈正相關，暗示此蛋白在巴金森病腸道中的表現於啟動致病機轉之可能角色。

Background Parkinson’s disease (PD) is the second most common neurodegenerative disorder coming from the interplay between genetic and environmental risk factors. According to Braak's hypothesis, Lewy pathology begins in the gut enteric nervous system (ENS) and further spreads to the brain in a caudal-to-rostral fashion. Recent evidence has suggested proinflammatory gut microbiota profile and increased fecal inflammatory marker and expression of colon mucosa proinflammatory cytokines in PD gut, implicating the possible role of low-grade bowel inflammation in the development of PD. Also, studies have indicated that the expression of α-synuclein (the main component of Lewy body) can be induced by certain infections or intestinal wall inflammation. Therefore, it is speculated that toll-like receptors (TLRs), the crucial players of our gut innate immune system, may be involved in the development of the disease. Interestingly, not only inflammatory bowel disease was found to be a risk factor for PD in many epidemiology studies, a whole-genome sequencing study also found shared LRRK2 genetic risk variants in inflammatory bowel disease and PD. LRRK2 is the most common known genetic cause of PD and has been found to modulate the inflammatory pathway downstream of TLRs. Because the pathophysiology of dysbiosis and gut inflammation to the development of PD is still largely unknown, our study aims to evaluate the expression of LRRK2, TLR2, TLR4, and α-synuclein in the colon biopsy specimens of patients with PD and to correlate with disease severity. Materials and Methods Patients with PD who fulfilled the UK PD Brain Bank Criteria and healthy subjects who received routine health check-up, including a routine colonoscopy at the National Taiwan University Hospital, were recruited after informed consent. Colon biopsy samples of recruited subjects were collected and processed for paraffin embedding. Immunohistochemistry (IHC) was performed using the BioGenex detection system. Selected antibodies of LRRK2, TLR2, TLR4 and phospho-Ser129 α-synuclein (p-α-syn) were used. Slides were mounted after staining and scanned to electronic files for analysis by image analysis software, StrataQuest, for quantitative analysis. Mann-Whitney U test is used for comparison between PD and controls. Kruskal-Wallis test was used for comparison between controls, early PD (H Y stage I, II, and late PD (H Y stage III-V)). Linear mixed-effects model was used to analyze repeat biopsies of the same individual at different time points. Results Forty-two colonic random mucosal biopsies from nineteen patients with PD and eight colonic mucosa biopsies from seven healthy age and gender-matched control subjects were collected and stained for selected antibodies. Slides with acceptable quality were selected for quantitative analysis. LRRK2 in the colonic mucosa is significantly increased in PD patients (LRRK2 positive cells/total cell counts: 2.63% vs. 0.13%, P=0.016), whereas the expressions of TLR2, TLR4, and p-α-syn were similar between PD and controls. When comparing the expression of different proteins in colonic mucosa between early (H Y stage I or II) and advanced (H Y stage III - V) stage PD, LRRK2 exhibits a trend of increase in correlation with disease severity (P=0.06). In patients with repeated biopsies, levels of LRRK2 and TLR2 were generally increased after motor symptoms onset as compared to the pre-symptomatic stage. Conclusion In our study, colonic LRRK2 expression was significantly increased in patients with PD compared to controls and had a trend to correlate with the motor severity of PD. Future studies to examine the role of LRRK2 in igniting gut dysbiosis leading to PD are needed.