肌萎縮側索硬化症生物標記與腦源性神經保護因子應用於治療神經退化性疾病之分子機轉

Biomarkers of Amyotrophic Lateral Sclerosis and Molecular Mechanisms of Brain Derived Neurotrophic Factor in theTherapy of Neurodegenerative Diseases

黃啟訓(Chi-Shin Hwang)

指導教授 ： 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2013年)

https://doi.org/10.6843/NTHU.2013.00277

摘要

中文摘要
肌萎縮側索硬化症是一種可怕且複雜的進行性神經退化疾病，屬於世界衛生組織公告包括癌症及愛滋病在內之二十一世紀五大重要且困難治療的疾病之ㄧ，本研究顯示此疾病在台灣的發生率約為十萬分之0.51、盛行率約為十萬分之 1.97 。到目前為止其致病原因尚不清楚，且臨床診斷的難度相當高；以往各國的研究顯示平均誤診率高達10%、確診延遲時間約13到18個月。因此尋找新穎的肌萎縮側索硬化症分子生物標記以幫助提高臨床診斷的正確率至為重要。
肌萎縮側索硬化症的致病機轉包括：神經性發炎反應、谷氨酸興奮性中毒、細胞結構性蛋白病變、神經軸性物質傳導障礙、氧化壓力等因素；據信其中氧化壓力扮演極重要的角色。此外，目前已發現Damage Associated Molecular Patterns (DAMP)可以做為許多神經退化性疾病的診斷性生物標記；本研究發現熱休克蛋白HSP60及HSC70反應域專一性單株抗體能準確地測出肌萎縮側索硬化症病患血清中的熱休克蛋白分子濃度，並能與健康對照組明顯地鑑別診斷，證實特殊的熱休克蛋白反應域專一性單株抗體確實可應用於肌萎縮側索硬化症的分子診斷。
進一步研究發現肌萎縮側索硬化症患者血清中High Motility Group Box 1 (HMGB1)自體抗體(autoantibody)濃度較阿茲海默症患者、巴金森氏症患者、及正常人明顯高出許多；同時肌萎縮側索硬化症患者血清中HMGB1自體抗體濃度的上昇程度也與其病情進展成正相關。本研究率先報導血清中HMGB1自體抗體可應用於肌萎縮側索硬化症的早期診斷與病情追蹤，未來此新穎分子生物標記可應用於臨床試驗中的療效評估，同時幫助臨床醫師更進一步了解肌萎縮側索硬化症的真正致病機轉。
研究神經保護作用的分子機轉對於肌萎縮側索硬化症乃至於其他神經退化性疾病治療策略的研發都具有關鍵性的角色，目前已知神經保護因子缺乏為肌萎縮側索硬化症、亨廷頓氏症、及其他神經退化性疾病的重要致病因素；同時已發現腦源性神經保護因子brain-derived neurotrophic factor (BDNF)可延緩各種神經退化性疾病造成的神經細胞死亡。
3-Nitropropionic acid (3-NP)是一種不可逆的粒線體Succinate dehydrogenase呼吸酶抑制劑，已廣泛應用於研究神經退化性疾病中粒線體功能異常導致細胞死亡的分子機轉。本研究培養老鼠胚胎腦細胞並使用3-NP及BDNF進行各種試驗，根據實驗結果我們提出一個BDNF神經保護作用之可能分子機轉路徑：BDNF → NO →PKG → Thioredoxin → Bcl-2→神經保護作用。然而本研究亦發現活化此BDNF分子機轉路徑尚不足以達到完全的神經保護作用，其原因可能是此分子機轉路徑只是BDNF神經保護作用複雜級聯路徑(cascade)中的一條。
我們曾經報導過老鼠胚胎腦細胞經由BDNF處理後可誘導產生sonic hedgehog (SHH)以達到其神經保護作用；進一步研究更發現BDNF 誘發產生SHH必須經由活化 erythropoietin (EPO)作用。據此，本研究提出BDNF神經保護作用分子級聯機轉的另一條可能路徑如下：BDNF → EPO → SHH →神經保護作用。綜合以上我們的發現為肌萎縮側索硬化症、亨廷頓氏症、及其他神經退化性疾病的治療研發提供了一個嶄新的發展方向。

關鍵字

肌萎縮側索硬化症 ； 生物標記 ； 高活動性群族蛋白-1型 ； 熱休克蛋白 ； 嗜伊紅白血球神經毒素 ； 腦源性神經保護因子 ； 神經保護作用

並列摘要

Abstract

Amyotrophic lateral sclerosis (ALS) is a complicate and progressive onset devastating neurodegenerative disease. According to the announcement of World Health Organization, it is one of the five most important incurable human diseases of this era, along with cancer and AIDS. Our study showed that average incidence and prevalence of ALS in Taiwan was 0.51 and 1.97 per 105, respectively. Till now, the etiology of ALS remains largely unclear and its diagnosis is difficult; on average 10% misdiagnosis rate for ALS is reported. In addition, the averaged delay period between symptom onset to confirmation of ALS diagnosis may be up to 13 to 18 months. Hence searching for novel molecular biomarkers to improve the clinical diagnostic ability for ALS is essentially important.
Neuroinflammation, glutamate excitotoxicity, altered cytoskeletal proteins, impaired axoplasmic transport, and oxidative stress are involved in the pathogenesis of ALS. Among which oxidative stress plays an important role. Damaged associated molecular patterns (DAMPs) represent potential diagnostic biomarkers for a number of neurodegenerative diseases. In our studies, we found that domain specific monoclonal antibodies against heat shock proteins (HSP60 and HSC70) could successfully detect serum HSPs level in ALS patients and prove the difference between healthy adults and ALS patients. These monoclonal antibodies could be useful molecular biomarkers for the diagnosis of ALS.
Interestingly, serum levels of autoantibody (autoAb) against high mobility group box 1 (HMGB1) in ALS patients were significantly higher as compared with those in patients with Alzheimer’s disease (AD), Parkinson’s disease (PD), and healthy control subjects. Meanwhile, serum levels of HMGB1 autoAb were correlated with ALS disease progression, indicating that HMGB1 autoAb served as an effective molecular biomarker for diagnosis and clinical evaluation of ALS. This biomarker could be used to diagnose early stages of ALS, monitor disease progression, and potentially evaluate therapeutic efficacy during clinical trials. Therefore, identification of these molecular biomarkers could eventually assist understanding of the disease by providing insights into the pathogenesis of ALS.
Investigation of neuroprotective mechanism is important in development of therapeutic strategies for ALS and other neurodegenerative diseases. Deficiency of neurotrophic factors and mitochondrial dysfunction has been implicated in the pathogenesis of ALS, Huntington's disease (HD) and many neurodegenerative diseases. Meanwhile, brain-derived neurotrophic factor (BDNF) is known to rescue neuronal death.
3-Nitropropionic acid (3-NP) is an irreversible inhibitor of mitochondrial succinate dehydrogenase for exploring molecular mechanisms of cell death associated with mitochondrial dysfunction in neurodegenerative diseases. Here primary culture of fetal rat cortical neurons with different treatments of 3-NP and BDNF was studied. We proposed a hypothetical signal transduction pathway potentially inducible by BDNF preconditioning, denoted as: “BDNF → NO →PKG → Thioredoxin → Bcl-2 → protect neurodegeneration”. However, we also found that full protection could not be achieved with these pathway activators, indicating that this proposed pathway is only part of the signaling cascade inducible by BDNF preconditioning.
On the other hand, our other previous studies have also found that preconditioning of neuronal culture with BDNF induced sonic hedgehog (SHH), meanwhile BDNF-dependent SHH expression and 3-NP resistance required prior induction of EPO. Based on these findings, we proposed another hypothetical signal transduction pathway of BDNF signaling cascade, denoted as: "BDNF → EPO → SHH → protect neurodegeneration". In conclusion, all the above findings may illustrate a clue to develop new therapeutic strategy for ALS, HD, and many other neurodegenerative diseases.

並列關鍵字

Amyotrophic lateral sclerosis (ALS) ； Biomarkers ； High mobility group box 1 (HMGB1) ； Heat shock protein (HSP) ； Eosinophil derived neurotoxin (EDN) ； Brain-derived neurotrophic factor (BDNF) ； Neuroprotection

參考文獻

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