| 摘要: | 本論文重點為治療高流於台灣和世界之兩種神經退化性疾病的新方法。 儘管創傷性腦損傷（TBI）和巴金森氏症（PD）在病理部位方面可能看起來不同，但在病理生理學和臨床數據方面存在許多共同的因素。這些共同要素在引言部分中引用，並在討論中更詳細地引用。第一篇關於PD之論文發表在國際分子科學雜誌（IF 3.8）上，第二篇關於TBI的論文最近在細胞移植（IF 2.9）中被接受，我是這兩篇論文的第一作者。關於輕度TBI的第三篇論文正在疾病神經生物學雜誌修訂中，我是本文的共同作者，在方法，結果和討論部分提供了重要的貢獻。我的研究結果如下。
A. 巴金森氏症
巴金森氏病（PD）通常以運動功能受損為特徵。在注射神經毒素6-羥基多巴胺（6-OHDA）以誘導單側黑質紋狀體多巴胺死亡後，可在動物中觀察到類似於在PD患者的步態異常。運動已被證明是一種具有潛力的非藥物治療方法，可降低神經退化性疾病的風險。在本論文中，我們研究了自主性滾輪運動對6-OHDA損傷的PD大鼠模型中步態、憂鬱、認知、旋轉行為表現以及組織學上的長期影響。我們發現，與非運動對照組相比，五週自主運動能緩解並延遲了6-OHDA所引起的步態異常，包括顯著改善的步行速度、步長、支撐寬度和足印長度。此外，我們也發現透過自主運動可以改善非運動功能，如新物體識別和強迫游泳測試。然而，與非運動組相比，運動組的旋轉行為沒有顯示出顯著差異。我們還詳細分析步態的時空變化，以研究PD大鼠模型長期運動後的潛在益處，這可能有助於未來客觀評估PD或其他神經退化性動物模型中的運動功能。此外，我們的研究結果顯示短期自主運動能緩解6-OHDA損傷大鼠的認知缺陷和憂鬱行為，而長期自主運動可減少運動症狀的發展，並提高酪氨酸羥化酶（TH），腦源性神經營養因子（BDNF），染色體X上骨髓酪氨酸激酶（BMX）的蛋白表現，而不影響該PD大鼠模型中的多巴胺能（DA）神經元損失。
B. 創傷性中度腦損傷 - 控制性皮層撞擊腦外傷模型
創傷性腦損傷（TBI）是死亡率和發病率的主要原因，影響全球1000萬人，且治療選擇有限。先前研究已發現，（-）- phenserine（Phen），一種乙酰膽鹼酯酶抑製劑，最初設計和測試在阿茲海默症（AD）的臨床III期臨床試驗中，能減少TBI後的神經變性，並減輕輕度TBI引起的認知障礙。在本論文中，我們使用控制性皮層撞擊腦外傷（CCI）中至重度TBI小鼠模型來評估Phen對創傷後組織化學和行為變化的影響。在損傷當天開始用臨床可轉換劑量Phen（2.5mg / kg，IP，BID）處理動物5天，並且在損傷後1週和2週透過動物行為和組織學檢測評估效果。Phen能顯著減弱TBI導致的挫傷體積，側腦室擴大，運動不對稱，感覺運動功能，運動協調和平衡功能等行為障礙。在TBI後，小膠質細胞的形態從休止轉變為活性形態，且Phen顯著降低了活化與休止型態小膠質細胞的比例，表示Phen能減輕TBI後的神經發炎反應。雖然Phen具有有效的抗乙酰膽鹼酯酶活性，但其（+）異構體Posiphen幾乎完全沒有抗乙酰膽鹼酯酶活性，卻仍具有許多神經保護特性。我們以與Phen相似的劑量評估Posiphen，發現側腦室大小增加，運動不對稱，運動協調和平衡功能有類似緩解現象，表示Phen對這些組織學和行為測試的改善是透過抑制乙酰膽鹼酯酶（AChE）以外的途徑達到。然而，Posiphen對病變大小的減少和感覺運動功能的改善遠小於相同劑量的Phen。總之，這些結果表示，在5天內用Phen治療顯著降低了TBI的損傷影響。這些數據表示該化合物可用於TBI治療的臨床應用。
C. 創傷性輕度腦損傷 - 落錘撞擊腦外傷模型與阿茲海默症
輕度創傷性腦損傷（mTBI）在臨床上佔TBI病例的80％-85％，且為神經退行性疾病（如阿爾茨海默病（AD））的危險因子。TBI衍生的神經病理學由發炎過程所促成：慢性小膠質細胞增生和促炎細胞因子的釋放，其進一步促使神經元功能障礙和喪失。我們還評估了兩種臨床可轉換劑量（2.5和5.0mg / kg，一天兩次），在野生型（WT）和AD APP / PSEN1轉基因小鼠的落錘撞擊腦外傷mTBI模型中對預先編程的細胞死亡/神經發炎/突觸完整性和Phen的功能的影響。在WT小鼠中，透過新物體識別和Y-迷宮行為評估，Phen能減輕mTBI誘導的認知障礙。通過計數WT小鼠的海馬和皮質中Fluoro-Jade C陽性（FJC +）細胞來評估，Phen完全減輕mTBI誘導的神經病變。在APP / PSEN1小鼠中，與WT小鼠相比，實驗組的退化細胞計數較大，且mTBI相對於AD對照組顯著提高FJC +細胞計數。而mTBI-Phen處理的小鼠與APP / PSEN1假性傷害組在退化細胞計數無顯著差異。此外，我們還評估了對小膠質細胞活化（Iba1免疫反應性（IR））和促炎細胞因子TNF-α的抗發炎作用。在WT和APP / PSEN1小鼠中，與假性傷害組相比，mTBI增加Iba1-IR和TNF-α/ Iba1共定位數量。 Phen降低了WT小鼠的海馬和皮質中的Iba1-IR，以及AD小鼠的皮質中的Iba1-IR。同樣，Phen在WT的所有區域中降低了Iba1 / TNF-α-IR共定位的表現範圍，在APP / PSEN1小鼠中具有類似的趨勢。透過定量PSD-95 +樹突棘和突觸素（Syn）-IR來評估突觸密度，在WT和APP / PSEN1小鼠中，mTBI與假性傷害組均顯著降低。 Phen能完全抵消PSD-95 +脊柱缺失在WT小鼠中和降低Syn-IR表現在APP / PSEN1和WT小鼠中。總而言之，臨床可轉換劑量的Phen能改善WT小鼠中mTBI誘導的預編程細胞死亡/神經發炎/突觸功能障礙，與完全減輕mTBI誘導的認知障礙。 Phen另外在AD小鼠腦微環境病理性現象，表現出積極的作用，進一步支持其作為mTBI治療的再利用。
This thesis focuses on novel approaches for treatment of two neurodegenerative disorders of high prevalence in Taiwan and worldwide. Although traumatic brain injury (TBI) and Parkinson’s Disease (PD) may seem dissimilar in terms of site of pathology, there are many common elements, both in terms of pathophysiology and clinical data. These elements of commonality are cited in the Introduction section and in more detail in the Discussion. The first paper, on PD, has been published in the International Journal of Molecular Sciences (IF 3.8). The second paper, on TBI, is recently accepted in Cell Transplantation (IF 2.9). I am first author on these two papers. A third paper on mild TBI is under revision in Neurobiology of Disease. I am a co-author on this paper and provided critical input to Methods, Results, and Discussion. A summary of my findings follows.
A. Parkinson’s Disease
Parkinson’s disease (PD) is typically characterized by impairment of motor function. Gait disturbances similar to those observed in patients with PD can be observed in animals after injection of neurotoxin 6-hydroxydopamine (6-OHDA) to induce unilateral nigrostriatal dopamine depletion. Exercise has been shown to be a promising non-pharmacological approach to reduce the risk of neurodegenerative disease. In this thesis, we investigated the long-term effects of voluntary running wheel exercise on gait phenotypes, depression, cognitive, rotational behaviors as well as histology in a 6-OHDA-lesioned rat model of PD. We observed that, when compared with the non-exercise controls, five-week voluntary exercise alleviated and postponed the 6-OHDA-induced gait deficits, including a significantly improved walking speed, step/stride length, base of support and print length. In addition, we found that the non-motor functions, such as novel object recognition and forced swim test, were also ameliorated by voluntary exercise. However, the rotational behavior of the exercise group did not show significant differences when compared with the non-exercise group. We also analyzed the detailed spatiotemporal changes of gait pattern to investigate the potential benefits after long-term exercise in this rat model of PD, which could be useful for future objective assessment of locomotor function in PD or other neurodegenerative animal models. Furthermore, our results suggest that short-term voluntary exercise is sufficient to alleviate cognition deficits and depressive behavior in 6-OHDA lesioned rats and long-term treatment reduces the progression of motor symptoms and elevates tyrosine hydroxylase (TH), brain-derived neurotrophic factor (BDNF), bone marrow tyrosine kinase in chromosome X (BMX) protein expression level without affecting dopaminergic (DA) neuron loss in this PD rat model.
B. Traumatic Moderate Brain Injury – Controlled Cortical Impact
Traumatic brain injury (TBI), a major cause of mortality and morbidity, affects 10 million people worldwide with limited treatment options. It has been previously shown that (-)-phenserine (Phen), an acetylcholinesterase inhibitor originally designed and tested in clinical Phase III trials for Alzheimer's disease (AD), can reduce neurodegeneration after TBI and reduce cognitive impairments induced by mild TBI using a weight-drop mouse model. In this thesis, we used a mouse model of moderate to severe TBI by controlled cortical impact (CCI) to assess the effects of Phen on post-trauma histochemical and behavioral changes. Animals were treated with Phen (2.5 mg/kg, IP, BID) for 5 days started on the day of injury at a clinically translatable dose and the effects were evaluated by behavioral and histological examinations at 1 and 2 weeks after injury. Phen significantly attenuated TBI-induced contusion volume, enlargement of the lateral ventricle, and behavioral impairments in motor asymmetry, sensorimotor functions, motor coordination and balance functions. The morphology of microglia was shifted to an active from a resting form after TBI, and Phen dramatically reduced the ratio of activated to resting microglia, suggesting that Phen also mitigates neuroinflammation after TBI. While Phen has potent anti-acetylcholinesterase activity, its (+) isomer Posiphen shares many neuroprotective properties but is almost completely devoid of anti-acetylcholinesterase activity. We evaluated Posiphen at a similar dose to Phen and found similar mitigation in lateral ventricular size increase, motor asymmetry, motor coordination, and balance function, suggesting the improvement of these histological and behavioral tests by Phen treatment occur via pathways other than anti-acetylcholinesterase (AChE) inhibition. However, the reduction of lesion size and improvement of sensorimotor function by Posiphen were much smaller than with equivalent doses of Phen. Taken together, these results show that post-injury treatment with Phen over 5 days significantly reduces the effects of TBI. These data suggest a potential development of this compound for clinical use in TBI therapy.
C. Traumatic Mild Brain Injury – Weight Drop Impact (WDI) and Alzheimer’s Disease
Mild traumatic brain injury accounts for 80%-85% of TBI cases clinically. Mild traumatic brain injury (mTBI) is a risk factor for neurodegenerative disorders, such as AD. TBI-derived neuropathologies are promoted by inflammatory processes: chronic microgliosis and release of pro-inflammatory cytokines that further promote neuronal dysfunction and loss. Herein, we also evaluated the effect on pre-programmed cell death/ neuroinflammation/synaptic integrity and function of Phen, an agent originally developed for AD, at two clinically translatable doses (2.5 and 5.0mg/kg, BID), in a weight drop (concussive) mTBI model in wild type (WT) and AD APP/PSEN1 transgenic mice. Phen mitigated mTBI-induced cognitive impairment, assessed by Novel Object Recognition and Y-maze behavioral paradigms, in WT mice. Phen fully abated mTBI-induced neurodegeneration, evaluated by counting Fluoro-Jade C-positive (FJC+) cells, in hippocampus and cortex of WT mice. In APP/PSEN1 mice, degenerating cell counts were consistently greater across all experimental groups vs. WT mice, and mTBI significantly elevated FJC+ cell counts vs. the AD control (sham) group. In contrast, mTBI-Phen treated mice had degenerating cell counts not significantly different from APP/PSEN1 shams. Anti-inflammatory effects on microglial activation (Ionized calcium binding adaptor molecule 1 (Iba1)-immunoreactivity (IR)) and the pro-inflammatory cytokine TNF-α were also evaluated. mTBI increased Iba1-IR and TNF-α/Iba1 colocalization vs. sham, in WT and APP/PSEN1 mice. Phen decreased Iba1-IR throughout hippocampi and cortices of WT mice, and in cortices of AD mice. Phen, likewise, reduced levels of Iba1/TNF-α-IR colocalization volume across all areas in WT, with a similar trend in APP/PSEN1 mice. Actions on astrocyte activation by mTBI were followed by evaluating GFAP, and were similarly mitigated by Phen. Synaptic density was evaluated by quantifying Postsynaptic density protein 95 (PSD-95) + dendritic spines and Synaptophysin (Syn)-IR. Both were significantly reduced in mTBI vs. sham in both WT and APP/PSEN1 mice. Phen fully counteracted the PSD-95+ spine loss in WT and Syn-IR decrease in both WT and APP/PSEN1 mice. In synopsis, clinically translatable doses of Phen ameliorated mTBI-mediated pre-programmed cell death/neuroinflammation/synaptic dysfunction in WT mice, consistent with fully mitigating mTBI-induced cognitive impairments. Phen additionally demonstrated positive actions in the more pathologic brain microenvironment of AD mice, further supporting its repurposing as a treatment for mTBI. |
