粒線體在細胞的代謝和存活上扮演一個很重要的角色，也因此粒線體的功能異常會導致細胞的死亡。從過去的研究顯示，許多人類的神經退化性疾病，例如Huntington病和粒線體腦病變等都與神經細胞的氧化磷酸化異常有關。然而造成這些神經細胞死亡的原因並不清楚。為了更深入探討線體功能異常時神經細胞死亡的機轉，我們使用一種粒線體毒物3-nitropropionic acid (3-NP)來研究粒線體功能異常所致腦病變的致病機轉。
3-NP本身為粒線體呼吸鏈第二複合體succinate dehydrogenase (SDH)的抑制劑。SDH本身也是Krebs循環的一個成份。動物實驗中發現，3-NP在大鼠和非人類的哺乳動物可造成動物行為的異常和選擇性的紋狀體病變，和人類的Huntington病相同。因此，我們希望藉由3-NP的動物模式來研究神經退化性疾病神經細胞死亡的機轉。由於過去的研究大多侷限於組織學或體外神經細胞培養的研究，所以首先我們希望建立一活體研究的動物模式。我們利用核磁共振影像和質譜的方法來追蹤注射3-NP所產生的腦病變。我們將2個月大Sprague-Dawley公鼠分為五組(各組n=10)，除第一組注射生理食鹽水為控制組外，第二至五組皆注射3-NP(15 mg/kg/d)連續五天。第三和四組除3-NP外另外注射Lamotrigine (第三組，10 mg/kg/d；第四組，20 mg/kg/d)而第五組則另外注射MK-801(2 mg/kg/d)。我們除每天觀察動物的行為改變以外，並於第六天以核磁共振影像(T2 圖譜)和活體質子磁共振質譜來評量鼠腦病變的大小和代謝物的變化。結果我們發現，連續注射3-NP的公鼠在第二天或第三天便開始出現典型的姿態障礙或後肢癱瘓的現象。在第六天10隻單純注射3-NP的大鼠中有9隻產生第4度或第5度的行為異常，亦即已四肢癱瘓無法行動，甚至倒在地上。相對的，同時注射3-NP和Lamotrigine 10 mg/kg/d的大鼠則除了兩隻產生輕微的神經障礙(第1度)外，其它接受3-NP和Lamotrigine或MK-801注射的大鼠，都沒有顯著的神經異常。磁共振影像和活體質子磁共振質譜的檢查也得到類似的結果。連續注射3-NP 5天的大鼠產生了明顯的選擇性紋狀體和海馬迴病變。T2 圖譜紋狀體部位的T2 值由原來的61.79 ±0.50(平均值±標準誤)增加到107.28 ± 28.12；而海馬迴的T2 值由64.40 ± 1.47增加到97.88 ± 19.22。但是皮質部位的T2 值則沒有改變，視丘部位也只有一隻大鼠產生輕微的病變，這與人類的Huntington病極為相似。質子磁共振質譜上腦代謝物的改變也反應出這個事實。注射3-NP 5天後磁共振質譜上，紋狀體部位N-acetylaspartate (NAA)/Creatine (Cr)的比值明顯由1.327 ± 0.066 (平均值±標準誤)減少到0.923 ±0.137，表示紋狀體部位的神經細胞部份死亡或功能異常。除此之外，紋狀體部位也出現一Lactate的波峰，顯示出粒線體的氧化磷酸化受到影響。這些T2 圖譜上的病變和代謝物的變化與大鼠產生的行為改變有很好的相關。當我們除3-NP外再同時注射Lamotrigine或MK-801時，除了前述的行為變化消失了，紋狀體和海馬迴的病變也減輕，且NAA/Cr的比值也接近正常值，而Lactate波峰也消失。這表示紋狀體神經細胞的功能因Lamotrigine和MK-801的治療而得到明顯改善。有趣的是高劑量Lamotrigine明顯比低劑量提供更好的保護作用，甚至比MK-801的效果還好。由這些研究的結果顯示核磁共振影像和活體磁共振質譜的方法可有效的用來追蹤和評估3-NP所引起的傷害和代謝物的改變，因此是一個很好的3-NP活體研究的動物模式。由於MK-801是NMDA受體的拮抗劑，而Lamotrigine本身可抑制突觸前glutamate的釋放，因此從它們所提供的保護作用可證明3-NP的神經毒性主要是透過glutamate受體，尤其是NMDA受體的活化來進行。
接著我們希望更進一步探討3-NP所導致的腦病變與代謝物變化的關係。結果在靜脈注射單劑的3-NP (30 mg/kg)以後， Succinate/Cr和Lactate/Cr的比值逐漸增加。相較於控制組，3-NP組的NAA/Cr 比值也由原來的1.137減少到135分時的0.974 (95% C.I. = 0.887－1.061)，並於195分降到最低點。令人感興趣的是在注射3-NP以後，當NNA的波峰慢慢下降時在其右側逐漸出現一小的波峰與其重疊在一起。我們利用體外質子磁共振質譜的方法證實這個小波峰便是acetate。Acetate的變化比Lactate和NAA的變化更早發生，在注射3-NP後75分便產生統計上的意義，並在165分時達最高值(平均值0.369；95 %C.I. = 0.309-0.430)。我們也同時利用擴散加權造影(diffusion-weighted image)的方法追蹤靜脈注射3-NP以後的腦病變。結果發現大鼠紋狀體的變化在3-NP注射後的145分鐘才開始出現顯著的差異，此約相當於磁共振質譜上NAA開始減少的時間。因此，注射3-NP以後腦內紋狀體代謝物的改變比影像的變化更早發生。研究中我們發現的Acetate的增加可能來自於3-NP毒性所導致的脂肪酸崩解，SDH的抑制或可能來自NAA的水解。所增加的Acetate可提供Acetyl基作為神經細胞毒性傷害時細胞膜的修復之用。因此，Acetate的增加可作為3-NP導致細胞傷害的最早證據。
我們接著希望更進一步了解3-NP對神經細胞的影響。我們利用一由NT-2細胞分化而來的人類神經細胞NT2-N細胞來作研究。當我們以3-NP處理NT2-N細胞時，NT2-N細胞產生兩種不同的死亡形式，亦即細胞凋亡和細胞壞死。當3-NP處理的時間越久時，NT2-N細胞發生細胞凋亡的比例越高。而當3-NP濃度的增加，發生細胞壞死的比例也增加。NT2-N細胞死亡的型態與細胞ATP的含量有關。高濃度的3-NP所造成的ATP減少較快且較嚴重，主要引起細胞壞死；而低濃度(0.1 mM)的3-NP所引起的ATP 減少較輕微，所以主要以細胞凋亡為主。當我們同時加入MK-801 10 µM處理時，在48小時後，細胞內ATP的含量由單純只加3-NP時的25 ± 4%回升到80 ± 2%(n=10)。由此證明3-NP所導致的細胞內ATP含量的減少只部份因3-NP抑制SDH所造成，而主要還是導因於NMDA受體的活化所造成的結果。
由我們活體動物的研究已發現，NMDA受體拮抗劑MK-801可減輕3-NP的毒性傷害。在NT2-N細胞我們也得到相同的結論，雖然培養液內glutamate的濃度並沒有增加，1 mM 3-NP處理48小時所引起的細胞死亡可顯著地被MK-801所減輕。相對地，非NMDA受體拮抗劑如CNQX或L型鈣離子通道的拮抗劑Nifedipine所提供的保護作用則較小。顯示出NMDA受體的活化在NT2-N細胞的死亡中比非NMDA受體的活化來得重要。除了這些藥物外，許多藥物也對這種NT2-N細胞的死亡提供保護作用。例如propyl gallate這種抗氧化劑便可減少3-NP所致LDH的釋放達44 ± 4%(n=8)；這表示自由基過度製造也是造成NT2-N細胞死亡的原因之一。同樣的道理，Bongkrekic acid和cyclosporin A這兩種藥皆可抑制粒線體通透性轉換(mitochondrial permeability transition)，也同樣明顯減少了3-NP所造成的細胞死亡，證明粒線體通透性轉換在3-NP所致的細胞死亡上也扮演重要的角色。另外，有趣的是，一種可抑制內質網鈣離子釋放的藥xestospongin C也提供了很好的保護作用。這表示3-NP造成NT2-N細胞死亡的機轉是多方面的。
由於細胞內鈣離子濃度([Ca2+]ί)的過度增加與NMDA受體活化所引起的神經毒性有關，我們接著探討3-NP對NT2-N細胞內[Ca2+]ί調控的影響。當我們加入1 mM 3-NP以後，NT2-N細胞的[Ca2+]ί會由原來的48 ± 2nM增加到2小時後的140 ± 12nM。如果我們同時加入MK-801則[Ca2+]ί的增加幅度明顯減慢，但是如果我們同時再加入Nifedipine，[Ca2+]ί增加的速度並沒有進一步減少，證明NMDA受體所形成的通道是造成[Ca2+]ί增加的主要管道。
由於xestospongin C也提供保護作用，因此我們接著探討細胞內鈣離子的增加是否與細胞內原有的鈣離子貯積有關。當我們在無Ca2+的溶液內加入1 mM 3-NP時，最初幾分內[Ca2+]ί增加的情形與含Ca2+溶液中的情形相同。但當時間更久，[Ca2+]ί增加的速度便減慢，這證明加入3-NP後剛開始幾分鐘 [Ca2+]ί增加主要來自於細胞內Ca2+貯積(從內質網或粒線體)的釋放，但細胞外Ca2+的內流對維持[Ca2+]ί的持續增加十分重要。當我們加入xestospongin C和dantrolene抑制內質網Ca2+的釋放，則最初幾分鐘內[Ca2+]ί的增加也被抑制。由這些結果證明，內質網內鈣離子的釋放才是造成最初[Ca2+]ί增加的主因。這一點在過去的研究是未曾被報告的。
我們接著探討這種[Ca2+]ί不平衡的現象是否會持續到24或48小時。結果我們發現只有分別17%和25%的細胞其[Ca2+]ί在24和48小時後維持在100 nM以上。如果除了3-NP外，再同時加上MK-801或xestospongin C則[Ca2+]ί在100 nM以上的細胞會明顯更少。如果除了MK-801再同時加上xestospongin C或CNQX，則[Ca2+]ί大於100 nM的細胞會比單單加MK-801時更少。因此， NT2-N細胞[Ca2+]ί的失去均衡與細胞外Ca2+內流和細胞內Ca2+的釋放有關，而[Ca2+]ί的調控出了問題是造成NT2-N死亡的一個重要因素。這是第一個以人類神經細胞為主的3-NP研究模式，也因此可提供我們更多3-NP對人類神經細胞影響的資訊。
除了[Ca2+]ί的不平衡外，近幾年粒線體內鈣離子濃度([Ca2+]m)的增加也被認為是造成細胞凋亡或細胞壞死的主因。因此，我們接著探討3-NP對[Ca2+]m的影響。相對於[Ca2+]ί的改變，[Ca2+]m在加入3-NP 2-4小時後才逐漸增加。加入10 µM ruthenium red抑制粒線體Ca2+的吸收則[Ca2+] m的增加明顯減少，同時也減少3-NP所引起的細胞凋亡的細胞數目達70%。由此證明[Ca2+]m的增加是導致3-NP神經毒性的一個重要步驟。
由前面的實驗證明，自由基的過度製造也是導致細胞死亡的原因之一。由實驗發現，reactive oxygen species (ROS)在加入3-NP後便慢慢增加。加入抗氧化劑propyl gallate不但防止了ROS的增加，且減少了3-NP所引起的細胞凋亡。當我們加入ruthenium red抑制[Ca2+]m的增加，ROS明顯減少；相反地，加入propyl gallate，[Ca2+]m只輕微減少。此表示[Ca2+]m的增加是ROS增加的一個主要來源，而早期ROS的產生並不會明顯影響粒線體的功能。
當我們加入3-NP 6-8小時後，粒線體膜電位開始慢慢減少。如果我們加入ruthenium red抑制[Ca2+]m的增加，膜電位則不會減少。由於粒線體膜電位的去極化與粒線體通透性轉換的發生有關，因此當我們加入cyclosporin A抑制粒線體通透性轉換後，粒線體膜電位便不會減少，同時也防止了細胞凋亡。然而cyclosporin A並不會影響[Ca2+]m的變化。這說明了[Ca2+]m的增加可能導致粒線體通透性轉換的發生，進一步引起粒線體膜電位的去極化。
Caspase的活化與細胞凋亡有關。3-NP的處理使caspase-3的活性由原先的8 ± 1增加到8小時後的42 ± 3。如果加入zVAD-fmk和DEVD-fmk分別抑制所有caspase和caspase-3的活化，則細胞的凋亡皆會減少，證明3-NP的毒性與caspase(包括caspase-3)的活化有關；加入zVAD-fmk和DEVD-fmk也減少了[Ca2+]m增加的幅度，同時粒線體膜電位的下降也減少。證明加入3-NP以後早期caspase (包括caspase-3)的活化可能與隨後[Ca2+]m的增加和粒線體膜電位的去極化有關。當我們同時加入MK-801時，[Ca2+]m的增加，自由基的產生，粒線體膜電位的去極化，和caspase-3的活化，幅度都明顯減少。由此證明，NMDA受體的活化與隨後粒線體功能的改變，caspase-3的活化和ROS的產生都有關。
雖然NMDA受體的活化是3-NP造成神經毒性的主因，這些受體拮抗物的毒性限制了它們臨床運用的價值。然而我們的研究顯示，抑制glutamate的釋放，自由基的產生，內質網Ca2+的釋放，和[Ca2+]m的過度負荷都可能可以運用於防止粒線體功能異常所造成的細胞死亡。此外，抑制粒線體膜電位的去極化與粒線體通透性轉換的發生也是未來可努力的目標。

Mitochondria play an important role in cellular metabolism and survival. Mitochondrial dysfunction was noted to be associated with some neurodegenerative diseases, such as mitochondrial encephalopathy, Parkinson disease, and Huntington’s disease. One of the challenges in understanding disorders with mitochondrial dysfunction is the neuropathogenic mechanism of the lesions in the brain, and their neuroprotective strategies. Systemic administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase (SDH), the complex II in mitochondrial respiratory chain, induces selective striatal lesions in rats and non-human primates mimicking those in Huntington’s disease. However, the pathogenic mechanism of the brain lesions also remained unknown. A pathogenic mechanism similar to hypoxic/ischemic damage has been suggested, and excitatory amino acids, especially glutamate, may play an important role, especially through the activation of NMDA receptors. The possibility that the impairment of mitochondrial metabolism might lead to secondary excitotoxic lesions via activation of NMDA receptor was first suggested by Novelli et al.. They found that neurons are more susceptible to glutamate when energy production is impaired. Because a significant proportion of energy in brain is used to support neurotransmission and maintain homeostasis, partial inhibition of energy metabolism may result in neuronal loss, which can be blocked by antagonists of NMDA receptors. Recent studies had shown that non-NMDA receptors may also been activated when the energy metabolism is severely impaired. Therefore, inhibition of glutamate release and blockage of glutamate neurotransmission may be some possible targets for the therapy of brain lesions induced by mitochondrial dysfunction.
In the past, most of the studies about mitochondrial dysfunction were performed by histological evaluation. Over the past several years, more and more evidence was accumulated to indicate that magnetic resonance imaging (MRI) and in vivo proton magnetic resonance spectroscopy (1H-MRS) can be used to non-invasively evaluate cerebral lesions and measure the change of regional cerebral metabolites in vivo. The images obtained in MRI are well correlated with histological changes. 1H-MRS, allowing non-invasive in vivo investigation of cerebral metabolites, has recently been widely used to evaluate a variety of cerebral diseases. Therefore, in the first part of the studies, we try to establish a non-invasive animal model using MRI to investigate the possible pathogenic mechanism of 3-NP-induced neurotoxicity.
We used MRI and 1H-MRS to evaluate the rat brain lesions induced by 3-NP. Systemic administration of 3-NP (15mg/kg/day) to two-month-old Sprague-Dawley rats (n=10 for each group) for five consecutive days induced selective striatal and hippocampal lesions and specific behavioral change mimicking those in Huntington disease. Rats developed behavioral changes 2-3 days after systemic administration of 3-NP. Nine of the ten 3-NP-treated rats developed Grade 4-5 behavioral changes at the 6th day. Pretreatment with lamotrigine (10mg/kg or 20mg/kg/day) or MK-801 (2mg/kg/day) attenuated the lesions and behavioral change. Except for two rats, treated with lamotrigine 10mg/kg/day, developing mild neurological deficits in movement, other rats pretreated with lamotrigine and MK-801 showed only mild decrease of daily activity without prominent limb paralysis. There were also no significant differences in T2 values of the striatum and hippocampus among rats pretreated with MK-801, lamotrigine (20mg/kg) and sham controls. In rats pretreated with lamotrigine 10mg/kg, only two rats still had prominent striatal and hippocampal lesions, and one had a mild hippocampal lesion; the others were free of lesions. The calculated T2 values in the striatum were attenuated from 107.28±28.12 ms in 3-NP-treated rats to 66.28±11.09 ms (lamotrigine 10mg/kg group), 62.21±2.36 ms (lamotrigine 20mg/kg group) and 63.19±1.91 ms (MK-801 group), respectively (P<0.001). In the hippocampus, the T2 values were attenuated from 97.88±19.20 ms in 3-NP-treated rats to 72.36±19.30 ms (lamotrigine 10mg/kg group), 63.41±2.30 ms (lamotrigine 20mg/kg group) and 66.39±2.27 ms (MK-801 group), respectively (P<0.001). Compared with only 3-NP-treated rats, the T2 values of the hippocampus were attenuated by 25-35% while the T2 values of the striatum were attenuated by 38-42% after pretreatment with lamotrigine or MK-801.
Significant elevations of Succinate/creatine (Cr) and Lactate/Cr ratios and decreases of N-acetylaspartate/Cr (NAA/Cr) and Choline/Cr ratios were observed after 3-NP injections for 5 days (P<0.001). The NAA/Cr ratio in the striata well correlated with the behavioral changes of rats. The lactate peaks completely disappeared after pretreatment with lamotrigine and MK-801. However, the changes of NAA/Cr and Choline/Cr ratios were different in various groups of rats. They were nearly prevented after pretreatment with lamotrigine (20mg/kg). However, the NAA/Cr in rats pretreated with lamotrigine (10mg/kg) (P<0.01) and MK-801 (P<0.05) still showed significant reduction as compared with sham controls. Therefore, both lamotrigine and MK-801 are effective in attenuation of brain lesions induced by 3-NP. A higher dose of lamotrigine provides better neuroprotective effect than MK-801. With better therapeutic effect and fewer side effects, lamotrigine is more promising for the potential clinical application. The present study demonstrated that the activation of glutamate receptors is possibly the pathogenic mechanism leading to selective striatal lesions in 3-NP intoxication. This non-invasive animal model can be used to investigate the potential therapeutic medications in the future.
Because 3-NP can lead to significant metabolic changes in rat striatum, to further investigate the mechanisms of neuronal death in neurodegeneration and the effect of 3-NP neurotoxicity on neurons, in vivo 1H-MRS and diffusion-weighted MRI (DWI) were used to evaluate temporal changes in rat striata after 3-NP administration. We found that NAA reduction was preceded by a progressive elevation of lactate peaks located at 1.33 ppm, indicating that impaired oxidative phosphorylation is the mechanism of neuronal death in acute 3-NP intoxication. In this study, NAA reduction with nearly simultaneous development of striatal lesions in DWI was also preceded by a significant and progressive elevation of acetate peak, which was finally confirmed by in vitro 1H-MRS. The elevation of acetate/Cr, which occurred much earlier than the changes of Lactate/Cr and NAA/Cr ratios, was first observed at 75 min after 3-NP injection (mean of Acetate/Cr, 0.243; 95%C.I., 0.182-0.303) (P=0.011). It reached a peak at 165 min after the beginning of NAA/Cr reduction (mean of Ac/Cr, 0.369; 95%C.I., 0.309-0.430), and then began to decline after 165 min when there was still a mild decrease in the NAA/Cr ratio, and recovered to the control level at 255 min. There were no significant changes in Ac/Cr ratio after that point. An early significant elevation of acetate, to our knowledge, has not previously been detected in brain lesions associated with impaired oxidative phosphorylation. There are at least three possible sources of acetate. Because acute administration of 3-NP in rats increases free fatty acids (FFAs) by 130-300% in selective brain regions within one to two hours, the early elevation of acetate may arise partially from the degradation of FFA as in cerebral ischemia or trauma. Besides, 3-NP can inhibit Krebs cycle''s function and lead to the accumulation of the Krebs cycle metabolites, including acetyl-CoA, which may contribute to the elevation of acetate. As a result of disruption of the cell membrane in neuronal injuries, NAA is also freely accessible to degrading enzyme asparto-acylase, leading to the hydrolysis of NAA to aspartate and acetate. These results suggest that acetate elevation may arise from fatty acid degradation, inhibition of SDH, and possible NAA hydrolysis. The elevated acetate may provide a source of acetyl group for membrane repair during excitotoxic brain injury. The present study showed that both DWI and 1H-MRS are sensitive methods for detecting early brain damage due to impaired oxidative phosphorylation. However, acetate and lactate elevations in 1H-MRS provide much earlier evidence of neuronal dysfunction when metabolic energy generation is impaired.
After establishing an in vivo animal model for evaluating the 3-NP-induced neurotoxicity, we would like further to investigate the pathogenic mechanism of 3-NP in cellular level. Human NT2-N neurons, prepared by retinoic acid-induced differentiation of the human teratocarcinoma line Ntera-2, are susceptible to NMDA and non-NMDA receptor-mediated excitotoxicity, and that oxygen/glucose deprivation causes NT2-N neuronal necrosis by an NMDA receptor-mediated mechanism. When 3-NP was used to treat human NT2-N neuron for a long time, it leads to ATP depletion and both necrosis and apoptosis in human NT2-N neurons. Necrosis occurred predominantly during the first 2 days in a dose-dependent manner, whereas apoptosis was observed after 24 hr or later at a low constant rate in different concentration of 3-NP. We assayed the intracellular concentration of Ca2+ ([Ca2+]i) during the first 48 hours in 1mM 3-NP, a period during which 10% of the neurons died by necrosis and 3% by apoptosis. During the first 2 hours in 3-NP, all NT2-N neurons showed [Ca2+]i rise from 48 + 2 to 140 + 12 nM (mean + SEM). After 24 and 48 hours in 3-NP, however, [Ca2+]i remained above 100nM in only 17% and 25% of the NT2-N neurons, respectively, suggesting that most neurons were able to correct this early rise in [Ca2+]i, despite severe ATP depletion, and to survive. Addition of NMDA receptor antagonist MK-801 in acute 3-NP exposure attenuated the rise in [Ca2+]i, while nifedipine, an inhibitor of L-type voltage-gated calcium channels, did not further attenuate the elevation. That indicates that extracellular Ca2+ influx through NMDA receptors rather than calcium channel may explain the major [Ca2+]i elevation in acute 3-NP exposure. Because the addition of xestospongin C and dantrolene prevented the early rise in [Ca2+]i in Ca2+-free solution, the release of internal Ca2+ store from the endoplasmic reticulum rather than mitochondria can explain the early rise in [Ca2+]i, while the elevation of [Ca2+]i in later stage depends on the extracellular Ca2+ influx.
In our study, we also showed that activation of NMDA receptors contributed substantially to 3-NP-induced ATP depletion, and subsequent chronic elevation of [Ca2+]i in the NT2-N neurons. ATP content of control NT2-N neurons was 12.2 + 1.5 nM/mg protein (mean + SEM, n=18). Neuronal ATP content did not fall during the first 6 hours of incubation with 0.1 mM 3-NP, but then declined to 48 + 9 % and 47 + 2% (mean + SEM, n=6) of the basal value at 24 and 48 hours, respectively. Higher concentrations of 3-NP caused an earlier and more profound decline. In 1 mM 3-NP, for example, ATP content varied between 67 and 86% of the basal value during the first 6 hours, and had fallen to 22 + 3% and 25 + 4% of the control value after 24 and 48 hours, respectively. Addition of 10 mM MK-801 concurrently with the 1mM 3-NP substantially diminished ATP depletion. ATP content remained at 80 + 2 % (n=10) of the control value at 48 hours, more than 3-fold higher than in parallel cultures in the absence of MK-801.
The pathogenic mechanisms of 3-NP-induced neuronal death in NT2-N neurons are multiple. Except for NMDA and non-NMDA receptor antagonists and calcium channel antagonists, we also demonstrated that blocking endoplasmic reticulum Ca2+ release enhanced the capacity of these human neurons to maintain [Ca2+]i homeostasis and resist necrosis while subjected to chronic energy deprivation. Two agents, bongkrekic acid and cyclosporin A, that block the mitochondrial permeability transition were modestly neuroprotective, indicating that induction of mitochondrial permeability transition is also important in 3-NP-induced neuronal death. The antioxidant, propyl gallate (10mM), previously reported to diminish 3-NP-induced neuronal apoptosis, also diminished 3-NP-induced NT2-N neuronal LDH release by 44% + 4% (mean + SEM, n=8, p<0.01) at 48 hours.
In the study mentioned above, we showed that 3-NP might produce neuronal death secondary to perturbed intracellular calcium homeostasis. Because studies also showed that Ca2+ influx thought NMDA receptors is more toxic than through calcium channels, there should be other factors in determining the selective toxicity of [Ca2+]i elevation. Intramitochondrial calcium ([Ca2+]m) overload had been found to be associated with both necrosis and apoptosis. Because mitochondria are located close to NMDA receptors, the selective toxicity of Ca2+ entering via NMDA receptors can, therefore, be interpreted as a privileged access of Ca2+ entry to the mitochondria, leading to [Ca2+]m overload. However, the response of [Ca2+]m to 3-NP in neurons remains unknown. In this study, we investigated the roles of and relationships among [Ca2+]m overload, mitochondrial reactive oxygen species, and mitochondrial membrane depolarization in 3-NP-induced neuronal death. Following 1mM 3-NP treatment on primary rat neuronal cultures, there was a gradual increase of [Ca2+]m beginning at 2-4 hrs post 3-NP application. Compared with the [Ca2+]i elevation in previous study, [Ca2+]m elevation occurred much later than the rise in [Ca2+]i. After application of 3-NP, there was also a 2-fold increase of mitochondrial reactive oxygen species (ROS) at 4 hrs. These were followed by mitochondrial membrane depolarization at 6-8 hrs post-treatment. By inhibiting [Ca2+]m uptake, ruthenium red attenuated the production of ROS, and prevented the 3-NP-induced mitochondrial membrane depolarization and 70% of apoptotic neuronal death (p<0.001). Therefore, the elevation of [Ca2+]m contributes to the major part of free radical production, and may lead to mitochondrial permeability transition and subsequent mitochondrial membrane depolarization. The caspase-3 was also activated since the first hour post 3-NP application. Inhibition of caspase or caspase-3 activation attenuated the elevation of [Ca2+]m and mitochondrial membrane depolarization (p<0.001), indicating that caspase (including caspase-3) activation plays a role in the elevation of [Ca2+]m and mitochondrial membrane depolarization.
In this study, MK-801, an antagonist of NMDA receptors, prevented 3-NP-induced [Ca2+]m elevation, ROS production, caspase-3 activation, mitochondrial depolarization, and finally neuronal death. It indicates that the activation of NMDA receptors play a most important role in 3-NP-induced neurotoxicity. Inhibition of its activation and [Ca2+]m overload with subsequent mitochondrial membrane depolarization can therefore attenuate the neuronal death induced by 3-nitropropionic acid.
From our serial studies about 3-NP neurotoxicity, we found that impaired oxidative phosphorylation with subsequent activation of glutamate receptors, especially NMDA receptors, may be the major pathogenic mechanism of 3-NP neurotoxicity. However, perturbed intracellular calcium homeostasis with overproduction of ROS, intramitochondrial calcium overload, mitochondrial permeability transition, and mitochondrial depolarization will interact to lead to neuronal death. Because there is significant neurotoxicity for NMDA receptor antagonist, inhibition of glutamate release, prevention of perturbed intracellular calcium homeostasis, including mitochondrial calcium overload, and inhibition of mitochondrial permeability transition and membrane depolarization may be other potential therapeutic targets. Both in vivo and in vitro research models established in our studies can be used to investigate the pathogenic mechanisms of other neurological diseases.
Key words: 3-nitropropionic acid, magnetic resonance spectroscopy, NMDA receptors, reactive oxygen species, mitochondrial permeability transition, caspase, mitochondrial membrane potential, apoptosis.

目 錄
一、 中文摘要 ---------------------------------- 6
二、 緒論 ------------------------------------- 14
第一節 研究的問題與動機---------------------------- 15
第二節 glutamate受體的活化扮演重要的角色 ------------15
第三節 Huntington病的化學動物模式 ------------------ 16
第四節 3-NP所導致神經細胞的死亡形式 ------------ 20
第五節 3-NP神經毒性的致病機轉 --------------------- 21
第六節 粒線體內鈣離子過度負荷的意義 --------------- 23
第七節 粒線體功能異常的治療方針：可能的藥物 -------- 24
第八節 人類NT2-N細胞是一個研究神經毒性的好模型 ----- 26
第九節 磁共振影像和質子磁共振質譜是追蹤神經退化的
好工具------------------------------------------------ 29
三、 材料與研究方法 ------------------------------ 31
第一部份 3-NP對鼠腦的亞急性傷害
第一節 動物的處理與藥物的給予 ---------------------- 32
第二節 動物行為的觀察 ------------------------------ 32
第三節 鼠腦磁振影像的取得 ------------------------- 33
第二部份 3-NP對鼠腦的急性傷害和腦代謝產物的影響
第一節 動物的處理與藥物的給予----------------------- 34
第二節 磁共振影像和質譜的取得 ---------------------- 34
第三部份 3-NP對人類神經細胞NT2-N細胞的影響
第一節 細胞培養 ------------------------------------ 35
第二節 細胞死亡的測量 ------------------------------ 35
第三節 神經細胞的ATP含量 --------------------------- 36
第四節 細胞內鈣離子濃度的測定 ---------------------- 37
第五節 培養液內glutamate濃度的測定 --------------- 38
第六節 Bcl-2蛋白的表現 ----------------------------- 38
第四部份 3-NP導致神經細胞凋亡的機制是多重性的
第一節 鼠腦神經元培養和藥物處理 ------------------- 38
第二節 神經元存活的評估 ---------------------------- 39
第三節 粒線體內鈣離子濃度的測定 ------------------- 39
第四節 自由基和粒線體膜電位的測定----------------- 40
第五節 caspase活性的測量 --------------------------- 40
四、 結果 ---------------------------------------- 42
第一部份 3-NP對大大鼠行為的影響和致病的機制
第一節 3-NP所引起的行為改變 ------------------------ 43
第二節 T2 圖譜的變化 ------------------------------- 43
第三節 3-NP所導致的腦代謝物的變化 ------------------ 44
第二部份 3-NP對神經細胞的急性傷害
第一節 擴散加權造影(DWI)的變化 --------------------- 50
第二節 活體質子磁共振質譜的變化 -------------------- 50
第三節 體外質子磁共振質譜 -------------------------- 51
第三部份 3-NP對人類神經細胞NT2-N細胞的影響
第一節 3-NP影響NT2-N細胞的存活和粒線體
還原能力 -------------------------------------------- 57
第二節 NT2-N細胞的死亡與許多機制有關 -------------- 58
第三節 3-NP導致細胞ATP含量的減少，這種減少
可被MK-801所抑制 ------------------------------------- 59
第四節 3-NP並沒有增加細胞外glutamate的濃度---------- 59
第五節 3-NP導致NT2-N細胞內鈣離子的增加 ------------- 60
第六節 內質網和粒線體在調節細胞上鈣離子增加
所扮演的角色 ----------------------------------------- 60
第七節 3-NP對NT2-N細胞內Ca2+平衡的慢性效應
------------------------------------------------------ 61
第四部份 3-NP造成神經細胞死亡的原因是多重的
第一節 3-NP可導致鼠腦神經細胞的死亡 ---------------- 71
第二節 粒線體內鈣離子的增加與神經細胞的死亡
有關 ------------------------------------------------ 71
第三節 [Ca2+]m的增加與粒線體自由基的增加，粒
線體通透性轉換的產生和隨後的粒線體膜電位去極化有關 --- 72
第四節 Caspase的活化是3-NP所致細胞死亡所須
且與粒線體功能的改變有關 ----------------------------- 74
第五節 NMDA受體的活化是導致3-NP神經毒性的必要步驟 -- 74
五、 討論 ---------------------------------------- 84
六、 結論與展望 --------------------------------- 98
七、 論文英文簡述 ------------------------------ 105
八、 參考文獻 ------------------------------------ 116
九、 圖表 ---------------------------------------- 147
十、 相關論文發表 ------------------------------- 149

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