神經保護藥物對粒線體功能異常時鼠腦病變的影響

3-Nitropropionic acid (3-NP)為粒線體 呼吸鏈複合體II 的抑制劑，它可在鼠導致 選擇性紋狀體病變，如同人類的Huntington 病。過去的許多研究包括我們的研究顯 示， 抑制興奮性氨基酸的釋放和施予 NMDA 受體的拮抗物，會減輕粒線體毒物 引起的腦病變。最近的研究也顯示， Dichloroacetate (DCA)對心臟衰竭及腦缺 血的鼠有保護作用。DCA 也被用於治療粒 線體腦病變。為了更進一步了解DCA 是否 也可用於減輕3-NP 所致的腦病變，及這些 藥物對腦代謝物的影響，在本計畫中，我 們利用磁共振影像(MRI)(T2 圖譜)和生物 活體磁共振質譜(in vivo 1H MRS)的變化來 評估DCA 對粒線體功能異常所引發腦病 變的治療效果。我們以3-NP 為實驗藥物， 以迷你注射器包埋於二個月大的Sprague- Dawley 株鼠腹部皮下， 並以MK- 801(2mg/kg)和DCA (100mg/kg)為治療藥 物，比較治療的效果。我們先觀察慢性3- NP 注射對鼠行為和腦病變的影響。然後觀 察DCA 對亞急性3-NP 腦病變的影響。結 果顯示慢性3-NP 注射可使鼠產生肢體障 礙類似Huntington 病，如同亞急性注射。 同時鼠腦紋狀體也會產生病變。生物活體 磁共振質譜也顯示NAA 下降，表示神經元 的死亡。於亞急性的動物模式中，DCA 的 治療並不減輕3-NP 對鼠腦紋狀體的傷 害。DCA 的臨床運用需更進一步的深入探 討其可行性。Systemic injection of 3-nitropropionic acid (3-NP), an irreversible inhibitor of complex II in mitochondrial respiratory chain, induces selective striatal lesions in rats and non-human primates mimicking those in Huntington’s disease. In recent studies, dichloroacetate (DCA) was shown to have protective effects in rat models of cerebral ischemia and myocardial dysfunction. However, its therapeutic effect on brain lesions induced by mitochondrial dysfunction is rarely investigated. In the past year, we established an in vivo animal model to evaluate the rat brain lesions in mitochondrial dysfuction. In the present study, we compared the therapeutic effect of DCA and MK-801 by in vivo animal model. Two-month-old Sprague-Dawley rats were treated with 3-NP by continuous drug release from mini-pump, implanted subcutaneously. MK-801 (2mg/kg) and DCA (100mg/kg) were given in the same way. The rats were then evaluated by MRI (T2 maps) and in vivo 1H-MRS at indicated time points. We first evaluated the effect of chronic 3-NP injection on rats, and then evaluated the effect of DCA on the striatal lesions induced by 5-day 3-NP injection. The results showed that chronic 3-NP injection produced behavioral change and selective striatal lesions on rats. MRS also showed the decline of NAA/Cr ratio, indicating the neuronal loss or dysfunction. The simultaneous application of DCA showed no attenuation of the striatal lesions. The present results suggest that the application of DCA in brain lesions induced by mitochondrial inhibitors need further investigation

粒線體功能異常對人體神經細胞的影響(2/2)

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, induced 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 0.1mM as well as 5mM 3-NP. We assayed the intracellular concentration of Ca24- ([Ca2j1) 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 [Ca2j1 rise from48¡Ó2 to 140¡Ó l2nM(mean¡ÓSEM¡¦). After 24 and 48 hours in 3-NP, however, [Ca24-J remained above IOOnM in only 17% and 25% of the NT2-N neurons, respectively, suggesting that most neurons were able to correct this early rise in [Ca24-], despite severe ATP depletion, and to survive. Activation of NMDA-GIuR contributed substantially to 3-NP-induced ATP depletion, and subsequent chronic elevation of [Ca24-]1 in the NT2-N neurons. We also demonstrated that blocking endoplasmic reticulum (ER) Ca24- release enhanced the capacity of these human neurons to maintain [Ca21], homeostasis and resist necrosis while subjected to chronic energy deprivation

粒線體與神經細胞死亡的研究(1/2)

粒線體功能異常會引起許多不同年齡層的神經病變。過去的研究顯示， 3-nitropropionic acid 所導致的神經細胞死亡與caspases 的活化有關。動物的研究顯示， 安非他命和甲基安非他命也會使紋狀體神經細胞退化死亡。然而這些神經細胞死亡的機 轉目前並不很清楚。產生過多的自由基是一個可能的機轉，另外也有研究顯示安非他命 和甲基安非他命可能影響粒線體的功能。因此，在本實驗中我們利用鼠腦初級皮質神經 細胞培養來探討導致安非他命和甲基安非他命神經毒性的原因。並比較與 3-nitropropionic acid 所導致神經細胞死亡的差異。我們發現安非他命和甲基安非他命都 可導致神經細胞死亡，且僅引起輕微caspase-3 的活化。證明安非他命和甲基安非他命 可以導致細胞壞死與細胞凋零。另外安非他命和甲基安非他命也使細胞自由基的產量增 加，尤其甲基安非他命所導致的增加更大，證明自由基的產量增加是導致神經細胞死亡 的主因之一。由於caspase-3 的活化發生於粒線體膜電位去極化後，因此主要發生於細 胞色素c 釋放之後。這一點與3-nitropropionic acid 有所差異。我們將繼續探討安非他命 和甲基安非他命的神經毒性，和釐清caspase-3 的活化是否的確會影響粒線體功能，而 更進一步了解3-nitropropionic acid 與安非他命和甲基安非他命神經毒性差異的地方。3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase. Previous studies had shown that 3-NP can lead to neuronal death following the activation of caspases. In the present study, we first investigate the neurotoxicity of methamphetamine and amphetamine in primary rat cortical neuronal cultures, and will compare the pathogenetic mechanisms of neuronal death in methamphetamine and amphetamine with those in 3-NP. We found that there was a dose- and time-dependent increase of neuronal death following the application of methamphetamine and amphetamine. Only mild activation of caspase-3 was found following the treatment, indicating that both methamphetamine and amphetamine can result in apoptosis and necrosis. The caspase-3 activation developed following mitochondrial depolarization, which was different from that in 3-NP. Significant elevation of reactive oxygen species was found post the application of the drugs, especially in methamphetamine. It suggests that increase of reactive oxygen species is one of the major pathogenic mechanisms of neuronal death for both amphetamine and methamphetamine. In the following year, works will be focused on the differences of neurotoxicity in both 3-NP and methamphetamine (and amphetamine). Whether caspase-3 activation can really affect mitochondrial function as that in 3-NP neurotoxicity will also be investigated

不同藥物對粒線體功能異常時鼠大腦病變的影響: 磁共振影像,質譜與組織變化的相關

粒線體功能異常會引起許多不同年齡層的神 經病變。最近的研究顯示，抑制興奮性氨基酸的釋 放和施予NMDA 受體的拮抗物，都會減輕粒線體毒 物引起的腦病變。在本計畫中，我們以粒線體呼吸 鏈複合體II 的抑制劑(3-Nitropropionic acid, 3-NP，15mg/kg)為實驗藥物，連續注射於八週大的 Sprague-Dawley 株鼠(n=10)五天，並於第六天以磁 共振影像(MRI)和質譜(1H NMR spectroscopy)來評 估其系列的變化。結果發現Sprague-Dawley 株鼠注 射3-NP 後，會產生紋狀體和海馬迴的病變，並出 現特別的行為變化，這種變化與組織學上的變化一 致。若接受MK-801(2mg/kg) 和Lamotrigine (10mg/kg 和 20mg/kg)的注射，腦病變則明顯改 善，並且行為的變化也消失；磁共振質譜上的變化 則可看到治療後原先NAA 的降低也減輕，並且 Lactate 的昇高也消失。整體而言，在治療的藥物 中lamotrigine(20mg/kg)較lamotrigine(10mg/kg) 和MK-801 能提供更好的保護作用。由於 lamotrigine 的副作用較少，因此更有臨床運用的 價值，值得未來更進一步的研究。Magnetic resonance imaging and in vivo proton magnetic resonance spectroscopy were used to evaluate the therapeutic effect of lamotrigine and MK- 801 on rat brain lesions induced by 3-nitropropionic acid. Systemic administration of 3-nitropropionic acid (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. Pretreatment with lamotrigine (10mg/kg or 20mg/kg/day) or MK-801 (2mg/kg/day) attenuated the lesions and behavioral change. There were no significant differences in T2 values of the striatum and hippocampus among rats pretreated with MK-801, lamotrigine (20mg/kg) and sham controls. Significant elevations of succinate/creatine and lactate/creatine ratios and decreases of N-acetylaspartate/creatine and choline/creatine ratios were observed after 3- nitropropionic acid injections (P<0.001). The changes were nearly prevented after pretreatment with lamotrigine (20mg/kg). However, the Nacetylaspartate/ creatine 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. We conclude that both lamotrigine and MK-801 are effective in attenuation of brain lesions induced by 3-nitropropionic acid. 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