L-肉毒碱在Alzheimer样tau蛋白过度磷酸化和空间记忆障碍中的保护作用
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摘要
阿尔茨海默病(Alzheimer’s disease, AD)是老年人群中常见的神经退行性疾病之一,随着社会人口老龄化其发病率逐年升高。神经原纤维缠结(neurofibrillary tangles, NFTs)是AD患者脑中典型的病理学特征之一,NFTs的主要成分是异常过度磷酸化的微管相关蛋白tau聚集形成的双螺旋细丝(paire helical filaments, PHFs)。研究表明,tau蛋白的过度磷酸化是AD病程发展的早期事件,而且AD患者的临床痴呆程度与NFTs的数量密切相关。因此调节tau蛋白过度磷酸化为靶点的药物可能是防治AD的有效方法之一。
肉毒碱(Carnitine)是赖氨酸的衍生物,其中左旋体具有生物学活性。本研究主要目的是探讨L-肉毒碱(L-Carnitine)在wotmannin(WT)及GF-109203 X(GFX)引起的AD样病理变化模型中的药理作用及其相关机制。我们给予50mg/只/天L-Carnitine溶于大鼠每日饮水中连续14天,在给药第8天时同时进行Morris水迷宫训练,在第15天给予大鼠侧脑室联合注射10μl浓度为100μM的PI3K特异性抑制剂wortmannin(WT)及PKC的特异性抑制剂GF-109203X(GFX)。侧脑室注射24h后进行Morris水迷宫测试检测大鼠空间记忆保留情况,之后取材进行免疫印迹及免疫组化检测。
1.L-Carnitine可改善WT/GFX引起的空间记忆保留障碍
结果显示:侧脑室注射WT/GFX24h后,大鼠找到隐藏平台的潜伏期明显延长,搜寻轨迹杂乱,而预先给予L-Carnitine的大鼠找到平台时间明显缩短,搜寻轨迹接近正常,表明预先给予L-Carnitine能有效改善由WT/GFX所致的大鼠空间记忆障碍。
2. L-Carnitine可减轻WT/GFX引起的大鼠海马tau蛋白过度磷酸化
免疫印迹结果显示:总tau蛋白水平,各个实验组无明显变化。侧脑室注射WT/GFX24h后tau蛋白磷酸化在Ser199/202、Thr205、Ser396及Ser404位点明显增强,预先给予L-Carnitine能减轻Ser199/202及Thr205位点的磷酸化水平,不能减轻Ser396及Ser404位点的磷酸化水平。Ser214位点tau蛋白磷酸化各个实验组无明显变化。
免疫组织化学检测tau蛋白磷酸化及在海马分布情况,结果显示:L-Carnitine明显降低由于侧脑室注射WT/GFX24h后导致的PT205在大鼠海马各区的阳性染色,而增强Tau-1在海马CA3区的阳性染色。以上结果表明预先给予L-Carnitine能减轻WT/GFX导致的tau蛋白在PT205及Tau-1位点的过度磷酸化。
3.L-Carnitine可减轻WT/GFX诱导的大鼠海马GSK-3β过度激活不依赖Akt激活
为了探讨L-Carnitine减弱WT/GFX诱导的tau蛋白过度磷酸化的机制,研究发现注射WT/GFX后GSK-3β的非活性形式Ser9磷酸化的水平明显降低,预先给予L-Carnitine可增加GSK-3β的Ser9磷酸化水平,而总GSK-3β水平在各组均无明显变化,表明L-Carnitine能抑制WT/GFX所致的GSK-3β过度激活。Akt已被确定为是GSK-3β的上游调节因子,我们检测大鼠海马中AKT的Ser473磷酸化水平在注射WT/GFX后明显降低,给予L-Carnitine后不能使其水平升高。以上结果提示:WT/GFX激活GSK-3β,从而导致tau蛋白过度磷酸化和空间记忆障碍,L-Carnitine下调GSK-3β的活性而减轻tau蛋白的过度磷酸化及改善记忆是不依赖于Akt的激活。
4.L-Carnitine增加c-Fos的表达从而提高记忆能力不是通过PKA/CREB通路实现的
我们进一步研究了L-Carnitine对记忆保护的分子机制。我们用免疫组化检测大鼠海马内c-Fos的表达水平及分布,结果显示,注射WT/GFX的大鼠海马内c-Fos的表达明显较对照组有所降低,预先给予L-Carnitine能增加其表达,尤其在CA1, CA3及DG区最为明显。c-Fos是一种立早基因,它参与学习记忆的过程。以上结果表明:L-Carnitine提高学习记忆与增加c-Fos的表达有关。为了研究L-Carnitine增加c-Fos表达的上游机制,我们检测了PKA催化亚基及调节亚基和转录因子CREB的Ser133位点磷酸化水平。结果发现:各实验组PKA的催化亚基无明显差异,注射WT/GFX后导致PKA调节亚基显著增高,给予L-Carnitine不能逆转PKA调节亚基的水平。大鼠海马锥体细胞内CREB的Ser133位点磷酸化阳性着色在各个实验组无明显差异。以上结果提示: L-Carnitine增加c-Fos表达的不是通过PKA/CREB通路实现的。
5. L-Carnitine对PP-2A催化亚单位酪氨酸307位点磷酸化的影响
我们检测了PP-2A催化亚基总量(PP-2AC)和PP-2A催化亚单位Tyr307位点磷酸化(PP-2A的非活性形式)的水平。结果表明:各实验组之间PP-2AC未发生明显变化;PP-2A催化亚单位Tyr307位点的磷酸化水平在WT/GFX处理后无明显变化,而给与L-Carnitine后与对照组相比该位点磷酸化水平明显下降。提示L-Carnitine对tau蛋白过度磷酸化及空间记忆障碍的保护作用可能与其上调了PP-2A有关。
Alzheimer’s disease (AD) is the most common neurodegenerative disease in the aged population. With the aged tendency of population, the morbidity of AD increases year after year. NFTs is the known neuropathological feature in AD, they are intracellular fibrillar structures composed of aggregations of PHFs, which are made up of abnormally phosphorylated tau. Hyperphosphorylated tau is believed to be an early pathological event of AD and the amount of NFTs is closely correlated with the clinical dementia degree of AD patients. Thus target the underlying pathogenic mechanisms of tau hyperphosphorylation in AD might be a potential therapeutic strategy for prevention or treatment of the diseae.
L-Carnitine is an L-lysine derivative, which was first discovered in beef by scientists from Russia in 1905. Its primary role appears to be facilitating the transport of long chain fatty acids into mitochondria in order to enter theβ-oxidation cycle. In our present study, we have investigated the pharmacological effect of L-Carnitine on WT/GFX induced AD-like pathological models and its involved mechanism of the effection. Each rat was fed orally with 50 mg/ day of L-Carnitine in drinking water or nomal drinking water as control for two weeks. At the day 8, the rats meanwhile were trained in Morris water maze everyday for 7 days. At the day15, wortmannin (WT, a specific PI3K inhibitor) and GF-109203X (GFX, a specific PKC inhibitor) (100Μm of each, total volume of 10μl) were co-injected into the left ventricle of the rats and the spatial memory retention was tested at 24h after the brain injection. The samples were prepared for western blotting and immunocytochemistry research after the spatial memory retention test.
1.L-Carnitine improves WT/GFX-induced spatial memory retention deficits
The results are shown as follows: after the co-injection of WT/GFX, the latency of finding the hidden platform increased remarkably and swimming path was tortuous. Pre-treatment with L-Carnitine significantly shortened the WT/GFX-induced increase of the latency and improved the searching strategy of the rats. These results suggest that L-Carnitine can effectively prevent the rats from WT/GFX-induced spatial memory deficits.
2.L-Carnitine attenuates WT/GFX-induced tau hyperphosphorylation in rat hippocampus
To detect the effect of L-Carnitine on WT/GFX-induced tau phosphorylation, the level of total tau using antibody Tau-5, no significant difference was observed. We carried out western blot by using phosphorylation-dependent Tau-1 (reacts with non-phosphorylated tau at Ser 199/202), PT205, PS396 and PS404. We found that the immunoreactivity of Tau-1 was remarkably weaker, and of PT205, PS396 and PS404 was obviously stronger after the co-injection of WT/GFX than in control rats. Pre-administration with L-Carnitine attenuates the WT/GFX-induced tau hyperphosphorylation at PT205 and Tau-1 epitopes, but not at PS396 and PS404 epitopes. The level of phosphorylation at PS214, no significant difference was observed
Immunohistochemistry was used to detect the distribution of the phosphorylated tau. It was shown that L-Carnitine prevented WT/GFX-induced hyperphosphorylation of tau at site of Thr205 and Ser199/202. Co-injection of WT/GFX caused a dramatically increasing PT205 staining of the hippocampus and a decreasing Tau-1 staining in neural fibers of the CA3 regions. L-Carnitine markedly reversed the hyperphosphorylation of tau at these regions.
3. L-Carnitine decreases WT/GFX-induced overactivation of GSK-3βwith Akt-independent manner
we measured the total level and the activity-dependent Ser9-phosphorylated level of GSK-3βin the hippocampal extracts by western blotting. No obvious change was seen in total level of GSK-3βin four groups. However, the level of pSer9 -GSK-3β(representing the inactivated form of the kinase) was significantly decreased in WT/GFX-injected group, L-Carnitine could increase the level of the pSer9-GSK-3β, which suggested that L-Carnitine could prevent GSK-3βfrom WT/GFX-induced overactivation.
To study whether the protective effect of L-Carnitine on GSK-3βand tau phosphorylation involves protein kinase B (Akt), an upstream effector of GSK-3β. We tested the activity-dependent Ser473-phosphorylated level of Akt and total AKT. WT/GFX treatment induced suppression of Akt demonstrated by a dramatic decline of the pAkt at Ser473. L-Carnitine cannot increase ratio of the pSer473Akt to total Akt. The results showed that L-Carnitine could attenuate WT/GFX-induced GSK-3βactivation with Akt-independent manner.
4. L-Carnitine enhances the expression of c-Fos independent of PKA/CREB
In order to investigate the molecular mechanism of memory protection of L-Carnitine, We used immunohistochemisty to detect the c-Fos expression and distribution in rat hippocampus, L-Carnitine could dramaticly increased the level of c-Fos expression in the hippocampus which decreased by WT/GFX. The expression of c-Fos is significantly increased in long-term memory. L-Carnitine improve learning and memory through targeting c-Fos .
In order to investigate the upstream mechanism, we measured PKA catalytic subunit , regulatory subunit and transcription factor CREB phosphorylation at Ser133. PKA catalytic subunit was no significance of four groups. Injection with WT/GFX induced the increasing of PKA regulatory subunit, L-Carnitine did not reverse WT/GFX- induced the increasing. The colour of pSer133-CREB- positive staining neurons was no significantly of four groups. It is suggested that L-Carnitine enhances the expression of c-Fos independent of PKA/CREB .
5. L-Carnitine attenuates phosphorylation of PP-2Ac at Tyr307
We also tested total PP-2AC and Tyr307-phosphorylated PP-2AC (the inactive form of PP-2A). It was shown that total PP-2AC was no significant difference among four groups and the level of Tyr307-phosphorylated PP-2A catalytic subunit did not change after the injection of WT/GFX, however, pre-administered with L-Carnitine can attenuates phosphorylation of PP-2Ac at Tyr307 as control group. The results suggested that L-Carnitine decreased the lever of tau hyperphosphorylation and improve spatial memory retention deficits partly because of upregulating PP-2A activity.
肉毒碱(Carnitine)是赖氨酸的衍生物,其中左旋体具有生物学活性。本研究主要目的是探讨L-肉毒碱(L-Carnitine)在wotmannin(WT)及GF-109203 X(GFX)引起的AD样病理变化模型中的药理作用及其相关机制。我们给予50mg/只/天L-Carnitine溶于大鼠每日饮水中连续14天,在给药第8天时同时进行Morris水迷宫训练,在第15天给予大鼠侧脑室联合注射10μl浓度为100μM的PI3K特异性抑制剂wortmannin(WT)及PKC的特异性抑制剂GF-109203X(GFX)。侧脑室注射24h后进行Morris水迷宫测试检测大鼠空间记忆保留情况,之后取材进行免疫印迹及免疫组化检测。
1.L-Carnitine可改善WT/GFX引起的空间记忆保留障碍
结果显示:侧脑室注射WT/GFX24h后,大鼠找到隐藏平台的潜伏期明显延长,搜寻轨迹杂乱,而预先给予L-Carnitine的大鼠找到平台时间明显缩短,搜寻轨迹接近正常,表明预先给予L-Carnitine能有效改善由WT/GFX所致的大鼠空间记忆障碍。
2. L-Carnitine可减轻WT/GFX引起的大鼠海马tau蛋白过度磷酸化
免疫印迹结果显示:总tau蛋白水平,各个实验组无明显变化。侧脑室注射WT/GFX24h后tau蛋白磷酸化在Ser199/202、Thr205、Ser396及Ser404位点明显增强,预先给予L-Carnitine能减轻Ser199/202及Thr205位点的磷酸化水平,不能减轻Ser396及Ser404位点的磷酸化水平。Ser214位点tau蛋白磷酸化各个实验组无明显变化。
免疫组织化学检测tau蛋白磷酸化及在海马分布情况,结果显示:L-Carnitine明显降低由于侧脑室注射WT/GFX24h后导致的PT205在大鼠海马各区的阳性染色,而增强Tau-1在海马CA3区的阳性染色。以上结果表明预先给予L-Carnitine能减轻WT/GFX导致的tau蛋白在PT205及Tau-1位点的过度磷酸化。
3.L-Carnitine可减轻WT/GFX诱导的大鼠海马GSK-3β过度激活不依赖Akt激活
为了探讨L-Carnitine减弱WT/GFX诱导的tau蛋白过度磷酸化的机制,研究发现注射WT/GFX后GSK-3β的非活性形式Ser9磷酸化的水平明显降低,预先给予L-Carnitine可增加GSK-3β的Ser9磷酸化水平,而总GSK-3β水平在各组均无明显变化,表明L-Carnitine能抑制WT/GFX所致的GSK-3β过度激活。Akt已被确定为是GSK-3β的上游调节因子,我们检测大鼠海马中AKT的Ser473磷酸化水平在注射WT/GFX后明显降低,给予L-Carnitine后不能使其水平升高。以上结果提示:WT/GFX激活GSK-3β,从而导致tau蛋白过度磷酸化和空间记忆障碍,L-Carnitine下调GSK-3β的活性而减轻tau蛋白的过度磷酸化及改善记忆是不依赖于Akt的激活。
4.L-Carnitine增加c-Fos的表达从而提高记忆能力不是通过PKA/CREB通路实现的
我们进一步研究了L-Carnitine对记忆保护的分子机制。我们用免疫组化检测大鼠海马内c-Fos的表达水平及分布,结果显示,注射WT/GFX的大鼠海马内c-Fos的表达明显较对照组有所降低,预先给予L-Carnitine能增加其表达,尤其在CA1, CA3及DG区最为明显。c-Fos是一种立早基因,它参与学习记忆的过程。以上结果表明:L-Carnitine提高学习记忆与增加c-Fos的表达有关。为了研究L-Carnitine增加c-Fos表达的上游机制,我们检测了PKA催化亚基及调节亚基和转录因子CREB的Ser133位点磷酸化水平。结果发现:各实验组PKA的催化亚基无明显差异,注射WT/GFX后导致PKA调节亚基显著增高,给予L-Carnitine不能逆转PKA调节亚基的水平。大鼠海马锥体细胞内CREB的Ser133位点磷酸化阳性着色在各个实验组无明显差异。以上结果提示: L-Carnitine增加c-Fos表达的不是通过PKA/CREB通路实现的。
5. L-Carnitine对PP-2A催化亚单位酪氨酸307位点磷酸化的影响
我们检测了PP-2A催化亚基总量(PP-2AC)和PP-2A催化亚单位Tyr307位点磷酸化(PP-2A的非活性形式)的水平。结果表明:各实验组之间PP-2AC未发生明显变化;PP-2A催化亚单位Tyr307位点的磷酸化水平在WT/GFX处理后无明显变化,而给与L-Carnitine后与对照组相比该位点磷酸化水平明显下降。提示L-Carnitine对tau蛋白过度磷酸化及空间记忆障碍的保护作用可能与其上调了PP-2A有关。
Alzheimer’s disease (AD) is the most common neurodegenerative disease in the aged population. With the aged tendency of population, the morbidity of AD increases year after year. NFTs is the known neuropathological feature in AD, they are intracellular fibrillar structures composed of aggregations of PHFs, which are made up of abnormally phosphorylated tau. Hyperphosphorylated tau is believed to be an early pathological event of AD and the amount of NFTs is closely correlated with the clinical dementia degree of AD patients. Thus target the underlying pathogenic mechanisms of tau hyperphosphorylation in AD might be a potential therapeutic strategy for prevention or treatment of the diseae.
L-Carnitine is an L-lysine derivative, which was first discovered in beef by scientists from Russia in 1905. Its primary role appears to be facilitating the transport of long chain fatty acids into mitochondria in order to enter theβ-oxidation cycle. In our present study, we have investigated the pharmacological effect of L-Carnitine on WT/GFX induced AD-like pathological models and its involved mechanism of the effection. Each rat was fed orally with 50 mg/ day of L-Carnitine in drinking water or nomal drinking water as control for two weeks. At the day 8, the rats meanwhile were trained in Morris water maze everyday for 7 days. At the day15, wortmannin (WT, a specific PI3K inhibitor) and GF-109203X (GFX, a specific PKC inhibitor) (100Μm of each, total volume of 10μl) were co-injected into the left ventricle of the rats and the spatial memory retention was tested at 24h after the brain injection. The samples were prepared for western blotting and immunocytochemistry research after the spatial memory retention test.
1.L-Carnitine improves WT/GFX-induced spatial memory retention deficits
The results are shown as follows: after the co-injection of WT/GFX, the latency of finding the hidden platform increased remarkably and swimming path was tortuous. Pre-treatment with L-Carnitine significantly shortened the WT/GFX-induced increase of the latency and improved the searching strategy of the rats. These results suggest that L-Carnitine can effectively prevent the rats from WT/GFX-induced spatial memory deficits.
2.L-Carnitine attenuates WT/GFX-induced tau hyperphosphorylation in rat hippocampus
To detect the effect of L-Carnitine on WT/GFX-induced tau phosphorylation, the level of total tau using antibody Tau-5, no significant difference was observed. We carried out western blot by using phosphorylation-dependent Tau-1 (reacts with non-phosphorylated tau at Ser 199/202), PT205, PS396 and PS404. We found that the immunoreactivity of Tau-1 was remarkably weaker, and of PT205, PS396 and PS404 was obviously stronger after the co-injection of WT/GFX than in control rats. Pre-administration with L-Carnitine attenuates the WT/GFX-induced tau hyperphosphorylation at PT205 and Tau-1 epitopes, but not at PS396 and PS404 epitopes. The level of phosphorylation at PS214, no significant difference was observed
Immunohistochemistry was used to detect the distribution of the phosphorylated tau. It was shown that L-Carnitine prevented WT/GFX-induced hyperphosphorylation of tau at site of Thr205 and Ser199/202. Co-injection of WT/GFX caused a dramatically increasing PT205 staining of the hippocampus and a decreasing Tau-1 staining in neural fibers of the CA3 regions. L-Carnitine markedly reversed the hyperphosphorylation of tau at these regions.
3. L-Carnitine decreases WT/GFX-induced overactivation of GSK-3βwith Akt-independent manner
we measured the total level and the activity-dependent Ser9-phosphorylated level of GSK-3βin the hippocampal extracts by western blotting. No obvious change was seen in total level of GSK-3βin four groups. However, the level of pSer9 -GSK-3β(representing the inactivated form of the kinase) was significantly decreased in WT/GFX-injected group, L-Carnitine could increase the level of the pSer9-GSK-3β, which suggested that L-Carnitine could prevent GSK-3βfrom WT/GFX-induced overactivation.
To study whether the protective effect of L-Carnitine on GSK-3βand tau phosphorylation involves protein kinase B (Akt), an upstream effector of GSK-3β. We tested the activity-dependent Ser473-phosphorylated level of Akt and total AKT. WT/GFX treatment induced suppression of Akt demonstrated by a dramatic decline of the pAkt at Ser473. L-Carnitine cannot increase ratio of the pSer473Akt to total Akt. The results showed that L-Carnitine could attenuate WT/GFX-induced GSK-3βactivation with Akt-independent manner.
4. L-Carnitine enhances the expression of c-Fos independent of PKA/CREB
In order to investigate the molecular mechanism of memory protection of L-Carnitine, We used immunohistochemisty to detect the c-Fos expression and distribution in rat hippocampus, L-Carnitine could dramaticly increased the level of c-Fos expression in the hippocampus which decreased by WT/GFX. The expression of c-Fos is significantly increased in long-term memory. L-Carnitine improve learning and memory through targeting c-Fos .
In order to investigate the upstream mechanism, we measured PKA catalytic subunit , regulatory subunit and transcription factor CREB phosphorylation at Ser133. PKA catalytic subunit was no significance of four groups. Injection with WT/GFX induced the increasing of PKA regulatory subunit, L-Carnitine did not reverse WT/GFX- induced the increasing. The colour of pSer133-CREB- positive staining neurons was no significantly of four groups. It is suggested that L-Carnitine enhances the expression of c-Fos independent of PKA/CREB .
5. L-Carnitine attenuates phosphorylation of PP-2Ac at Tyr307
We also tested total PP-2AC and Tyr307-phosphorylated PP-2AC (the inactive form of PP-2A). It was shown that total PP-2AC was no significant difference among four groups and the level of Tyr307-phosphorylated PP-2A catalytic subunit did not change after the injection of WT/GFX, however, pre-administered with L-Carnitine can attenuates phosphorylation of PP-2Ac at Tyr307 as control group. The results suggested that L-Carnitine decreased the lever of tau hyperphosphorylation and improve spatial memory retention deficits partly because of upregulating PP-2A activity.
引文
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