【作者】 王少辉；

【导师】 王建枝；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease, AD)的两大重要的病理学特征是神经细胞内的大量神经原纤维缠结(neurofibrillary tangles, NFT)和细胞间沉积的大量老年斑(senile plaques, SP)。神经原纤维缠结主要由异常过度磷酸化的骨架蛋白tau构成,而老年斑的核心成分是由β-淀粉样前体蛋白(β-amyloid precursor protein, APP)降解产生的β-淀粉样多肽(β-amyloid, Aβ)。目前研究认为蛋白激酶和蛋白磷酸酯酶系统的失衡是导致AD中tau蛋白过度磷酸化的主要原因,然而对磷酸化系统失衡的上游调控因子尚知之甚少。有报道证明老年人褪黑素(melatonin, MT)水平下降,在AD病人尤为明显,我们推测老年人褪黑素水平下降可能和AD发生有关,但是尚缺乏直接证据。我们最近发现褪黑素可以对抗花萼海绵诱癌素诱导的神经细丝损伤和神经毒性作用。本实验中,给予褪黑素合成抑制剂——氟哌啶醇,观察对大鼠的空间记忆的影响,tau蛋白磷酸化水平,蛋白磷酸酯酶PP2A活性改变并探讨其可能机制。结果如下:(1)血清中褪黑素含量测定:为了检测给予氟哌啶醇对大鼠褪黑素合成的抑制作用,我们采用高压液相(HPLC)检测大鼠血清中褪黑素的含量。结果显示:和对照组相比,给予氟哌啶醇26小时以后,血清中褪黑素的含量明显下降;外源性补充褪黑素,并未发现褪黑素水平显著升高,可能和褪黑素的代谢半衰期较短有关,具体原因见讨论部分。(2)抑制褪黑素的生物合成对大鼠空间记忆的影响:为了检测抑制大鼠褪黑素的生物合成对大鼠空间记忆的影响,我们采用Morris水迷宫检测大鼠的空间记忆。结果表明,给予氟哌啶醇后,大鼠找到平台的潜伏期明显变长;外源性补充褪黑素可以部分改善大鼠的学习障碍,但不同剂量之间没有明显差异。(3)抑制大鼠褪黑素的生物合成对tau蛋白磷酸化的作用:为了检测抑制大鼠褪黑素的生物合成对tau蛋白磷酸化的影响,我们发现给予氟哌啶醇以后,免疫印迹和免疫组化结果显示总的tau蛋白表达水平没有明显变化,在PHF-1(Ser396/404)位点磷酸化水平增强,而非磷酸化的tau蛋白在tau-1(Ser198/199/202)位点的免疫反应减弱,表明抑制大鼠的褪黑素生物合成可引起tau蛋白发生过度磷酸化。(4)抑制大鼠褪黑素的生物合成对PP2A活性的影响:为阐明抑制大鼠褪黑素的生物合成tau蛋白发生过度磷酸化的机制,我们监测了PP2A的活性变化。采用γ-32P标记的特殊底物法进行活性分析。与对照组相比,给予氟哌啶醇以后,PP2A的活性下降明显,补充低剂量的褪黑素,PP2A的活性未发现明显改善,高剂量组PP2A活性则显著性提高。(5)抑制大鼠褪黑素的生物合成对氧化应激的影响:褪黑素作为体内的抗氧化剂,其合成被抑制后,体内的氧化应激可能增强,氧化应激可能影响PP2A的活性,从而影响tau蛋白的磷酸化。具体检测结果如下:给予氟哌啶醇以后,SOD活性显著下降,MDA的含量增多,补充褪黑素以后,SOD的活性和MDA的含量可以恢复到正常水平,二者之间没有明显差异。结论:以上结果提示:给予大鼠氟哌啶醇以后,可以抑制大鼠褪黑素的生物合成,抑制PP2A的生物活性,使大鼠的空间记忆发生障碍,tau蛋白在PHF-1位点发生过度磷酸化;氧化应激的增强可能是PP2A活性下降的机制之一;外源性补充褪黑素,可以抑制氧化应激,增强PP2A的活性,改善大鼠的空间记忆障碍,降低tau蛋白的过度磷酸化。过度磷酸化的tau蛋白作为AD两大病理特征之一的神经原纤维缠结(NFTs)的主要成分,在AD的发病过程中起重要作用。有研究表明,NFT的水平和痴呆程度正相关,而蛋白激酶(如Gsk3, cdk5, MAPK, PKA等)和蛋白磷酸酯酶(PP1, PP2A等)系统的失衡是导致AD中tau蛋白过度磷酸化的主要原因。本文主要探讨抑制大鼠褪黑素的生物合成以后,蛋白激酶PKA和GSK-3的活性改变,探讨tau蛋白不同位点磷酸化的可能发生机制。用氟哌啶醇抑制褪黑素的生物合成,通过γ-P32标记的酶的活性测定方法检测PKA和GSK-3的活性,结果显示:(1)抑制大鼠褪黑素的生物合成后,PKA的活性比正常水平升高1.9倍,外源性补充褪黑素,可以明显抑制氟哌啶醇引起的PKA活性升高,同时基础水平的PKA也被抑制。抑制褪黑素生物合成时,GSK-3的活性与对照组相比下降不明显,而外源性补充褪黑素可以抑制GSK-3的活性。(2)抑制大鼠褪黑素的生物合成对不同位点tau蛋白磷酸化的影响:抑制褪黑素的生物合成以后,tau蛋白在pS214和M4位点发生过度磷酸化,而总的tau蛋白水平变化不明显。(3)结论:抑制大鼠褪黑素的生物合成,可以激活PKA引起tau蛋白在pS214和M4位点发生过度磷酸化,而GSK-3活性没有明显改变。直接补充外源性褪黑素可以同时抑制PKA和GSK-3的活性。在阿尔茨海默病(Alzheimer’s Disease, AD)患者脑内神经细胞骨架蛋白神经细丝(Neurofilament, NF)发生了过度磷酸化,同时褪黑素的水平下降,但二者的内在关系不明。本部分探讨了抑制大鼠褪黑素的生物合成和神经细丝磷酸化之间的关系并探讨其可能机制。结果如下:(1)抑制大鼠褪黑素的生物合成对大鼠海马神经细丝磷酸化的影响:抑制大鼠褪黑素的生物合成以后,我们用免疫印迹和免疫组化分别检测了海马神经细丝的磷酸化情况。给予氟哌啶醇以后,与对照组相比,大鼠海马神经元磷酸化的神经细丝明显增多,非磷酸化的神经细丝下降;外源性补充褪黑素,则可减轻神经细丝的磷酸化。免疫组化的结果与免疫印迹的结果相似。(2)抑制大鼠褪黑素的生物合成对大鼠皮质神经细丝磷酸化的影响:免疫组化与免疫印迹的结果显示:给予氟哌啶醇以后,与对照组相比,大鼠海马神经元磷酸化的神经细丝明显增多,非磷酸化的神经细丝下降;外源性补充褪黑素,则可减轻神经细丝的磷酸化。(3)抑制大鼠褪黑素的生物合成对大鼠cdk-5活性的影响:结果显示,给予氟哌啶醇以后,海马和皮质的cdk-5的活性比对照组升高了大约1.5倍,外源性补充褪黑素能逆转cdk-5的活性。结论:抑制褪黑素的生物合成可以激活cdk-5导致神经细丝的过度磷酸化,而外源性补充褪黑素可以抑制cdk-5的活性并抑制氟哌啶醇诱导的神经细丝过度磷酸化。长期记忆的形成需要基因表达和新的蛋白质合成,海马突触可塑性则是信息存储和长期记忆的基础。大量研究资料表明神经元的兴奋可以影响细胞的形态和功能,但是神经元的兴奋如何保持持久性,目前尚不清楚。Cohen-Armon等在Aplysia上研究发现,PARP-1(polyADP-ribose-polymerase-1,聚ADP-核糖聚合酶-1)可参与基因的调控和长期记忆的形成,然而,对PARP-1是否影响海马依赖的记忆目前还不清楚。本研究通过给予3-Aminobenzamide(3AB)抑制PARP-1或NGF(Nerve growth factor)激活PARP-1,观察PARP-1对大鼠海马参与的记忆,包括长期记忆(long-term memory, LTM)和短期记忆(short-term memory, STM)的影响,探讨其可能的分子机制和信号途径。结果显示:(1)PARP-1抑制或激活可影响大鼠海马依赖的联想记忆(Contextual memory),而不依赖海马的(Cue memory)记忆则不受到影响。(2)采用不同浓度的3AB抑制PARP-1,用电跳台观察大鼠的短期记忆和长期记忆的影响,发现低浓度(1 mM)3AB对大鼠的短期记忆和长期记忆无明显作用;当浓度增加(50 mM, 100 mM)时,大鼠的长期记忆受到抑制,但短期记忆仍未见明显改变。(3)抑制PARP-1降低大鼠的空间记忆能力;同时给予PARP-1激活剂NGF,大鼠的空间记忆得到改善;单独给予NGF可促进大鼠的空间记忆。(4)采用双向电泳加抗体检测PARP-1等电点偏移的情况以评估PARP-1的活性,发现3AB使PARP-1活性下降,而NGF激活PARP-1。(5)给予3AB抑制PARP-1时,蛋白polyADP-ribosylation作用减弱,再给予NGF,则部分逆转该效应;单独NGF处理可使蛋白polyADP-ribosylation作用明显增强。(6)抑制PARP-1可以损伤大鼠的长时程增强(long-term potentiation, LTP)。不同浓度的3AB,都可以抑制大鼠LTP的斜率和幅度。(7)给予大鼠3AB时,PKA的含量与对照组相比没有明显改变,而再给予NGF或单独给予NGF,PKA的含量明显增加。免疫组织化学的结果则显示,给予3AB组与对照组相比,海马CA3区着色增强,CA1区海马着色反而减弱;先给予3AB以后再给予NGF,与3AB组相比,海马CA3区着色减弱,CA1区海马着色反而增强;NGF组结果类似。提示PARP-1对海马的不同分区作用可能不尽相同。而3AB组于对照组相比,PKA的活性轻微下降,再给予NGF,PKA的活性趋于正常,单独给予NGF,可以显著升高PKA的活性。(8)利用放射免疫实时测定法检测cAMP的含量,发现给予3AB以后,cAMP的含量明显下降;再给予NGF以后,cAMP的含量有所增加;单独给予NGF以后,cAMP的含量明显高于对照组和3AB组。(9)给予3AB导致CREB的磷酸化受到抑制,再给予NGF以后,CREB的磷酸化恢复正常,单独给予NGF,与3AB组相比,磷酸化增强,但对照组相比没有明显差异。(10)采用PCR检测记忆相关基因PKA和NF-κB的mRNA的表达情况,结果显示,抑制PARP-1对PKA和NF-κB的表达没有明显影响,推测可能和其他基因有关。结论:(i) 3AB(50 mM)可抑制大鼠PARP-1的活性,NGF则可激活PARP-1;(ii)抑制PARP-1的活性可损伤大鼠海马依赖的长期记忆和空间记忆;(iii)抑制PARP-1可降低大鼠的LTP;(iv)抑制PARP-1时,cAMP含量下降;反之cAMP含量升高,同时PKA的活性相应发生变化;(v)抑制PARP-1的活性时,CREB的磷酸化受到抑制;(vi)与记忆相关基因PKA和NF-κB mRNA水平未受到影响。更多还原

【Abstract】 Alzheimer’s disease is characterized by intracellular neurofibrillary tangles and extracellular senile plaques. Neurofibrillary tangles are composed of aberrantly hyperphosphorylated tau protein, andβ-amyloid (Aβ), a peptide derived from cleavage ofβ-amyloid precursor protein (APP), is the major component of senile plaques. Numerous studies have demonstrated that an imbalanced regulation of specific phosphatases and kinases in neurons represents a critical step for the cytoskeleton hyperphosphorylation. But till now the upstream factors leading to cytoskeleton hyperphosphorylation are not defined. Lots of evidences suggest that melatonin deficit may serve as an upstream effecter in AD-like pathology and melatonin may be a potent drug for the therapy of AD. Recent studies show that the level of melatonin in serum is decreased in aged people, especially in AD patients and the cognitive function of the patients is improved after melatonin supplement. We have found recently that melatonin protects SH-SY5Y neuroblastoma cells from calyculin A-induced neurofilament impairment and neurotoxicity. Growing evidences suggest that melatonin deficit may serve as an upstream effector in AD-like pathology. The aim of the present study is to investigate the in vivo effect of inhibiting melatonin biosynthesis on spatial memory retention of rats and tau phosphorylation, and the possible underlying mechanisms. The results as follows: (1) Effect of haloperidol on serum melatonin level: To confirm the suppression of melatonin synthesis by haloperidol, the serum level of melatonin was measured by HPLC. The melatonin level decreased dramatically compared with vehicle control after administration of haloperidol. We also observed that exogenous supplementation of melatonin did not significantly restore the serum level of melatonin. (2) Effect of inhibited biosynthesis of melatonin on spatial memory of rats: To detect the effect of inhibited biosynthesis of melatonin on spatial memory of rats, Morris water maze test was used. Compared with vehicle-control rats, injection of haloperidol significantly prolonged the latency of rats to find the hidden platform, and these rats took a random searching strategy for the platform in the maze. Supplementation of melatonin with both low and high doses before and during haloperidol injection partially restored the haloperidol-induced impairment in spatial memory. (3) Effect of inhibition melatonin synthesis on tau phosphorylation: To detect the effect of inhibited melatonin synthesis on tau phosphorylation, we carried out western blot by using phosphorylation-dependent mAb tau-1 and PHF-1, as well as nonphosphorylation dependent pAb R134d. We found that the immunoreactivity of tau-1 was remarkably weaker, and of PHF-1 was obviously stronger in haloperidoltreated rats than in vehicle control rats. The immunoreactivity of tau was partially restored to the vehicle control level by supplement of melatonin both with low and high doses. The treatments used in the study did not alter the immunoreactivity of total tau stained by R134d. These results suggested that tau was hyperphosphorylated at Ser199/202 (tau-1 epitope) and Ser396/404 (PHF-1 epitope) when synthesis of melatonin was inhibited, and the supplement of melatonin partially arrested the haloperidol-induced hyperphosphorylation of tau at the above epitopes. (4) Effect of haloperidol and melatonin on the activity of PP2A: To determine the connection of PP2A with haloperidol-induced melatonin deficits and tau hyperphosphorylation, we measured the activity of PP2A by 32P-labeling assay using specific substrates. We found that the infusion of haloperidol significantly inhibited PP2A activity. The supplementation with melatonin at the high dose not only restored but also significantly elevated PP2A activity. An obvious increased activity of PP2A was also observed in low dose melatonin supplemented group, but it was not statistically significant. (5) Effect of inhibition of melatonin biosynthesis on the oxidative stress: Melatonin is a well-known antioxidant. It was shown that treatment of the rats with haloperidol significantly depressed the activity of SOD with a concomitant elevation of MDA. Supplementation of high and low doses of melatonin significantly restored SOD activity and arrested MDA overproduction. Conclusion: In summary, we have found in the present study (i) that inhibition of melatonin biosynthesis by haloperidol induces hyperphosphorylation of tau at both PHF-1 and tau-1 epitopes and compromises spatial memory retention in rats; (ii) that inhibition of melatonin biosynthesis inactivates PP2A, which may play a major role in haloperidol-induced tau hyperphosphorylation; (iii) that administration of haloperidol inhibits SOD and elevates MDA which might be the upstream alterations of decreased PP2A activity, and as well as tau hyperphosphorylation and spatial memory impairment; (iv) that melatonin supplementation can at least partially reverse elevated haloperidol-induced oxidative stress, tau hyperphosphorylation/PP2A inhibition, and as well as behavioral changes. Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is characterized by the presence of two pathological hallmarks: extracellular b-amyloid deposits in senile plaques and intracellular neurofibrillary tangles (NFTs). It is reported that the abundance of NFTs correlates well with the clinical degree of dementia and numerous studies have demonstrated that tau hyperphosphorylation plays a crucial role in the formation of NFTs. An imbalanced regulation of specific phosphatases and kinases in neurons represents a critical step for the initiation of tau hyperphosphorylation. The aim of this study is to investigate the effect of inhibiting melatonin biosynthesis on activities of protein kinase A (PKA), glycogen synthase kinase-3 (GSK-3) and tau phosphorylation at PS214 and M4 epitopes using haloperidol, a specific inhibitor of 5-hydroxyindole-O-methyltransferase.By 32P-labeling assays of protein kinase activities and western blot, we found that: Haloperidol injection through the lateral ventricle and intraperitoneal reinforcement significantly stimulated PKA activity with a concurrent hyperphosphorylation of tau at M4 (Thr231/Ser235) and pS214 (Ser214) sites. Prior treatment of the rats using melatonin supplement for one week and reinforcement during the haloperidol administration arrested PKA activity and attenuated tau hyperphosphorylation. GSK-3 activity showed no obvious change after haloperidol injection, however, melatonin supplements and reinforcements during haloperidol infusion inactivated basal activity of GSK-3. We concluded that: Decreased melatonin may be involved in Alzheimer-like tau hyperphosphorylation, and overactivation of PKA may play a crucial role in this process. Decreased level of melatonin and hyperphosphorylation of neurofilament proteins have been reported in Alzheimer’s disease (AD). However, the direct evidence linking melatonin and neurofilament phosphorylation is still lacking. Here, we investigated the effect of inhibiting melatonin biosynthesis on phosphorylation of neurofilament proteins and the involvement of cyclin-dependent kinase 5 (cdk-5) in rats. We observed that injection of haloperidol, a specific inhibitor of 5-hydroxyindole-O-methyltransferase, resulted in significantly decreased level of serum melatonin with a concomitantly increased phosphorylation of neurofilament proteins and activation of cdk-5 in rats. Exogenous supplementation of melatonin partially arrested the hyperphosphorylation of neurofilament and the activation of cdk-5. These results suggest that inhibition of melatonin biosynthesis may activate cdk-5 and thus induces Alzheimer-like hyperphosphorylation of neurofilament proteins. Synaptic activity-dependent de novo gene transcription is crucial for long-lasting neuronal plasticity and long-term memory (LTM), while short term memory (STM) involves gene expression-dependent synaptic plasticity in the hippocampus. PolyADP-ribose-polymerase-1 (PARP-1) is a multifunctional enzyme that can modulate gene expression and LTM. The cAMP-PKA pathway is involved in CREB activation, which is recognized as a molecular switch for LTM formation in hippocampus. Little is known whether PARP-1 is involved in the formation of hippocampus-dependent memory. Here, we studied the effect of PARP-1 on hippocampus-dependent memory in rats and the underlying mechanisms. The PARP-1 inhibitor (3-Aminobenzamide) and activator (NGF) were adiministered respectively to rats through lateral ventricle injection. The results were as follows: (1) Inhibition of PARP-1 by injection of 3AB (50 mM, 20μl) impaired the hippocampus-dependent contextual memory but not the cue memory measured by fear conditioning tasks (2) Injection of 3AB at 50 mM and 100 mM inhibited LTM but not STM measured by inhibitory avoidance tasks, and 3AB at 1mM did not affect the memory of the rats. (3) 3AB (50 mM, 20μl) impaired the spatial memory measured by Morris water maze, and the impairment was partially improved by simultaneous administration of NGF. Adminitration of NGF alone stimulated the memory functions. (4) The relative amount of polyADP-ribosylated PARP-1, representing activated PARP-1, in 3AB (50 mM, 20μl) treated hippocampal neurons was significantly decreased, and a significant shift to acid PI was found in samples treated with NGF. (5) Western blotting results showed that polyADP-ribosylation protein decreased when the activity of PARP-1 was inhibited by 3AB. Activition of PARP-1 by NGF increased the level of polyADP-ribosylation proteins. Immunocytochemical results also demonstrated that after treated 3AB, staining with PAR was much weaker in hippocampus and cortex, while staining of 3AB+NGF group was stronger than group treated with 3AB alone. (6) Inhibition of PARP-1 impaired the LTP. When the high-dose 3AB was given (3AB50, 50mM; 3AB100, 100mM), the ampitude and slope of LTP were decreased compared with the controls. No statistical significance between 3AB1 and control group was observed. (7) Western blotting and quantitative analysis of PKAcαshowed that there was no significance between 3AB group and control. The level of PKAcαwas increased after NGF was administered. The activity of PKA was significantly decreased after inhibition of PARP-1 by 3AB, whereas NGF could provent 3AB-induced inhibition of the kinase. Additionally, NGF alone could increase significantly the activity of PKA. The results of immunohistochemistry in hippoampus showed that the staining of PKA was much stronger in region CA3, while weaker in region CA1 compared with control when 3AB was adiministered. When the animals were then treated with NGF, the staining became weaker in CA3 but stronger in CA1 region than that of 3AB group; In animals treated with NGF alone, the staining of PKA was the weakest in CA3 and the strongest in CA1 region. (8) The cAMP level was significantly decreased in the group treated with 3AB while NGF could increase the level of cAMP. cAMP level was significantly increased when NGF was administrated alone. (9) Western blots of homogenized tissue from rat hippocampal neurons were probed with anti-CREB and anti-phospho-CREB (p-CREB) antibodies. The total amount of CREB did not change in each group. The phosphorylated CREB level was decreased in 3AB-treated animals, and NGF supplementation could provent the inhibition of CREB phosphorylation. But when NGF was given alone, the phosphorylation level of CREB did not increase compared with control. (10) The mRNA levels of memory-associated genes, including PKA cαand NF-κB, were measured by RT-PCR and no change was found. Conclusion: these results disclose a novel hierarchical transcriptional network involving PARP-1, PKA, and CREB that leads to concerted nuclear transduction of synaptic signals in neurons, accounting for the critical function of PARP-1 in learning and memory.更多还原