The effect of cdk-5 overexpression on tau phosphorylation and spatial memory of rat

In Alzheimer’s disease (AD), hyperphosphorylation of tau may be the underlying mechanism for the cytoskeletal abnormalities and neuronal death. It was reported that cyclin-dependent kinase5 (cdk-5) could phosphorylate tau at most AD-related epitopesin vitro. In this study, we investigated the effect of cdk-5 overexpression on tau phosphorylation and spatial memory in rat. We demonstrated that 24 h after transfection into rat hippocampus, cdk-5 was overexpressed and induced a reduced staining with antibody tau-1 and an enhanced staining with antibodies 12e8 and PHF-1, suggesting hyperphosphorylation of tau at Ser199/202, Ser262/356 and Ser396/404 sites. Additionally, the cdk-5 transfected rats showed long latency to find the hidden platform in Morris water maze compared to the control rat. 48 h after transfection, the level of cdk-5 was decreased significantly, and the latency of rats to find the hidden platform was prolonged. It implies thatin vivo overexpression of cdk-5 leads to impairment of spatial memory in rat and tau hyperphosphorylation may be the underlying mechanism.     

The effect of cdk-5 overexpression on tau phosphorylation and spatial memory of rat

Neurofibrillarytangle(NFT),primarilycom-posedofthehyperphosphorylatedmicrotubule-asso-ciatedproteintau[1],isoneofthemajorneuropa-thologichallmarksinAD.Intheaffectedneurons,themajormechanismoftauhyperphosphorylationistheabnormalregulationofproteinkinasesandproteinphosphatases.TherearemanyproteinkinasesthatcanphosphorylatetauatAD-relatedepitopessuchaspro-teinkinaseC(PKC),glycogensynthasekinase3b(GSK3b),Ca2+/calmodulin-dependentkinaseII(Ca-MKII).Amongthem,cdk-5isactivepredominantlyinneur…     

抑制大鼠褪黑素的合成对神经细丝过度磷酸化的影响

目的探讨抑制褪黑素的生物合成对大鼠海马神经细丝过度磷酸化的影响及其可能机制.方法侧脑室注射氟哌啶醇并腹腔注射加强,HPLC检测血清中褪黑素水平;利用免疫组化检测大鼠海马区域神经细丝磷酸化情况;微量测定法测定超氧化物歧化酶(SOD)活性和脂质过氧化产物丙二醛的含量.结果①模型组大鼠血清中褪黑素的含量明显下降.②海马区域神经细丝发生聚集和异常过度磷酸化,补充不同剂量的褪黑素的治疗组较模型组的磷酸化程度有所改善.③模型组SOD活性降低,丙二醛(MDA)含量明显升高.结论褪黑素水平的降低引起AD-样神经细丝异常过度磷酸化,外源性补充褪黑素可以一定程度上减轻神经细丝的异常过度磷酸化.     

抑制褪黑素生物合成对大鼠Tau蛋白磷酸化的影响

目的 研究抑制褪黑素的生物合成对大鼠海马Tau蛋白磷酸化的影响。方法 侧脑室注射氟哌啶醇并腹腔注射加强,利用免疫组化检测大鼠海马区域Tau蛋白磷酸化情况;HPLC检测血清中褪黑素水平。结果 模型组大鼠海马Tau蛋白在Ser199/Ser202和Ser396/Ser404位点均发生异常过度磷酸化,褪黑素治疗组较模型组的磷酸化程度轻。结论 褪黑素水平的降低可能与AD样Tau蛋白异常过度磷酸化相关,外源性补充褪黑素可以减轻Tau蛋白的异常过度磷酸化。     

Differential Cellular Phosphorylation of Neurofilament Heavy Side-Arms by Glycogen Synthase Kinase-3 and Cyclin-Dependent Kinase-5

Abstract: To investigate the cellular mechanisms regulating neurofilament-heavy subunit (NF-H) side-arm phosphorylation, we studied the ability of three putative neurofilament kinases, glycogen synthase kinase-3 (GSK-3)α, GSK-3β, and cyclin-dependent kinase-5 (cdk-5), to phosphorylate NF-H in transfected cells. We analysed NF-H phosphorylation by using a panel of phosphorylation-dependent antibodies and also by monitoring the electrophoretic mobility of the transfected NF-H on sodium dodecyl sulphate-polyacrylamide gel electrophoresis because this is known to be affected by side-arm phosphorylation. Our results demonstrate that whereas GSK-3α, GSK-3β, and cdk-5 will all phosphorylate NF-H, they generate different antibody reactivity profiles. GSK-3α and GSK-3β induce a partial retardation of a proportion of the transfected NF-H, but only cdk-5 alters the rate of electrophoretic migration to that of NF-H from brain. We conclude that cdk-5 and GSK-3 phosphorylate different residues or sets of residues within NF-H sidearms in cells. We further show that cdk-5 is active in both the CNS and the PNS but that this activity is not dependent on expression of its activator, p35. This suggests that there are other activators of cdk-5.     

褪黑素对异丙肾上腺素诱导大鼠tau蛋白过度磷酸化的预防作用

异常过度磷酸化的微管相关蛋白tau是阿尔茨海默病(Alzheimer's disease,AD)患者大脑中神经原纤维缠结的主要组成部分.迄今为止,尚无有效的措施阻止tau蛋白的过度磷酸化.为探讨褪黑素(melatonin,Mel)对AD样tau蛋白过度磷酸化的预防作用,我们以β受体激动剂异丙肾上腺素(isoproterenol,IP)来复制AD样tau蛋白过度磷酸化的动物模型,在大鼠双侧海马注射IP前,以褪黑素作为保护组药物,于腹腔连续注射5 d.应用磷酸化位点特异性抗体(PHF-1和Tau-1)作免疫印迹和免疫组织化学检测tau蛋白的磷酸化水平,并用非磷酸化依赖的总tau蛋白抗体(111e)进行标准化.免疫印迹结果显示在注射IP 48 h后,tau蛋白在PHF-1表位的免疫反应显著增强,在Tau-1表位显著减弱,表明tau蛋白在Ser396/Ser404(PHF-1)和Ser199/Ser202(Tau-1)位点有过度磷酸化.免疫组织化学染色结果与免疫印迹结果相似,主要检测到在大鼠海马CA3区的神经纤维有tau蛋白过度磷酸化.褪黑素预处理大鼠可有效地阻止IP诱导tau蛋白在Tau-1和PHF-1位点的过度磷酸化.上述结果提示褪黑素可预防大鼠脑组织中由异丙肾上腺素引起的AD样tau蛋白的过度磷酸化.     

Phosphorylation of Neurofilament Heavy-Chain Side-Arm Fragments by Cyclin-Dependent Kinase-5 and Glycogen Synthase Kinase-3α in Transfected Cells

Abstract: The side-arm domain of neurofilament heavy-chain (NF-H) is heavily phosphorylated in axons. Much of this phosphate is located within a multiphosphorylation repeat (MPR) domain situated toward the carboxy terminus of the molecule. The MPR domain contains the repeat motif KSP of which there are two broad categories, KSPXX and KSPXK. In mouse NF-H, the KSPXK repeats are situated toward the latter part of the MPR domain. We have expressed in mammalian cells fragments of mouse NF-H side-arm containing all of the MPR domain, the latter part of the MPR domain containing the KSPXK repeats, and the complementary amino-terminal part of the MPR domain, which contains the KSPXX repeats. By cotransfecting these fragments with the neurofilament kinases cyclin-dependent kinase-5 (cdk-5)/p35 and glycogen synthase kinase-3α (GSK-3α), we show that cdk-5 induces cellular phosphorylation of the KSPXK-containing fragment of NF-H. Using the transfected fragments, we also map the epitopes for several commonly utilised NF-H monoclonal antibodies and describe the effects that phosphorylation by cdk-5 and GSK-3α have on their reactivities.     

褪黑素与维生素 E 对抗花萼海绵诱癌素毒性作用的差异

最近的研究发现,褪黑素对花萼海绵诱癌素 (calyculin A , CA) 引起的骨架蛋白神经细丝异常过度磷酸化有保护作用 . 为进一步探讨褪黑素对骨架蛋白τ异常过度磷酸化的保护作用及其机制,分别用 CA, CA+ 褪黑素或 CA+ 维生素 E 处理鼠野生型成神经瘤细胞 (N2awt) ,采用 MTT 法测定细胞存活率,用免疫印迹法测定τ蛋白磷酸化水平,用 ³²P- 特异底物标记技术检测 GSK-3 和 PP-2A 活性,并进一步测定了细胞内脂质过氧化产物丙二醛含量,细胞内过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶活性 . 结果显示：褪黑素不仅对 CA 引起的抗氧化酶活性降低和脂质过氧化的保护作用强于经典抗氧化剂维生素 E ,而且对τ蛋白磷酸化的保护作用也强于经典抗氧化剂维生素 E ;褪黑素可同时激活 PP-2A 又抑制 GSK-3 ,而维生素 E 同时抑制两种酶的活性 . 研究提示：褪黑素既通过抗氧化作用,也通过调节细胞内磷酸化平衡对抗 CA 对神经细胞的毒性作用 .     

Effect of Melatonin and Melatonylvalpromide on β-amyloid and Neurofilaments in N2a Cells

In the present study, we have studied the effect of melatonin (Mt) and melatonin derivative, i.e., melatonylvalpromide (Mtv), on cell viability, β-amyloid (Aβ) production, cell morphology, and expression and phosphorylation of neurofilament proteins in wild-type murine neuroblastoma N2a (N2a/wt) and N2a stably transfected with amyloid precursor protein (N2a/APP) cell lines. The study used MTT assay, Sandwich ELISA, immunocytochemistry and Western blots techniques. The results showed that both Mt and Mtv could increase cell viability, but Mtv did so more effectively. The N2a/APP showed shorter and less amount of cell processes than N2a/wt, and Mtv but not Mt slightly improved the morphological changes in N2A/APP. Both Mt and Mtv suppressed the Aβ level in cell lysates, but the effect of Mtv was stronger than Mt. The immunoreaction to the non-phosphorylated neurofilament proteins probed by SMI32 and SMI33 were remarkably weaker in N2a/APP than N2a/wt, while the immunoreaction to the phosphorylated neurofilament proteins at SMI34 epitopes was slightly stronger in N2a/APP than N2a/wt, suggesting higher phosphorylation level of neurofilament proteins in N2a/APP. Treatment of the cells with Mt and Mtv increased the immunoreaction at SMI32 and SMI33 epitopes, while only Mtv but not Mt decreased the staining at SMI34 epitope, suggesting both Mt and Mtv promote dephosphorylation of neurofilament at SMI32 and SMI33 epitopes, while Mtv stimulates dephosphorylation of neurofilament at SMI34 epitope. These results suggest that Mtv may be a better candidate in arresting the intracellular accumulation of Aβ and protecting the cells from Aβ-related toxicity. Xiao-Chuan Wang and Yin-Chun Zhang equally contributed to the work.     

Melatonin and its metabolite N(1)‐acetyl‐N(1)‐formyl‐5‐methoxykynuramine improve learning and memory impairment related to Alzheimer's disease in rats

The aim of this study was to investigate the effect of melatonin (MT) and its metabolite N(1)‐acetyl‐N(2)‐formyl‐5‐methoxykynuramine (AFMK) on Alzheimer‐like learning and memory impairment in rats intracerebroventricularly injected with streptozotocin (STZ). The results showed that the escape latency of the STZ group was longer than that of the control (CON), MT, and AFMK groups. Increased levels of hyperphosphorylated tau, neurofilament proteins, and malondialdehyde and decreased superoxide dismutase levels were observed in the brains of the rats from the STZ group compared with the brains of the rats from the CON, MT, AFMK high and low group. These results suggest that exogenous MT and AFMK can improve memory impairment and downregulate AD‐like hyperphosphorylation induced by STZ, most likely through their antioxidation function. Meanwhile, we found that an equal dose of AFMK had a stronger effect than that of MT. Our results indicate that MT and its metabolite AFMK represent novel treatment strategies for Alzheimer's disease.     

Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions

Recent studies suggest that increased activity of cyclin-dependent kinase 5 (Cdk5) may contribute to neuronal death and cytoskeletal abnormalities in Alzheimer's disease. We report here such deregulation of Cdk5 activity associated with the hyperphosphorylation of tau and neurofilament (NF) proteins in mice expressing a mutant superoxide dismutase (SOD1(G37R)) linked to amyotrophic lateral sclerosis (ALS). A Cdk5 involvement in motor neuron degeneration is supported by our analysis of three SOD1(G37R) mouse lines exhibiting perikaryal inclusions of NF proteins. Our results suggest that perikaryal accumulations of NF proteins in motor neurons may alleviate ALS pathogenesis by acting as a phosphorylation sink for Cdk5 activity, thereby reducing the detrimental hyperphosphorylation of tau and other neuronal substrates.     

反复饥饿对大鼠空间学习及海马神经元骨架蛋白磷酸化的影响

为了进一步研究饥饿处理对大鼠空间学习、记忆的影响,通过饥饿2 d、恢复喂食3 d的方法,连续60 d,用Morris水迷宫检测大鼠的空间学习能力.免疫印迹检测神经元骨架蛋白—tau蛋白和神经细丝(Neurofilament,NF)磷酸化水平与分布变化,以及骨架蛋白磷酸化调节的关键酯酶磷酸酯酶PP-2A催化亚单位蛋白水平与分布.反复饥饿的大鼠空间学习能力明显差于对照组(P0.05),tau蛋白在Ser199/202位点和Ser396/404位点发生了过度磷酸化(P0.05),NF磷酸化水平无明显改变,PP-2A的催化亚单位蛋白水平下调(P0.05).反复饥饿可以引起大鼠出现空间学习记忆障碍,下调PP-2A催化亚单位蛋白水平,PP-2A活性抑制及tau蛋白发生过度磷酸化.     

Hyperphosphorylation and accumulation of neurofilament proteins in Alzheimer disease brain and in okadaic acid-treated SY5Y cells.

We investigated the role of neurofilament (NF) proteins in Alzheimer disease (AD) neurofibrillary degeneration. The levels and degree of phosphorylation of NF proteins in AD neocortex were determined by Western blots developed with a panel of phosphorylation-dependent NF antibodies. Levels of all three NF subunits and the degree of phosphorylation of NF-H and NF-M were significantly increased in AD as compared to Huntington disease brains used as control tissue. The increase in the levels of NF-H and NF-M was 1.7- and 1.5-fold (P<0.01) as determined by monoclonal antibody SMI33, and was 1.6-fold (P<0.01) in NF-L using antibody NR4. The phosphorylation of NF-H and NF-M in AD was increased respectively at the SMI31 epitope by 1.6- and 1.9-fold (P<0.05) and at the SMI33 epitope by 2.7- and 1.3-fold (P<0.01 and P<0.05). Essentially similar effects were observed in SY5Y human neuroblastoma cells when treated with okadaic acid, an inhibitor of protein phosphatase (PP)-2A and -1. This is the first biochemical evidence which unambiguously demonstrates the hyperphosphorylation and the accumulation of NF subunits in AD brain, and shows that the inhibition of PP-2A/PP-1 activities can lead to the hyperphosphorylation of NF-H and NF-M subunits.     

Attenuation of okadaic acid-induced hyperphosphorylation of cytoskeletal proteins by heat preconditioning and its possible underlying mechanisms

An imbalanced phosphorylation system is recognized to be one of the main reasons for Alzheimer-like hyperphosphorylation of cytoskeletal proteins. However, little is known about the strategies rectifying the lesions caused by this disrupted phosphorylation. To search for the means to arrest Alzheimer-like damages and explore the underlying mechanisms, in this study we treated N2a/peuht40 cells with okadaic acid (OA), a specific inhibitor of protein phosphatase-2A (PP-2A) and PP-1, to mimic an Alzheimer-like phosphatase-deficient system and then used heat preconditioning (42 degrees C for 1 hour) to induce the expression of inducible heat shock protein 70 (Hsp70) in the cells. We observed that heat preconditioning arrested OA-induced hyperphosphorylation of neurofilament (NF) protein at SMI34 and SMI33 epitopes as well as hyperphosphorylation of tau at Tau-1 and PHF-1 epitopes. It counteracted OA-induced decrease in PP-2A activity with a concurrent inhibition in constitutive activity of mitogen-activated protein kinases (MAPKs) and cyclic adenosine 5'-monophosphate-dependent protein kinase A (PKA). Conversely, quercetin, a recognized blocker of stress-responsive Hsp70 expression, diminished the effects caused by heat preconditioning. These results suggested that Hsp70 antagonized OA-induced Alzheimer-like NF and tau hyperphosphorylation, and the restoration of PP-2A and inhibition of MAPKs-PKA activity might be part of the underlying mechanisms for the rectification of OA-induced hyperphosphorylation.     

Suppression of Extensive Neurofilament Phosphorylation Rescues α-Internexin/Peripherin-Overexpressing PC12 Cells from Neuronal Cell Death

Intermediate filament (IF) overproduction induces abnormal accumulation of neuronal IF, which is a pathological indicator of some neurodegenerative disorders. In our study, α-Internexin- and peripherin-overexpressing PC12 cells (pINT-EGFP and pEGFP-peripherin) were used as models to study neuropathological pathways responsible for neurodegenerative diseases. Microarray data revealed that Cdk5-related genes were downregulated and Cdk5 regulatory subunit-associated protein 3 (GSK-3α and GSK-3β) were upregulated in pINT-EGFP cells. Increased expression of phosphorylated neurofilament and aberrant activation of Cdk5 and GSK-3β were detected in both pEGFP-peripherin and pINT-EGFP cells by Western blotting. In addition, pharmacological approaches to retaining viability of pINT-EGFP and pEGFP-peripherin cells were examined. Treatment with Cdk5 inhibitor and GSK-3β inhibitor significantly suppressed neuronal death. Dynamic changes of disaggregation of EGFP-peripherin and decrease in green fluorescence intensity were observed in pEGFP-peripherin and pINT-EGFP cells by confocal microscopy after GSK-3β inhibitor treatment. We conclude that inhibition of Cdk5 and GSK-3β suppresses neurofilament phosphorylation, slows down the accumulation of neuronal IF in the cytoplasm, and subsequently avoids damages to cell organelles. The results suggest that suppression of extensive neurofilament phosphorylation may be a potential strategy for ameliorating neuron death. The suppression of hyperphosphorylation of neuronal cytoskeletons with kinase inhibitors could be one of potential therapeutic treatments for neurodegenerative diseases.     

Age-related characteristics of the effects of epithalamin on serotonin metabolism in the pineal gland of rats]

The biosynthesis of serotonin into melatonin was decreased in old (18-20-month) in comparison to young (4-5-month) male Wistar rats. 5-day morning injections to young and old rats with polypeptide pineal preparation (epithalamin) in a dose of 2.5 mg/kg of body weight induced the increase in the night peak of serotonin, N-acetylserotonin and melatonin in young and melatonin alone in old rats and did not influence 5-methoxytryptamine, 5-oxy- and 5-methoxyindoleacetic acids level. These data support suggestion of ultrashort loop between pineal peptides and indoles and that epithalamin increases the metabolism of serotonin into melatonin.     

Alpha-Ketoisocaproic Acid Increases Phosphorylation of Intermediate Filament Proteins from Rat Cerebral Cortex by Mechanisms Involving Ca<Superscript>2+</Superscript> and cAMP

We have previously described that α-ketoisocaproic acid (KIC), the main metabolite accumulating in maple syrup urine disease (MSUD), increased the in vitro phosphorylation of cytoskeletal proteins in cerebral cortex of 17- and 21-day-old rats through NMDA glutamatergic receptors. In the present study we investigated the protein kinases involved in the effects of KIC on the phosphorylating system associated with the cytoskeletal fraction and provided an insight on the mechanisms involved in such effects. Results showed that 1 mM KIC increased the in vitro incorporation of ³²P into intermediate filament (IF) proteins in slices of 21-day-old rats at shorter incubation times (5 min) than previously reported. Furthermore, this effect was prevented by 10 μM KN-93 and 10 μM H-89, indicating that KIC treatment increased Ca²⁺/calmodulin- (PKCaMII) and cAMP- (PKA) dependent protein kinases activities, respectively. Nifedipine (100 μM), a blocker of voltage-dependent calcium channels (VDCC), DL-AP5 (100 μM), a NMDA glutamate receptor antagonist and BAPTA-AM (50 μM), a potent intracellular Ca²⁺ chelator, were also able to prevent KIC-induced increase of in vitro phosphorylation of IF proteins. In addition, KIC treatment was able to significantly increase the intracellular cAMP levels. This data support the view that KIC increased the activity of the second messenger-dependent protein kinases PKCaMII and PKA through intracellular Ca²⁺ levels. Considering that hyperphosphorylation of cytoskeletal proteins is related to neurodegeneration it is presumed that the Ca²⁺-dependent hyperphosphorylation of IF proteins caused by KIC may be involved to the neuropathology of MSUD patients.     

Tau becomes a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain

Microtubule-associated protein tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) and other tauopathies and is believed to lead to neurodegeneration in this family of diseases. Here we show that infusion of forskolin, a specific cAMP-dependent protein kinase A (PKA) activator, into the lateral ventricle of brain in adult rats induced activation of PKA by severalfold and concurrently enhanced the phosphorylation of tau at Ser-214, Ser-198, Ser-199, and or Ser-202 (Tau-1 site) and Ser-396 and or Ser-404 (PHF-1 site), which are among the major abnormally hyperphosphorylated sites seen in AD. PKA activation positively correlated to the extent of tau phosphorylation at these sites. Infusion of forskolin together with PKA inhibitor or glycogen synthase kinase-3 (GSK-3) inhibitor revealed that the phosphorylation of tau at Ser-214 was catalyzed by PKA and that the phosphorylation at both the Tau-1 and the PHF-1 sites is induced by basal level of GSK-3, because forskolin activated PKA and not GSK-3 and inhibition of the latter inhibited the phosphorylation at Tau-1 and PHF-1 sites. Inhibition of cdc2, cdk5, or MAPK had no significant effect on the forskolin-induced hyperphosphorylation of tau. Forskolin inhibited spatial memory in a dose-dependent manner in the absence but not in the presence of R(p)-adenosine 3',5'-cyclic monophosphorothioate triethyl ammonium salt, a PKA inhibitor. These results demonstrate for the first time that phosphorylation of tau by PKA primes it for phosphorylation by GSK-3 at the Tau-1 and the PHF-1 sites and that an associated loss in spatial memory is inhibited by inhibition of the hyperphosphorylation of tau. These data provide a novel mechanism of the hyperphosphorylation of tau and identify both PKA and GSK-3 as promising therapeutic targets for AD and other tauopathies.