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Role of a novel neuropeptide (p5/Tp5/TFp5) derived from a neuronal cell cycle kinase (Cdk5),p35 activator protein, in neurobiology

源自神经元细胞周期激酶 (Cdk5)、p35 激活蛋白的新型神经肽 (p5/Tp5/TFp5) 在神经生物学中的作用

基本信息

批准号：
10263012
负责人：
HARISH C PANT
金额：
$ 45.42万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

Research Accomplishments: Our laboratory have shown that cyclin-dependent kinase 5 Cdk5), a critical and multifunctional neuronal kinase activity is tightly regulated and involved in nervous system development, regulates large number of synaptic and other processes including survival under physiological conditions. However, due to neuronal insults is deregulated and hyperactivated in Alzheimers disease (AD) and other neuropathologies may be, in part responsible for the hallmark pathology of amyloid plaques, neurofibrillary tangles (NFTs) and neuronal death. It has been proposed that hyperactive Cdk5 results from the overexpression of p25, (a truncated fragment of p35, the normal Cdk5 regulator), which, when complexes to Cdk5, induces deregulation, hyperactivity, hyper phosphorylates tau, neurofilament and other cytoskeletal proteins forms tangles (NFTs), A-beta plaques and neuronal death. ATP analogs have been used to inhibit the kinase activity but all these compounds are non-specific and toxic. Therefore, it was important to inhibit selectively inhibit the deregulated and hyperactive Cdk5/p25 not the Cdk5/p35. Cdk5/p35 is the physiological and Cdk5/p25 the pathological targets. We asked the question what will be the effect of smaller truncated peptides of p25. This study led to the invention of a small peptide p5, a 24 amino acid selectively inhibited the Cdk5/p25, the pathological but not Cdk5/p35 physiological activity. Recently our laboratory has shown that intraperitoneal (i.p.) injections of a modified truncated p5 peptide (TFP5), derived from the Cdk5 activator p35, penetrated the blood-brain barrier and rescued AD-like pathology in 5XFAD model mice. The principal pathology in the 5XFAD mutant, however, is extensive amyloid plaques; hence, as a proof of concept, we believe it is essential to demonstrate the peptides efficacy in a mouse model expressing high levels of p25, such as the inducible CK-p25Tg model mouse that overexpresses p25 in CamKII positive neurons. Using a modified TFP5 treatment, we show that peptide i.p. injections in these mice decrease Cdk5 hyperactivity, tau, neurofilament-M/H hyper phosphorylation, and restore synaptic function (LTP) and behavior (i.e., spatial working memory). It is noteworthy that TFP5 does not inhibit endogenous Cdk5/p35 activity, or other Cdks in vivo suggesting it might have no toxic side effects, and may serve as an excellent therapeutic candidate for neurodegenerative disorders expressing abnormally high brain levels of p25 and hyperactive Cdk5. As a proof of concept, we have demonstrated that the peptide, injected into an AD model mouse overexpressing Cdk5/p25 (P25Tg mice), specifically targets the hyperactive kinase, reduces or eliminates AD pathology, and restores normal behavior in both in vivo and in vitro. We suggest that the peptide may serve as a potential therapeutic candidate for those neurodegenerative disorders that overexpress p25, the hyperactive activator form of p35. TP5; Protective & Restoractive Preliminary data (Sharda Yadav Current Studies) TP5 modulates the dendritic morphology Development of the neurite outgrowth and spine density is critical for the neuronal function. Previous studies revealed that Cdk5 activity is required for the development of neurite outgrowth, establishment of synaptic connections and neuronal morphology. Recently we developed, TP5 derived from the CDK5 activator p35, as an inhibitor of the hyperactive Cdk5 in vitro. To test the specificity of the TP5 we screened several kinases and noted almost 20 other kinases including Cdk5 were inhibited to some extent in test tube experiments. However, their physiological relevance has not yet established. To understand the overall impact of inhibition of these kinases by TP5 we decided to test its effect on the cultured rat cortical neurons. We focused on to observe morphological changes specifically neurite outgrowth, number of dendritic spines and establishment of the mature synaptic connections. For this cultured rat cortical neurons were transfected with ER-GFP reporter alone (to visualize the morphological changes) or in addition to Rab8-mCherry (to mark the vesicles required for neurite out growth). Neurons were treated with TP5 (500nM) from DIV3 till harvested on DIV17. Scrambled peptide (500nM) and mock treated neurons were used as negative control. Cultured neurons on the cover slips were harvested at DIV17. Neurons were fixed with 4% PFA. Images were obtained using confocal microscope LSM510. Morphological changes and number of dendritic spines were counted using Image J. We observed significant increase in the dendritic morphology of the TP5 treated cortical neurons at DIV17. We also noted a slight increase in dendritic morphology at DIV6 which was further enhanced at DIV12. However, these changes were not observed in cortical neurons treated with scrambled peptide or in blank. Our results suggest that TP5 promote the neurite outgrowth in the cultured cortical neurons. TP5 enhanced the mature synaptic connections Functional synaptic connections between neurons needed for effective transfer of the electrochemical signals between neurons. Furthermore, development of functional synapses in neurons require restrained Cdk5 activity. In pathological conditions altered Cdk5 activity leads to aberrant synapse formation. To evaluate the role of the TP5 and its impact on the establishment of the synaptic connections we treated the cultured rat cortical neurons with TP5 peptide from DIV3 till DIV17. Without peptide treated neurons were used as control ( + media). Both group of the neurons were transfected with ER-GFP reporter to mark the morphological changes. We immunostained cortical neurons with synaptophysin and PSD95 (mature synaptic markers). Furthermore, quantification of the synaptophysin and PSD95 positive staining revealed an increase in the number of synaptic connection in TP5 treated neurons compared to blank (+media). Our data revealed that TP5 treated neurons establish more mature synaptic connections compared to corresponding control treated neurons. This suggest that TP5 has a role in establishing the mature synaptic connections and possibly explains part of the rescue phenotype of 5XFAD mice and p25 transgenic mice reported earlier. Future Plans; Effect of the TP5 on the transport and distribution of different organelles In neurons distribution of the different organelle is precisely regulated and represents their functional status. Aberrant organelle trafficking has been reported in several neurodegenerative disorders such as ALS, PD and AD. As Cdk5 has a wide range of substrates, many of them are involved in different cellular processes including, components of structural proteins present in different organelles, molecular motor complex protein subunits and integral part of the molecular tracks such as microtubules and their accessory factors that regulate the trafficking of the cargo from neuronal soma to axons and dendrites. Cargo trafficking occurs in both retrograde and anterograde directions and highly controlled by phosphorylation events by Cdk5. In fact, activity of the many of the anterograde and retrograde components are directly regulated by Cdk5 phosphorylation. However, the impact of the TP5 on these processes need to be further evaluated in normal and animal models of the neurodegenerative diseases. In this context, we have developed several fluorescent markers to tag different organelles (e.g. mitochondria, peroxisome, lysosome) and different vesicular compartments such as Rab5, Rab7 and Rab11). In addition, we have also obtained previously reported molecular motor complex proteins (such as Kif1a and Kif5b) fused with GFP to visualize the motors and microtubule tracks will be stained for the endogenous alpha tubulin.

研究成果：我们的实验室已经表明，细胞周期蛋白依赖性激酶5（Cdk5）是一种关键的多功能神经元激酶活性，受到严格调控并参与神经系统发育，调节大量突触和其他过程，包括生理条件下的生存。然而，由于阿尔茨海默病（AD）中的神经元损伤失调和过度激活，其他神经病理学可能是淀粉样蛋白斑、神经原纤维缠结（NFT）和神经元死亡的标志性病理学的部分原因。有人提出，过度活跃的 Cdk5 是由于 p25（正常 Cdk5 调节因子 p35 的截短片段）过度表达所致，当 p25 与 Cdk5 复合时，会导致失调、过度活跃、过度磷酸化 tau、神经丝和其他细胞骨架蛋白，形成缠结 (NFT)、A-β 斑块和神经元死亡。 ATP 类似物已被用来抑制激酶活性，但所有这些化合物都是非特异性且有毒的。因此，重要的是选择性抑制失调和过度活跃的 Cdk5/p25，而不是 Cdk5/p35。 Cdk5/p35 是生理目标，Cdk5/p25 是病理目标。我们提出了这样的问题：p25 的较小截短肽会产生什么影响。这项研究导致发明了一种小肽p5，它由24个氨基酸组成，选择性抑制Cdk5/p25的病理活性，但不抑制Cdk5/p35的生理活性。最近，我们的实验室表明，腹膜内 (i.p.) 注射源自 Cdk5 激活剂 p35 的修饰截短 p5 肽 (TFP5) 可以穿透血脑屏障，并在 5XFAD 模型小鼠中挽救 AD 样病理。然而，5XFAD 突变体的主要病理是广泛的淀粉样斑块。因此，作为概念证明，我们认为有必要在表达高水平 p25 的小鼠模型中证明肽的功效，例如在 CamKII 阳性神经元中过度表达 p25 的诱导型 CK-p25Tg 模型小鼠。使用改良的 TFP5 处理，我们表明肽腹腔注射。对这些小鼠进行注射可降低 Cdk5 过度活跃、tau 蛋白、神经丝 M/H 过度磷酸化，并恢复突触功能 (LTP) 和行为（即空间工作记忆）。值得注意的是，TFP5 不会抑制内源性 Cdk5/p35 活性或其他体内 Cdk 活性，这表明它可能没有毒副作用，并且可能作为表达异常高的脑部 p25 水平和过度活跃的 Cdk5 的神经退行性疾病的优秀治疗候选者。作为概念证明，我们已经证明，将这种肽注射到过度表达 Cdk5/p25 的 AD 模型小鼠（P25Tg 小鼠）中，可以特异性靶向过度活跃的激酶，减少或消除 AD 病理，并在体内和体外恢复正常行为。我们认为，该肽可以作为过度表达 p25（p35 的过度活跃激活剂形式）的神经退行性疾病的潜在治疗候选物。 TP5；保护和恢复初步数据（Sharda Yadav 当前研究） TP5 调节树突形态神经突生长和脊柱密度的发育对于神经元功能至关重要。先前的研究表明，Cdk5 活性是神经突生长、突触连接和神经元形态发育所必需的。最近我们开发了源自 CDK5 激活剂 p35 的 TP5，作为体外高活性 Cdk5 的抑制剂。为了测试 TP5 的特异性，我们筛选了几种激酶，并注意到包括 Cdk5 在内的近 20 种其他激酶在试管实验中受到一定程度的抑制。然而，它们的生理相关性尚未确定。为了了解 TP5 抑制这些激酶的总体影响，我们决定测试其对培养的大鼠皮质神经元的影响。我们重点观察形态变化，特别是神经突生长、树突棘数量和成熟突触连接的建立。对于这种培养的大鼠皮层神经元，单独用 ER-GFP 报告基因转染（以可视化形态变化），或添加 Rab8-mCherry（以标记神经突生长所需的囊泡）。用来自 DIV3 的 TP5 (500nM) 处理神经元，直至在 DIV17 上收获。乱序肽（500nM）和模拟处理的神经元用作阴性对照。在 DIV17 收获盖玻片上培养的神经元。神经元用 4% PFA 固定。使用共焦显微镜 LSM510 获得图像。使用 Image J 计算形态变化和树突棘数量。我们观察到 DIV17 时 TP5 处理的皮层神经元的树突形态显着增加。我们还注意到 DIV6 的树突形态略有增加，在 DIV12 进一步增强。然而，在用乱序肽或空白处理的皮质神经元中没有观察到这些变化。我们的结果表明 TP5 促进培养的皮质神经元中的神经突生长。 TP5增强成熟的突触连接神经元之间的功能性突触连接需要神经元之间电化学信号的有效传递。此外，神经元功能性突触的发育需要抑制 Cdk5 活性。在病理条件下，Cdk5 活性的改变会导致异常的突触形成。为了评估 TP5 的作用及其对突触连接建立的影响，我们用 DIV3 至 DIV17 的 TP5 肽处理培养的大鼠皮质神经元。使用未经肽处理的神经元作为对照（+培养基）。两组神经元均转染ER-GFP报告基因以标记形态变化。我们用突触素和 PSD95（成熟突触标记物）对皮质神经元进行免疫染色。此外，突触素和 PSD95 阳性染色的定量显示，与空白（+介质）相比，TP5 处理的神经元中突触连接的数量有所增加。我们的数据显示，与相应的对照处理神经元相比，TP5 处理的神经元建立了更成熟的突触连接。这表明TP5在建立成熟突触连接方面发挥着作用，并可能解释了先前报道的5XFAD小鼠和p25转基因小鼠的部分拯救表型。未来计划； TP5对不同细胞器转运和分布的影响在神经元中，不同细胞器的分布受到精确调节并代表它们的功能状态。据报道，ALS、PD 和 AD 等多种神经退行性疾病中存在细胞器运输异常。由于 Cdk5 具有广泛的底物，其中许多底物参与不同的细胞过程，包括不同细胞器中存在的结构蛋白的成分、分子运动复合体蛋白亚基和分子轨道的组成部分，例如微管及其调节从神经元胞体到轴突和树突的货物运输的辅助因子。货物运输沿逆行和顺行方向发生，并受到 Cdk5 磷酸化事件的高度控制。事实上，许多顺行和逆行成分的活性直接受到 Cdk5 磷酸化的调节。然而，TP5 对这些过程的影响需要在正常和神经退行性疾病动物模型中进一步评估。在这种情况下，我们开发了几种荧光标记物来标记不同的细胞器（例如线粒体、过氧化物酶体、溶酶体）和不同的囊泡区室（例如 Rab5、Rab7 和 Rab11）。此外，我们还获得了先前报道的与 GFP 融合的分子运动复合体蛋白（例如 Kif1a 和 Kif5b），以可视化运动和微管轨道，并将对内源 α 微管蛋白进行染色。