Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases¹. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials^2-4. The rapid iterative negative geotaxis (RING) assay⁵ has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.     

Computer-aided molecular design of pyrazolotriazines targeting glycogen synthase kinase 3

Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several processes associated with Alzheimer’s disease (AD). Therefore, GSK-3 has become a crucial therapeutic target for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep synthesis of ethyl 4-oxo-pyrazolo[4,3-d][1–3]triazine-7-carboxylates and their biological evaluation as GSK-3 inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical structure. Potential binding mode determination in the enzyme and the analysis of the key features in the catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood–brain barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and permeation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphorylation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo studies and may be considered as new leads for further optimisation for the treatment of AD.     

GSK-3在阿尔茨海默病样细胞骨架蛋白过度磷酸化中的作用(英)

神经原纤维缠结是阿尔茨海默病（Alzheimer disease, AD）的特征性病理改变.蛋白激酶和蛋白磷酸酯酶失衡可导致骨架蛋白的异常过度磷酸化,而异常过度磷酸化的tau 和神经丝 (neurofilament, NF) 是神经原纤维缠结的组成部分.在众多激酶中,糖原合酶激酶-3（glycogen synthase kinase-3,GSK-3）可能是AD神经退行性变起重要作用.为深入探讨GSK-3在AD样神经退行性变中的作用,以磷酯酰肌醇三磷酸激酶（phosphatidylinositol 3-kinase,PI3K）的特异性抑制剂渥曼青霉素（wortmannin,WT）处理野生型鼠成神经瘤细胞株（wild type mouse neuroblastoma cell lines, N2a wt）,系统观察WT处理N2a wt不同时间点（1 h、3 h、6 h）细胞代谢率、细胞形态、细胞骨架蛋白tau和NF的磷酸化状态改变以及细胞的命运,并分析了GSK-3活性与上述参数改变之间的相关性.结果发现：1 μmol/L WT处理细胞1 h,GSK-3活性与未经WT处理的对照组相比明显增高,并伴有Ser9磷酸化的GSK-3水平的降低; NF磷酸化程度增强,tau在Ser198/Ser199/Ser202位点的磷酸化增强. 1 μmol/L WT处理细胞3 h,GSK-3活性与对照组和处理1 h 组相比明显下降,NF磷酸化程度较1 h降低,但仍高于正常水平.1 μmol/L WT处理细胞6 h,细胞形态、GSK-3活性、Ser9磷酸化形式的GSK-3β的表达、NF磷酸化程度与对照组相比均无明显改变.WT呈剂量依赖性降低细胞代谢率.1 μmol/L WT处理细胞1 h和3 h导致细胞变圆,突起变短甚至消失.1 μmol/L WT处理细胞1 h,用TUNEL法和电子显微镜技术未观察到细胞凋亡.研究结果提示：在N2a细胞中过度激活GSK-3可导致神经细丝和tau蛋白的AD样过度磷酸化,从而引起神经细胞的AD样退行性变.     

Tissue-specific targeting of Hsp26 has no effect on heat resistance of neural function in larval <Emphasis Type="Italic">Drosophila</Emphasis>

Hsp26 belongs to the small heat-shock protein family and is normally expressed in all cells during heat stress. We aimed to determine if overexpression of this protein protects behavior and neural function in Drosophila melanogaster during heat stress, as has previously been shown for Hsp70. We used the UAS-GAL4 expression system to drive expression of Hsp26 in the whole animal (ubiquitously), in the motoneurons, and in the muscles of wandering third-instar larvae. There were slight increases in time to crawling failure and normalized excitatory junction potential (EJP) area for some of the transgenic lines, but these were not consistent. In addition, Hsp26 had no effect on the temperature at failure of EJPs, normalized EJP peak amplitude, and normalized EJP half-width. Overexpression larvae had a similar number of motoneuronal boutons and length of nerve terminals as controls, indicating that the occasional protective effects on locomotion were not due to changes at the synapse. We conclude that overexpression had a small thermoprotective effect on locomotion and no effect on neural function. As it has been shown that Hsp26 requires action of other Hsps to reactivate the denatured proteins to which it binds, we propose that at least in larvae, the function of Hsp26 was masked in the relative absence of other Hsps.     

E-pharmacophore-based virtual screening to identify GSK-3β inhibitors

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase which has attracted significant attention during recent years in drug design studies. The deregulation of GSK-3β increased the loss of hippocampal neurons by triggering apoptosis-mediating production of neurofibrillary tangles and alleviates memory deficits in Alzheimer’s disease (AD). Given its role in the formation of neurofibrillary tangles leading to AD, it has been a major therapeutic target for intervention in AD, hence was targeted in the present study. Twenty crystal structures were refined to generate pharmacophore models based on energy involvement in binding co-crystal ligands. Four common e-pharmacophore models were optimized from the 20 pharmacophore models. Shape-based screening of four e-pharmacophore models against nine established small molecule databases using Phase v3.9 had resulted in 1800 compounds having similar pharmacophore features. Rigid receptor docking (RRD) was performed for 1800 compounds and 20 co-crystal ligands with GSK-3β to generate dock complexes. Interactions of the best scoring lead obtained through RRD were further studied with quantum polarized ligand docking (QPLD), induced fit docking (IFD) and molecular mechanics/generalized Born surface area. Comparing the obtained leads to 20 co-crystal ligands resulted in 18 leads among them, lead1 had the lowest docking score, lower binding free energy and better binding orientation toward GSK-3β. The 50?ns MD simulations run confirmed the stable nature of GSK-3β-lead1 docking complex. The results from RRD, QPLD, IFD and MD simulations confirmed that lead1 might be used as a potent antagonist for GSK-3β.     

GSK-3 phosphorylation of the Alzheimer epitope within collapsin response mediator proteins regulates axon elongation in primary neurons

Elevated glycogen synthase kinase-3 (GSK-3) activity is associated with Alzheimer disease. We have found that collapsin response mediator proteins (CRMP) 2 and 4 are physiological substrates of GSK-3. The amino acids targeted by GSK-3 comprise a hyperphosphorylated epitope first identified in plaques isolated from Alzheimer brain. Expression of wild type CRMP2 in primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, a GSK-3-insensitive CRMP2 mutant has dramatically reduced ability to promote axon elongation, a similar effect to pharmacological inhibition of GSK-3. Hence, we propose that phosphorylation of CRMP proteins by GSK-3 regulates axon elongation. This work provides a direct connection between hyperphosphorylation of these residues and elevated GSK-3 activity, both of which are observed in Alzheimer brain.     

MicroRNA-153对靶基因下游信号分子GSK-3β表达水平及细胞抗损伤能力的影响

目的 mir-153可负调控阿尔茨海默病（Alzheimer’s disease,AD）主要致病基因APP及APLP2的蛋白表达,降低其胞内降解片段（intracellular domains,ICDs）的生成。因ICDs具有转录活化及促凋亡活性,本研究旨在探讨mir-153对这两个靶基因下游信号分子GSK-3β表达水平及细胞抗损伤能力的影响,以期进一步阐明mir-153在阿尔茨海默病发病机制中的作用。方法构建mir-153稳转细胞系及mir-153转基因小鼠,Western blot检测该细胞系及小鼠脑内磷酸化GSK-3β、Tau及其总蛋白的表达;Aβ42肽和H2O2分别处理mir-153稳转细胞系,MTS法检测细胞增殖活性的改变,流式细胞术检测细胞凋亡水平的改变。结果 mir-153稳转细胞系中磷酸化GSK-3β及其总蛋白的表达下调,Tau磷酸化水平降低。mir-153转基因小鼠脑内,磷酸化GSK-3β及其总蛋白的表达降低,磷酸化Tau及其总蛋白水平均无明显变化。Aβ42肽和H2O2损伤作用下,mir-153稳转细胞系的增殖活性显著降低,凋亡水平增加。结论 mir-153可负调控靶基因下游信号分子GSK-3β的表达;高表达mir-153可降低细胞抗损伤的能力。     

Design,synthesis and structure–activity relationships of 1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3β

Glycogen synthase kinase-3β (GSK-3β) is implicated in abnormal hyperphosphorylation of tau protein and its inhibitors are expected to be a promising therapeutic agents for the treatment of Alzheimer’s disease. Here we report design, synthesis and structure–activity relationships of a novel series of oxadiazole derivatives as GSK-3β inhibitors. Among these inhibitors, compound 20x showed highly selective and potent GSK-3β inhibitory activity in vitro and its binding mode was determined by obtaining the X-ray co-crystal structure of 20x and GSK-3β.     

GSK-3β在阿尔茨海默症病理特征形成中的作用

糖原合成酶激酶3β(glycogen synthase kinase-3β,GSK-3β)是糖原合成酶激酶3的一种亚型。GSK-3β不仅参与淀粉样蛋白质前体(amyloid precursor protein,APP)代谢,还在tau蛋白过度磷酸化过程中发挥作用,GSK-3β表达及活性的异常会导致神经元细胞的凋亡。APP异常代谢和tau蛋白异常磷酸化是阿尔茨海默病(Alzheimer’s disease,AD)发展的重要因素,因此GSK-3β可能与AD的病理变化密切相关,明确其在AD中的作用及其机制对AD的治疗有重要的意义。     

Drosophila eye color mutants as therapeutic tools for Huntington disease

Huntington disease (HD) is a fatal inherited neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (htt). A pathological hallmark of the disease is the loss of a specific population of striatal neurons, and considerable attention has been paid to the role of the kynurenine pathway (KP) of tryptophan (TRP) degradation in this process. The KP contains three neuroactive metabolites: 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA). 3-HK and QUIN are neurotoxic, and are increased in the brains of early stage HD patients, as well as in yeast and mouse models of HD. Conversely, KYNA is neuroprotective and has been shown to be decreased in HD patient brains. We recently used a Drosophila model of HD to measure the neuroprotective effect of genetic and pharmacological inhibition of kynurenine monoxygenase (KMO)—the enzyme catalyzing the formation of 3-HK at a pivotal branch point in the KP. We found that KMO inhibition in Drosophila robustly attenuated neurodegeneration, and that this neuroprotection was correlated with reduced levels of 3-HK relative to KYNA. Importantly, we showed that KP metabolites are causative in this process, as 3-HK and KYNA feeding experiments modulated neurodegeneration. We also found that genetic inhibition of the upstream KP enzyme tryptophan-2,3-dioxygenase (TDO) was neuroprotective in flies. Here, we extend these results by reporting that genetic impairment of KMO or TDO is protective against the eclosion defect in HD model fruit flies. Our results provide further support for the possibility of therapeutic KP interventions in HD.     

Ginsenoside Rd Attenuates Tau Protein Phosphorylation Via the PI3K/AKT/GSK-3β Pathway After Transient Forebrain Ischemia

Phosphorylated tau was found to be regulated after cerebral ischemia and linked to high risk for the development of post-stroke dementia. Our previous study showed that ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, decreased tau phosphorylation in Alzheimer model. As an extending study, here we investigated whether Rd could reduce tau phosphorylation and sequential cognition impairment after ischemic stroke. Sprague–Dawley rats were subjected to focal cerebral ischemia. The tau phosphorylation of rat brains were analyzed following ischemia by Western blot and animal cognitive functions were examined by Morris water maze and Novel object recognition task. Ischemic insults increased the levels of phosphorylated tau protein at Ser199/202 and PHF-1 sites and caused animal memory deficits. Rd treatment attenuated ischemia-induced enhancement of tau phosphorylation and ameliorated behavior impairment. Furthermore, we revealed that Rd inhibited the activity of Glycogen synthase kinase-3β (GSK-3β), the most important kinase involving tau phosphorylation, but enhanced the activity of protein kinase B (PKB/AKT), a key kinase suppressing GSK-3β activity. Moreover, we found that LY294002, an antagonist for phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, abolished the inhibitory effect of Rd on GSK-3β activity and tau phosphorylation. Taken together, our findings provide the first evidence that Rd may reduce cerebral ischemia-induced tau phosphorylation via the PI3K/AKT/GSK-3β pathway.     

Structure-based design of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3β inhibitors to activate Wnt/β-catenin pathway

Deregulation of Wnt/β-catenin pathway is closely related to the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD), and glycogen synthase kinase 3β (GSK-3β), the central negative regulator of Wnt pathway, is regarded as an important target for these diseases. Here, we report a series of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3β inhibitors by rational-design and synthesis, which show high selectivity against GSK-3β over Cyclin-dependent kinase 2 (CDK2), and significantly activate the cellular Wnt/β-catenin pathway. The structure–activity relationship of these GSK-3β inhibitors was also explored by in silico molecular docking.     

Docking,molecular dynamics,binding energy-MM-PBSA studies of naphthofuran derivatives to identify potential dual inhibitors against BACE-1 and GSK-3β

BACE-1 and GSK-3β both are potential therapeutic drug targets for Alzheimer’s disease. Recently, both these targets received attention for designing dual inhibitors. Till now only two scaffolds (triazinone and curcumin) derivatives have been reported as BACE-1 and GSK-3β dual inhibitors. In our previous work, we have reported first in class dual inhibitor for BACE-1 and GSK-3β. In this study, we have explored other naphthofuran derivatives for their potential to inhibit BACE-1 and GSK-3β through docking, molecular dynamics, binding energy (MM-PBSA). These computational methods were performed to estimate the binding affinity of naphthofuran derivatives towards the BACE-1 and GSK-3β. In the docking results, two derivatives (NS7 and NS9) showed better binding affinity as compared to previously reported inhibitors. Hydrogen bond occupancy of NS7 and NS9 generated from MD trajectories showed good interaction with the flap residues Gln73, Thr72 of BACE-1 and Arg141, Thr138 residues of GSK-3β. MM-PBSA and energy decomposition per residue revealed different components of binding energy and relative importance of amino acid involved in binding. The results showed that the binding of inhibitors was majorly governed by the hydrophobic interactions and suggesting that hydrophobic interactions might be the key to design dual inhibitors for BACE1-1 and GSK-3β. Distance between important pair of amino acid residues indicated that BACE-1 and GSK-3β adopt closed conformation and become inactive after ligand binding. The results suggested that naphthofuran derivatives might act as dual inhibitor against BACE-1 and GSK-3β.     

Docosahexaenoic acid: one molecule diverse functions

Docosahexaenoic acid (DHA, C22:6, ω-3) is a highly polyunsaturated omega-3 fatty acid. It is concentrated in neuronal brain membranes, for which reason it is also referred to as a “brain food”. DHA is essential for brain development and function. It plays an important role in improving antioxidant and cognitive activities of the brain. DHA deficiency occurs during aging and dementia, impairs memory and learning, and promotes age-related neurodegenerative diseases, including Alzheimer’s disease (AD). For about two decades, we have reported that oral administration of DHA increases spatial memory acquisition, stimulates neurogenesis, and protects against and reverses memory impairment in amyloid β peptide-infused AD rat models by decreasing amyloidogenesis and protects against age-related cognitive decline in the elderly. These results demonstrate a robust link between DHA and cognitive health. Rodents that were fed a diet low in ω-3 polyunsaturated fatty acids, particularly those that were DHA-deficient, frequently suffered from anxiety, depression and memory impairment. Although the exact mechanisms of action of DHA in brain functions are still elusive, a host of mechanisms have been proposed. For example, DHA, which inherently has a characteristic three-dimensional structure, increases membrane fluidity, strengthens antioxidant activity and enhances the expression of several proteins that act as substrates for improving memory functions. It reduces the brain amyloid burden and inhibits in vitro fibrillation and amyloid-induced neurotoxicity in cell-culture model. In this review, we discuss how DHA acts as a molecule with diverse functions.     

β淀粉样蛋白对线粒体作用的研究进展

阿尔茨海默病的一个重要病理特征是胞外β淀粉样蛋白沉积形成的老年斑,β淀粉样蛋白可以引起氧化损伤以及神经细胞凋亡等。随着研究的深入,在细胞内也发现了β淀粉样蛋白的存在。线粒体是细胞内ATP和活性氧自由基产生的主要部位,在氧化损伤和细胞凋亡过程中起到重要的作用。近年的研究表明,β淀粉样蛋白对线粒体有很重要的作用。该文主要针对这一领域的进展,介绍了阿尔茨海默病中β淀粉样蛋白对线粒体多个生理过程的作用以及这些作用在阿尔茨海默病中产生的影响。     

Shaggy/GSK-3beta kinase localizes to the centrosome and to specialized cytoskeletal structures in Drosophila

The assembly of a functional bipolar mitotic spindle requires an exquisite regulation of microtubule behavior in time and space. To characterize new elements of this machinery we carried out a GFP based "protein trap" screen and selected fusion proteins which localized to the spindle apparatus. By this method we identified Shaggy, the Drosophila homologue of glycogen synthase kinase-3beta (GSK-3beta), as a component of centrosomes. GSK-3beta acting in the Wingless signaling pathway is involved in a vast range of developmental processes, from pattern formation to cell-fate specification, and is a key factor for cell proliferation in most animals. We exploited our Shaggy::GFP Drosophila line to analyze the subcellular localizations of GSK-3beta/Shaggy and shed light on its multiple roles during embryogenesis. We found that Shaggy becomes enriched transiently in a variety of specialized cytoskeletal structures of the embryo, including centrosomes throughout mitosis, suggesting that this kinase is involved in the regulation of many aspects of the cytoskeleton function.     

Potentiation of GSK-3-catalyzed Alzheimer-like phosphorylation of human tau by cdk5

Tau protein from Alzheimer disease (AD) brain is hyperphosphorylated by both proline-dependent protein kinases (PDPKs) and non-PDPKs. It is presently unclear how PDPKs and non-PDPKs interact in tau hyperphosphorylation. Previously we have shown that non-PDPKs can positively modulate the activity of a PDPK (GSK-3) in tau phosphorylation (Singh et al. (1995) FEBS Lett. 358, 267-272). In this study we have investigated whether (A) non-PDPKs can also modulate the activity of the PDPK, cdk5, (B) a PDPK can modulate the activities of another PDPK, as well as non-PDPKs. We found that, like GSK-3, the activity of cdk5 is stimulated if tau were first prephosphorylated by any of several non-PDPKs (A-kinase, C-kinase, CK-1, CaM-kinase II). Prephosphorylation of tau by cdk5 stimulated both the rate and extent of a subsequent phosphorylation catalyzed by GSK-3. Under these conditions thr 231 phosphorylation was especially enhanced (9-fold). No significant stimulation of phosphorylation was obser ved when the order of these kinases was reversed (i.e. GSK-3 followed by cdk5). By contrast, prephosphorylation of tau by cdk5 served to inhibit subsequent phosphorylation catalyzed by C-kinase and CK-1, but not by A-kinase or CaM-kinase II. Our results suggest that in tau hyperphosphorylation in AD brain, cdk5-catalyzed phosphorylation may serve to up-regulate the activity of GSK-3 and down-regulate the activities of C-kinase and CK-1. (Mol Cell Biochem 167: 99-105, 1997)     

Acetyl-L-carnitine ameliorates spatial memory deficits induced by inhibition of phosphoinositol-3 kinase and protein kinase C

Glycogen synthase kinase-3β (GSK-3β) plays a crucial role in memory deficits and tau hyperphosphorylation as seen in Alzheimer's disease, the most common dementia in the aged population. We reported that ventricular co-injection of wortmannin and GF-109203X (WT/GFX) can induce tau hyperphosophorylation and memory impairment of rats through activation of GSK-3 [Liu S. J., Zhang A. H., Li H. L., Wang Q., Deng H. M., Netzer W. J., Xu H. X. and Wang J. Z. (2003) J. Neurochem. 87, 1333]. In the present study, we found that feeding the rats with Acetyl-L-Carnitine (ALCAR, 50 mg/day·rat, per os) for 2 weeks rescued the WT/GFX-induced spatial memory retention impairment of the rats by antagonizing GSK-3β activation independent of Akt, PKCζ and Erk1/2. We also found that ALCAR arrested microtubule-associated protein tau hyperphosphorylation at multiple Alzheimer's disease sites in vivo and in vitro. Moreover, ALCAR enhanced the expression of several memory-associated proteins including c-Fos, synapsin I in rat hippocampus. These results suggest that ALCAR could ameliorate WT/GFX-induced spatial memory deficits through inhibition tau hyperphosphorylation and modulation of memory-associated proteins.