GSK 264220A

GSK-3beta Overexpression Alters the Dendritic Spines of Developmentally Generated Granule Neurons in the Mouse Hippocampal Dentate Gyrus.[Pubmed:28344548]

Front Neuroanat. 2017 Mar 10;11:18.

The dentate gyrus (DG) plays a crucial role in hippocampal-related memory. The most abundant cellular type in the DG, namely granule neurons, are developmentally generated around postnatal day P6 in mice. Moreover, a unique feature of the DG is the occurrence of adult hippocampal neurogenesis, a process that gives rise to newborn granule neurons throughout life. Adult-born and developmentally generated granule neurons share some maturational aspects but differ in others, such as in their positioning within the granule cell layer. Adult hippocampal neurogenesis encompasses a series of plastic changes that modify the function of the hippocampal trisynaptic network. In this regard, it is known that glycogen synthase kinase 3beta (GSK-3beta) regulates both synaptic plasticity and memory. By using a transgenic mouse overexpressing GSK-3beta in hippocampal neurons, we previously demonstrated that the overexpression of this kinase has deleterious effects on the maturation of newborn granule neurons. In the present study, we addressed the effects of GSK-3beta overexpression on the morphology and number of dendritic spines of developmentally generated granule neurons. To this end, we performed intracellular injections of Lucifer Yellow in developmentally generated granule neurons of wild-type and GSK-3beta-overexpressing mice and analyzed the number and morphologies of dendritic spines (namely, stubby, thin and mushroom). GSK-3beta overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased.

Transient Cerebral Ischemia Alters GSK-3beta and p-GSK-3beta Immunoreactivity in Pyramidal Neurons and Induces p-GSK-3beta Expression in Astrocytes in the Gerbil Hippocampal CA1 Area.[Pubmed:28349361]

Neurochem Res. 2017 Aug;42(8):2305-2313.

Glycogen synthase kinase 3beta (GSK-3beta) is a key downstream protein in the PI3K/Akt pathway. Phosphorylation of serine 9 of GSK-3beta (GSK-3beta activity inhibition) promotes cell survival. In this study, we examined changes in expressions of GSK-3beta and phosphorylation of GSK-3beta (p-GSK-3beta) in the gerbil hippocampal CA1 area after 5 min of transient cerebral ischemia. GSK-3beta immunoreactivity in the CA1 area was increased in pyramidal cells at 6 h after ischemia-reperfusion. It was decreased in CA1 pyramidal cells from 12 h after ischemia-reperfusion, and hardly detected in the CA1 pyramidal cells at 5 days after ischemia-reperfusion. p-GSK-3beta immunoreactivity was slightly decreased in CA1 pyramidal cells at 6 and 12 h after ischemia-reperfusion. It was significantly increased in these cells at 1 and 2 days after ischemia-reperfusion. Five days after ischemia-reperfusion, p-GSK-3beta immunoreactivity was hardly found in CA1 pyramidal cells. However, p-GSK-3beta immunoreactivity was strongly expressed in astrocytes primarily distributed in strata oriens and radiatum. In conclusion, GSK-3beta and p-GSK-3beta were significantly changed in pyramidal cells and/or astrocytes in the gerbil hippocampal CA1 area following 5 min of transient cerebral ischemia. This finding indicates that GSK-3beta and p-GSK-3beta are closely related to delayed neuronal death.

Activation of Ras-ERK Signaling and GSK-3 by Amyloid Precursor Protein and Amyloid Beta Facilitates Neurodegeneration in Alzheimer's Disease.[Pubmed:28374012]

eNeuro. 2017 Mar 27;4(2). pii: eN-NWR-0149-16.

It is widely accepted that amyloid beta (Abeta) generated from amyloid precursor protein (APP) oligomerizes and fibrillizes to form neuritic plaques in Alzheimer's disease (AD), yet little is known about the contribution of APP to intracellular signaling events preceding AD pathogenesis. The data presented here demonstrate that APP expression and neuronal exposure to oligomeric Abeta42 enhance Ras/ERK signaling cascade and glycogen synthase kinase 3 (GSK-3) activation. We find that RNA interference (RNAi)-directed knockdown of APP in B103 rat neuroblastoma cells expressing APP inhibits Ras-ERK signaling and GSK-3 activation, indicating that APP acts upstream of these signal transduction events. Both ERK and GSK-3 are known to induce hyperphosphorylation of tau and APP at Thr668, and our findings suggest that aberrant signaling by APP facilitates these events. Supporting this notion, analysis of human AD brain samples showed increased expression of Ras, activation of GSK-3, and phosphorylation of APP and tau, which correlated with Abeta levels in the AD brains. Furthermore, treatment of primary rat neurons with Abeta recapitulated these events and showed enhanced Ras-ERK signaling, GSK-3 activation, upregulation of cyclin D1, and phosphorylation of APP and tau. The finding that Abeta induces Thr668 phosphorylation on APP, which enhances APP proteolysis and Abeta generation, denotes a vicious feedforward mechanism by which APP and Abeta promote tau hyperphosphorylation and neurodegeneration in AD. Based on these results, we hypothesize that aberrant proliferative signaling by APP plays a fundamental role in AD neurodegeneration and that inhibition of this would impede cell cycle deregulation and neurodegeneration observed in AD.

SLM, a novel carbazole-based fluorophore attenuates okadaic acid-induced tau hyperphosphorylation via down-regulating GSK-3beta activity in SH-SY5Y cells.[Pubmed:28359686]

Eur J Pharm Sci. 2017 Dec 15;110:101-108.

Phosphorylated tau dissociates from microtubules and aggregates to form neurofibrillary tangles resulting in neuronal toxicity and cognitive deficits. Attenuating tau hyperphosphorylation is considered as an effective therapeutic approach for Alzheimer's disease (AD). From our previous study, SLM, a carbazole-based fluorophore prevents Abeta aggregation, reduced glycogen synthase kinase-3beta (GSK-3beta) activity and tau hyperphosphorylation in triple transgenic mouse model of AD. However, the mechanism by which SLM attenuates tau hyperphosphorylation warrants further investigation. In the current study, we intend to evaluate the effects of SLM against okadaic acid (OA)-induced tau hyperphosphorylation and microtubules instability in human neuroblastoma (SH-SY5Y) cells. The results showed that, SLM reduced the OA-induced cell neurotoxicity and tau hyperphosphorylation in SH-SY5Y cells. SLM treatment down-regulated GSK-3beta activity. However, in the presence of GSK-3beta inhibitor (SB216763, 10muM), SLM treatment could not reduce GSK-3beta activity and tau hyperphosphorylation as compared with SB216763 treatment alone. Furthermore, SLM treatment also ameliorated OA-induced microtubules instability and cytoskeleton damage. Collectively, SLM attenuated OA-induced tau hyperphosphorylation via down-regulating GSK-3beta activity in SH-SY5Y cells. Therefore, this study supports SLM as a potential compound for AD and other tau pathology-related neurodegenerative disorders.

A high-throughput screen for endothelial lipase using HDL as substrate.[Pubmed:18566479]

J Biomol Screen. 2008 Jul;13(6):468-75.

Endothelial lipase (EL) is a 482-amino-acid protein from the triglyceride lipase gene family that uses a Ser-His-Asp triad for catalysis. Its expression in endothelial cells and preference for phospholipids rather than triglycerides are unique. Animal models in which it is overexpressed or knocked out indicate EL levels are inversely correlated with high-density lipoprotein cholesterol (HDL-C). HDL-C is commonly referred to as the good form of cholesterol because it is involved in the reverse cholesterol transport pathway, in which excess cholesterol is effluxed from peripheral tissues for excretion or reabsorption. Thus, EL inhibition in humans is expected to lead to increases in HDL levels and possibly a decrease in cardiovascular disease. To discover inhibitors of EL, a coupled assay for EL has been developed, using its native substrate, HDL. Hydrolysis of HDL by EL yields free fatty acids, which are coupled through acyl-CoA synthetase, acyl-CoA oxidase, and horseradish peroxidase to produce the fluorescent species resorufin. This assay was developed into a 5-microL, 1536-well assay format, and a high-throughput screen was executed against the GSK collection. In addition to describing the screening results, novel post-HTS mechanism-of-action studies were developed for EL and applied to 1 of the screening hits as an example.