Ainsley A. Culbert

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription

BACKGROUND Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro, with K(i)s of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase. CONCLUSIONS SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease.

Selective small‐molecule inhibitors of glycogen synthase kinase‐3 activity protect primary neurones from death

The phosphatidylinositol 3‐kinase (PI 3‐kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase‐3 (GSK‐3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK‐3 in response to insulin and growth factors, which inhibits GSK‐3 activity and leads to the modulation of multiple GSK‐3 regulated cellular processes. We show that the novel potent and selective small‐molecule inhibitors of GSK‐3; SB‐415286 and SB‐216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3‐kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK‐3 activity and modulation of GSK‐3 substrates tau and β‐catenin. Thus, in addition to the previously assigned roles of GSK‐3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK‐3 activity in primary neurones is sufficient to prevent death, implicating GSK‐3 as a physiologically relevant principal regulatory target of the PI 3‐kinase/PKB neuronal survival pathway.

MAPK-activated Protein Kinase 2 Deficiency in Microglia Inhibits Pro-inflammatory Mediator Release and Resultant Neurotoxicity RELEVANCE TO NEUROINFLAMMATION IN A TRANSGENIC MOUSE MODEL OF ALZHEIMER DISEASE

MAPK-activated protein kinase 2 (MAPKAP K2 or MK2) is one of several kinases directly regulated by p38 MAPK. A role for p38 MAPK in the pathology of Alzheimer disease (AD) has previously been suggested. Here, we provide evidence to suggest that MK2 also plays a role in neuroinflammatory and neurodegenerative pathology of relevance to AD. MK2 activation and expression were increased in lipopolysaccharide (LPS) + interferon γ-stimulated microglial cells, implicating a role for MK2 in eliciting a pro-inflammatory response. Microglia cultured ex vivo from MK2-deficient (MK2–/–) mice demonstrated significant inhibition in release of tumor necrosis factor α, KC (mouse chemokine with highest sequence identity to human GROs and interleukin-8), and macrophage inflammatory protein 1α on stimulation with LPS + interferon γ or amyloid-β peptide (1–42) compared with MK2+/+ wild-type microglia. Consistent with an inhibition in pro-inflammatory mediator release, cortical neurons co-cultured with LPS + interferon γ-stimulated or amyloid-β peptide (1–42)-stimulated MK2–/– microglia were protected from microglial-mediated neuronal cell toxicity. In a transgenic mouse model of AD in which amyloid precursor protein and presenilin-1 harboring familial AD mutations are overexpressed in specific regions of the brain, elevated activation and expression of MK2 correlated with β-amyloid deposition, microglial activation, and up-regulation of tumor necrosis factor α, macrophage inflammatory protein 1α, and KC gene expression in the same brain regions. Our data propose a role for MK2 in AD brain pathology, for which neuroinflammation involving cytokines and chemokines and overt neuronal loss have been documented.

Expression and Characterization of GSK-3 Mutants and Their Effect on β-Catenin Phosphorylation in Intact Cells

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates β-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of β-catenin by GSK-3 does not require primingin vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3β. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321–1327). Here we have investigated whether phosphorylation of Ser33, Ser37, and Thr41 in β-catenin requires priming through prior phosphorylation at Ser45 in intact cells. We have shown that the Arg96 mutant does not induce β-catenin degradation but instead stabilizes β-catenin, indicating that it is unable to phosphorylate β-catenin in intact cells. Furthermore, if Ser45 in β-catenin is mutated to Ala, β-catenin is markedly stabilized, and phosphorylation of Ser33, Ser37, and Thr41 in β-catenin by wild type GSK-3β is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate β-catenin in intact cells although it has reduced activity. Mutation of Tyr216 to Phe markedly reduces the ability of GSK-3β to phosphorylate and down-regulate β-catenin. In conclusion, we have found that the Arg96 mutant has a dominant-negative effect on GSK-3β-dependent phosphorylation of β-catenin and that targeting of β-catenin for degradation requires prior priming through phosphorylation of Ser45.

GSK-3 inhibition by adenoviral FRAT1 overexpression is neuroprotective and induces Tau dephosphorylation and β-catenin stabilisation without elevation of glycogen synthase activity

Glycogen synthase kinase 3 (GSK‐3) has previously been shown to play an important role in the regulation of apoptosis. However, the nature of GSK‐3 effector pathways that are relevant to neuroprotection remains poorly defined. Here, we have compared neuroprotection resulting from modulation of GSK‐3 activity in PC12 cells using either selective small molecule ATP‐competitive GSK‐3 inhibitors (SB‐216763 and SB‐415286), or adenovirus overexpressing requently earranged in dvanced ‐cell lymphomas 1 (FRAT1), a protein proposed as a negative regulator of GSK‐3 activity towards Axin and β‐catenin. Our data demonstrate that cellular overexpression of FRAT1 is sufficient to confer neuroprotection and correlates with inhibition of GSK‐3 activity towards Tau and β‐catenin, but not modulation of glycogen synthase (GS) activity. By comparison, treatment with SB‐216763 and SB‐415286 proved more potent in terms of neuroprotection, and correlated with inhibition of GSK‐3 activity towards GS in addition to Tau and β‐catenin.

P2X7 receptors on microglial cells mediate injury to cortical neurons in vitro

The P2X7 receptor has been implicated in the release of cytokines and in the induction of cell death, and is up‐regulated in a transgenic mouse model of Alzheimers disease. Using cocultures of rat cortical neurons and microglia, we show that ATP and the more potent P2X7 agonist benzoylbenzoyl‐ATP (BzATP) cause neuronal cell injury. The deleterious effects of BzATP‐treated microglia were prevented by nonselective P2X antagonists (PPADS and oxidized ATP) and by the more selective P2X7 antagonist Brilliant Blue G. Similar concentrations of BzATP caused release of superoxide and nitric oxide from isolated microglia, and neuronal cell injury was attenuated by a superoxide dismutase mimetic and by a peroxynitrite decomposition catalyst, suggesting a role for reactive oxide species. Cocultures composed of wild‐type cortical neurons, and microglia from P2X7 receptor‐deficient mice failed to exhibit neuronal cell injury in the presence of BzATP, but retained sensitivity to injury when microglia were derived from genotypically matched normal (P2X7+/+ mice), thereby establishing P2X7 involvement in the injury process. P2X7 receptor activation on microglia thus appears necessary for microglial‐mediated injury of neurons, and proposes that targeting P2X7 receptors may constitute a novel approach for the treatment of acute and chronic neurodegenerative disorders where a microglial component is evident.

Repeated administration of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) modulates neuroinflammation and amyloid plaque load in mice bearing amyloid precursor protein and presenilin-1 mutant transgenes

BackgroundData indicates anti-oxidant, anti-inflammatory and pro-cognitive properties of noradrenaline and analyses of post-mortem brain of Alzheimers disease (AD) patients reveal major neuronal loss in the noradrenergic locus coeruleus (LC), the main source of CNS noradrenaline (NA). The LC has projections to brain regions vulnerable to amyloid deposition and lack of LC derived NA could play a role in the progression of neuroinflammation in AD. Previous studies reveal that intraperitoneal (IP) injection of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) can modulate neuroinflammation in amyloid over-expressing mice and in one study, DSP-4 exacerbated existing neurodegeneration.MethodsTASTPM mice over-express human APP and beta amyloid protein and show age related cognitive decline and neuroinflammation. In the present studies, 5 month old C57/BL6 and TASTPM mice were injected once monthly for 6 months with a low dose of DSP-4 (5 mg kg-1) or vehicle. At 8 and 11 months of age, mice were tested for cognitive ability and brains were examined for amyloid load and neuroinflammation.ResultsAt 8 months of age there was no difference in LC tyrosine hydroxylase (TH) across all groups and cortical NA levels of TASTPM/DSP-4, WT/Vehicle and WT/DSP-4 were similar. NA levels were lowest in TASTPM/Vehicle. Messenger ribonucleic acid (mRNA) for various inflammatory markers were significantly increased in TASTPM/Vehicle compared with WT/Vehicle and by 8 months of age DSP-4 treatment modified this by reducing the levels of some of these markers in TASTPM. TASTPM/Vehicle showed increased astrocytosis and a significantly larger area of cortical amyloid plaque compared with TASTPM/DSP-4. However, by 11 months, NA levels were lowest in TASTPM/DSP-4 and there was a significant reduction in LC TH of TASTPM/DSP-4 only. Both TASTPM groups had comparable levels of amyloid, microglial activation and astrocytosis and mRNA for inflammatory markers was similar except for interleukin-1 beta which was increased by DSP-4. TASTPM mice were cognitively impaired at 8 and 11 months but DSP-4 did not modify this.ConclusionThese data reveal that a low dose of DSP-4 can have varied effects on the modulation of amyloid plaque deposition and neuroinflammation in TASTPM mice dependent on the duration of dosing.

FRAT1, a Substrate-specific Regulator of Glycogen Synthase Kinase-3 Activity, Is a Cellular Substrate of Protein Kinase A

FRAT1, like its Xenopus homolog glycogen synthase kinase-3 (GSK-3)-binding protein, is known to inhibit GSK-3-mediated phosphorylation of β-catenin. It is currently unknown how FRAT-GSK-3-binding protein activity toward GSK-3 is regulated. FRAT1 has recently been shown to be a phosphoprotein in vivo; however, the responsible kinase(s) have not been determined. In this study, we identified Ser188 as a phosphorylated residue in FRAT1. The identity of the kinase that catalyzes Ser188 phosphorylation and the significance of this phosphorylation to FRAT1 function were investigated. Protein kinase A (PKA) was found to phosphorylate Ser188 in vitro as well as in intact cells. Importantly, activation of endogenous cAMP-coupled β-adrenergic receptors with norepinephrine stimulated the phosphorylation of FRAT1 at Ser188. GSK-3 was also able to phosphorylate FRAT1 at Ser188 and other residues in vitro or when overexpressed in intact cells. In contrast, endogenous GSK-3 did not lead to significant FRAT1 phosphorylation in cells, suggesting that GSK-3 is not a major FRAT1 kinase in vivo. Phosphorylation of Ser188 by PKA inhibited the ability of FRAT1 to activate β-catenin-dependent transcription. In conclusion, PKA phosphorylates FRAT1 in vitro as well as in intact cells and may play a role in regulating the inhibitory activity of FRAT1 toward GSK-3.

P1-027: Assessment of the consequences of noradrenaline manipulation in the APP/PS1 mutant TASTPM mouse

before and directly after bolus injection of ultra small paramagnetic particles of iron oxide (USPIO). With deuterium magnetic resonance spectroscopy (MRS) techniques, rCBV of the whole brain was determined. Further, A , sterol and fatty acid levels were determined in brain tissue. Results: Results show that a diet enriched with 1% cholesterol increases A plaque burden in the dentate gyrus of the hippocampus of 19 months old APP/PS1 mice, and in addition also tends to decrease rCBV of these TWD(t) mice. No changes in fatty acid profiles or cholesterol could be observed in brain tissue, whereas a DHA diet significantly increased cortex rCBV and the n3 fatty acid concentration, indicating increased membrane fluidity. Conclusions: Together these results suggest that changes in hemodynamics, such as rCBV, show association with amyloid deposition, indicating a more solid ground for AD as a vascular disorder. To elucidate these findings, experiments are repeated earlier in the course of the disease (8 and 15 months) to strengthen the present results and hypothesis. Except rCBV and Alzheimer’s pathology, cognitive/behavioural studies will be exerted as well.