Neuronal apoptosis and reversible motor deficit in dominant-negative GSK-3 conditional transgenic mice

Increased glycogen synthase kinase-3 (GSK-3) activity is believed to contribute to the etiology of chronic disorders like Alzheimer's disease and diabetes, thus supporting therapeutic potential of GSK-3 inhibitors. However, sustained GSK-3 inhibition might induce tumorigenesis through beta-catenin-APC dysregulation. Besides, sustained in vivo inhibition by genetic means (constitutive knock-out mice) revealed unexpected embryonic lethality due to massive hepatocyte apoptosis. Here, we have generated transgenic mice with conditional (tetracycline system) expression of dominant-negative-GSK-3 as an alternative genetic approach to predict the outcome of chronic GSK-3 inhibition, either per se, or in combination with mouse models of disease. By choosing a postnatal neuron-specific promoter, here we specifically address the neurological consequences. Tet/DN-GSK-3 mice showed increased neuronal apoptosis and impaired motor coordination. Interestingly, DN-GSK-3 expression shut-down restored normal GSK-3 activity and re-established normal incidence of apoptosis and motor coordination. These results reveal the importance of intact GSK-3 activity for adult neuron viability and physiology and warn of potential neurological toxicity of GSK-3 pharmacological inhibition beyond physiological levels. Interestingly, the reversibility data also suggest that unwanted side effects are likely to revert if excessive GSK-3 inhibition is halted.     

Novel indolylmaleimide acts as GSK-3β inhibitor in human neural progenitor cells

The Wnt pathway is involved in cellular processes linked to either proliferation or differentiation. Therefore small molecules offer an attractive opportunity to modulate this pathway, whereas the key enzyme GSK-3β is of special interest. In this study, non-symmetrically substituted indolylmaleimides have been synthesized and their ability to function as GSK-3β inhibitors has been investigated in a human neural progenitor cell line. Among the newly synthesized compounds, the substance IM-12 showed a significant activity in several biological tests which was comparable or even outplayed the effects of the known GSK-3β inhibitor SB-216763. Furthermore the treatment of human neural progenitor cells with IM-12 resulted in an increase of neuronal cells. Therefore we conclude that indolylmaleimides act via the canonical Wnt signalling pathway by inhibition of the key enzyme GSK-3β.     

Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3beta and its upstream regulators

Axon-dendrite polarity is a cardinal feature of neuronal morphology essential for information flow. Here we report a differential distribution of GSK-3beta activity in the axon versus the dendrites. A constitutively active GSK-3beta mutant inhibited axon formation, whereas multiple axons formed from a single neuron when GSK-3beta activity was reduced by pharmacological inhibitors, a peptide inhibitor, or siRNAs. An active mechanism for maintaining neuronal polarity was revealed by the conversion of preexisting dendrites into axons upon GSK-3 inhibition. Biochemical and functional data show that the Akt kinase and the PTEN phosphatase are upstream of GSK-3beta in determining neuronal polarity. Our results demonstrate that there are active mechanisms for maintaining as well as establishing neuronal polarity, indicate that GSK-3beta relays signaling from Akt and PTEN to play critical roles in neuronal polarity, and suggest that application of GSK-3beta inhibitors can be a novel approach to promote generation of new axons after neural injuries.     

The new iminothiadiazole derivative VP1.14 ameliorates hippocampal damage after an excitotoxic injury

J. Neurochem. (2012) 122, 1193-1202. ABSTRACT: Increased levels of glutamate causing excitotoxic damage accompany many neurological disorders. A well-characterized model of excitotoxic damage involves administration of kainic acid (KA), which causes limbic seizure activity and subsequent neuronal death, particularly in the CA1 and CA3 areas of the hippocampus. Inhibition of the enzyme glycogen synthase kinase-3 (GSK-3) and cAMP levels might play an important role in neuroprotection. As intracellular cAMP levels depend, in part, on the activity of the phosphodiesterase enzymes (PDEs), these enzymes have recently emerged as potential therapeutic targets for the treatment of several diseases. In previous works, we have shown a potent anti-inflammatory and neuroprotective effect of GSK-3 inhibition in a model of excitotoxicity, as well as a reduction of nigrostriatal dopaminergic neuronal cell death after phosphodiesterase 7 inhibition, which leads to an increase in cAMP levels. This study was undertaken to determine whether simultaneous inhibition of GSK-3 and PDE-7 by a novel 5-imino-1,2,4-thiadiazole compound, named VP1.14, could prevent the massive neuronal loss in the hippocampus evoked by intrahippocampal injection of KA. Here, we show that rats treated with VP1.14 showed a reduced inflammatory response after KA injection, and exhibited a significant reduction in pyramidal cell loss in the CA1 and CA3 areas of the hippocampus. Studies with hippocampal HT22 cells in vitro also showed a clear neuroprotective effect of VP1.14 and an anti-inflammatory effect shown by a decrease in the nitrite liberation and in the expression of pro-inflammatory cytokines by primary cultures of astrocytes treated with lipopolysaccharide.     

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.     

Once and again: retinoic acid signaling in the developing and regenerating olfactory pathway

Retinoic acid (RA), a member of the steroid/thyroid superfamily of signaling molecules, is an essential regulator of morphogenesis, differentiation, and regeneration in the mammalian olfactory pathway. RA-mediated teratogenesis dramatically alters olfactory pathway development, presumably by disrupting retinoid-mediated inductive signaling that influences initial olfactory epithelium (OE) and bulb (OB) morphogenesis. Subsequently, RA modulates the genesis, growth, or stability of subsets of OE cells and OB interneurons. RA receptors, cofactors, and synthetic enzymes are expressed in the OE, OB, and anterior subventricular zone (SVZ), the site of neural precursors that generate new OB interneurons throughout adulthood. Their expression apparently accommodates RA signaling in OE cells, OB interneurons, and slowly dividing SVZ neural precursors. Deficiency of vitamin A, the dietary metabolic RA precursor, leads to cytological changes in the OE, as well as olfactory sensory deficits. Vitamin A therapy in animals with olfactory system damage can accelerate functional recovery. RA-related pathology as well as its potential therapeutic activity may reflect endogenous retinoid regulation of neuronal differentiation, stability, or regeneration in the olfactory pathway from embryogenesis through adulthood. These influences may be in register with retinoid effects on immune responses, metabolism, and modulation of food intake.     

Regulation and function of glycogen synthase kinase-3 isoforms in neuronal survival

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase consisting of two isoforms, alpha and beta. The activities of GSK-3 are regulated negatively by serine phosphorylation but positively by tyrosine phosphorylation. GSK-3 inactivation has been proposed as a mechanism to promote neuronal survival. We used GSK-3 isoform-specific small interfering RNAs, dominant-negative mutants, or pharmacological inhibitors to search for functions of the two GSK-3 isoforms in regulating neuronal survival in cultured cortical neurons in response to glutamate insult or during neuronal maturation/aging. Surprisingly, RNA interference-induced depletion of either isoform was sufficient to block glutamate-induced excitotoxicity, and the resulting neuroprotection was associated with enhanced N-terminal serine phosphorylation in both GSK-3 isoforms. However, GSK-3beta depletion was more effective than GSK-3alpha depletion in suppressing spontaneous neuronal death in extended culture. This phenomenon is likely due to selective and robust inhibition of GSK-3beta activation resulting from GSK-3beta Ser9 dephosphorylation during the course of spontaneous neuronal death. GSK-3alpha silencing resulted in reduced tyrosine phosphorylation of GSK-3beta, suggesting that tyrosine phosphorylation is also a critical autoregulatory event. Interestingly, GSK-3 inhibitors caused a rapid and long-lasting increase in GSK-3alpha Ser21 phosphorylation levels, followed by a delayed increase in GSK-3beta Ser9 phosphorylation and a decrease in GSK-3alpha Tyr279 and GSK-3beta Tyr216 phosphorylation, thus implying additional levels of GSK-3 autoregulation. Taken together, our results underscore important similarities and dissimilarities of GSK-3alpha and GSK-3beta in the roles of cell survival as well as their distinct modes of regulation. The development of GSK-3 isoform-specific inhibitors seems to be warranted for treating GSK-3-mediated pathology.     

Neural Precursor Cycling at Sonic Speed: N-Myc Pedals,GSK-3 Brakes

Signaling by the sonic hedgehog (Shh) pathway is essential for neural precursor population expansion during normal central nervous system (CNS) development, and is implicated in the childhood brain tumor, medulloblastoma. The proto-oncogene N-myc plays essential roles as a downstream effector of Shh proliferative effects in neural precursors of the cerebellum, where medulloblastomas arise. It is likely that N-Myc has analogous functions in medulloblastomas and other CNS tumors where it is highly expressed due to altered regulation or gene amplification. Myc destabilization occurs in response to phosphorylation by GSK-3β. N-Myc degradation is required for cerebellar neural precursors to exit the cell cycle. During mitosis in cerebellar neural precursors, levels of N-Myc primed for phosphorylation by GSK-3β increase, due to cdk1 complex activity towards N-Myc. GSK-3β is kept in check by insulin-like growth factor signaling, which also plays critical roles in brain development and cancer. These findings indicate that therapeutic strategies targeting N-myc and the IGF pathway might be effective against medulloblastoma.     

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 beta-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.     

Zinc induces cell death in immortalized embryonic hippocampal cells via activation of Akt-GSK-3beta signaling

Zinc is an essential catalytic and structural element of many proteins and a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes severe neuronal cell death. We have previously observed that zinc-induced neuronal cell death is accompanied by Akt activation in embryonic hippocampal progenitor (H19-7) cells. In the present study, we examined the role of Akt activation and its downstream signaling events during extracellular zinc-induced neuronal cell death. Treatment of H19-7 cells with 10 microM of zinc plus zinc ionophore, pyrithione, led to increased phosphorylation of Akt at Ser-473/Thr-308 and increased Akt kinase activity. Zinc-induced Akt activation was accompanied by increased Tyr-phosphorylated GSK-3beta as well as increased GSK-3beta kinase activity. Transient overexpression of a kinase-deficient Akt mutant remarkably suppressed GSK-3beta activation and cell death. Furthermore, tau phosphorylation, but not the degradation of beta-catenin, was dependent upon zinc-induced GSK-3beta activation and contributed to cell death. The current data suggest that, following exposure to zinc, the sequential activation of Akt and GSK-3beta plays an important role directing hippocampal neural precursor cell death.     

Regulation of AMPA Receptor Trafficking and Function by Glycogen Synthase Kinase 3

Accumulating evidence suggests that glycogen synthase kinase 3 (GSK-3) is a multifunctional kinase implicated in neuronal development, mood stabilization, and neurodegeneration. However, the synaptic actions of GSK-3 are largely unknown. In this study, we examined the impact of GSK-3 on AMPA receptor (AMPAR) channels, the major mediator of excitatory transmission, in cortical neurons. Application of GSK-3 inhibitors or knockdown of GSK-3 caused a significant reduction of the amplitude of miniature excitatory postsynaptic current (mEPSC), a readout of the unitary strength of synaptic AMPARs. Treatment with GSK-3 inhibitors also decreased surface and synaptic GluR1 clusters on dendrites and increased internalized GluR1 in cortical cultures. Rab5, the small GTPase controlling the transport from plasma membrane to early endosomes, was activated by GSK-3 inhibitors. Knockdown of Rab5 prevented GSK-3 inhibitors from regulating mEPSC amplitude. Guanyl nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab5 between membrane and cytosol, formed an increased complex with Rab5 after treatment with GSK-3 inhibitors. Blocking the function of GDI occluded the effect of GSK-3 inhibitors on mEPSC amplitude. In cells transfected with the non-phosphorylatable GDI mutant, GDI(S45A), GSK-3 inhibitors lost the capability to regulate GDI-Rab5 complex, mEPSC amplitude, and AMPAR surface expression. These results suggest that GSK-3, via altering the GDI-Rab5 complex, regulates Rab5-mediated endocytosis of AMPARs. It provides a potential mechanism underlying the role of GSK-3 in synaptic transmission and plasticity.     

Glycogen synthase kinase 3: an emerging therapeutic target

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that has recently emerged as a key target in drug discovery. It has been implicated in multiple cellular processes and linked with the pathogenesis of several diseases. GSK-3 inhibitors might prove useful as therapeutic compounds in the treatment of conditions associated with elevated levels of enzyme activity, such as type 2 diabetes and Alzheimer's disease. The pro-apoptotic feature of GSK-3 activity suggests a potential role for its inhibitors in protection against neuronal cell death, and in the treatment of traumatic head injury and stroke. Finally, selective inhibitors of GSK-3 could mimic the action of mood stabilizers such as lithium and valproic acid and be used in the treatment of bipolar mood disorders.     

Neurogenesis and cell death in olfactory epithelium

The olfactory epithelium (OE) of the mammal is uniquely suited as a model system for studying how neurogenesis and cell death interact to regulate neuron number during development and regeneration. To identify factors regulating neurogenesis and neuronal death in the OE, and to determine the mechanisms by which these factors act, investigators studied OE using two major experimental paradigms: tissue culture of OE; and ablation of the olfactory bulb or severing the olfactory nerve in adult animals, procedures that induce cell death and a subsequent surge of neurogenesis in the OE in vivo. These studies characterized the cellular stages in the olfactory receptor neuron (ORN) lineage, leading to the realization that at least three distinct stages of proliferating neuronal precursor cells are employed in generating ORNs. The identification of a number of factors that act to regulate proliferation and survival of ORNs and their precursors suggests that these multiple developmental stages may serve as control points at which cell number is regulated by extrinsic factors. In vivo surgical studies, which have shown that all cell types in the neuronal lineage of the OE undergo apoptotic cell death, support this idea. These studies, and the possible coregulation of neuronal birth and apoptosis in the OE, are discussed. © 1996 John Wiley & Sons, Inc.     

Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3 （GSK-3） might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3β may contribute to NF-κB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3β activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.