Correction: Nonsense mutation suppression is enhanced by targeting different stages of the protein synthesis process.

[This corrects the article DOI: 10.1371/journal.pbio.3002355.].

Nonsense mutation suppression is enhanced by targeting different stages of the protein synthesis process.

The introduction of premature termination codons (PTCs), as a result of splicing defects, insertions, deletions, or point mutations (also termed nonsense mutations), lead to numerous genetic diseases, ranging from rare neuro-metabolic disorders to relatively common inheritable cancer syndromes and muscular dystrophies. Over the years, a large number of studies have demonstrated that certain antibiotics and other synthetic molecules can act as PTC suppressors by inducing readthrough of nonsense mutations, thereby restoring the expression of full-length proteins. Unfortunately, most PTC readthrough-inducing agents are toxic, have limited effects, and cannot be used for therapeutic purposes. Thus, further efforts are required to improve the clinical outcome of nonsense mutation suppressors. Here, by focusing on enhancing readthrough of pathogenic nonsense mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, we show that disturbing the protein translation initiation complex, as well as targeting other stages of the protein translation machinery, enhances both antibiotic and non-antibiotic-mediated readthrough of nonsense mutations. These findings strongly increase our understanding of the mechanisms involved in nonsense mutation readthrough and facilitate the development of novel therapeutic targets for nonsense suppression to restore protein expression from a large variety of disease-causing mutated transcripts.

Physiological expression of mutated TAU impaired astrocyte activity and exacerbates ß-amyloid pathology in 5xFAD mice.

BACKGROUND: Alzheimer's disease (AD) is the leading cause of dementia in the world. The pathology of AD is affiliated with the elevation of both tau (τ) and ß-amyloid (Aß) pathologies. Yet, the direct link between natural τ expression on glia cell activity and Aß remains unclear. While experiments in mouse models suggest that an increase in Aß exacerbates τ pathology when expressed under a neuronal promoter, brain pathology from AD patients suggests an appearance of τ pathology in regions without Aß. METHODS: Here, we aimed to assess the link between τ and Aß using a new mouse model that was generated by crossing a mouse model that expresses two human mutations of the human MAPT under a mouse Tau natural promoter with 5xFAD mice that express human mutated APP and PS1 in neurons. RESULTS: The new mouse model, called 5xFAD TAU, shows accelerated cognitive impairment at 2 months of age, increased number of Aß depositions at 4 months and neuritic plaques at 6 months of age. An expression of human mutated TAU in astrocytes leads to a dystrophic appearance and reduces their ability to engulf Aß, which leads to an increased brain Aß load. Astrocytes expressing mutated human TAU showed an impairment in the expression of vascular endothelial growth factor (VEGF) that has previously been suggested to play an important role in supporting neurons. CONCLUSIONS: Our results suggest the role of τ in exacerbating Aß pathology in addition to pointing out the potential role of astrocytes in disease progression. Further research of the crosstalk between τ and Aß in astrocytes may increase our understanding of the role glia cells have in the pathology of AD with the aim of identifying novel therapeutic interventions to an otherwise currently incurable disease.

Inhibition of GSK-3 ameliorates the pathogenesis of Huntington's disease.

In Huntington's disease (HD), the mutant huntingtin (mHtt) accumulates as toxic aggregates in the striatum tissue, with deleterious effects on motor-coordination and cognitive functions. Reducing the levels of mHtt is therefore a promising therapeutic strategy. We have previously reported that GSK-3 is a negative regulator of the autophagy/lysosome pathway, which is responsible for intracellular degradation, and is critically important for maintaining neuronal vitality. Thus, we hypothesized that inhibition of GSK-3 may trigger mHtt clearance thereby reducing mHtt cytotoxicity and improving HD symptoms. Here, we demonstrate that depletion or suppression of autophagy results in a massive accumulation of mHtt aggregates. Accordingly, mHtt aggregates were localized in lysosomes, but, mostly mislocalized from lysosomes in the absence of functional autophagy. Overexpression of GSK-3, particularly the α isozyme, increased the number of mHtt aggregates, while silencing GSK-3α/ß, or treatment with a selective GSK-3 inhibitor, L807mts, previously described by us, reduced the amounts of mHtt aggregates. This effect was mediated by increased autophagic and lysosomal activity. Treating R6/2 mouse model of HD with L807mts, reduced striatal mHtt aggregates and elevated autophagic and lysosomal markers. The L807mts treatment also reduced hyperglycemia and improved motor-coordination functions in these mice. In addition, L807mts restored the expression levels of Sirt1, a critical neuroprotective factor in the HD striatum, along with its targets BDNF, DRPP-32, and active Akt, all provide neurotrophic/pro-survival support and typically decline in the HD brain. Our results provide strong evidence for a role for GSK-3 in the regulation of mHtt dynamics, and demonstrate the benefits of GSK-3 inhibition in reducing mHtt toxicity, providing neuroprotective support, and improving HD symptoms.

Discovery and Design of Novel Small Molecule GSK-3 Inhibitors Targeting the Substrate Binding Site.

The serine/threonine kinase, GSK-3, is a promising drug discovery target for treating multiple pathological disorders. Most GSK-3 inhibitors that were developed function as ATP competitive inhibitors, with typical limitations in specificity, safety and drug-induced resistance. In contrast, substrate competitive inhibitors (SCIs), are considered highly selective, and more suitable for clinical practice. The development of SCIs has been largely neglected in the past because the ambiguous, undefined nature of the substrate-binding site makes them difficult to design. In this study, we used our previously described structural models of GSK-3 bound to SCI peptides, to design a pharmacophore model and to virtually screen the "drug-like" Zinc database (~6.3 million compounds). We identified leading hits that interact with critical binding elements in the GSK-3 substrate binding site and are chemically distinct from known GSK-3 inhibitors. Accordingly, novel GSK-3 SCI compounds were designed and synthesized with IC50 values of~1-4 µM. Biological activity of the SCI compound was confirmed in cells and in primary neurons that showed increased ß-catenin levels and reduced tau phosphorylation in response to compound treatment. We have generated a new type of small molecule GSK-3 inhibitors and propose to use this strategy to further develop SCIs for other protein kinases.

Regulation of Th1 cells and experimental autoimmune encephalomyelitis by glycogen synthase kinase-3.

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis (MS), a debilitating autoimmune disease of the CNS, for which only limited therapeutic interventions are available. Because MS is mediated in part by autoreactive T cells, particularly Th17 and Th1 cells, in the current study, we tested whether inhibitors of glycogen synthase kinase-3 (GSK3), previously reported to reduce Th17 cell generation, also alter Th1 cell production or alleviate EAE. GSK3 inhibitors were found to impede the production of Th1 cells by reducing STAT1 activation. Molecularly reducing the expression of either of the two GSK3 isoforms demonstrated that Th17 cell production was sensitive to reduced levels of GSK3ß and Th1 cell production was inhibited in GSK3α-deficient cells. Administration of the selective GSK3 inhibitors TDZD-8, VP2.51, VP0.7, or L803-mts significantly reduced the clinical symptoms of myelin oligodendrocyte glycoprotein35-55-induced EAE in mice, nearly eliminating the chronic progressive phase, and reduced the number of Th17 and Th1 cells in the spinal cord. Administration of TDZD-8 or L803-mts after the initial disease episode alleviated clinical symptoms in a relapsing-remitting model of proteolipid protein139-151-induced EAE. Furthermore, deletion of GSK3ß specifically in T cells was sufficient to alleviate myelin oligodendrocyte glycoprotein35-55-induced EAE. These results demonstrate the isoform-selective effects of GSK3 on T cell generation and the therapeutic effects of GSK3 inhibitors in EAE, as well as showing that GSK3 inhibition in T cells is sufficient to reduce the severity of EAE, suggesting that GSK3 may be a feasible target for developing new therapeutic interventions for MS.

GSK-3ß Inhibition Affects Singing Behavior and Neurogenesis in Adult Songbirds.

GSK-3 (glycogen synthase kinase-3) is a serine/threonine kinase which is a critical regulator in neuronal signaling, cognition, and behavior. We have previously shown that unlike other vertebrates that harbor both α and ß GSK-3 genes, the α gene is missing in birds. Therefore, birds can be used as a new animal model to study the roles of GSK-3ß in behavior and in regulating adult neurogenesis. In the present study, we inhibited GSK-3ß in brains of adult male zebra finches (Taeniopygia guttata) and accordingly investigated how this inhibition affects behavior and cell proliferation. Our results show that GSK-3 inhibition: (1) affects specific aspects of singing behavior, which might be related to social interactions in birds, and (2) differentially affects cell proliferation in various parts of the ventricular zone. Taken together, our study demonstrates a role of GSK-3ß in regulating singing behavior and neuronal proliferation in birds and highlights the importance of GSK-3ß in modulating cognitive abilities as well as social behavior.

Inhibition of glycogen synthase kinase-3 ameliorates ß-amyloid pathology and restores lysosomal acidification and mammalian target of rapamycin activity in the Alzheimer disease mouse model: in vivo and in vitro studies.

Accumulation of ß-amyloid (Aß) deposits is a primary pathological feature of Alzheimer disease that is correlated with neurotoxicity and cognitive decline. The role of glycogen synthase kinase-3 (GSK-3) in Alzheimer disease pathogenesis has been debated. To study the role of GSK-3 in Aß pathology, we used 5XFAD mice co-expressing mutated amyloid precursor protein and presenilin-1 that develop massive cerebral Aß loads. Both GSK-3 isozymes (α/ß) were hyperactive in this model. Nasal treatment of 5XFAD mice with a novel substrate competitive GSK-3 inhibitor, L803-mts, reduced Aß deposits and ameliorated cognitive deficits. Analyses of 5XFAD hemi-brain samples indicated that L803-mts restored the activity of mammalian target of rapamycin (mTOR) and inhibited autophagy. Lysosomal acidification was impaired in the 5XFAD brains as indicated by reduced cathepsin D activity and decreased N-glycoyslation of the vacuolar ATPase subunit V0a1, a modification required for lysosomal acidification. Treatment with L803-mts restored lysosomal acidification in 5XFAD brains. Studies in SH-SY5Y cells confirmed that GSK-3α and GSK-3ß impair lysosomal acidification and that treatment with L803-mts enhanced the acidic lysosomal pool as demonstrated in LysoTracker Red-stained cells. Furthermore, L803-mts restored impaired lysosomal acidification caused by dysfunctional presenilin-1. We provide evidence that mTOR is a target activated by GSK-3 but inhibited by impaired lysosomal acidification and elevation in amyloid precursor protein/Aß loads. Taken together, our data indicate that GSK-3 is a player in Aß pathology. Inhibition of GSK-3 restores lysosomal acidification that in turn enables clearance of Aß burdens and reactivation of mTOR. These changes facilitate amelioration in cognitive function.

GSK-3 inhibition: achieving moderate efficacy with high selectivity.

Inhibiting glycogen synthase kinase-3 (GSK-3) activity has become an attractive approach for treatment of neurodegenerative and psychiatric disorders. Diverse GSK-3 inhibitors have been reported and used in cellular and in vivo models. A major challenge, however, is achieving selectivity. In addition, it is increasingly recognized that a moderate inhibition of a cellular target, particularly for long-term treatment, provides more favorable outcome than complete inhibition. Substrate competitive inhibitors can fulfill the requirement for selectivity and allow fine tuning of the degree of inhibition. Here we describe the therapeutic potential of GSK-3 inhibitors and highlight our progress in the development of substrate competitive inhibitors. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Ataxia Telangiectasia Mutated Signaling Delays Skin Pigmentation upon UV Exposure by Mediating MITF Function toward DNA Repair Mode.

Skin pigmentation is paused after sun exposure; however, the mechanism behind this pausing is unknown. In this study, we found that the UVB-induced DNA repair system, led by the ataxia telangiectasia mutated (ATM) protein kinase, represses MITF transcriptional activity of pigmentation genes while placing MITF in DNA repair mode, thus directly inhibiting pigment production. Phosphoproteomics analysis revealed ATM to be the most significantly enriched pathway among all UVB-induced DNA repair systems. ATM inhibition in mouse or human skin, either genetically or chemically, induces pigmentation. Upon UVB exposure, MITF transcriptional activation is blocked owing to ATM-dependent phosphorylation of MITF on S414, which modifies MITF activity and interactome toward DNA repair, including binding to TRIM28 and RBBP4. Accordingly, MITF genome occupancy is enriched in sites of high DNA damage that are likely repaired. This suggests that ATM harnesses the pigmentation key activator for the necessary rapid, efficient DNA repair, thus optimizing the chances of the cell surviving. Data are available from ProteomeXchange with the identifier PXD041121.

Distinct molecular regulation of glycogen synthase kinase-3alpha isozyme controlled by its N-terminal region: functional role in calcium/calpain signaling.

Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and ß. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3ß, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of ß-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/ß-catenin pathway.

Prospects in GSK-3 Signaling: From Cellular Regulation to Disease Therapy.

Over the last decade, there has been continuous progress in our understanding of the biology of the protein kinase GSK-3 [...].

GSK-3ß Inhibition in Birds Affects Social Behavior and Increases Motor Activity.

Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase that plays a central role in a wide variety of cellular processes, cognition and behaviour. In a previous study we showed that its α and ß isozymes are highly conserved in vertebrates, however the α gene is missing in birds. This selective loss offers a unique opportunity to study the role of GSK-3ß independently. Accordingly, in the present study we aimed to investigate the role of GSK-3ß in social behaviour, motivation, and motor activity in zebra finches (Taeniopygia guttata). We did that by selective inhibition of GSK-3ß and by using tests that were specifically designed in our laboratory. Our results show that GSK-3ß inhibition: 1) Affected social recognition, because the treated birds tended to move closer towards a stranger, unlike the control birds that stood closer to a familiar bird. 2) Caused the treated birds to spend more time in the more middle parts of the cage compared to controls, a behaviour that might indicate anxiety. 3) As the experiment progressed, the treated birds took less time to make a decision where to stand in the cage compared to controls, suggesting an effect on decision-making. 4) Increased in the motor activity of the treated birds compared to the controls, which can be regarded as hyperactivity. 5) Caused the treated birds to pass through a barrier in order to join their flock members faster compared to controls, and regardless of the increase in the level of difficulty, possibly suggesting increased motivation. Our study calls for further investigation, because GSK-3 is well acknowledged as a central player in regulating mood behaviour, cognitive functions, and neuronal viability. Therefore, studying its impact on normal behaviour as we did in the current study, unlike most studies that were done in diseases models, can advance our understanding regarding GSK-3 various roles and can contribute to the discovery and development of effective treatments to repair cognition and behaviour.

An Exercise-Induced Metabolic Shield in Distant Organs Blocks Cancer Progression and Metastatic Dissemination.

Exercise prevents cancer incidence and recurrence, yet the underlying mechanism behind this relationship remains mostly unknown. Here we report that exercise induces the metabolic reprogramming of internal organs that increases nutrient demand and protects against metastatic colonization by limiting nutrient availability to the tumor, generating an exercise-induced metabolic shield. Proteomic and ex vivo metabolic capacity analyses of murine internal organs revealed that exercise induces catabolic processes, glucose uptake, mitochondrial activity, and GLUT expression. Proteomic analysis of routinely active human subject plasma demonstrated increased carbohydrate utilization following exercise. Epidemiologic data from a 20-year prospective study of a large human cohort of initially cancer-free participants revealed that exercise prior to cancer initiation had a modest impact on cancer incidence in low metastatic stages but significantly reduced the likelihood of highly metastatic cancer. In three models of melanoma in mice, exercise prior to cancer injection significantly protected against metastases in distant organs. The protective effects of exercise were dependent on mTOR activity, and inhibition of the mTOR pathway with rapamycin treatment ex vivo reversed the exercise-induced metabolic shield. Under limited glucose conditions, active stroma consumed significantly more glucose at the expense of the tumor. Collectively, these data suggest a clash between the metabolic plasticity of cancer and exercise-induced metabolic reprogramming of the stroma, raising an opportunity to block metastasis by challenging the metabolic needs of the tumor. SIGNIFICANCE: Exercise protects against cancer progression and metastasis by inducing a high nutrient demand in internal organs, indicating that reducing nutrient availability to tumor cells represents a potential strategy to prevent metastasis. See related commentary by Zerhouni and Piskounova, p. 4124.

Substrate competitive GSK-3 inhibitors - strategy and implications.

Glycogen synthase kinase-3 (GSK-3) is a highly conserved protein serine/threonine kinase ubiquitously distributed in eukaryotes as a constitutively active enzyme. Abnormally high GSK-3 activity has been implicated in several pathological disorders, including diabetes and neuron degenerative and affective disorders. This led to the hypothesis that inhibition of GSK-3 may have therapeutic benefit. Most GSK-3 inhibitors developed so far compete with ATP and often show limited specificity. Our goal is to develop inhibitors that compete with GSK-3 substrates, as this type of inhibitor is more specific and may be useful for clinical applications. We have employed computational, biochemical, and molecular analyses to gain in-depth understanding of GSK-3's substrate recognition. Here we argue that GSK-3 is a promising drug discovery target and describe the strategy and practice for developing specific substrate-competitive inhibitors of GSK-3.

Wnt signaling pathway overcomes the disruption of neuronal differentiation of neural progenitor cells induced by oligomeric amyloid ß-peptide.

Neural stem cells give rise to new hippocampal neurons throughout adulthood. Defects in neurogenesis are associated with cognitive dysfunctions, such as Alzheimer disease (AD). Our understanding of the signals controlling this process is limited. The present in vitro study explored the manner in which the Wnt signaling pathway regulates the differentiation of hippocampal progenitors (HPs) into neurons under the influence of amyloid ß(42) (Aß(42) ). The results showed that oligomeric Aß(42) reduced neuronal differentiation. This process was accompanied by a reduction in active ß-catenin levels and proneural gene expression. The addition of Wnt3a increased the neuronal differentiation of Aß(42) -treated HPs, at the expense of astrocyte differentiation. The effect of Wnt signaling was attributable to progenitor cell differentiation to the neuronal lineage, and not to increased proliferation or rescue of neurons. The interruption of Wnt signaling by oligomeric Aß(42) may have clinical implications for the treatment of impaired neurogenesis in AD.

Synthesis and biological evaluation of glycogen synthase kinase 3 (GSK-3) inhibitors: an fast and atom efficient access to 1-aryl-3-benzylureas.

The glycogen synthase kinase 3 (GSK-3) is implicated in multiple cellular processes and has been linked to the pathogenesis of Alzheimer's disease (AD). In the course of our research topic we synthesized a library of potent GSK-3 inhibitors. We utilized the urea scaffold present in the potent and highly selective GSK-3 inhibitor AR-A014418 (AstraZeneca). This moiety suits both (a) a convergent approach utilizing readily accessible building blocks and (b) a divergent approach based on a microwave heating assisted Suzuki coupling. We established a chromatography-free purification method to generate products with sufficient purity for the biological assays. The structure-activity relationship of the library provided the rationale for the synthesis of the benzothiazolylurea 66 (IC(50)=140 nM) and the pyridylurea 62 (IC(50)=98 nM), which displayed two to threefold enhanced activity versus the reference compound 18 (AR-A014418: IC(50)=330 nM) in our assays.

Identification of eukaryotic elongation factor-2 as a novel cellular target of lithium and glycogen synthase kinase-3.

Inhibition of glycogen synthase kinase-3 (GSK-3) is thought to be a major consequence of the biological and clinical activity of the mood stabilizer lithium, however, lithium and GSK-3 may activate distinct cellular pathways. We employed a proteomic method to uncover new downstream targets of lithium, and then examined how these proteins are related to GSK-3. Proteomic analysis identified eukaryotic elongation factor-2 (eEF-2) as a cellular target of lithium. This was verified in SH-SY5Y cells and animal models. In cells, lithium decreased eEF-2 phosphorylation at its key inhibitory site, threonine 56, and blocked the enhancement of eEF-2 phosphorylation normally coupled with stress conditions such as nutrient and serum deprivation. Unexpectedly, inhibition of GSK-3 enhanced eEF-2 phosphorylation, and overexpression of GSK-3α or GSK-3ß resulted in a strong reduction in eEF-2 phosphorylation. Chronic administration of lithium reduced the hippocampal fraction of phospho-eEF-2 (phospho-eEF-2/total eEF-2) twofold in two different mouse strains. In summary, unexpectedly eEF-2 is activated by both lithium and GSK-3, whereas, lithium treatment and inhibition of GSK-3 have opposing effects on eEF-2.

Mechanisms and Therapeutic Implications of GSK-3 in Treating Neurodegeneration.

Neurodegenerative disorders are spreading worldwide and are one of the greatest threats to public health. There is currently no adequate therapy for these disorders, and therefore there is an urgent need to accelerate the discovery and development of effective treatments. Although neurodegenerative disorders are broad ranging and highly complex, they may share overlapping mechanisms, and thus potentially manifest common targets for therapeutic interventions. Glycogen synthase kinase-3 (GSK-3) is now acknowledged to be a central player in regulating mood behavior, cognitive functions, and neuron viability. Indeed, many targets controlled by GSK-3 are critically involved in progressing neuron deterioration and disease pathogenesis. In this review, we focus on three pathways that represent prominent mechanisms linking GSK-3 with neurodegenerative disorders: cytoskeleton organization, the mammalian target of rapamycin (mTOR)/autophagy axis, and mitochondria. We also consider the challenges and opportunities in the development of GSK-3 inhibitors for treating neurodegeneration.

Glycogen Synthase Kinase-3 Inhibitors: Preclinical and Clinical Focus on CNS-A Decade Onward.

The protein kinase, GSK-3, participates in diverse biological processes and is now recognized a promising drug discovery target in treating multiple pathological conditions. Over the last decade, a range of newly developed GSK-3 inhibitors of diverse chemotypes and inhibition modes has been developed. Even more conspicuous is the dramatic increase in the indications that were tested from mood and behavior disorders, autism and cognitive disabilities, to neurodegeneration, brain injury and pain. Indeed, clinical and pre-clinical studies were largely expanded uncovering new mechanisms and novel insights into the contribution of GSK-3 to neurodegeneration and central nerve system (CNS)-related disorders. In this review we summarize new developments in the field and describe the use of GSK-3 inhibitors in the variety of CNS disorders. This remarkable volume of information being generated undoubtedly reflects the great interest, as well as the intense hope, in developing potent and safe GSK-3 inhibitors in clinical practice.