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.

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.

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.

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.

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.

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.

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.

GSK-3-TSC axis governs lysosomal acidification through autophagy and endocytic pathways.

Impaired lysosomal activity, which results in defective protein processing, waste accumulation, and protein aggregation, is implicated in a number of disease pathologies. Acidification of lysosomes is a crucial process required for lysosome function. Previously we showed that inhibition of glycogen synthase kinase-3 (GSK-3) enhanced lysosomal acidification in both normal and pathological conditions. However, how GSK-3 integrates into the lysosome networking is unknown. Here we show that inhibition of mTORC1 and increased autophagic activity are downstream to GSK-3 inhibition and contribute to lysosomal acidification. Strikingly, lysosomal acidification is also restored by GSK-3 inhibition in the absence of functional autophagy, and, independently of mTORC1. This is facilitated by increased endocytic traffic: We show that GSK-3 inhibition enhanced material internalization, increased recruitment of active Rab5 into endosomes, and increased Rab7/RILP clustering into lysosomes, all processes required for late endosome maturation. Consistently, in cells defective in endocytic traffic caused by either constitutively active Rab5, or, deletion of the Niemann-Pick C1 protein, GSK-3 inhibition could not restore lysosomal acidification. Finally we found that the tuberous sclerosis complex, TSC, is required for lysosomal acidification and is activated by GSK-3 inhibition. Thus, the GSK-3/TSC axis regulates lysosomal acidification via both the autophagic and endocytic pathways. Our study provides new insights into the therapeutic potential of GSK-3 inhibitors in treating pathological conditions associated with impaired cellular clearance.

Intranasal siRNA administration reveals IGF2 deficiency contributes to impaired cognition in Fragile X syndrome mice.

Molecular mechanisms underlying learning and memory remain imprecisely understood, and restorative interventions are lacking. We report that intranasal administration of siRNAs can be used to identify targets important in cognitive processes and to improve genetically impaired learning and memory. In mice modeling the intellectual deficiency of Fragile X syndrome, intranasally administered siRNA targeting glycogen synthase kinase-3ß (GSK3ß), histone deacetylase-1 (HDAC1), HDAC2, or HDAC3 diminished cognitive impairments. In WT mice, intranasally administered brain-derived neurotrophic factor (BDNF) siRNA or HDAC4 siRNA impaired learning and memory, which was partially due to reduced insulin-like growth factor-2 (IGF2) levels because the BDNF siRNA- or HDAC4 siRNA-induced cognitive impairments were ameliorated by intranasal IGF2 administration. In Fmr1-/- mice, hippocampal IGF2 was deficient, and learning and memory impairments were ameliorated by IGF2 intranasal administration. Therefore intranasal siRNA administration is an effective means to identify mechanisms regulating cognition and to modulate therapeutic targets.

Ketamine up-regulates a cluster of intronic miRNAs within the serotonin receptor 2C gene by inhibiting glycogen synthase kinase-3.

OBJECTIVES: We examined mechanisms that contribute to the rapid antidepressant effect of ketamine in mice that is dependent on glycogen synthase kinase-3 (GSK3) inhibition. METHODS: We measured serotonergic (5HT)-2C-receptor (5HTR2C) cluster microRNA (miRNA) levels in mouse hippocampus after administering an antidepressant dose of ketamine (10 mg/kg) in wild-type and GSK3 knockin mice, after GSK3 inhibition with L803-mts, and in learned helpless mice. RESULTS: Ketamine up-regulated cluster miRNAs 448-3p, 764-5p, 1264-3p, 1298-5p and 1912-3p (2- to 11-fold). This up-regulation was abolished in GSK3 knockin mice that express mutant constitutively active GSK3. The GSK3 specific inhibitor L803-mts was antidepressant in the learned helplessness and novelty suppressed feeding depression-like behaviours and up-regulated the 5HTR2C miRNA cluster in mouse hippocampus. After administration of the learned helplessness paradigm mice were divided into cohorts that were resilient (non-depressed) or were susceptible (depressed) to learned helplessness. The resilient, but not depressed, mice displayed increased hippocampal levels of miRNAs 448-3p and 1264-3p. Administration of an antagonist to miRNA 448-3p diminished the antidepressant effect of ketamine in the learned helplessness paradigm, indicating that up-regulation of miRNA 448-3p provides an antidepressant action. CONCLUSIONS: These findings identify a new outcome of GSK3 inhibition by ketamine that may contribute to antidepressant effects.

Up-regulation of insulin-like growth factor 2 by ketamine requires glycogen synthase kinase-3 inhibition.

An antidepressant dose of the rapidly-acting ketamine inhibits glycogen synthase kinase-3 (GSK3) in mouse hippocampus, and this inhibition is required for the antidepressant effect of ketamine in learned helplessness depression-like behavior. Here we report that treatment with an antidepressant dose of ketamine (10mg/kg) increased expression of insulin-like growth factor 2 (IGF2) in mouse hippocampus, an effect that required ketamine-induced inhibition of GSK3. Ketamine also inhibited hippocampal GSK3 and increased expression of hippocampal IGF2 in mice when administered after the induction of learned helplessness. Treatment with the specific GSK3 inhibitor L803-mts was sufficient to up-regulate hippocampal IGF2 expression. Administration of IGF2 siRNA reduced ketamine's antidepressant effect in the learned helplessness paradigm. Mice subjected to the learned helplessness paradigm were separated into two groups, those that were resilient (non-depressed) and those that were susceptible (depressed). Non-depressed resilient mice displayed higher expression of IGF2 than susceptible mice. These results indicate that IGF2 contributes to ketamine's antidepressant effect and that IGF2 may confer resilience to depression-like behavior.

Science Signaling Podcast for 15 November 2016: A new type of kinase inhibitor.

This Podcast features an interview with Hagit Eldar-Finkelman, author of a Research Article that appears in the 15 November 2016 issue of Science Signaling, about a newly developed inhibitor of glycogen synthase kinase 3 (GSK-3). GSK-3 participates in several signaling networks and has been implicated in various pathologies, including neurodegenerative diseases, cognitive impairments, and cancer. Licht-Murava et al developed L807mts, a substrate-competitive peptide inhibitor that blocks GSK-3 activity through an unusual mechanism. L807mts not only bound to the substrate recognition domain of GSK-3, it was also phosphorylated by the kinase. This phosphorylated form of L807mts remained associated with GSK-3 and inhibited GSK-3 activity. L807mts treatment reduced cellular, cognitive, and behavioral symptoms in a mouse model of Alzheimer's disease. L807mts is an advance in kinase inhibitor development because it is both highly specific and very potent.Listen to Podcast.

A unique type of GSK-3 inhibitor brings new opportunities to the clinic.

Development of protein kinase inhibitors is a focus of many drug discovery programs. A major problem, however, is the limited specificity of the commonly used adenosine triphosphate-competitive inhibitors and the weak inhibition of the more selective substrate-competitive inhibitors. Glycogen synthase kinase-3 (GSK-3) is a promising drug target for treating neurodegenerative disorders, including Alzheimer's disease (AD), but most GSK-3 inhibitors have not reached the clinic. We describe a new type of GSK-3 inhibitor, L807mts, that acts through a substrate-to-inhibitor conversion mechanism that occurs within the catalytic site of the enzyme. We determined that L807mts was a potent and highly selective GSK-3 inhibitor with reasonable pharmacological and safety properties when tested in rodents. Treatment with L807mts enhanced the clearance of ß-amyloid loads, reduced inflammation, enhanced autophagic flux, and improved cognitive and social skills in the 5XFAD AD mouse model. This new modality of GSK-3 inhibition may be therapeutic in patients with AD or other central nervous system disorders associated with dysregulated GSK-3.

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.

Design, synthesis, and biological evaluation of 1-phenylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-diones as new glycogen synthase kinase-3ß inhibitors.

Compound 5 was selected from our in-house library as a suitable starting point for the rational design of new GSK-3ß inhibitors. MC/FEP calculations of 5 led to the identification of a structural class of new GSK-3ß inhibitors. Compound 18 inhibited GSK-3ß with an IC50 of 0.24 µM and inhibited tau phosphorylation in a cell-based assay. It proved to be a selective inhibitor of GSK-3 against a panel of 17 kinases and showed >10-fold selectivity against CDK2. Calculated physicochemical properties and Volsurf predictions suggested that compound 18 has the potential to diffuse passively across the blood-brain barrier.

GSK-3 and lysosomes meet in Alzheimer's disease.

Aberrant regulation of glycogen synthase kinase-3 (GSK-3) is implicated in Alzheimer's disease (AD), but the mechanisms involved remain elusive. Our recent study shows that GSK-3 impairs lysosomal acidification and that inhibition of GSK-3 re-acidified lysosomes in brains of AD mice. This effect was accompanied by reductions in ß-amyloid pathology and amelioration of cognitive deficits. Presenilin-1 (PS1) is an essential factor in lysosomal acidification. To determine whether the inhibition of GSK-3 restores lysosomal malfunction caused by dysfunctional PS1, we treated MEF cells deficient in presenilin proteins (MEF-PS1/2(-/-)) with a selective substrate competitive GSK-3 inhibitor, L803-mts. L803-mts enhanced the acidic lysosomal pool in MEF-PS1/2(-/-) cells and increased levels of activated cathepsin D in the lysosomes. We conclude that GSK-3 and PS1 operate via similar mechanisms to disrupt lysosomal acidification. Importantly, these data indicate that GSK-3 inhibitors have potential in treatment of conditions associated with defective PS1.