Elraglusib (formerly 9-ING-41) possesses potent anti-lymphoma properties which cannot be attributed to GSK3 inhibition.

Elraglusib (formerly 9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3ß (GSK3ß) undergoing clinical trials for the treatment of various cancers including non-Hodgkin lymphoma (NHL). The drug reduces proliferation of several NHL cell lines and has efficacy in xenograft models of the disease. To confirm the importance of its action on GSK3ß, we treated 3 lymphoma cell lines with selective, structurally distinct GSK3 inhibitors: CT99021, SB216763, LY2090314, tideglusib, and elraglusib. Stabilization of ß-catenin and reduced phosphorylation of CRMP2, two validated targets of GSK3, were used as functional read-outs for GSK3 inhibition. CT99021, SB216763, and LY2090314 failed to reduce proliferation or viability in any cell line at concentrations that stabilized ß-catenin and reduced CRMP2 phosphorylation. There was partial reduction of CRMP2 phosphorylation but no significant effect on ß-catenin at cytotoxic doses of elraglusib. There was no indication of GSK3 inhibition at doses of tideglusib that affected cell viability and apoptosis. Cell-free kinase screening confirmed several other targets of elraglusib, distinct from the GSK3 inhibitors with no anti-lymphoma actions, including PIM kinases and MST2. These data question GSK3 as the target of elraglusib in lymphoma, and hence the utility of GSK3 expression as a 'stand-alone', therapeutic biomarker in NHL. Video Abstract.

Investigation of salicylate hepatic responses in comparison with chemical analogues of the drug.

Anti-hyperglycaemic effects of the hydroxybenzoic acid salicylate might stem from effects of the drug on mitochondrial uncoupling, activation of AMP-activated protein kinase, and inhibition of NF-κB signalling. Here, we have gauged the contribution of these effects to control of hepatocyte glucose production, comparing salicylate with inactive hydroxybenzoic acid analogues of the drug. In rat H4IIE hepatoma cells, salicylate was the only drug tested that activated AMPK. Salicylate also reduced mTOR signalling, but this property was observed widely among the analogues. In a sub-panel of analogues, salicylate alone reduced promoter activity of the key gluconeogenic enzyme glucose 6-phosphatase and suppressed basal glucose production in mouse primary hepatocytes. Both salicylate and 2,6 dihydroxybenzoic acid suppressed TNFα-induced IκB degradation, and in genetic knockout experiments, we found that the effect of salicylate on IκB degradation was AMPK-independent. Previous data also identified AMPK-independent regulation of glucose but we found that direct inhibition of neither NF-κB nor mTOR signalling suppressed glucose production, suggesting that other factors besides these cell signalling pathways may need to be considered to account for this response to salicylate. We found, for example, that H4IIE cells were exquisitely sensitive to uncoupling with modest doses of salicylate, which occurred on a similar time course to another anti-hyperglycaemic uncoupling agent 2,4-dinitrophenol, while there was no discernible effect at all of two salicylate analogues which are not anti-hyperglycaemic. This finding supports much earlier literature suggesting that salicylates exert anti-hyperglycaemic effects at least in part through uncoupling.

Dual regulation of transcription factor Nrf2 by Keap1 and by the combined actions of ß-TrCP and GSK-3.

Nuclear factor-erythroid 2 p45 (NF-E2 p45)-related factor 2 (Nrf2) is a master regulator of redox homoeostasis that allows cells to adapt to oxidative stress and also promotes cell proliferation. In this review, we describe the molecular mechanisms by which oxidants/electrophilic agents and growth factors increase Nrf2 activity. In the former case, oxidants/electrophiles increase the stability of Nrf2 by antagonizing the ability of Kelch-like ECH-associated protein 1 (Keap1) to target the transcription factor for proteasomal degradation via the cullin-3 (Cul3)-RING ubiquitin ligase CRL(Keap1). In the latter case, we speculate that growth factors increase the stability of Nrf2 by stimulating phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB)/Akt signalling, which in turn results in inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) and in doing so prevents the formation of a DSGIS motif-containing phosphodegron in Nrf2 that is recognized by the ß-transducin repeat-containing protein (ß-TrCP) Cul1-based E3 ubiquitin ligase complex SCF(ß-TrCP). We present data showing that in the absence of Keap1, the electrophile tert-butyl hydroquinone (tBHQ) can stimulate Nrf2 activity and induce the Nrf2-target gene NAD(P)H: quinone oxidoreductase-1 (NQO1), whilst simultaneously causing inhibitory phosphorylation of GSK-3ß at Ser(9). Together, these observations suggest that tBHQ can suppress the ability of SCF(ß-TrCP) to target Nrf2 for proteasomal degradation by increasing PI3K-PKB/Akt signalling. We also propose a scheme that explains how other protein kinases that inhibit GSK-3 could stimulate induction of Nrf2-target genes by preventing formation of the DSGIS motif-containing phosphodegron in Nrf2.

Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis.

BACKGROUND: Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. METHODS: Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. RESULTS: The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. CONCLUSIONS: For the first time this data associates altered CDK5 substrate phosphorylation with oncogenesis in some but not all tumour types, implicating altered CDK5 activity in aspects of pathogenesis. These data identify a novel oncogenic mechanism where CDK5 activation induces CRMP2A phosphorylation in the nuclei of tumour cells.

Invited commentary on Lithium treatment and risk for dementia in adults with bipolar disorder.

Alzheimer's disease clinical trials are failing at an alarming rate, highlighting the desperate need for novel thinking to combat this escalating health crisis. A recent large-scale population study indicates that lithium treatment reduces dementia development, supporting preclinical mechanistic evidence that this commonly used agent might be clinically valuable in dementia.

Neuronal deletion of GSK3ß increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2.

BACKGROUND: In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity. RESULTS: Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3ß (GSK3ß) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3ß regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3ß activity in neurons. Following spinal cord injury, reduced GSK3ß levels or complete neuronal deletion of GSK3ß led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3ß substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3ß-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3ß-CRMP-2 relevance during axon regeneration. CONCLUSIONS: Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3ß-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth.

Point-of-Care testing of HbA1c levels in community settings for people with established diabetes or people at risk of developing diabetes: A systematic review and meta-analysis.

BACKGROUND: Glycated haemoglobin (HbA1c) measurement using Point of Care (POC) testing may be of huge utility, providing convenient testing for early diagnosis and regular monitoring of hard-to-reach patient groups. This systematic review aimed to identify evidence for the successful deployment of these devices to improve patient outcomes in diabetes. METHODS: A systematic review and meta-analysis was undertaken in February 2023, to identify all relevant articles: (CINAHL, Cochrane, PubMed, Scopus, and Web of Science). Studies were included if they reported outcomes of community POC testing for HbA1c for people with diabetes or at risk of diabetes. The Prospero database and trial registers were searched. Only English language articles were included. Title, abstract screening and full text review was carried out by two reviewers (AG/AR). The Cochrane risk of bias tool for randomised studies and the NIH Quality Assessment tool for observational cohort and cross-sectional studies were used. Publication bias was assessed visually using funnel plot and statistical assessment. We performed a meta-analysis on appropriate studies, applying a fixed effect model. We investigated heterogeneity using visual inspection of forest plots along with evaluative approaches (χ2, I2). Strength of evidence was assessed using GRADE. FINDINGS: 24 studies fulfilled the criteria to be included in the narrative synthesis and 5 could be included in quantitative analysis. 13 studies evaluated HbA1c POC testing in non-diabetic patients, 9 reported results for diabetic patients and 2 included both groups. The narrative synthesis was constructed around 6 key themes: increased test access, diagnosis of people who would otherwise go undiagnosed, intervention/lifestyle change, POC testing effect on HbA1c and glycaemic control, follow-up time and patient satisfaction. INTERPRETATION: The available published data supports the proposed use of POC devices in a community setting, with positive effects on diabetic care with limited evidence that patients can achieve better glycaemic control.

Inhibition of glycogen synthase kinase-3 enhances NRF2 protein stability, nuclear localisation and target gene transcription in pancreatic beta cells.

Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. NRF2 (NF-E2 p45-related factor-2) regulates the adaptation to oxidative stress, and its activity is negatively regulated by the redox-sensitive CUL3 (cullin-3) ubiquitin ligase substrate adaptor KEAP1 (Kelch-like ECH-associated protein-1). Additionally, NRF2 is repressed by the insulin-regulated Glycogen Synthase Kinase-3 (GSK3). We have demonstrated that phosphorylation of NRF2 by GSK3 enhances ß-TrCP (beta-transducin repeat-containing protein) binding and ubiquitylation by CUL1 (cullin-1), resulting in increased proteasomal degradation of NRF2. Thus, we hypothesise that inhibition of GSK3 activity or ß-TrCP binding upregulates NRF2 and so protects beta cells against oxidative stress. We have found that treating the pancreatic beta cell line INS-1 832/13 with the KEAP1 inhibitor TBE31 significantly enhanced NRF2 protein levels. The presence of the GSK3 inhibitor CT99021 or the ß-TrCP-NRF2 protein-protein interaction inhibitor PHAR, along with TBE31, resulted in prolonged NRF2 stability and enhanced nuclear localisation (P < 0.05). TBE31-mediated induction of NRF2-target genes encoding NAD(P)H quinone oxidoreductase 1 (Nqo1), glutamate-cysteine ligase modifier (Gclm) subunit and heme oxygenase (Hmox1) was significantly enhanced by the presence of CT99021 or PHAR (P < 0.05) in both INS-1 832/13 and in isolated mouse islets. Identical results were obtained using structurally distinct GSK3 inhibitors and inhibition of KEAP1 with sulforaphane. In summary, we demonstrate that GSK3 and ß-TrCP/CUL1 regulate the proteasomal degradation of NRF2, enhancing the impact of KEAP1 regulation, and so contributes to the redox status of pancreatic beta cells. Inhibition of GSK3, or ß-TrCP/CUL1 binding to NRF2 may represent a strategy to protect beta cells from oxidative stress.

Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling.

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

Point-of-care testing of HbA1c levels in community settings for people with established diabetes or people at risk of developing type 2 diabetes: a systematic review and meta-analysis protocol.

INTRODUCTION: Diabetes mellitus has increased in prevalence worldwide and is causing an increasing burden on health services. The best patient outcomes occur with early diagnosis to prevent health complications. Glycated haemoglobin (HbA1c) is used to assess glycaemic control over 3-6 months and inform clinical management. Point-of-care (POC) HbA1c devices can be used in community settings, independent of clinical laboratories. This review aims to evaluate how these devices have been implemented in community settings and what patient outcomes have been documented. METHODS AND ANALYSIS: This protocol follows the Preferred Reporting Items for Systematic Review and Meta-Analysis guidance. A systematic search was undertaken in October 2022, using the defined PICOS (population, intervention, comparison, outcomes, study type) statement to identify all relevant articles: CINAHL, Cochrane, PubMed, Scopus and Web of Science were searched (updated February 2023). Studies will be included if they report outcomes of community POC testing for HbA1c for people with diabetes or at risk of diabetes. We will review the PROSPERO database and trial registers.Title, abstract screening and full-text review will be carried out by two reviewers. The Cochrane risk-of-bias tool will be used to assess randomised studies and the National Institutes of Health (NIH) Quality Assessment tool for observational cohort and cross-sectional studies. Publication bias will be assessed visually with a funnel plot and statistical approaches if necessary. If a group of sufficiently comparable studies are identified, we will perform a meta-analysis applying a fixed or random effects model as appropriate. We will investigate heterogeneity using visual inspection of forest plots along with review of evaluative approaches such as Χ2 and the I2 statistic. Strength of evidence will be assessed using Grading of Recommendations, Assessment, Development and Evaluation. ETHICS AND DISSEMINATION: Ethics approval is not required for this literature review. The results will be disseminated through peer-reviewed publication and conference presentations. Furthermore, this systematic review will be used to inform the design of a community pharmacy-based prediabetes intervention. PROSPERO REGISTRATION NUMBER: CRD42023383784.

Phosphorylation of the N-terminus of Syntaxin-16 controls interaction with mVps45 and GLUT4 trafficking in adipocytes.

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the 'insulin responsive vesicles' are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro. Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes.

Implementation of point-of-care HbA1C instruments into community pharmacies: Initial development of a pathway for robust community testing.

BACKGROUND: Point-of-care (POC) analysers in community settings can provide opportunistic and regular HbA1c monitoring. Community pharmacies in NHS Scotland are utilised by populations at greatest risk of type two diabetes (T2D). This study describes initial development of an HbA1c pathway using a POC analyser in community pharmacies. METHODS: The Abbott Afinion analyser was compared in (i) NHS Tayside's Blood Sciences Service and (ii) community pharmacies from four Scottish Health Boards. A side by side comparison with standard operating procedures for HbA1c quantification using 80 T2D patient venous samples. The machine was implemented into 11 community pharmacies and 144 samples obtained from patients for comparison to their recent laboratory HbA1c. Four focus groups examined themes around the intervention and an exit questionnaire was administered. RESULTS: Laboratory assessment verified the efficacy of the POC test machine. The value for level 1 quality control was 44 mmol/mol and the mean during testing 42.7 mmol/mol. The greatest percent coefficient of variation (cv) was within-run for both levels of quality control material, at a value of 1.63% and 1.62%, respectively. The analyser performed robustly within the pharmacy assessment, with a mean difference of 1.68 and a standard deviation of 0.71 (CV 0.423). Patients with T2D reported positive experiences of using a pharmacy. The focus groups identified an appreciation of the convenience of pharmacies and of the longitudinal relationships with pharmacy staff. CONCLUSION: POC HbA1c analysers can be successfully established in community pharmacies. The target patient group responded positively to the opportunity to use a pharmacy service.

Metformin increases the uptake of glucose into the gut from the circulation in high-fat diet-fed male mice, which is enhanced by a reduction in whole-body Slc2a2 expression.

OBJECTIVES: Metformin is the first line therapy recommended for type 2 diabetes. However, the precise mechanism of action remains unclear and up to a quarter of patients show some degree of intolerance to the drug, with a similar number showing poor response to treatment, limiting its effectiveness. A better understanding of the mechanism of action of metformin may improve its clinical use. SLC2A2 (GLUT2) is a transmembrane facilitated glucose transporter, with important roles in the liver, gut and pancreas. Our group previously identified single nucleotide polymorphisms in the human SLC2A2 gene, which were associated with reduced transporter expression and an improved response to metformin treatment. The aims of this study were to model Slc2a2 deficiency and measure the impact on glucose homoeostasis and metformin response in mice. METHODS: We performed extensive metabolic phenotyping and 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG)-positron emission tomography (PET) analysis of gut glucose uptake in high-fat diet-fed (HFD) mice with whole-body reduced Slc2a2 (Slc2a2+/-) and intestinal Slc2a2 KO, to assess the impact of metformin treatment. RESULTS: Slc2a2 partial deficiency had no major impact on body weight and insulin sensitivity, however mice with whole-body reduced Slc2a2 expression (Slc2a2+/-) developed an age-related decline in glucose homoeostasis (as measured by glucose tolerance test) compared to wild-type (Slc2a2+/+) littermates. Glucose uptake into the gut from the circulation was enhanced by metformin exposure in Slc2a2+/+ animals fed HFD and this action of the drug was significantly higher in Slc2a2+/- animals. However, there was no effect of specifically knocking-out Slc2a2 in the mouse intestinal epithelial cells. CONCLUSIONS: Overall, this work identifies a differential metformin response, dependent on expression of the SLC2A2 glucose transporter, and also adds to the growing evidence that metformin efficacy includes modifying glucose transport in the gut. We also describe a novel and important role for this transporter in maintaining efficient glucose homoeostasis during ageing.

Bioinformatic prediction and confirmation of beta-adducin as a novel substrate of glycogen synthase kinase 3.

It is important to identify the true substrates of protein kinases because this illuminates the primary function of any kinase. Here, we used bioinformatics and biochemical validation to identify novel brain substrates of the Ser/Thr kinase glycogen synthase kinase 3 (GSK3). Briefly, sequence databases were searched for proteins containing a conserved GSK3 phosphorylation consensus sequence ((S/T)PXX(S/T)P or (S/T)PXXX(S/T)P), as well as other criteria of interest (e.g. brain proteins). Importantly, candidates were highlighted if they had previously been reported to be phosphorylated at these sites by large-scale phosphoproteomic studies. These criteria identified the brain-enriched cytoskeleton-associated protein ß-adducin as a likely substrate of GSK3. To confirm this experimentally, it was cloned and subjected to a combination of cell culture and in vitro kinase assays that demonstrated direct phosphorylation by GSK3 in vitro and in cells. Phosphosites were mapped to three separate regions near the C terminus and confirmed using phosphospecific antibodies. Prior priming phosphorylation by Cdk5 enhanced phosphorylation by GSK3. Expression of wild type, but not non-phosphorylatable (GSK3 insensitive), ß-adducin increased axon and dendrite elongation in primary cortical neurons. Therefore, phosphorylation of ß-adducin by GSK3 promotes efficient neurite outgrowth in neurons.

Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP)•collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation.

Neuron polarization is essential for the formation of one axon and multiple dendrites, establishing the neuronal circuitry. Phosphoinositide 3-kinase (PI3K) signaling promotes axon selection and elongation. Here we report in hippocampal neurons siRNA knockdown of the proline-rich inositol polyphosphate 5-phosphatase (PIPP), which degrades PI3K-generated PtdIns(3,4,5)P(3), results in multiple hyperelongated axons consistent with a polarization defect. We identify collapsin response mediator protein 2 (CRMP2), which regulates axon selection by promoting WAVE1 delivery via Kinesin-1 motors to the axon growth cone, as a PIPP-interacting protein by Y2H screening, direct binding studies, and coimmunoprecipitation of an endogenous PIPP, CRMP2, and Kinesin-1 complex from brain lysates. The C-terminal growth cone-targeting domain of PIPP facilitates its interaction with CRMP2. PIPP growth cone localization is CRMP2-dependent. PIPP knockdown in PC12 cells promotes neurite elongation, WAVE1 and Kinesin-1 growth cone localization, whereas knockdown of CRMP2 exhibits the opposite phenotype, with shorter neurites and decreased WAVE1/Kinesin-1 at the growth cone. In contrast, CRMP2 overexpression promotes neurite elongation, a phenotype rescued by full-length PIPP, or expression of the CRMP2-binding PIPP domain. Therefore this study identifies PIPP and CRMP2 exert opposing roles in promoting axon selection and neurite elongation and the complex between these proteins serves to regulate the localization of effectors that promote neurite extension.

Pleiotropic effects of Syntaxin16 identified by gene editing in cultured adipocytes.

Adipocytes play multiple roles in the regulation of glucose metabolism which rely on the regulation of membrane traffic. These include secretion of adipokines and serving as an energy store. Central to their energy storing function is the ability to increase glucose uptake in response to insulin, mediated through translocation of the facilitative glucose transporter GLUT4 to the cell surface. The trans-Golgi reticulum localized SNARE protein syntaxin 16 (Sx16) has been identified as a key component of the secretory pathway required for insulin-regulated trafficking of GLUT4. We used CRISPR/Cas9 technology to generate 3T3-L1 adipocytes lacking Sx16 to understand the role of the secretory pathway on adipocyte function. GLUT4 mRNA and protein levels were reduced in Sx16 knockout adipocytes and insulin stimulated GLUT4 translocation to the cell surface was reduced. Strikingly, neither basal nor insulin-stimulated glucose transport were affected. By contrast, GLUT1 levels were upregulated in Sx16 knockout cells. Levels of sortilin and insulin regulated aminopeptidase were also increased in Sx16 knockout adipocytes which may indicate an upregulation of an alternative GLUT4 sorting pathway as a compensatory mechanism for the loss of Sx16. In response to chronic insulin stimulation, Sx16 knockout adipocytes exhibit elevated insulin-independent glucose transport and significant alterations in lactate metabolism. We further show that the adipokine secretory pathways are impaired in Sx16 knockout cells. Together this demonstrates a role for Sx16 in the control of glucose transport, the response to elevated insulin, cellular metabolic profiles and adipocytokine secretion.

GSK3 beta regulates myelin-dependent axon outgrowth inhibition through CRMP4.

Myelin-associated inhibitors (MAIs) contribute to failed regeneration in the CNS. The intracellular signaling pathways through which MAIs block axonal repair remain largely unknown. Here, we report that the kinase GSK3beta is directly phosphorylated and inactivated by MAIs, consequently regulating protein-protein interactions that are critical for myelin-dependent inhibition. Inhibition of GSK3beta mimics the neurite outgrowth inhibitory effect of myelin. The inhibitory effects of GSK3beta inhibitors and myelin are not additive indicating that GSK3beta is a major effector of MAIs. Consistent with this, overexpression of GSK3beta attenuates myelin inhibition. MAI-dependent phosphorylation and inactivation of GSK3beta regulate phosphorylation of CRMP4, a cytosolic regulator of myelin inhibition, and its ability to complex with RhoA. Introduction of a CRMP4 antagonist attenuates the neurite outgrowth inhibitory properties of GSK3beta inhibitors. We describe the first example of GSK3beta inactivation in response to inhibitory ligands and link the neurite outgrowth inhibitory effects of GSK3beta inhibition directly to CRMP4. These findings raise the possibility that GSK3beta inhibition will not effectively promote long-distance CNS regeneration following trauma such as spinal cord injury.

Models of dementia: an introductory overview.

The analysis of the molecular development of AD (Alzheimer's disease) is technically challenging, due to the chronic nature of the disease, the lack of early and definitive clinical diagnosis, and the fact that the abnormal molecular pathology occurs in the brain. Therefore appropriate animal models of AD are essential if we are to dissect the processes leading to molecular pathology, and ultimately to test the efficacy of potential therapies before clinical studies. Unfortunately, there is controversy over the benefits of the available models, the only consensus of opinion being that no perfect model currently exists. The investigation of animal models is extremely costly and time-consuming, therefore researchers tend to focus on one or two models. For scientists entering the AD research field, it can be difficult to identify the most appropriate model for their needs. Therefore the Models of Dementia: the Good, the Bad and the Future Biochemical Society Focused Meeting provided a platform for discussion and debate on the use and limitations of current models, the most appropriate methods for their characterization and identification of the most pressing needs of the field in general.

The genetic association of the transcription factor NPAT with glycemic response to metformin involves regulation of fuel selection.

The biguanide, metformin, is the first-choice therapeutic agent for type-2 diabetes, although the mechanisms that underpin metformin clinical efficacy remain the subject of much debate, partly due to the considerable variation in patient response to metformin. Identification of poor responders by genotype could avoid unnecessary treatment and provide clues to the underlying mechanism of action. GWAS identified SNPs associated with metformin treatment success at a locus containing the NPAT (nuclear protein, ataxia-telangiectasia locus) and ATM (ataxia-telangiectasia mutated) genes. This implies that gene sequence dictates a subsequent biological function to influence metformin action. Hence, we modified expression of NPAT in immortalized cell lines, primary mouse hepatocytes and mouse tissues, and analysed the outcomes on metformin action using confocal microscopy, immunoblotting and immunocytochemistry. In addition, we characterised the metabolic phenotype of npat heterozygous knockout mice and established the metformin response following development of insulin resistance. NPAT protein was localised in the nucleus at discrete loci in several cell types, but over-expression or depletion of NPAT in immortalised cell models did not change cellular responses to biguanides. In contrast, metformin regulation of respiratory exchange ratio (RER) was completely lost in animals lacking one allele of npat. There was also a reduction in metformin correction of impaired glucose tolerance, however no other metabolic abnormalities, or response to metformin, were found in the npat heterozygous mice. In summary, we provide methodological advancements for the detection of NPAT, demonstrate that minor reductions in NPAT mRNA levels (20-40%) influence metformin regulation of RER, and propose that the association between NPAT SNPs and metformin response observed in GWAS, could be due to loss of metformin modification of cellular fuel usage.

Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain.

Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3α and GSK3ß, while a splice variant of GSK3ß (GSK3ß2), containing a 13 amino acid insert, is enriched in neurons. GSK3α and GSK3ß deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, ß-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking GSK3ß, yet normal in cortex lacking GSK3α). Furthermore, the neuron-enriched GSK3ß2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3ß1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3ß isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3ß splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of GSK3ß1 and GSK3ß2 are similar.