Restoring neuronal chloride extrusion reverses cognitive decline linked to Alzheimer's disease mutations.

Disinhibition during early stages of Alzheimer's disease is postulated to cause network dysfunction and hyperexcitability leading to cognitive deficits. However, the underlying molecular mechanism remains unknown. Here we show that, in mouse lines carrying Alzheimer's disease-related mutations, a loss of neuronal membrane potassium-chloride cotransporter KCC2, responsible for maintaining the robustness of GABAA-mediated inhibition, occurs pre-symptomatically in the hippocampus and prefrontal cortex. KCC2 downregulation was inversely correlated with the age-dependent increase in amyloid-ß 42 (Aß42). Acute administration of Aß42 caused a downregulation of membrane KCC2. Loss of KCC2 resulted in impaired chloride homeostasis. Preventing the decrease in KCC2 using long term treatment with CLP290 protected against deterioration of learning and cortical hyperactivity. In addition, restoring KCC2, using short term CLP290 treatment, following the transporter reduction effectively reversed spatial memory deficits and social dysfunction, linking chloride dysregulation with Alzheimer's disease-related cognitive decline. These results reveal KCC2 hypofunction as a viable target for treatment of Alzheimer's disease-related cognitive decline; they confirm target engagement, where the therapeutic intervention takes place, and its effectiveness.

Sex-Specific Retinal Anomalies Induced by Chronic Social Defeat Stress in Mice.

Major depressive disorder (MDD) is one of the most common consequences of chronic stress. Still, there is currently no reliable biomarker to detect individuals at risk to develop the disease. Recently, the retina emerged as an effective way to investigate psychiatric disorders using the electroretinogram (ERG). In this study, cone and rod ERGs were performed in male and female C57BL/6 mice before and after chronic social defeat stress (CSDS). Mice were then divided as susceptible or resilient to stress. Our results suggest that CSDS reduces the amplitude of both oscillatory potentials and a-waves in the rods of resilient but not susceptible males. Similar effects were revealed following the analysis of the cone b-waves, which were faster after CSDS in resilient mice specifically. In females, rod ERGs revealed age-related changes with no change in cone ERGs. Finally, our analysis suggests that baseline ERG can predict with an efficacy up to 71% the expression of susceptibility and resilience before stress exposition in males and females. Overall, our findings suggest that retinal activity is a valid biomarker of stress response that could potentially serve as a tool to predict whether males and females will become susceptible or resilient when facing CSDS.

FXR1P is a GSK3ß substrate regulating mood and emotion processing.

Inhibition of glycogen synthase kinase 3ß (GSK3ß) is a shared action believed to be involved in the regulation of behavior by psychoactive drugs such as antipsychotics and mood stabilizers. However, little is known about the identity of the substrates through which GSK3ß affects behavior. We identified fragile X mental retardation-related protein 1 (FXR1P), a RNA binding protein associated to genetic risk for schizophrenia, as a substrate for GSK3ß. Phosphorylation of FXR1P by GSK3ß is facilitated by prior phosphorylation by ERK2 and leads to its down-regulation. In contrast, behaviorally effective chronic mood stabilizer treatments in mice inhibit GSK3ß and increase FXR1P levels. In line with this, overexpression of FXR1P in the mouse prefrontal cortex also leads to comparable mood-related responses. Furthermore, functional genetic polymorphisms affecting either FXR1P or GSK3ß gene expression interact to regulate emotional brain responsiveness and stability in humans. These observations uncovered a GSK3ß/FXR1P signaling pathway that contributes to regulating mood and emotion processing. Regulation of FXR1P by GSK3ß also provides a mechanistic framework that may explain how inhibition of GSK3ß can contribute to the regulation of mood by psychoactive drugs in mental illnesses such as bipolar disorder. Moreover, this pathway could potentially be implicated in other biological functions, such as inflammation and cell proliferation, in which FXR1P and GSK3 are known to play a role.

A role for cytosolic isocitrate dehydrogenase as a negative regulator of glucose signaling for insulin secretion in pancreatic ß-cells.

Cytosolic NADPH may act as one of the signals that couple glucose metabolism to insulin secretion in the pancreatic ß-cell. NADPH levels in the cytoplasm are largely controlled by the cytosolic isoforms of malic enzyme and isocitrate dehydrogenase (IDHc). Some studies have provided evidence for a role of malic enzyme in glucose-induced insulin secretion (GIIS) via pyruvate cycling, but the role of IDHc in ß-cell signaling is unsettled. IDHc is an established component of the isocitrate/α-ketoglutarate shuttle that transfers reducing equivalents (NADPH) from the mitochondrion to the cytosol. This shuttle is energy consuming since it is coupled to nicotinamide nucleotide transhydrogenase that uses the mitochondrial proton gradient to produce mitochondrial NADPH and NAD(+) from NADP(+) and NADH. To determine whether flux through IDHc is positively or negatively linked to GIIS, we performed RNAi knockdown experiments in ß-cells. Reduced IDHc expression in INS 832/13 cells and isolated rat islet ß-cells resulted in enhanced GIIS. This effect was mediated at least in part via the KATP-independent amplification arm of GIIS. IDHc knockdown in INS 832/13 cells did not alter glucose oxidation but it reduced fatty acid oxidation and increased lipogenesis from glucose. Metabolome profiling in INS 832/13 cells showed that IDHc knockdown increased isocitrate and NADP(+) levels. It also increased the cellular contents of several metabolites linked to GIIS, in particular some Krebs cycle intermediates, acetyl-CoA, glutamate, cAMP and ATP. The results identify IDHc as a component of the emerging pathways that negatively regulate GIIS.

Spatial intensity distribution analysis (SpIDA): a new tool for receptor tyrosine kinase activation and transactivation quantification.

This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G protein-coupled receptors. A custom graphical user interface developed for MATLAB is used to extract quantal brightness and receptor density information from intensity histograms calculated from single fluorescence microscopy images. This approach allows measurement of monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. Application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In addition, because of its compatibility with conventional immunostaining approaches, SpIDA is suitable not only for use in recombinant systems but also for the characterization of mechanisms involving endogenous proteins. Therefore, SpIDA enables these biological processes to be monitored directly in their native cellular environment.

Quantification of receptor tyrosine kinase activation and transactivation by G-protein-coupled receptors using spatial intensity distribution analysis (SpIDA).

This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to the pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G-protein-coupled receptors. Intensity histograms are generated from single fluorescence microscopy images. These histograms are then fit with Poissonian distributions to obtain density maps and quantal brightness values of the labeled proteins underlying the images. This approach allows resolving monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. The application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In contrast to methods based on resonance energy transfer, SpIDA is suitable not only for use in recombinant systems but also for the characterization of mechanisms involving endogenous proteins. Therefore, SpIDA enables these biological processes to be monitored directly in their native cellular environment.

Glucolipotoxicity alters lipid partitioning and causes mitochondrial dysfunction, cholesterol, and ceramide deposition and reactive oxygen species production in INS832/13 ss-cells.

Elevated glucose and saturated fatty acids synergize in inducing apoptosis in INS832/13 cells and in human islet cells. In order to gain insight into the molecular mechanism(s) of glucolipotoxicity (Gltox), gene profiling and metabolic analyses were performed in INS832/13 cells cultured at 5 or 20 mm glucose in the absence or presence of palmitate. Expression changes were observed for transcripts involved in mitochondrial, lipid, and glucose metabolism. At 24 h after Gltox, increased expression of lipid partitioning genes suggested a promotion of fatty acid esterification and reduced lipid oxidation/detoxification, whereas changes in the expression of energy metabolism genes suggested mitochondrial dysfunction. These changes were associated with decreased glucose-induced insulin secretion, total insulin content, ATP levels, AMP-kinase activity, mitochondrial membrane potential and fat oxidation, unchanged de novo fatty acid synthesis, and increased reactive oxygen species, cholesterol, ceramide, and triglyceride levels. However, the synergy between elevated glucose and palmitate to cause ss-cell toxicity in term of apoptosis and reduced glucose-induced insulin secretion only correlated with triglyceride and ceramide depositions. Overexpression of endoplasmic reticulum glycerol-3-phosphate acyl transferase to enhance lipid esterification amplified Gltox at intermediate glucose (11 mm), whereas reducing acetyl-coenzyme A carboxylase 1 expression by small interfering RNA to shift lipid partitioning to fat oxidation reduced Gltox. The results suggest that Gltox entails alterations in lipid partitioning, sterol and ceramide accumulation, mitochondrial dysfunction, and reactive oxygen species production, all contributing to altering ss-cell function. The data also suggest that the early promotion of lipid esterification processes is instrumental in the Gltox process.

A role for the malonyl-CoA/long-chain acyl-CoA pathway of lipid signaling in the regulation of insulin secretion in response to both fuel and nonfuel stimuli.

The malonyl-CoA/long-chain acyl-CoA (LC-CoA) model of glucose-induced insulin secretion (GIIS) predicts that malonyl-CoA derived from glucose metabolism inhibits fatty acid oxidation, thereby increasing the availability of LC-CoA for lipid signaling to cellular processes involved in exocytosis. For directly testing the model, INSr3 cell clones overexpressing malonyl-CoA decarboxylase in the cytosol (MCDc) in a tetracycline regulatable manner were generated, and INS(832/13) and rat islets were infected with MCDc-expressing adenoviruses. MCD activity was increased more than fivefold, and the malonyl-CoA content was markedly diminished. This was associated with enhanced fat oxidation at high glucose, a suppression of the glucose-induced increase in cellular free fatty acid (FFA) content, and reduced partitioning at elevated glucose of exogenous palmitate into lipid esterification products. MCDc overexpression, in the presence of exogenous FFAs but not in their absence, reduced GIIS in all beta-cell lines and in rat islets. It also markedly curtailed the stimulation of insulin secretion by other fuel and nonfuel secretagogues. In the absence of MCDc overexpression, the secretory responses to all types of secretagogues were amplified by the provision of exogenous fatty acids. In the presence of exogenous FFAs, the fatty acyl-CoA synthetase inhibitor triacsin C reduced secretion in response to glucose and nonfuel stimuli. The data show the existence of important links between the metabolic coupling factor malonyl-CoA, the partitioning of fatty acids, and the stimulation of insulin secretion to both fuel and nonfuel stimuli.