AKAP79/150 coordinates leptin-induced PKA activation to regulate KATP channel trafficking in pancreatic β-cells

The adipocyte hormone leptin regulates glucose homeostasis both centrally and peripherally. A key peripheral target is the pancreatic β-cell, which secretes insulin upon glucose stimulation. Leptin suppresses glucose-stimulated insulin secretion by promoting trafficking of KATP channels to the β-cell surface, which increases K+ conductance and causes β-cell hyperpolarization. Here we investigate the signaling mechanism underlying leptin-induced KATP channel translocation with a focus on protein kinase A (PKA). Using FRET-based PKA activity reporters, we show that leptin increases PKA activity at the cell membrane via a signaling pathway involving NMDA receptors, CaMKKβ and AMPK. Genetic knockdown and rescue experiments reveal that leptin activation of PKA requires tethering of PKA to the membrane-targeted PKA-anchoring protein AKAP79/150. Interestingly, disrupting protein phosphatase 2B (PP2B) anchoring to AKAP79/150, known to elevate basal PKA signaling, increases surface KATP channels. Our findings uncover a novel role of AKAP79/150 in coordinating leptin and PKA signaling to regulate β-cell function.

Brain sterol flux mediated by cytochrome P450 46A1 affects membrane properties and membrane-dependent processes

Cytochrome P450 46A1 encoded by CYP46A1 catalyzes cholesterol 24-hydroxylation and is a CNS-specific enzyme that controls cholesterol removal and turnover in the brain. Accumulating data suggest that increases in cytochrome P450 46A1 activity in mouse models of common neurodegenerative diseases affect various, apparently unlinked biological processes and pathways. Yet, the underlying reason for these multiple enzyme activity effects is currently unknown. Herein, we tested the hypothesis that cytochrome P450 46A1-mediated sterol flux alters physico-chemical properties of the plasma membranes and thereby membrane-dependent events. We used 9-month-old 5XFAD mice (an Alzheimer’s disease model) treated for 6 months with the anti-HIV drug efavirenz. These animals have previously been shown to have improved behavioural performance, increased cytochrome P450 46A1 activity in the brain, and increased sterol flux through the plasma membranes. We further examined 9-month-old Cyp46a1−/− mice, which have previously been observed to have cognitive deficits and decreased sterol flux through brain membranes. Synaptosomal fractions from the brain of efavirenz-treated 5XFAD mice had essentially unchanged cholesterol levels as compared to control 5XFAD mice. However with efavirenz treatment in these mice, there were changes in the membrane properties (increased cholesterol accessibility, ordering, osmotic resistance and thickness) as well as total glutamate content and ability to release glutamate in response to mild stimulation. Similarly, the cholesterol content in synaptosomal fractions from the brain of Cyp46a1−/− mice was essentially the same as in wild-type mice but knockout of Cyp46a1 was associated with changes in membrane properties and glutamate content and its exocytotic release. Changes in Cyp46a1−/− mice were in the opposite direction to those observed in efavirenz-treated versus control 5XFAD mice. Incubation of synaptosomal fractions with the inhibitors of glycogen synthase kinase 3, cyclin-dependent kinase 5, protein phosphatase 1/2 A, and protein phosphatase 2B revealed that increased sterol flux in efavirenz-treated versus control 5XFAD mice affected the ability of all four enzymes to modulate glutamate release. In contrast, in Cyp46a1−/− versus wild-type mice, decreased sterol flux altered the ability of only cyclin-dependent kinase 5 and protein phosphatase 2B to regulate the glutamate release. Collectively, our results support cytochrome P450 46A1-mediated sterol flux as an important contributor to the fundamental properties of the membranes, protein phosphorylation and synaptic transmission. Also, our data provide an explanation of how one enzyme, cytochrome P450 46A1, can affect multiple pathways and processes and serve as a common potential target for several neurodegenerative disorders.

Binding of Filamentous Actin to CaMKII as a Potential Mechanism for the Regulation of Bidirectional Synaptic Plasticity by βCaMKII in Cerebellar Purkinje Cells

Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but little is known about its functional role during plasticity induction in the cerebellum. Experiments have indicated that the β isoform of CaMKII controls the bidirectional inversion of plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses in cerebellar cortex. Because the cellular events that underlie these experimental findings are still poorly understood, we developed a simple computational model to investigate how βCaMKII regulates the direction of plasticity in cerebellar PCs. We present the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. Our simulation results suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors can determine whether LTD or LTP occurs in cerebellar PCs. The model replicates experimental observations that indicate that βCaMKII controls the direction of plasticity at PF-PC synapses, and demonstrates that the binding of filamentous actin to CaMKII can enable the β isoform of the kinase to regulate bidirectional plasticity at these synapses.Author SummaryMany molecules and the complex interactions between them are involved in synaptic plasticity in the cerebellum. However, the exact relationship between cerebellar plasticity and the different signalling cascades remains unclear. Calcium-calmodulin dependent protein kinase II (CaMKII) is an important component of the signalling network that is responsible for plasticity in cerebellar Purkinje cells (PCs). The CaMKII holoenzyme contains different isoforms such as αCaMKII and βCaMKII. Experiments with Camk2b knockout mice that lack the β isoform of CaMKII demonstrated that βCaMKII regulates the direction of plasticity at parallel fibre (PF)-PC synapses. Stimulation protocols that induce long-term depression in wild-type mice, which contain both α and βCaMKII, lead to long-term potentiation in knockout mice without βCaMKII, and vice versa. We developed a kinetic simulation of the phosphorylation and dephosphorylation of AMPA receptors by CaMKII and protein phosphatase 2B to investigate how βCaMKII can control bidirectional synaptic plasticity in cerebellar PCs. Our simulation results demonstrate that the binding of filamentous actin to βCaMKII can contribute to the regulation of bidirectional plasticity at PF-PC synapses. Our computational model of intracellular signalling significantly advances the understanding of the mechanisms of synaptic plasticity induction in the cerebellum.

Binding of Filamentous Actin to CaMKII as a Potential Mechanism for the Regulation of Bidirectional Synaptic Plasticity by βCaMKII in Cerebellar Purkinje Cells

Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but little is known about its functional role during plasticity induction in the cerebellum. Experiments have indicated that the β isoform of CaMKII controls the bidirectional inversion of plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses in cerebellar cortex. Because the cellular events that underlie these experimental findings are still poorly understood, we developed a simple computational model to investigate how βCaMKII regulates the direction of plasticity in cerebellar PCs. We present the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. Our simulation results suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors can determine whether LTD or LTP occurs in cerebellar PCs. The model replicates experimental observations that indicate that βCaMKII controls the direction of plasticity at PF-PC synapses, and demonstrates that the binding of filamentous actin to CaMKII can enable the β isoform of the kinase to regulate bidirectional plasticity at these synapses.Author SummaryMany molecules and the complex interactions between them are involved in synaptic plasticity in the cerebellum. However, the exact relationship between cerebellar plasticity and the different signalling cascades remains unclear. Calcium-calmodulin dependent protein kinase II (CaMKII) is an important component of the signalling network that is responsible for plasticity in cerebellar Purkinje cells (PCs). The CaMKII holoenzyme contains different isoforms such as αCaMKII and βCaMKII. Experiments with Camk2b knockout mice that lack the β isoform of CaMKII demonstrated that βCaMKII regulates the direction of plasticity at parallel fibre (PF)-PC synapses. Stimulation protocols that induce long-term depression in wild-type mice, which contain both α and βCaMKII, lead to long-term potentiation in knockout mice without βCaMKII, and vice versa. We developed a kinetic simulation of the phosphorylation and dephosphorylation of AMPA receptors by CaMKII and protein phosphatase 2B to investigate how βCaMKII can control bidirectional synaptic plasticity in cerebellar PCs. Our simulation results demonstrate that the binding of filamentous actin to βCaMKII can contribute to the regulation of bidirectional plasticity at PF-PC synapses. Our computational model of intracellular signalling significantly advances the understanding of the mechanisms of synaptic plasticity induction in the cerebellum.