Increased prefrontal cortex neurogranin enhances plasticity and extinction learning.

Increasing plasticity in neurons of the prefrontal cortex (PFC) has been proposed as a possible therapeutic tool to enhance extinction, a process that is impaired in post-traumatic stress disorder, schizophrenia, and addiction. To test this hypothesis, we generated transgenic mice that overexpress neurogranin (a calmodulin-binding protein that facilitates long-term potentiation) in the PFC. Neurogranin overexpression in the PFC enhanced long-term potentiation and increased the rates of extinction learning of both fear conditioning and sucrose self-administration. Our results indicate that elevated neurogranin function within the PFC can enhance local plasticity and increase the rate of extinction learning across different behavioral tasks. Thus, neurogranin can provide a molecular link between enhanced plasticity and enhanced extinction.

Neurogranin enhances synaptic strength through its interaction with calmodulin.

Learning-correlated plasticity at CA1 hippocampal excitatory synapses is dependent on neuronal activity and NMDA receptor (NMDAR) activation. However, the molecular mechanisms that transduce plasticity stimuli to postsynaptic potentiation are poorly understood. Here, we report that neurogranin (Ng), a neuron-specific and postsynaptic protein, enhances postsynaptic sensitivity and increases synaptic strength in an activity- and NMDAR-dependent manner. In addition, Ng-mediated potentiation of synaptic transmission mimics and occludes long-term potentiation (LTP). Expression of Ng mutants that lack the ability to bind to, or dissociate from, calmodulin (CaM) fails to potentiate synaptic transmission, strongly suggesting that regulated Ng-CaM binding is necessary for Ng-mediated potentiation. Moreover, knocking-down Ng blocked LTP induction. Thus, Ng-CaM interaction can provide a mechanistic link between induction and expression of postsynaptic potentiation.

Protective effect of 20-HETE inhibition in a model of oxygen-glucose deprivation in hippocampal slice cultures.

Recent studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) may have direct neuroprotective actions since they reduce infarct volume after ischemia reperfusion in the brain without altering blood flow. To explore this possibility, the present study used organotypic hippocampal slice cultures subjected to oxygen-glucose deprivation (OGD) and reoxygenation to examine whether 20-HETE is released by organotypic hippocampal slices after OGD and whether it contributes to neuronal death through the generation of ROS and activation of caspase-3. The production of 20-HETE increased twofold after OGD and reoxygenation. Blockade of the synthesis of 20-HETE with N-hydroxy-N'-(4-butyl-2-methylphenol)formamidine (HET0016) or its actions with a 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, reduced cell death, as measured by the release of lactate dehydrogenase and propidium iodide uptake. Administration of a 20-HETE mimetic, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (5,14-20-HEDE), had the opposite effect and increased injury after OGD. The death of neurons after OGD was associated with an increase in the production of ROS and activation of caspase-3. These effects were attenuated by HET0016 and potentiated after the administration of 5,14-20-HEDE. These findings indicate that the production of 20-HETE by hippocampal slices is increased after OGD and that inhibitors of the synthesis or actions of 20-HETE protect neurons from ischemic cell death. The protective effect of 20-HETE inhibitors is associated with a decrease in superoxide production and activation of caspase-3.

An IQSEC2 Mutation Associated With Intellectual Disability and Autism Results in Decreased Surface AMPA Receptors.

We have recently described an A350V mutation in IQSEC2 associated with intellectual disability, autism and epilepsy. We sought to understand the molecular pathophysiology of this mutation with the goal of developing targets for drug intervention. We demonstrate here that the A350V mutation results in interference with the binding of apocalmodulin to the IQ domain of IQSEC2. We further demonstrate that this mutation results in constitutive activation of the guanine nucleotide exchange factor (GEF) activity of IQSEC2 resulting in increased production of the active form of Arf6. In a CRISPR generated mouse model of the A350V IQSEC2 mutation, we demonstrate that the surface expression of GluA2 AMPA receptors in mouse hippocampal tissue was significantly reduced in A350V IQSEC2 mutant mice compared to wild type IQSEC2 mice and that there is a significant reduction in basal synaptic transmission in the hippocampus of A350V IQSEC2 mice compared to wild type IQSEC2 mice. Finally, the A350V IQSEC2 mice demonstrated increased activity, abnormal social behavior and learning as compared to wild type IQSEC2 mice. These findings suggest a model of how the A350V mutation in IQSEC2 may mediate disease with implications for targets for drug therapy. These studies provide a paradigm for a personalized approach to precision therapy for a disease that heretofore has no therapy.

Pull-down of calmodulin-binding proteins.

Calcium (Ca(2+)) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca(2+) signaling is mediated through the calcium-binding protein known as calmodulin (CaM). CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca(2+) (Ca(2+)-CaM) as opposed to its Ca(2+)-free state (ApoCaM). While most target proteins bind Ca(2+)-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin, neurogranin (Ng), and certain myosins. These proteins have been shown to play important roles in presynaptic function, postsynaptic function, and muscle contraction, respectively. Their ability to bind and release CaM in the absence or presence of Ca(2+) is pivotal in their function. In contrast, many proteins only bind Ca(2+)-CaM and require this binding for their activation. Examples include myosin light chain kinase, Ca(2+)/CaM-dependent kinases (CaMKs) and phosphatases (e.g. calcineurin), and spectrin kinase, which have a variety of direct and downstream effects. The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca(2+)-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca(2+)-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM. Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca(2+), using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca(2+) on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca(2+)/CaM signaling and proteins that interact with CaM.