Glu60 of α-Calcium/calmodulin dependent protein kinase II mediates crosstalk between the regulatory T-site and protein substrate binding region of the active site.

Memory formation transpires to be by activation and persistent modification of synapses. A chain of biochemical events accompany synaptic activation and culminate in memory formation. These biochemical events are steered by interplay and modulation of various synaptic proteins, achieved by conformational changes and phosphorylation/dephosphorylation of these proteins. Calcium/calmodulin dependent protein kinase II (CaMKII) and N-methyl-d-aspartate receptors (NMDARs) are synaptic proteins whose interactions play a pivotal role in learning and memory process. Catalytic activity of CaMKII is modulated upon its interaction with the GluN2B subunit of NMDAR. The structural basis of this interaction is not clearly understood. We have investigated the role of Glu60 of α-CaMKII, a conserved residue present in the ATP binding region of kinases, in the regulation of catalysis of CaMKII by GluN2B. Mutation of Glu60 to Gly exerts different effects on the kinetic parameters of phosphorylation of GluN2B and GluN2A, of which only GluN2B binds to the T-site of CaMKII. GluN2B induced modulation of the kinetic parameters of peptide substrate was altered in the E60G mutant. The mutation almost abolished the modulation of the apparent Km value for protein substrate. However, although kinetic parameters for ATP were altered by mutating Glu60, modulation of the apparent Km value for ATP by GluN2B seen in WT was exhibited by the E60G mutant of α-CaMKII. Hence our results posit that the communication of the T-site of CaMKII with protein substrate binding region of active site is mediated through Glu60 while the communication of the T-site with the ATP binding region is not entirely dependent on Glu60.

miR-146a and miR-200b alter cognition by targeting NMDA receptor subunits.

MicroRNAs fine-tune gene regulation and can be targeted for therapeutic purposes. We investigated the physiological roles of miR-146a and miR-200b that are differentially expressed in neurological disorders such as Alzheimer's disease and schizophrenia, particularly in learning and memory mechanisms. Using bioinformatics tools and luciferase assay, we show interaction of these miRNAs with transcripts of N-methyl-D-aspartate receptor (NMDAR) subunits Grin2A and Grin2B. Overexpression of these miRNAs in primary hippocampal neurons caused downregulation of GluN2B and GluN2A proteins. Stereotactic injections of these miRNAs into rat hippocampus caused cognitive deficits in multiple behavioral tests with decreased protein levels of GluN1, GluN2A, GluN2B, AMPAR subunit GluR1, and Neuregulin 1. In pharmacologically treated rat models [MK-801 treated and methylazoxymethanol acetate (MAM) treated], we found upregulated levels of these miRNAs, implying their involvement in downregulating NMDAR subunits in these models. These results suggest the importance of miR-146a-5p and miR-200b-3p in hippocampus-dependent learning and memory.

Differential expression of miR-148b, miR-129-2 and miR-296 in animal models of schizophrenia-Relevance to NMDA receptor hypofunction.

Hypofunction of N-methyl-d-aspartate receptors (NMDAR) is a key component in the pathophysiology of schizophrenia. Alterations in the regulation of NMDARs by microRNAs (miRNAs) are possible since numerous miRNAs are differentially expressed in post mortem schizophrenia brain samples. We screened the miRNAs that are altered in schizophrenia against the targets, Grin2A and Grin2B subunits of NMDAR using bioinformatic tools. Among the predicted miRNAs some interacted with the 3'-UTR sequences of Grin2A (miR-296, miR-148b, miR-129-2, miR-137) and Grin2B (miR-296, miR-148b, miR-129-2, miR-223) in dual luciferase assays. This was supported by downregulation of the GluN2B protein in primary hippocampal neurons upon overexpressing Grin2B targeting miRNAs. In two models of schizophrenia-pharmacological MK-801 model and neurodevelopmental methylazoxymethanol acetate (MAM) model which showed cognitive deficits - protein levels of GluN2A and GluN2B were downregulated but their transcript levels were upregulated. miR-296-3p, miR-148b-5p and miR-137-3p levels showed upregulation in both models which could have interacted with Grin2A/Grin2B transcripts resulting in translational arrest. In MAM model, reciprocal changes in the expression of the 3p and 5p forms of miR-148b and miR-137 were observed. Expression of some genes implicated in schizophrenia such as neuregulin 1, BDNF and CaMKIIα, were also altered in these models. This is the first report showing downregulation of GluN2A and GluN2B by miR-296, miR-148b and miR-129-2 in vitro and association between them in animal models. Mining miRNAs regulating NMDA receptors might give insights into the pathophysiology of this disorder, providing avenues in therapeutics.

Role of Ca2+/Calmodulin-Dependent Protein Kinase Type II in Mediating Function and Dysfunction at Glutamatergic Synapses.

Glutamatergic synapses harbor abundant amounts of the multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMKII). Both in the postsynaptic density as well as in the cytosolic compartment of postsynaptic terminals, CaMKII plays major roles. In addition to its Ca2+-stimulated kinase activity, it can also bind to a variety of membrane proteins at the synapse and thus exert spatially restricted activity. The abundance of CaMKII in glutamatergic synapse is akin to scaffolding proteins although its prominent function still appears to be that of a kinase. The multimeric structure of CaMKII also confers several functional capabilities on the enzyme. The versatility of the enzyme has prompted hypotheses proposing several roles for the enzyme such as Ca2+ signal transduction, memory molecule function and scaffolding. The article will review the multiple roles played by CaMKII in glutamatergic synapses and how they are affected in disease conditions.

Cellular calcium signaling in the aging brain.

Aging in the biological system is an irreversible process. In the initial stages of lifespan aging improves survival skills of an organism while in the later stages aging reduce the survival skills. Aging is associated with changes in several cellular and molecular functions among which calcium signaling is a prominent one. Calcium signaling is essential for many vital functions of the brain and even minor impairments in calcium signaling can lead to deleterious consequences including neuronal death. Calcium signaling proteins are pursued as promising drug targets for many aging related diseases. This review attempts to summarize changes in calcium signaling in the brain as a result of aging.