Ramakrishnapillai Vyomakesannair Omkumar

Background: Neuronal transmission through the N -methyl- d -aspartate receptor (NMDAR) is a key event in synaptic plasticity and excitotoxicity. The channel properties and biochemical signalling activities of the receptor are regulated by phosphatases such as protein phosphatase 1. While the immediate consequences of NMDAR activation have been reported previously, the long-term behavioural changes remain unclear. Purpose: We attempted to investigate the long-term behavioural effects of N -methyl- d -aspartic acid (NMDA) injection and the role of phosphatases during NMDAR signalling. Methods: NMDAR was activated by stereotaxic injection of NMDA into the prefrontal cortex of adult rats. To elucidate the role of phosphatases in mediating NMDAR signalling and associated animal behaviour, okadaic acid (OA), a phosphatase inhibitor, was administered before NMDA injection. The animals were tested for their general locomotion and cognitive function using behavioural assays. Results: A single injection of NMDA impaired cognition in the long term. Interestingly, intracortical OA injection resulted in increased locomotor activity and decreased anxiety-like behaviour in animals without major cognitive effects. Conclusion: We demonstrate that the inhibition of phosphatases during NMDAR signalling can affect locomotion and anxiety-like behaviour in adult male rats. Our study underscores the potential of modulating phosphatases as a pharmacological target for anxiety disorders.

MicroRNAs (miRNAs) play pivotal roles in fine-tuning gene regulation. Understanding the mechanism of action of such miRNAs might help in manipulating the respective pathways thus providing therapeutic options. We have investigated the physiological roles of two miRNAs, miR-146a and miR-200b, that are differentially expressed in neurological disorders such as Alzheimer’s disease andschizophrenia.We specifically studied their involvement in learning and memory mechanisms. We show through bioinformatics prediction tools that these miRNAs can interact with transcripts of the N-methyl-D-aspartate receptor (NMDAR) subunits Grin2A and Grin2B . This was further supported by showing interaction of the miRNAs to the 3’UTR sequences of Grin2A and Grin2B through luciferase assay. Overexpression of these miRNAs in primary hippocampal neurons caused downregulation of GluN2B and GluN2A protein levels. Stereotactic injections of these miRNAs into rat hippocampus caused cognitive deficits in multiple behavioural tests along with decreased protein levels of the NMDAR subunits, GluN1, GluN2A and GluN2B, AMPAR subunit GluR1 and Neuregulin 1 (NRG1). During downregulation of NMDAR subunits by other physiological stimuli as in pharmacologically treated rat models [MK-801 treated and methylazoxymethanol acetate (MAM) treated], we found upregulated levels of miR-146a-5p and miR-200b-3p implying their involvement in downregulating NMDAR subunits. These results suggest the importance of miR-146a-5p and miR-200b-3p in mediating gene regulation in the hippocampus and their involvement in hippocampus dependent learning and memory.

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.

N-methyl-D-aspartate receptors (NMDAR) are downregulated in schizophrenia possibly through microRNAs (miRNAs) that are differentially expressed in this condition. 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 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 neuregulin 1 ( NRG1 ), BDNF and CaMKIIα , genes implicated in schizophrenia, were also altered in these models. This is the first report of downregulation of GluN2A and GluN2B by miR-296, miR-148b and miR-129-2. Mining miRNAs regulating NMDA receptors might give insights into the pathophysiology of this disorder, providing avenues in therapeutics.

The complex and multifactorial nature of neuropsychiatric diseases demands multi-target drugs that can intervene with various sub-pathologies underlying disease progression. Targeting the impairments in cholinergic and glutamatergic neurotransmissions with small molecules has been suggested as one of the potential disease-modifying approaches for Alzheimer’s disease (AD). Tacrine, a potent inhibitor of acetylcholinesterase (AChE) is the first FDA approved drug for the treatment of AD. Tacrine is also a low affinity antagonist of N-methyl-D-aspartate receptor (NMDAR). However, tacrine was withdrawn from its clinical use later due to its hepatotoxicity. With an aim to develop novel high affinity multi-target directed ligands (MTDLs) against AChE and NMDAR, with reduced hepatotoxicity, we performed in silico structure-based modifications on tacrine, chemical synthesis of the derivatives and in vitro validation of their activities. Nineteen such derivatives showed inhibition with IC₅₀ values in the range of 18.53 ± 2.09 – 184.09 ± 19.23 nM against AChE and 0.27 ± 0.05 – 38.84 ± 9.64 μM against NMDAR. Some of the selected compounds also protected rat primary cortical neurons from glutamate induced excitotoxicity. Two of the tacrine derived MTDLs, 201 and 208 exhibited in vivo efficacy in rats by protecting against behavioral impairment induced by administration of the excitotoxic agent, monosodium glutamate. Additionally, several of these synthesized compounds also exhibited promising inhibitory activitiy against butyrylcholinesterase. MTDL-201 was also devoid of hepatotoxicity in vivo. Given the therapeutic potential of MTDLs in disease-modifying therapy, our studies revealed several promising MTDLs among which 201 appears to be a potential candidate for immediate preclinical evaluations.

The complex and multifactorial nature of neuropsychiatric diseases demands multi-target drugs that can intervene with various sub-pathologies underlying disease progression. Targeting the impairments in cholinergic and glutamatergic neurotransmissions with small molecules has been suggested as one of the potential disease-modifying approaches for Alzheimer’s disease (AD). Tacrine, apotent inhibitor of acetylcholinesterase (AChE) is the first FDA approved drug for the treatment of AD. Tacrine is also a low affinity antagonist of N-methyl-D-aspartate receptor (NMDAR). However, tacrine was withdrawn from its clinical use later due to its hepato-toxicity. With an aim to develop novel high affinity multi-target directed ligands (MTDLs) against AChE and NMDAR, with reduced hepatotoxicity, we performed in silico structure-based modifications on tacrine, chemical synthesis of the derivatives and in vitro validation of their activities. Nineteen such derivatives showed inhibition with IC ₅₀ values in the range of 18.53±2.09 to 184.09±19.23 nM against AChE and 0.27±0.05 to 38.84±9.64 μM against NMDAR. Some of the selected compounds also protected rat primary cortical neurons from glutamate induced excitotoxicity. Two of the tacrine derived MTDLs, 201 and 208 exhibited in vivo efficacy in rats by protecting against behavioral impairment induced by administration of the excitotoxic agent, monosodium glutamate. Additionally, several of these synthesized compounds also exhibited promising inhibitory activities against butyrylcholinesterase and β-secretase. Given the therapeutic potential of MTDLs in disease-modifying therapy, our studies revealed several promising MTDLs of which 201 appears to be a potential candidate for immediate preclinical and clinical evaluations.