Phillips, M. B. | bioRxiv Science

bioRxiv2024

State-specific inhibition of NMDA receptors by memantine depends on intracellular calcium and provides insights into NMDAR channel blocker tolerability

NMDA receptors (NMDARs) are key mediators of neuronal Ca2+ influx. NMDAR-mediated Ca2+ influx plays a central role in synaptogenesis, synaptic plasticity, dendritic integration, and neuronal survival. However, excessive NMDAR-mediated Ca2+ influx initiates cellular signaling pathways that result in neuronal death and is broadly associated with neurological disease. Drugs targeting NMDARs are of great clinical interest, but widespread alteration of NMDAR activity can generate negative side effects. The NMDAR channel blocker memantine is a well-tolerated Alzheimers disease medication that shows promise in treatment of other neurological disorders. Memantine enhances desensitization of NMDARs in a subtype- and Ca2+-dependent manner, thereby more effectively inhibiting NMDARs on neurons that experience increased buildup of intracellular Ca2+. However, little is known about the properties or implications of the interaction between intracellular Ca2+ and NMDAR inhibition by memantine or other NMDAR channel blockers. Utilizing customized Ca2+ buffering solutions and whole-cell patch-clamp recordings, we demonstrated that memantine inhibition of both recombinant and native NMDARs increases with increasing intracellular Ca2+ and that the effect of intracellular Ca2+ on memantine action depends on NMDAR subtype. Neuroprotection assays and recordings of postsynaptic currents revealed that memantine preferentially inhibits NMDARs under neurotoxic conditions whereas ketamine, a clinically useful NMDAR channel blocker with strong side effects, inhibits strongly across contexts. Our results present a previously unexamined form of state-specific antagonism, Ca2+-dependent NMDAR channel block, that could have a profound impact on the design of drugs that selectively target NMDAR subpopulations involved in disease.

neuroscience↗

bioRxiv2021

The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis

Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein 1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was non-lethal in wild-type mice, whereas greater than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, and extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 compared to T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a non-lethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo, which potentiates the capacity of reovirus to cause myocarditis. IMPORTANCEReovirus is a non-enveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the non-lethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.

microbiology↗