Grin2A (N596S): a new murine model for audiogenic seizure (AGS) and attention-deficit hyperactivity disorder (ADHD)

The voltage dependent Mg2+ block of GluN2A-containing NMDARs is essential for activity-induced neuroplasticity. The GRIN2A gene, encoding the NMDAR subunits GluN2A, was found to be disrupted or mutated in individuals with mental retardation and/or epilepsy. In my thesis work I performed behavioral test, genotyping, western-blot analysis and many different histological technics to analyze protein expression and anatomical structures of neurons and glia of mice carrying a gene targeted point mutation, Grin2A(N596S), in the gene encoding NMDAR subunit GluN2A. This mutation causes the loss of the Mg2+ block and a decrease in Ca2+ permeability and it is homologous to a human de novo point mutation Grin2A(N615K), found in a young patient with severe mental retardation and epileptic seizures (Endele S. et al., 2010). As in humans, mice homozygous for the mutation (Grin2aS/S) have profound cognitive impairments and, analyzing their performance in a battery of behavioral test such as the Burrowing test, the Crawley’s sociability test, the Cliff avoidance reaction (CAR) test and the passive avoidance (PA) test, emerged that Grin2aS/S mice express also features related to ADHD, which are hyperactivity, inattention and impulsivity. On the other hand, in heterozygous Grin2aS/N mice the mutation had a lower penetrance, resulting in an intermediate behavior and ambiguous ADHD-like phenotype compared to the other two groups of mice, Grin2aS/S and Grin2aN/N courts phenotype. Grin2aS/S mice exhibited a 100% penetrance of generalized convulsive seizure induced by an external stimulus (11 kHz tone), namely Audiogenic Seizure (AGS). The AGS ended always with respiratory arrest and the death, resembling the same human process called SUDEP (sudden unexpected death in epilepsy). The epileptic phenotype could be genetically rescued in a different genetic background of FVB mice or pharmacologically by low doses of memantine. Western blot analysis reveled no differences in NMDAR subunits protein expression and other kinds of scaffolding proteins enriched in the PSD in forebrain membrane fraction of P28 Grin2aS/S, Grin2aS/N and Grin2aN/N mice. Moreover, c-fos and arc expression were verified by immunofluorescence after tone induction, confirming published data about AGS-related brain circuit. I also examined the presence of gliosis and neurodegeneration, 24 hours and 8 days after seizure induction, discovering a GFAP (glial fibrillary acidic protein) reduction in some brain areas and gliosis-plaque in the cortex of Grin2aS/S mice; furthermore, Silver and NeuN stainings highlighted signs of neurodegeneration in the same brain areas related to AGS. Finally, using the Golgi impregnation technique I compared the normal brain development and neuronal structure in mutated and wild-type mice. In summary, the results of my research revealed an ADHD-like phenotype in Grin2aS/S mice, coupled with a strong epileptic phenotype, which caused the death of the animal and some molecular adaptive responses in the brain circuit related to the seizure, but that it could be prevented by memantine, a drug commonly used in therapy