Abstracts

Rationale: A gain-of-function missense mutation in GRIN2D (V667I) was previously identified in several children with severe developmental and epileptic encephalopathy. By 12 months, children experience intervention-refractory seizures, including atypical absence, complex partial and generalized, accompanied by developmental delay, axial hypotonia and speech disorder. To understand how this mutation leads to altered brain development and severe neurological phenotypes and obtain a model for potential therapy, we developed constitutive and conditional (cKI) mouse lines carrying the orthologous mutation, V664I.

Methods: The V664I mutation was generated by CRISPR/Cas9 mutagenesis in C57BL/6J zygotes. Acute iEEG was performed in mouse pups using Neurogrid arrays. Chronic EEG in adult mice was done using 3 subdural cortical electrodes and a cerebellum reference electrode. Histology, immunostaining, whole cell patch-clamp and LFP recordings used standard procedures.

Results: Global knockin of V664I produced pups in normal Mendelian ratios, with a smaller thalamus, hypothalamus, amygdala and caudate putamen but no significant cell death. Modest growth delay was observed by postnatal day 10 (P10), hindlimb clasping by P14, and premature lethality with a median survival of P40. Acute intracranial EEG of mouse pups revealed spontaneous epileptic discharges between P7 and P14. Adult heterozygotes display three forms of severe seizure: unusually frequent spike-wave discharges and interictal epileptiform discharges both of which lengthen with age (often lasting several minutes) and terminal generalized tonic-clonic seizures. To evaluate neuronal cell types that mediate these severe phenotypes, cKI mice were crossed to cell-type specific Cre driver mice, revealing that inhibitory neurons are necessary and sufficient for hindlimb clasping and seizure activity. Assessment of synapse distribution in hippocampal neurons from V667I primary neuron culture and from brain slices showed decreased synaptic density in proximal dendrites, accompanied by large synaptic clumps. Assessment of synaptic properties in CA1 inhibitory neurons from hippocampal slices revealed a prolonged decay time constant, and local field potential measures showed a 1.5-fold increase in evoked network response and a 1.7-fold increase in spiking following stimulation. Immunostaining showed no significant difference in the number of PV+ inhibitory neuron in hippocampus but increased number of PV+ neurons in cortex.

Conclusions: Grin2d-V664I/+ mice display multiple significant neurological features consistent with severe DEE, notably including chronic seizure activity more persistent than previously observed in genetic rodent models. V664I expression in inhibitory neurons accounts for most, if not all in vivo phenotypes. The impact of the mutation on neurodevelopment was apparent in altered brain morphology and epileptiform activity in mouse pups, with evidence of widespread cellular and network imbalance.

Funding: NS031348, NS111619, OD020351