GRIN2D Recurrent De Novo Dominant Mutation Causes a Severe Epileptic Encephalopathy Treatable with NMDA Receptor Channel Blockers.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated cation channels that mediate excitatory synaptic transmission. Genetic mutations in multiple NMDAR subunits cause various childhood epilepsy syndromes. Here, we report a de novo recurrent heterozygous missense mutation-c.1999G>A (p.Val667Ile)-in a NMDAR gene previously unrecognized to harbor disease-causing mutations, GRIN2D, identified by exome and candidate panel sequencing in two unrelated children with epileptic encephalopathy. The resulting GluN2D p.Val667Ile exchange occurs in the M3 transmembrane domain involved in channel gating. This gain-of-function mutation increases glutamate and glycine potency by 2-fold, increases channel open probability by 6-fold, and reduces receptor sensitivity to endogenous negative modulators such as extracellular protons. Moreover, this mutation prolongs the deactivation time course after glutamate removal, which controls the synaptic time course. Transfection of cultured neurons with human GRIN2D cDNA harboring c.1999G>A leads to dendritic swelling and neuronal cell death, suggestive of excitotoxicity mediated by NMDAR over-activation. Because both individuals' seizures had proven refractory to conventional antiepileptic medications, the sensitivity of mutant NMDARs to FDA-approved NMDAR antagonists was evaluated. Based on these results, oral memantine was administered to both children, with resulting mild to moderate improvement in seizure burden and development. The older proband subsequently developed refractory status epilepticus, with dramatic electroclinical improvement upon treatment with ketamine and magnesium. Overall, these results suggest that NMDAR antagonists can be useful as adjuvant epilepsy therapy in individuals with GRIN2D gain-of-function mutations. This work further demonstrates the value of functionally evaluating a mutation, enabling mechanistic understanding and therapeutic modeling to realize precision medicine for epilepsy.

Zinc bells rang in Jerusalem!

"Oh, Jerusalem of gold, and of light, and of bronze..." goes the popular song. But it was another metal that towered above the Jerusalem landscape during the meeting of the International Society for Zinc Biology (ISZB; http://www.iszb.org/), held at Mishkenot Sha'ananim, a whisper away from the Old City walls. More than 100 scientists gathered on 1 to 5 December 2009 to discuss their research on the biology of this metal. Zinc is a double-edged sword. Zinc supplementation accelerates wound healing and growth and promotes an effective immune response. On the other hand, zinc deficiency leads to growth retardation and impaired learning and memory function, and has been linked to mood disorders. At the cellular level, however, uncontrolled increases in zinc concentrations can lead to neuronal cell death and may be involved in neurodegenerative disorders. Through regulation of various intracellular signaling pathways, zinc can accelerate cell growth and possibly contribute to cancer. However, despite the physiological and clinical importance of this metal, research on the molecular basis of these effects is still in its infancy. The 2009 ISZB meeting provided a venue for investigators working on various zinc-related issues to share their thoughts and ideas and to promote the growth of this field.

Zinc accumulation after target loss: an early event in retrograde degeneration of thalamic neurons.

Accumulation of cytoplasmic zinc is linked with a cascade of events leading to neuronal death. In many in vivo models of zinc-induced cell death, toxic concentrations of synaptically released zinc enter vulnerable neurons via neurotransmitter- or voltage-gated ion channels. In vitro studies demonstrate, in addition, that zinc can be liberated from intracellular stores following oxidative stress and contribute to cell death processes, including apoptosis. Here we describe accumulation of intracellular zinc in an in vivo model of cell death in the absence of presynaptic zinc release. We focused on the lateral geniculate nucleus (LGN) because LGN neurons undergo apoptosis when separated from their target, the primary visual cortex (V1), and the LGN is mostly devoid of zinc-containing presynaptic terminals. Infant and adult rats and adult mice received unilateral ablation of V1, either by aspiration or kainate injection. One to 14 days later, brain sections were stained with selenium autometallography or fluorescently labeled to localize zinc, or stained immunochemically for activated caspase-3. V1 lesions led to zinc accumulation in LGN neurons in infant and adult subjects. Zinc-containing neurons were evident 1-3 days after aspiration lesions, depending on age, but not until 14 days after kainate injection. Zinc accumulation was followed rapidly by immunostaining for activated caspase-3. Our data indicate that like neurotrauma and excitotoxicity, target deprivation leads to accumulation of zinc in apoptotic neurons. Moreover, zinc accumulation in vivo can occur in the absence of presynaptic zinc release. Together these findings suggest that accumulation of intracellular zinc is a ubiquitous component of the cell death cascade in neurons.

Novel neuroprotective K+ channel inhibitor identified by high-throughput screening in yeast.

Discovery of K+ channel modulators is limited by low-throughput capacity of existing K+ channel assays. To enable high-throughput screening for novel pharmacological modulators of K+ channels, we developed an assay based on growth of yeast that functionally expresses mammalian Kir2.1 channels. Screening of 10,000 small molecules from a combinatorial chemical library yielded 42 potential Kir2.1 inhibitors. One compound, 3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol, was confirmed to inhibit K+ channels in patch-clamp measurements in mammalian cells with EC50 values of 60 and 1 microM for Kir2.1 and Kv2.1 channels, respectively. Inhibition of Kv2.1 channels decreased in the presence of the external pore blocker tetraethylammonium (TEA) and depended on a residue required for extracellular TEA action, suggesting that the identified compound targets the external mouth of the channel. Furthermore, at the nontoxic concentration of 3 microM, the identified compound completely abolished in vitro neuronal apoptosis mediated by Kv2.1 channels. Therefore, yeast-based screening has identified a novel uncharged neuroprotective mammalian K+ channel inhibitor.