Ca2+ currents, especially those activated at low voltages (LVA), influence burst generation in thalamocortical circuitry and enhance the abnormal rhythmicity associated with absence epilepsy. Mutations in several genes for high-voltage-activated (HVA) Ca2+ channel subunits are linked to spike-wave seizure phenotypes in mice; however, none of these mutations are predicted to increase intrinsic membrane excitability or directly enhance LVA currents. We examined biophysical properties of both LVA and HVA Ca2+ currents in thalamic cells of tottering (tg; Cav2.1/alpha1A subunit), lethargic (lh; beta4 subunit), and stargazer (stg; gamma2 subunit) brain slices. We observed 46, 51, and 45% increases in peak current densities of LVA Ca2+ currents evoked at -50 mV from -110 mV in tg, lh, and stg mice, respectively, compared with wild type. The half-maximal voltages for steady-state inactivation of LVA currents were shifted in a depolarized direction by 7.5-13.5 mV in all three mutants, although no alterations in the time-constant for recovery from inactivation of LVA currents were found. HVA peak current densities in tg and stg were increased by 22 and 45%, respectively, and a 5 mV depolarizing shift of the activation curve was observed in lh. Despite elevated LVA amplitudes, no alterations in mRNA expression of the genes mediating T-type subunits, Cav3.1/alpha1G, Cav3.2/alpha1H, or Cav3.3/alpha1I, were detected in the three mutants. Our data demonstrate that mutation of Cav2.1 or regulatory subunit genes increases intrinsic membrane excitability in thalamic neurons by potentiating LVA Ca2+ currents. These alterations increase the probability for abnormal thalamocortical synchronization and absence epilepsy in tg, lh, and stg mice.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a progressive neurodegenerative disorder that has been diagnosed in a substantial fraction of older male fragile X premutation carriers. Patients affected by FXTAS have elevated levels of ribo-rCGG repeat containing FMR1 mRNA with normal to slightly reduced levels of FMRP in blood leukocytes. Coupled with the absence of FXTAS in fragile X syndrome patients, this suggests premutation-sized elongated rCGG repeats in the FMR1 transcript rather than alterations in the levels of FMRP are responsible for the FXTAS pathology. Mice expressing rCGG in the context of Fmr1 or the enhanced green fluorescent protein specifically in Purkinje neurons were generated to segregate the effects of rCGG from alterations in Fmr1 and to provide evidence that rCGG is necessary and sufficient to cause pathology similar to human FXTAS. The models exhibit the presence of intranuclear inclusions in Purkinje neurons, Purkinje neuron cell death and behavioral deficits. These results demonstrate that rCGG expressed in Purkinje neurons outside the context of Fmr1 mRNA can result in neuronal pathology in a mammalian system and demonstrate that expanded CGG repeats in RNA are the likely cause of the neurodegeneration in FXTAS.

MAGI-2 [also known as S-SCAM (synaptic scaffolding molecule)] is a multi-PDZ domain scaffolding protein that interacts with several different ligands in brain, including PTEN (phosphatase and tensin homolog), dasm1(dendrite arborization and synapse maturation 1), dendrin, axin, ␤-and ␦-catenin, neuroligin, hyperpolarization-activated cation channels, ␤1-adrenergic receptors, and NMDA receptors. We confirmed that MAGI-2 coimmunoprecipitated with stargazin in vivo from mouse cerebral cortex and used in vitro assays to localize the interaction to the C-terminal -TTPV amino acid motif of stargazin and the PDZ1, PDZ3, and PDZ5 domains of MAGI-2. Expression of stargazin recruited MAGI-2 to cell membranes and cell-cell contact sites in transfected HEK-293T cells dependent on the presence of the stargazin -TTPV motif. These experiments identify MAGI-2 as a strong candidate for linking TARP/AMPA receptor complexes to a wide range of other postsynaptic molecules and pathways and advance our knowledge of protein interactions at mammalian CNS synapses.

Mcl1 is a Bcl2-related antiapoptotic protein originally isolated from human myeloid leukemia cells. Unlike Bcl2, expression has not been reported in CNS neurons. We isolated Mcl1 in a direct screen for candidate modifier genes of neuronal vulnerability by differential display of mRNAs upregulated following prolonged seizures in two mouse strains with contrasting levels of hippocampal cell death. Mcl1 is widely expressed in neurons, and transcription is rapidly induced in both strains. In resistant C57Bl/6J mice, Mcl1 protein levels remain persistently elevated in hippocampal pyramidal neurons after seizures, but fall rapidly in C3H/HeJ hippocampus, coinciding with extensive neuronal apoptosis. DNA damage and caspase-mediated cell death were strikingly increased in Mcl1-deficient mice when compared to þ / þ littermates after similar seizures. We identify Mcl1 as a neuronal gene responsive to excitotoxic insult in the brain, and link relative levels of Mcl1 expression to inherited differences in neuronal thresholds for apoptosis.