Altered inhibitory synapses in de novo GABRA5 and GABRA1 mutations associated with early onset epileptic encephalopathies.

We performed next generation sequencing on 1696 patients with epilepsy and intellectual disability using a gene panel with 480 epilepsy-related genes including all GABAA receptor subunit genes (GABRs), and we identified six de novo GABR mutations, two novel GABRA5 mutations (c.880G>T, p.V294F and c.1238C>T, p.S413F), two novel GABRA1 mutations (c.778C>T, p.P260S and c.887T>C, p.L296S/c.944G>T, p.W315L) and two known GABRA1 mutations (c.335G>A, p.R112Q and c.343A>G, p.N115D) in six patients with intractable early onset epileptic encephalopathy. The α5(V294F and S413F) and α1(P260S and L296S/W315L) subunit residue substitutions were all in transmembrane domains, while the α1(R112Q and N115R) subunit residue substitutions were in the N-terminal GABA binding domain. Using multidisciplinary approaches, we compared effects of mutant GABAA receptor α5 and α1 subunits on the properties of recombinant α5ß3γ2 and α1ß3γ2 GABAA receptors in both neuronal and non-neuronal cells and characterized their effects on receptor clustering, biogenesis and channel function. GABAA receptors containing mutant α5 and α1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Our study identified GABRA5 as a causative gene for early onset epileptic encephalopathy and expands the mutant GABRA1 phenotypic spectrum, supporting growing evidence that defects in GABAergic neurotransmission contribute to early onset epileptic encephalopathy phenotypes.

GABA beyond the synapse: defining the subtype-specific pharmacodynamics of non-synaptic GABAA receptors.

KEY POINTS: Physiologically relevant combinations of recombinant GABAA receptor (GABAR) subunits were expressed in HEK293 cells. Using whole-cell voltage clamp and rapid drug application, we measured the GABAR-subtype-specific properties to convey either synaptic or extrasynaptic signalling in a range of physiological contexts. α4ßδ GABARs are optimally tuned to submicromolar tonic GABA and transient surges of micromolar GABA concentrations. α5ß2γ2l GABARs are better suited to higher tonic GABA levels, but also convey robust responses to brief synaptic and perisynaptic GABA fluctuations. α1ß2/3δ GABARs function well at prolonged, micromolar (>2 µm) GABA levels, but not to low tonic (<1 µm GABA) or synaptic/transient GABAergic signalling. These results help illuminate the context- and isoform-specific modes of GABAergic signalling in the brain. ABSTRACT: GABAA receptors (GABARs) mediate a remarkable diversity of signalling modalities in vivo. Yet most published work characterizing responses to GABA has focused on the properties needed to convey fast, phasic synaptic inhibition. We therefore aimed to characterize the most prevalent (α4ßδ, α5ß3γ2L) and least prevalent (α1ß2δ) non-synaptic GABAR currents, using whole-cell voltage clamp recordings of recombinant GABAR expressed in HEK293 cells and drug application protocols to recapitulate the GABA concentration profiles occurring during both fast synaptic and slow extrasynaptic signalling. We found that α4ßδ GABARs were very sensitive to submicromolar GABA, with a rank order potency of α4ß2δ ≥ α4ß1δ ≈ α4ß3δ GABARs. In comparison, the GABA EC50 was up to 20 times higher for α1ß2γ2L GABARs, with α1ß2δ and α5ß3γ2L GABARs having intermediate GABA potency. Both α4ßδ and α5ß3γ2L GABAR currents exhibited slow, but substantial, desensitization as well as prolonged rates of deactivation. These GABAR current properties defined distinct 'dynamic ranges' of responsiveness to changing GABA for α4ß2δ (0.1-1 µm), α5ß3γ2L (0.5-7 µm) and α1ß2γ2L (0.6-9 µm) GABARs. Finally, α1ß2δ GABARs were notable for their relative lack of desensitization and extremely quick deactivation. In summary, our results help delineate the roles that specific GABARs may play in mediating non-synaptic GABA signals. Since ambient GABA levels may be altered during development as well as by drugs and disease states, these findings may help future efforts to understand disrupted inhibition underlying a variety of neurological illnesses, such as epilepsy.

Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αßγ and αßδ Expression Using Flow Cytometry and Electrophysiology: EVIDENCE FOR ALTERNATIVE SUBUNIT STOICHIOMETRIES AND ARRANGEMENTS.

The subunit stoichiometry and arrangement of synaptic αßγ GABAA receptors are generally accepted as 2α:2ß:1γ with a ß-α-γ-ß-α counterclockwise configuration, respectively. Whether extrasynaptic αßδ receptors adopt the analogous ß-α-δ-ß-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or ß2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and ß2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of ß2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1ß2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αßγ and αßδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αßγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical ß-α-γ/δ-ß-α receptors, including ß-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and ß-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.

Generalized onset seizures with focal evolution (GOFE) - A unique seizure type in the setting of generalized epilepsy.

PURPOSE: We report clinical and electrographic features of generalized onset seizures with focal evolution (GOFE) and present arguments for the inclusion of this seizure type in the seizure classification. METHODS: The adult and pediatric Epilepsy Monitoring Unit databases at Vanderbilt Medical Center and Children's Hospital were screened to identify generalized onset seizures with focal evolution. We reviewed medical records for epilepsy characteristics, epilepsy risk factors, MRI abnormalities, neurologic examination, antiepileptic medications before and after diagnosis, and response to medications. We also reviewed ictal and interictal EEG tracings, as well as video-recorded semiology. RESULTS: Ten patients were identified, 7 males and 3 females. All of the patients developed generalized epilepsy in childhood or adolescence (ages 3-15years). Generalized onset seizures with focal evolution developed years after onset in 9 patients, with a semiology concerning for focal seizures or nonepileptic events. Ictal discharges had a generalized onset on EEG, described as either generalized spike-and-wave and/or polyspike-and-wave discharges, or generalized fast activity. This electrographic activity then evolved to focal rhythmic activity most commonly localized to one temporal or frontal region; five patients had multiple seizures evolving to focal activity in different regions of both hemispheres. The predominant interictal epileptiform activity included generalized spike-and-wave and/or polyspike-and-wave discharges in all patients. Taking into consideration all clinical and EEG data, six patients were classified with genetic (idiopathic) generalized epilepsy, and four were classified with structural/metabolic (symptomatic) generalized epilepsy. All of the patients had modifications to their medications following discharge, with three becoming seizure-free and five responding with >50% reduction in seizure frequency. CONCLUSION: Generalized onset seizures may occasionally have focal evolution with semiology suggestive of focal seizures, leading to a misdiagnosis of focal onset. This unique seizure type may occur with genetic as well as structural/metabolic forms of epilepsy. The identification of this seizure type may help clinicians choose appropriate medications, avoiding narrow spectrum agents known to aggravate generalized onset seizures.

Video-EEG results and clinical characteristics in patients with psychogenic nonepileptic spells: The effect of a coexistent epilepsy.

RATIONALE: Epilepsy and psychogenic nonepileptic spells (PNES) can coexist, often posing diagnostic and therapeutic challenges. We sought to identify clinical and historical characteristics of two groups of patients, those with coexisting epilepsy and PNES and those with PNES alone, and determine the prevalence of coexisting epilepsy/PNES with strict diagnostic criteria in a large group of epilepsy monitoring unit (EMU) patients. METHODS: We reviewed the medical records of all consecutive patients admitted to the Vanderbilt University Medical Center Adult EMU between July 1, 2007 and June 30, 2012. We identified patients with recorded PNES and classified them as having coexisting epilepsy/PNES or PNES alone and then systematically compared the clinical characteristics of these two groups. RESULTS: A total of 1567 patient medical records were reviewed. The prevalence rate of coexisting epilepsy/PNES was 5.2% among all EMU admissions (12.3% of all patients with epilepsy and 14.8% of all patients with PNES). These rates were lower when patients with interictal epileptiform activity (IEA) alone and no recorded ictal discharges were not included in the group with epilepsy (2.6%, 6.2%, and 7.4%, respectively). The accuracy of pre-EMU clinical suspicion was significantly higher in the group with PNES-only. Patients with epilepsy/PNES were significantly more likely to require more than one EMU admission for definitive diagnosis. The first PNES event preceded an epileptic seizure (ES) in 94.4% of patients with epilepsy/PNES. The group with PNES-only had significantly higher suggestibility, and the group with epilepsy/PNES had a significantly higher presence of epilepsy risk factors. Abnormal neurological examination and abnormal brain MRI were also significantly more common in the group with epilepsy/PNES. CONCLUSIONS: Our study defined the prevalence of coexisting epilepsy/PNES in a large cohort with strict diagnostic criteria and outlined specific clinical and historical characteristics differentiating the two groups of patients with coexisting epilepsy/PNES and PNES-only. These findings should help guide clinicians to reach the correct diagnosis faster and provide appropriate treatment earlier.

Temporal lobe origin is common in patients who have undergone epilepsy surgery for hypermotor seizures.

RATIONALE: Hypermotor seizures are most often reported from the frontal lobe but may also have temporal, parietal, or insular origin. We noted a higher proportion of patients with temporal lobe epilepsy in our surgical cohort who had hypermotor seizures. We evaluated the anatomic localization and surgical outcome in patient with refractory hypermotor seizures who had epilepsy surgery in our center. METHODS: We identified twenty three patients with refractory hypermotor seizures from our epilepsy surgery database. We analyzed demographics, presurgical evaluation including semiology, MRI, PET scan, interictal/ictal scalp video-EEG, intracranial recording, and surgical outcomes. We evaluated preoperative variables as predictors of outcome. RESULTS: Most patients (65%) had normal brain MRI. Intracranial EEG was required in 20 patients (86.9%). Based on the presurgical evaluation, the resection was anterior temporal in fourteen patients, orbitofrontal in four patients, cingulate in four patients, and temporoparietal in one patient. The median duration of follow-up after surgery was 76.4months. Fourteen patients (60%) had been seizure free at the last follow up while 3 patients had rare disabling seizures. CONCLUSIONS: Hypermotor seizures often originated from the temporal lobe in this series of patients who had epilepsy surgery. This large proportion of temporal lobe epilepsy may be the result of a selection bias, due to easier localization and expected better outcome in temporal lobe epilepsy. With extensive presurgical evaluation, including intracranial EEG when needed, seizure freedom can be expected in the majority of patients.

Co-expression of γ2 subunits hinders processing of N-linked glycans attached to the N104 glycosylation sites of GABAA receptor ß2 subunits.

GABAA receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and ß subunits with or without a γ2 subunit. The α1ß2γ2 receptor is the most abundant GABAA receptor in brain. Co-expression of γ2 with α1 and ß2 subunits causes conformational changes, increases GABAA receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three ß2 subunit glycosylation sites, N32, N104 and N173, was important for α1ß2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering ß2 subunits. We found that co-expression of γ2 subunits hindered processing of ß2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2(+/-)) mice led to appreciable changes in the endoglycosidase H digestion pattern of neuronal ß2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the ß2 subunit glycosylation sites. The ß2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with ß2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the ß2 subunit N104 N-glycans were involved in altering the function of surface GABAA receptors.

Region and layer-specific expression of GABAA receptor isoforms and KCC2 in developing cortex.

Introduction: γ-Aminobutyric acid (GABA) type A receptors (GABAARs) are ligand-gated Cl-channels that mediate the bulk of inhibitory neurotransmission in the mature CNS and are targets of many drugs. During cortical development, GABAAR-mediated signals are significantly modulated by changing subunit composition and expression of Cl-transporters as part of developmental processes and early network activity. To date, this developmental evolution has remained understudied, particularly at the level of cortical layer-specific changes. In this study, we characterized the expression of nine major GABAAR subunits and K-Cl transporter 2 (KCC2) in mouse somatosensory cortex from embryonic development to postweaning maturity. Methods: We evaluated expression of α1-5, ß2-3, γ2, and δ GABAAR subunits using immunohistochemistry and Western blot techniques, and expression of KCC2 using immunohistochemistry in cortices from E13.5 to P25 mice. Results: We found that embryonic cortex expresses mainly α3, α5, ß3, and γ2, while expression of α1, α2, α4, ß2, δ, and KCC2 begins at later points in development; however, many patterns of nuanced expression can be found in specific lamina, cortical regions, and cells and structures. Discussion: While the general pattern of expression of each subunit and KCC2 is similar to previous studies, we found a number of unique temporal, regional, and laminar patterns that were previously unknown. These findings provide much needed knowledge of the intricate developmental evolution in GABAAR composition and KCC2 expression to accommodate developmental signals that transition to mature neurotransmission.

Loss of KCC2 in GABAergic Neurons Causes Seizures and an Imbalance of Cortical Interneurons.

K-Cl transporter KCC2 is an important regulator of neuronal development and neuronal function at maturity. Through its canonical transporter role, KCC2 maintains inhibitory responses mediated by γ-aminobutyric acid (GABA) type A receptors. During development, late onset of KCC2 transporter activity defines the period when depolarizing GABAergic signals promote a wealth of developmental processes. In addition to its transporter function, KCC2 directly interacts with a number of proteins to regulate dendritic spine formation, cell survival, synaptic plasticity, neuronal excitability, and other processes. Either overexpression or loss of KCC2 can lead to abnormal circuit formation, seizures, or even perinatal death. GABA has been reported to be especially important for driving migration and development of cortical interneurons (IN), and we hypothesized that properly timed onset of KCC2 expression is vital to this process. To test this hypothesis, we created a mouse with conditional knockout of KCC2 in Dlx5-lineage neurons (Dlx5 KCC2 cKO), which targets INs and other post-mitotic GABAergic neurons in the forebrain starting during embryonic development. While KCC2 was first expressed in the INs of layer 5 cortex, perinatal IN migrations and laminar localization appeared to be unaffected by the loss of KCC2. Nonetheless, the mice had early seizures, failure to thrive, and premature death in the second and third weeks of life. At this age, we found an underlying change in IN distribution, including an excess number of somatostatin neurons in layer 5 and a decrease in parvalbumin-expressing neurons in layer 2/3 and layer 6. Our research suggests that while KCC2 expression may not be entirely necessary for early IN migration, loss of KCC2 causes an imbalance in cortical interneuron subtypes, seizures, and early death. More work will be needed to define the specific cellular basis for these findings, including whether they are due to abnormal circuit formation versus the sequela of defective IN inhibition.

Glycosylation of {beta}2 subunits regulates GABAA receptor biogenesis and channel gating.

γ-aminobutyric acid type A (GABA(A)) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABA(A) receptor ß2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted ß2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1ß2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of ß2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-ß-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1ß2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1ß2 receptor current amplitudes and altered the gating properties of α1ß2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-ß-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface ß2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1ß2 channel, we propose mechanisms for regulation of GABA(A) receptors by glycosylation.

Transient improvement after brief antiepileptic drug withdrawal in the epilepsy monitoring unit--possible relationship to AED tolerance.

PURPOSE: A drug holiday seems to produce seizure interval prolongation (SIP) after reinstitution of antiepileptic drugs (AEDs). This effect was demonstrated mainly with carbamazepine. We evaluated SIP with newer AEDs and tested the relationship of SIP to history of AED tolerance. METHODS: We prospectively studied patients with refractory epilepsy admitted to the Vanderbilt epilepsy monitoring unit (EMU) over a period of 12 months. We included only patients on levetiracetam, lamotrigine, or oxcarbazepine who had their AEDs withdrawn on admission and reinstituted without change upon discharge. We defined SIP as the interval from EMU discharge to first seizure minus the interval between the last two seizures before EMU admission. RESULTS: A total of 43 patients completed the study; 15 were on monotherapy. SIP was greater than zero in this patient group (p < 0.0001), with a mean prolongation of 19.4 +/- 28.0 days. The average SIP was higher (p = 0.01) in patients on monotherapy (29.7 +/- 23.8 days) than patients on polytherapy (13.9 +/- 29.0 days). SIP tended to be greater in patients with a prior history of AED tolerance (25.7 +/- 36.8 days) compared to patient with no prior history of AED tolerance (14.0 +/- 16.3 days). DISCUSSION: SIP does occur after brief AED withdrawal. This effect is greater in patients on monotherapy and tends to be larger in patients with a history of AED tolerance. The SIP effect may be related to the phenomenon of tolerance, clinically seen as resistance to AED therapeutic effect.

Developmental modulation of GABA(A) receptor function by RNA editing.

Adenosine-to-inosine (A-to-I) editing of RNA transcripts is an increasingly recognized cellular strategy to modulate the function of proteins involved in neuronal excitability. We have characterized the editing of transcripts encoding the alpha3 subunit of heteromeric GABA(A) receptors (Gabra3), in which a genomically encoded isoleucine codon (ATA) is converted to a methionine codon (ATI) in a region encoding the predicted third transmembrane domain of this subunit. Editing at this position (I/M site) was regulated in a spatiotemporal manner with approximately 90% of the Gabra3 transcripts edited in most regions of adult mouse brain, but with lower levels of editing in the hippocampus. Editing was low in whole-mouse brain at embryonic day 15 and increased during development, reaching maximal levels by postnatal day 7. GABA-evoked current in transfected cells expressing nonedited alpha3(I)beta3gamma2L GABA(A) receptors activated more rapidly and deactivated much more slowly than edited alpha3(M)beta3gamma2L receptors. Furthermore, currents from nonedited alpha3(I)beta3gamma2L receptors were strongly outwardly rectifying (corresponding to chloride ion influx), whereas currents from edited alpha3(M)beta3gamma2L receptors had a more linear current/voltage relationship. These studies suggest that increased expression of the nonedited alpha3(I) subunit during brain development, when GABA is depolarizing, may allow the robust excitatory responses that are critical for normal synapse formation. However, the strong chloride ion influx conducted by receptors containing the nonedited alpha3(I) subunit could act as a shunt to prevent excessive excitation, providing the delicate balance necessary for normal neuronal development.

Generalized-onset seizures with secondary focal evolution.

The international seizure classification recognizes that partial-onset seizures can become secondarily generalized, but generalized-onset seizures are expected to remain generalized. We report six patients who had recorded seizures with generalized onset, but subsequent evolution into a focal discharge. The clinical seizure onset was generalized absence or myoclonic, and the most common subsequent clinical pattern was prolonged behavioral arrest with mild automatisms, and then postictal confusion. The ictal discharge started with generalized spike-and-wave activity and then acquired a focal predominance. Interictal epileptiform activity was generalized. There were no focal magnetic resonance imaging abnormalities. Four patients were misdiagnosed with complex partial seizures. All patients were initially refractory, but three became seizure-free and three improved after treatment with antiepileptic medications appropriate for absence or myoclonic seizures. Generalized-onset seizures that acquire focal features are easily misdiagnosed as complex partial. These seizures have a more favorable response to medications effective against generalized absence and myoclonic seizures.

Folic acid supplementation for women with epilepsy who might become pregnant.

Low folate levels are associated with an increased risk of spontaneous abortion and congenital malformations. Up to half of all pregnancies are unplanned, so official guidelines in a number of countries, including the US, recommend folic acid supplementation for all women who might become pregnant, before they actually start planning to have children. Children born to women who are taking antiepileptic medications, many of which antagonize folate action, are at an increased risk of birth defects. This article highlights a study by Pittschieler et al. that has suggested that supplementation with folic acid might be particularly important in women with epilepsy who are taking antiepileptic medications--especially valproate. However, the sample size in the study was too small to determine the optimum dose or to detect whether folic acid protects against birth defects. Nonetheless, it is clear that all women of reproductive age, especially those taking certain antiepileptic medications, should take folic acid supplements.

Loss of mTORC2 signaling in oligodendrocyte precursor cells delays myelination.

Myelin abnormalities are increasingly being recognized as an important component of a number of neurologic developmental disorders. The integration of many signaling pathways and cell types are critical for correct myelinogenesis. The PI3-K and mechanistic target of rapamycin (mTOR) pathways have been found to play key roles. mTOR is found within two distinct complexes, mTORC1 and mTORC2. mTORC1 activity has been shown to play a major role during myelination, while the role of mTORC2 is not yet well understood. To determine the role of mTORC2 signaling in myelinogenesis, we generated a mouse lacking the critical mTORC2 component Rictor in oligodendrocyte precursors (OPCs). Targeted deletion of Rictor in these cells decreases and delays the expression of myelin related proteins and reduces the size of cerebral white matter tracts. This is developmentally manifest as a transient reduction in myelinated axon density and g-ratio. OPC cell number is reduced at birth without detectable change in proliferation with proportional reductions in mature oligodendrocyte number at P15. The total number of oligodendrocytes as well as extent of myelination, does improve over time. Adult conditional knock-out (CKO) animals do not demonstrate a behavioral phenotype likely due in part to preserved axonal conduction velocities. These data support and extend prior studies demonstrating an important but transient contribution of mTORC2 signaling to myelin development.

Mutations in the GABRA1 and EFHC1 genes are rare in familial juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME), accounting for approximately 25% of idiopathic generalized epilepsies, is genetically heterogeneous. Mutations in the alpha-1 subunit of the GABAA receptor (GABRA1) and EFHC1 genes have been reported in a few families with autosomal dominant (AD) JME. We have investigated the contribution of these two genes to familial JME in our cohort of 54 JME Caucasian families. Syndromic classification of JME was based on previously published criteria. We considered kindreds with at least one affected first-degree relative and the evidence of a vertical transmission as definite AD JME, and families with at least one affected second-degree relative as probable AD JME. We included 33 families meeting criteria for definitive AD JME and 21 that were classified as probable AD JME. None of these families were considered informative enough to analyze candidate loci for JME using linkage analysis. We have systematically screened coding exons of these two genes using temperature gradient capillary electrophoresis. Every heteroduplex with an abnormal mobility was sequenced. No disease-causing mutations in the GABRA1 gene were identified. Analysis of EFHC1 gene found one putative disease-causing mutation R221H that was previously reported as a tandem mutation. Several synonymous and non-synonymous coding polymorphisms were identified but the allelic frequency did not differ between controls and affected individuals. Our data suggests that the majority of familial AD JME is not caused by mutations in the GABRA1 and EFHC1 genes.

Of mothers and myelin: Aberrant myelination phenotypes in mouse model of Angelman syndrome are dependent on maternal and dietary influences.

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by a number of neurological problems, including developmental delay, movement disorders, and epilepsy. AS results from the loss of UBE3A (an imprinted gene) expressed from the maternal chromosome in neurons. Given the ubiquitous expression of Ube3a and the devastating nature of AS, the role of environmental and maternal effects has been largely ignored. Severe ataxia, anxiety-like behaviors and learning deficits are well-documented in patients and AS mice. More recently, clinical imaging studies of AS patients suggest myelination may be delayed or reduced. Utilizing a mouse model of AS, we found disrupted expression of cortical myelin proteins, the magnitude of which is influenced by maternal status, in that the aberrant myelination in the AS pups of AS affected mothers were more pronounced than those seen in AS pups raised by unaffected (Ube3a (m+/p-)) Carrier mothers. Furthermore, feeding the breeding mothers a higher fat (11% vs 5%) diet normalizes these myelin defects. These effects are not limited to myelin proteins. Since AS mice have abnormal stress responses, including altered glucocorticoid receptor (GR) expression, we measured GR expression in pups from Carrier and affected AS mothers. AS pups had higher GR expression than their WT littermates. However, we also found an effect of maternal status, with reduced GR levels in pups from affected mothers compared to genotypically identical pups raised by unaffected Carrier mothers. Taken together, our findings suggest that the phenotypes observed in AS mice may be modulated by factors independent of Ube3a genotype.

Estrogen modulation of K(+) channel activity in hypothalamic neurons involved in the control of the reproductive axis.

Here we report on the progress we have made in elucidating the mechanisms through which estrogen alters synaptic responses in hypothalamic neurons. We examined the modulation by estrogen of the coupling of various receptor systems to inwardly rectifying and small conductance, Ca(2+)-activated K(+) (SK) channels. We used intracellular sharp-electrode and whole-cell recordings in hypothalamic slices from ovariectomized female guinea pigs. Estrogen rapidly uncouples mu-opioid receptors from G protein-gated inwardly rectifying K(+) (GIRK) channels in beta-endorphin neurons, manifest by a reduction in the potency of mu-opioid receptor agonists to hyperpolarize these cells. This effect is blocked by inhibitors of protein kinase A and protein kinase C. Estrogen also uncouples gamma-aminobutyric acid (GABA)(B) receptors from the same population of GIRK channels coupled to mu-opioid receptors. At 24 h after steroid administration, the GABA(B)/GIRK channel uncoupling observed in GABAergic neurons of the preoptic area (POA) is associated with reduced agonist efficacy. Conversely, estrogen enhances the efficacy of alpha(1)-adrenergic receptor agonists to inhibit apamin-sensitive SK currents in these POA GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we observed a direct, steroid-induced hyperpolarization of both arcuate and POA neurons, among which gonadotropin-releasing hormone (GnRH) neurons are particularly sensitive. These findings indicate a richly complex yet coordinated steroid modulation of K(+) channel activity that serves to control the excitability of hypothalamic neurons involved in regulating the reproductive axis.

Diagnostic and therapeutic aspects of Hashimoto's encephalopathy.

OBJECTIVE: To share our experience on clinical presentation and management of patients diagnosed with Hashimoto's Encephalopathy (HE) at Vanderbilt Medical Center between 1999 and 2012. BACKGROUND: HE is a rare disorder characterized by encephalopathy and central nervous system (CNS) dysfunction, elevated antithyroid antibodies, the absence of infection or structural abnormalities in the CNS, and a response to treatment with steroids. The relationship between thyroid antibodies and encephalopathy has remained unresolved. DESIGN/METHODS: Retrospective chart review. RESULTS: We identified 13 patients who met the criteria for the diagnosis of HE. The median age was 49 years (range, 2-66) and all except one were women. Encephalopathy in the form of altered mental status, stroke-like symptoms or seizures, with prompt resolution of symptoms upon receiving steroids, was the commonest presentation, seen in 7 patients. The second commonest presentation was subacute progressive decrease in cognitive function, which reversed within days to weeks after steroid therapy, seen in 4 patients. Electroencephalogram (EEG) was available in 12 patients and was abnormal in 8, showing nonspecific cerebral dysfunction in all 8 and epileptiform activity in 3. Treatment consisted of steroids in the acute phase for 12 of 13 patients with rapid improvement in symptoms. Maintenance therapy was rituximab in 7 patients, intravenous immunoglobulin (IVIg) in 7, azathioprine in 4, mycophenolate mofetil in 3, and methotrexate in 1 (some patients received sequential therapy with different agents). There was complete or near complete resolution of symptoms in 12 of the 13 patients. CONCLUSIONS: We present a cohort of patients in whom CNS dysfunction was associated with elevated antithyroid antibodies and reversal of disease followed immunomodulatory therapies.