Andre H. Lagrange

Rapid effects of estrogen to modulate G protein-coupled receptors via activation of protein kinase A and protein kinase C pathways.

17Beta-estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic neurons. We have used intracellular recordings from female guinea pig hypothalamic slices to characterize the receptor and intracellular pathway(s) mediating E2s rapid effects. E2 acts stereospecifically with physiologically relevant concentration-dependence (EC50 = 8 nM) to cause a fourfold reduction in the potency of the mu-opioid agonist (D-Ala2-N-Me-Phe4-Gly5-ol)-enkephalin and the GABA(B) agonist baclofen to activate an inwardly rectifying K+ conductance in hypothalamic neurons. Both the nonsteroidal estrogen diethylstilbestrol and the anti-estrogen ICI 164,384 blocked E2 actions to uncouple mu-opioid receptors. Using a pharmacological Schild analysis, we found that ICI 164,384 competed for this E2 receptor with a Ke of approximately 0.3 nM. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a rapid, nongenomic mechanism of E2 action. The effects of E2 were mimicked by the bath application of the protein kinase A (PKA) activators, forskolin and Sp-cAMP, and the protein kinase C (PKC) activator phorbol-12,13-dibutyrate. Furthermore, the selective PKA antagonists Rp-cAMP and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. In addition, the actions of E2 were blocked by the selective PKC inhibitor Calphostin C. Therefore, it appears that E2 can activate both PKA and PKC to cause a heterologous desensitization of both mu-opioid and GABA(B) receptors, which has the potential to alter synaptic transmission in many regions of the CNS.

Estrogen rapidly attenuates a GABAB response in hypothalamic neurons.

GABA is a predominant neurotransmitter in the hypothalamus and an important regulator of hypothalamic function. To elucidate the cellular basis for GABAergic action in this region, we used intracellular recordings from identified hypothalamic neurons. Ninety-three percent of the mediobasal hypothalamic neurons responded to GABAB receptor stimulation, and the presence of bicuculline-sensitive synaptic potentials indicated a tonic, GABAA receptor-mediated input. Stimulation of GABAB receptors hyperpolarized these cells by activating an inwardly rectifying potassium conductance. We characterized GABAB responses by generating concentration-response curves to the GABAB agonist baclofen. There was heterogeneity in the responses to baclofen, with one third of the cells having low baclofen potency (EC50 = 5.0 microM). Two thirds of the neurons had a 4-fold higher potency (EC50 = 1.2 microM), larger somas and a more lateral distribution. Previous work has shown that hypothalamic GABAB and mu-opioid receptors open the same K+ channels and that the response to mu-opioid agonists is rapidly attenuated by 17 beta-estradiol (E2). In order to test the hypothesis that the coupling of GABAB receptors to K+ channels is also altered, baclofen concentration-response curves were generated before and after an E2 challenge (100 nM, 20 min). Consistent with our hypothesis, the potency of baclofen was decreased nearly 4-fold in a subset of the cells that had a high potency response to baclofen. Furthermore, decreased baclofen potency only occurred in those cells in which E2 also altered the mu-opioid responses. Therefore, our findings suggest that a discrete subpopulation of hypothalamic neurons is sensitive to estrogen actions to alter inhibitory transmission. We propose that the alteration of GABAB and mu-opioid input is consistent with estrogens rapid inhibition of the reproductive axis.

Enhanced macroscopic desensitization shapes the response of α4 subtype‐containing GABAA receptors to synaptic and extrasynaptic GABA

Up‐regulation of the GABAA receptor α4 subunit subtype has been consistently shown in multiple animal models of chronic epilepsy. This isoform is expressed in both thalamus and hippocampus and is likely to play a significant role in regulating corticothalamic and hippocampal rhythms. However, little is known about its physiological properties, thus limiting understanding of the role of α4 subtype‐containing GABAA receptors in normal and abnormal physiology. We used rapid GABA application to recombinant GABAA receptors expressed in HEK293T cells to compare the macroscopic kinetic properties of α4β3γ2L receptors to those of the more widely distributed α1β3γ2L receptors. These receptor currents had similar peak current amplitudes and GABA EC50 values. However, α4β3γ2L currents activated more slowly when exposed to submaximal GABA concentrations, had more fast desensitization (τ= 15–100 ms), and had less residual current during long GABA applications. In addition, α4β3γ2L currents deactivated more slowly than α1β3γ2L currents. Peak currents evoked by repetitive, brief GABA applications were more strongly attenuated for α4β3γ2L currents than α1β3γ2L currents. Moreover, the time required to recover from desensitization was prolonged in α4β3γ2L currents compared to α1β3γ2L currents. We also found that exposure to prolonged low levels of GABA, similar to those that might be present in the extrasynaptic space, greatly suppressed the response of α4β3γ2L currents to higher concentrations of GABA, while α1β3γ2L currents were less affected by exposure to low levels of GABA. Taken together, these data suggest that α4β3γ2L receptors have unique kinetic properties that limit the range of GABA applications to which they can respond maximally. While similar to α1β3γ2L receptors in their ability to respond to brief and low frequency synaptic inputs, α4β3γ2L receptors are less efficacious when exposed to prolonged tonic GABA or during repetitive stimulation, as may occur during learning and seizures.

Developmental Modulation of GABAA 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 α3 subunit of heteromeric GABAA 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 ∼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 α3(I)β3γ2L GABAA receptors activated more rapidly and deactivated much more slowly than edited α3(M)β3γ2L receptors. Furthermore, currents from nonedited α3(I)β3γ2L receptors were strongly outwardly rectifying (corresponding to chloride ion influx), whereas currents from edited α3(M)β3γ2L receptors had a more linear current/voltage relationship. These studies suggest that increased expression of the nonedited α3(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 α3(I) subunit could act as a shunt to prevent excessive excitation, providing the delicate balance necessary for normal neuronal development.

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 Hashimotos 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.

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.

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

γ-Aminobutyric acid type A (GABAA) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABAA receptor β2 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 β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 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 α1β2 channel, we propose mechanisms for regulation of GABAA receptors by glycosylation.

Benzodiazepine modulation of GABAA receptor opening frequency depends on activation context: A patch clamp and simulation study

Benzodiazepines (BDZs) are GABA(A) receptor modulators with anxiolytic, hypnotic, and anticonvulsant properties. BDZs are understood to potentiate GABA(A) receptor function by increasing channel opening frequency, in contrast to barbiturates, which increase channel open duration. However, the in vitro evidence demonstrating increased opening frequency involved prolonged exposure to sub-saturating GABA concentrations, conditions most similar to those found in extrasynaptic areas. In contrast, synaptic GABA(A) receptors are transiently activated by high GABA concentrations. To determine if BDZ modulation of single-channel opening frequency would be different for BDZ-sensitive receptors activated under synaptic versus extrasynaptic conditions, a combination of patch clamp recording and kinetic modeling was used. Consistent with the original experimental findings, BDZs were found to increase receptor affinity for GABA by decreasing the unbinding rate. While this mechanism was predicted to increase opening frequency under extrasynaptic conditions, simulations predicted that the same mechanism under synaptic conditions would increase the number, but not the frequency, of single-channel openings. Thus, a single mechanism (slower GABA unbinding) can produce differential changes in opening frequency under synaptic versus extrasynaptic conditions. The functional impact of BDZs on GABA(A) receptors therefore depends upon the physiological context of receptor activation.

Rasmussen’s syndrome and new-onset narcolepsy, cataplexy, and epilepsy in an adult

We report a case of new-onset seizures and narcolepsy in a previously healthy 40-year-old man. He developed severe daytime somnolence and cataplexy over the course of a few months. Brain MRI was normal, and polysomnography with multiple sleep latency testing confirmed a diagnosis of narcolepsy. His HLA haplotype is DQB1*0602 and cerebrospinal fluid analysis showed no detectable hypocretin. Approximately 18 months later, he developed complex partial seizures. Further MRI showed a progressively enlarging lesion involving the left frontotemporal and insular areas. Pathology from a partial resection was consistent with Rasmussens syndrome. Evaluation for tumor, infectious, and paraneoplastic etiologies was negative. There was no further progression of the residual lesion on serial MRI. Although the pathophysiologic bases of narcolepsy and Rasmussens syndrome are unknown, they may have an autoimmune basis. This unique case of both disorders in a single patient suggests the possibility of a common underlying disease process.