Endoplasmic reticulum stress increases inflammatory cytokines in an epilepsy mouse model Gabrg2+/Q390X knockin: A link between genetic and acquired epilepsy?

OBJECTIVE: Neuroinflammation is a major theme in epilepsy, which has been characterized in acquired epilepsy but is poorly understood in genetic epilepsy. γ-Aminobutyric acid type A receptor subunit gene mutations are significant causes of epilepsy, and we have studied the pathophysiology directly resulting from defective receptor channels. Here, we determined the proinflammatory factors in a genetic mouse model, the Gabrg2+/Q390X knockin (KI). We have identified increased cytokines in multiple brain regions of the KI mouse throughout different developmental stages and propose that accumulation of the trafficking-deficient mutant protein may increase neuroinflammation, which would be a novel mechanism for genetic epilepsy. METHODS: We used enzyme-linked immunosorbent assay, immunoprecipitation, nuclei purification, immunoblot, immunohistochemistry, and confocal microscopy to characterize increased neuroinflammation and its potential causes in a Gabrg2+/Q390X KI mouse and a Gabrg2+/- knockout (KO) mouse, each associated with a different epilepsy syndrome with different severities. RESULTS: We found that proinflammatory cytokines such as tumor necrosis factor alpha, interleukin 1-beta (IL-1ß), and IL-6 were increased in the KI mice but not in the KO mice. A major underlying basis for the discrepancy in cytokine expression between the two mouse models is likely chronic mutant protein accumulation and endoplasmic reticulum (ER) stress. The presence of mutant protein dampened cytokine induction upon further cellular stimulation or external stress such as elevated temperature. Pharmacological induction of ER stress upregulated cytokine expression in the wild-type and KO but not in the KI mice. The increased cytokine expression was independent of seizure occurrence, because it was upregulated in both mice and cultured neurons. SIGNIFICANCE: Together, these data demonstrate a novel pathophysiology for genetic epilepsy, increased neuroinflammation, which is a common mechanism for acquired epilepsy. The findings thus provide the first link of neuroinflammation between genetic epilepsy associated with an ion channel gene mutation and acquired epilepsy.

γ-Aminobutyric acid receptor alpha 1 subunit loss of function causes genetic generalized epilepsy by impairing inhibitory network neurodevelopment.

OBJECTIVE: In humans, mutations of the γ-aminobutyric acid receptor subunit 1 (GABRA1) cause either mild or severe generalized epilepsy. Although these epilepsy-causing mutations have been shown to disrupt the receptor activity in vitro, their in vivo consequences on brain development and activity are not known. Here, we aim at unraveling the epileptogenesis mechanisms of GABRA1 loss of function. METHODS: We generated a gabra1-/- zebrafish mutant line displaying highly penetrant epileptic seizures. We sought to identify the underlying molecular mechanisms through unbiased whole transcriptomic assay of gabra1-/- larval brains. RESULTS: Interestingly, mutant fish show fully penetrant seizures at juvenile stages that accurately mimic tonic-clonic generalized seizures observed in patients. Moreover, highly penetrant seizures can be induced by light stimulation, thus providing us with the first zebrafish model in which evident epileptic seizures can be induced by nonchemical agents. Our transcriptomic assay identified misregulated genes in several pathways essential for correct brain development. More specifically, we show that the early development of the brain inhibitory network is specifically affected. Although the number of GABAergic neurons is not altered, we observed a drastic reduction in the number of inhibitory synapses and a decreased complexity of the GABAergic network. This is consistent with the disruption in expression of many genes involved in axon guidance and synapse formation. SIGNIFICANCE: Together with the role of GABA in neurodevelopment, our data identify a novel aspect of epileptogenesis, suggesting that the substratum of GABRA1-deficiency epilepsy is a consequence of early brain neurodevelopmental defects, in particular at the level of inhibitory network wiring.

Lack of GABAB receptors modifies behavioural and biochemical alterations induced by precipitated nicotine withdrawal.

The nicotine (NIC) withdrawal syndrome is considered to be a major cause of the high relapse rate among individuals undergoing smoking cessation. The aim of the present study was to evaluate a possible role of GABAB receptors in NIC withdrawal, by comparing GABAB1 knockout mice and their wild-type littermates. We analysed the time course of the global withdrawal score, the anxiety-like effects, monoamine concentrations, the brain-derived neurotrophic factor (BDNF) expression, the corticosterone plasmatic levels and [(3)H]epibatidine binding sites during NIC withdrawal precipitated by mecamylamine, a nicotinic receptor antagonist (MEC). In NIC withdrawn wild-type mice, we observed a global withdrawal score, an anxiety-like effect in the elevated plus maze, a decrease of the striatal dopamine and 3,4-dihydroxyphenylacetic acid concentrations, an increase of corticosterone plasma levels, a reduction of BDNF expression in several brain areas and an increase of [(3)H]epibatidine binding sites in specific brain regions. Interestingly, the effects found in NIC withdrawn wild-type mice were absent in GABAB1 knockout mice, suggesting that GABAB1 subunit of the GABAB receptor is involved in the regulation of the behavioural and biochemical alterations induced by NIC withdrawal in mice. These results reveal an interaction between the GABAB receptors and the neurochemical systems through which NIC exerts its long-term effects.

GABAB Receptors Regulate Extrasynaptic GABAA Receptors.

Tonic inhibitory GABA(A) receptor-mediated currents are observed in numerous cell types in the CNS, including thalamocortical neurons of the ventrobasal thalamus, dentate gyrus granule cells, and cerebellar granule cells. Here we show that in rat brain slices, activation of postsynaptic GABA(B) receptors enhances the magnitude of the tonic GABA(A) current recorded in these cell types via a pathway involving G G proteins, adenylate cyclase, and cAMP-dependent protein kinase. Using a combination of pharmacology and knockout mice, we show that this pathway is independent of potassium channels or GABA transporters. Furthermore, the enhancement in tonic current is sufficient to significantly alter the excitability of thalamocortical neurons. These results demonstrate for the first time a postsynaptic crosstalk between GABA(B) and GABA(A) receptors.

Differential effects of two major neurosteroids on cerebellar and cortical GABA(A) receptor binding and function.

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA(A)) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [(3)H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3µM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [(3)H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC(50)=150nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA(A) receptors containing α6 and δ subunits. In contrast, [(3)H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC(50) ~80nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [(3)H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [(3)H]EBOB binding with EC(50) ~0.5µM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1µM GABA in cerebellar granule cells, for 5α-THDOC with EC(50,1) ~16nM and EC(50,2) ~1.3µM. Differences in peak current enhancements in the absence minus presence of 0.1mM furosemide corresponding to α6ßδ GABA(A) receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA(A) receptor populations.

Altered ultrasonic vocalization and impaired learning and memory in Angelman syndrome mouse model with a large maternal deletion from Ube3a to Gabrb3.

Angelman syndrome (AS) is a neurobehavioral disorder associated with mental retardation, absence of language development, characteristic electroencephalography (EEG) abnormalities and epilepsy, happy disposition, movement or balance disorders, and autistic behaviors. The molecular defects underlying AS are heterogeneous, including large maternal deletions of chromosome 15q11-q13 (70%), paternal uniparental disomy (UPD) of chromosome 15 (5%), imprinting mutations (rare), and mutations in the E6-AP ubiquitin ligase gene UBE3A (15%). Although patients with UBE3A mutations have a wide spectrum of neurological phenotypes, their features are usually milder than AS patients with deletions of 15q11-q13. Using a chromosomal engineering strategy, we generated mutant mice with a 1.6-Mb chromosomal deletion from Ube3a to Gabrb3, which inactivated the Ube3a and Gabrb3 genes and deleted the Atp10a gene. Homozygous deletion mutant mice died in the perinatal period due to a cleft palate resulting from the null mutation in Gabrb3 gene. Mice with a maternal deletion (m-/p+) were viable and did not have any obvious developmental defects. Expression analysis of the maternal and paternal deletion mice confirmed that the Ube3a gene is maternally expressed in brain, and showed that the Atp10a and Gabrb3 genes are biallelically expressed in all brain sub-regions studied. Maternal (m-/p+), but not paternal (m+/p-), deletion mice had increased spontaneous seizure activity and abnormal EEG. Extensive behavioral analyses revealed significant impairment in motor function, learning and memory tasks, and anxiety-related measures assayed in the light-dark box in maternal deletion but not paternal deletion mice. Ultrasonic vocalization (USV) recording in newborns revealed that maternal deletion pups emitted significantly more USVs than wild-type littermates. The increased USV in maternal deletion mice suggests abnormal signaling behavior between mothers and pups that may reflect abnormal communication behaviors in human AS patients. Thus, mutant mice with a maternal deletion from Ube3a to Gabrb3 provide an AS mouse model that is molecularly more similar to the contiguous gene deletion form of AS in humans than mice with Ube3a mutation alone. These mice will be valuable for future comparative studies to mice with maternal deficiency of Ube3a alone.

Retinal vascular leakage occurring in GABA Rho-1 subunit deficient mice.

Recent studies demonstrate that GABAergic activity elicits relaxation of retinal arterioles leading to an increase in blood flow. It has also been found that GABAnergic activity in the retina of mice with diabetic retinopathy is suppressed. In this study, we provide further evidence that lack of GABAergic activity significantly alters vasculature development as well as the hypoxia-induced angiogenic response. Using GABA(C) receptor rho(1) subunit-knockout mice (rho-1(-/-)), our results demonstrate that in hypoxia-induced retinas a severe vascular leakage occurred in 2 week-old rho-1(-/-) mice compared with their wildtype counterparts. In addition, our results also showed that all of the rho-1(-/-) mice developed significant retinal vascular leakages by 48 weeks-of-age. Microarray and real-time PCR experiments revealed a unique angiogenesis-related gene expression pattern. This suggests that retinal vascular disorders of rho-1(-/-) mice results from significant up-regulation of angiogenic genes and concomitant down-regulation of anti-angiogenic genes. The study results are consistent with the pathological changes of the retinal vascular leakage seen in diabetic retinopathy. Our data indicate that the GABA(C) rho(1) subunit plays a role in maintaining both homeostasis and balance of retinal neurotransmitter function. Knockout of the retinal GABA(C) rho(1)-subunit leads to changes in vascular permeability similar to the pathological changes induced by retinal hypoxic conditions.

Progesterone receptor antagonists Org 31710 and RU 486 increase apoptosis in human periovulatory granulosa cells.

OBJECTIVE: To investigate if progesterone receptor (PR)-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated human luteinizing granulosa cells. DESIGN: Laboratory study. SETTING: Göteborg University and an in vitro fertilization laboratory of a university hospital. PATIENT(S): Women undergoing oocyte retrieval for in vitro fertilization after ovulation induction with gonadotropins. INTERVENTION(S): Luteinizing granulosa cells were isolated from follicular aspirates after oocyte removal. The cells were treated with or without RU 486 (1 microM-100 microM), Org 31710 (1 microM-100 microM), progesterone (1 nM-10 microM), dexamethasone (0.5 microM-100 microM), dihydrotestosterone (1 nM-25 microM), RU 486 (10 microM-100 microM) + dexamethasone (50 microM), and picrotoxin (1 microM-100 microM) and were cultured under serum-free conditions. MAIN OUTCOME MEASURE(S): Measurement of caspase-3 activity; detection of internucleosomal DNA fragmentation using gel electrophoresis and fluorospectrophotometry; progesterone analysis of spent medium. RESULT(S): Addition of the PR antagonists RU 486 or Org 31710 in vitro to human luteinizing granulosa cells caused an increase in caspase-3 activity and a dose-dependent increase in internucleosomal DNA fragmentation. No effect on DNA fragmentation was seen after addition of dexamethasone, dihydrotestosterone, or picrotoxin. CONCLUSION(S): Nuclear PR-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated human luteinizing granulosa cells.