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Slow degradation and aggregation in vitro of mutant GABAA receptor gamma2(Q351X) subunits associated with epilepsy.

ABSTRACT: The GABA(A) receptor ?2 subunit nonsense mutation Q351X has been associated with the genetic epilepsy syndrome generalized epilepsy with febrile seizures plus, which includes a spectrum of seizures types from febrile seizures to Dravet syndrome. Although most genetic epilepsy syndromes are mild and remit with age, Dravet syndrome has a more severe clinical course with refractory seizures associated with developmental delay and cognitive impairment. The basis for the broad spectrum of seizure phenotypes is uncertain. We demonstrated previously that the GABA(A) receptor ?2 subunit gene Q351X mutation suppressed biogenesis of wild-type partnering ?1 and ?2 subunits in addition to its loss of function. Here we show that ?2S(Q351X) subunits have an additional impairment of biogenesis. Mutant ?2(Q351X) subunits were degraded more slowly than wild-type ?2 subunits and formed SDS-resistant, high-molecular-mass complexes or aggregates in multiple cell types, including neurons. The half-life of ?2S(Q351X) subunits was ?4 h, whereas that of ?2S subunits was ?2 h. Mutant subunits formed complexes rapidly after synthesis onset. Using multiple truncated subunits, we demonstrated that aggregate formation was a general phenomenon for truncated ?2S subunits and that their Cys-loop cysteines were involved in aggregate formation. Protein aggregation is a hallmark of neurodegenerative diseases, but the effects of the mutant ?2S(Q351X) subunit aggregates on neuronal function and survival are unclear. Additional validation of the mutant subunit aggregation in vivo and determination of the involved signaling pathways will help reveal the pathological effects of these mutant subunit aggregates in the pathogenesis of genetic epilepsy syndromes.

SUBMITTER: Kang JQ

PROVIDER: S-EPMC2976503 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Slow degradation and aggregation in vitro of mutant GABAA receptor gamma2(Q351X) subunits associated with epilepsy.

Kang Jing-Qiong JQ Shen Wangzhen W Lee Melissa M Gallagher Martin J MJ Macdonald Robert L RL

The Journal of neuroscience : the official journal of the Society for Neuroscience 20101001 41

The GABA(A) receptor γ2 subunit nonsense mutation Q351X has been associated with the genetic epilepsy syndrome generalized epilepsy with febrile seizures plus, which includes a spectrum of seizures types from febrile seizures to Dravet syndrome. Although most genetic epilepsy syndromes are mild and remit with age, Dravet syndrome has a more severe clinical course with refractory seizures associated with developmental delay and cognitive impairment. The basis for the broad spectrum of seizure phe ...[more]

PMID: 20943930

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Project description:Phasic GABAergic inhibition in hippocampus and neocortex falls into two kinetically distinct categories, GABA(A,fast) and GABA(A,slow). In hippocampal area CA1, GABA(A,fast) is generally believed to underlie gamma oscillations, whereas the contribution of GABA(A,slow) to hippocampal rhythms has been speculative. Hypothesizing that GABA(A) receptors containing the beta(3) subunit contribute to GABA(A,slow) inhibition and that slow inhibitory synapses control excitability as well as contribute to network rhythms, we investigated the consequences of this subunit's absence on synaptic inhibition and network function. In pyramidal neurons of GABA(A) receptor beta(3) subunit-deficient (beta(3)(-/-)) mice, spontaneous GABA(A,slow) inhibitory postsynaptic currents (IPSCs) were much less frequent, and evoked GABA(A,slow) currents were much smaller than in wild-type mice. Fittingly, long-lasting recurrent inhibition of population spikes was less powerful in the mutant, indicating that receptors containing beta(3) subunits contribute substantially to GABA(A,slow) currents in pyramidal neurons. By contrast, slow inhibitory control of GABA(A,fast)-producing interneurons was unaffected in beta(3)(-/-) mice. In vivo hippocampal network activity was markedly different in the two genotypes. In beta(3)(-/-) mice, epileptiform activity was observed, and theta oscillations were weaker, slower, less regular and less well coordinated across laminae compared with wild-type mice, whereas gamma oscillations were weaker and faster. The amplitude modulation of gamma oscillations at theta frequency ("nesting") was preserved but was less well coordinated with theta oscillations. With the caveat that seizure-induced changes in inhibitory circuits might have contributed to the changes observed in the mutant animals, our results point to a strong contribution of beta(3) subunits to slow GABAergic inhibition onto pyramidal neurons but not onto GABA(A,fast) -producing interneurons and support different roles for these slow inhibitory synapses in the generation and coordination of hippocampal network rhythms.

Project description:Presynaptic α2δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α2δ-2 can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ-2 is essential for the trans-synaptic mechanism. The novel function of α2δ-2 identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α2δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α2δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α2δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.

Dataset Information

Slow degradation and aggregation in vitro of mutant GABAA receptor gamma2(Q351X) subunits associated with epilepsy.

Publications

Slow degradation and aggregation in vitro of mutant GABAA receptor gamma2(Q351X) subunits associated with epilepsy.

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