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Testing the silence of mutations: Transcriptomic and behavioral studies of GABA(A) receptor ?1 and ?2 subunit knock-in mice.

ABSTRACT: Knock-in mice were constructed with mutations in the ?1 (H(270), A(277)) and ?2 (H(270), A(277)) subunits of the GABAA receptor, which resulted in receptors that lacked modulation by ethanol but retained normal responses to GABA in vitro. A key question is whether these mutant receptors also function normally in vivo. Perturbation of brain function was evaluated by gene expression profiling in the cerebral cortex and by behavioral pharmacology experiments with GABAergic drugs. Analysis of individual transcripts found only six transcripts that were changed in ?1 knock-in mice and three in the ?2 mutants (p<0.05, corrected for multiple comparisons). Two transcripts that are sensitive to neuronal activity, Arc and Fos, increased about 250% in the ?2 mutants, and about 50% in the ?1 mutants. Behavioral effects (loss of righting reflex, rotarod) of flurazepam and pentobarbital were not different between ?2 mutants and wild-type, but they were enhanced for ?1 knock-in mice. These results indicate that introduction of these mutations in the ?2 subunit of the GABAA receptor does not produce marked perturbation of brain function, as measured by gene expression and GABAergic behavioral responses, but the same mutations in the ?1 subunit produce more pronounced changes, especially in GABAergic function.

SUBMITTER: Harris RA

PROVIDER: S-EPMC3033563 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Testing the silence of mutations: Transcriptomic and behavioral studies of GABA(A) receptor α1 and α2 subunit knock-in mice.

Harris R A RA Osterndorff-Kahanek E E Ponomarev I I Homanics G E GE Blednov Y A YA

Neuroscience letters 20101105 1

Knock-in mice were constructed with mutations in the α1 (H(270), A(277)) and α2 (H(270), A(277)) subunits of the GABAA receptor, which resulted in receptors that lacked modulation by ethanol but retained normal responses to GABA in vitro. A key question is whether these mutant receptors also function normally in vivo. Perturbation of brain function was evaluated by gene expression profiling in the cerebral cortex and by behavioral pharmacology experiments with GABAergic drugs. Analysis of indivi ...[more]

PMID: 21056629

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Project description:Glycine receptors (GlyRs) containing the α2 subunit govern cell fate, neuronal migration and synaptogenesis in the developing cortex and spinal cord. Rare missense variants and microdeletions in the X-linked GlyR α2 subunit gene (GLRA2) have been associated with human autism spectrum disorder (ASD), where they typically cause a loss-of-function via protein truncation, reduced cell-surface trafficking and/or reduced glycine sensitivity (e.g., GLRA2Δex8-9 and extracellular domain variants p.N109S and p.R126Q). However, the GlyR α2 missense variant p.R323L in the intracellular M3-M4 domain results in a gain-of-function characterized by slower synaptic decay times, longer duration active periods and increases in channel conductance. This study reports the functional characterization of four missense variants in GLRA2 associated with ASD or developmental disorders (p.V-22L, p.N38K, p.K213E, p.T269M) using a combination of bioinformatics, molecular dynamics simulations, cellular models of GlyR trafficking and electrophysiology in artificial synapses. The GlyR α2V-22L variant resulted in altered predicted signal peptide cleavage and a reduction in cell-surface expression, suggestive of a partial loss-of-function. Similarly, GlyR α2N38K homomers showed reduced cell-surface expression, a reduced affinity for glycine and a reduced magnitude of IPSCs in artificial synapses. By contrast, GlyR α2K213E homomers showed a slight reduction in cell-surface expression, but IPSCs were larger, with faster rise/decay times, suggesting a gain-of-function. Lastly, GlyR α2T269M homomers exhibited a high glycine sensitivity accompanied by a substantial leak current, suggestive of an altered function that could dramatically enhance glycinergic signaling. These results may explain the heterogeneity of clinical phenotypes associated with GLRA2 mutations and reveal that missense variants can result in a loss, gain or alteration of GlyR α2 function. In turn, these GlyR α2 missense variants are likely to either negatively or positively deregulate cortical progenitor homeostasis and neuronal migration in the developing brain, leading to changes in cognition, learning, and memory.

Project description:BackgroundSeizures are associated with a decrease in γ-aminobutyric type A acid receptors (GABAaRs) on the neuronal surface, which may be regulated by enhanced internalization of GABAaRs. When interactions between GABAaR subunit α-1 (GABRA1) and postsynaptic scaffold proteins are weakened, the α1-containing GABAaRs leave the postsynaptic membrane and are internalized. Previous evidence suggested that neuroplastin (NPTN) promotes the localization of GABRA1 on the postsynaptic membrane. However, the association between NPTN and GABRA1 in seizures and its effect on the internalization of α1-containing GABAaRs on the neuronal surface has not been studied before.MethodsAn in vitro seizure model was constructed using magnesium-free extracellular fluid, and an in vivo model of status epilepticus (SE) was constructed using pentylenetetrazole (PTZ). Additionally, in vitro and in vivo NPTN-overexpression models were constructed. Electrophysiological recordings and internalization assays were performed to evaluate the action potentials and miniature inhibitory postsynaptic currents of neurons, as well as the intracellular accumulation ratio of α1-containing GABAaRs in neurons. Western blot analysis was performed to detect the expression of GABRA1 and NPTN both in vitro and in vivo. Immunofluorescence co-localization analysis and co-immunoprecipitation were performed to evaluate the interaction between GABRA1 and NPTN.ResultsThe expression of GABRA1 was found to be decreased on the neuronal surface both in vivo and in vitro seizure models. In the in vitro seizure model, α1-containing GABAaRs showed increased internalization. NPTN expression was found to be positively correlated with GABRA1 expression on the neuronal surface both in vivo and in vitro seizure models. In addition, NPTN overexpression alleviated seizures and NPTN was shown to bind to GABRA1 to form protein complexes that can be disrupted during seizures in both in vivo and in vitro models. Furthermore, NPTN was found to inhibit the internalization of α1-containing GABAaRs in the in vitro seizure model.ConclusionOur findings provide evidence that NPTN may exert antiepileptic effects by binding to GABRA1 to inhibit the internalization of α1-containing GABAaRs.

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Testing the silence of mutations: Transcriptomic and behavioral studies of GABA(A) receptor ?1 and ?2 subunit knock-in mice.

Publications

Testing the silence of mutations: Transcriptomic and behavioral studies of GABA(A) receptor α1 and α2 subunit knock-in mice.

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