Impaired GABAA receptor endocytosis and its correlation to spatial memory deficits

Abstract

GABA_A receptors mediate the majority of fast synaptic inhibition in the mammalian brain. Mechanisms that regulate GABA_A function are thus of critical importance in modulating overall synaptic inhibition. Phosphorylation of GABA_A receptor subunits is one such mechanism that leads to the dynamic modulation of GABA_A receptor function. In particular, phosphorylation of tyrosine residues 365 and 367 (Y365, Y367) within the γ2 subunit of GABAA receptors has been shown in previous in vitro studies to negatively regulate clathrin-dependent endocytosis of GABA_A receptors and to enhance the efficacy of synaptic inhibition. With the aim of investigating the impact of this phosphorylation-dependent modulation of GABA_A receptors on animal behavior, we recently developed a knock-in mouse in which these critical tyrosine residues within the γ2 subunit have been mutated to phenylalanines (Y365/7F). These animals exhibited enhanced GABA_A receptor accumulation at postsynaptic inhibitory synaptic specializations on pyramidal neurons within the hippocampus, primarily due to aberrant trafficking within the endocytic pathway. We found that this enhanced inhibition correlated with a specific deficit in spatial memory in these mice, without modifying a number of other behavioral paradigms. Here, we summarize our recently reported observations and further discuss their possible implications.

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GABA_A receptors are heteropentameric chloride-selective ligand-gated ion channels that mediate fast synaptic inhibition in the adult central nervous system. Regulation of the number of GABA_A receptors at synapses is critical for maintaining the correct level of synaptic inhibitory transmission and physiological function.1 Recent studies have suggested that dysregulation in GABA_A receptor trafficking may underlie pathophysiological conditions, including pharmaco-resistance and self-sustaining seizures in status epilepticus,2,3 increased excitotoxicity in ischemia4 and trait anxiety.5 Understanding the endogenous mechanisms that regulate GABA_A receptor membrane trafficking is thus of clinical significance.

GABA_A receptor trafficking can be regulated, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles. Endocytosis of GABA_A receptors is facilitated by the clathrin adaptor protein 2 (AP2) complex, which interacts directly with neuronal GABA_A receptor subunits (Fig. 1A). A classical tyrosine-based (Yxxφ) binding motif for AP2 is located within the intracellular domain of the γ2 subunit of the GABA_A receptor, centered on tyrosine residues 365 and 367.6,7 This motif binds the γ2 subunit of the AP2 complex with highaffinity.6 Consistent with studies of other tyrosine-based internalization motifs,8 the tyrosine residues Y365/7 in the γ2 subunit can be phosphorylated by Src family tyrosine kinases.9–11 Phosphorylation of these residues has been demonstrated in prior in vitro studies to negatively regulate clathrin-dependent endocytosis of GABA_A receptors and to enhance the efficacy of synaptic inhibition.6,9,10

In our recent study,12 we aimed to investigate the impact of this phosphorylation- dependent modulation of GABA_A receptors on animal behavior. We used a genetic-engineering approach to generate a knock-in mouse in which tyrosine residues 365 and 367 within the GABA_A receptor γ2 subunit were mutated to phenylalanines. Surprisingly, homozygous mice for this mutation exhibited a lethal phenotype, with morbidity occurring at a very early embryonic developmental stage. One intriguing speculation is that GABA_A receptor signaling plays a key role in neurogenesis/ early developmental maturation of the nervous system, however, further studies will be needed to determine the exact underlying cause of the embryonic lethality.

We then focused our study on male mice heterozygous for the γ2(Y365/7F) mutation. We observed a dramatic reduction in the level of tyrosine phosphorylation of the γ2 subunit in γ2(Y365/7F), which was an expected result based on the mutation of these critical tyrosine phosphorylation sites. We also observed significant increases in the cell surface expression levels of the γ2 subunit in the hippocampus of heterozygous mice, an effect that was mediated by a reduction in the level of endocytosis of the mutant γ2 subunit. Thus, Y365/7 residues play a central role in regulating GABA_A receptor endocytosis in vivo. This is consistent with our in vitro studies demonstrating that mutation of these tyrosine residues to phenylalanines significantly reduces binding to μ2-AP26,12 (Fig. 1B).

One interesting observation was that the increase in γ2 subunit levels in the mutants was not uniform throughout the hippocampus. It appeared to be particularly dominant in specific neuronal populations, such as CA3 pyramidal neurons. The reasons underlying this region-specific disparity are currently unknown, however it will be an interesting question to address in future work. Differences in the subunit composition of GABA_A receptors between the different hippocampal subregions may be one underlying cause.13 Regional specificity in the particular tyrosine kinases and/or phosphatases that regulate phosphorylation of endogenous γ2 subunits may also contribute to the differential regulation of GABA_A receptor trafficking within distinct hippocampal subregions.

We next assessed whether these region-specific increases in the γ2 subunit expression had an impact on hippocampal-dependent behavior. After confirming that the γ2(Y365/7F) heterozygotes have no deficits in basal locomotor or exploratory behavior, we then assessed the cognitive abilities of these mice. We did this by using the spatial version of the object recognition task, which is based on the ability of animals to discriminate between familiar and novel spatial locations of objects. Strikingly, the mutant mice failed to discriminate between the displaced and nondisplaced objects, whereas wild-type mice displayed selectivity for the newly displaced object. This difference was not due to a deficit in object recognition memory, since the mutants performed similarly to wild-type mice when subjected to the novel object recognition task. Thus, our behavioral studies demonstrate that γ2(Y365/7F) heterozygote animals have specific deficits in spatial memory, a task that is critically dependent on the CA3 region of the hippocampus.14 Moreover, this behavioral deficit is mirrored by an enhancement in the efficacy of synaptic inhibition in CA3, which presumably underlies this behavioral deficit.12 This result is reminiscent of animal models of Down syndrome, where excessive levels of neuronal inhibition are believed to contribute to cognitive deficits.15

The possibility of improving memory performance and cognition by altering the balance between excitatory and inhibitory neurotransmission is a working hypothesis that is currently being investigated by many groups for its therapeutic potential.16 For example, a reversal of the cognitive impairments observed in a mouse model of Down syndrome was achieved by pharmacological treatment with GABA_A receptor antagonists.15 Other studies have proposed that partial inverse agonists with a preference for α5-containing GABA_A receptors may be particularly good candidates for memory-enhancing drugs.17,18 Thus, drugs that target specific GABA_A receptor populations, or alternatively, pharmacological manipulation of endogenous GABA_A receptor trafficking mechanisms, may prove useful for achieving cognitive enhancement in the near future.

In summary, our results demonstrate that two tyrosine residues (Y365/7) within the GABA_A receptor γ2 subunit play a critical role in shaping hippocampal neuronal activity by regulating the cell surface accumulation of GABA_A receptors at inhibitory synapses and consequently the efficacy of synaptic inhibition. Mutation of these residues results in aberrant synaptic GABA_A receptor trafficking and dysfunctional hippocampal-dependent learning. Based on our results, and recent results from other investigators, it thus appears that alterations in the number and/or function of GABA_A receptors can have significant effects on memory and cognition.

Figures and Tables

Figure 1 Model summarizing the regulation of GABA_A receptor endocytosis by the Yxxφ motif within the GABA_A receptor γ2 subunit. (A) Endocytosis via clathrin-coated vesicles (CCV) is the major internalization mechanism for neuronal GABA_A receptors (GABA_ARs). The intracellular domain of the γ2 subunit of the GABA_AR interacts with the μ2 subunit of the clathrin-adaptor protein 2 (AP2). This binding is mediated via a Yxxφ motif contained within the γ2 subunit that is centered around tyrosine residues 365 and 367. (B) Binding of the AP2 complex is negatively regulated by mutation of these tyrosine residues to phenylalanines. γ2(Y365/7F) heterozygous mutant mice have decreased levels of receptor endocytosis and increased cell-surface levels of γ2-containing GABA_ARs. We have also demonstrated that these mutant mice display an enhanced efficacy of inhibitory synaptic transmission in the CA3 region of the hippocampus.12

Acknowledgements

S.J.M. is supported by National Institute of Neurological Disorders and Stroke Grants (NS047478, NS051195, NS056359, NS054900), the Medical Research Council, and the Wellcome Trust. R.J. is supported by the Maltz Family Foundation as a National Alliance for Research on Schizophrenia and Depression Young Investigator.

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