Genetic and functional analyses demonstrate a role for abnormal glycinergic signaling in autism

Abstract

Autism spectrum disorder (ASD) is a common neurodevelopmental condition characterized by marked genetic heterogeneity. Recent studies of rare structural and sequence variants have identified hundreds of loci involved in ASD, but our knowledge of the overall genetic architecture and the underlying pathophysiological mechanisms remains incomplete. Glycine receptors (GlyRs) are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system but exert an excitatory action in immature neurons. GlyRs containing the α2 subunit are highly expressed in the embryonic brain, where they promote cortical interneuron migration and the generation of excitatory projection neurons. We previously identified a rare microdeletion of the X-linked gene GLRA2, encoding the GlyR α2 subunit, in a boy with autism. The microdeletion removes the terminal exons of the gene (GLRA2(Δex8-9)). Here, we sequenced 400 males with ASD and identified one de novo missense mutation, p.R153Q, absent from controls. In vitro functional analysis demonstrated that the GLRA2(Δex8)(-)(9) protein failed to localize to the cell membrane, while the R153Q mutation impaired surface expression and markedly reduced sensitivity to glycine. Very recently, an additional de novo missense mutation (p.N136S) was reported in a boy with ASD, and we show that this mutation also reduced cell-surface expression and glycine sensitivity. Targeted glra2 knockdown in zebrafish induced severe axon-branching defects, rescued by injection of wild type but not GLRA2(Δex8-9) or R153Q transcripts, providing further evidence for their loss-of-function effect. Glra2 knockout mice exhibited deficits in object recognition memory and impaired long-term potentiation in the prefrontal cortex. Taken together, these results implicate GLRA2 in non-syndromic ASD, unveil a novel role for GLRA2 in synaptic plasticity and learning and memory, and link altered glycinergic signaling to social and cognitive impairments.

Figure 1. Microdeletion and mutation of GLRA2 in ASD

(a) Physical map of Xp22.2. CNV analysis showed a GLRA2 deletion removing exons 8 and 9 in Patient 1, inherited from his unaffected mother (red). (b) Gel image of long-range PCR products showing an abnormal 1.1 kb allele in Patient 1 and his mother. (c) Breakpoint sequence analysis in Patient 1 revealed a 151055 bp deletion starting in intron 7 of GLRA2. A G>A transition precedes the deletion. (d) Identification of a de novo missense mutation in exon 4 of GLRA2 in Patient 2 (c.458G>A, p.153R>Q). (e) Schematic representation of the GLRA2 protein with the deletion and the mutation identified in ASD. The exons are indicated by brackets; the signal peptide and the four transmembrane domains are shown in dark blue and orange, respectively. The alignment of protein sequences of human GlyRs and orthologs from other species is shown below. The highly conserved arginine at position 153, mutated in Patient 2, is boxed in red.

Figure 2. GLRA2 mutations identified in individuals with ASD abolish normal protein function

(a) Schematic representation of deleted and mutated GLRA2 constructs co-transfected with EGFP cDNA in CHO cells. The truncated mutant (GLRA2^Δex8-9) was produced to end at amino acid 310, immediately followed by six amino acids (VRNLA*), resulting in loss of two transmembrane domains, as in Patient 1. In the mutant construct (GLRA2^R153Q), Arg153 was mutated to glutamine as in Patient 2. The first and last amino acids are numbered; the signal peptide and the four transmembrane domains are shown in dark blue and orange, respectively. Confocal cross-sections show surface expression of wild-type and mutant GLRA2 proteins in non-permeabilized cells. GLRA2^R153Q localized properly to the plasma membrane, as shown by the punctiform staining of GlyR α2 at the cell surface, whereas GLRA2^Δex8-9 failed to traffic to the membrane. Scale bar, 10 μm. (b) Representative traces of currents evoked by application of glycine on CHO cells expressing wild-type and mutated GLRA2 proteins. Bars represent application of glycine at concentrations noted. (c) Fit of data in (b) to the Hill equation. Errors indicate SEM (n = 7 GLRA2^R153Q, 13 wild-type). (d, e) Predicted structure of the homomeric GlyR α2 subunit. The blue and yellow α2 subunits contain residues on the plus and minus sides of the glycine-binding site, respectively. A single glycine-binding site is represented in the native state (d) and the R153Q mutant (e). Predicted residues of importance are labeled by residue number. Dashed lines indicate hydrogen bonds. Elimination of the hydrogen bonds stabilizing the backbone of the binding-site residue T238 is predicted to cause direct disruption of glycine binding.

Figure 3. ASD-associated mutations in GLRA2 fail to rescue the axon branching phenotype caused by knockdown of the zebrafish orthologue

(a, b) Immunostaining of 28-h post-fertilization control (a), morphant (MO^α2) (a), and rescued (MO^α2 + mRNA^α2 or mRNA^α2R153Q or mRNA^α2Δex8-9) (b) larvae labeled with Znp1 antibody; lateral views of the trunk (anterior to the left). Higher magnifications are shown on the right. Arrows indicate aberrant supernumerary branches of spinal motor axons. Scale bars for all images, 10 μm. (c) Quantification of axon branching in 16 somites centered around the anus in control, morphant, and rescued larvae. The number of branches was significantly higher in MO^α2 larvae than in controls and was rescued by concomitant overexpression of human GLRA2 (mRNA^α2), whereas deleted (mRNA^α2Δex8-9) or mutated (mRNA^α2R153Q) versions of the transcript failed to rescue the abnormal axon phenotype. Data represent mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Glra2^−/Y mice exhibit deficits in recognition memory and impaired cortical synaptic plasticity

(a) In the novel object recognition task, adult wild-type (WT) mice spent more time exploring the novel object, whereas Glra2^−/Y mice had no preference for either object after a 10 min or 24 h memory delay, as measured by exploration time and discrimination index (n = 11 WT, 12 Glra2^−/Y). (b) Glra2^−/Y mice exhibited no deficits when assessed on the spatial version of the task (the novel location recognition test) and showed normal preference for the object placed in a new location (n = 9 WT, 10 Glra2^−/Y). (c) In the Morris water maze, Glra2^−/Y mice displayed normal acquisition and reversal learning. Both genotypes showed a significant preference for the target quadrant during a probe trial conducted 10 min following the final training session (n = 5 WT, 7 Glra2^−/Y). AdjL, adjacent left quadrant; Target, target quadrant; AdjR, adjacent right quadrant; Opp, opposite to target quadrant. (d) Glra2^−/Y mice exhibited impaired long-term potentiation in the prefrontal cortex (n = 14 slices from 9 WT mice, 10 slices from 6 Glra2^−/Y mice). fEPSP, field excitatory postsynaptic potential. Data represent mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.