Autism-related deficits via dysregulated eIF4E-dependent translational control

Abstract

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)-an eIF4E repressor downstream of mTOR-or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

Figure 1. Social interaction deficits, repetitive behavior and elevated USVs in4E-BP2-KO mice

(a) Three-chamber social interaction test, showing time spent in each chamber, number of entries and time spent sniffing the wire-cage; n=12 for each group; ***p<0.001, **p<0.01, *p<0.03, two-way ANOVA with Bonferroni’s post-hoc. (b) Homecage and (c) reciprocal social interactions tests showing total time in contact and number of contacts; n=18 per group **p<0.02, *p<0.03; one way ANOVA with Bonferroni’s post-hoc. (d) Self-grooming test showing total time spent grooming. (e) Marble-burying test showing marbles buried; n=12 per group *p<0.03; Student's t-test. (f–h) Isolation induced USVs at various post-natal days (pnd) (f) number of calls/min, (g) duration of calls and (h) amplitude of USVs in pups; n=12 per group *p<0.05; two-way ANOVA, with Bonferroni’s post-hoc. Error-bars mean ±SEM.

Figure 2. Enhanced eIF4E-dependent translation of neuroligins

(a) Polysome profiles from hippocampal lysates of WT and 4E-BP2-KO mice and (b) WT and βT-eIF4E mice. Positions of 40S, 60S and 80S ribosome peaks and polysomes are indicated. (c) RT-PCR on RNA extracted from polysome fractions from WT and 4E-BP2-KO and (d) WT and βT-eIF4E (4E) hippocampi. Representative gel images are shown; n=4 per group. (e) Representative immunoblots of crude (cr.) and synaptosomal (syn.) fractions from hippocampal lysates of WT or 4E-BP2-KO and (f) WT or βT-eIF4E (4E) mice, probed with antibodies against the indicated proteins. (g–h) Quantification of immunoblots from (e) and (f) for neuroligins; n=4 per group *p<0.05; Student's t-test. Error-bars mean ±SEM.

Figure 3. Excitation is increased to a larger extent than inhibition in 4E-BP2-KO mice

(a, c) Representative traces (top) and cumulative distribution plots (bottom) of mEPSCs (a) and mIPSCs (c) from CA1 pyramidal in acute slices from WT and 4E-BP2^−/− mice, illustrating changes in amplitude and frequency. (b, d) Bar graphs showing mEPSC (b) and (d) IPSC amplitude and frequency (n=number of cells). (e) Bar graphs showing the synaptic total charge transfer in 4E-BP2-KO relative to WT slices. For a-e, *p<0.05; one-way ANOVA with Bonferroni’s post-hoc. (f) Relative changes in mEPSC and mIPSC total charge transfer, normalized to the mean WT value, for each neuron from 4E-BP2-KO slices; Kolmogorov-Smirnov test, p=0.005, KS statistic=0.916. Error-bars mean ±SEM.

Figure 4. Rescue of excitatory/inhibitory synaptic activity imbalance and social deficits in 4E-BP2-KO by inhibiting the eIF4E-eIF4G interaction

(a) Schematic of 4EGI-1 treatment in the three-chamber test (Top) and slice-culture recordings (Bottom). (b) Immunoblot analysis of cap column pull-down in 4E-BP2-KO and WT mice treated with 4EGI-1 or vehicle. Total and m⁷GDP bound extracts were probed for eIF4GI, eIF4E and 4E-BP2; n=4 - quantification in Supplementary Fig. 16a (c, d) Effects of 4EGI-1 on mEPSCs (c) or mIPSCs (d) amplitude and frequency in 4E-BP2-KO slices. (e) Effects of 4EGI-1 on total charge transfer of mEPSCs (Top) and mIPSCs (Middle) in 4E-BP2-KO slices. Bottom: Relative changes in mEPSC and mIPSC total charge transfer, normalized to the mean WT value, for 4E-BP2-KO neurons. Kolmogorov-Smirnov test. For c-e, n=6 per group; *p< 0.05; one-way ANOVA with Bonferroni’s post-hoc. (f, g) Rescue effects of 4EGI-1 infusion in the three-chamber test showing (f) time spent in chambers and (g) time spent sniffing the wire cage in 4E-BP2-KO and WT mice. For f, g, n=12 per group; *p<0.03; two-way ANOVA, with a Bonferroni’s post-hoc. Error-bars mean ±SEM.

Figure 5. Knockdown of neuroligin 1 rescues the excitatory/inhibitory synaptic activity imbalance and social deficits in 4E-BP2-KO mice

(a) Schematic of the three-chamber test in mice icv-injected with lentiviruses expressing shRNAs (Top) and recordings from siRNA-transfected slice-cultures (Bottom). (b) Western blot analysis of hippocampal lysates from WT or 4E-BP2-KO mice injected with lentiviruses expressing shRNAs against a non-targeting sequence or Nlgn1-2. Extracts were probed for NLGN1-4, β -actin, eIF4E and 4E-BP2; n=4-quantification in Supplementary Fig. 16b,c. (c,d) Effects of Nlgn1 or Nlgn2 knockdown on mEPSCs (c) or mIPSCs (d) amplitude and frequency in transfected pyramidal cells. (e) mEPSCs (Top) or mIPSCs (Middle) total charge transfer in Nlgn1-2 knockdown cells. Bottom: Relative changes in mEPSC and mIPSC total charge transfer, normalized to the mean WT value, for each neuron from 4E-BP2-KO slices; Kolmogorov-Smirnov test. For c-e, n=5 per group; *p<0.05; one-way ANOVA with Bonferroni’s post-hoc. (f,g) Knockdown of Nlgn1-2 in the three-chamber test showing (f) time spent in chambers and (g) time spent sniffing the wire-cage. For f–g, n=12 per group; *p<0.02; two-way ANOVA, with a Bonferroni’s post-hoc. Error-bars mean ±SEM.