Negative allosteric modulation of the mGluR5 receptor reduces repetitive behaviors and rescues social deficits in mouse models of autism

Abstract

Neurodevelopmental disorders such as autism and fragile X syndrome were long thought to be medically untreatable, on the assumption that brain dysfunctions were immutably hardwired before diagnosis. Recent revelations that many cases of autism are caused by mutations in genes that control the ongoing formation and maturation of synapses have challenged this dogma. Antagonists of metabotropic glutamate receptor subtype 5 (mGluR5), which modulate excitatory neurotransmission, are in clinical trials for fragile X syndrome, a major genetic cause of intellectual disabilities. About 30% of patients with fragile X syndrome meet the diagnostic criteria for autism. Reasoning by analogy, we considered the mGluR5 receptor as a potential target for intervention in autism. We used BTBR T+tf/J (BTBR) mice, an established model with robust behavioral phenotypes relevant to the three diagnostic behavioral symptoms of autism--unusual social interactions, impaired communication, and repetitive behaviors--to probe the efficacy of a selective negative allosteric modulator of the mGluR5 receptor, GRN-529. GRN-529 reduced repetitive behaviors in three cohorts of BTBR mice at doses that did not induce sedation in control assays of open field locomotion. In addition, the same nonsedating doses reduced the spontaneous stereotyped jumping that characterizes a second inbred strain of mice, C58/J. Further, GRN-529 partially reversed the striking lack of sociability in BTBR mice on some parameters of social approach and reciprocal social interactions. These findings raise the possibility that a single targeted pharmacological intervention may alleviate multiple diagnostic behavioral symptoms of autism.

Fig. 1

GRN-529 chemical structure, plasma and brain concentrations, and receptor occupancy in B6, BTBR, and C58 mice. (A) Chemical structure of GRN-529 and binding properties [K_i = 5.4 nM and median inhibitory concentration (IC₅₀) = 3.1 nM] at rat mGluR5. (B to D) Unbound plasma and brain concentrations of GRN-529 30 or 60 min after systemic administration in B6, BTBR, and C58 mice. s.c., subcutaneously; i.p., intraperitoneally. (E) Time course of unbound plasma and brain concentrations (nM) of GRN-529 for 2 hours after systemic intraperitoneal administration in B6 mice. (F) Relationship of the concentration of unbound (Cb,u) GRN-529 concentrations (nM) and mGluR5 occupancy in brains from BTBR, B6, and C58 mice. n = 3 to 5 per dose and strain. Data are expressed as the mean for each group.

Fig. 2

Effect of GRN-529 on repetitive self-grooming in BTBR and stereotyped jumping in C58 mice. Cumulative time spent self-grooming by BTBR and B6 mice was scored over a 10-min session in a clean, closed, empty cage after a 10-min acclimation period. Observations of stereotyped jumping behavior in C58 mice were quantified for a period of 10 min. GRN-529 was tested in two independent laboratory environments across three cohorts. (A) B6 mice did not display any significant differences in the amount of time spent self-grooming after treatment with vehicle (10% Tween 80/saline) or GRN-529 at doses of 0.3, 1.0, or 3.0 mg/kg intraperitoneally (n = 8 to 10 per dose, cohort 1, tested at NIMH, *P < 0.05 versus vehicle). (B) BTBR displayed significant reductions in their innately high levels of repetitive self-grooming after treatment with GRN-529 at doses of 1.0 and 3.0 mg/kg (n = 11 to 14 per dose, cohort 1, tested at NIMH, *P < 0.05 versus vehicle). (C) B6 mice displayed significant reductions in the amount of time spent self-grooming after treatment with GRN-529 at doses of 1.0 and 3.0 mg/kg compared to vehicle (cohort 2, tested at Pfizer). (D) BTBR again displayed significant reductions in high levels of repetitive self-grooming after treatment with GRN-529 at doses of 1.0 and 3.0 mg/kg intraperitoneally (n = 17 to 25 per dose for each strain, cohort 2, tested at Pfizer, *P < 0.05 versus vehicle). (E) Stereotyped vertical jumping in C58 mice was significantly reduced after GRN-529 administration at doses of 0.3, 1.0, and 3.0 mg/kg intraperitoneally versus vehicle (*P < 0.05, tested at Pfizer). (F) No adverse or sedating effects on the general activity of C58 mice were observed during open field locomotion (P > 0.05, tested at Pfizer).

Fig. 3

Effect of GRN-529 on social approach in adult BTBR mice. Social approach was assessed in an automated photocell-equipped three-chambered arena with observer scoring of direct sniffing interactions from videotapes of the social approach. (A) B6 mice displayed sociability on the more sensitive parameter, time spent sniffing the novel mouse compared to time spent sniffing the novel object, at each dose of GRN-529 and vehicle. (B) BTBR exhibited its characteristic lack of sociability on the sniff parameter after vehicle administration. BTBR treated with a single acute dose of GRN-529, 0.3, 1.0, or 3.0 mg/kg intraperitoneally, exhibited significant sociability on the sniff time parameter. (C) The B6 control strain displayed normal sociability, defined as spending more time in the chamber with the novel mouse than in the chamber with the novel object, after a single intraperitoneal dose of vehicle (10% Tween 80/saline) or GRN-529 at doses of 0.3, 1.0, and 3 mg/kg. (D) BTBR exhibited its characteristic lack of sociability, that is, did not spend more time in the novel mouse chamber than in the novel object chamber, after treatment with vehicle or the two lower doses of GRN-529. At the highest dose, 3.0 mg/kg, BTBR displayed significant sociability. (E and F) B6 (E) and BTBR (F) displayed a greater number of entries into the side chambers after treatment with GRN-529 at the highest dose, 3.0 mg/kg intraperitoneally, indicating a general increase of exploratory activity during the social approach task at that dose. *P < 0.05, novel mouse versus novel object in (A) to (D); *P < 0.05 versus vehicle in (E) and (F). n = 10 per dose for each strain, cohort 1, assayed at NIMH. See fig. S2 for replicated findings in cohort 2.

Fig. 4

Effect of GRN-529 on dyadic reciprocal social interactions in BTBR mice. Social interactions were digitally recorded in dyads of mice in the Noldus PhenoTyper 3000 arena. Coded videos were subsequently scored by an observer uninformed of the treatment condition using Noldus Observer 8.0XT software. (A) The B6 control strain displayed normal sociability, as illustrated by high levels of nose-to-nose sniffing with the 129/SvImJ partner stimulus mouse, after a single intraperitoneal dose of vehicle (10% Tween 80/saline) or GRN-529 (3.0 mg/kg). (B) BTBR treated with vehicle exhibited its characteristic low sociability, displaying fewer bouts of nose-to-nose sniffing with the 129/SvImJ partner stimulus mouse. GRN-529 (3.0 mg/kg) increased nose-to-nose sniffing bouts in the BTBR. (C) B6 displayed high sociability on the parameter, time spent in social contact after GRN-529 or vehicle. (D) BTBR exhibited its characteristic low sociability on time spent in social contact after vehicle administration. BTBR treated with a single acute dose of GRN-529 (3.0 mg/kg) exhibited increased time in social contact. (E) Cumulative time spent self-grooming was calculated during the 10-min reciprocal social interaction test session. B6 mice treated with either vehicle or GRN-529 (3.0 mg/kg) did not display any significant differences in the amount of time spent self-grooming during the session. (F) BTBR treated with GRN-529 (3.0 mg/kg) displayed significant reductions in their high levels of repetitive self-grooming versus BTBR treated with vehicle. (G) Cumulative time spent digging in the arena floor bedding during the social task was calculated during the 10-min test session. B6 mice treated with either vehicle or GRN-529 (3.0 mg/kg) displayed similar time spent digging during the session. (H) BTBR treated with GRN-529 (3.0 mg/kg) displayed significant reductions in their high levels of repetitive digging behavior versus BTBR treated with vehicle. n = 9 to 11 per treatment group, GRN-529 (3.0 mg/kg) and vehicle, for each strain, *P < 0.05 versus vehicle.

Fig. 5

Effect of GRN-529 on open field locomotion at doses that reversed repetitive and social deficits. Exploratory locomotion was assayed in a standard automated open field arena, in 5-min time bins across a 30-min session after the identical GRN-529 treatments. (A) B6 displayed a significant increase in total distance traversed after GRN-529 at the highest dose, 3.0 mg/kg, intraperitoneally, compared to vehicle. (B and C) GRN-529 administration had no significant effect on (B) time spent in the center of the arena or (C) vertical activity in B6 tested at NIMH. n = 9 to 10 per dose. (D) BTBR displayed significant increases in total distance traversed after GRN-529 at doses of 1.0 and 3.0 mg/kg intraperitoneally compared to vehicle, indicating increased exploratory activity. (E and F) GRN-529 administration had no significant effect on (E) time spent in the center of the arena or (F) vertical activity in BTBR. *P < 0.05, n = 10 per dose, cohort 1 tested at NIMH. See fig. S4 for open field results replicated at NIMH and fig. S5 for open field results replicated at Pfizer. See fig. S6 for additional open field parameters in C58 mice.