JHU-083 selectively blocks glutaminase activity in brain CD11b+ cells and prevents depression-associated behaviors induced by chronic social defeat stress

Abstract

There are a number of clinically effective treatments for stress-associated psychiatric diseases, including major depressive disorder (MDD). Nonetheless, many patients exhibit resistance to first-line interventions calling for novel interventions based on pathological mechanisms. Accumulating evidence implicates altered glutamate signaling in MDD pathophysiology, suggesting that modulation of glutamate signaling cascades may offer novel therapeutic potential. Here we report that JHU-083, our recently developed prodrug of the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) ameliorates social avoidance and anhedonia-like behaviors in mice subjected to chronic social defeat stress (CSDS). JHU-083 normalized CSDS-induced increases in glutaminase activity specifically in microglia-enriched CD11b⁺ cells isolated from the prefrontal cortex and hippocampus. JHU-083 treatment also reverses the CSDS-induced inflammatory activation of CD11b⁺ cells. These results support the importance of altered glutamate signaling in the behavioral abnormalities observed in the CSDS model, and identify glutaminase in microglia-enriched CD11b⁺ cells as a pharmacotherapeutic target implicated in the pathophysiology of stress-associated psychiatric conditions such as MDD.

Fig. 1

In vivo brain and plasma pharmacokinetics of compound DON following oral administration of JHU-083 in mice. a Chemical structure of JHU-083 is shown. b JHU-083 (1.82 mg/kg, p.o., q.o.d.) exhibited sufficient oral bioavailability and favorable brain penetration for efficacy testing in mice. Data are presented as the mean ± SEM. p.o., per os; q.o.d, every other day; C_max, maximum concentration observed; T_max, time at which the maximum concentration is observed; AUC, area under the concentration-time curve; AUC_0-5h, AUC up to the last measurable concentration

Fig. 2

Experimental timeline for CSDS followed by chronic and acute administration of JHU-083. a Male 7-to 8-week-old C57BL/6J (C57) mice were exposed to chronic social defeat stress (CSDS) for 12 days and were subsequently treated with JHU-083 (1.82 mg/kg, p.o.) or vehicle every other day for 12 days. One day after the completed treatment of JHU-083 or vehicle, behavioral characterization was conducted by social interaction test (SIT) followed by sucrose preference test (SPT). Brain extraction for biochemical experiments was also performed one day after JHU-083 treatment. b To explore acute effects of JHU-083, mice were administered a single dose of JHU-083 (1.82 mg/kg, p.o.) or vehicle one day after exposure to 12 days of CSDS which was followed by behavioral assessments

Fig. 3

Chronic JHU-083 treatment ameliorates deficits in social behavior and anhedonia-associated behavior induced by CSDS. a, b CSDS impairs sociability assessed by time spent in each chamber and time spent sniffing the wire cage in three-chambered social interaction test. Chronic treatment of JHU-083 (1.82 mg/kg, p.o.) alleviates social avoidance induced by CSDS, but has no effect of JHU-083 on sociability in control mice (Ctr). **p < 0.01, determined by Student’s t-test. Data are presented as the mean ± SEM. c, d CSDS leads to reduced preference for social novelty assessed by measurement of time spent in each chamber and time spent sniffing the stranger 1 and stranger 2 in three-chambered social interaction test. Chronic treatment of JHU-083 (1.82 mg/kg, p.o.) has no effect on altered preference for social novelty induced by CSDS, and none on social novelty recognition in control mice. *p < 0.05, **p < 0.01, determined by Student’s t-test. a–d Representative heat map images (lower panels) represent movements of the CSDS mice that treated with vehicle or JHU-083. e CSDS induced anhedonia-associated behavior reflected by decreased sucrose consumption when given a choice between 1.5% sucrose and water. JHU-083 rescued CSDS-induced sucrose preference deficit, while having no effect on sucrose preference in control mice. CSDS + vehicle vs. CSDS + JHU-083: **p < 0.01, by three-way repeated measures ANOVA followed by Bonferroni’s post-hoc comparisons. a–e Data are presented as the mean ± SEM. Ctr+ vehicle, n = 6; Ctr + JHU-083, n = 6; CSDS + vehicle, n = 11; CSDS + JHU-083, n = 10

Fig. 4

Acute JHU-083 treatment ameliorates deficits in social behavior, but not anhedonia-associated behavior induced by CSDS. a, b Acute treatment of JHU-083 (1.82 mg/kg, p.o.) one day after CSDS alleviates social avoidance induced by CSDS, but has no effect on sociability in control mice (Ctr). **p < 0.01, determined by Student’s t-test. c, d CSDS leads to reduced preference for social novelty assessed by measurement of time spent in each chamber and time spent sniffing stranger 1 and stranger 2 mouse in the three-chambered social interaction test. Acute treatment of JHU-083 (1.82 mg/kg, p.o.) has no effect on altered preference for social novelty induced by CSDS nor on social novelty recognition in control mice. *p < 0.05, **p < 0.01, determined by Student’s t-test. a–d Representative heat map images (lower panels) represent movements of the CSDS mice treated with vehicle or JHU-083. e CSDS induced anhedonia-associated behavior reflected by decreased sucrose consumption when given a choice between 1.5% sucrose and water. JHU-083 did not rescue CSDS-induced sucrose preference deficit nor have effect on sucrose preference in control mice. CSDS + Vehicle vs. CSDS + JHU-083: **p < 0.01, by three-way repeated measures ANOVA followed by Bonferroni’s post-hoc comparisons. a–e Data are presented as the mean ± SEM. Ctr + vehicle, n = 10; Ctr + JHU-083, n = 10; CSDS + vehicle, n = 11; CSDS + JHU-083, n = 11

Fig. 5

JHU-083 attenuates CSDS-induced increase in glutaminase activity in CD11b⁺ cells in the prefrontal cortex and hippocampus, but not in the cerebellum. a–c CSDS induced significant elevation in ex vivo glutaminase activity specifically in CD11b⁺ cells in the prefrontal cortex (PFC) and hippocampus (HPC), but not in the cerebellum, that was completely normalized by JHU-083 administration (1.82 mg/kg, p.o., q.o.d.). There was no effect of JHU-083 on glutaminase activity in CD11b⁺ cells harvested from the prefrontal cortex in control mice, whereas down-regulated effect in the hippocampus in control mice (Ctr). d–f There was no effect of CSDS or JHU-083 treatment on glutaminase activity in non-CD11b⁺ cells in the prefrontal cortex and hippocampus. Expression of glutaminase was slightly increased in the cerebellum by CSDS. Bars represent means of each group in three independent experiments. Ctr + Vehicle vs CSDS + Vehicle: **p < 0.01; CSDS + Vehicle vs CSDS + JHU-083: ^##p < 0.01, comparison between groups by two-way ANOVA with Tukey’s post-hoc test. Data are presented as the mean ± SEM

Fig. 6

JHU-083 attenuates CSDS-induced inflammatory activation of CD11b⁺ cells. a–c CSDS induces increased mRNA expression of TNF-α and IL-1β, but not IL-6 in CD11b⁺ cells assessed by Quantitative real-time PCR (qPCR). Chronic treatment of JHU-083 (1.82 mg/kg, p.o.) suppresses increased TNF-α and IL-1β induced by CSDS, whereas there is no effect of JHU-083 on expression of inflammatory cytokines in CD11b⁺ cells isolated from control mice (Ctr). d No effect of CSDS or JHU-083 treatment on mRNA expression of glutaminase in CD11b⁺ cells. Neither CSDS nor chronic treatment of JHU-083 (1.82 mg/kg, p.o.) affected mRNA expression of glutaminase in CD11b⁺ cells assessed by qPCR. Bars represent means of each group in three independent experiments. Ctr + Vehicle vs CSDS + Vehicle: *p < 0.05; CSDS + Vehicle vs. CSDS + JHU-083: ^#p < 0.05, comparison between groups by two-way ANOVA with Tukey’s post-hoc test. Data are presented as the mean ± SEM