Reversal of autism-like behaviors and metabolism in adult mice with single-dose antipurinergic therapy

Abstract

Autism spectrum disorders (ASDs) now affect 1-2% of the children born in the United States. Hundreds of genetic, metabolic and environmental factors are known to increase the risk of ASD. Similar factors are known to influence the risk of schizophrenia and bipolar disorder; however, a unifying mechanistic explanation has remained elusive. Here we used the maternal immune activation (MIA) mouse model of neurodevelopmental and neuropsychiatric disorders to study the effects of a single dose of the antipurinergic drug suramin on the behavior and metabolism of adult animals. We found that disturbances in social behavior, novelty preference and metabolism are not permanent but are treatable with antipurinergic therapy (APT) in this model of ASD and schizophrenia. A single dose of suramin (20 mg kg(-1) intraperitoneally (i.p.)) given to 6-month-old adults restored normal social behavior, novelty preference and metabolism. Comprehensive metabolomic analysis identified purine metabolism as the key regulatory pathway. Correction of purine metabolism normalized 17 of 18 metabolic pathways that were disturbed in the MIA model. Two days after treatment, the suramin concentration in the plasma and brainstem was 7.64 μM pmol μl(-1) (±0.50) and 5.15 pmol mg(-1) (±0.49), respectively. These data show good uptake of suramin into the central nervous system at the level of the brainstem. Most of the improvements associated with APT were lost after 5 weeks of drug washout, consistent with the 1-week plasma half-life of suramin in mice. Our results show that purine metabolism is a master regulator of behavior and metabolism in the MIA model, and that single-dose APT with suramin acutely reverses these abnormalities, even in adults.

Figure 1

Single-dose correction of behavioral abnormalities. (a) Social abnormalities in male MIA animals were found at the earliest ages of testing at 2.25 months of age. (Student's t-test ****P<0.0002; N=19 Saline and 25 Poly(IC)). (b) A single dose of suramin given to adult MIA mice restored normal social behavior (PIC-Sur). two-way ANOVA was first used to test for the presence of interaction between drug treatment and experimental groups. This revealed an interaction consistent with the observation that suramin benefited social behavior in the MIA animals but had no effect on normal controls (F(1,39)=13.48); P=0.0007). We then performed one-way ANOVA to test for suramin effects. A single treatment with suramin (20 mg kg⁻¹ i.p.) given 2–4 days before testing restored normal social behavior (one-way ANOVA F(3,40)=8.95; P<0.0001; Tukey post hoc PIC-Sal versus PIC-Sur ****P<0.0001; N=8–13 per group). (c) After 5 weeks of suramin washout, the social behavior remained improved compared with saline-treated animals but was decreased from the first week after treatment. (F(3,40)=10.5; Tukey post hoc PIC-Sal versus PIC-Sur *P<0.05; N=8–13 per group). Values are expressed as means±s.e.m. (d) We estimated the strength of novelty preference as spontaneous alternation in the T-maze. MIA mice showed deficits in spontaneous alternation from the age of earliest testing at 4 months of age (Student's t-test; ****P<0.0001; N=19 Saline and 25 PIC). (e) Two-way ANOVA was first used to test for the presence of interaction between drug treatment and experimental groups. This revealed an interaction consistent with the observation that suramin restored spontaneous alternation in the MIA animals but had no effect on normal controls (F(1,40)=7.609; P=0.0087). We then performed one-way ANOVA to test for suramin effects. A single dose of suramin (20 mg kg⁻¹ i.p.) injected 2–4 days before testing corrected the deficits in young adult animals that were 5.25 months of age. (F(3,40)=9.46; ; Tukey post hoc Sal-Sal versus PIC-Sal **P<0.01; PIC-Sal versus PIC-Sur ***P<0.001); N=8–13 per group). (f) This benefit was lost after a drug washout period of 5 weeks, leaving a significant difference between control (Sal) and MIA (PIC) groups (F(3,39)=18.05; P<0.0001), but no remaining effect of suramin by post hoc testing. (Tukey post hoc PIC-Sal versus PIC-Sur P=ns; N=8–13 per group). Values are expressed as means±s.e.m. (g) Motor coordination abnormalities were quantified on the rotarod as latency to fall. Performance was abnormal from the earliest age of testing at 2.5 months of age (Student's t-test ****P<0.0001; N=19 Saline and 25 Poly(IC)). (h) Suramin did not improve performance after two doses (20 mg kg⁻¹ i.p.) given at 6.5 and 6.75 months of age and tested 2–4 days after the second dose. (two-way ANOVA interaction F(1,39)=0.1227; P=0.728 (ns); Poly(IC) effect F(1,39)=25.06; ****P<0.0001; treatment effect F(1,39)=0.01; P=0.908 (ns)). Values are expressed as means±s.e.m.

Figure 2

Plasma and brainstem suramin quantitation. (a) Suramin is a polysulphonated napthylurea with a molecular weight of 1297 g mol⁻¹. (b) Suramin was present in the plasma and brainstem but was not detectable in the cerebrum or cerebellum. Two days after a single 20 mg kg⁻¹ i.p. dose of suramin, drug levels were measured in plasma (7.64 μM±0.50), brainstem (5.15 pmol mg⁻¹±0.49), cerebrum (<0.1 pmol mg⁻¹) and cerebellum (<0.1 pmol mg⁻¹) in both controls (Sal) and maternal immune activation (PIC) animals. (N=4–6 per tissue). Values are expressed as means±s.e.m.

Figure 3

Metabolomic analysis. (a) APT rescues widespread metabolic abnormalities. Plasma samples were collected 2 days after a single dose of suramin (20 mg kg⁻¹ i.p.) or saline (5 μl g⁻¹ i.p.). This analysis shows that a single dose of suramin (PIC-Sur; green) drives the metabolism of MIA animals (PIC-Sal; red) strongly in the direction of controls (Sal-Sal; blue). Metabolomic profiles consisted of 478 metabolites from 44 biochemical pathways measured with LC-MS/MS. N=6, 6.5-month-old males per group. (b) Metabolic memory preserves metabolic rescue by APT. This analysis shows that 5 weeks after a single dose of suramin (PIC-Sur W/O; green) the metabolism of treated animals has drifted back toward that of untreated, MIA animals (PIC-Sal; red; N=6 males per group). (c) Hierarchical clustering of suramin-treated and suramin-washout metabotypes. This analysis illustrates the metabolic similarity of control (Sal-Sal; light blue) and MIA animals treated with one dose of suramin (PIC-Sur; green) compared with saline-treated MIA animals (PIC-Sal; red) and ASD-like animals tested 5 weeks after suramin washout (PIC-Sur W/O; dark blue). The numbers listed along the x axis are animal ID numbers. (d) Rank Order of metabolites disturbed in the MIA model. Multivariate analysis across the four treatment groups (PIC-Sal=MIA; PIC-Sur=acute suramin treatment; PIC-Sur w/o=5 weeks post-suramin washout; Sal-Sal=Controls). Biochemical pathway assignments are listed on the left. Relative magnitudes of each metabolite disturbance are listed on the right as high (red), intermediate (yellow or light green) and low (dark green). Variable importance in projection (VIP) scores are a multivariate statistic that reflects the impact of each metabolite on the partial least squares discriminant analysis model. VIP scores above 1.5 are significant.