Antipurinergic therapy corrects the autism-like features in the poly(IC) mouse model

Abstract

Background: Autism spectrum disorders (ASDs) are caused by both genetic and environmental factors. Mitochondria act to connect genes and environment by regulating gene-encoded metabolic networks according to changes in the chemistry of the cell and its environment. Mitochondrial ATP and other metabolites are mitokines-signaling molecules made in mitochondria-that undergo regulated release from cells to communicate cellular health and danger to neighboring cells via purinergic signaling. The role of purinergic signaling has not yet been explored in autism spectrum disorders.

Objectives and methods: We used the maternal immune activation (MIA) mouse model of gestational poly(IC) exposure and treatment with the non-selective purinergic antagonist suramin to test the role of purinergic signaling in C57BL/6J mice.

Results: We found that antipurinergic therapy (APT) corrected 16 multisystem abnormalities that defined the ASD-like phenotype in this model. These included correction of the core social deficits and sensorimotor coordination abnormalities, prevention of cerebellar Purkinje cell loss, correction of the ultrastructural synaptic dysmorphology, and correction of the hypothermia, metabolic, mitochondrial, P2Y2 and P2X7 purinergic receptor expression, and ERK1/2 and CAMKII signal transduction abnormalities.

Conclusions: Hyperpurinergia is a fundamental and treatable feature of the multisystem abnormalities in the poly(IC) mouse model of autism spectrum disorders. Antipurinergic therapy provides a new tool for refining current concepts of pathogenesis in autism and related spectrum disorders, and represents a fresh path forward for new drug development.

Conflict of interest statement

Competing Interests: The authors declare no competing financial interests.

Figures

Figure 1. Correction of the Core Behavioral Features of the Maternal Immune Activation (MIA) Mouse Model of Autism Spectrum Disorders (ASD).

(A) Social Preference. MIA males had a 54% decrease in social preference compared to controls (PIC-Sal 12.5+/−4.2% vs Sal-Sal 27.6+/−2.6%; one-way ANOVA F(3,42) = 3.74; with Newman-Keuls post-hoc testing; n = 9–15 males per group; age = 10-weeks; p

Figure 2. Relative Hypothermia Was Corrected, and Aerobic Metabolism was Increased by Antipurinergic Therapy.

(A) Relative Hypothermia in the MIA Model was Corrected by Antipurinergic Therapy. (Linear mixed effects model analysis; F(1,47) = 25.3; n = 9–16 males per group; ages 8–16 weeks; p2 = 3552+/−47.6 ml/kg/hour; Treated MIA = PIC-Sur = 3938+/−45.9 (one-way ANOVA F(3,44) = 8.0; n = 6 males per group; age = 14 weeks; p = 0.0002). Antipurinergic therapy had no significant effect on oxygen consumption in the control animals; Saline-treated Controls = Sal-Sal VO2 = 3652+/−72.8; Treated Controls = Sal-Sur = 3821+/−71.5 (n = 6 males per group; p = 0.11). Values are expressed as mean +/− SEM.

Figure 3. Plasma Immunoglobulins and Corticosterone.

(A) Plasma immunoglobulins were increased 18% by antipurinergic therapy. (PIC-Sal = 1.6+/−0.05 mg/dl; PIC-Sur = 1.9+/−0.07 mg/dl; n = 6–10 males per group; age = 12 weeks; one-way ANOVA F(3,37) = 5.72; p

Figure 4. Cerebral Synaptosomal Ultrastructural Abnormalities Were Corrected by Antipurinergic Therapy.

(A) Control (Sal-Sal) synaptosome illustrating normal post-synaptic density (PSD) morphology (arrow), and normal electron lucency of the matrix (92,000× magnification; scale bar = 200 µm). (B) Treated controls (Sal-Sur) with an included mitochondrion (“m”; scale bar = 500 µm). (C) Untreated MIA (PIC-Sal) with an included mitochondrion (“m”) and malformed, hypomorphic PSD (arrow; scale bar = 500 µm). Note the abnormal accumulation of electron-dense matrix material. (D) Treated MIA (PIC-Sur) with restoration of near-normal PSD morphology (arrow), an included mitochondrion (“m”), and reduction in abnormal accumulations of electron-dense matrix material within the synaptosomes (scale bar = 500 µm). Representative fields from n = 3–4 males per group; age = 16 weeks.

Figure 5. Cerebral Mitochondrial Respiratory Chain Subunit Mass was Unchanged in the MIA Model.

Cerebral mitochondria were isolated by Percoll gradient centrifugation and quantified by Western Analysis. Each lane contains the mitochondria from 2–3 males isolated at 16-weeks of age (n = 4–5 per group).

Figure 6. Cerebral Mitochondrial Respiratory Chain Hyperfunction Abnormalities Were Corrected by Antipurinergic Therapy.

(A) Mitochondrial Respiratory Chain Complex I Enzymatic Activity was Increased 34% by gestational poly(IC) exposure and corrected by suramin treatment (Sal-Sal = 152+/−2.3 U/CS; PIC-Sal = 205+/−2.9 U/CS; one-way ANOVA F(3,12) = 137; p

Figure 7. Cerebral Synaptosomal Purinergic Receptors were Downregulated in the MIA Model and Restored to Normal by Antipurinergic Therapy.

(A) Western Analysis of Metabotropic P2Y2 and Ionotropic P2X7 receptors. Each lane contains the synaptosomes from 2–3 males isolated at 16-weeks of age (n = 4–5 per group). (B) P2Y2 receptor expression was decreased by over 50% by gestational poly(IC) exposure and normalized by suramin treatment (Sal-Sal = 100+/−7.3%; Sal-Sur = 62+/−4.6%; PIC-Sal = 48+/−4.7%; PIC-Sur = 84+/−4.7%; one-way ANOVA F(3,12) = 18.1; p

Figure 8. Cerebral Synaptosomal Hypophosphorylation of Extracellular Response Kinase 1 and 2 (ERK1/2), Calcium-Calmodulin Kinase II (CAMKII), and Downregulation Fragile X Protein (FMRP) in the MIA Model Were Corrected, and Nicotinic Acetylcholine Receptor subunit α7 (nAchRα7) Expression was Increased by Antipurinergic Therapy.

(A) Western analysis of phosphorylated ERK1/2 (pERK1/2Thr202/Tyr204), total ERK1/2, phosphorylated CAMKII (pCAMKIIThr286), total CAMKII (CAMKIIpan), FMRP (Fragile X Syndrome protein), Nicotinic Acetylcholine Receptor subunit α7 (nAchRα7), and PSD95 (post-synaptic density protein 95) was used as a loading control for synaptosomes. Each lane contains the synaptosomes from 2–3 males isolated at 16-weeks of age (n = 4–5 per group).

Figure 9. Quantitative Analysis of the Extracellular Response Kinase 1 and 2 (ERK1/2), Calcium-Calmodulin Kinase II (CAMKII), Fragile X Protein (FMRP), and the Nicotinic Acetylcholine Receptor subunit α7 (nAchRα7) in Cerebral Synaptosomes in the MIA Model.

(A) ERK1 (MAPK3) and ERK2 (MAPK1) phosphorylation was decreased by over 90% by gestational poly(IC) exposure and normalized by suramin treatment (Sal-Sal = 100+/−5%; Sal-Sur = 6.2+/−0.8%; PIC-Sal = 9.9+/−2.6%; PIC-Sur = 84+/−2.3%; one-way ANOVA F(3,12) = 241; p

Figure 10. Purkinje Cell Dropout Was Prevented by Antipurinergic Therapy.

(A) Mosaic reconstruction of a representative parasagittal section of the cerebellar vermis in an untreated MIA animal (PIC-Sal). Sections were stained for calbindin (green) and neuN (red). Purkinje cells are the bright, large neurons located at the margins of the molecular (green) and granular (red) layers of the cerebellum. Lobules III though X are indicated. MIA animals at 16 weeks of age, showed patchy loss of Purkinje cells that was most striking in lobules VI and VII. (B) Higher magnification of lobule VII in a control (Sal-Sal) animal illustrating normal Purkinje cell numbers. (C) Higher magnification of lobule VII in MIA illustrating nearly complete Purkinje cell dropout, with scattered cells at the boundary between lobules VII and VIII in an animal exposed to poly(IC) during gestation. (D) Quantitation of Lobule VII Purkinje Cells. Animals exposed to poly(IC) during gestation had a 63% reduction in Purkinje cell numbers. This was prevented by suramin treatment (10–20 mg/kg ip qWeek) started at 6 weeks of age (Sal-Sal = 16.0+/−1.8 cells/mm; Sal-Sur = 19.6+/−2.5; PIC-Sal = 5.8+/−1.6, PIC-Sur = 20.4+/−5.4; one-way ANOVA F(3,13) = 5.3; p = 0.013; Newman-Keuls post hoc test; n = 4–5 males per group; age = 16 weeks). Values are expressed as mean +/− SEM.