Metabolic and behavioral features of acute hyperpurinergia and the maternal immune activation mouse model of autism spectrum disorder

Abstract

Since 2012, studies in mice, rats, and humans have suggested that abnormalities in purinergic signaling may be a final common pathway for many genetic and environmental causes of autism spectrum disorder (ASD). The current study in mice was conducted to characterize the bioenergetic, metabolomic, breathomic, and behavioral features of acute hyperpurinergia triggered by systemic injection of the purinergic agonist and danger signal, extracellular ATP (eATP). Responses were studied in C57BL/6J mice in the maternal immune activation (MIA) model and controls. Basal metabolic rates and locomotor activity were measured in CLAMS cages. Plasma metabolomics measured 401 metabolites. Breathomics measured 98 volatile organic compounds. Intraperitoneal eATP dropped basal metabolic rate measured by whole body oxygen consumption by 74% ± 6% (mean ± SEM) and rectal temperature by 6.2˚ ± 0.3˚C in 30 minutes. Over 200 metabolites from 37 different biochemical pathways where changed. Breathomics showed an increase in exhaled carbon monoxide, dimethylsulfide, and isoprene. Metabolomics revealed an acute increase in lactate, citrate, purines, urea, dopamine, eicosanoids, microbiome metabolites, oxidized glutathione, thiamine, niacinamide, and pyridoxic acid, and decreased folate-methylation-1-carbon intermediates, amino acids, short and medium chain acyl-carnitines, phospholipids, ceramides, sphingomyelins, cholesterol, bile acids, and vitamin D similar to some children with ASD. MIA animals were hypersensitive to postnatal exposure to eATP or poly(IC), which produced a rebound increase in body temperature that lasted several weeks before returning to baseline. Acute hyperpurinergia produced metabolic and behavioral changes in mice. The behaviors and metabolic changes produced by ATP injection were associated with mitochondrial functional changes that were profound but reversible.

Fig 1. Study overview.

Abbreviations: GC—gas chromatography, FID—flame ionization detection, ECD—electron capture detection, MSD—mass selective detection, LC—high performance liquid chromatography, MS/MS—triple quadrupole mass spectrometry.

Fig 2. Metabolomic analysis of acute hyperpurinergia.

A. Ranking of metabolites changed 30 minutes after ATP injection by partial least squares discriminant analysis (PLSDA). B. Bubble impact plot of pathways most changed 30 minutes after ATP injection, C. Venn diagram of pathways increased, decreased, or contained increased and decreased metabolites after 30 minutes, D. Ranking of metabolites changed 4 hours after ATP injection by partial least squares discriminant analysis (PLSDA), E. Bubble impact plot of pathways most changed 4 hours after ATP injection, F. Venn diagram of pathways and metabolites increased or decreased after 4 hours, G. Two-dimensional separation of the metabolomes by multivariate PLSDA components after saline and 0.5 and 4 hours post ATP injection, H. Dendrogram showing sharp separation of the metabolome at 30 minutes and the heterogeneous and incomplete return to baseline by 4 hours after ATP injection, I. Heatmap of the top 30 most-changed metabolites 30 minutes and 4 hours after ATP injection. ATP dose = 0.5 μmol/g body weight, n = 7–8 C57BL/6J males per group, age = 12–13 weeks. Abbreviations: VIP—variable importance in projection.

Fig 3. Breathomics, chemokines, cytokines, and corticosterone response to acute hyperpurinergia.

Breathomics captured and analyzed exhaled breath at 1–10 minutes after ATP injection (A-H; n = 3 C57BL/6J males per group, 3 samples per animal), A. Carbon monoxide, B. Methanol, C. Methane, D. Dimethylsulfide, E. Acetaldehyde, F. Acetone, G. Butyraldehyde, H. Isoprene. Plasma chemokine and cytokine analysis 30 min and 4 hours after ATP injection (I-K; n = 6–7 C57BL/6J males per group), I. CXCL1/KC/GRO J. IL10, K. IL6. Plasma corticosterone levels 30 minutes and 4 hours after ATP injection (ATP dose = 0.5 μmol/g body weight, n = 7 C57BL/6J females per group), L. Corticosterone. Abbreviations: CXCL1—chemokine (C-X-C motif) ligand 1, KC—keratinocyte-derived chemokine, GRO—growth related oncogene alpha, IL10—interleukin 10, IL6—interleukin 6. P-values: * = 0.05, ** = 0.01, *** = 0.001, **** = 0.0001.

Fig 4. Body temperature, bioenergetic, and behavioral responses to acute hyperpurinergia.

A. Male C57BL/6J mice (nucleotide dose = 0.5 μmol/g body weight, n = 6 per group, 5–6 months old), B. Female C57BL/6J mice (n = 6 per group, 5–6 months old). Intravenous (i.v.) vs intraperitoneal (i.p.) dosing (C and D, ATP dose = 0.5 μmol/g, n = 6 females per group, 5 months old), C. Body temperature response, D. Behavioral response. Sex-specific differences (E-H, temperatures measured at 15 minutes post-injection with 0–0.2 μmol/g ATP, n = 6–8 mice/group) E. Females were more sensitive to the hypothermic effects of ATP, F. Males were more sensitive to the hypothermic effects of ADP, G. Males and females were equally sensitive to the hypothermic effects of AMP, H. Males and females were equally sensitive to the hypothermic effects of adenosine. Behavioral responses (I and J), I. The behavioral response to high-dose ATP was the same in males and females (dose = 0.5 μmol/g, n = 10 per group), J. Dose-response curves at non-saturating ATP doses revealed that males were more sensitive to the behavioral effects of hyperpurinergia (PBRS scores measured at 15 minutes post-ATP, n = 6 per group, 5–6 months old). CLAMS cage analysis of bioenergetics (K-N, ATP dose = 0.5 μmol/g, n = 6 per group, 28-week old C57BL/6J females), K. The basal metabolic rate measured as the rate of oxygen utilization (VO₂) was decreased by 74% after ATP injection, L. The rate of CO₂ production was decreased by 76% after ATP injection, M. The respiratory exchange ratio (RER) dropped from 0.84 to 0.70 after ATP injection compared to saline, N. ATP injection decreased locomotor activity as measured by light beam breaks compared to saline. Abbreviations: PBRS—purinergic behavioral response scale, SAL—saline, CLAMS—comprehensive laboratory animal monitoring system. RER—respiratory exchange ratio = VCO₂/VO₂. Ambient temperature for all experiments was 22˚-24˚C.

Fig 5. Thermoregulation and the latent memory response to ATP and poly(IC) in the MIA mouse model.

The acute 1-hour response to postnatal challenge with ATP (A and B, n = 6 per group) A. Acute response in males, B. Acute response in females. The subacute 5-day response to postnatal challenge with ATP (C and D, n = 6 per group, 8–9 months old) C. Five-day response to ATP in males, D. Five-day response to ATP in females, E. The triphasic temperature response to postnatal challenge with poly(IC) (dose = 2 mg/kg, n = 6 males per group, 8–9 months old). Abbreviations: MIA—maternal immune activation mouse model, Poly(IC)—poly inosinic:cytosinic acid double strand RNA. Ambient temperature for all experiments was 22˚-24˚C.