Parental kynurenine 3-monooxygenase genotype in mice directs sex-specific behavioral outcomes in offspring

Abstract

Background: Disruptions in brain development can impact behavioral traits and increase the risk of neurodevelopmental conditions such as autism spectrum disorder, attention-deficit/hyperactivity disorder (ADHD), schizophrenia, and bipolar disorder, often in sex-specific ways. Dysregulation of the kynurenine pathway (KP) of tryptophan metabolism has been implicated in cognitive and neurodevelopmental disorders. Increased brain kynurenic acid (KYNA), a neuroactive metabolite implicated in cognition and sleep homeostasis, and variations in the Kmo gene, which encodes kynurenine 3-monooxygenase (KMO), have been identified in these patients. We hypothesize that parental Kmo genetics influence KP biochemistry, sleep behavior and brain energy demands, contributing to impairments in cognition and sleep in offspring through the influence of parental genotype and genetic nurture mechanisms.

Methods: A mouse model of partial Kmo deficiency, Kmo heterozygous (HET-Kmo^+/-), was used to examine brain KMO activity, KYNA levels, and sleep behavior in HET-Kmo^+/- compared to wild-type control (WT-Control) mice. Brain mitochondrial respiration was assessed, and KP metabolites and corticosterone levels were measured in breast milk. Behavioral assessments were conducted on wild-type offspring from two parental groups: (i) WT-Control from WT-Control parents, (ii) wild-type Kmo (WT-Kmo^+/+) from Kmo heterozygous parents (HET-Kmo^+/-). All mice were C57Bl/6J background strain. Adult female and male offspring underwent behavioral testing for learning, memory, anxiety-like behavior and sleep-wake patterns.

Results: HET-Kmo^+/- mice exhibited reduced brain KMO activity, increased KYNA levels, and disrupted sleep architecture and electroencephalogram (EEG) power spectra. Mitochondrial respiration (Complex I and Complex II activity) and electron transport chain protein levels were impaired in the hippocampus of HET-Kmo^+/- females. Breast milk from HET-Kmo^+/- mothers increased kynurenine exposure during lactation but corticosterone levels were unchanged. Compared to WT-Control offspring, WT-Kmo^+/+ females showed impaired spatial learning, heightened anxiety, reduced sleep and abnormal EEG spectral power. WT-Kmo^+/+ males had deficits in reversal learning but no sleep disturbances or anxiety-like behaviors.

Conclusions: These findings suggest that Kmo deficiency impacts KP biochemistry, sleep behavior, and brain mitochondrial function. Even though WT-Kmo^+/+ inherit identical genetic material as WT-Control, their development might be shaped by the parent's physiology, behavior, or metabolic state influenced by their Kmo genotype, leading to phenotypic sex-specific differences in offspring.

Keywords: Cognition; Kynurenine pathway; Neurodevelopment; Parental genotype; Sleep.

Fig. 1

Schematic diagram of the experimental groups to study the role for parental KMO of the kynurenine pathway in the offspring’s behaviors. A Schematic representation of the kynurenine pathway denoting the pivotal enzyme KMO (in purple color) that is genetically modified in our present mouse model. B Schematic representation of the experimental parental and offspring groups. Wild-type control (WT-Control) breeding pairs and heterozygous Kmo (HET-Kmo^+/–) breeding pairs generated wild-type (WT-Control) offspring and wild-type (WT-Kmo^+/+) offspring, respectively

Fig. 2

Decreased KMO activity and increased KYNA in the brain of Kmo heterozygous female and male mice. A Brain KMO activity in HET-Kmo^+/– mice is reduced compared to WT-Control mice (Unpaired t test ***P < 0.001, ****P < 0.0001). B Brain KYNA levels in HET-Kmo^+/– mice are increased compared to WT-Control mice (Unpaired t test **P < 0.01, ****P < 0.0001). Data are mean ± SEM. N = 5–7 per group

Fig. 3

HET-Kmo^+/– mice have decreased NREM sleep and more wake in comparison to wild-type (WT-Control) mice during the light phase. A Percentage of total time spent in each vigilance state (Unpaired t test **P < 0.01). B Total number of bouts for each vigilance state (Unpaired t test *P < 0.05). C Average bout duration for each vigilance state (Unpaired t test *P < 0.05). D Number of vigilance state transitions (Unpaired t test *P < 0.05). E REM sleep spectral power (Females: Two-way RM ANOVA Genotype x Frequency interaction F_{(38, 380)} = 3.063, ^^^^P < 0.0001; Males: Two-way RM ANOVA Genotype × Frequency interaction F_{(38, 532)} = 3.109, ^^^^P < 0.0001). F NREM sleep spectral power (Females: Two-way RM ANOVA Genotype x Frequency interaction F_{(38, 380)} = 3.799, ^^^^P < 0.0001; Males: Two-way RM ANOVA Genotype × Frequency interaction F_{(38, 532)} = 5.967, ^^^^P < 0.0001). G Relative cage activity (Unpaired t test *P < 0.05). Data are mean ± SEM. N = 6–8 per group

Fig. 4

HET-Kmo^+/– females display substrate-specific changes in hippocampal mitochondrial respiration. A Cortex Complex I-dependent (glutamate/malate) respiration was unchanged between genotypes. B Hippocampus mitochondrial Complex I-dependent State 3 (+ ADP) respiration was significantly decreased in female HET-Kmo^+/– versus WT-Control (Welch’s t test *P < 0.05). C Cortex Complex II-dependent (succinate/rotenone) respiration was unchanged between genotypes. D Hippocampus mitochondrial Complex II-dependent respiration was significantly increased in both basal and State 3 respiration in female HET-Kmo^+/– versus WT-Control (Welch’s t test *P < 0.05). E Profiling of select mitochondrial oxidative phosphorylation subunits in isolated mitochondria demonstrated a significant upregulation of the ATP5A component of Complex V (CV) in both the cortex and hippocampus of HET-Kmo^+/– female mice versus WT-Control. HET-Kmo^+/– hippocampal mitochondria also showed a significant decrease in Complex II (CII) SDHB subunit, whereas the CII SDHA subunit increased significantly. F Quantification of mitochondrial proteins measured in cortex (Welch’s t test *P < 0.05). G Quantification of mitochondrial proteins measured in hippocampus (Welch’s t test *P < 0.05, **P < 0.01). Data are mean ± SEM. N = 5 per group for respiratory data, N = 4 per group for immunoblot analysis. Mitochondrial subunit levels were normalized to total protein load as determined by Amido Black staining

Fig. 5

Breast milk content of kynurenine is elevated in HET-Kmo^+/– compared to WT-Control mothers. A Maternal breast milk tryptophan. B Maternal breast milk kynurenine (Unpaired t test ****P < 0.0001). C Maternal breast milk corticosterone. Data are mean ± SEM. N = 7–11 per group

Fig. 6

Impaired spatial learning in female wild-type (WT-Kmo^+/+) offspring from HET-Kmo^+/– parents. A Schematic representation of the experimental offspring groups. WT-Control offspring are derived from WT-Control parents. WT-Kmo^+/+ offspring are derived from heterozygous Kmo (HET-Kmo^+/–) parents. B Distance traveled in the Barnes maze (Female: Two-way RM ANOVA Genotype effect F_{(1, 36)} = 11.08, ^^P < 0.01 with Bonferroni’s post hoc test **P < 0.01). C Errors in the Barnes maze (Female: Two-way RM ANOVA Genotype effect F_{(1, 36)} = 5.311, ^P < 0.05; Male: Two-way RM ANOVA Genotype effect F_{(1, 40)} = 4.391, ^P < 0.05). D Percent of time immobile in the Barnes maze (Female: Two-way RM ANOVA Genotype effect F_{(1, 24)} = 7.1, ^P < 0.05 with Bonferroni’s post hoc test *P < 0.05, **P < 0.01). E Percent of time spent in each search strategy on the Barnes maze (Chi-square distribution test *P < 0.05). F Representative images of video tracking in the Barnes maze across learning trials. Data are mean ± SEM. N = 19–21 per group

Fig. 7

Impaired reversal learning in male wild-type (WT-Kmo^+/+) offspring from HET-Kmo^+/– parents. A Distance traveled in the reversal trial of the Barnes maze (Unpaired t-test *P < 0.05). B Errors in the reversal trial of the Barnes maze (Unpaired t-test *P < 0.05). C Percent of time spent in each search strategy in the reversal trial of the Barnes maze. D Representative images of video tracking in the Barnes maze during the reversal trial. Data are mean ± SEM. N = 19–21 per group

Fig. 8

Increased anxiety-like behavior in female wild-type (WT-Kmo^+/+) offspring from HET-Kmo^+/– parents. A Distance traveled in the elevated zero maze (Two-way RM ANOVA Genotype effect F_{(1, 70)} = 44.25, ^^^^P < 0.0001 with Fisher’s LSD post hoc test ***P < 0.001, ****P < 0.0001). B Entries to open and closed area in the elevated zero maze (Females: Two-way RM ANOVA Genotype effect F_{(1, 64)} = 14.71, ^^^P < 0.001 with Fisher’s LSD post hoc test *P < 0.05, **P < 0.01). C Number of grooming events in the sucrose splash test (Two-way RM ANOVA Sex effect F_{(1, 65)} = 4.093, ^P < 0.05). D Latency to grooming in the sucrose splash test. Data are mean ± SEM. N = 11–24 per group

Fig. 9

Female wild-type (WT-Kmo^+/+) from HET-Kmo^+/– parents have reduced NREM sleep and more wake in comparison to wild-type (WT-Control) mice during the light phase. A Total NREM duration (Females: Two-way RM ANOVA Genotype effect F_{(1, 10)} = 4.776, P = 0.054). B Total wake duration (Females: Two-way RM ANOVA Genotype effect F_{(1, 10)} = 5.584, ^P < 0.05). C NREM sleep spectral power (Females: Two-way RM ANOVA Genotype effect F_{(1, 10)} = 8.711, ^P < 0.05, Genotype × Frequency interaction F_{(38, 380)} = 2.022, ^^^P < 0.001). D REM sleep spectral power (Females: Two-way RM ANOVA Genotype × Frequency interaction F_{(38, 380)} = 1.487, ^P < 0.05). E Relative cage activity (Unpaired t test: *P < 0.05). Data are mean ± SEM. N = 6–10 per group

Fig. 10

Schematic representation of major findings supporting the conclusion that parental HET-Kmo^+/– genotype influences wild-type offspring phenotypes. Disruptions in sleep were observed in female and male HET-Kmo^+/– mice, and HET-Kmo^+/– female mice had altered hippocampal mitochondria respiration and increased kynurenine levels in breast milk. Even though WT-Kmo^+/+ inherit identical genetic material as WT-Control, the present data suggest that behavioral phenotypes of offspring could be shaped by parental physiology, behavior, or metabolic state influenced by their Kmo genotype. Sex-specific differences are noted in offspring. Female WT-Kmo^+/+ offspring from HET-Kmo^+/– parents exhibit spatial learning impairments, increased anxiety-like behavior, and sleep disturbances. Male WT-Kmo^+/+ offspring from HET-Kmo^+/– parents display impaired reversal learning, but intact spatial learning, anxiety-like behavior, and sleep patterns