In the present study, we found a critical NPY dependent change in microbiota-intestinal-brain communication in a a mouse model of ASD. We first characterized the levels of NPY system mRNA along the microbiota-gut-brain in this in relation neurobehavioral and sexual dimorphic changes. Our findings, provide first-time evidence that biological sex shaped NPY system expression along microbiota-gut-brain pathways in Nf1+/− mice, in association with anxious phenotypic differences which are sex dependent.
Distinct behavioral and neurobiological profiles have been identified in autistic women and men2. However, the mechanisms by which sex leads to distinct vulnerabilities and symptoms of ASD, remain largely unknown. Women and girls with autism are often underdiagnosed, in part due to their capacity for “social camouflaging”. Based on our previous works 10,50, we hypothesized that sex may influence the presentation of ASD symptomology. Emerging research has demonstrated that disruptions in the microbiota-gut-brain axis could be implicated in ASD48, but to date the exact mechanism by which this happens is unclear. Since NPY is an important anxiolytic player involved in gut-brain communication51, it could be a putative molecular mediator implicated in sex-related phenotypic differences observed in ASD. In this work, we evaluated the levels of NPY mRNA in the pre-frontal cortex, amygdala, and hippocampus, three main regions involved in anxiogenic neurocircuitry.
Our data revealed that only transgenic males showed overexpression of NPY in hippocampus, which could explain cognitive impairments, already reported by our group10. Indeed, we found that juvenile Nf1+/- male tend to develop an impaired learning and memory profile, which could be associated with hippocampal morphological changes and decreased hippocampal levels of GABA(A) receptors observed in these animals 10. Under physiological conditions, central NPY acts as a modulator of neuroplasticity, neurotransmission, and memory 52 by regulating synaptic plasticity and cognitive functions, including spatial memory in mice53. Importantly, NPY is expressed by different populations of GABAergic neurons within the mouse dentate gyrus of the hippocampus, affecting specific emotional-affective and cognitive aspects of a fear memory52.
Our results might suggest that changes in NPY expression could be an adaptive functional response to counterbalance the cognitive impairment observed in Nf1+/− males10.
On the other hand, Nf1+/− females displayed more severe anxiety-related traits, which was supported by lower mRNA expression of NPY in PFC and higher expression of Y2 in the amygdala. Our data also corroborates previous studies where reduced cortical NPY54 and amygdala Y2 activation is associated with an increase in anxiogenic phenotype23,24,26,55. It is also known that NPY levels were consistently reduced in pre-clinical and clinical subjects with depression, chronic stress, and post-traumatic stress disorder56–58. Indeed, postmortem studies have also observed a significant decrease in NPY mRNA and upregulation in Y2 mRNA in the hippocampus and PFC regions of subjects with depression and suicide behaviors 59. Accordingly, animal studies suggested that NPY may be involved in adaptive responses to stress, depending on the duration and nature of the stressor, stress procedure, and recovery phase60. Recently, a study conducted by Carbona et al. pointed out important sex-dependent changes in the relative levels of NPY at PFC of young juvenile rats in anxiety-related behavior61. A few numbers of reports have described the causality between Y2 in the amygdala and anxiogenesis. In the basolateral amygdala, activation of Y2 receptors that are predominantly expressed on presynaptic terminals increased anxiety behavior23. Using an adult conditional knock-out mouse model for Y2 receptor in the basolateral amygdala (BLA), anxiety-like behavior was reduced in the elevated plus maze paradigm, suggesting that region-site specific Y2 in the amygdala has a prone role in an anxiogenic circuit through a GABA-dependent mechanism 24,62.
Specifically, the NPY system is known to bear sex-dependent changes and is sensitive to gonadal steroids 63,64. Although the sex-biased effect of NPY on metabolic functions has been extensively investigated, the role of gonadal hormones on NPY-driven emotional behavior was not fully disclosed. Here, we found that NPY and Y2 receptors mRNA expression varies along female menstrual phases, where Y2 in the hippocampus and NPY in the amygdala showed distinct expression patterns in transgenic females compared to their sex-matched controls. Pivotal studies implicated the importance of NPY in the control of GnRH via Y265. Interestingly, by using immortalized hypothalamic neurons, Belsham et al. described that estradiol induces transcriptional changes in NPY gene expression suggesting that regulation of NPY expression might mediate estrogen-distinct effects on reproductive functions and energy homeostasis64.
In the context of microbiota-gut-brain axis, the NPY system has an impact on the interoceptive regulation of anxiety and mood and had been suggested to impact the composition and function of the gut microbiota 46. Furthermore, gut dysbiosis may also be related to anxious phenotypes because one of the communication pathways involving the gut and the brain implies the hypothalamic-pituitary axis, which is be involved in the anxiogenic process. Here, for the first time, we found that females Nf1+/− are particularly prone to transcriptomic changes in NPY and PYY of the colonic portion of the intestine. Transgenic females exhibited increased expression of NPY, PYY and Y2. Intriguingly, males Nf1+/− showed decreased expression of Y2. Intestinal Y2 receptor is strongly involved in gut contractility, feeding behavior, electrolyte secretion, and colonic transit. Its role in inhibiting diarrhea in mice has been extensively studied66. In addition, changes in the Y2 receptor could be correlated with major gastrointestinal issues observed among autistic children such as abdominal pain, constipation, and diarrhea67. Thus, based on our last results, sex-dependent differential expression of intestinal Y2 may potentially represent a novel sex-specific biomarker in ASD. The fact that there are no data implicating directly NPY/PYY/Y2 in autism and anxiety, provides further support for the novelty of our work. We suggest that sex-biased abnormal dysfunction of this peptidergic system could reflect an important framework to address gut-brain abnormalities in ASD.
As up to 70% of children with ASD have impaired gastrointestinal function68, an imbalance in the composition of microbiota can contribute to the prevalence of GI disturbances. Biological sex is also thought to play a prominent role in the sexual dimorphism of gut microbiota 19,48,69. Here, we characterized the gut relative abundance in Nf1+/− and we have also reported a sex-dependent dysbiosis in Lactobacillus genus and L. reuteri. We found that transgenic females exhibited decreased levels of L. reuteri, independent of the menstrual cycle, and males presented decreased levels of Lactobacillus genus, which corroborates the findings reported by other authors. Tabouy et al. found that dysregulation of Lactobacillus and L.reuteri in the gut of both males and females of Shank3−/− mice is associated with social impairments and stereotypic-like behaviors18. Furthermore, Costa-Mattioli et al. found that administration of a specific strain L. reuteri ameliorated social deficits in several ASD mouse models48.
In summary, our work first-time establishes that gut-brain NPY/Y2 receptor systems may represent a molecular contributor of anxiety-like behavior in a sex- and region-dependent manner, paving the mechanistic route between anxiety-gastrointestinal dysfunction and ASD pathophysiology. Our results suggest a sexually dimorphic impact of autistic Nf1+/− mutation in NPY neurochemistry, particularly regarding the Y2 receptor. Indeed, mutant females display dysfunction in the NPY system not only in the brain but also in the gut, which is accompanied by microbiome dysbiosis. Further investigation of these sex differences in the NPY system could shed further light on the mechanisms behind the heightened susceptibility of females to anxiety-triggered neuropsychiatric disorders. The present study also highlights the crucial importance of considering biological sex for new therapeutic approaches for ASD- and neuropsychiatric disorders, opening new avenues to define the relationship between genetics, the microbiome, and neurodevelopment.