The microbial metabolite p-Cresol induces autistic-like behaviors in mice by remodeling the gut microbiota

Brain development and behavioral responses are influenced by gut microbiota. Perturbations of the microbiota-gut-brain axis have been identified in autism spectrum disorders (ASD), suggesting that the microbiota could be involved in abnormal social and stereotyped behaviors in ASD patients. Notably, changes in microbiota composition and fecal, serum or urine levels of microbial metabolites are associated with ASD. Yet, a causal relationship between abnormal microbiota composition, altered microbial metabolite production, and ASD remains to be demonstrated. We hypothesized that p-Cresol (also known as 4-Cresol), a microbial metabolite that was described as more abundant in ASD patients, contributes to ASD core behavioral symptoms. Here we show that mice exposed to p-Cresol for 4 weeks in drinking water presented social behavior deficits, stereotypies, and perseverative behaviors, but no changes in anxiety, locomotion, or cognition. Abnormal social behavior induced by p-Cresol was associated with decreased activity of central dopamine neurons involved in the social reward circuit. Further, p-Cresol modified the relative abundance of specific bacterial taxa which correlated with social behavior. In addition, social behavior deficits were transferrable from p-Cresol-treated mice to control mice by fecal matter transplantation. In contrast, the microbiota from control mice restored both social interactions and dopamine neurons excitability when transplanted to p-Cresol-treated mice. Altogether, our results suggest that microbial metabolites p-Cresol could be involved in the development of core autistic behaviors via a gut microbiota-dependent mechanism. Further, this study paves the way for therapeutic interventions targeting the production of p-Cresol by gut bacteria to treat patients with ASD.


INTRODUCTION
Autism spectrum disorders (ASD) are frequent (1:100) neurodevelopmental pathologies characterized by social interaction and communication deficits, perseverative/stereotyped behaviors and restricted interests, as well as abnormal sensory processing 1 . Also, ASD often co-occur with anxiety, hyperactivity and intellectual disability 1 . ASD is also associated with gastrointestinal (GI) dysfunction and increased intestinal permeability 2 . Children with ASD and concurrent GI symptoms exhibit more pronounced social impairments, sensory overresponsivity and anxiety compared to ASD peers without GI symptoms [3][4][5] . In addition, ASD patients exhibit gut microbiota dysbiosis characterized by reduced bacterial b-diversity and changes in the relative abundances of several bacterial taxa [6][7][8] . Dysbiosis in ASD patients is associated with altered urinary, plasmatic or fecal levels of microbial metabolites such as shortchain fatty acids (SCFA), indoles and tyrosine-derived metabolites [9][10][11][12][13][14][15][16] . In a pilot study, fecal microbiota transplantation (FMT) from healthy individuals to ASD patients durably alleviated both GI symptoms and ASD core symptoms 17 . Dysbiosis [18][19][20][21][22] and altered levels of microbial metabolites 18,21,22 have also been observed in rodent models of ASD : the maternal immune activation (MIA), the diet-induced obesity (DIO) and the valproate environmental models, the BTBR T+tf/J idiopathic model, and the Shank3b-KO genetic model. Furthermore, changes in microbiota composition induced by FMT or probiotic treatment alleviated behavioral alterations in several of these ASD models [18][19][20] . Finally, mice born from mothers transplanted with feces from ASD patients exhibited social behavior deficits, dysbiosis and abnormal patterns of microbial metabolites 23 .
The link between altered levels of microbial metabolites and behavioral impairments in ASD remains mostly unknown. However, several microbial metabolites can induce behavioral changes when administered to rodents. Treatment with the SCFA propionate induced social interaction deficits, stereotypies, cognitive deficits and anxiety in rats 14 . The tyrosine derivative 4-ethylphenylsulfate (4-EPS) induced anxiety in mice 18 . Finally, indoles impacted 4 socioemotional behavior in rats 24 . Altogether, these data suggested that dysbiosis could contribute to ASD core and associated symptoms via the production of microbial metabolites.
Among the microbial metabolites linked to ASD, the small aromatic metabolite p-Cresol (para-Cresol, 4-Cresol, 4-methylphenol) is one of those for which the results are the more consistent across studies. Urinary 11, 12 and fecal 15,16 levels of p-Cresol were found to be increased ASD patients in four independent studies. Also, p-Cresol urinary levels correlated with the severity of ASD behavioral alterations 11, 12 . p-Cresol is the product of tyrosine degradation by the intestinal microbiota and mainly by Clostridioides difficile and other species from the Clostridioides genus 25,26 , which is more abundant in ASD patients [6][7][8] .
Based on these findings, we hypothesized that increased p-Cresol levels contribute to the induction or maintenance of ASD core symptoms. To test this hypothesis, we investigated in mice the impact of p-Cresol exposure on behavior, dopamine neurons electrophysiology and microbiota composition. 5

METHODS AND MATERIALS
Extended methods and materials are available in Supplementary Information. Animals treatment. Weaned C57BL/6J mice (21 to 28 day-old) were ordered to Charles Rivers (France). This developmental stage was chosen to avoid confounding effects of maternal exposure to p-Cresol, while still being in the time window for late neurodevelopment during which the microbiota-gut-brain axis can intervene 27 . Since the sex ratio for ASD is biased towards 3 males diagnosed for 1 female suggesting a higher susceptibility of males 28 , only males were considered in this study. Mice were randomly assigned to experimental groups. After 6 days of acclimation to the animal facility, mice were treated for at least 4 weeks with p-Cresol (Sigma-Aldrich) dispensed in drinking water at a concentration of 0.25g/l for a target dose of 50 mg/Kg/24 h. This dose per day corresponded to 7 times less the reported LD50 dose of 350 mg/Kg for acute oral administration of p-Cresol by gavage in mice, based on available toxicity data 29 .
Ex vivo patch-clamp electrophysiological recordings. Spontaneous excitatory postsynaptic currents (sEPSCs) or excitability were measured in 250 μm brain sections encompassing the ventral tegmented area (VTA) using visualized whole-cell voltage-clamp and current-clamp recordings, respectively, as described 35    with Benjamini-Hochberg's multiple testing correction. Statistical significance was set at an adjusted p-value below 0.05. Only significant differences are displayed.

p-Cresol induces ASD core symptoms in mice
To mimic exposure to p-Cresol through the GI tract, we treated C57BL/6J male mice with p-Cresol in drinking water starting at 4.5 weeks of age (Supplementary Figure 1A). We then analyzed p-Cresol-treated mice for social interaction deficits and repetitive/perseverative behaviors (as proxies for ASD core symptoms) as well as anxiety, hyperactivity and cognitive deficits (as proxies of ASD comorbidities). In the 3-chamber test, p-Cresol-treated mice presented reduced sociability ( Figure 1B) and no preference for the mouse interactor towards the toy mouse ( Figure 1C) compared to control mice. Although the number of close contacts with the mouse interactor was higher than with the toy mouse (Supplementary Figure 2G), their mean duration was reduced ( Figure 1D). During dyadic social interactions, p-Cresol-treated mice displayed a decrease in time spent in social contact compared to control mice ( Figure 1E). The time, number and mean duration of both nose and paw contacts as well as the number of followings were also reduced ( Figure 1F Thus, the occurrence of stereotyped behaviors was increased in p-Cresol-treated mice, as confirmed in the marble burying test ( Figure 1M). Consistently, p-Cresol treatment increased the frequency of perseverative same arm returns in the Y-maze spontaneous alternation task ( Figure 1N). Finally, p-Cresol-treated mice were clearly separated from control mice along the PC1 axis in a PCA analysis of scores recorded in the dyadic social interaction and stereotypies tests ( Figure 1O). Altogether, these results suggest that p-Cresol selectively induces ASD core symptoms which persist in the absence of p-Cresol, but does not impact the other behavioral dimensions investigated here.

p-Cresol impairs dopamine neurons excitability in the VTA and induces long-lasting autistic-like behaviors that persist after washout
We then sought to identify the neuronal circuits that were impacted by p-Cresol. We focused on dopamine neurons from the VTA that are part of a 'socially engaged reward circuit' 39 .
Altered VTA connectivity and impaired VTA dopamine neurons activity were demonstrated in ASD patients 40 and in ASD models 19,20,[41][42][43][44] respectively. We used whole-cell patch-clamp to record VTA dopamine neurons in acute brain slices from control and p-Cresol-treated animals

p-Cresol induces microbiota dysbiosis
Previous studies suggested that p-Cresol could have bacteriostatic properties 26 . We reasoned that irreversible changes in microbiota composition could explain the persistence of p-Cresol behavioral effects after a 4-week washout. We therefore analyzed the bacterial composition and community structure of the fecal microbiota from p-Cresol-treated and control mice using bacterial 16S ribosomal RNA sequencing. Bacteroidia and Clostridia were the dominant classes in both groups (Supplementary Figure 4A). Bacterial a-diversity indices did not reveal differences in richness or evenness between groups (Supplementary Figure 4B-E). However, the b-diversity index based on Jaccard's distances revealed changes in microbial taxonomic abundance profiles upon p-Cresol treatment (Supplementary Figure 4F). We then sought to identify the bacterial taxa and OTU that discriminated p-Cresol-treated and control mice.
Among the 91 OTU identified, 70 were discriminant (|Log10LDA score|>2; p<0.05) and the largest effect sizes (|Log10LDA score|>3) were observed for OTU related to the Bacteroidales and Clostridiales orders (Supplementary Table 1). We identified 29 OTU associated with p-Cresol exposure, mostly from the Lachnospiraceae and Muribaculaceae families. We also identified 41 OTUs associated with the control group, mostly from the Lachnospiraceae, Ruminococcaceae and Muribaculaceae families.
We then analyzed the association between the relative abundance of the 70 discriminant bacterial OTU (Supplementary Table 1) and social behavior and stereotypies scores impacted by p-Cresol ( Figure 1E-L). Correlation analyzes revealed moderate to strong associations (0.38 < |r| < 0.74) between bacterial abundances and behavior, in particular social behavior (Table 1, Supplementary Table 2). Among the OTU that were associated with p-Cresol treatment, the abundance of 4 OTU from the Bacteroidales and the Clostridiales orders correlated with social interaction deficits. Among the OTU that were associated with the control group, the abundance of 20 OTU from the Clostridiales order (Ruminococaceae and Lachnospiraceae families) correlated with higher sociability. The abundance of two OTU 11 correlated with reduced numbers of head shakes. All these OTU were differentially impacted by p-Cresol treatment, either downregulated if they correlated positively with behavioral outcome or upregulated if they were correlated with the severity of behavioral impairments (Figure 2A-C). This supports a tight link between microbiota dysbiosis induced by p-Cresol and behavioral impairments relative to core ASD symptoms.

The microbiota from p-Cresol-treated mice induces social behavior deficits when transplanted to normal recipients
Having shown that p-Cresol remodeled the intestinal microbiota, we investigated whether these changes were responsible for p-Cresol-induced behavioral alterations. To this aim, we transplanted the fecal microbiota from p-Cresol-treated mice to normal recipients ( Figure 3A). were clearly separated along the PC1 axis using PCA analysis of social interaction and stereotypies scores ( Figure 3J). In conclusion, the microbiota from p-Cresol-treated mice transfers social behavior deficits and to some extent stereotypies when transplanted to recipient mice.

Transfer of a normal microbiota to p-Cresol-treated recipient mice restores social behavior deficits and VTA dopamine neurons excitability
Having shown that the microbiota from p-Cresol-treated mice induced social behavior deficits when transplanted into normal recipients, we investigated whether p-Cresol-induced behavioral alterations could be restored by a normal microbiota. We therefore transplanted a 12 normal microbiota to either control mice or mice that had been treated for 4 weeks with p-Cresol ( Figure 4A). Before FMT, p-Cresol-treated mice displayed social behavior deficits and stereotypies ( Figure 4B-K) consistent with our previous results (Figures 1, 2). Recolonization of p-Cresol-treated mice with a normal microbiota restored all parameters to control levels in the dyadic social interaction test ( Figure 4B, C, E, G, Supplementary Figure 6I, J). The quality of social contacts was also restored as the mean duration of both nose and paw contacts were rescued ( Figure 4D, F). In the three-chamber test, the sociability index was also normalized ( Figure 4H, Supplementary Figure 6F), as well as the preference for the interactor mouse (Supplementary Figure 6G, H). The quality of social interactions was also rescued as assessed by normalization of contact mean duration ( Figure 4I). In contrast, reversal of stereotyped behaviors was incomplete, since head shakes were only modestly reduced and circling episodes were still present after FMT in recipient mice that had been treated with p-Cresol

p-Cresol induces ASD core behavioral deficits
ASD result from interactions between genes and environment. While 10-25% of ASD cases are explained by mutations in specific genetic loci, twin studies have revealed that genetic and environmental factors share equal influence on ASD risk 45 . The identification of environmental factors, including microbiota-linked factors, contributing to ASD is therefore critical to better understand the etiology of these multi-factorial pathologies.
Here we show that p-Cresol-treated mice exhibit social behavior deficits and stereotypies, demonstrating a possible causal relationship between elevated p-Cresol levels and ASD core symptoms. Like p-Cresol, other microbial metabolites are possibly linked to ASD. While several of these metabolites were demonstrated to modify behavior when administered to rodents, none of them selectively induced ASD core symptoms. For example, the microbial SCFA propionate did not only induce social interaction deficits and stereotypies, but also anxiety and hyperlocomotion 14 . Indoles increased social contact and anxiety but reduced locomotor activity in rats 24 . Lastly, 4-EPS increased anxiety and startle reflex in mice but had no impact on social behavior or stereotypies 18 . Therefore, in contrast to mice treated with other microbial metabolites, p-Cresol has selective effects on ASD core behaviors by inducing social behavior deficits and repetitive/perseverative behaviors, while not impacting behaviors related to frequent ASD comorbidities (stress, anxiety, hyperactivity, cognition).
Mice displaying social avoidance exhibited an increase in p-Cresol levels measured in gut tissue 46 . However, the causal relationship between social behavior and p-Cresol levels was not investigated. Also, a recent study showed that a single acute intravenous injection of p-Cresol exacerbated anxiety and reduced social preference in the BTBR idiopathic model of ASD 47 . Nonetheless, the intravenous administration of p-Cresol did not recapitulate chronic intestinal exposure and the broad observed effects may have resulted from acute activity of the compound. The specific induction of ASD core symptoms upon p-Cresol intestinal exposure supports the face validity of our environmental ASD model.

p-Cresol-induced dysbiosis modulates the social behavior of the host
How microbial metabolites linked to ASD such as propionate, indoles or 4-EPS impact the brain remains unknown. Some of these metabolites are ligands to the host's receptors: 3indoxylsulfate and indole-3-propionate are ligands to the aryl-hydrocarbon receptor 48 and the xenobiotic sensor pregnane X receptor 49 respectively, while propionate binds to GPR41 and GPR43 50 . Yet, their signaling role has been mostly investigated in the context of metabolic, GI or autoimmune disorders [48][49][50] , and not in the context of neuropsychiatric disorders. It appears unlikely that p-Cresol exerts per se a direct systemic or even central effect on a receptor in our experimental paradigm. In support of this, its circulating serum levels are not increased in p-Cresol-treated mice, making it unlikely to reach bioactive levels in the brain. Also, p-Cresol effects on behavior persist even in its absence after a 4-week washout period. Rather, we propose that GI exposure to p-Cresol triggers ASD behaviors by inducing a selective microbiota dysbiosis, which is sufficient to fully induce social behavior deficits and partially stereotypies upon transplantation to control mice. We indeed show that a p-Cresol-rich environment reduces bacterial b-diversity, as described in ASD patients [6][7][8] , and provides a selective growth advantage for several taxa, in line with a previous in vitro study 26  correlate negatively with social behavior abilities. We anticipate that the selective overgrowth of yet-to-be-identified bacterial species in a p-Cresol rich environment could contribute to social behavior deficits in our model. Finally, our model displays a 4-fold increase in p-Cresol urinary excretion, within the 2.5-fold increase range identified in ASD patients 11, 12 . Elevated urinary p-Cresol 11, 12 and dysbiosis 6-8 are observed both in ASD patients and in our model, underpinning its construct validity. Furthermore, its predictive validity is supported by our findings that FMT 15 of a normal microbiota normalizes behavior in p-Cresol-treated mice, as already shown in a pilot study in ASD patients 17 .

p-Cresol-induced dysbiosis dysregulates central dopamine pathways and the social reward system
Social interactions are pleasurable events for humans and animals, as shown by the activation of the reward circuit by social stimuli -activation that is blunted in ASD patients 51 . The VTA is a key subcortical node of the mesolimbic reward pathway 52 and ASD patients display defects in VTA connectivity 40 . Moreover, blocking VTA dopamine neurons that are part of a 'socially engaged reward circuit' is sufficient to diminish social interactions in rodents 39,53 . Further, impaired dopamine neurons activity in the VTA has been previously described in models targeting the ASD genes Shank3b, Nlgn3 and Ube3a 20, 41-43 and in the MIA and DIO environmental models of ASD 19,44 . The disruption of the social reward pathway both in ASD patients and in our model also argues for its construct validity. Dopaminergic circuits are sensitive to changes in gut microbiota composition 54 and gut-innervating vagal sensory neurons control striatal dopamine release 55 . Further, normalization of microbiota composition by FMT abolished social interaction deficits and restored VTA dopamine neurons excitability in p-Cresol-treated mice. Taken together, these results support a model in which p-Cresolinduced dysbiosis results in impaired VTA dopamine neurons activity which eventually disrupts the social reward pathway and causes social behavior deficits.

Conclusions and perspectives
We found that p-Cresol-treated mice exhibited social interaction deficits and stereotypies reminiscent of ASD core symptoms in humans. These behaviors were dependent on changes in microbiota composition and were associated with a reduced activity of dopamine neurons in the VTA. This environmental model of ASD exhibited face, construct and predictive validity.
Because p-Cresol levels are elevated in ASD patients, our study suggests that increased p-Cresol levels could contribute to ASD core symptoms in humans. Further, the ability of a 16 normal microbiota to normalize social interaction deficits when transplanted into p-Cresoltreated mice supports the rationale for the ongoing clinical trials aiming at ascertaining the beneficial impact of FMT in ASD, as highlighted in a pilot study 17 . Finally, prebiotic treatment with oligofructose-enriched inulin or probiotic treatment with Lactobacillus casei decreased p-Cresol urinary excretion in healthy volunteers 56 . Future clinical studies could evaluate the benefits of such interventions to reduce the microbial production of p-Cresol and alleviate the core symptoms of ASD.    Table 1. The abundance of bacterial OTU discriminating p-Cresol-treated mice from control mice correlate with social behavior/stereotypies scores. Spearman's rank r correlation coefficients are annotated and color-coded: positive correlations (red), negative (blue). LEfSE score and associated p-value are also reported for each OTU. Only significant correlations are displayed (FDR-adjusted p-value < 0.05). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 (n=30 animals: n=15 Control, n=15 p-Cresol). See also Supplementary Table 1 for complete LefSE analysis and Supplementary Table 2