Male BTBR Research Articles

Spatial Single-Cell Mapping of Transcriptional Differences Across Genetic Backgrounds in Mouse Brains.

Genetic variation can alter brain structure and, consequently, function. Comparative statistical analysis of mouse brains across genetic backgrounds requires spatial, single-cell, atlas-scale data, in replicates-a challenge for current technologies. We introduce A tlas-scale T ranscriptome L ocalization using A ggregate S ignatures (ATLAS), a scalable tissue mapping method. ATLAS learns transcriptional signatures from scRNAseq data, encodes them in situ with tens of thousands of oligonucleotide probes, and decodes them to infer cell types and imputed transcriptomes. We validated ATLAS by comparing its cell type inferences with direct MERFISH measurements of marker genes and quantitative comparisons to four other technologies. Using ATLAS, we mapped the central brains of five male and five female C57BL/6J (B6) mice and five male BTBR T+ tf/J (BTBR) mice, an idiopathic model of autism, collectively profiling over 40 million cells across over 400 coronal sections. Our analysis revealed over 40 significant differences in cell type distributions and identified 16 regional composition changes across male-female and B6-BTBR comparisons. ATLAS thus enables systematic comparative studies, facilitating organ-level structure-function analysis of disease models.

Alleviation of Autophagic Deficits and Neuroinflammation by Histamine H3 Receptor Antagonist E159 Ameliorates Autism-Related Behaviors in BTBR Mice

Background/Objectives: Autism spectrum disorder (ASD) is a neurodevelopmental condition marked by social interaction difficulties, repetitive behaviors, and immune dysregulation with elevated pro-inflammatory markers. Autophagic deficiency also contributes to social behavior deficits in ASD. Histamine H3 receptor (H3R) antagonism is a potential treatment strategy for brain disorders with features overlapping ASD, such as schizophrenia and Alzheimer’s disease. Methods: This study investigated the effects of sub-chronic systemic treatment with the H3R antagonist E159 on social deficits, repetitive behaviors, neuroinflammation, and autophagic disruption in male BTBR mice. Results: E159 (2.5, 5, and 10 mg/kg, i.p.) improved stereotypic repetitive behavior by reducing self-grooming time and enhancing spontaneous alternation in addition to attenuating social deficits. It also decreased pro-inflammatory cytokines in the cerebellum and hippocampus of treated BTBR mice. In BTBR mice, reduced expression of autophagy-related proteins LC3A/B and Beclin 1 was observed, which was elevated following treatment with E159, attenuating the disruption in autophagy. The co-administration with the H3R agonist MHA (10 mg/kg, i.p.) reversed these effects, highlighting the role of histaminergic neurotransmission in observed behavioral improvements. Conclusions: These preliminary findings suggest the therapeutic potential of H3R antagonists in targeting neuroinflammation and autophagic disruption to improve ASD-like behaviors.

Potential Neuroprotective Effect of Melatonin in the Hippocampus of Male BTBR Mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder identified by impairments in common social interactions and repetitive behaviors. In ASD patients, substantial morphological alterations have been observed in the hippocampus, which represents an important region for the development of social skills. Melatonin, commonly found in many foods and plants, is also produced by the pineal gland. This indolamine, known to regulate the circadian rhythm, shows antioxidant and anti-inflammatory properties. We therefore hypothesized that melatonin may reduce oxidative stress and inflammation in the hippocampus of ASD patients. We explored our hypothesis using the BTBR mouse, a well-regarded murine transgenic model for ASD. Immediately after weaning, male BTBR and C57BL/6 mice underwent an 8-week treatment with melatonin or vehicle. Later, through immunohistochemistry and the immunoblotting analysis of the hippocampus, we evaluated the overall expression and cellular localization of Nrf2 and SOD1, two enzymes involved in the oxidative stress response. Similarly, we evaluated NLRP3 and NFkB, two mediators of inflammation, and GAD67, an enzyme responsible for the synthesis of GABA. Ultimately, we addressed melatonin's potential to regulate iron metabolism through a DAB-enhanced Perls reaction assay. Results showed melatonin's potential for modulating the analyzed markers in BTBR mice, suggesting a potential neuroprotective effect in ASD patients.

Cannabidiol is a behavioral modulator in BTBR mouse model of idiopathic autism.

The prevalence of Autism Spectrum Disorder (ASD) has drastically risen over the last two decades and is currently estimated to affect 1 in 36 children in the U.S., according to the center for disease control (CDC). This heterogenous neurodevelopmental disorder is characterized by impaired social interactions, communication deficits, and repetitive behaviors plus restricted interest. Autistic individuals also commonly present with a myriad of comorbidities, such as attention deficit hyperactivity disorder, anxiety, and seizures. To date, a pharmacological intervention for the treatment of core autistic symptoms has not been identified. Cannabidiol (CBD), the major nonpsychoactive constituent of Cannabis sativa, is suggested to have multiple therapeutic applications, but its effect(s) on idiopathic autism is unknown. We hypothesized that CBD will effectively attenuate the autism-like behaviors and autism-associated comorbid behaviors in BTBR T+Itpr3tf/J (BTBR) mice, an established mouse model of idiopathic ASD. Male BTBR mice were injected intraperitoneally with either vehicle, 20 mg/kg CBD or 50 mg/kg CBD daily for two weeks beginning at postnatal day 21 ± 3. On the final treatment day, a battery of behavioral assays were used to evaluate the effects of CBD on the BTBR mice, as compared to age-matched, vehicle-treated C57BL/6 J mice. High dose (50 mg/kg) CBD treatment attenuated the elevated repetitive self-grooming behavior and hyperlocomotion in BTBR mice. The social deficits exhibited by the control BTBR mice were rescued by the 20 mg/kg CBD treatment. Our data indicate that different doses for CBD are needed for treating specific ASD-like behaviors. Together, our results suggest that CBD may be an effective drug to ameliorate repetitive/restricted behaviors, social deficits, and autism-associated hyperactivity.

Deletion, but Not Heterozygosity, of eNOS Raises Blood Pressure and Aggravates Nephropathy in BTBR ob/ob Mice

Introduction: ob/ob mice are a leptin-deficient type 2 diabetes mellitus model, which, on a BTBR background, mimics the glomerular pathophysiology of diabetic nephropathy (DN). Since leptin deficiency reduces blood pressure (BP) and endothelial nitric oxide synthase (eNOS) lowers BP and is kidney protective, we attempted to develop a more robust DN model by introducing eNOS deficiency in BTBR ob/ob mice. Methods: Six experimental groups included littermate male and female BTBR ob/ob or wild-type for ob (control) as well as wild-type (WT), heterozygote (HET), or knockout (KO) for eNOS. Systolic BP (by automated tail-cuff) and GFR (by FITC-sinistrin plasma kinetics) were determined in awake mice at 27–30 weeks of age, followed by molecular and histological kidney analyses. Results: Male and female ob/ob WT presented hyperglycemia and larger body and kidney weight, GFR, glomerular injury, and urine albumin to creatinine ratio (UACR) despite modestly lower BP versus control WT. These effects were associated with a higher tubular injury score and renal mRNA expression of NGAL only in males, whereas female ob/ob WT unexpectedly had lower KIM-1 and COL1A1 expression versus control WT, indicating sex differences. HET for eNOS did not consistently alter BP or renal outcome in control or ob/ob. In comparison, eNOS KO increased BP (15–25 mm Hg) and worsened renal markers of injury, inflammation and fibrosis, GFR, UACR, and survival rates, as observed in control and, more pronouncedly, in ob/ob mice and independent of sex. Conclusions: Deletion, but not heterozygosity, of eNOS raises blood pressure and aggravates nephropathy in BTBR ob/ob mice.

Comprehensive analysis of the gene expression profile of the male and female BTBR mice with diabetic nephropathy

Comprehensive insight into the gender-based gene expression-related omics data in a rodent model of diabetic nephropathy (DN) is scarce. In the present study, the gender-based genes regulating different pathways involved in the progression of DN were explored through an unbiased RNA sequence of kidneys from BTBR mice with DN. We identified 17,739 and 17,981 genes in male and female DN mice; 1121 and 655 genes were expressed differentially (DEGs, differentially expressed genes) in male and female DN mice; both genders displayed only 195 DEGs. In the male DN mice, the number of upregulated genes was nearly the same as that of the down-regulated genes. In contrast, the number of upregulated genes was lesser than that of the down-regulated genes in the female DN mice, manifesting a remarkable gender disparity during the progression of DN in this animal model. Gene Ontology (GO) and KEGG-enriched results showed that most of these DEGs were related to the critical biological processes, including metabolic pathways, natural oxidation, bile secretion, and PPAR signaling; all are highly associated with DN. Notably, the DEGs significantly enriched for steroid hormone biosynthesis pathway were identified in both genders; the number of DEGs increased was 22 in male DN mice and 14 in female DN mice. Specifically, the Ugt1a10, Akr1c12, and Akr1c14 were upregulated in both genders. Interestingly, the Hsd11b1 gene was upregulated in female DN mice but downregulated in male DN mice. These results suggest that a significant gender-based variance in the gene expression occurs during the progression of DN and may be playing a role in the advancement of DN in the BTBR mouse model.

TNF-α Pathway and Podocyte Dysfunction in Male and Female BTBR ob/ob Mice

TNF-α Pathway and Podocyte Dysfunction in Male and Female BTBR ob/ob Mice

Revisiting sociability: Factors facilitating approach and avoidance during the three-chamber test

The three-chamber test, the so-called sociability test, has been widely used to assess social deficits based on impaired socially oriented investigations in rodent models. An innate motivation for investigating conspecifics is theoretically a prerequisite for gaining sociability scores in this paradigm. However, several relevant factors mediating investigatory motives, such as familiarity, attractiveness, and aggression, may affect sociability scores, which must be verified to obtain an adequate evaluation of the psychiatric phenotypes exhibited by disease-relevant rodent models. We assessed the social and non-social factors that mediate proximity preference by the three-chamber test with standard C57BL/6 J (B6) mice and low sociability BTBR+ltpr3tf/J (BTBR) mice. Strains of the opponents had no effect. Sexual cues (i.e., opposite sex) increased proximity preference in both strains of mice; in contrast, novel objects induced an approach in B6 mice but avoidance in BTBR mice. Single-housing before testing, stimulated social motive, affected BTBR mice but not B6 mice. BTBR females showed increased proximity preference across the sessions, and BTBR males showed increased preference toward a male B6 stimulus, but not a male BTBR stimulus. The male preference was restored when the male BTBR stimulus was anesthetized. In addition, self-grooming was facilitated by social and non-social novelty cues in both strains. B6 mice predominantly exhibited an investigatory approach toward social or non-social stimuli, whereas BTBR mice recognized social cues but tended to show avoidance. The three-chamber test could evaluate approach-avoidance strategies in target mouse strains that comprise innate social distance between mice.

LC-MS-based untargeted metabolomics reveals the mechanism underlying prostate damage in a type 2 diabetes mouse model

Type 2 diabetes mellitus (T2DM) can cause prostate damage and affect male reproductive function, but the underlying mechanisms are not completely understood. In this study, we used liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics to identify endogenous metabolites in the prostate of a T2DM mouse model. The selected endogenous metabolites were then subjected to bioinformatics analysis and metabolic pathway studies to understand their role in the development of T2DM-induced prostate damage. We used male homozygous BTBR ob/ob mice (n = 12) and BTBR WT mice (n = 11) in this study. We monitored changes in blood glucose, body weight, prostate weight, and prostate index, as well as performed hematoxylin and eosin (H&E) staining and observed that the prostate of the BTBR ob/ob was damaged. We then used ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) for metabolomics analysis. The stability of the model was validated using principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Using variable importance in projection (VIP) > 1, false discovery rate (FDR) < 0.05, and coefficient of variation (CV) < 30 as criteria, a total of 149 differential metabolites (62 upregulated and 87 downregulated) were identified between the prostates of the two groups of mice. Topological pathway analysis showed that these differential metabolites were mainly involved in sphingolipid (SP) and glycerophospholipid (GP) metabolism. In conclusion, our study not only emphasizes the damage caused by T2DM to the prostate but also provides new insights into the potential mechanisms of T2DM-induced male reproductive dysfunction.

Serotonergic circuit dysregulation underlying autism-related phenotypes in BTBR mouse model of autism

The inbred mouse strain, BTBR T+Itpr3tf/J (BTBR), possesses neuronal and circuit abnormalities that underlie atypical behavioral profiles resembling the major symptoms of human autism spectrum disorder (ASD). Forebrain serotonin (5-HT) transmission has been implicated in ASD-related behavioral alterations. In this study, we assessed 5-HT signals and the functional responsiveness in BTBR mice compared to standard C57BL/6J (B6) control mice to elucidate how 5-HT alterations contribute to behavioral abnormalities in BTBR mice. A lower number of 5-HT neurons in the median raphe, but not in the dorsal raphe, was observed in male and female BTBR mice. Acute systemic injection of buspirone, a 5-HT1A receptor agonist, induced c-Fos in several brain regions in both B6 and BTBR mice; however, blunted c-Fos induction in BTBR mice was documented in the cingulate cortex, basolateral amygdala (BLA), and ventral hippocampus (Hipp). Decreased c-Fos responses in these regions are associated with a lack of buspirone effects on anxiety-like behavior in BTBR mice. Analysis of mRNA expression following acute buspirone injection indicated that 5HTR1a gene downregulation (or upregulation) occurred in the BLA and Hipp of B6 mice, respectively, but not BTBR mice. The mRNA expression of factors associated with neurogenesis or the pro-inflammatory state was not consistently altered by acute buspirone injection. Therefore, 5-HT responsivity via 5-HT1A receptors in the BLA and Hipp are linked to anxiety-like behavior, in which circuits are disrupted in BTBR mice. Other distinct 5-HT circuits from the BLA and Hipp that regulate social behavior are restricted but preserved in BTBR mice.

Faded neural projection from the posterior bed nucleus of the stria terminalis to the lateral habenula contributes to social signaling deficit in male BTBR mice as a mouse model of autism

BTBR T+ Itpr3tf/J (BTBR) mice display several behavioral characteristics, including social deficits resembling the core symptoms of human autism. Atypical social behaviors include sequential processes of assembled cognitive-behavior components, such as recognition, investigatory assessment, and signaling response. This study aimed to elucidate the neural circuits responsible for the regulation of the social signaling response, as shown by scent marking behavior in male mice. We first assessed the recognition and investigatory patterns of male BTBR mice compared to those of C57BL/6 J (B6) mice. Next, we examined their scent-marking behavior as innate social signaling responses adjusted to a confronted feature of social stimuli and situations, along with the expression of c-Fos as a marker of neuronal activity in selected brain areas involved in the regulation of social behavior. The function of the targeted brain area was confirmed by chemogenetic manipulation. We also examined the social peptides, oxytocin and vasopressin neurons of the major brain regions that are associated with the regulation of social behavior. Our data indicate that male BTBR mice are less responsive to the presentation of social stimuli and the expression of social signaling responses, which is paralleled by blunted c-Fos responsivity and vasopressin neurons morphological changes in selected brain areas, including the posterior bed nucleus of the stria terminalis (pBnST) and lateral habenula (LHb) in BTBR mice. Further investigation of LHb function revealed that chemogenetic inhibition and activation of LHb activity can induce a change in scent marking responses in both B6 and BTBR mice. Our elucidation of the downstream LHb circuits controlling scent marking behavior indicates intact function in BTBR mice. The altered morphological characteristics of oxytocin neurons in the paraventricular nucleus of the hypothalamus and vasopressin-positive neurons and axonal projections in the pBnST and LHb appear to underlie the dysfunction of scent marking responses in BTBR mice.(300/300 words)

Deficits in cerebellum-dependent learning and cerebellar morphology in male and female BTBR autism model mice.

Recently, there has been increased interest in the role of the cerebellum in autism spectrum disorders (ASD). To better understand the pathophysiological role of the cerebellum in ASD, it is necessary to have a variety of mouse models that have face validity for cerebellar disruption in humans. Here, we add to the literature on the cerebellum transgenic and induced mouse models of autism with the characterization of the cerebellum in the BTBR T+Itpr3tf/J (BTBR) inbred mouse strain, which has behavioral phenotypes that are suggestive of ASD in patients. When we examined both male and female BTBR mice in comparison to C57BL/6J (C57) controls, we noted that both sexes of BTBR mice showed motor coordination deficits characteristic of cerebellar dysfunction, but only the male mice showed differences in delay eyeblink conditioning, a cerebellum-dependent learning task that is also disrupted in ASD patients. Both male and female BTBR mice showed considerable expansion of and abnormal foliation in the cerebellum vermis--including significant expansion of specific lobules in the anterior cerebellum. In addition, we found a slight but significant decrease in Purkinje cell density in both male and female BTBR mice, irrespective of lobule. Furthermore, there was a marked reduction of Purkinje cell dendritic spines density in both male and female BTBR mice. These findings suggest that, for the most part, the BTBR mouse model successfully phenocopies many of the characteristics of the subpopulation of ASD patients that have a hypertrophic cerebellum. We discuss the significance of strain differences in the cerebellum as well as the importance of this first effort to identify both concordances and difference between male and female BTBR mice with regard to the cerebellum.

Impairments in operant probabilistic reversal learning in BTBR T+tf/J male and female mice

Autism spectrum disorder (ASD) presents with two core symptoms, impairments in social communication and the presence of restricted, repetitive behaviors (RRBs). RRBs are commonly linked to a lack of behavioral flexibility, having a significant negative impact on daily functioning for ASD individuals and their caregivers. Commonly utilized tests of behavioral flexibility employ a traditional deterministic reward approach where choices are either correct or incorrect throughout testing. The incorporation of an 80 %/20 % probabilistic reversal learning paradigm allows for the examination of flexible behavior in the face of variable outcomes, a more ecologically relevant approach. In this task, one specific choice is reinforced on 80 % of trials and the opposite or incorrect choice is reinforced on 20% of trials. Upon successful discrimination learning, the reward contingencies are switched so that the correct choice is now reinforced 20% of trials and the incorrect choice reinforced 80 % of trials, making it the new optimal choice. This translational task has been previously validated in ASD individuals and animal models of ASD, including the BTBR T + tf/J strain. Our lab and others have demonstrated that male BTBR T + tf/J mice have higher expression of lower order RRBs and display deficits in spatial probabilistic reversal learning tasks using a T-maze apparatus. Instead, female BTBR mice do not express the same lower order RRBs and results are mixed on whether females demonstrate similar probabilistic reversal learning deficits in a T-maze. Therefore, the purpose of this study was to assess the validity of using operant chambers to examine BTBR mouse performance on an 80 %/20 % probabilistic reversal learning task and to also examine the sex-specific differences in reversal learning performance in both mouse strains. Results show that BTBR mice, irrespective of sex, were impaired on the reversal learning, requiring more days and trials to reach reversal criterion compared to C57BL/6J mice. These results parallel previous strain findings in the spatial dependent T-maze task in male mice. Further error analysis showed that the impaired behavioral flexibility was due to elevated regressive errors and lose-shift probabilities. BTBR mice have more difficulty maintaining new choice patterns compared to C57BL/6J mice, which supports findings utilizing a spatial T-maze task. Together, these findings further support the use of the BTBR mouse as preclinical models of ASD due to their validity as an ASD model.

Selective Probiotic Treatment Positively Modulates the Microbiota-Gut-Brain Axis in the BTBR Mouse Model of Autism.

Recent studies have shown promise for the use of probiotics in modulating behaviour through the microbiota–gut–brain axis. In the present study, we assessed the impact of two probiotic strains in mitigating autism-related symptomology in the BTBR T+ Itpr3tf/J mouse model of autism spectrum disorder (ASD). Male juvenile BTBR mice were randomized into: (1) control, (2) Lr probiotic (1 × 109 CFU/mL Lacticaseibacillus rhamnosus HA-114), and (3) Ls probiotic groups (1 × 109 CFU/mL Ligilactobacillus salivarius HA-118) (n = 18–21/group), receiving treatments in drinking water for 4 weeks. Gut microbiota profiling by 16S rRNA showed Lr, but not Ls supplementation, to increase microbial richness and phylogenetic diversity, with a rise in potential anti-inflammatory and butyrate-producing taxa. Assessing serum and brain metabolites, Lr and Ls supplementation produced distinct metabolic profiles, with Lr treatment elevating concentrations of potentially beneficial neuroactive compounds, such as 5-aminovaleric acid and choline. As mitochondrial dysfunction is often observed in ASD, we assessed mitochondrial oxygen consumption rates in the prefrontal cortex and hippocampus. No differences were observed for either treatment. Both Lr and Ls treatment reduced behavioural deficits in social novelty preference. However, no changes in hyperactivity, repetitive behaviour, and sociability were observed. Results show Lr to impart positive changes along the microbiota–gut–brain axis, exhibiting beneficial effects on selected behaviour, gut microbial diversity, and metabolism in BTBR mice.

CXCR2 antagonist SB332235 mitigates deficits in social behavior and dysregulation of Th1/Th22 and T regulatory cell-related transcription factor signaling in male BTBR T+ Itpr3tf/J mouse model of autism

Autism spectrum disorders is a complex neurodevelopmental disorder characterized by abnormal social interaction, defective communication, repetitive and stereotyped patterns of behaviors or interests. The BTBR T+ Itpr3tf/J (BTBR) inbred mice are generally used as a model for ASD, display a range of autistic phenotypes. Recent studies suggest that the CXCR2 antagonist is crucial for targets in the treatment of inflammatory and neurodegenerative diseases. In this study, we investigated the potential effects of the CXCR2 antagonist SB332235 on sociability behaviors, marble burying, and self-grooming, we also explored the treatment of SB332235 on Th1 (IFN-γ, Stat1, and T-bet), Th22 (IL-22, TNF-α, and AhR), and T regulatory (Treg, IL-10, Helios and Foxp3) production in CD4+ T cells in male BTBR and C57BL/6 (C57) mice in spleen. We also investigated the effects of SB332235 on IFN-γ, IL-10, IL-22, T-bet, AhR, and Foxp3 mRNA expression levels in the brain tissues. The SB332235-treated mice significantly improve behavioral abnormalities in BTBR mice. In addition, SB332235 administration causes a significantly decreases in IFN-γ, Stat1, T-bet, IL-22, TNF-α, and AhR, and increases in IL-10, Foxp3 and Helios production CD4+ T cells in BTBR mice. We further observed that SB332235 downregulated IFN-γ, IL-10, IL-22, T-bet, and AhR, and upregulated IL-10 and Foxp3 mRNA expression in the brain tissues. Our findings demonstrated that SB332235 treatment attenuated behavior deficits, through inhibiting Th1/Th22 and upregulating Treg cell-related transcription factors signaling pathway. Therefore, CXCR2 antagonist administration may be a promising therapeutic agent to attenuate behavior deficits via its anti-inflammatory effect.

Postweaning social isolation and autism-like phenotype: A biochemical and behavioral comparative analysis

Adolescence is a critical period for brain development. In most mammalian species, disturbances experienced during adolescence constitute a risk factor for several neuropsychiatric disorders. In this study, we compared the biochemical and behavioral profile induced by postweaning social isolation (PWSI) in inbred C57BL/6 N mice with that of BTBR mice, a rodent model of autism spectrum disorders. Male C57BL/6 N mice were either housed in groups of four or isolated from weaning (postnatal day 21) for four weeks before experimental analyses. After weaning, male BTBR mice were housed four per cage and analyzed at 48 days of age. PWSI reduced hippocampal levels of type 2 metabotropic glutamate (mGlu2) receptors, and glucocorticoid and mineralocorticoid receptors. A similar reduction was seen in group-housed BTBR mice. Plasma corticosterone levels in basal conditions were not influenced by PWSI, but were increased in BTBR mice. Social investigation (total and head sniffing) and the number of ultrasonic vocalizations were reduced in both PWSI mice and age-matched group-housed BTBR mice, indicating a lower social responsiveness in both groups of mice. These results suggest that absence of social stimuli during adolescence induces an endophenotype with social deficit features, which mimics the phenotype of a mouse model of autism spectrum disorders.

Gut-Microbiome Composition in Response to Phenylketonuria Depends on Dietary Phenylalanine in BTBR Pahenu2 Mice.

Phenylketonuria (PKU) is a metabolic disorder caused by a hepatic enzyme deficiency causing high blood and brain levels of the amino acid Phenylalanine (Phe), leading to severe cognitive and psychological deficits that can be prevented, but not completely, by dietary treatment. The behavioral outcome of PKU could be affected by the gut-microbiome-brain axis, as diet is one of the major drivers of the gut microbiome composition. Gut-microbiome alterations have been reported in treated patients with PKU, although the question remains whether this is due to PKU, the dietary treatment, or their interaction. We, therefore, examined the effects of dietary Phe restriction on gut-microbiome composition and relationships with behavioral outcome in mice. Male and female BTBR Pahenu2 mice received either a control diet (normal protein, “high” Phe), liberalized Phe-restricted (33% natural protein restriction), or severe Phe-restricted (75% natural protein restriction) diet with protein substitutes for 10 weeks (n = 14 per group). Their behavioral performance was examined in an open field test, novel and spatial object location tests, and a balance beam. Fecal samples were collected and sequenced for the bacterial 16S ribosomal RNA (rRNA) region. Results indicated that PKU on a high Phe diet reduced Shannon diversity significantly and altered the microbiome composition compared with wild-type animals. Phe-restriction prevented this loss in Shannon diversity but changed community composition even more than the high-Phe diet, depending on the severity of the restriction. Moreover, on a taxonomic level, we observed the highest number of differentially abundant genera in animals that received 75% Phe-restriction. Based on correlation analyses with differentially abundant taxa, the families Entereococacceae, Erysipelotrichaceae, Porphyromonadaceae, and the genus Alloprevotella showed interesting relationships with either plasma Phe levels and/or object memory. According to our results, these bacterial taxa could be good candidates to start examining the microbial metabolic potential and probiotic properties in the context of PKU. We conclude that PKU leads to an altered gut microbiome composition in mice, which is least severe on a liberalized Phe-restricted diet. This may suggest that the current Phe-restricted diet for PKU patients could be optimized by taking dietary effects on the microbiome into account.

Prebiotic, Probiotic, and Synbiotic Consumption Alter Behavioral Variables and Intestinal Permeability and Microbiota in BTBR Mice.

Given that prebiotics have been shown to improve gut microbiota composition, gastrointestinal symptoms and select behaviors in autism spectrum disorder (ASD), we hypothesized that prebiotic supplementation would improve sociability, communication, and repetitive behaviors in a murine model of ASD. We also examined the effect of a synbiotic (probiotic + prebiotic). Juvenile male BTBR mice were randomized to: (1) control; (2) probiotic (1 × 1010 CFU/d Lactobacillus reuteri RC-14®; now known as Limosilactobacillus reuteri); (3) prebiotic (10% oligofructose-enriched inulin); (4) prebiotic + probiotic (n = 12/group) administered through food for 3 weeks. Sociability, communication, repetitive behavior, intestinal permeability and gut microbiota were assessed. Probiotic and symbiotic treatments improved sociability (92 s and 70 s longer in stranger than empty chamber) and repetitive behaviors (50% lower frequency), whereas prebiotic intake worsened sociability (82 s less in stranger chamber) and increased the total time spent self-grooming (96 s vs. 80 s CTR), but improved communication variables (4.6 ms longer call duration and 4 s higher total syllable activity). Mice consuming probiotics or synbiotics had lower intestinal permeability (30% and 15% lower than CTR). Prebiotic, probiotic, and symbiotic treatments shifted gut microbiota to taxa associated with improved gut health. L.reuteri may help alleviate ASD behavioral symptom severity and improve gut health. The potential use of prebiotics in an ASD population warrants further research.

Evaluation of Poorly-Bioavailable Cocoa Flavanols and Their Gut Microbial Metabolites in Potentiating Anti-diabetic Activities Through BTBR.Cg-Lepob/ob/WiscJ Mice

Abstract Objectives Cocoa (Theobroma cacao) is a concentrated dietary source of flavanols that have beneficial activities against type-2 diabetes. These compounds have limited small intestinal absorption and are metabolized by the microbiota to bioavailable metabolites that may exert anti-diabetic effects locally and in peripheral tissues. Our objectives were to 1) determine the role of the gut microbiome in facilitating protective effects of cocoa flavanols, and 2) evaluate these effects in a novel mouse model of progressive type-2 diabetes. Methods A small pilot study (n = 3) of male and female BTBR mice (wild-type and homozygous for the Lepob mutation) received either control or cocoa extract-supplemented diet for 10 weeks. Half the animals were administered antibiotics orally to knock down the commensal gut microbiota. Glucose and insulin tolerance tests were conducted at weeks 1 and 5 and 2 and 6, respectively. Weight gain and food intake were monitored weekly. Biomarkers of gut integrity and inflammation were assessed by ELISA. Results Baseline fasting blood glucose (FBG) levels in five-week-old homozygous males and females were measured at 211–271 mg/dL and 112–234 mg/dL, respectively. After five weeks, FBG measured at 281–438 mg/dL and 177–562 mg/dL, respectively. Cocoa provided moderate, yet not significant, protection against weight gain in homozygous males when compared to homozygous males fed control diet. Cocoa provided no significant protection against hypoglycemia in homozygous male or female mice when compared to homozygous controls. In treatment comparisons with and without antibiotics, knocking out the commensal gut microbiota appeared to have minimal effect on weight gain and glycemic control in both males and females. Conclusions Cocoa did provide a moderate level of protection for homozygous males when directly comparing weight gain and FBG across sex. While the microbiome has displayed a promising role in the bioavailability of large flavanols, in this particular model, the impact was minimal. Overall, cocoa was ineffective against the mediation of advanced diabetes and further work must be conducted to understand if this conclusion is isolated to this model of progressive type-2 diabetes. Funding Sources This work was supported by the US Department of Agriculture by AFRI grant 2020–67,017-30,846.

Deletion of eNOS enhances kidney injury in BTBR ob/ob mice

Background ob/ob mice are a leptin deficient murine model of type 2 diabetes mellitus (T2DM), characterized by hyperphagia, obesity and hyperglycemia. ob/ob mice on a BTBR background have enhanced insulin resistance and mimic the glomerular pathophysiology of diabetic nephropathy (DN) in humans. However, leptin deficiency reduces blood pressure (BP). Since reduced activity of endothelial nitric oxide synthase (eNOS) enhances BP and the progression of kidney disease, we attempted to develop a more robust DN model by introducing eNOS deficiency in BTBR ob/ob mice. Methods BTBR mice heterozygous for both ob and eNOS were bred to generate 6 experimental groups being wild-type for Ob (Con) or ob/ob (Ob) as well as wild-type (WT), heterozygote (HET) or knockout (KO) for eNOS: Con/WT, Con/HET, Con/KO, Ob/WT, Ob/HET & Ob/KO. Systolic BP (automated tail-cuff) and GFR (FITC sinistrin plasma kinetics) were determined in awake mice in weeks 27-30 followed by kidney harvest for RTqPCR analysis of injury markers. Statistics: 2-way ANOVA for Ob and eNOS followed by pairwise comparisons if interaction was significant. 13-24 mice (males plus females)/group for WT and HET, n= 5-8 mice/group for KO. Results In WT, Ob increased blood glucose (BG), body weight (Bw), kidney weight (Kw), GFR, and urinary albumin to creatinine ratio (UACR), and reduced BP vs Con. These responses to Ob were not different in HET. KO increased BP and UACR and reduced Kw and GFR vs WT in both Con and Ob. In Ob, KO reduced BG and Bw vs WT. All effects were independent of gender. In male mice: In WT, Ob increased renal mRNA expression of Ngal, Ccl2, and Timp1, whereas Kim1, Tnfa and Col1a1 were not significantly different vs Con. These responses to Ob were similar in HET. KO increased renal mRNA expression of Ngal, Timp1, Col1a1 vs WT in both Con and Ob; and KO, in addition, also increased Kim1, Ccl2 and Tnfa vs WT in Ob. In female mice: In WT, Ob did not change renal mRNA expression of Ngal, Ccl2, Timp1, and Tnfa and the expression of Kim1 and Col1a1 were actually reduced vs Con. Similar responses to Ob were observed in HET. KO increased renal mRNA expression of Ngal, Ccl2, Timp1, and Col1a1 vs WT in both Con and Ob; and KO in addition increased Tnfa vs WT in Ob. Conclusions Male and female BTBR ob/ob mice at 27-30 weeks of age presented hyperglycemia and increased Bw associated with larger kidneys, glomerular hyperfiltration and a 100x higher UACR vs Con. These effects were associated with modest increases in renal mRNA expression of Ngal, Ccl2, and Timp1 only in male mice, whereas female Ob mice unexpectedly had lesser expression of Kim1 and Col1a1 vs Con. eNOS heterozygosity did not induce robust renal changes vs WT in Con or Ob. In contrast, eNOS KO, which increased BP by 15-25 mmHg, enhanced kidney injury as indicated by lesser Kw and GFR, associated with higher UACR and renal mRNA expression of markers of injury, inflammation and fibrosis, and lower survival rates; these effects of eNOS KO were detectable in Con and more pronounced in Ob.