Adult Inception of Ketogenic Diet Therapy Increases Sleep during the Dark Cycle in C57BL/6J Wild Type and Fragile X Mice.

Sleep problems are a significant phenotype in children with fragile X syndrome. Our prior work assessed sleep-wake cycles in Fmr1KO male mice and wild type (WT) littermate controls in response to ketogenic diet therapy where mice were treated from weaning (postnatal day 18) through study completion (5-6 months of age). A potentially confounding issue with commencing treatment during an active period of growth is the significant reduction in weight gain in response to the ketogenic diet. The aim here was to employ sleep electroencephalography (EEG) to assess sleep-wake cycles in mice in response to the Fmr1 genotype and a ketogenic diet, with treatment starting at postnatal day 95. EEG results were compared with prior sleep outcomes to determine if the later intervention was efficacious, as well as with published rest-activity patterns to determine if actigraphy is a viable surrogate for sleep EEG. The data replicated findings that Fmr1KO mice exhibit sleep-wake patterns similar to wild type littermates during the dark cycle when maintained on a control purified-ingredient diet but revealed a genotype-specific difference during hours 4-6 of the light cycle of the increased wake (decreased sleep and NREM) state in Fmr1KO mice. Treatment with a high-fat, low-carbohydrate ketogenic diet increased the percentage of NREM sleep in both wild type and Fmr1KO mice during the dark cycle. Differences in sleep microstructure (length of wake bouts) supported the altered sleep states in response to ketogenic diet. Commencing ketogenic diet treatment in adulthood resulted in a 15% (WT) and 8.6% (Fmr1KO) decrease in body weight after 28 days of treatment, but not the severe reduction in body weight associated with starting treatment at weaning. We conclude that the lack of evidence for improved sleep during the light cycle (mouse sleep time) in Fmr1KO mice in response to ketogenic diet therapy in two studies suggests that ketogenic diet may not be beneficial in treating sleep problems associated with fragile X and that actigraphy is not a reliable surrogate for sleep EEG in mice.

Ketogenic Diet Affects Sleep Architecture in C57BL/6J Wild Type and Fragile X Mice.

Nearly half of children with fragile X syndrome experience sleep problems including trouble falling asleep and frequent nighttime awakenings. The goals here were to assess sleep-wake cycles in mice in response to Fmr1 genotype and a dietary intervention that reduces hyperactivity. Electroencephalography (EEG) results were compared with published rest-activity patterns to determine if actigraphy is a viable surrogate for sleep EEG. Specifically, sleep-wake patterns in adult wild type and Fmr1KO littermate mice were recorded after EEG electrode implantation and the recordings manually scored for vigilance states. The data indicated that Fmr1KO mice exhibited sleep-wake patterns similar to wild type littermates when maintained on a control purified ingredient diet. Treatment with a high-fat, low-carbohydrate ketogenic diet increased the percentage of non-rapid eye movement (NREM) sleep in both wild type and Fmr1KO mice during the dark cycle, which corresponded to decreased activity levels. Treatment with a ketogenic diet flattened diurnal sleep periodicity in both wild type and Fmr1KO mice. Differences in several sleep microstructure outcomes (number and length of sleep and wake bouts) supported the altered sleep states in response to a ketogenic diet and were correlated with altered rest-activity cycles. While actigraphy may be a less expensive, reduced labor surrogate for sleep EEG during the dark cycle, daytime resting in mice did not correlate with EEG sleep states.

Rescue of Fmr1KO phenotypes with mGluR5 inhibitors: MRZ-8456 versus AFQ-056.

Metabotropic glutamate receptor 5 (mGluR5) is a drug target for central nervous system disorders such as fragile X syndrome that involve excessive glutamate-induced excitation. We tested the efficacy of a novel negative allosteric modulator of mGluR5 developed by Merz Pharmaceuticals, MRZ-8456, in comparison to MPEP and AFQ-056 (Novartis, a.k.a. mavoglurant) in both in vivo and in vitro assays in a mouse model of fragile X syndrome, Fmr1KO mice. The in vivo assays included susceptibility to audiogenic-induced seizures and pharmacokinetic measurements of drug availability. The in vitro assays included dose response assessments of biomarker expression and dendritic spine length and density in cultured primary neurons. Both MRZ-8456 and AFQ-056 attenuated wild running and audiogenic-induced seizures in Fmr1KO mice with similar pharmacokinetic profiles. Both drugs significantly reduced dendritic expression of amyloid-beta protein precursor (APP) and rescued the ratio of mature to immature dendritic spines. These findings demonstrate that MRZ-8456, a drug being developed for the treatment of motor complications of L-DOPA in Parkinson's disease and which completed a phase I clinical trial, is effective in attenuating both well-established (seizures and dendritic spine maturity) and exploratory biomarker (APP expression) phenotypes in a mouse model of fragile X syndrome.

Effects of Soy Protein Isolate on Fragile X Phenotypes in Mice.

Obesity is a pediatric epidemic that is more prevalent in children with developmental disabilities. We hypothesize that soy protein-based diets increase weight gain and alter neurobehavioral outcomes. Our objective herein was to test matched casein- and soy protein-based purified ingredient diets in a mouse model of fragile X syndrome, Fmr1KO mice. The experimental methods included assessment of growth; 24-7 activity levels; motor coordination; learning and memory; blood-based amino acid, phytoestrogen and glucose levels; and organ weights. The primary outcome measure was body weight. We find increased body weight in male Fmr1KO from postnatal day 6 (P6) to P224, male wild type (WT) from P32-P39, female Fmr1KO from P6-P18 and P168-P224, and female Fmr1HET from P9-P18 as a function of soy. Activity at the beginning of the light and dark cycles increased in female Fmr1HET and Fmr1KO mice fed soy. We did not find significant differences in rotarod or passive avoidance behavior as a function of genotype or diet. Several blood-based amino acids and phytoestrogens were significantly altered in response to soy. Liver weight was increased in WT and adipose tissue in Fmr1KO mice fed soy. Activity levels at the beginning of the light cycle and testes weight were greater in Fmr1KO versus WT males irrespective of diet. DEXA analysis at 8-months-old indicated increased fat mass and total body area in Fmr1KO females and lean mass and bone mineral density in Fmr1KO males fed soy. Overall, dietary consumption of soy protein isolate by C57BL/6J mice caused increased growth, which could be attributed to increased lean mass in males and fat mass in females. There were sex-specific differences with more pronounced effects in Fmr1KO versus WT and in males versus females.

On-Tissue Spatial Proteomics Integrating MALDI-MS Imaging with Shotgun Proteomics Reveals Soy Consumption-Induced Protein Changes in a Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability and autism, is caused by the transcriptional silencing of the FMR1 gene, which encodes the fragile X messenger ribonucleoprotein (FMRP). FMRP interacts with numerous brain mRNAs that are involved in synaptic plasticity and implicated in autism spectrum disorders. Our published studies indicate that single-source, soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for an unbiased protein marker identification in FXS in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging with label-free proteomics in the FXS mouse model to map the spatial distribution and quantify levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1250 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in glycolysis, synaptic transmission, and coexpression network analysis suggest a significant association between soy proteins and metabolic and synaptic processes in the Fmr1KO brain. Ultimately, this spatial proteomics work represents a crucial step toward identifying potential candidate protein markers and novel therapeutic targets for FXS.

Time of day regulates subcellular trafficking, tripartite synaptic localization, and polyadenylation of the astrocytic Fabp7 mRNA.

The astrocyte brain fatty acid binding protein (Fabp7) has previously been shown to have a coordinated diurnal regulation of mRNA and protein throughout mouse brain, and an age-dependent decline in protein expression within synaptoneurosomal fractions. Mechanisms that control time-of-day changes in expression and trafficking Fabp7 to the perisynaptic process are not known. In this study, we confirmed an enrichment of Fabp7 mRNA and protein in the astrocytic perisynaptic compartment, and observed a diurnal change in the intracellular distribution of Fabp7 mRNA in molecular layers of hippocampus. Northern blotting revealed a coordinated time-of-day-dependent oscillation for the Fabp7 mRNA poly(A) tail throughout murine brain. Cytoplasmic polyadenylation element-binding protein 1 (CPEB1) regulates subcellular trafficking and translation of synaptic plasticity-related mRNAs. Here we show that Fabp7 mRNA coimmunoprecipitated with CPEB1 from primary mouse astrocyte extracts, and its 3'UTR contains phylogenetically conserved cytoplasmic polyadenylation elements (CPEs) capable of regulating translation of reporter mRNAs during Xenopus oocyte maturation. Given that Fabp7 expression is confined to astrocytes and neural progenitors in adult mouse brain, the synchronized cycling pattern of Fabp7 mRNA is a novel discovery among known CPE-regulated transcripts. These results implicate circadian, sleep, and/or metabolic control of CPEB-mediated subcellular trafficking and localized translation of Fabp7 mRNA in the tripartite synapse of mammalian brain.

Toward an understanding of the role of the exposome on fragile X phenotypes.

Fragile X syndrome (FXS) is the leading known monogenetic cause of autism with an estimated 21-50% of FXS individuals meeting autism diagnostic criteria. A critical gap in medical care for persons with autism is an understanding of how environmental exposures and gene-environment interactions affect disease outcomes. Our research indicates more severe neurological and metabolic outcomes (seizures, autism, increased body weight) in mouse and human models of autism spectrum disorders (ASD) as a function of diet. Thus, early-life exposure to chemicals in the diet could cause or exacerbate disease outcomes. Herein, we review the effects of potential dietary toxins, i.e., soy phytoestrogens, glyphosate, and polychlorinated biphenyls (PCB) in FXS and other autism models. The rationale is that potentially toxic chemicals in the diet, particularly infant formula, could contribute to the development and/or severity of ASD and that further study in this area has potential to improve ASD outcomes through dietary modification.

Diet in treatment of autism spectrum disorders.

Altering the diet to treat disease dates to c. 400 BC when starvation was used to reduce seizures in persons with epilepsy. The current diversity of symptomology and mechanisms underlying autism spectrum disorders (ASDs) and a corresponding lack of disorder-specific effective treatments prompts an evaluation of diet as a therapeutic approach to improve symptoms of ASDs. In this review article, we summarize the main findings of nutritional studies in ASDs, with an emphasis on the most common monogenic cause of autism, Fragile X Syndrome (FXS), and the most studied dietary intervention, the ketogenic diet as well as other dietary interventions. We also discuss the gut microbiota in relation to pre- and probiotic therapies and provide insight into future directions that could aid in understanding the mechanism(s) underlying dietary efficacy.

Improving Reproducibility to Enhance Scientific Rigor through Consideration of Mouse Diet.

Animal husbandry conditions, including rodent diet, constitute an example highlighting the importance of reporting experimental variables to enhance scientific rigor. In the present study, we examine the effects of three common rodent diets including two chows (Purina 5015 and Teklad 2019) and one purified ingredient diet (AIN-76A) on growth anthropometrics (body weight), behavior (nest building, actigraphy, passive avoidance) and blood biomarkers (ketones, glucose, amino acid profiles) in male and female C57BL/6J mice. We find increased body weight in response to the chows compared to purified ingredient diet albeit selectively in male mice. We did not find significantly altered behavior in female or male wild type C57BL/6J mice. However, amino acid profiles changed as an effect of sex and diet. These data contribute to a growing body of knowledge indicating that rodent diet impacts experimental outcomes and needs to be considered in study design and reporting.

Effects of Soy-Based Infant Formula on Weight Gain and Neurodevelopment in an Autism Mouse Model.

Mice fed soy-based diets exhibit increased weight gain compared to mice fed casein-based diets, and the effects are more pronounced in a model of fragile X syndrome (FXS; Fmr1KO). FXS is a neurodevelopmental disability characterized by intellectual impairment, seizures, autistic behavior, anxiety, and obesity. Here, we analyzed body weight as a function of mouse age, diet, and genotype to determine the effect of diet (soy, casein, and grain-based) on weight gain. We also assessed plasma protein biomarker expression and behavior in response to diet. Juvenile Fmr1KO mice fed a soy protein-based rodent chow throughout gestation and postnatal development exhibit increased weight gain compared to mice fed a casein-based purified ingredient diet or grain-based, low phytoestrogen chow. Adolescent and adult Fmr1KO mice fed a soy-based infant formula diet exhibited increased weight gain compared to reference diets. Increased body mass was due to increased lean mass. Wild-type male mice fed soy-based infant formula exhibited increased learning in a passive avoidance paradigm, and Fmr1KO male mice had a deficit in nest building. Thus, at the systems level, consumption of soy-based diets increases weight gain and affects behavior. At the molecular level, a soy-based infant formula diet was associated with altered expression of numerous plasma proteins, including the adipose hormone leptin and the ß-amyloid degrading enzyme neprilysin. In conclusion, single-source, soy-based diets may contribute to the development of obesity and the exacerbation of neurological phenotypes in developmental disabilities, such as FXS.

Consumption of Breast Milk Is Associated with Decreased Prevalence of Autism in Fragile X Syndrome.

Breastfeeding is associated with numerous health benefits, but early life nutrition has not been specifically studied in the neurodevelopmental disorder fragile X syndrome (FXS). Herein, I evaluate associations between the consumption of breast milk during infancy and the prevalence of autism, allergies, diabetes, gastrointestinal (GI) problems and seizures in FXS. The study design was a retrospective survey of families enrolled in the Fragile X Online Registry and Accessible Research Database (FORWARD). There was a 1.7-fold reduction in the prevalence of autism in FXS participants who were fed breast milk for 12 months or longer. There were strong negative correlations between increased time the infant was fed breast milk and the prevalence of autism and seizures and moderate negative correlations with the prevalence of GI problems and allergies. However, participants reporting GI problems or allergies commenced these comorbidities significantly earlier than those not fed breast milk. Parsing the data by sex indicated that males exclusively fed breast milk exhibited decreased prevalence of GI problems and allergies. These data suggest that long-term or exclusive use of breast milk is associated with reduced prevalence of key comorbidities in FXS, although breast milk is associated with the earlier development of GI problems and allergies.

Diet in the Treatment of Epilepsy.

This Special Issue for Nutrients focuses on the effects of diet on brain function with a special emphasis on epileptic disorders [...].

Parental Reports on Early Autism Behaviors in Their Children with Fragile X Syndrome as a Function of Infant Feeding.

This study evaluates the prevalence of autistic behaviors in fragile X syndrome as a function of infant diet. Retrospective survey data from the Fragile X Syndrome Nutrition Study, which included data on infant feeding and caregiver-reported developmental milestones for 190 children with fragile X syndrome enrolled in the Fragile X Online Registry with Accessible Database (FORWARD), were analyzed. Exploratory, sex-specific associations were found linking the use of soy-based infant formula with worse autistic behaviors related to language in females and self-injurious behavior in males. These findings prompt prospective evaluation of the effects of soy-based infant formula on disease comorbidities in fragile X syndrome, a rare disorder for which newborn screening could be implemented if there was an intervention. Gastrointestinal problems were the most common reason cited for switching to soy-based infant formula. Thus, these findings also support the study of early gastrointestinal problems in fragile X syndrome, which may underly the development and severity of disease comorbidities. In conjunction with comorbidity data from the previous analyses of the Fragile X Syndrome Nutrition Study, the findings indicate that premutation fragile X mothers should be encouraged to breastfeed.

Testing Fmr1 KO Phenotypes in Response to GSK3 Inhibitors: SB216763 versus AFC03127.

Glycogen synthase kinase 3 (GSK3) is a proline-directed serine-threonine kinase that is associated with several neurological disorders, including Alzheimer's disease and fragile X syndrome (FXS). We tested the efficacy of a novel GSK3 inhibitor AFC03127, which was developed by Angelini Pharma, in comparison to the metabotropic glutamate receptor 5 inhibitor 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and the GSK3 inhibitor SB216763 in in vivo and in vitro assays in Fmr1 KO mice, a mouse model useful for the study of FXS. The in vivo assay tested susceptibility to audiogenic-induced seizures (AGS) whereas the in vitro assays assessed biomarker expression and dendritic spine length and density in cultured primary neurons as a function of drug dose. MPEP and SB216763 attenuated AGS in Fmr1 KO mice, whereas AFC03127 did not. MPEP and AFC03127 significantly reduced dendritic expression of amyloid-beta protein precursor (APP). All drugs rescued spine length and the ratio of mature dendritic spines. Spine density was not statistically different between vehicle and GSK3 inhibitor-treated cells. The drugs were tested over a wide concentration range in the in vitro assays to determine dose responses. A bell-shaped dose response decrease in APP expression was observed in response to AFC03127, which was more effective than SB216763. These findings confirm previous studies demonstrating differential effects of various GSK3 inhibitors on AGS propensity in Fmr1 KO mice and confirm APP as a downstream biomarker that is responsive to GSK3 activity.

FMRP mediates mGluR5-dependent translation of amyloid precursor protein.

Amyloid precursor protein (APP) facilitates synapse formation in the developing brain, while beta-amyloid (Abeta) accumulation, which is associated with Alzheimer disease, results in synaptic loss and impaired neurotransmission. Fragile X mental retardation protein (FMRP) is a cytoplasmic mRNA binding protein whose expression is lost in fragile X syndrome. Here we show that FMRP binds to the coding region of APP mRNA at a guanine-rich, G-quartet-like sequence. Stimulation of cortical synaptoneurosomes or primary neuronal cells with the metabotropic glutamate receptor agonist DHPG increased APP translation in wild-type but not fmr-1 knockout samples. APP mRNA coimmunoprecipitated with FMRP in resting synaptoneurosomes, but the interaction was lost shortly after DHPG treatment. Soluble Abeta40 or Abeta42 levels were significantly higher in multiple strains of fmr-1 knockout mice compared to wild-type controls. Our data indicate that postsynaptic FMRP binds to and regulates the translation of APP mRNA through metabotropic glutamate receptor activation and suggests a possible link between Alzheimer disease and fragile X syndrome.

Reply to "The Fallacy of Using Administrative Data in Assessing the Effectiveness of Food Fortification. Comment on: Folic Acid Fortification and Neural Tube Defect Risk: Analysis of the Food Fortification Initiative Dataset. Nutrients 2020, 12, 247".

We would like to thank Kancherla et al [...].

Folic Acid Fortification and Neural Tube Defect Risk: Analysis of the Food Fortification Initiative Dataset.

The United States implemented mandatory fortification of cereal grains with folic acid in 1998 to prevent neural tube defects (NTDs) during pregnancy. The health benefits of folate (vitamin B9) are well documented; however, there are potential risks of exceeding the upper tolerable limit, particularly in vulnerable populations. We conducted a population-based analysis of the Food Fortification Initiative dataset to determine the strength of the evidence regarding reports of decreased NTDs at the national level in response to mandatory folic acid fortification of cereal grains. We found a very weak correlation between NTD prevalence and the level of folic acid fortification, irrespective of the cereal grain fortified (wheat, maize or rice). Stratification of the data based on socioeconomic status (SES) indicated a strong linear relationship between reduced NTDs and better SES. We conclude that national fortification with folic acid is not associated with a significant decrease in the prevalence of NTDs at the population level.