Molecular Biomarkers Predictive of Sertraline Treatment Response in Young Children With Autism Spectrum Disorder.

Sertraline is one among several selective serotonin reuptake inhibitors (SSRIs) that exhibited improvement of language development in Autism Spectrum Disorder (ASD); however, the molecular mechanism has not been elucidated. A double blind, randomized, 6-month, placebo-controlled, clinical trial of low-dose sertraline in children ages (3-6 years) with ASD was conducted at the UC Davis MIND Institute. It aimed at evaluating the efficacy and benefit with respect to early expressive language development and global clinical improvement. This study aimed to identify molecular biomarkers that might be key players in the serotonin pathway and might be predictive of a clinical response to sertraline. Fifty eight subjects with the diagnosis of ASD were randomized to sertraline or placebo. Eight subjects from the sertraline arm and five from the placebo arm discontinued from the study. Furthermore, four subjects did not have a successful blood draw. Hence, genotypes for 41 subjects (20 on placebo and 21 on sertraline) were determined for several genes involved in the serotonin pathway including the serotonin transporter-linked polymorphic region (5-HTTLPR), the tryptophan hydroxylase 2 (TPH2), and the Brain-Derived Neurotrophic Factor (BDNF). In addition, plasma levels of BDNF, Matrix metallopeptidase 9 (MMP-9) and a selected panel of cytokines were determined at baseline and post-treatment. Intent-to-treat analysis revealed several primary significant correlations between molecular changes and the Mullen Scales of Early Learning (MSEL) and Clinical Global Impression Scale - Improvement (CGI-I) of treatment and control groups but they were not significant after adjustment for multiple testing. Thus, sertraline showed no benefit for treatment of young children with ASD in language development or changes in molecular markers in this study. These results indicate that sertraline may not be beneficial for the treatment of children with ASD; however, further investigation of larger groups as well as longer term follow-up studies are warranted

A Drug Combination Rescues Frataxin-Dependent Neural and Cardiac Pathophysiology in FA Models

Friedreich's ataxia (FA) is an inherited multisystemic neuro- and cardio-degenerative disorder. Seventy-four clinical trials are listed for FA (including past and present), but none are considered FDA/EMA-approved therapy. To date, FA therapeutic strategies have focused along two main lines using a single-drug approach: a) increasing frataxin and b) enhancing downstream pathways, including antioxidant levels and mitochondrial function. Our novel strategy employed a combinatorial approach to screen approved compounds to determine if a combination of molecules provided an additive or synergistic benefit to FA cells and/or animal models. Eight single drug molecules were administered to FA fibroblast patient cells: nicotinamide riboside, hemin, betamethasone, resveratrol, epicatechin, histone deacetylase inhibitor 109, methylene blue, and dimethyl fumarate. We measured their individual ability to induce FXN transcription and mitochondrial biogenesis in patient cells. Single-drug testing highlighted that dimethyl fumarate and resveratrol increased these two parameters. In addition, the simultaneous administration of these two drugs was the most effective in terms of FXN mRNA and mitobiogenesis increase. Interestingly, this combination also improved mitochondrial functions and reduced reactive oxygen species in neurons and cardiomyocytes. Behavioral tests in an FA mouse model treated with dimethyl fumarate and resveratrol demonstrated improved rotarod performance. Our data suggest that dimethyl fumarate is effective as a single agent, and the addition of resveratrol provides further benefit in some assays without showing toxicity. Therefore, they could be a valuable combination to counteract FA pathophysiology. Further studies will help fully understand the potential of a combined therapeutic strategy in FA pathophysiology

Dyclonine rescues frataxin deficiency in animal models and buccal cells of patients with Friedreich's ataxia.

Inherited deficiency in the mitochondrial protein frataxin (FXN) causes the rare disease Friedreich's ataxia (FA), for which there is no successful treatment. We identified a redox deficiency in FA cells and used this to model the disease. We screened a 1600-compound library to identify existing drugs, which could be of therapeutic benefit. We identified the topical anesthetic dyclonine as protective. Dyclonine increased FXN transcript and FXN protein dose-dependently in FA cells and brains of animal models. Dyclonine also rescued FXN-dependent enzyme deficiencies in the iron-sulfur enzymes, aconitase and succinate dehydrogenase. Dyclonine induces the Nrf2 [nuclear factor (erythroid-derived 2)-like 2] transcription factor, which we show binds an upstream response element in the FXN locus. Additionally, dyclonine also inhibited the activity of histone methyltransferase G9a, known to methylate histone H3K9 to silence FA chromatin. Chronic dosing in a FA mouse model prevented a performance decline in balance beam studies. A human clinical proof-of-concept study was completed in eight FA patients dosed twice daily using a 1% dyclonine rinse for 1 week. Six of the eight patients showed an increase in buccal cell FXN levels, and fold induction was significantly correlated with disease severity. Dyclonine represents a novel therapeutic strategy that can potentially be repurposed for the treatment of FA

Urine-Derived Epithelial Cell Lines: A New Tool to Model Fragile X Syndrome (FXS)

Fragile X syndrome (FXS) is an X-linked neurodevelopmental condition associated with intellectual disability and behavioral problems due to the lack of the Fragile X mental retardation protein (FMRP), which plays a crucial role in synaptic plasticity and memory. A desirable in vitro cell model to study FXS would be one that can be generated by simple isolation and culture method from a collection of a non-invasive donor specimen. Currently, the various donor-specific cells can be isolated mainly from peripheral blood and skin biopsy. However, they are somewhat invasive methods for establishing cell lines from the primary subject material. In this study, we characterized a cost-effective and straightforward method to derive epithelial cell lines from urine samples collected from participants with FXS and healthy controls (TD). The urine-derived cells expressed epithelial cell surface markers via fluorescence-activated cell sorting (FACS). We observed inter, and the intra-tissue CGG mosaicism in the PBMCs and the urine-derived cells from participants with FXS potentially related to the observed variations in the phenotypic and clinical presentation FXS. We characterized these urine-derived epithelial cells for FMR1 mRNA and FMRP expression and observed some expression in the lines derived from full mutation mosaic participants. Further, FMRP expression was localized in the cytoplasm of the urine-derived epithelial cells of healthy controls. Deficient FMRP expression was also observed in mosaic males, while, as expected, no expression was observed in cells derived from participants with a hypermethylated full mutation

Profiling Genome-Wide DNA Methylation in Children with Autism Spectrum Disorder and in Children with Fragile X Syndrome

Autism spectrum disorder (ASD) is an early onset, developmental disorder whose genetic cause is heterogeneous and complex. In total, 70% of ASD cases are due to an unknown etiology. Among the monogenic causes of ASD, fragile X syndrome (FXS) accounts for 2–4% of ASD cases, and 60% of individuals with FXS present with ASD. Epigenetic changes, specifically DNA methylation, which modulates gene expression levels, play a significant role in the pathogenesis of both disorders. Thus, in this study, using the Human Methylation EPIC Bead Chip, we examined the global DNA methylation profiles of biological samples derived from 57 age-matched male participants (2–6 years old), including 23 subjects with ASD, 23 subjects with FXS with ASD (FXSA) and 11 typical developing (TD) children. After controlling for technical variation and white blood cell composition, using the conservatory threshold of the false discovery rate (FDR ≤ 0.05), in the three comparison groups, TD vs. AD, TD vs. FXSA and ASD vs. FXSA, we identified 156, 79 and 3100 differentially methylated sites (DMS), and 14, 13 and 263 differential methylation regions (DMRs). Interestingly, several genes differentially methylated among the three groups were among those listed in the SFARI Gene database, including the PAK2, GTF2I and FOXP1 genes important for brain development. Further, enrichment analyses identified pathways involved in several functions, including synaptic plasticity. Our preliminary study identified a significant role of altered DNA methylation in the pathology of ASD and FXS, suggesting that the characterization of a DNA methylation signature may help to unravel the pathogenicity of FXS and ASD and may help the development of an improved diagnostic classification of children with ASD and FXSA. In addition, it may pave the way for developing therapeutic interventions that could reverse the altered methylome profile in children with neurodevelopmental disorders

Profiling Genome-Wide DNA Methylation in Children with Autism Spectrum Disorder and in Children with Fragile X Syndrome

Autism spectrum disorder (ASD) is an early onset, developmental disorder whose genetic cause is heterogeneous and complex. In total, 70% of ASD cases are due to an unknown etiology. Among the monogenic causes of ASD, fragile X syndrome (FXS) accounts for 2-4% of ASD cases, and 60% of individuals with FXS present with ASD. Epigenetic changes, specifically DNA methylation, which modulates gene expression levels, play a significant role in the pathogenesis of both disorders. Thus, in this study, using the Human Methylation EPIC Bead Chip, we examined the global DNA methylation profiles of biological samples derived from 57 age-matched male participants (2-6 years old), including 23 subjects with ASD, 23 subjects with FXS with ASD (FXSA) and 11 typical developing (TD) children. After controlling for technical variation and white blood cell composition, using the conservatory threshold of the false discovery rate (FDR ≤ 0.05), in the three comparison groups, TD vs. AD, TD vs. FXSA and ASD vs. FXSA, we identified 156, 79 and 3100 differentially methylated sites (DMS), and 14, 13 and 263 differential methylation regions (DMRs). Interestingly, several genes differentially methylated among the three groups were among those listed in the SFARI Gene database, including the PAK2, GTF2I and FOXP1 genes important for brain development. Further, enrichment analyses identified pathways involved in several functions, including synaptic plasticity. Our preliminary study identified a significant role of altered DNA methylation in the pathology of ASD and FXS, suggesting that the characterization of a DNA methylation signature may help to unravel the pathogenicity of FXS and ASD and may help the development of an improved diagnostic classification of children with ASD and FXSA. In addition, it may pave the way for developing therapeutic interventions that could reverse the altered methylome profile in children with neurodevelopmental disorders

Frataxin deficiency impairs mitochondrial biogenesis in cells, mice and humans.

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by inherited deficiency of the mitochondrial protein Frataxin (FXN), which has no approved therapy and is an area in which biomarkers are needed for clinical development. Here, we investigated the consequences of FXN deficiency in patient-derived FRDA fibroblast cell models, the FRDA mouse model KIKO, and in whole blood collected from patients with FRDA. We observed decreased mitochondrial copy number in all the three FRDA models tested: cells, mice and patient blood. In addition, we observed 40% residual mitochondrial gene expression in FRDA patient blood. These deficiencies of mitochondrial biogenesis in FRDA cells and patient blood are significantly correlated with FXN expression, consistent with the idea that the decreased mitochondrial biogenesis is a consequence of FXN deficiency. The observations appear relevant to the FRDA pathophysiological mechanism, as FXN-dependent deficiency in mitochondrial biogenesis and consequent mitochondrial bioenergetic defect could contribute to the neurodegenerative process. The observations may also have translational potential, as mitochondrial biogenesis could now be followed as a clinical biomarker of FRDA as a correlate of disease severity, progression, and therapeutic effect. Also, mitochondrial copy number in blood is objective, scalar and more investigator-independent than clinical-neurological patient rating scales. Thus, FXN deficiency causes mitochondrial deficiency in FRDA cells, the KIKO mouse model, and in whole blood of patients with FRDA, and this deficiency could potentially be used in clinical trial design