Biomarkers of mitochondrial dysfunction in autism spectrum disorder: A systematic review and meta-analysis.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting 1 in 36 children and is associated with physiological abnormalities, most notably mitochondrial dysfunction, at least in a subset of individuals. This systematic review and meta-analysis discovered 204 relevant articles which evaluated biomarkers of mitochondrial dysfunction in ASD individuals. Significant elevations (all p < 0.01) in the prevalence of lactate (17%), pyruvate (41%), alanine (15%) and creatine kinase (9%) were found in ASD. Individuals with ASD had significant differences (all p < 0.01) with moderate to large effect sizes (Cohen's d' ≥ 0.6) compared to controls in mean pyruvate, lactate-to-pyruvate ratio, ATP, and creatine kinase. Some studies found abnormal TCA cycle metabolites associated with ASD. Thirteen controlled studies reported mitochondrial DNA (mtDNA) deletions or variations in the ASD group in blood, peripheral blood mononuclear cells, lymphocytes, leucocytes, granulocytes, and brain. Meta-analyses discovered significant differences (p < 0.01) in copy number of mtDNA overall and in ND1, ND4 and CytB genes. Four studies linked specific mtDNA haplogroups to ASD. A series of studies found a subgroup of ASD with elevated mitochondrial respiration which was associated with increased sensitivity of the mitochondria to physiological stressors and neurodevelopmental regression. Lactate, pyruvate, lactate-to-pyruvate ratio, carnitine, and acyl-carnitines were associated with clinical features such as delays in language, social interaction, cognition, motor skills, and with repetitive behaviors and gastrointestinal symptoms, although not all studies found an association. Lactate, carnitine, acyl-carnitines, ATP, CoQ10, as well as mtDNA variants, heteroplasmy, haplogroups and copy number were associated with ASD severity. Variability was found across biomarker studies primarily due to differences in collection and processing techniques as well as the intrinsic heterogeneity of the ASD population. Several studies reported alterations in mitochondrial metabolism in mothers of children with ASD and in neonates who develop ASD. Treatments targeting mitochondria, particularly carnitine and ubiquinol, appear beneficial in ASD. The link between mitochondrial dysfunction in ASD and common physiological abnormalities in individuals with ASD including gastrointestinal disorders, oxidative stress, and immune dysfunction is outlined. Several subtypes of mitochondrial dysfunction in ASD are discussed, including one related to neurodevelopmental regression, another related to alterations in microbiome metabolites, and another related to elevations in acyl-carnitines. Mechanisms linking abnormal mitochondrial function with alterations in prenatal brain development and postnatal brain function are outlined. Given the multisystem complexity of some individuals with ASD, this review presents evidence for the mitochondria being central to ASD by contributing to abnormalities in brain development, cognition, and comorbidities such as immune and gastrointestinal dysfunction as well as neurodevelopmental regression. A diagnostic approach to identify mitochondrial dysfunction in ASD is outlined. From this evidence, it is clear that many individuals with ASD have alterations in mitochondrial function which may need to be addressed in order to achieve optimal clinical outcomes. The fact that alterations in mitochondrial metabolism may be found during pregnancy and early in the life of individuals who eventually develop ASD provides promise for early life predictive biomarkers of ASD. Further studies may improve the understanding of the role of the mitochondria in ASD by better defining subgroups and understanding the molecular mechanisms driving some of the unique changes found in mitochondrial function in those with ASD.

Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells.

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology.

Bioenergetic signatures of neurodevelopmental regression.

Background: Studies have linked autism spectrum disorder (ASD) to physiological abnormalities including mitochondrial dysfunction. Mitochondrial dysfunction may be linked to a subset of children with ASD who have neurodevelopmental regression (NDR). We have developed a cell model of ASD which demonstrates a unique mitochondrial profile with mitochondrial respiration higher than normal and sensitive to physiological stress. We have previously shown similar mitochondrial profiles in individuals with ASD and NDR. Methods: Twenty-six ASD individuals without a history of NDR (ASD-NoNDR) and 15 ASD individuals with a history of NDR (ASD-NDR) were recruited from 34 families. From these families, 30 mothers, 17 fathers and 5 typically developing (TD) siblings participated. Mitochondrial respiration was measured in peripheral blood mononuclear cells (PBMCs) with the Seahorse 96 XF Analyzer. PBMCs were exposed to various levels of physiological stress for 1 h prior to the assay using 2,3-dimethoxy-1,4-napthoquinone. Results: ASD-NDR children were found to have higher respiratory rates with mitochondria that were more sensitive to physiological stress as compared to ASD-NoNDR children, similar to our cellular model of NDR. Differences in mitochondrial respiration between ASD-NDR and TD siblings were similar to the differences between ASD-NDR and ASD-NoNDR children. Interesting, parents of children with ASD and NDR demonstrated patterns of mitochondrial respiration similar to their children such that parents of children with ASD and NDR demonstrated elevated respiratory rates with mitochondria that were more sensitive to physiological stress. In addition, sex differences were seen in ASD children and parents. Age effects in parents suggested that mitochondria of older parents were more sensitive to physiological stress. Conclusion: This study provides further evidence that children with ASD and NDR may have a unique type of mitochondrial physiology that may make them susceptible to physiological stressors. Identifying these children early in life before NDR occurs and providing treatment to protect mitochondrial physiology may protect children from experiencing NDR. The fact that parents also demonstrate mitochondrial respiration patterns similar to their children implies that this unique change in mitochondrial physiology may be a heritable factor (genetic or epigenetic), a result of shared environment, or both.

Nitrosative Stress in Autism: Supportive Evidence and Implications for Mitochondrial Dysfunction.

A recent study by the Amal team published in this journal in May 2023 proved for the first time the link of nitric oxide (NO) with autism spectrum disorder (ASD), thereby opening new venues for the potential use of neuronal nitric oxide synthase (nNOS) inhibitors as therapeutics for improving the neurological and behavioral symptoms of ASD. The authors conclude that their findings demonstrate that NO plays a significant role in ASD. Indeed, earlier studies support elevated NO and its metabolites, nitrite, and peroxynitrite, in individuals diagnosed with ASD. Dysregulated NOS activity may underlie the well-documented mitochondrial dysfunction in a subset of individuals with ASD. Strategies for treating ASD shall also consider NO effects on mitochondrial respiration in modulating NOS activity. Further experimental evidence and controlled clinical trials with NOS modifiers are required for assessing their therapeutic potential for individuals with ASD.

Folate Receptor Alpha Autoantibodies in the Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS) and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) Population.

The folate receptor alpha autoantibodies (FRAAs) are associated with cerebral folate deficiency (CFD) and autism spectrum disorder (ASD). Both of these syndromes have overlapping characteristics with Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) and Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS). Thus, we propose that the FRAAs may contribute to the symptomatology of PANS/PANDAS. To test this hypothesis, 1 mL of serum from 47 patients (age range = 6-18 years old) clinically diagnosed with PANS/PANDAS was sent to Vascular Strategies (Plymouth Meeting, PA, USA) for analysis of FRAAs. Moreover, 63.8% of PANS/PANDAS patients (male = 15; female = 15) were found to have either the blocking and/or blinding FRAAs, with 25 (83.3%; male = 14; female = 11) having binding FRAAs, two (6.7%; all female = 2) having blocking FRAAs, and 3 (10%; male = 1; female = 2) having both binding and blocking. Furthermore, surprisingly, ASD was associated with a 0.76 lower binding titer (p = 0.02), and severe tics were associated with a 0.90 higher binding titer (p = 0.01). A case of a FRAA-positive patient is provided to illustrate that a treatment plan including leucovorin can result in symptom improvement in patients with PANS/PANDAS who are FRAA-positive. These data, for the first time, demonstrate that PANS/PANDAS is associated with FRAAs and suggest folate metabolism abnormalities may contribute to PANS/PANDAS symptomatology. Further studies investigating the therapeutic nature of leucovorin in the treatment of PANS/PANDAS are needed. Such studies may open up an alternative, safe, and well-tolerated treatment for those with the PANS/PANDAS diagnosis.

Reanalysis of Trio Whole-Genome Sequencing Data Doubles the Yield in Autism Spectrum Disorder: De Novo Variants Present in Half.

Autism spectrum disorder (ASD) is a common condition with lifelong implications. The last decade has seen dramatic improvements in DNA sequencing and related bioinformatics and databases. We analyzed the raw DNA sequencing files on the Variantyx® bioinformatics platform for the last 50 ASD patients evaluated with trio whole-genome sequencing (trio-WGS). "Qualified" variants were defined as coding, rare, and evolutionarily conserved. Primary Diagnostic Variants (PDV), additionally, were present in genes directly linked to ASD and matched clinical correlation. A PDV was identified in 34/50 (68%) of cases, including 25 (50%) cases with heterozygous de novo and 10 (20%) with inherited variants. De novo variants in genes directly associated with ASD were far more likely to be Qualifying than non-Qualifying versus a control group of genes (p = 0.0002), validating that most are indeed disease related. Sequence reanalysis increased diagnostic yield from 28% to 68%, mostly through inclusion of de novo PDVs in genes not yet reported as ASD associated. Thirty-three subjects (66%) had treatment recommendation(s) based on DNA analyses. Our results demonstrate a high yield of trio-WGS for revealing molecular diagnoses in ASD, which is greatly enhanced by reanalyzing DNA sequencing files. In contrast to previous reports, de novo variants dominate the findings, mostly representing novel conditions. This has implications to the cause and rising prevalence of autism.

Binding Folate Receptor Alpha Autoantibody Is a Biomarker for Leucovorin Treatment Response in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) affects up to 1 in 36 children in the United States. It is a heterogeneous neurodevelopmental disorder with life-long consequences. Patients with ASD and folate pathway abnormalities have demonstrated improved symptoms after treatment with leucovorin (folinic acid), a reduced form of folate. However, biomarkers for treatment response have not been well investigated and clinical trials are lacking. In this retrospective analysis, a cohort of prospectively collected data from 110 consecutive ASD clinic patients [mean (SD) age: 10.5 (6.2) years; 74% male] was examined. These patients all underwent testing for folate receptor alpha autoantibodies (FRAAs) and soluble folate binding proteins (sFBPs) biomarkers and were treated with leucovorin, if appropriate. Analyses examined whether these biomarkers could predict response to leucovorin treatment as well as the severity of ASD characteristics at baseline. The social responsiveness scale (SRS), a measure of core ASD symptoms, and the aberrant behavior checklist (ABC), a measure of disruptive behavior, were collected at each clinic visit. Those positive for sFBPs had more severe ASD symptoms, and higher binding FRAA titers were associated with greater ABC irritability. Treatment with leucovorin improved most SRS subscales with higher binding FRAA titers associated with greater response. Leucovorin treatment also improved ABC irritability. These results confirm and expand on previous studies, underscore the need for biomarkers to guide treatment of folate pathways in ASD, and suggest that leucovorin may be effective for children with ASD.

Editorial: Mitochondrial Gene Variations Increase Autism Risk: Uncovering the Complex Polygenetic Landscape of Autism.

Autism spectrum disorder (ASD) is a behaviorally defined disorder with a complex, mostly unknown, etiology. Although many neurodevelopmental genetic disorders are associated with ASD, single gene mutations and copy number variations do not account for the majority of ASD cases. In fact, when found, genetic alterations are usually de novo rather than inherited.1 Scientists are starting to consider polygenetic influences in the etiology of ASD whereby changes in multiple genes might add up to a threshold that disrupts cellular pathways. Further studies have implicated many environmental factors, particularly the prenatal maternal environment, suggesting that many cases of ASD might be associated with complex interactions between polygenetic predisposition and environmental factors.2.

Quantitative assessment of mitochondrial morphology relevant for studies on cellular health and environmental toxicity.

Mitochondria are essential organelles that play crucial roles in cellular energy metabolism, calcium signaling and apoptosis. Their importance in tissue homeostasis and stress responses, combined to their ability to transition between various structural and functional states, make them excellent organelles for monitoring cellular health. Quantitative assessment of mitochondrial morphology can therefore provide valuable insights into environmentally-induced cell damage. High-content screening (HCS) provides a powerful tool for analyzing organelles and cellular substructures. We developed a fully automated and miniaturized HCS wet-plus-dry pipeline (MITOMATICS) exploiting mitochondrial morphology as a marker for monitoring cellular health or damage. MITOMATICS uses an in-house, proprietary software (MitoRadar) to enable fast, exhaustive and cost-effective analysis of mitochondrial morphology and its inherent diversity in live cells. We applied our pipeline and big data analytics software to assess the mitotoxicity of selected chemicals, using the mitochondrial uncoupler CCCP as an internal control. Six different pesticides (inhibiting complexes I, II and III of the mitochondrial respiratory chain) were tested as individual compounds and five other pesticides present locally in Occitanie (Southern France) were assessed in combination to determine acute mitotoxicity. Our results show that the assayed pesticides exhibit specific signatures when used as single compounds or chemical mixtures and that they function synergistically to impact mitochondrial architecture. Study of environment-induced mitochondrial damage has the potential to open new fields in mechanistic toxicology, currently underexplored by regulatory toxicology and exposome research. Such exploration could inform health policy guidelines and foster pharmacological intervention, water, air and soil pollution control and food safety.

E2F1 Mediates SOX17 Deficiency-Induced Pulmonary Hypertension.

BACKGROUND: Rare genetic variants and genetic variation at loci in an enhancer in SOX17 (SRY-box transcription factor 17) are identified in patients with idiopathic pulmonary arterial hypertension (PAH) and PAH with congenital heart disease. However, the exact role of genetic variants or mutations in SOX17 in PAH pathogenesis has not been reported. METHODS: SOX17 expression was evaluated in the lungs and pulmonary endothelial cells (ECs) of patients with idiopathic PAH. Mice with Tie2Cre-mediated Sox17 knockdown and EC-specific Sox17 deletion were generated to determine the role of SOX17 deficiency in the pathogenesis of PAH. Human pulmonary ECs were cultured to understand the role of SOX17 deficiency. Single-cell RNA sequencing, RNA-sequencing analysis, and luciferase assay were performed to understand the underlying molecular mechanisms of SOX17 deficiency-induced PAH. E2F1 (E2F transcription factor 1) inhibitor HLM006474 was used in EC-specific Sox17 mice. RESULTS: SOX17 expression was downregulated in the lung and pulmonary ECs from patients with idiopathic PAH. Mice with Tie2Cre-mediated Sox17 knockdown and EC-specific Sox17 deletion induced spontaneously mild pulmonary hypertension. Loss of endothelial Sox17 in EC exacerbated hypoxia-induced pulmonary hypertension in mice. Loss of SOX17 in lung ECs induced endothelial dysfunctions including upregulation of cell cycle programming, proliferative and antiapoptotic phenotypes, augmentation of paracrine effect on pulmonary arterial smooth muscle cells, impaired cellular junction, and BMP (bone morphogenetic protein) signaling. E2F1 signaling was shown to mediate the SOX17 deficiency-induced EC dysfunction. Pharmacological inhibition of E2F1 in Sox17 EC-deficient mice attenuated pulmonary hypertension development. CONCLUSIONS: Our study demonstrated that endothelial SOX17 deficiency induces pulmonary hypertension through E2F1. Thus, targeting E2F1 signaling represents a promising approach in patients with PAH.

Postinfectious Inflammation, Autoimmunity, and Obsessive-Compulsive Disorder: Sydenham Chorea, Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcal Infection, and Pediatric Acute-Onset Neuropsychiatric Disorder.

Postinfectious neuroinflammation has been implicated in multiple models of acute-onset obsessive-compulsive disorder including Sydenham chorea (SC), pediatric acute-onset neuropsychiatric syndrome (PANS), and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS). These conditions are associated with a range of autoantibodies which are thought to be triggered by infections, most notably group A streptococci (GAS). Based on animal models using huma sera, these autoantibodies are thought to cross-react with neural antigens in the basal ganglia and modulate neuronal activity and behavior. As is true for many childhood neuroinflammatory diseases and rheumatological diseases, SC, PANS, and PANDAS lack clinically available, rigorous diagnostic biomarkers and randomized clinical trials. In this review article, we outline the accumulating evidence supporting the role neuroinflammation plays in these disorders. We describe work with animal models including patient-derived anti-neuronal autoantibodies, and we outline imaging studies that show alterations in the basal ganglia. In addition, we present research on metabolites, which are helpful in deciphering functional phenotypes, and on the implication of sleep in these disorders. Finally, we encourage future researchers to collaborate across medical specialties (e.g., pediatrics, psychiatry, rheumatology, immunology, and infectious disease) in order to further research on clinical syndromes presenting with neuropsychiatric manifestations.

Synchrony 2022: The Role of Neuroinflammation in Behavioral Exacerbations in Autism Spectrum Disorder.

The BRAIN Foundation (Pleasanton, CA) hosted Synchrony 2022, a medical conference focusing on research for treatments to benefit individuals with neurodevelopmental disorders (NDD), including those with autism spectrum disorders (ASD) [...].

Age-Related Changes in Epilepsy Characteristics and Response to Antiepileptic Treatment in Autism Spectrum Disorders.

Despite the high prevalence of epilepsy in individuals with autism spectrum disorder (ASD), there is little information regarding whether seizure characteristics and treatment effectiveness change across age. Using an online survey, seizure characteristics, effectiveness of antiepileptic treatments, comorbidities, potential etiologies, and ASD diagnosis were collected from individuals with ASD and seizures. We previously reported overall general patterns of treatment effectiveness but did not examine the effect of seizure characteristics or age on antiepileptic treatment effectiveness. Such information would improve the personalized medicine approach to the treatment of seizures in ASD. Survey data from 570 individuals with ASD and clinical seizures were analyzed. Seizure severity (seizure/week) decreased with age of onset of seizures, plateauing in adolescence, with a greater reduction in generalized tonic-clonic (GTC) seizures with age. Seizure severity was worse in those with genetic disorders, neurodevelopmental regression (NDR) and poor sleep maintenance. Carbamazepine and oxcarbazepine were reported to be more effective when seizures started in later childhood, while surgery and the Atkins/modified Atkins Diet (A/MAD) were reported to be more effective when seizures started early in life. A/MAD and the ketogenic diet were reported to be more effective in those with NDR. Interestingly, atypical Landau-Kleffner syndrome was associated with mitochondrial dysfunction and NDR, suggesting a novel syndrome. These interesting findings need to be verified in independent, prospectively collected cohorts, but nonetheless, these data provide insights into novel relationships that may assist in a better understanding of epilepsy in ASD and provide insight into personalizing epilepsy care in ASD.

Resting State EEG Correlates of Suicide Ideation and Suicide Attempt.

Suicide is a global phenomenon that impacts individuals, families, and communities from all income groups and all regions worldwide. While it can be prevented if personalized interventions are implemented, more objective and reliable diagnostic methods are needed to complement interview-based risk assessments. In this context, electroencephalography (EEG) might play a key role. We systematically reviewed EEG resting state studies of adults with suicide ideation (SI) or with a history of suicide attempts (SAs). After searching for relevant studies using the PubMed and Web of Science databases, we applied the PRISMA method to exclude duplicates and studies that did not match our inclusion criteria. The selection process yielded seven studies, which suggest that imbalances in frontal and left temporal brain regions might reflect abnormal activation and correlate with psychological distress. Furthermore, asymmetrical activation in frontal and posterior cortical regions was detected in high-risk depressed persons, although the pattern in the frontal region was inverted in non-depressed persons. The literature reviewed suggests that SI and SA may be driven by separate neural circuits and that high-risk persons can be found within non-depressed populations. More research is needed to develop intelligent algorithms for the automated detection of high-risk EEG anomalies in the general population.

Synchrony 2022: Roundtable Discussion on the Pathways and the Challenges of Getting Medications FDA Approved.

The BRAIN Foundation (Pleasanton, CA, USA) hosted a medicine conference, Synchrony 2022, for research into treatments to benefit individuals with neurodevelopmental disorders (NDDs), including autism spectrum disorders (ASD) [...].

Biochemical, Genetic and Clinical Diagnostic Approaches to Autism-Associated Inherited Metabolic Disorders.

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders characterized by impaired social interaction, limited communication skills, and restrictive and repetitive behaviours. The pathophysiology of ASD is multifactorial and includes genetic, epigenetic, and environmental factors, whereas a causal relationship has been described between ASD and inherited metabolic disorders (IMDs). This review describes biochemical, genetic, and clinical approaches to investigating IMDs associated with ASD. The biochemical work-up includes body fluid analysis to confirm general metabolic and/or lysosomal storage diseases, while the advances and applications of genomic testing technology would assist with identifying molecular defects. An IMD is considered likely underlying pathophysiology in ASD patients with suggestive clinical symptoms and multiorgan involvement, of which early recognition and treatment increase their likelihood of achieving optimal care and a better quality of life.

Deciduous tooth biomarkers reveal atypical fetal inflammatory regulation in autism spectrum disorder.

Atypical regulation of inflammation has been proposed in the etiology of autism spectrum disorder (ASD); however, measuring the temporal profile of fetal inflammation associated with future ASD diagnosis has not been possible. Here, we present a method to generate approximately daily profiles of prenatal and early childhood inflammation as measured by developmentally archived C-reactive protein (CRP) in incremental layers of deciduous tooth dentin. In our discovery population, a group of Swedish twins, we found heightened inflammation in the third trimester in children with future ASD diagnosis relative to controls (n = 66; 14 ASD cases; critical window: -90 to -50 days before birth). In our replication study, in the US, we observed a similar increase in CRP in ASD cases during the third trimester (n = 47; 23 ASD cases; -128 to -21 days before birth). Our results indicate that the third trimester is a critical period of atypical fetal inflammatory regulation in ASD.

Synchrony 2022: Catalyzing Research and Treatments to Benefit Individuals with Neurodevelopmental Disorders including Autism Spectrum Disorders.

A unique translational medicine conference for research into treatments that can benefit individuals with neurodevelopmental disorders (NDD), including autism spectrum disorder (ASD), has been developed and hosted by The BRAIN Foundation (Pleasanton, CA, USA) since 2019 [...].

Synchrony 2022: Epilepsy and Seizures in Autism Spectrum Disorder Roundtable.

The BRAIN Foundation (Pleasanton, CA, USA) hosted Synchrony 2022, a translational medicine conference focused on research into treatments for individuals with neurodevelopmental disorders (NDD), including those with autism spectrum disorders (ASD) [...].