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.

Nerve to subclavius for neurotization procedures: an anatomical feasibility study.

Injuries to the inferior trunk of the brachial plexus and its components can be debilitating. As such injuries are prioritized by surgeons during repair, an additional nerve transfer is considered herein. In the supine position, 13 adult cadavers (26 sides) underwent dissection of the nerve to subclavius and the proximal brachial plexus in the supraclavicular region. Once the nerve was located and isolated from origin to termination, measurements of its length and diameter were made. Next, the C8 and T1 roots and inferior trunk were identified. The distal end of the nerve to subclavius was transected and swung to these roots and the inferior trunk. Once the nerves to subclavius were transposed and in a tension-free manner, the length of excess nerve following being brought to each of these nerves was measured. A nerve to subclavius was identified on all sides. The nerve originated from the superior trunk and traveled anterior to the middle and inferior trunks on all sides. The mean diameter of the nerve to subclavius was 0.8 mm, and the mean length was 57 mm. After cutting the nerve to subclavius at its entrance into the subclavius muscle, the distal nerve could be transferred tension free to the inferior trunk of the brachial plexus and T1 ventral ramus on all sides. The distal nerve to subclavius reached the T1 ventral ramus with an average of 18 mm of additional length and to the C8 ventral ramus with an average of 19 mm. The nerve also could be transferred to the inferior trunk of the brachial plexus with an average of 20 mm of additional length. The nerve to subclavius was found to have approximately 3000 axons. To our knowledge, use of the nerve to subclavius has previously not been used for nerve transfer procedures. Based on our cadaveric study, this often-overlooked nerve can be easily transposed to other regional nerves such as the inferior trunk of the brachial plexus.

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.

Clinical, radiological, and laboratory factors associated with mortality and functional outcomes in pediatric patients presenting with intracranial gunshot wounds.

OBJECTIVE: Firearm injuries are now the leading cause of death in children and young adults younger than 25 years of age in the US. Current management of these injuries is extrapolated from adult blunt and penetrating traumatic brain injury guidelines. The objectives of this study were to investigate and analyze the clinical, radiological, and laboratory factors associated with mortality and functional outcomes in pediatric patients presenting with intracranial gunshot wounds (GSWs). METHODS: Medical records were screened for all patients younger than 21 years of age with an intracranial GSW who presented to the University Medical Center in New Orleans, Louisiana, from 2012 to 2022. Demographics and radiological, clinical, and laboratory data were extracted, and chi-square and Fisher's exact tests were used to evaluate individual association with mortality and functional outcome. Odds ratios were calculated from the cross tabulations for categorical variables and univariate binary logistic regression models for continuous variables. Multivariate binary logistic regression was used to adjust for effects of covariates and isolate the contributions of predictor variables for mortality and functional outcome. RESULTS: Ninety-six patients (82 male, 14 female) had a median age of 18 (interquartile range [IQR] 15-20) years. The 30-day inpatient, 60-day, and 6-month mortality rates among these patients were 53.1%, 0%, and 2.4%, respectively. Those who died were more likely to have an initial Glasgow Coma Scale score ≤ 8 (p < 0.001), bilateral fixed pupils (p < 0.001), transventricular trajectory (p < 0.001), deep nuclear/third ventricle involvement (p = 0.004), bihemispheric trajectory (p = 0.025), injury to ≥ 3 lobes (p = 0.015), parietal lobe involvement (p = 0.023), base deficit < -5 mEq/L (p = 0.013), international normalized ratio (INR) > 1.5 (p = 0.007), and a St. Louis Scale (SLS) score ≥ 5 (p < 0.001). The survivors with favorable functional outcome were more likely to have lower median SLS scores (p = 0.016) and injury to < 3 lobes (p < 0.001). In a multivariate analysis, bilaterally fixed nonreactive pupils were positively associated with mortality and negatively associated with favorable functional outcome, whereas the Injury Severity Score (ISS) and injury to ≥ 3 lobes were negatively associated with favorable functional outcome only. CONCLUSIONS: This is one of the largest series of pediatric intracranial GSWs to date. The authors identified certain clinical (bilateral fixed pupils, SLS score ≥ 5, ISS > 16), laboratory (INR > 1.5, base deficit < -5 mEq/L), and radiological (transventricular trajectory, deep nuclear/third ventricle involvement, parietal lobe involvement) factors that were associated with death and poor functional outcome in this pediatric cohort.

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.

Time dependent changes in the bioenergetics of peripheral blood mononuclear cells: processing time, collection tubes and cryopreservation effects.

OBJECTIVES: Bioenergetic measurements in peripheral blood mononuclear cells (PBMCs) using high-throughput respirometry is a promising minimally invasive approach to studying mitochondrial function in humans. However, optimal methods for collecting PBMCs are not well studied. METHODS: Bioenergetics and viability were measured across processing delays, tube type and cryopreservation. RESULTS: Storage of collection tubes on dry ice resulted in unrecoverable samples and using the Cell Preparation Tube (CPTTM) significantly reduced viability. Thus, storage in Sodium Citrate (NaC) and ethylenediaminetetraacetic acid (EDTA) tubes were studied in detail. Cell viability decreased by 0.5% for each hour the samples remained on wet ice prior to processing while cryopreservation decreased viability by 9.6% with viability remaining stable for about one month in liquid nitrogen. Adenosine triphosphate linked respiration (ALR) and proton-leak respiration (PLR) changed minimally while maximal respiratory capacity (MRC) and reserve capacity (RC) decreased markedly with collection tubes stored on wet ice over 24 hrs. Changes in respiratory parameters were more modest over the first 8 hours. Manipulations to replace media did not attenuate changes in respiratory parameters. Cryopreservation decreased ALR, MRC and RC by 17.20, 95.30 and 54.92 pmol/min, respectively and increased PLR by 2.65 pmol/min. PLR, MRC and RC changed moderately during the first month in liquid nitrogen for freshly frozen PBMCs. CONCLUSIONS: Our results suggest that bioenergetics in PBMCs vary based on the processing time from specimen collection and preservation method. Changes in bioenergetics can be minimized by processing samples with a minimal time delay. Changes in viability are minimal and may not correspond to changes in bioenergetics.

The Soluble Folate Receptor in Autism Spectrum Disorder: Relation to Autism Severity and Leucovorin Treatment.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder with life-long consequences that affects up to 1 in 44 children. Treatment with leucovorin (folinic acid), a reduced form of folate, has been shown to improve symptoms in those with ASD and folate pathway abnormalities in controlled clinical trials. Although soluble folate binding proteins (sFBPs) have been observed in the serum of some patients with ASD, the significance of this finding has not been studied. Here, we present a cohort of ASD patients with sFBPs. These patients had severe ASD and were medically complex. Using baseline controlled open-label methodology and standardized assessments, these patients were found to improve in both core and associated ASD symptoms with leucovorin treatment. No adverse effects were related to leucovorin treatment. This is the first report of the sFBPs in ASD. This study complements ongoing controlled clinical trials and suggests that leucovorin may be effective for children with ASD who are positive for sFBPs. Further, sFBPs might be important biomarkers for treatment response to leucovorin in children with ASD. This study paves the way for further controlled studies for patients with sFBPs.

Air Pollution and Maximum Temperature Are Associated with Neurodevelopmental Regressive Events in Autism Spectrum Disorder.

Neurodevelopmental regression (NDR) is an enigmatic event associated with autism spectrum disorder (ASD) during which a child loses previously acquired skills and develops ASD symptoms. In some, a trigger which precedes the NDR event, such as a fever, can be identified, but in many cases no trigger is obvious. We hypothesize that air pollution (PM2.5) may trigger NDR, especially in those children without an identified trigger. Average daily PM2.5, ozone, precipitation and maximum temperature (Tmax) were derived from Environmental Protection Agency models and National Oceanic and Atmospheric Administration monitors based on zip-code information from 83 ASD participants during the six-weeks following the onset month of an NDR event and a reference period defined as one year before and one year after the event. Seasonally adjusted logistic regression (LR) and linear mixed models (LMM) compared cases (with a history of NDR) and matched controls (without a history of NDR). LR models found that the risk of NDR was related to higher PM2.5 during 3 to 6 weeks of the NDR event period, particularly in those without a trigger. Overall, both models converged on NDR being related to a higher PM2.5 and lower Tmax both during the NDR event period as well as the reference period, particularly in those without a known trigger. This temporal pattern suggests that environmental triggers, particularly PM2.5, could be related to NDR, especially in those without an identifiable trigger. Further studies to determine the underlying biological mechanism of this observation could help better understand NDR and provide opportunities to prevent NDR.

Central Nervous System Metabolism in Autism, Epilepsy and Developmental Delays: A Cerebrospinal Fluid Analysis.

Neurodevelopmental disorders are associated with metabolic pathway imbalances; however, most metabolic measurements are made peripherally, leaving central metabolic disturbances under-investigated. Cerebrospinal fluid obtained intraoperatively from children with autism spectrum disorder (ASD, n = 34), developmental delays (DD, n = 20), and those without known DD/ASD (n = 34) was analyzed using large-scale targeted mass spectrometry. Eighteen also had epilepsy (EPI). Metabolites significantly related to ASD, DD and EPI were identified by linear models and entered into metabolite-metabolite network pathway analysis. Common disrupted pathways were analyzed for each group of interest. Central metabolites most involved in metabolic pathways were L-cysteine, adenine, and dodecanoic acid for ASD; nicotinamide adenine dinucleotide phosphate, L-aspartic acid, and glycine for EPI; and adenosine triphosphate, L-glutamine, ornithine, L-arginine, L-lysine, citrulline, and L-homoserine for DD. Amino acid and energy metabolism pathways were most disrupted in all disorders, but the source of the disruption was different for each disorder. Disruption in vitamin and one-carbon metabolism was associated with DD and EPI, lipid pathway disruption was associated with EPI and redox metabolism disruption was related to ASD. Two microbiome metabolites were also detected in the CSF: shikimic and cis-cis-muconic acid. Overall, this study provides increased insight into unique metabolic disruptions in distinct but overlapping neurodevelopmental disorders.

Resting State Functional Magnetic Resonance Imaging Elucidates Neurotransmitter Deficiency in Autism Spectrum Disorder.

Resting-state functional magnetic resonance imaging provides dynamic insight into the functional organization of the brains' intrinsic activity at rest. The emergence of resting-state functional magnetic resonance imaging in both the clinical and research settings may be attributed to recent advancements in statistical techniques, non-invasiveness and enhanced spatiotemporal resolution compared to other neuroimaging modalities, and the capability to identify and characterize deep brain structures and networks. In this report we describe a 16-year-old female patient with autism spectrum disorder who underwent resting-state functional magnetic resonance imaging due to late regression. Imaging revealed deactivated networks in deep brain structures involved in monoamine synthesis. Monoamine neurotransmitter deficits were confirmed by cerebrospinal fluid analysis. This case suggests that resting-state functional magnetic resonance imaging may have clinical utility as a non-invasive biomarker of central nervous system neurochemical alterations by measuring the function of neurotransmitter-driven networks. Use of this technology can accelerate and increase the accuracy of selecting appropriate therapeutic agents for patients with neurological and neurodevelopmental disorders.

Locked-in Intact Functional Networks in Children with Autism Spectrum Disorder: A Case-Control Study.

Resting-state functional magnetic resonance imaging (rs-fMRI) has the potential to investigate abnormalities in brain network structure and connectivity on an individual level in neurodevelopmental disorders, such as autism spectrum disorder (ASD), paving the way toward using this technology for a personalized, precision medicine approach to diagnosis and treatment. Using a case-control design, we compared five patients with severe regressive-type ASD to five patients with temporal lobe epilepsy (TLE) to examine the association between brain network characteristics and diagnosis. All children with ASD and TLE demonstrated intact motor, language, and frontoparietal (FP) networks. However, aberrant networks not usually seen in the typical brain were also found. These aberrant networks were located in the motor (40%), language (80%), and FP (100%) regions in children with ASD, while children with TLE only presented with aberrant networks in the motor (40%) and language (20%) regions, in addition to identified seizure onset zones. Fisher's exact test indicated a significant relationship between aberrant FP networks and diagnosis (p = 0.008), with ASD and atypical FP networks co-occurring more frequently than expected by chance. Despite severe cognitive delays, children with regressive-type ASD may demonstrate intact typical cortical network activation despite an inability to use these cognitive facilities. The functions of these intact cognitive networks may not be fully expressed, potentially because aberrant networks interfere with their long-range signaling, thus creating a unique "locked-in network" syndrome.