Clinical chemistry tests for autism spectrum disorder (ASD) are currently unavailable. The aim of this study was to explore the diagnostic utility of proteotoxic biomarkers in plasma and urine, plasma protein glycation, oxidation, and nitration adducts, and related glycated, oxidized, and nitrated amino acids (free adducts), for the clinical diagnosis of ASD. Thirty-eight children with ASD (29 male, 9 female; age 7.6 ± 2.0 years) and 31 age-matched healthy controls (23 males, 8 females; 8.6 ± 2.0 years) were recruited for this study. Plasma protein glycation, oxidation, and nitration adducts and amino acid metabolome in plasma and urine were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Machine learning methods were then employed to explore and optimize combinations of analyte data for ASD diagnosis. We found that children with ASD had increased advanced glycation endproducts (AGEs), Nε-carboxymethyl-lysine (CML) and Nω-carboxymethylarginine (CMA), and increased oxidation damage marker, dityrosine (DT), in plasma protein, with respect to healthy controls. We also found that children with ASD had increased CMA free adduct in plasma ultrafiltrate and increased urinary excretion of oxidation free adducts, alpha-aminoadipic semialdehyde and glutamic semialdehyde. From study of renal handling of amino acids, we found that children with ASD had decreased renal clearance of arginine and CMA with respect to healthy controls. Algorithms to discriminate between ASD and healthy controls gave strong diagnostic performance with features: plasma protein AGEs—CML, CMA—and 3-deoxyglucosone-derived hydroimidazolone, and oxidative damage marker, DT. The sensitivity, specificity, and receiver operating characteristic area-under-the-curve were 92%, 84%, and 0.94, respectively. Changes in plasma AGEs were likely indicative of dysfunctional metabolism of dicarbonyl metabolite precursors of AGEs, glyoxal and 3-deoxyglucosone. DT is formed enzymatically by dual oxidase (DUOX); selective increase of DT as an oxidative damage marker implicates increased DUOX activity in ASD possibly linked to impaired gut mucosal immunity. Decreased renal clearance of arginine and CMA in ASD is indicative of increased arginine transporter activity which may be a surrogate marker of disturbance of neuronal availability of amino acids. Data driven combination of these biomarkers perturbed by proteotoxic stress, plasma protein AGEs and DT, gave diagnostic algorithms of high sensitivity and specificity for ASD.

The wide spectrum of unique needs and strengths of Autism Spectrum Disorders (ASD) is a challenge for the worldwide healthcare system. With the plethora of information from research, a common thread is required to conceptualize an exhaustive pathogenetic paradigm. The epidemiological and clinical findings in ASD cannot be explained by the traditional linear genetic model, hence the need to move towards a more fluid conception, integrating genetics, environment, and epigenetics as a whole. The embryo-fetal period and the first two years of life (the so-called ‘First 1000 Days’) are the crucial time window for neurodevelopment. In particular, the interplay and the vicious loop between immune activation, gut dysbiosis, and mitochondrial impairment/oxidative stress significantly affects neurodevelopment during pregnancy and undermines the health of ASD people throughout life. Consequently, the most effective intervention in ASD is expected by primary prevention aimed at pregnancy and at early control of the main effector molecular pathways. We will reason here on a comprehensive and exhaustive pathogenetic paradigm in ASD, viewed not just as a theoretical issue, but as a tool to provide suggestions for effective preventive strategies and personalized, dynamic (from womb to adulthood), systemic, and interdisciplinary healthcare approach.

Background: It has been established that children with Autism Spectrum Disorders (ASD) are affected by oxidative stress, the origin of which is still under investigation. In the present work, we evaluated inflammatory and pro-oxidant soluble signature in non-syndromic ASD and age-matched typically developing (TD) control children. Methods: We analyzed leukocyte gene expression of inflammatory cytokines and inflammation/oxidative-stress related molecules in 21 ASD and 20 TD children. Moreover, in another—comparable—group of non-syndromic ASD (N = 22) and TD (N = 21) children, we analyzed for the first time the protein expression of the four members of the antioxidant enzyme family of peroxiredoxins (Prx) in both erythrocyte membranes and in plasma. Results: The gene expression of IL6 and of HSP70i, a stress protein, was increased in ASD children. Moreover, gene expression of many inflammatory cytokines and inflammation/oxidative stress-related proteins correlated with clinical features, and appeared to be linked by a complex network of inter-correlations involving the Aryl Hydrocarbon Receptor signaling pathway. In addition, when the study of inter-correlations within the expression pattern of these molecules was extended to include the healthy subjects, the intrinsic physiological relationships of the inflammatory/oxidative stress network emerged. Plasma levels of Prx2 and Prx5 were remarkably increased in ASD compared to healthy controls, while no significant differences were found in red cell Prx levels. Conclusions: Previous findings reported elevated inflammatory cytokines in the plasma of ASD children, without clearly pointing to the presence of neuro-inflammation. On the other hand, the finding of microglia activation in autoptic specimens was clearly suggesting the presence of neuro-inflammation in ASD. Given the role of peroxiredoxins in the protection of brain cells against oxidative stress, the whole of our results, using peripheral data collected in living patients, support the involvement of neuro-inflammation in ASD, and generate a rational for neuro-inflammation as a possible therapeutic target and for plasma Prx5 as a novel indicator of ASD severity.

In a previous study, the whole transcriptome of the vastus lateralis muscle from sedentary elderly and from age-matched athletes with an exceptional record of high-intensity, life-long exercise training was compared—the two groups representing the two extremes on a physical activity scale. Exercise training enabled the skeletal muscle to counteract age-related sarcopenia by inducing a wide range of adaptations, sustained by the expression of protein-coding genes involved in energy handling, proteostasis, cytoskeletal organization, inflammation control, and cellular senescence. Building on the previous study, we examined here the network of non-coding RNAs participating in the orchestration of gene expression and identified differentially expressed micro- and long-non-coding RNAs and some of their possible targets and roles. Unsupervised hierarchical clustering analyses of all non-coding RNAs were able to discriminate between sedentary and trained individuals, regardless of the exercise typology. Validated targets of differentially expressed miRNA were grouped by KEGG analysis, which pointed to functional areas involved in cell cycle, cytoskeletal control, longevity, and many signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), which had been shown to be pivotal in the modulation of the effects of high-intensity, life-long exercise training. The analysis of differentially expressed long-non-coding RNAs identified transcriptional networks, involving lncRNAs, miRNAs and mRNAs, affecting processes in line with the beneficial role of exercise training.

Physical exercise is deemed the most efficient way of counteracting the age-related decline of skeletal muscle. Here we report a transcriptional study by next-generation sequencing of vastus lateralis biopsies from elderly with a life-long high-level training practice (n = 9) and from age-matched sedentary subjects (n = 5). Unsupervised mixture distribution analysis was able to correctly categorize trained and untrained subjects, whereas it failed to discriminate between individuals who underwent a prevalent endurance (n = 5) or a prevalent resistance (n = 4) training, thus showing that the training mode was not relevant for sarcopenia prevention. KEGG analysis of transcripts showed that physical exercise affected a high number of metabolic and signaling pathways, in particular those related to energy handling and mitochondrial biogenesis, where AMPK and AKT-mTOR signaling pathways are both active and balance each other, concurring to the establishment of an insulin-sensitive phenotype and to the maintenance of a functional muscle mass. Other pathways affected by exercise training increased the efficiency of the proteostatic mechanisms, consolidated the cytoskeletal organization, lowered the inflammation level, and contrasted cellular senescence. This study on extraordinary individuals who trained at high level for at least thirty years suggests that aging processes and exercise training travel the same paths in the opposite direction.