Abstract
BackgroundAutism spectrum disorder (ASD) has been associated with brain inflammation as indicated by the activation of microglia, but the triggers are not known. Extracellular vesicles (EVs) are secreted from many cells in the blood and other biological fluids and carry molecules that could influence the function of target cells. EVs have been recently implicated in several diseases, but their presence or function in ASD has not been studied.MethodsEVs were isolated from the serum of children with ASD (n = 20, 16 males and 4 females, 4–12 years old) and unrelated age and sex-matched normotypic controls (n = 8, 6 males and 2 females, 4–12 years old) using the exoEasy Qiagen kit. EVs were characterized by determining the CD9 and CD81 membrane-associated markers with Western blot analysis, while their morphology and size were assessed by transmission electron microscopy (TEM). Human microglia SV40 were cultured for 24 h and then stimulated with EVs (1 or 5 μg/mL), quantitated as total EV-associated protein, for 24 or 48 h. IL-1β secretion was measured by ELISA. The results were analyzed using the Mann-Whitney U non-parametric test, and all statistical analyses were performed using Graph Pad Prism 5.ResultsEVs were isolated and shown to be spherical structures (about 100 nm) surrounded by a membrane. Total EV-associated protein was found to be significantly increased (p = 0.02) in patients as compared to normotypic controls. EVs (5 μg/mL) isolated from the serum of patients with ASD stimulated cultured human microglia to secrete significantly more of the pro-inflammatory cytokine interleukin IL-1β (163.5 ± 13.34 pg/mL) as compared to the control (117.7 ± 3.96 pg/mL, p < 0.0001). The amount of mitochondrial DNA (mtDNA7S) contained in EVs from children with ASD was found to be increased (p = 0.046) compared to the normotypic controls.ConclusionsThese findings provide novel information that may help explain what triggers inflammation in the brain of children with ASD and could lead to novel effective treatments.
Highlights
Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by deficits in communication, as well as the presence of restricted, repetitive behaviors [1,2,3]
Serum Extracellular vesicles (EVs)-associated protein is increased in children with ASD The total protein concentration in EV-enriched serum samples was significantly higher in children with ASD (204.6 ± 10.6 μg/mL) as compared to the normotypic controls (161.4 ± 9.6 μg/mL), p = 0.02 (Fig. 2a)
The amount of Mitochondrial-specific DNA for 7S (mtDNA7S) contained in EVs from children with ASD was found to be increased (p = 0.046) compared to the normotypic controls (Fig. 2b)
Summary
Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by deficits in communication, as well as the presence of restricted, repetitive behaviors [1,2,3]. The prevalence of ASD in the USA was recently estimated to be 1 in about 59 children [4]. Abnormal microglial growth and activation indicating inflammation has been reported in the brain of patients with ASD since 2005 [12,13,14,15,16]. One trigger of microglia could be mitochondrial DNA (mtDNA), which we had previously reported to be secreted from stimulated MC [20], and shown to be increased in the serum of children with ASD [20]. Autism spectrum disorder (ASD) has been associated with brain inflammation as indicated by the activation of microglia, but the triggers are not known. EVs have been recently implicated in several diseases, but their presence or function in ASD has not been studied
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