Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by varying degrees of dysfunctional communication and social abilities, repetitive and stereotypic behaviors, as well as attention, cognitive, learning and sensory deficits [1]. The prevalence of ASD has increased impressively during the last two decades with the most current estimates being about 1/100 children [2]. In spite of numerous clues regarding the possible underlying pathophysiology, there is major disagreement among scholars as to the significance of such clues for either the pathogenesis or diagnosis of autism [1]. Moreover, there are no reliable biomarkers or effective treatment of the core symptoms [3, 4].

A number of papers have suggested that ASD may be associated with some immune dysfunction in the patients [5], or the mother during gestation [6, 7]. However, these papers do not provide support of direct relationship. Additional evidence suggests that ASD may have a neuroimmune component [8]. In particular, it was recently shown that the peptide neurotensin (NT) is significantly increased in young children with autistic disorder [9]. A number of studies reporting mitochondrial (mt) dysfunction in autism have focused on altered energy metabolism [10], and concluded that it may involve a subset of children with autism [11]. Mitochondria are the primary energy-generating organelles in eukaryotic cells, and they participate in multiple intracellular processes, including calcium buffering [12]. However, mitochondria evolved from bacteria that became symbiotic with eukaryotic cells and are typically prevented from being released extracellularly by autophagy [13].

We hypothesized that mitochondrial components, such as mtDNA may be released extracellularly early in life and induce an "autoimmune" response that may contribute to the pathogenesis of autism.

We tested serum samples from autistic patients for mtDNA (n = 20; 16 males and 4 females; mean age 3.0 ± 0.4 years) and AMA-M2 antibodies (n = 14; 11 males and 3 females; mean age 3.0 ± 0.4 years) compared to controls (n = 12; 11 males and 1 female; mean age 3 ± 1.2 years). The number of patients analyzed for AMA-M2 was smaller only because of the lack of availability of the EIA kit that would have allowed us to assay the rest of the patients.

We show that serum from young autistic children contains amount of mtDNA significantly higher for mt-CytB (p = 0.0002) and for mt-7S (p = 0.006) (Figure 1A and 2B). Linear regression shows an excellent correlation (R^2 = 0.89) between mt-CytB and 7S (Figure 1C. No presence of GAPDH DNA was detected indicating there was no genomic DNA release.

Serum of autistic patients also contains AMA-M2 antibodies (p = 0.001) compared to unrelated, normal controls (Figure 2). However, there was no correlation between mt-CytB DNA and AMA-M2 level (Figure 2B).

It is obvious from the scattergrams that the values corresponding to the autistic patients seem to segregate in 2 groups, indicating the possible presence of different endophenotypes. However, nothing in the clinical presentation of these children allowed for obvious separation of the subjects in subgroups.

We further hypothesized that NT may be able to trigger release of extracellular mt components from human mast cells. Stimulation of human cultured LAD2 cells with NT (1, 5 and 10 μM, for 1 h at 37°C) resulted in significant release of CytB and 7S mtDNA in the supernatant fluid (Figure 3).

Here we report the presence of serum extracellular mtDNA and AMA-M2 in young children with autism. Serum mtDNA has never been associated with any neuropsychiatric disease. Moreover, AMA-M2 have been clinically detected only in primary biliary cirrhosis [15]. Consequently, our findings are unique even though there are no available data as to what serum levels of either mtDNA or AMA-M2 may constitute an index of a pathological process. However, mitochondria evolved from bacteria that became symbiotic with eukaryotic cells and are typically prevented from being released extracellularly by autophagy [13]. Consequently, mtDNA released extracellularly would be extremely immunogenic. It was recently reported that damage-associated mitochondrial patterns (DAMPs), which contain mtDNA, are present in the blood of patients with trauma-induced sepsis in the absence of any apparent infection and are able to activate toll-like receptor 9(TLR9) on human peripheral polymorphonuclear leukocytes leading to release of IL-8 [16].

The presence of extacellular mtDNA in children with autism suggests that it may be one source of "autoimmune" triggers, and may potentially explain some aspects of immune dysregulation reported in autistic patients. For instance, mtDNA (or other extracellular mitochondrial components not measured in this study) could activate TLRs on immune or glial cells to release pro-inflammatory cytokines, such as IL-6, IL-8 or TNF, high gene expression of which was reported in brains of autistic children [17].

Our present results do not imply any mitochondrial dysfunction. Moreover, we cannot definitely state that subjects didn't have any mitochondrial dysfunction, since such confirmation requires extensive clinical and laboratory evaluation [18, 19] that was not performed in this case. Mitochondrial dysfunction has been reported in a subset of children with autism [11, 20], but apparently is not linked to altered energy metabolism [10]. Such subsets of ASD children with mitochondrial dysfunction may be more vulnerable to regression following a febrile episode [21].

The source of the extracellular mtDNA and other mitochondrial components in the serum of autistic patients is not presently known. There is no reason to suspect that these derive from apoptotic or necrotic cells because no GAPDH DNA was detected. Moreover, there is no apparent cell damage, at least outside the brain, in autism. One possibility is that mt components are secreted from immune cells, as was recently reported for activated neutrophils [22]. Another possibility could be activated tissue mast cells in the gut, where NT [23], is abundant and may induce mucosal permeability [24]. Our current findings showing that NT can trigger mtDNA release from human mast cells is supported by our previous report that NT, found both in the brain and the gut was elevated in autistic children [9]

The present results may be limited only to autistic disorder and only to the young age of the subjects studied. Nevertheless, these results suggest that serum mt components may induce autoimmune responses, as previously reported for TLR9 activation on human peripheral polymorphonuclear leukocytes [16], and may help with early diagnosis of at least a subgroup of autistic patients.