Autism spectrum disorders (ASD) are neurodevelopmental diseases with complex symptoms and whose neurobiological basis remains poorly understood. Research suggests that a combination of genetic, autoimmune, environmental, and perhaps in utero risk factors leading to neuroinflammation can contribute to the pathogenesis of ASD [1–6] as well as other neurobehavioral and/or neuropsychiatric disorders including schizophrenia (SCZ) [7–13]. Over-expression of acute phase proteins in the serum, brain, and cerebrospinal fluid of ASD as well as SCZ individuals suggests that inflammation is involved in the pathophysiology of these diseases [4, 6, 14–21]. Autoimmune diseases have been associated with increased risk of ASD and SCZ [10, 22–28] suggesting altered or inappropriate immune responses.

Furthermore, many ASD patients experience gastrointestinal (GI) symptoms and/or dysfunctions [29–38]. Clinical observations describe increased intestinal permeability in ASD [39–42], and permeability to food antigens derived from the partial digestion of wheat (gliadorphins) and cow’s milk (casomorphins) has been reported in both ASD and SCZ [41, 43–46].

Despite research efforts, there are no defined explanations of how environmental triggers can lead to these neurobehavioral conditions. One hypothesis, based on the interconnectivity of the gut–brain axis, suggests that inappropriate antigen trafficking through an impaired intestinal barrier, followed by passage of antigens or activated immune complexes through a permissive blood–brain barrier (BBB), can be part of the chain of events leading to neuroinflammation and thereby subsequent disease.

The BBB plays a critical role in the central nervous system (CNS) defense through limiting the access of circulating solutes, macromolecules, and cells that could negatively impact neuronal activity. Dysfunctions of the BBB have been associated with numerous neurological disorders, such as stroke, epilepsy, multiple sclerosis, and Parkinson’s and Alzheimer’s disease [47–56].

The overall goal of this study was to assess whether a dysfunctional BBB or gut barrier could contribute pathophysiologically to ASD. To address this, we conducted an in-depth molecular analysis of the components associated with the BBB and gut barrier integrity in postmortem brain tissue and small intestinal biopsies obtained from ASD subjects. In addition, we assessed changes in the BBB integrity of patients with SCZ as SCZ is a psychiatric illness in which the involvement of inflammation, immunity and altered BBB integrity have been postulated [22, 23, 57, 58].

To the best of our knowledge, this is the first study addressing the molecular signature of BBB dysfunctions in ASD and SCZ in human samples. In spite of the obvious small sample number, due to the nature of the samples and the consequent procurement difficulty, we observed statistically significant differences among the study groups. However, it is also worthwhile to acknowledge that the small sample number may have affected some otherwise possibly significant results, which could have emerged if a larger number of tissues had been available.

Our molecular analysis of the BBB integrity and function shows an altered BBB in the ASD subjects evaluated. Indeed, the increased gene expression of MMP-9 we detected in ASD subjects supports our hypothesis of an impaired BBB, most likely associated with neuroinflammation. Several pieces of evidence indicate that MMP-9 secretion induces BBB disruption and this is an important step in the development of inflammatory diseases of the nervous system [66–74]. Although we did not observe elevated gene expression levels of pro-inflammatory cytokines in the ASD subjects analyzed in this study, we found a significantly high expression of TSPO in the ASD brain. TSPO increased expression level in activated microglia and reactive astrocytes [75–81] is associated with brain injury and neuroinflammation, making TSPO a reliable marker of brain inflammation.

Of the four claudins (i.e., CLDN-1, -3, -5 and -12) that to date are thought to be incorporated in the BBB [82–85], we found that two were significantly more expressed in the ASD brain as compared in HC. CLDN-5, the major and more abundant cell adhesion molecule of TJs in brain endothelial cells, is expressed 5.1 and 3.6 times higher in the cortex and CBL of ASD subjects, respectively, than in those of HC (Table 3). Similarly, CLDN-12 is expressed about 7.7 and 3.6 times more in the ASD cortex and CBL, respectively, than in the control group. CLDN-3, on the other hand, appears elevated by about 2.7 and 3.0 times in the cortex of both SCZ and ASD subjects, respectively, compared in that of HC. TRIC, another component of the TJs associated with increased epithelial tightness and decreased permeability to macromolecules [86], appears significantly elevated in the ASD cortex, suggesting either a more competent BBB or a compensatory mechanism for disrupted endothelial barrier integrity.

At the protein level, CLDN-5 is elevated in the ASD cortex, consistent with its mRNA expression, whereas, on the other hand, CLDN-12 is significantly reduced. An increased gene expression of sealing TJ components (CLDN-5, -12, and -3 and TRIC) should imply a tighter BBB and a better control in the diffusion of macromolecules. However, our data on MMP9 and TSPO expression compatible with ongoing neuroinflammation suggest a compensatory mechanism to repair a compromised BBB. Another plausible explanation for the CLDN-5 data is that, once produced, the protein is not capable of being incorporated into the TJ complex because of impaired trafficking to the endothelial cell–cell interface or because the protein has some mutations that prevent it from establishing homophilic and/or heterophilic protein–protein interactions, leading to a sustained compensatory gene expression and protein accumulation. On the other hand, the discrepancy between gene upregulation and decreased protein expression observed for CLDN-12 might suggest the existence of a compensatory mechanism for post-translational destruction/degradation of the protein itself. While the sealing function of CLDN-5 in BBB TJs is well established [83, 87], that of CLDN-3 and -12 are less understood. CLDN-1 and -3 sequences are highly homologous to CLDN-5, suggesting similar properties. Indeed, several studies strongly suggest that CLDN-3 contributes to BBB tightness [85, 88–90]. Conversely, CLDN-12 sequence is quite dissimilar from those of CLDN-1 and -3, and this might indicate a different function. Piontek et al. [91] showed that CLDN-12 cannot form homophilic trans-interactions or homopolymeric strands in vitro, suggesting that “heterophilic cis- and/or trans-interactions with another TJ-protein are necessary for the incorporation of CLDN-12 in strands.” It may be hypothesized that the increased CLDN-5 we observed in ASD subjects might be compensatory to a decreased CLDN-12 in a heterophilic cis- and/or trans-interaction between the two claudins.

Our cohort of SCZ individuals did not show major changes in the genes analyzed compared to HC, except for an increased IL-1b in the CBL and significantly reduced levels of tPA and IBA-1 in the cortex. Whereas we do not know the physiological relevance of downregulated IBA-1, our results are in agreement with data from other studies indicating activation of pro-inflammatory networks and reduced tPA activity in SCZ [92–102], this latter feature leading to a dysfunctional coagulation system and increased risk of thrombotic events in SCZ patients [101]. Other genes analyzed in SCZ appeared to follow a trend similar to ASD subjects (i.e., increased CLDN-3 and reduced CLDN-12 protein level in the cortex). Furthermore, although non-statistically significant (p = 0.07), we did observe a trend toward reduced CLDN-5 protein level in the SCZ CBL as well, suggesting a possibly leaking BBB. Previous clinical and postmortem research in SCZ has suggested that an impaired BBB could be a contributing factor to the pathogenesis of the disease in only a subgroup of SCZ patients [103–105]. It is important to mention that the SCZ and ASD subjects studied were not selected on the basis of specific immune biomarkers and, therefore, may represent a mixed group of patients, which thereby limits the statistical significance of some of our findings. A sample stratification approach based on specific immunological features might be a better way of analyzing pathways of interest, and this may be particularly true for SCZ, a disorder characterized by marked clinical and neurobiological heterogeneity. Alternatively, SCZ physiopathology may lie in subtle changes that chronically affect brain function.

As for the intestine, due to the high heterogeneity intrinsic of ASD, our goal was to elucidate the TJ component profile from a functional perspective. Toward this aim, we have analyzed the expression level of TJ elements dividing them into two major functional groups: pore-forming (CLDN-1, OCLN, TRIC) and barrier-forming components (CLDN-2, -10, -15). These results, showing increased expression levels of pore-forming (66% of the ASD samples) and decreased levels of barrier-forming (75% of the ASD samples) TJ components in the duodenal samples, suggest an impaired gut barrier and serve as a proof of concept to support the hypothesis of a gut–brain axis dysfunction in a subgroup of ASD patients. According to this hypothesis, non-self antigens crossing a damaged intestinal barrier elicit a local and/or systemic inflammatory reaction that, associated with a breach of the BBB, may lead to ASD in genetically predisposed subjects.

This study addressed for the first time, at the molecular level, some relevant and still open questions regarding the gut–brain axis in neurobehavioral disorders and furthermore demonstrated that both the BBB and gut barrier may be affected in ASD. However, these findings present with a few limitations. The first lies in the postmortem nature of the samples with potential confounding factors deriving from tissue preservation and the personal history of each subject, including exposure to medications, health conditions, cause of death, and postmortem interval (PMI). Our statistical analysis of the PMI showed no differences among groups suggesting that PMI might not have affected our results. Similarly, data from medical records or postmortem toxicology analysis showed that about the same number of subjects in ASD and SCZ groups were on antipsychotics/antidepressants, so it is unlikely that the differences we observed among groups could be explained by the effects of the drugs. For clarity, a few studies addressing the impact of antipsychotics on the BBB function do suggest a disturbance effect on its sealing property [106, 107], whereas other studies on the blood–cerebrospinal (BCS) fluid barrier show divergent results [103, 108]. Furthermore, there are numerous studies reporting the suppressing effect of antipsychotics on the blood cytokine profile in SCZ and ASD [98, 109–118], as well as on microglia activation [119, 120], that could explain our data on IBA-1 and the cytokine gene expression profiles. However, our IBA-1 results are well in agreement with the published studies on postmortem brain tissues showing no significant changes in its expression in ASD [121].

The second limitation of this study is represented by its descriptive nature that does not allow us to draw conclusions on the causality of a breach of the intestinal/BBB integrity supporting neuroinflammation and ASD and SCZ symptoms. However, our data is in line with animal studies suggesting causality between loss of barrier function and neurobehavioral changes compatible with ASD [122–124]. Finally, as already mentioned, a third limitation is represented by the small number of samples analyzed due to limited availability which might impact on the statistical significance of some of our results. However, given the complexity of the studies performed and the difficulty associated with the procurement of these samples, our study represents a solid foundation to justify a more robust sample collection and analysis to further investigate the gut–brain axis in ASD.