Gastrointestinal (GI) symptoms are among the most prevalent comorbidities associated with ASD27. A shift in gut microbiome and their by-products in autistic individuals has been reported6,7,8,9,10,11,12,30. Specifically, autistic gut seems to have an increase in Clostridia spp., Bacteriodetes, and Desulfovibrio spp. which are known to be active fermenters and producers of SCFAs including PPA and BA7,8,9,10,11,12. We were intrigued by MacFade et al finding that intracerebroventricular injection of PPA in rat’s brains induced reactive gliosis11. In the current study, we are linking maternal PPA exposure to disturbed neural patterning during early stages of embryonic neural development leading to over proliferation of glial cells, abnormal neural architecture, and increased inflammatory profile; possible precursors for autism. We employed a three-dimensional neurosphere assay to evaluate how SCFAs affect hNSC proliferation in vitro. Neurospheres are 3D progenitor cell conglomerates, representing a valuable in vitro model as they mirror the earliest stages of neural development32. They are useful to study cell proliferation, migration and differentiation making them a neurotoxic test of choice for plethora of agents and chemicals, particularly hormones, pesticides, or to study chemotherapy-induced neurotoxicity33. Our data unequivocally show that both PPA and BA promote hNSC self-renewal and proliferation in vitro, as evidenced by increased neurosphere number and diameter following exposure to either PPA or BA (Fig. 1). These results support earlier reports suggesting gut microbiota promote proliferation and maturation of enteric progenitor cells34. Our study provided additional evidence to support the proliferative role of gut by-products such as SCFAs on enteric progenitor cells. Furthermore, we demonstrated that such proliferative role is mediated through GPR41 receptor since its inactivation with β-HB voided SCFAs effect (Fig. 1).

Although disturbance in neuro/glia ratio in the autistic brain has been reported21,22, it was not clear how, when and why this dys-balance occurs. In this study, exposing differentiating hNSCs to 2 mM PPA induced a shift towards glial phenotype (Figs 2 and 3). This is an intriguing finding and a first in the field. Surprisingly, exposing differentiating hNSCs to BA favored the opposite, with increased neural proliferation (Figs 2 and 3). The SCFAs effect was confirmed following blocking GPR41 receptor with β-HB. This indicates that such effect is triggered by the specific binding of PPA and BA to GPR41 followed by a downstream molecular machinery leading to either glial or neural proliferation. Of importance here, the ratio of glia/neuron in BA treated cells is not as significant as that of PPA, possibly because PPA is the most potent activator of GPR4129. Over expression of GPR41 in PPA-treated hNSCs confirmed differentiation shift to gliosis (Fig. 4).

PTEN was reported to regulate radial glia cell proliferation in the early stages of neural development through inhibition of Akt pro-survival pathway26. Recent studies reported that PTEN is downregulated in autistic glial cells26,27, however, what triggers PTEN inhibition in ASD remains uncertain. In this study, data suggest that PPA binding to its receptor may lead to GPR41-induced PTEN inhibition, thereof allowing Akt survival pathway to proceed. As we demonstrated in Fig. 5, PPA seems to tamper with both PTEN and activated p-Akt levels. PTEN expression decreased with increased PPA concentration and vice versa for p-Akt. Noteworthy, PPA interfered with the amount of activated p-Akt but not Akt expression. This result further validates that PPA has no direct effect on Akt expression but rather downregulates PTEN expression. Consequently, this allows p-Akt to remain active which results in over-proliferation of glia-committed neural progenitor cells.

To understand the inflammatory response and GI disorder in individuals with ASD, we studied the effect of PPA on gliosis and inflammatory cytokines in differentiated hNSCs. Our data showed that PPA seems to upregulate TNF-α and IL-10 and increase the level of the cytokines (Fig. 6). Since PPA induced glial cell differentiation and increase in TNF-α and IL-10 transcription and translation, we propose that exposure to PPA during gestation may be related to gliosis and inflammation as reported in multiple neuro-developmental diseases including ASD. Specifically, exposure to high dose of PPA during early stages of neural stem cell development promotes proliferation and activation of glial cells, recapitulating the state of neuro-inflammation as reported in the post-partum autistic brain18,19,20,35.

In the developing brain, neurons are produced in the ventricular zone (VZ) and migrate into the developing neocortex guided by adjacent glial cells along the way21,24. Once settled, they undergo terminal differentiation in which long axons and dendrites extend to connect with adjacent neurons to form the final brain network, supported by glial cells14,24. It is therefore of outmost importance that the number and positioning of supporting glial cells be at chirurgical precision in order to achieve this delicate neuro-architecture. In the autistic brain, reports indicate that short and long distance inter-neuronal communication is disturbed, causing delays in information processing, increased repetitive behaviors and idiosyncrasies, as well as distortion in brain regions, such as the prefrontal cortex (PFC), associated with higher functioning36. However and as of latest data, it remained unclear what maybe causing neuronal circuitry disruption in ASD. We here stipulate that glial cells outnumbering neurons may constitute a physical barrier to the extending neurites, therefore accounting for a decrease in overall axonal growth. This phenomenon was clearly reflected in our data showing that PPA increased glial cell count and resulted in decreased neurite growth (Fig. 7). PPA may also block the molecular machinery involved in axonal expansion, nevertheless, further studies are needed in this area.

Overall, the data in this study suggest that microbiome shift in maternal gut leads to formation of by-product such as PPA which then interferes with neural patterning during the early stages of the fetus’ neural development. This favors glial progenitor cells proliferation and survival leading to increased inflammatory profile and perturbed neural architecture. The data further suggests that such process is achieved through modulation of PTEN/Akt pathway within the growing glial cells but not neurons (Fig. 8).

Figure 8 Overall Diagram for Hypothesized PPA Effect on hNSC in vitro. During the early stages of pregnancy, increased consumption of PPA-rich processed foods combined with pre-existent dysbiosis may lead to accumulation of PPA in the maternal GI, travel through general circulation, cross the placental barrier, and interfere with neural differentiation through binding to GPR41 receptor preferably expressed on glial progenitor cells. This will activate a downstream molecular pathway resulting in PTEN inhibition and activation of pro-survival Akt pathway, therefore favoring glial progenitor cells proliferation and differentiation. Mature glial cells will move on to produce inflammatory cytokines and release GFAP, all of which mimic gliosis and neuro-inflammation observed in ASD. Some illustrations used in this figure were originated from leased Motifolio (Scientific Illustration Toolkits for Presentations and Publications) materials. Full size image

Unexpectedly, BA, another SCFA used in this study as a control, seems to play a potential pro-neural role by virtue of increased neural cell count upon BA treatment (Figs 2 and 3). Interestingly, excess neural proliferation and macrocephaly were also linked to ASD21,24,37. However, what may be causing this shift remains largely debatable. We might be tempted to speculate that excess BA could be the culprit for the macrocephaly observed in ASD; however, more studies are warranted to make a better guided guess. Instead, it is safe assuming that the normal developing brain comes equipped with a set of neurons and supportive glial cells and any stirring away from this homeostatic ratio, towards glial overgrowth or neuronal over-proliferation, may disrupt the brain architecture potentially causing ASD. Therefore, and in light of these preliminary BA data, exploratory studies are more than warranted to elucidate BA role, if any, in ASD.

Solving the conundrical etiology of ASD is critical for any future prevention or treatment strategies. There is no doubt that genetic polymorphisms and environmental triggers are both involved in ASD development or at least in ASD complications. Because of the fact that autistic individuals who undergo antibiotic treatment seem to demonstrate a provisory yet noticeable relief from GI symptoms and some ASD behavior amelioration, and they may benefit from fecal replacement as a method to restore their microbiota pool38,39, there are good reasons to suggest that gut-brain axis is a potential culprit in ASD pathogenesis. This study is the first to link PPA and ASD-microbiome by-product to gliosis, disturbed neural architecture, and increase in inflammatory response, all of which may translate into dramatic neuro-complications including ASD.