Propionic Acid Induces Gliosis and Neuro-inflammation through Modulation of PTEN/AKT Pathway in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by glia over-proliferation, neuro-inflammation, perturbed neural circuitry, and gastrointestinal symptoms. The role of gut dys-biosis in ASD is intriguing and should be elucidated. We investigated the effect of Propionic acid (PPA), a short-chain fatty acid (SCFA) and a product of dys-biotic ASD gut, on human neural stem cells (hNSCs) proliferation, differentiation and inflammation. hNSCs proliferated to 66 neuropsheres when exposed to PPA versus 45 in control. The neurosphere diameter also increased at day 10 post PPA treatment to (Mean: 193.47 um ± SEM: 6.673 um) versus (154.16 um ± 9.95 um) in control, p < 0.001. Pre-treatment with β-HB, SCFA receptor inhibitor, hindered neurosphere expansion (p < 0.001). While hNSCs spontaneously differentiated to (48.38% ± 6.08%) neurons (Tubulin-IIIβ positive) and (46.63% ± 2.5%) glia (GFAP positive), PPA treatment drastically shifted differentiation to 80% GFAP cells (p < 0.05). Following 2 mM PPA exposure, TNF-α transcription increased 4.98 fold and the cytokine increased 3.29 fold compared to control (P < 0.001). Likewise, GPR41 (PPA receptor) and pro-survival p-Akt protein were elevated (p < 0.001). PTEN (Akt inhibitor) level decreased to (0.42 ug/ul ± 0.04 ug/ul) at 2 mM PPA compared to (0.83 ug/ul ± 0.09 ug/ul) in control (p < 0.001). PPA at 2 mM decreased neurite outgrowth to (80.70 um ± 5.5 um) compared to (194.93 um ± 19.7 um) in control. Clearly, the data supports a significant role for PPA in modulating hNSC patterning leading to gliosis, disturbed neuro-circuitry, and inflammatory response as seen in ASD.

In circulation, PPA passes through the blood brain barrier to modulate multiple cell signaling processes including energy metabolism, neurotransmitter synthesis and release, and lipid metabolism 9 . Meanwhile, excessive PPA level might be toxic. In neonatal Propionic Acidemia (PA), Propionyl CoA Carboxylase (PCC), involved in the metabolism of amino and fatty acids, is not functional due to a mutation in one of the two genes that code for its Alpha and Beta subunits; PCCA and PCCB. As a result, PPA accumulates in the blood causing severe seizures, movement disorders, gastrointestinal issues, aloofness, and overall developmental delays 13 . Interestingly, PA and ASD share most of their core symptoms with multiple case studies reporting ASD as a comorbidity to PA [13][14][15] . Furthermore, high levels of PPA, but not BA, acetate, or other SCFAs, have been reported in the stools of ASD individuals; however, how PPA is involved in the development of ASD remains largely unknown 9,15 .
PPA is believed to cause systematic mitochondrial dysfunction (MD), as evidenced by increased free acyl-carnitine (cofactor used to transport long-chain and very-long-chain fatty-acids into the mitochondria) in rats exposed to PPA 16 . Interestingly, more than 30% of ASD patients were also reported to have MD, and elevations in carnitine-bound unprocessed long-chain and very-long-chain fatty-acids; thus providing further evidence for the association between PPA and ASD 15 . However, it remains unclear how MD and/or disturbed fatty acid metabolism may cause autistic phenotype.
Attempts to resume autistic-like behavior in rodents by exposure to PPA at different developmental stages have been reported 14,15 . For instance, intracerebroventricular delivery of PPA in rats resulted in increased IL-6, TNF-α, and interferon-γ cytokine levels, disturbed fatty acid metabolism, and marked microglia (neuro-inflammatory macrophages) over-proliferation 14 . Nonetheless, it remains unclear how PPA may affect the other neuronal cell types (Neurons and glia) particularly during the most sensitive stages of neural development.
Neural stem cells give rise to neuroepithelial progenitor cells (NPCs) which then differentiate into neuronal or glial cells 17 . Glial cells including oligodendrocytes and astrocytes, play a role in neurons development, connectivity, and protection [18][19][20] . During traumatic brain injury, reactive glial cells proliferate and release fibrillary acidic protein (GFAP) to inhibit damaged axonal regeneration causing gliosis 17,18 . Furthermore, glial and microglial cells release inflammatory cytokines to clean up damaged cells and toxins, therefore causing neuro-inflammation [18][19][20] . Some investigators regard gliosis as a protective process as it clears up damaged cells and blocks regrowth of damaged axons 19 . However, it is safe assuming that if gliosis occurs during the earliest stages of brain development it will greatly affect neural architecture and connectivity. In the ASD brain, disturbed neuronal circuitry with increased regional cell density in cortical, limbic, and cerebellar regions were reported [21][22][23][24] . In the meantime, glial cell count far exceeded that of neurons 23,24 . Nevertheless, it remains unclear if premature gliosis may play a role in ASD.
Concurring evidence suggests that ASD may stem from a disorder in glial cells [19][20][21][22][23] . Specifically, GFAP was shown to be highly expressed in the ASD brain compared to age matched healthy controls 20,25 . Other studies reported that anti-tumoral, pro-apoptotic Phosphatase and tensin homolog (PTEN), was elevated in autistic astroglial cells 26 . This suggests an over-proliferation of glial cells in ASD. Wen Yi, et al. reported that transgenic mice with astroglial specific deletion of PTEN, demonstrated altered radial glia cell proliferation and disturbed neuronal patterning 27 . Most intriguingly, reduced microbiota complexity was directly linked with impaired microglial proliferation and maturation in germ free mice 28 . In contrast, upon re-introduction of Clostridium cluster XIV, Bacteroides distasonis, and Lactobacillus salivarius strains to the GI tract, microglial phenotype was partially restored 28 . It was further established that such effect is facilitated by SCFA produced by these bacteria. Nevertheless, the paper does not provide cues on the proliferation state of the main brain cells; neurons and glia.
PPA interacts with brain cells via G-protein-coupled SCFA receptors including GPR41 29 . Since PPA (3 carbons) is the most potent activator of GPR41, and due to the prevalence of Clostridia spp., Bacteriodetes, and Desulfovibrio spp. in ASD microbiome and its association with elevated PPA and gliosis 30 , we hypothesize that elevated PPA may tamper with neural cell plasticity and differentiation in vitro leading to gliosis, increased inflammatory profile, and disturbed neural connectivity, similar to ASD.
Treatment with PPA, BA and GPR41 inhibitor. NSCs were treated with sodium propionate (PPA), and sodium butyrate (BA) (Sigma) at 0.1 mM, 0.5 mM, 1 mM, and 2 mM final concentration. Control cells were treated with 1X PBS. Another set of NSCs were pre-treated for 24 h with 2 mM ketone body β-hydroxybutyrate (β-HB), a potent inhibitor of GPR41 receptor, prior to PPA and BA treatments 31 . All treatments were done in triplicates (n = 3). Cells were then incubated for up to 10 days to form neurospheres. For differentiation purposes, intact neurospheres from each set were further plated on Geltrex (Cat# A14133; Fisher Scientific) pre-coated 24 well plates or 8 well chambers for an additional 7 to 10 days in a humidified 37 °C and 5% CO 2 incubator.
Differentiation media consisted of the regular StemPro ® complete media without growth factors. Neurosphere count and diameter measurements. Neurospheres were imaged using a digital camera (Amscope MU130, USB 2.0 DC 5V, 250 mA) mounted on an inverted tissue culture microscope (40X-800x Amscope, Japan) at 10x magnification every other day for 4 time points post plating (Days 2, 4, 8, and 10). The www.nature.com/scientificreports www.nature.com/scientificreports/ total number of neurospheres with a minimum diameter of 25 um were counted. The diameter of at least 15 Neurosphere per condition was measured using Amscope software version x64, 3.7.7303. The average diameter of neurospheres per condition was reported.

Neurite outgrowth measurements.
To evaluate the effect of PPA on neurite outgrowth, three 25X fluorescent images of Tubulin-IIIβ positive neurons were obtained using Amscope IN480TC-FL-MF603 fluorescent microscope mounted with an MF603C-CCD digital camera. Each experiment was repeated 3 times for accuracy and neurite outgrowth length (µm) from 15 random neurons per treatment setting were measured using Amscope software version x64, 3.7.7303. and averaged.
Evaluation of gene expression using RT-PCR. Cells were plated in 24-well plates as described earlier and allowed to differentiate for 7 days followed by RNA extraction and cDNA synthesis.
RNA isolation. Total RNA was extracted using the TRI ® reagent (Invitrogen) in RNase free environment. RNA was then separated from the aqueous phase in chloroform and precipitated in isopropyl alcohol followed by a washing step in 75% ethanol. Dried RNA pellets were suspended in TE buffer or RNAse free water and saved in −20 °C. Measurement of protein and cytokine levels. Cells were plated in 24-well plates as described earlier and allowed to differentiate for 7 days. Next, they were harvested by centrifugation at 300 RPM for 5 min and supernatant saved for cytokine analysis. To extract cellular proteins, cell pellets were incubated for 15 min in shilled RIPA lysis buffer (Thermo-Fisher; Cat# 89901), and then centrifuged at 14,000 RCF for 10 min. Supernatant containing protein homogenates were subjected to commercially available ELISAs specific to GFAP Statistical analysis. Statistical analysis was performed using GraphPad Prism 7.02 software. Significance among experiments was assessed by either Unpaired Two-tailed t test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test and cross checked with Wilcoxon matched-pairs test for non-parametric tests (# of (n) repetitions superior to 5 and inferior to 20; 5 < n < 20). Data is presented as (Mean ± Standard Error of the Mean (SEM)). Statistical significance is assigned for p-value < 0.05 and confidence interval exceeding 95%. P-values < 0.001, and p-values < 0.0001 are also mentioned when achieved. F-Statistic value is presented as F (DFn, DFd).

PPA and BA enhance neural stem cell proliferation in vitro.
To understand the effect of PPA on hNSCs proliferation in vitro, we treated the cells with PPA (2 mM), BA (2 mM), or PBS (1x) and monitored neurosphere formation for 10 days. As shown in Fig. 1A, at day 2 following treatments, neurosphere diameter averaged (Mean: 58.45 um ± SEM: 4.1 um) across all treatments and the diameter differentially increased by day 10. When www.nature.com/scientificreports www.nature.com/scientificreports/ cells were pre-treated with 2 mM of β-HB (GPR41 inhibitor) before exposure to PPA or BA, the neurosphere diameter on day 10 averaged (56.53 um ± 4.74 um) for β-HB + PPA, (78.41 um ± 4.51 um) for β-HB + BA, and (56.26 um ± 5.73 um) for β-HB alone. Figure 1B illustrates the progress of the neurosphere expansion at 4 intervals within 10 days treatment. In absence of β-HB pre-treatment, PPA and BA increased neurosphere diameter significantly when compared to untreated cells (One-way ANOVA followed by Tukey's multiple comparison test. p < 0.0001, F (6, 286) = 48.71). We also measured the neurosphere counts following each treatment (Fig. 1C). At Day 2, the neurospheres average count was (24 ± 3) for all treatments. At day 10, the neurospheres average count increased to 66 and 65 for PPA and BA, respectively. Cells treated with PBS had an average of 48 neurospheres. Cells pre-treated with β-HB contained 17, 32, and 20 total neurospheres after 10 days of treatment with PPA, BA, and β-HB alone, respectively.

PPA promotes glial cells differentiation in vitro.
The effect of PPA and BA on hNSCs differentiation was evaluated by quantifying GFAP versus Tubulin-IIIβ positive immunostained cells in three different areas from each treatment. Figure 2A depicts confocal representative images of differentiated cells from hNSCs with blue color for DAPI positive cells, green for GFAP positive, and red for Tubulin-IIIβ positive cells, scale bar 25 um. As shown in Fig. 2B, in vitro plated untreated hNSCs have spontaneously differentiated to equal number of neurons and glia with (48.38% ± 6.08%) neurons and (46.53% ± 2.5%) glia (p < 0.05). There was no change when cells were treated with 1x PBS. Surprisingly, treatment with PPA drastically shifted hNSCs differentiation to more than 80% glia whereas Tubulin-IIIβ positive cells were only 13.20% of the total number of cells. On the contrary, BA shifted hNSCs differentiation to (77.78% ± 1.29%) neurons whereas GFAP positive cells drastically decreased (17.26% ± 3.23%). Interestingly, pre-treatment of hNSCs with β-HB resulted in cell differentiation similar to control cells (p < 0.05, Fig. 2B). Data was collected from a minimum of (n = 3) random areas per duplicated treatment setting. Statistical significance was tested using One-way ANOVA followed by Tukey multiple comparison test and confirmed with Wilcoxon matched-pairs tests. (*p < 0.05, F (13, 28) = 2.520), for GFAP vs Tubulin-IIIβ positive cells within the ([) limited treatments.
Effect of PPA on tubulin-IIIβ and GFAP. We measured protein and gene expression of both Tubulin-IIIβ and GFAP in each cell group. As shown in Fig. 3A, Tubulin-IIIβ protein level gradually decreased up to 5X following treatment with 2 mM PPA (*p < 0.0001, F (9, 10) = 17.91). Similarly, Tubulin-IIIβ relative mRNA expression significantly decreased after PPA treatment (Two-tailed Unpaired t test, p < 0.05, Fig. 3B). On the other hand, GFAP protein level and gene expression increased significantly following PPA treatment. Statistical significance was ran using One-Way ANOVA followed by Tukey's multiple comparison tests (Protein *P < 0.05, F (9, 10) = 3.890 and gene expression p < 0.0001, F (9, 10) = 253.0), Fig. 3C,D.  Figure 3A,B illustrates the positive increase in Tubulin-IIIβ protein and gene expression in cells treated with BA compared to significant decrease in GFAP protein and gene expression in these cells (*p < 0.05 Fig. 3C,D).

Expression pattern of GPR41 on neurons versus glial cells.
To study the effect of PPA and BA on expression of GPR41 in differentiated hNSCs, we measured GPR41 protein level and relative mRNA expression. Figure 4A,B show confocal images representative of colocalization of GPR41 receptor on GFAP positive cells (Panel 4A) and Tubulin-IIIβ (Panel 4B). As shown in Fig. 4A, GFAP strongly co-localized with GPR41 in cells treated with PPA (image A-i and enlarged in image A-j) compared to untreated cells (image A-d and enlarged in image A-e). There was minimum co-localization between Tubulin-IIIβ and GPR41 following BA treatment (images B-d and B-i). Of note, although all groups were immune-stained and analyzed, we only chose to depict PPA and BA treatments along with their controls for accurate representation of the co-localization. We also measured GPR41 protein level and relative mRNA expression following treatment with PPA and BA. As shown in Fig. 4C, PPA increased GPR41 protein at least 3X following treatment with 2 mM PPA (*P < 0.01, F (9, 10) = 8.66). Similarly, GPR41 relative gene expression increased several folds following PPA treatment (Fig. 3D, p < 0.05). There was no change on GPR41 level for cells treated with BA or PBS Fig. 4D). Statistical significance was ran using One-way ANOVA followed by Tukey's multiple comparison tests (*P < 0.0001, F (9, 10) = 718.4).

PPA Induces GPR41-mediated p-Akt survival pathway in differentiating glial cells. ELISA and
RT-PCR were used to determine the in vitro effect of PPA and BA on expression levels of pro-survival Akt and its direct inheritor PTEN. As shown in Fig. 5, only 1.0 mM PPA was needed to significantly decrease PTEN both protein (A) and gene expression levels (B) compared to untreated cells (p < 0.0001). Although there was no change in PTEN expression following BA treatment (Fig. 5B), PTEN protein level increased significantly and seems to be dose-dependent in BA-treated cells (Fig. 5A) compared to controls (p < 0.0001 was achieved at 2 mM BA treatment, F (9, 10) = 49.86). www.nature.com/scientificreports www.nature.com/scientificreports/ Similarly, ELISA results for the activated form of Akt (p-Akt) showed that ascending concentrations of PPA promotes an increase in available p-Akt to reach significance against control at 2 mM PPA (Fig. 5C). BA on the other hand achieved the exact opposite with a significant decrease in available p-Akt to reach the lowest significant (P < 0.0001, F (9, 10) = 18.42) level at 2 mM BA (Fig. 5C). Interestingly, there was no effect on Akt gene expression following any of the SCFAs treatments (Fig. 5D). Statistical significance was ran using One-way ANOVA followed by Tukey's multiple comparison test.

PPA promotes gliosis and Pro-Inflammatory cytokines release.
To determine the function of PPA-induced Glia cell differentiation, we measured TNF-α and IL-10 released into the cell culture media and their corresponding mRNA in cells lysates. As shown in Fig. 6, TNF-α increased significantly at both the protein (P < 0.001, F (9, 10) = 9.174) and RNA (P < 0.0001, F (9, 10) = 15.72) levels when hNSCs were treated with a minimum of 0.5 mM PPA (Fig. 6A,B, respectively). Similarly, the anti-inflammatory IL-10 levels increased under ascending concentrations of PPA ( Fig. 6C: P < 0.02, F (9, 10) = 4.147, and Fig. 6D: P < 0.0001, F (9, 10) = 81.23). Overall, TNF-α level has super exceeded the increase in IL-10 level leading to a net increase in the pro-inflammatory TNF-α. Although treatment with BA seems to increase TNF-α protein level, there was minimum change in TNF-α or IL-10 gene expression (Fig. 6). Statistical significance was ran using One-way ANOVA followed by Tukey's multiple comparison test. To test whether PPA or BA modulate neurite growth through GPR41 receptor, we measured neurite outgrowth in hNSCs pre-treated with β-HB. Clearly, β-HB blocked the effect of PPA and BA (Panel 7A-c and A-e). Specifically, β-HB pre-treatment resulted in neurite outgrowth measurements equivalent to controls (Fig. 7C). Statistical significance was ran using Wilcoxon matched-pairs and One-way ANOVA followed by Tukey post-hoc test). (*P < 0.0001, F (10, 111) = 53.15). Black bars represent the controls (no treatment other than media) and media supplemented with 1x PBS. Data is represented as Mean + SEM (n = 3 per group) and statistical significance (*p < 0.0001 for Tubulin-IIIβ and p < 0.05 for GFAP) was obtained using either (Two-tailed Unpaired t test, Wilcoxon matched-pairs, and/or One-way ANOVA followed by Tukey's post-hoc test) vs. Controls.

Discussion
Gastrointestinal (GI) symptoms are among the most prevalent comorbidities associated with ASD 27 . A shift in gut microbiome and their by-products in autistic individuals has been reported [6][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 BA 7-12 . We were intrigued by MacFade et al finding that intracerebroventricular injection of PPA in rat's brains induced reactive gliosis 11 . 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 development 32 . 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 neurotoxicity 33 . 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 cells 34 . 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 reported 21,22 , 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 GPR41 29 . Over expression of GPR41 in PPA-treated hNSCs confirmed differentiation shift to gliosis (Fig. 4). www.nature.com/scientificreports www.nature.com/scientificreports/ PTEN was reported to regulate radial glia cell proliferation in the early stages of neural development through inhibition of Akt pro-survival pathway 26 . Recent studies reported that PTEN is downregulated in autistic glial cells 26,27 , 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 brain [18][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 way 21,24 . 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 cells 14,24 . 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 functioning 36 . 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 . Black bars represent the controls (no treatment other than media) and media supplemented with 1x PBS. Data is represented as Mean + SEM (n = 3 per group) and statistical significance (*p < 0.001 for TNF-α and p < 0.05 for IL-10) was calculated using One-way ANOVA followed by Tukey's post-hoc tests vs. Controls. www.nature.com/scientificreports www.nature.com/scientificreports/ 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).
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 ASD 21,24,37 . 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 pool 38,39 , 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.

Data Availability
Raw data is available upon request. 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 prosurvival 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 neuroinflammation observed in ASD. Some illustrations used in this figure were originated from leased Motifolio (Scientific Illustration Toolkits for Presentations and Publications) materials.