Prostaglandin E2 alters Wnt-dependent migration and proliferation in neuroectodermal stem cells: implications for autism spectrum disorders
© Wong et al.; licensee BioMed Central Ltd. 2014
Received: 13 September 2013
Accepted: 13 March 2014
Published: 23 March 2014
Prostaglandin E2 (PGE2) is a natural lipid-derived molecule that is involved in important physiological functions. Abnormal PGE2 signalling has been associated with pathologies of the nervous system. Previous studies provide evidence for the interaction of PGE2 and canonical Wnt signalling pathways in non-neuronal cells. Since the Wnt pathway is crucial in the development and organization of the brain, the main goal of this study is to determine whether collaboration between these pathways exists in neuronal cell types. We report that PGE2 interacts with canonical Wnt signalling through PKA and PI-3K in neuroectodermal (NE-4C) stem cells. We used time-lapse microscopy to determine that PGE2 increases the final distance from origin, path length travelled, and the average speed of migration in Wnt-activated cells. Furthermore, PGE2 alters distinct cellular phenotypes that are characteristic of Wnt-induced NE-4C cells, which corresponds to the modified splitting behaviour of the cells. We also found that in Wnt-induced cells the level of β-catenin protein was increased and the expression levels of Wnt-target genes (Ctnnb1, Ptgs2, Ccnd1, Mmp9) was significantly upregulated in response to PGE2 treatment. This confirms that PGE2 activated the canonical Wnt signalling pathway. Furthermore, the upregulated genes have been previously associated with ASD. Our findings show, for the first time, evidence for cross-talk between PGE2 and Wnt signalling in neuronal cells, where PKA and PI-3K might act as mediators between the two pathways. Given the importance of PGE2 and Wnt signalling in prenatal development of the nervous system, our study provides insight into how interaction between these two pathways may influence neurodevelopment.
KeywordsProstaglandin E2 Wnt signalling Neuroectodermal stem cells Cell motility Proliferation Autism
The plasma membrane phospholipids play a fundamental role in the nervous system and act as a reservoir for second messenger molecules important for the development and normal functioning of the brain. Prostaglandin E2 (PGE2) is a bioactive fatty acid that is derived from arachidonic acid, a major structural component of plasma membrane phospholipids, through the enzymatic metabolism of cyclooxygenases −1 and −2 (COX-1,-2) and then prostaglandin synthases . Extracellular stimuli such as immunological and infectious agents [2–4], environmental toxins such as mercury and lead , and exposure to drugs including misoprostol and valproic acid  can trigger the local production of PGE2 via specific biosynthetic pathways, resulting in altered cell signal transmission that modulates biological functions such as sleep, fever, inflammation, and pain .
The diverse action of PGE2 is achieved through the activation of 4 different G-protein coupled E-prostanoid receptors (EP1 through 4) [8, 9]. The divergent role of PGE2 is amplified by the variety of different kinase-mediated signalling cascades that can be activated through its EP receptors, such as the protein kinase A (PKA), phosphatidylinositide 3-kinases (PI-3K), and protein kinase C (PKC) pathways .
During the early stages of pregnancy, there are elevated levels of COX-2 and PGE synthases, enzymes responsible for the production of PGE2, which is indicative of the involvement of PGE2 in prenatal development . We have previously shown that the expression profiles of EP receptors during mouse embryonic development changes depending on the embryonic stage, with EP receptor expression highest during E7 (Embryonic day 7) and E15, which corresponds to peak periods of neurogenesis . It has been shown that PGE2 plays a regulatory role in membrane excitability and synaptic transmission in neurons . PGE2 increases the dendritic length and complexity of Purkinje neurons, and can also alter neuronal firing activity in the developing brain . PGE2 is involved in synaptic plasticity and neuroprotection , and can also be involved in neuronal cell death and apoptosis [16, 17]. Prostaglandins have also been reported to induce the differentiation of neuronal cells . Moreover, the inhibition of COX-2, can suppress neurogenesis and proliferation of neural progenitor cells . These studies show the important role PGE2 can play during normal development of the nervous system. Furthermore, previous research found that PGE2 can exert various effects on cell development, proliferation, and migration in a diversity of cell lines. It has been shown that PGE2 stimulates cell growth and motility in osteoblasts , prostate cancer cells , and pancreatic stellate cells . The migration of vascular smooth muscle cells , intestinal subepithelial myofibroblasts , dendritic cells , hepatocellular carcinoma cells , and mesangial cells  can all be regulated by PGE2. However, the effects of PGE2 on neural stem cell behaviour and movement are not well characterized. Our previous studies provide some insight into the molecular mechanisms of abnormal PGE2 signalling on neuronal cells. We have found that exposure to PGE2 results in the retraction of neurites and the elevation of calcium amplitude fluctuations in growth cones of differentiated Neuro-2A cells [12, 28].
Abnormal fatty acid metabolism through the PGE2 pathway may contribute to the pathology of neurodevelopmental disorders such as Autism Spectrum Disorders (ASD) . Abnormal levels of PGE2 and other fatty acid metabolites have been identified as potential biomarkers for ASD . PGE2 can act as an endogenous modulator for cerebellar development in the rat brain affecting social interaction and sensory behaviour, which are characteristic behaviours altered in ASD . A clinical study showed that maternal exposure to the drug misoprostol (prostaglandin E analogue), has been associated with the development of Moebius syndrome and autistic-like symptoms [32–34].
Current literature also provides evidence that PGE2 signalling interacts with another crucial developmental pathway, the canonical Wnt (wingless-related MMTV integration site) signalling pathway in various non-neuronal cells  such as osteocytes , prostate and colon cancer cells , hematopoietic stem cells , and mesenchymal stem cells . Wnt signalling is tightly regulated in early development and is required for the formation of the nervous system . The canonical Wnt signalling pathway is composed of a network of proteins that modify cell communication and interactions with other cells. Wnt proteins bind to cell surface Frizzled (FZD) receptors, where the signal is then transduced to β-catenin, activating the transcription of Wnt target genes. The Wnt molecules are vital to embryonic development since they can moderate cell proliferation and differentiation by participating in the determination of cell fates . Previous literature shows that convergence of PGE2-dependent signalling with the canonical Wnt pathway can occur at the level of β-catenin through EP1-4 receptors, including the association of the Gαs subunit with Axin, the stimulation of the cAMP/PKA pathway, or the phosphorylation of GSK-3β by PI-3K . However, the interaction of PGE2 and Wnt signalling is not well-characterized in the nervous system. To activate and study canonical Wnt signalling in an in vitro model system, Wnt Agonist (WntA), 2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl) pyrimidine, can serve as a useful reagent. WntA is a cell-permeable pyrimidine compound that mimics the effects of Wnt by functioning through the canonical pathway via upregulating TCF-activity without inhibiting the activity of GSK-3β . This is important because GSK-3β plays a regulatory role in many signalling pathways other than Wnt so an agonist that blocks GSK-3β could have disparate effects in cellular models.
This study investigates the effects of PGE2 interaction with the Wnt signalling pathway on the behaviour of murine neuroectodermal (NE-4C) stem cells. We demonstrate that PGE2 modifies canonical Wnt signalling in NE-4C stem cells by altering components of cell motility such as final distance travelled, path length travelled, average speed of migration, as well as cell splitting behaviour. We also reveal that PGE2 can alter the protein expression of non-phospho (active) β-catenin (Ser33/37/Thr41), as well as the expression of specific Wnt-target genes. Interestingly, the genes implicated in our study have been previously associated with ASD. To our knowledge, we show for the first time, that PGE2 signalling interacts with the Wnt pathway in neural stem cells to affect cell behaviour and gene transcription. Our study furthers our understanding of the possible mechanisms by which intracellular cross-talk between PGE2 and Wnt signalling may contribute to cell migration and proliferation during prenatal development of the nervous system.
Expression of EP1-4 receptors in NE-4C cells
Prostaglandin E2 increases the cell motility of Wnt-induced NE-4C cell migration
The effect of PGE2 on Wnt-dependent migration of NE-4C cells was determined using Nikon Eclipse Ti-E microscope with NIS Elements time-lapse tracking software over a 24 hour period. Final distance, path length, and average speed were quantified after exposure to 1 μM PGE2, 2 μM Wnt Agonist (WntA), or 2 μM WntA with the addition of 1 μM PGE2. The final distance was defined as the distance between the initial and final positions of the cell, represented as a straight line distance. The path length was the total distance travelled from the initial to the final cell position. The average speed of a cell was calculated by dividing the total distance travelled by the time it took to travel between the two positions.
Quantification of average speed showed that PGE2 treated cells travelled at a speed of 10.5 nm/s, which was not significantly different from untreated NE-4C cells that moved at a speed of 11.0 nm/s (Figure 3B). WntA only treatment resulted in a decreased average cell speed of 1.65 nm/s (p = 0.00065). Addition of PGE2 to WntA-treated cells resulted in an average speed of 7.34 nm/s. This suggests that addition of PGE2 elevated the average speed by 439%; an increase of 5.59 nm/s (p = 0.00946) when compared to WntA only-treatment.
In summary, administration of PGE2 treatment leads to significant changes in WntA-regulated cell behaviours such as final distance, path length, and average speed. PGE2 treatment significantly restored the cell kinematic measures which were suppressed by WntA treatment.
Prostaglandin E2 modulates Wnt-induced cell behaviour through PKA and PI-3K kinases
Previous studies in embryonic kidney and colon cancer cells determined that the convergence of PGE2 signalling on the Wnt pathway occurred through the activation of PKA or PI-3K [44–46]. To determine whether PGE2 treatment alters Wnt-induced cell migration behaviour via these kinases in NE-4C cells, we used dihydrochloride hydrate (H89) to block PKA and Wortmannin (Wort) to block PI-3K. Our results show a trend across final distance, path length, and average speed (Figures 2 and 3). With the addition of H89 to WntA + PGE2 treated cells, all cell motility measures significantly decreased compared to the WntA + PGE2 treated cells, resulting in movement profiles that were not statistically different from the WntA-only condition. Specifically, H89-treated cells travelled a final distance of 20.32 μm from the origin (p = 0.02477), path length of 116.01 μm (p = 0.00567), and at an average speed of 1.37 nm/s (p = 0.00073) (Figure 2A and 2B).
With the addition of Wort to WntA + PGE2 treated cells, there was a decreasing trend in final distance and path length but it was not significantly different from PGE2 + WntA treated cells. Only average speed significantly decreased to 2.76 nm/s (N = 3; p = 0.00422) compared to the WntA + PGE2 treatment. Post hoc Dunnett t-test revealed that measurements from the H89 and Wort conditions were not significantly different from the WntA-only treatment, indicating that H89 and Wort significantly diminished the effect of PGE2 on WntA-treated cells. This indicates that PGE2 likely acts through PKA and PI-3K to elicit effects on the WntA-dependent cell motility. However, it appears that H89 may have had a greater effect, suggesting that PGE2 may predominately act through PKA.
Prostaglandin E2 alters cell proliferation behaviour of NE-4C cells induced by Wnt agonist treatment
However, we observed distinct differences in cell phenotype between the WntA, WntA + PGE2 and WntA + PGE2 with H89 or Wort treatment. A majority of the cells treated with WntA adopted a shiny circular shape (indicated by black arrows, Figure 4D). This was not as prevalent in the WntA + PGE2 condition. However, the cells treated with WntA + PGE2 and Wort blocker, adopted the shiny circular phenotype seen in the WntA condition. Cells treated with WntA + PGE2 and H89 blocker adopted a circular appearance as well but a smaller population of these round cells were shiny.
Our experiments showed that cell viability was not affected but a distinct shiny circular cell appearance was observed, which is characteristic of a cell just prior to splitting into two daughter cells. Therefore, we also quantified the split percentage, defined as the percentage of cells that successfully divided into two daughter cells during the recorded time period. As expected, the NE-4C untreated cells demonstrated a split percentage of 100% (Figure 4C), indicating that all cells entering a mitotic phase resulted in cell division. A similar pattern was seen in PGE2-treated cells (97.5%). However, treatment of WntA resulted in a significant decrease of split percentage to 0% (p < 0.001), where mitotic cells appeared to become arrested in a round stage denoted in Figure 4D (WntA Image) with black arrows. The addition of 1 μM PGE2 to WntA-treated cells produced a significant increase in split percentage to 14.7% (p < 0.001, Figure 4C) as compared to WntA only treatment. The cells appear to resume their flat morphology. These results suggest that PGE2 treatment can modify Wnt-induced proliferation behaviour such as split percentage. Following treatment with either H89 or Wort, cells returned to a split percentage of 0% as seen with WntA only treatment (Figure 4C, D). This again indicates that PGE2 likely acts on the Wnt pathway through PKA and PI-3K to modify cell proliferation.
Prostaglandin E2 increases active β-catenin expression in Wnt-induced NE-4C cells
Prostaglandin E2 regulates expression of Wnt-target genes in Wnt-induced NE-4C cells
Ctnnb1 (beta-catenin) levels were not altered with the addition of PGE2 when compared to untreated NE-4C cells, but cells treated with WntA showed a significant increase of RQ value 1.25 (p = 0.0372). Addition of PGE2 to WntA-induced cells led to a further increase of Ctnnb1 level to an RQ value of 1.55, which was significantly different from the WntA-only condition (p = 0.0131). This pattern was consistent with the expression of phospho (active) β-catenin (Ser33/37/Thr41) protein quantified earlier using Western blot analysis. Addition of H89 or Wort to PGE2 + WntA treated cells resulted in RQ values to 0.83 and 0.60, respectively, compared to untreated cells which was a significant decrease compared to the PGE2 + WntA condition (p > 0.001, p > 0.001). The PKA and PI3K blockers, H89 and Wort, appeared to remove the effect of PGE2 on Ctnnb1 expression in WntA-induced cells, while also reversing the influence on Ctnnb1 levels from WntA-only treatment. This suggests that PKA and PI3K signalling may modify Ctnnb1 expression through PGE2 signalling.
NE-4C cells treated with PGE2 alone had a significant decrease in Ptgs2 (prostaglandin-endoperoxide synthase 2; gene encoding COX-2) mRNA levels compared to untreated cells (RQ = 0.56, p < 0.001), while cells treated with WntA had a significant increase of RQ value 2.99 (p = 0.00286). In contrast, when PGE2 was added to WntA-induced NE-4C cells, Ptgs2 expression was further elevated with an RQ value of 4.59 compared to untreated. This value was significantly different from the PGE2 + WntA condition (p = 0.015). Addition of H89 or Wort to PGE2 + WntA treated cells resulted in RQ values of 2.16 and 4.22, but only the H89 treatment was significantly different from the PGE2 + WntA condition (p < 0.001). This suggests that the effect of PGE2 on WntA-induced cells may be through PKA.
Expression of Ccnd1 (cyclin D1) was also altered. Administration of PGE2 treatment to NE-4C cells correlated with a significant increase of an RQ value to 3.68 (p = 0.045) compared to untreated cells, while WntA-treated cells had a significant increase of RQ value to 1.50 (p = 0.048). Addition of PGE2 to WntA-activated cells was associated with a further increase of Ccnd1 expression, with an RQ value 1.99 compared to untreated cells, which was significantly different from WntA-only treated cells (p = 0.047). H89 or Wort added to PGE2 + WntA treated cells had RQ values of 0.74 and 1.42, respectively, which was significantly different from the PGE2 + WntA condition (p = 0.0054, p = 0.0078). The blockers, H89 and Wort, seemed to attenuate the increase of Ccnd1 levels associated with the addition of PGE2 to WntA-induced cells.
In comparison to untreated NE-4C cells, PGE2 treatment did not change levels of Mmp9 (matrix metalloproteinase 9). However, when compared to WntA-induced NE-4C cells, addition of PGE2 treatment to WntA-treated cells caused a significant increase in expression level (p < 0.001). Specifically, with WntA treatment, Mmp9 expression was significantly elevated to an RQ value of 2.19 (p < 0.001) compared to untreated cells, but addition of PGE2 to WntA-induced cells resulted in a further rise of Mmp9 expression with an RQ value of 3.00. H89 and Wort were added to PGE2 + WntA treated cells and RQ values for Mmp9 were 2.16 and 2.68, respectively, compared to the untreated condition. These values were significantly different from the PGE2 + WntA condition. This indicates that the use of H89 and Wort diminished the increase in Mmp9 expression as a result of PGE2 treatment on WntA-induced cells.
Overall, these results demonstrate that PGE2 can raise the expression of Wnt-target genes, specifically, Ctnnb1, Ptgs2, Ccnd1, and Mmp9, in WntA-induced NE-4C cells. Since H89 and Wort attenuated the changes caused by PGE2, PKA and PI3K likely serve as a molecular link for the interaction between the PGE2 and canonical Wnt signalling pathways.
Cell migration and proliferation are crucial components of neural development. Previous studies have shown that elevated levels of PGE2 can result in increased cell motility and proliferation in various non-neuronal cells [46, 54–56]. Recent evidence indicates that abnormalities in cell behaviour can result from the interaction between PGE2 with Wnt signalling pathways [44, 57]. Our current study provides evidence, for the first time, for the cross-talk between these two pathways in neural stem cells. We report that PGE2 treatment elicits changes in cell behaviour such as an increase in components of cell motility and proliferation, as well as expression of Wnt-target genes, in Wnt-activated NE-4C stem cells. Moreover, the stimulatory effects of PGE2 are subdued through the inhibition of downstream pathway kinases, PKA and PI-3K, suggesting that PGE2 acts through these particular kinases to converge with the Wnt pathway.
Previous studies have shown that PGE2 can increase or decrease the activity of canonical Wnt signalling. PGE2 activates several components of the canonical Wnt pathway in colorectal cancer cells (reviewed in ). Specifically in these cells, PGE2 stimulated a significant increase in the activity of Wnt transcription factors, T cell factor-4 (Tcf-4), as well as elevated protein levels of Wnt-target genes . PGE2 acted through its EP2 receptor to modulate β-catenin activity of the Wnt pathway, promoting the growth of colon cancer cells . Wnt activation induced by PGE2 also contributed to abnormal proliferation resulting in enhanced gastric tumorigenesis . Furthermore, PGE2–regulated Wnt signalling had a hepatoprotective effect, aiding in liver regeneration . In pre-osteoblastic cells, concentration-dependent treatment of PGE2 modulated Wnt signalling by altering protein expression of pathway activators, β-catenin and low-density lipoprotein receptor-related protein 5/6 (LRP 5/6), as well as Wnt inhibitor, dickkopf-1 (DKK-1); low doses of PGE2 promoted the Wnt pathway while high doses inhibited it . PGE2 also modified Tcf-luciferase activity of Wnt signalling through the same dose effect . Additionally, in human colorectal adenoma and carcinoma cells, PGE2 treatment up-regulated the protein expression of the Wnt target gene, leucine-rich G-protein coupled receptor 5 (LGR5), which internalizes FZD co-receptor LRP6 and decreases Wnt activity . Altogether, these studies reveal that the interaction between PGE2 and Wnt signalling can have different effects depending on the dose of PGE2 administered and the specific cell type.
We reveal that PGE2 increases the final distance and path length travelled, as well as the average speed of migration in Wnt-activated neuroectodermal stem NE-4C cells. We also show that PGE2 alters the phenotype of Wnt-treated cells, which corresponds to an increase in split percentage. Aberrations in cell motility and proliferation behaviour could have detrimental effects to early development of the nervous system. This is because proper neural development requires an orchestrated system of cellular events, such as migration and proliferation, to occur over particular windows of time . Careful control of these crucial neurobiological processes during prenatal development is required for the formation of complex layered structures in the brain like the cerebral cortex, hippocampus, and cerebellum [62, 63].
Our study adds to the current body of research by showing that PGE2 interferes with the Wnt pathway by attenuating Wnt-dependent cell behaviour in NE-4C cells. This is important because Wnt signalling is involved in a myriad of regulatory processes important for the development and organization of the nervous system . It is thoroughly established that Wnt signalling is instrumental to normal anterior-posterior patterning of the embryo . Wnt proteins are key regulators for the formation of the neural tube, as well as neuronal migration and differentiation [40, 64]. Wnt signalling also modulates neurite outgrowth , axon growth and guidance [67–70], dendritic development and arborization [71, 72], radial migration , and synapse formation and plasticity [74, 75]. Moreover, Wnt signalling is crucial in neuronal fate determination, particularly in the specification and differentiation of neuronal precursors in the midbrain  and forebrain [77, 78]. Furthermore, epithelial stem cells require Wnt/β-catenin signalling for proliferation and quiescent division  and the balance between re-entry and exit of the cell cycle can be altered by Wnt/β-catenin signalling . Additionally, aberrant cortical progenitor cell proliferation patterns and defective hippocampus development can result due to abnormal Wnt signalling . Interestingly, recent findings provide evidence that defective Wnt signalling could contribute to the pathogenesis of psychiatric disorders like schizophrenia and ASD [82–84]. Specifically, Wnt2, located in the putative speech and language region at chromosome 7q31-33, has been identified as a susceptibility gene for autism. [85, 86]. Given the importance of Wnt signalling in prenatal development and the existing interaction between Wnt and PGE2 pathways in NE-4C stem cells, alterations in levels of PGE2 via endogenous and exogenous means may have profound effects on nervous system development.
In addition to quantifying cell behaviour, we also demonstrate that PGE2 can affect the expression of non-phospho (active) β-catenin (Ser33/37/Thr41). Wnt/β-catenin signalling occurs through a complex, highly regulated pathway that involves the phosphorylation of multiple sites on β-catenin, which may promote its degradation or activation and subsequent nuclear internalization. For instance, the phosphorylation of sites Ser33, 37, and Thr41 targets β-catenin for ubiquitination and proteasomal degradation [87, 88]. Quantification of β-catenin that is non-phosphorylated at these sites has become a common measurement for active or stabilized β-catenin expression. Phosphorylation of β-catenin at the site Ser552 has also been correlated with increased β-catenin/TCF mediated transcriptional activity [89, 90]. We found that PGE2 treatment administered to Wnt-activated cells increased the expression of non-phospho (active) β-catenin (Ser33/37/Thr41) protein. In contrast, the phospho- β-catenin (Ser552) levels remained unchanged. It has been established that the regulation of glycogen synthase kinase 3 beta (GSK3β) activity may control stabilization of β-catenin and increased levels of non-phospho (active) β-catenin (Ser33/37/Thr41) protein . It is possible that PGE2 signalling may modify GSK3β activity but this remains to be determined. Nonetheless, the increased levels of non-phospho (active) β-catenin (Ser33/37/Thr41) quantified were in line with our gene expression results that also showed an increase in Ctnnb1 expression as well as other Wnt-target genes. Ctnnb1 encodes for the β-catenin protein, which can regulate cell growth and adhesion and is also a key downstream component of the canonical Wnt pathway. It has also been shown to regulate cortical size; enlarged cortices with increased cortical folds were observed in Ctnnb1 transgenic mice . Interestingly, brain overgrowth and abnormal excess in number of neurons was measured in children with autism . Gene expression of Ctnnb1 was altered in both young and adult autistic cases . Furthermore, de novo mutations of this gene and its relevant network have been ranked significantly as potential autism candidate genes [94, 95]. Within the canonical Wnt pathway, the β-catenin/TCF complex can promote the transcription of target genes including Ptgs2, Ccnd1[97, 98], and Mmp9[99, 100]. Expression of these genes was increased as an effect of elevated PGE2 signalling in our study, and interestingly, previous studies have reported a link between these genes and ASD as described below.
Ptgs2, also known as COX-2, is the key enzyme in prostaglandin biosynthesis, including the production of PGE2. COX-2 is a crucial mediator of inflammation and prostanoid signalling [101, 102]. Polymorphism of Ptgs2 has been associated with ASD . A recent clinical study proved the efficacy of a COX-2 inhibitor drug, celecoxib, as an adjunctive therapy in the treatment of autism: the treatment was superior for treating irritability, social withdrawal, and stereotypy of children with autism .
Another gene affected was Ccnd1. This gene encodes for a protein in the cyclin family, which are important regulators in cell cycle progression, transcription, and neuronal function [105, 106]. The increased levels of Ccnd1, as a result of added PGE2, may be involved with the altered proliferation behaviour visualized in this study. Aberrant Ccnd1 levels have also been associated with ASD. In autistic rat pups (model encompassed administration of valproic acid), Ccnd1 expression was atypical in the cerebellum compared to controls . Another study showed that the dysregulation of Ccnd1 lead to abnormal cell cycle and proliferation, neuronal and network excitability and behaviour, and revealed its potential link to human neuro-cardio-facial-cutaneous and related syndromes, which are associated with developmental abnormalities, cognitive deficits, and autism . Diminished expression of 22q11 genes, which disrupts cortical neurogenesis and cell migration, led to alterations in Ccnd1 levels . The authors explain that a developmental disruption, as such, may alter cortical circuitry and establish vulnerability for developmental disorders, including schizophrenia and autism.
Mmp9 is a membrane of the matrix metalloproteinase (MMP) family, which can target many extracellular proteins including proteases, growth factors, and adhesion molecules  and are involved with the breakdown of the extracellular matrix in normal physiological processes such as embryonic development and tissue remodelling . MMPs are also important in neuronal development, plasticity, and maintenance of neuronal health . Mmp9 has also been shown to regulate the proliferation and migration of embryonic neural stem cells  and participate in neuronal differentiation by regulating neurite elongation and neuronal cell migration [113–115]. Therefore, altered Mmp9 expression may contribute to the deviant behaviour observed in our study. Mmp9 has also been associated with ASD . Elevated levels of MMP9 protein were found in the amniotic fluid of ASD cases compared to controls . Findings from this study provide evidence that molecular and physiological abnormalities in ASD may begin prenatally. Mmp9 has also been implicated in Fragile X syndrome (FXS) , which is characterized by behaviours at the extreme of the autistic spectrum. Using in a mouse model of fragile x (Fmr1 KO), levels of MMP9 was found to be elevated in the hippocampus of Fmr1 KO mice . Furthermore, Minocycline, a drug that inhibits MMP9 activity, has been shown to promote dendrite spine maturation and improve behavioural performance in Fmr1 KO mice . These researchers continued their work in human trials and found that Minocycline taken as a daily dose for 8 weeks led to behavioural improvements in FXS patients. This was consistent with their fmr1 KO mouse model results, indicating that MMP9 activity alters underlying neural defects that contribute to behavioural abnormalities seen in ASD .
Taken altogether, our gene expression results not only show a potential interaction of the PGE2 and canonical Wnt pathway in the nervous system, but also provide further evidence for a link to ASD.
Increasing evidence for the contribution of environmental factors in the etiology of neurodevelopmental disorders like ASD has prompted urgency to reveal their potential exogenous causes and underlying mechanisms . Environmental factors like exposure to drugs, toxins or infectious agents cause disruptions in PGE2 signalling by increasing the levels of oxidative stress, consequent lipid peroxidation, and the immunological response; these factors and consequences that disturb normal PGE2 signalling have all been linked to ASD . We show that increased PGE2 signalling can modify cell migration, proliferation behaviour, and gene expression in Wnt-activated NE-4C stem cells. Aberrant cell migration and proliferation are pathophysiologic mechanisms that impact the brain broadly, and could be possible factors that contribute to the origination of neurodevelopment disorders. Abnormalities in structure, organization, and connectivity of the brain are all indicators of irregular neural cell migration and proliferation. Local distortions in neural cytoarchitecture, dysplasia, and hypoplasia have been described in brains of autistic subjects . Moreover, structural abnormalities and atypical connectivity of the brain in ASD has been identified by a number of research groups [123, 125–128]. Noteworthy, areas of the brain that would be most impacted by dysregulation in neuronal migration and proliferation—that is the cerebellum, cerebral cortex, and hippocampus—are also implicated in ASD [124, 129–131]. Despite the assumptions that can be made from our in vitro results, in vivo models must be employed to further describe the possible effects of PGE2 and its interaction with morphogenic signalling pathways, such as Wnt, during prenatal development.
PGE2 is an important bioactive lipid signalling molecule and its interaction with Wnt signalling pathway could have significant effects on prenatal development. Our study shows for the first time that PGE2 can affect Wnt-dependent cell behaviours and gene expression in neuroectodermal stem cells through PKA and PI-3K. Aberrant PGE2 and Wnt signalling have been linked to ASD. Moreover, altered migration and proliferation due to irregular gene expression during embryonic development in ASD have been suggested in previous studies. Our in vitro study provided further evidence that these aberrations may be potential mechanisms in the genesis of neurodevelopment disorders like ASD.
Mouse NE-4C cells were obtained from American Tissue Culture Collection (ATCC) and grown in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 X penicillin-streptomycin mixture (Invitrogen). Cells were maintained in an incubator containing 5% CO2 at 95% humidity 37°C. Cells were plated on 0.01% poly-L-lysine (Sigma) coated 100 mm culture plates (BD Falcon) and were subcultured at a 1:10 ratio. Supplemented MEM was changed every 2–3 days.
NE-4C cells (ATCC) were dissociated with 0.05% trypsin-EDTA, pelleted and resuspended in complete medium as described above. The cells were plated on poly-L-lysine 0.01% (Sigma, MW 70000–150000 kDa) on 35 mm tissue culture dishes (Sarstedt). Plated cells were incubated in 5% CO2 at 95% humidity 37°C overnight before treatment with Wnt Agonist (WntA, Calbiochem), prostaglandin E2 (PGE2, Sigma) and/or blockers. WntA (2 μM), PGE2 (1 μM), H89 dihydrochloride hydrate (H89, 10 μM, Sigma), Wortmannin (WORT, 1 μM, Sigma) or an equivalent volume of vehicle were added to each well. Cells were treated for 24 hours.
Reverse transcription and real-time PCR
Forward and reverse primer sequences for real-time-PCR
Primer sequences (5′→ 3′)
Amplicon size (base pair)
R: GAGAATAAAGCAACTG CACAAACAA
Western blot analysis
Total protein was extracted from NE-4C cells using the NucleoSpin RNA/Protein Kit (Macherey-Nagel). Samples were separated by polyacrylamide gel electrophoresis (PAGE). Primary antibodies used for EP expression levels include rabbit polyclonal anti-EP1, −EP2, −EP3, −EP4 (1:200; Santa Cruz Biotechnology). Detection of rabbit monoclonal anti-Phospho-Histone H3 (Ser10) (1:1000; Cell Signaling) was used as a measure of cell splitting behaviour. Primary antibodies used for β-catenin expression levels were rabbit monoclonal anti-non-phospho (Active) β-catenin (Ser33/37/Thr41) and rabbit polyclonal anti-phospho-β-catenin (Ser552) (1:1000; Cell Signaling). Blots were reprobed with mouse monoclonal anti-β-Actin (1:10,000; Abcam). Visualization of bound anti-rabbit and anti-mouse horseradish peroxidise-conjugated secondary antibodies was achieved by incubation with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and detection by Geliance 600 Imaging System (Perkin Elmer).
NE-4C cells were seeded onto 35 mm culture plates containing poly-L-lysine coated coverslips and grown overnight at 37°C. The cells were fixed with 50% acetone and 50% methanol for 20 minutes at −20°C and washed with phosphate buffered saline (4.3 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, 1.4 mM KH2PO4). Cells were then incubated with primary antibodies in PBS with 0.3% Triton-X 100 and 2% Normal Goat Serum. Cellular localization of the EP receptors was determined by incubation with anti-EP primary antibodies as described above along with mouse monoclonal anti-Lamin A + C nuclear envelope marker (1:200; Abcam), anti-58 K Golgi marker [58 K-9] (1:100; Abcam), anti-PDI endoplasmic reticulum marker [RL90] (1:100; Abcam) or β-Actin (1:1000; Abcam) at room temperature for 1 hour. Following primary antibody incubation, cells were washed three times with PBS-T for 15 min and incubated with secondary antibodies in PBS-T and 2% NGS for 1 hour at room temperature in the dark. Secondary antibodies used were anti-rabbit fluorescein isothiocyanate (FITC) (1:100; Jackson ImmunoResearch Laboratories) and anti-mouse Texas Red (1:200; Jackson ImmunoResearch Laboratories). Cells were then washed twice with PBS-T for 10 min, followed by a 20 minute incubation of 4′,6-diamidino-2-phenylindole (DAPI) (1:2000; Molecular Probes) at room temperature. Cells were washed twice with PBS-T for 5 min and coverslips were mounted on glass microscope slides with mounting media (Vectashield). The staining was visualized and captured using an Eclipse 80i upright fluorescent microscope with DS-5MC camera (Nikon).
Time-lapse imaging and analysis
Cell behaviour was recorded using Nikon Eclipse Ti-E microscope. Three biological replicates of each treatment condition were performed (N = 3), where an average of 150 cells were tracked. Micrographs were automatically captured every 10 minutes for a 24 hour period from a minimum of three fields. To maintain conditions physiologically suitable for the cells, an enclosed chamber was mounted to the microscope, which was equipped with CO2 supply and temperature thermostat. Cells were kept at 5% CO2, 95% humidity, 37°C. Measurements were completed using NIS Elements software, including a specialized tracking module. Final distance from origin, path length, and average speed were tracked and calculated from an average of 150 cells per treatment condition. Initial and final cell counts were used to determine fold change as a measurement of proliferation. Split percentage was quantified as a measurement of proliferation behaviour. Split percentage was defined as the percentage of cells that fulfilled the complete cell cycle, which was evaluated based on whether the parent cell could successfully split into two daughter cells.
Cell viability analysis
Cells were disassociated and diluted with equal volumes of trypan blue dye (4%). Cell count averages were taken from a minimum of four hemacytometer squares to determine cell number and viability.
All numerical data were presented as mean + SEM of three individual experiments. Statistical analysis was performed using student t-test or one-way analysis of variance (ANOVA) followed by Tukey post-hoc comparisons or Dunnett t-test (2-sided). Differences were considered statistically significant at *p < 0.05, **p < 0.01, or ***p < 0.001.
Autism spectrum disorders
-2: Cyclooxygenases −1 and −2
E-prostanoid 1 through 4
Matrix metalloproteinase 9
- NE-4C stem cell:
Neuroectrodermal stem cells
Protein kinase A
Protein kinase C
Prostaglandin-endoperoxide synthase 2
Wingless-related MMTV integration site
This research work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).
- Breyer RM, Bagdassarian CK, Myers SA, Breyer MD: Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol. 2001, 41: 661-690. 10.1146/annurev.pharmtox.41.1.661.PubMedView ArticleGoogle Scholar
- Brown AS: Epidemiologic studies of exposure to prenatal infection and risk of schizophrenia and autism. Dev Neurobiol. 2012, 72 (10): 1272-1276. 10.1002/dneu.22024.PubMedPubMed CentralView ArticleGoogle Scholar
- Parker-Athill EC, Tan J: Maternal immune activation and autism spectrum disorder: interleukin-6 signaling as a key mechanistic pathway. Neurosignals. 2010, 18 (2): 113-128. 10.1159/000319828.PubMedPubMed CentralView ArticleGoogle Scholar
- Patterson PH: Maternal infection and immune involvement in autism. Trends Mol Med. 2011, 17 (7): 389-394. 10.1016/j.molmed.2011.03.001.PubMedPubMed CentralView ArticleGoogle Scholar
- Grandjean P, Landrigan PJ: Developmental neurotoxicity of industrial chemicals. Lancet. 2006, 368 (9553): 2167-2178. 10.1016/S0140-6736(06)69665-7.PubMedView ArticleGoogle Scholar
- Arndt TL, Stodgell CJ, Rodier PM: The teratology of autism. Int J Dev Neurosci. 2005, 23 (2–3): 189-199.PubMedView ArticleGoogle Scholar
- Sugimoto Y, Narumiya S: Prostaglandin E receptors. J Biol Chem. 2007, 282 (16): 11613-11617. 10.1074/jbc.R600038200.PubMedView ArticleGoogle Scholar
- Coleman RA, Smith WL, Narumiya S: International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev. 1994, 46 (2): 205-229.PubMedGoogle Scholar
- Furuyashiki T, Narumiya S: Stress responses: the contribution of prostaglandin E(2) and its receptors. Nat Rev Endocrinol. 2011, 7 (3): 163-175. 10.1038/nrendo.2010.194.PubMedView ArticleGoogle Scholar
- Andreasson K: Emerging roles of PGE2 receptors in models of neurological disease. Prostaglandins Other Lipid Mediat. 2010, 91 (3–4): 104-112.PubMedPubMed CentralView ArticleGoogle Scholar
- Saint-Dizier M, Guyader-Joly C, Charpigny G, Grimard B, Humblot P, Ponter AA: Expression of enzymes involved in the synthesis of prostaglandin E2 in bovine in vitro-produced embryos. Zygote. 2011, 19 (3): 277-283. 10.1017/S0967199410000596.PubMedView ArticleGoogle Scholar
- Tamiji J, Crawford DA: Prostaglandin E(2) and misoprostol induce neurite retraction in Neuro-2a cells. Biochem Biophys Res Commun. 2010, 398 (3): 450-456. 10.1016/j.bbrc.2010.06.098.PubMedView ArticleGoogle Scholar
- Chen C, Bazan NG: Endogenous PGE2 regulates membrane excitability and synaptic transmission in hippocampal CA1 pyramidal neurons. J Neurophysiol. 2005, 93 (2): 929-941.PubMedView ArticleGoogle Scholar
- Dean SL, Wright CL, Hoffman JF, Wang M, Alger BE, McCarthy MM: Prostaglandin E2 stimulates estradiol synthesis in the cerebellum postnatally with associated effects on Purkinje neuron dendritic arbor and electrophysiological properties. Endocrinology. 2012, 153 (11): 5415-5427. 10.1210/en.2012-1350.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen C, Bazan NG: Lipid signaling: sleep, synaptic plasticity, and neuroprotection. Prostaglandins Other Lipid Mediat. 2005, 77 (1–4): 65-76.PubMedView ArticleGoogle Scholar
- Shimamura M, Zhou P, Casolla B, Qian L, Capone C, Kurinami H, Iadecola C, Anrather J: Prostaglandin E type 1 receptors contribute to neuronal apoptosis after transient forebrain ischemia. J Cereb Blood Flow Metab. 2013, 33 (8): 1207-1214. 10.1038/jcbfm.2013.69.PubMedPubMed CentralView ArticleGoogle Scholar
- Miyagishi H, Kosuge Y, Yoneoka Y, Ozone M, Endo M, Osada N, Ishige K, Kusama-Eguchi K, Ito Y: Prostaglandin E2-induced cell death is mediated by activation of EP2 receptors in motor neuron-like NSC-34 cells. J Pharmacol Sci. 2013, 121 (4): 347-350. 10.1254/jphs.12274SC.PubMedView ArticleGoogle Scholar
- Choi SY, Choi BH, Suh BC, Chae HD, Kim JS, Shin MJ, Kang SS, Negishi M, Kim KT: Potentiation of PGE(2)-mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cells. J Neurochem. 2001, 79 (2): 303-310.PubMedView ArticleGoogle Scholar
- Goncalves MB, Williams EJ, Yip P, Yanez-Munoz RJ, Williams G, Doherty P: The COX-2 inhibitors, meloxicam and nimesulide, suppress neurogenesis in the adult mouse brain. Br J Pharmacol. 2010, 159 (5): 1118-1125. 10.1111/j.1476-5381.2009.00618.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Raisz LG, Pilbeam CC, Fall PM: Prostaglandins: mechanisms of action and regulation of production in bone. Osteoporos Int. 1993, 3 (Suppl 1): 136-140.PubMedView ArticleGoogle Scholar
- Tjandrawinata RR, Dahiya R, Hughes-Fulford M: Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells. Br J Cancer. 1997, 75 (8): 1111-1118. 10.1038/bjc.1997.192.PubMedPubMed CentralView ArticleGoogle Scholar
- Charo C, Holla V, Arumugam T, Hwang R, Yang P, Dubois RN, Menter DG, Logsdon CD, Ramachandran V: Prostaglandin E2 regulates pancreatic stellate cell activity via the EP4 receptor. Pancreas. 2013, 42 (3): 467-474. 10.1097/MPA.0b013e318264d0f8.PubMedPubMed CentralView ArticleGoogle Scholar
- Lin CC, Lin WN, Cheng SE, Tung WH, Wang HH, Yang CM: Transactivation of EGFR/PI3K/Akt involved in ATP-induced inflammatory protein expression and cell motility. J Cell Physiol. 2012, 227 (4): 1628-1638. 10.1002/jcp.22880.PubMedView ArticleGoogle Scholar
- Iwanaga K, Okada M, Murata T, Hori M, Ozaki H: Prostaglandin E2 promotes wound-induced migration of intestinal subepithelial myofibroblasts via EP2, EP3, and EP4 prostanoid receptor activation. J Pharmacol Exp Ther. 2012, 340 (3): 604-611. 10.1124/jpet.111.189845.PubMedView ArticleGoogle Scholar
- Joo HJ, Kim HS, Choi YS, Kim H, Kim SJ, Moon WK: Detection of prostaglandin E2-induced dendritic cell migration into the lymph nodes of mice using a 1.5 T clinical MR scanner. NMR Biomed. 2012, 25 (4): 570-579. 10.1002/nbm.1774.PubMedView ArticleGoogle Scholar
- Mayoral R, Fernandez-Martinez A, Bosca L, Martin-Sanz P: Prostaglandin E2 promotes migration and adhesion in hepatocellular carcinoma cells. Carcinogenesis. 2005, 26 (4): 753-761. 10.1093/carcin/bgi022.PubMedView ArticleGoogle Scholar
- Jaffer S, Mattana J, Singhal PC: Effects of prostaglandin E2 on mesangial cell migration. Am J Nephrol. 1995, 15 (4): 300-305. 10.1159/000168853.PubMedView ArticleGoogle Scholar
- Tamiji J, Crawford DA: Misoprostol elevates intracellular calcium in Neuro-2a cells via protein kinase A. Biochem Biophys Res Commun. 2010, 399 (4): 565-570. 10.1016/j.bbrc.2010.07.112.PubMedView ArticleGoogle Scholar
- Tamiji J, Crawford DA: The neurobiology of lipid metabolism in autism spectrum disorders. Neurosignals. 2010, 18 (2): 98-112. 10.1159/000323189.PubMedView ArticleGoogle Scholar
- El-Ansary A, Al-Ayadhi L: Lipid mediators in plasma of autism spectrum disorders. Lipids Health Dis. 2012, 11: 160-10.1186/1476-511X-11-160.PubMedPubMed CentralView ArticleGoogle Scholar
- Dean SL, Knutson JF, Krebs-Kraft DL, McCarthy MM: Prostaglandin E2 is an endogenous modulator of cerebellar development and complex behavior during a sensitive postnatal period. Eur J Neurosci. 2012, 35 (8): 1218-1229. 10.1111/j.1460-9568.2012.08032.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Bandim JM, Ventura LO, Miller MT, Almeida HC, Costa AE: Autism and Mobius sequence: an exploratory study of children in northeastern Brazil. Arq Neuropsiquiatr. 2003, 61 (2A): 181-185. 10.1590/S0004-282X2003000200004.PubMedView ArticleGoogle Scholar
- Miller MT, Stromland K, Ventura L, Johansson M, Bandim JM, Gillberg C: Autism associated with conditions characterized by developmental errors in early embryogenesis: a mini review. Int J Dev Neurosci. 2005, 23 (2–3): 201-219.PubMedView ArticleGoogle Scholar
- Miller G: Neurological disorders. The mystery of the missing smile. Science. 2007, 316 (5826): 826-827. 10.1126/science.316.5826.826.PubMedView ArticleGoogle Scholar
- Wong C, Crawford D: Lipid Signalling in the Pathology of Autism Spectrum Disorders. Comprehensive Guide to Autism. Edited by: Patel VB, Preedy VR, Martin CR. 2014, New York: Springer, 1259-1283.View ArticleGoogle Scholar
- Kitase Y, Barragan L, Qing H, Kondoh S, Jiang JX, Johnson ML, Bonewald LF: Mechanical induction of PGE2 in osteocytes blocks glucocorticoid-induced apoptosis through both the beta-catenin and PKA pathways. J Bone Miner Res. 2010, 25 (12): 2657-2668. 10.1002/jbmr.168.PubMedPubMed CentralView ArticleGoogle Scholar
- Liu XH, Kirschenbaum A, Weinstein BM, Zaidi M, Yao S, Levine AC: Prostaglandin E2 modulates components of the Wnt signaling system in bone and prostate cancer cells. Biochem Biophys Res Commun. 2010, 394 (3): 715-720. 10.1016/j.bbrc.2010.03.057.PubMedView ArticleGoogle Scholar
- Goessling W, North TE, Loewer S, Lord AM, Lee S, Stoick-Cooper CL, Weidinger G, Puder M, Daley GQ, Moon RT, Zon LI: Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration. Cell. 2009, 136 (6): 1136-1147. 10.1016/j.cell.2009.01.015.PubMedPubMed CentralView ArticleGoogle Scholar
- Kleiveland CR, Kassem M, Lea T: Human mesenchymal stem cell proliferation is regulated by PGE2 through differential activation of cAMP-dependent protein kinase isoforms. Exp Cell Res. 2008, 314 (8): 1831-1838. 10.1016/j.yexcr.2008.02.004.PubMedView ArticleGoogle Scholar
- Ciani L, Salinas PC: WNTs in the vertebrate nervous system: from patterning to neuronal connectivity. Nat Rev Neurosci. 2005, 6 (5): 351-362.PubMedView ArticleGoogle Scholar
- Buechling T, Boutros M: Wnt signaling signaling at and above the receptor level. Curr Top Dev Biol. 2011, 97: 21-53.PubMedView ArticleGoogle Scholar
- Buchanan FG, DuBois RN: Connecting COX-2 and Wnt in cancer. Cancer Cell. 2006, 9 (1): 6-8. 10.1016/j.ccr.2005.12.029.PubMedView ArticleGoogle Scholar
- Liu J, Wu X, Mitchell B, Kintner C, Ding S, Schultz PG: A small-molecule agonist of the Wnt signaling pathway. Angew Chem Int Ed Engl. 2005, 44 (13): 1987-1990. 10.1002/anie.200462552.PubMedView ArticleGoogle Scholar
- Castellone MD, Teramoto H, Williams BO, Druey KM, Gutkind JS: Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis. Science. 2005, 310 (5753): 1504-1510. 10.1126/science.1116221.PubMedView ArticleGoogle Scholar
- Fujino H, West KA, Regan JW: Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. J Biol Chem. 2002, 277 (4): 2614-2619. 10.1074/jbc.M109440200.PubMedView ArticleGoogle Scholar
- Sheng H, Shao J, Washington MK, DuBois RN: Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem. 2001, 276 (21): 18075-18081. 10.1074/jbc.M009689200.PubMedView ArticleGoogle Scholar
- Goto H, Tomono Y, Ajiro K, Kosako H, Fujita M, Sakurai M, Okawa K, Iwamatsu A, Okigaki T, Takahashi T, Inagaki M: Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation. J Biol Chem. 1999, 274 (36): 25543-25549. 10.1074/jbc.274.36.25543.PubMedView ArticleGoogle Scholar
- Liokatis S, Stutzer A, Elsasser SJ, Theillet FX, Klingberg R, van Rossum B, Schwarzer D, Allis CD, Fischle W, Selenko P: Phosphorylation of histone H3 Ser10 establishes a hierarchy for subsequent intramolecular modification events. Nat Struct Mol Biol. 2012, 19 (8): 819-823. 10.1038/nsmb.2310.PubMedView ArticleGoogle Scholar
- Nowak SJ, Corces VG: Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet. 2004, 20 (4): 214-220.PubMedView ArticleGoogle Scholar
- Cadigan KM, Nusse R: Wnt signaling: a common theme in animal development. Genes Dev. 1997, 11 (24): 3286-3305. 10.1101/gad.11.24.3286.PubMedView ArticleGoogle Scholar
- Wu G, He X: Threonine 41 in beta-catenin serves as a key phosphorylation relay residue in beta-catenin degradation. Biochemistry. 2006, 45 (16): 5319-5323. 10.1021/bi0601149.PubMedView ArticleGoogle Scholar
- Fang D, Hawke D, Zheng Y, Xia Y, Meisenhelder J, Nika H, Mills GB, Kobayashi R, Hunter T, Lu Z: Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J Biol Chem. 2007, 282 (15): 11221-11229. 10.1074/jbc.M611871200.PubMedPubMed CentralView ArticleGoogle Scholar
- He XC, Yin T, Grindley JC, Tian Q, Sato T, Tao WA, Dirisina R, Porter-Westpfahl KS, Hembree M, Johnson T, Wiedemann LM, Barrett TA, Hood L, Wu H, Li L: PTEN-deficient intestinal stem cells initiate intestinal polyposis. Nat Genet. 2007, 39 (2): 189-198. 10.1038/ng1928.PubMedPubMed CentralView ArticleGoogle Scholar
- Aso H, Ito S, Mori A, Suganuma N, Morioka M, Takahara N, Kondo M, Hasegawa Y: Differential regulation of airway smooth muscle cell migration by E-prostanoid receptor subtypes. Am J Respir Cell Mol Biol. 2013, 48 (3): 322-329. 10.1165/rcmb.2012-0158OC.PubMedView ArticleGoogle Scholar
- Bai XM, Zhang W, Liu NB, Jiang H, Lou KX, Peng T, Ma J, Zhang L, Zhang H, Leng J: Focal adhesion kinase: important to prostaglandin E2-mediated adhesion, migration and invasion in hepatocellular carcinoma cells. Oncol Rep. 2009, 21 (1): 129-136.PubMedGoogle Scholar
- Yen JH, Khayrullina T, Ganea D: PGE2-induced metalloproteinase-9 is essential for dendritic cell migration. Blood. 2008, 111 (1): 260-270. 10.1182/blood-2007-05-090613.PubMedPubMed CentralView ArticleGoogle Scholar
- Oshima H, Oshima M: The role of PGE2-associated inflammatory responses in gastric cancer development. Semin Immunopathol. 2013, 35 (2): 139-150. 10.1007/s00281-012-0353-5.PubMedView ArticleGoogle Scholar
- Shao J, Jung C, Liu C, Sheng H: Prostaglandin E2 Stimulates the beta-catenin/T cell factor-dependent transcription in colon cancer. J Biol Chem. 2005, 280 (28): 26565-26572. 10.1074/jbc.M413056200.PubMedView ArticleGoogle Scholar
- North TE, Babu IR, Vedder LM, Lord AM, Wishnok JS, Tannenbaum SR, Zon LI, Goessling W: PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury. Proc Natl Acad Sci U S A. 2010, 107 (40): 17315-17320. 10.1073/pnas.1008209107.PubMedPubMed CentralView ArticleGoogle Scholar
- Al-Kharusi MR, Smartt HJ, Greenhough A, Collard TJ, Emery ED, Williams AC, Paraskeva C: LGR5 promotes survival in human colorectal adenoma cells and is upregulated by PGE2: implications for targeting adenoma stem cells with NSAIDs. Carcinogenesis. 2013, 34 (5): 1150-1157. 10.1093/carcin/bgt020.PubMedPubMed CentralView ArticleGoogle Scholar
- Stiles J, Jernigan TL: The basics of brain development. Neuropsychol Rev. 2010, 20 (4): 327-348. 10.1007/s11065-010-9148-4.PubMedPubMed CentralView ArticleGoogle Scholar
- Evsyukova I, Plestant C, Anton ES: Integrative mechanisms of oriented neuronal migration in the developing brain. Annu Rev Cell Dev Biol. 2013, 29: 299-353. 10.1146/annurev-cellbio-101512-122400.PubMedPubMed CentralView ArticleGoogle Scholar
- Hatten ME: Central nervous system neuronal migration. Annu Rev Neurosci. 1999, 22: 511-539. 10.1146/annurev.neuro.22.1.511.PubMedView ArticleGoogle Scholar
- Ille F, Sommer L: Wnt signaling: multiple functions in neural development. Cell Mol Life Sci. 2005, 62 (10): 1100-1108. 10.1007/s00018-005-4552-2.PubMedView ArticleGoogle Scholar
- Arkell RM, Fossat N, Tam PP: Wnt signalling in mouse gastrulation and anterior development: new players in the pathway and signal output. Curr Opin Genet Dev. 2013, 23 (4): 454-460. 10.1016/j.gde.2013.03.001.PubMedView ArticleGoogle Scholar
- Lu W, Yamamoto V, Ortega B, Baltimore D: Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth. Cell. 2004, 119 (1): 97-108. 10.1016/j.cell.2004.09.019.PubMedView ArticleGoogle Scholar
- Bovolenta P, Rodriguez J, Esteve P: Frizzled/RYK mediated signalling in axon guidance. Development. 2006, 133 (22): 4399-4408. 10.1242/dev.02592.PubMedView ArticleGoogle Scholar
- Lyuksyutova AI, Lu CC, Milanesio N, King LA, Guo N, Wang Y, Nathans J, Tessier-Lavigne M, Zou Y: Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling. Science. 2003, 302 (5652): 1984-1988. 10.1126/science.1089610.PubMedView ArticleGoogle Scholar
- Zou Y: Wnt signaling in axon guidance. Trends Neurosci. 2004, 27 (9): 528-532. 10.1016/j.tins.2004.06.015.PubMedView ArticleGoogle Scholar
- Sanchez-Camacho C, Rodriguez J, Ruiz JM, Trousse F, Bovolenta P: Morphogens as growth cone signalling molecules. Brain Res Brain Res Rev. 2005, 49 (2): 242-252. 10.1016/j.brainresrev.2004.10.004.PubMedView ArticleGoogle Scholar
- Rosso SB, Sussman D, Wynshaw-Boris A, Salinas PC: Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development. Nat Neurosci. 2005, 8 (1): 34-42. 10.1038/nn1374.PubMedView ArticleGoogle Scholar
- Wayman GA, Impey S, Marks D, Saneyoshi T, Grant WF, Derkach V, Soderling TR: Activity-dependent dendritic arborization mediated by CaM-kinase I activation and enhanced CREB-dependent transcription of Wnt-2. Neuron. 2006, 50 (6): 897-909. 10.1016/j.neuron.2006.05.008.PubMedView ArticleGoogle Scholar
- Zhou CJ, Zhao C, Pleasure SJ: Wnt signaling mutants have decreased dentate granule cell production and radial glial scaffolding abnormalities. J Neurosci. 2004, 24 (1): 121-126. 10.1523/JNEUROSCI.4071-03.2004.PubMedView ArticleGoogle Scholar
- Chen J, Park CS, Tang SJ: Activity-dependent synaptic Wnt release regulates hippocampal long term potentiation. J Biol Chem. 2006, 281 (17): 11910-11916. 10.1074/jbc.M511920200.PubMedView ArticleGoogle Scholar
- Gogolla N, Galimberti I, Deguchi Y, Caroni P: Wnt signaling mediates experience-related regulation of synapse numbers and mossy fiber connectivities in the adult hippocampus. Neuron. 2009, 62 (4): 510-525. 10.1016/j.neuron.2009.04.022.PubMedView ArticleGoogle Scholar
- Prakash N, Brodski C, Naserke T, Puelles E, Gogoi R, Hall A, Panhuysen M, Echevarria D, Sussel L, Weisenhorn DM, Martinez S, Arenas E, Simeone A, Wurst W: A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo. Development. 2006, 133 (1): 89-98. 10.1242/dev.02181.PubMedView ArticleGoogle Scholar
- Hirabayashi Y, Gotoh Y: Stage-dependent fate determination of neural precursor cells in mouse forebrain. Neurosci Res. 2005, 51 (4): 331-336. 10.1016/j.neures.2005.01.004.PubMedView ArticleGoogle Scholar
- Zhou CJ, Borello U, Rubenstein JL, Pleasure SJ: Neuronal production and precursor proliferation defects in the neocortex of mice with loss of function in the canonical Wnt signaling pathway. Neuroscience. 2006, 142 (4): 1119-1131. 10.1016/j.neuroscience.2006.07.007.PubMedView ArticleGoogle Scholar
- Lowry WE, Blanpain C, Nowak JA, Guasch G, Lewis L, Fuchs E: Defining the impact of beta-catenin/Tcf transactivation on epithelial stem cells. Genes Dev. 2005, 19 (13): 1596-1611. 10.1101/gad.1324905.PubMedPubMed CentralView ArticleGoogle Scholar
- Chenn A, Walsh CA: Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science. 2002, 297 (5580): 365-369. 10.1126/science.1074192.PubMedView ArticleGoogle Scholar
- Lee SM, Tole S, Grove E, McMahon AP: A local Wnt-3a signal is required for development of the mammalian hippocampus. Development. 2000, 127 (3): 457-467.PubMedGoogle Scholar
- Kalkman HO: A review of the evidence for the canonical Wnt pathway in autism spectrum disorders. Mol Autism. 2012, 3 (1): 10-10.1186/2040-2392-3-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Okerlund ND, Cheyette BN: Synaptic Wnt signaling-a contributor to major psychiatric disorders?. J Neurodev Disord. 2011, 3 (2): 162-174. 10.1007/s11689-011-9083-6.PubMedPubMed CentralView ArticleGoogle Scholar
- Cotter D, Kerwin R, Al-Sarraji S, Brion JP, Chadwich A, Lovestone S, Anderton B, Everall I: Abnormalities of Wnt signalling in schizophrenia--evidence for neurodevelopmental abnormality. Neuroreport. 1998, 9 (7): 1379-1383. 10.1097/00001756-199805110-00024.PubMedView ArticleGoogle Scholar
- Lin PI, Chien YL, Wu YY, Chen CH, Gau SS, Huang YS, Liu SK, Tsai WC, Chiu YN: The WNT2 gene polymorphism associated with speech delay inherent to autism. Res Dev Disabil. 2012, 33 (5): 1533-1540. 10.1016/j.ridd.2012.03.004.PubMedView ArticleGoogle Scholar
- Wassink TH, Piven J, Vieland VJ, Huang J, Swiderski RE, Pietila J, Braun T, Beck G, Folstein SE, Haines JL, Sheffield VC: Evidence supporting WNT2 as an autism susceptibility gene. Am J Med Genet. 2001, 105 (5): 406-413. 10.1002/ajmg.1401.PubMedView ArticleGoogle Scholar
- Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y, Zhang Z, Lin X, He X: Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell. 2002, 108 (6): 837-847. 10.1016/S0092-8674(02)00685-2.PubMedView ArticleGoogle Scholar
- Kimelman D, Xu W: Beta-catenin destruction complex: insights and questions from a structural perspective. Oncogene. 2006, 25 (57): 7482-7491. 10.1038/sj.onc.1210055.PubMedView ArticleGoogle Scholar
- Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO: Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase. J Biol Chem. 2006, 281 (15): 9971-9976. 10.1074/jbc.M508778200.PubMedView ArticleGoogle Scholar
- Zhao J, Yue W, Zhu MJ, Sreejayan N, Du M: AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of beta-catenin at Ser 552. Biochem Biophys Res Commun. 2010, 395 (1): 146-151. 10.1016/j.bbrc.2010.03.161.PubMedPubMed CentralView ArticleGoogle Scholar
- Gao C, Chen G, Romero G, Moschos S, Xu X, Hu J: Induction of Gsk3beta-beta-TrCP Interaction Is Required for Late Phase Stabilization of beta-catenin in Canonical Wnt Signaling. J Biol Chem. 2014, Epub ahead of printGoogle Scholar
- Courchesne E, Mouton PR, Calhoun ME, Semendeferi K, Ahrens-Barbeau C, Hallet MJ, Barnes CC, Pierce K: Neuron number and size in prefrontal cortex of children with autism. JAMA. 2011, 306 (18): 2001-2010. 10.1001/jama.2011.1638.PubMedView ArticleGoogle Scholar
- Chow ML, Pramparo T, Winn ME, Barnes CC, Li HR, Weiss L, Fan JB, Murray S, April C, Belinson H, Fu XD, Wynshaw-Boris A, Schork NJ, Courchesne E: Age-dependent brain gene expression and copy number anomalies in autism suggest distinct pathological processes at young versus mature ages. PLoS Genet. 2012, 8 (3): e1002592-10.1371/journal.pgen.1002592.PubMedPubMed CentralView ArticleGoogle Scholar
- Krumm N, O’Roak BJ, Shendure J, Eichler EE: A de novo convergence of autism genetics and molecular neuroscience. Trends Neurosci. 2014, 37 (2): 95-105. 10.1016/j.tins.2013.11.005.PubMedPubMed CentralView ArticleGoogle Scholar
- O’Roak BJ, Vives L, Girirajan S, Karakoc E, Krumm N, Coe BP, Levy R, Ko A, Lee C, Smith JD, Turner EH, Stanaway IB, Vernot B, Malig M, Baker C, Reilly B, Akey JM, Borenstein E, Rieder MJ, Nickerson DA, Bernier R, Shendure J, Eichler EE: Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature. 2012, 485 (7397): 246-250. 10.1038/nature10989.PubMedPubMed CentralView ArticleGoogle Scholar
- Nunez F, Bravo S, Cruzat F, Montecino M, De Ferrari GV: Wnt/beta-catenin signaling enhances cyclooxygenase-2 (COX2) transcriptional activity in gastric cancer cells. PLoS One. 2011, 6 (4): e18562-10.1371/journal.pone.0018562.PubMedPubMed CentralView ArticleGoogle Scholar
- Klein EA, Assoian RK: Transcriptional regulation of the cyclin D1 gene at a glance. J Cell Sci. 2008, 121 (Pt 23): 3853-3857.PubMedPubMed CentralView ArticleGoogle Scholar
- Shtutman M, Zhurinsky J, Simcha I, Albanese C, D’Amico M, Pestell R, Ben-Ze’ev A: The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A. 1999, 96 (10): 5522-5527. 10.1073/pnas.96.10.5522.PubMedPubMed CentralView ArticleGoogle Scholar
- Ingraham CA, Park GC, Makarenkova HP, Crossin KL: Matrix metalloproteinase (MMP)-9 induced by Wnt signaling increases the proliferation and migration of embryonic neural stem cells at low O2 levels. J Biol Chem. 2011, 286 (20): 17649-17657. 10.1074/jbc.M111.229427.PubMedPubMed CentralView ArticleGoogle Scholar
- Wu B, Crampton SP, Hughes CC: Wnt signaling induces matrix metalloproteinase expression and regulates T cell transmigration. Immunity. 2007, 26 (2): 227-239. 10.1016/j.immuni.2006.12.007.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen C: COX-2’s new role in inflammation. Nat Chem Biol. 2010, 6 (6): 401-402. 10.1038/nchembio.375.PubMedView ArticleGoogle Scholar
- Seibert K, Masferrer JL: Role of inducible cyclooxygenase (COX-2) in inflammation. Receptor. 1994, 4 (1): 17-23.PubMedGoogle Scholar
- Yoo HJ, Cho IH, Park M, Cho E, Cho SC, Kim BN, Kim JW, Kim SA: Association between PTGS2 polymorphism and autism spectrum disorders in Korean trios. Neurosci Res. 2008, 62 (1): 66-69. 10.1016/j.neures.2008.05.008.PubMedView ArticleGoogle Scholar
- Asadabadi M, Mohammadi MR, Ghanizadeh A, Modabbernia A, Ashrafi M, Hassanzadeh E, Forghani S, Akhondzadeh S: Celecoxib as adjunctive treatment to risperidone in children with autistic disorder: a randomized, double-blind, placebo-controlled trial. Psychopharmacology (Berl). 2013, 225 (1): 51-59. 10.1007/s00213-012-2796-8.View ArticleGoogle Scholar
- Bloom J, Cross FR: Multiple levels of cyclin specificity in cell-cycle control. Nat Rev Mol Cell Biol. 2007, 8 (2): 149-160. 10.1038/nrm2105.PubMedView ArticleGoogle Scholar
- Lim S, Kaldis P: Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development. 2013, 140 (15): 3079-3093. 10.1242/dev.091744.PubMedView ArticleGoogle Scholar
- Kim JE, Shin MS, Seo TB, Ji ES, Baek SS, Lee SJ, Park JK, Kim CJ: Treadmill exercise ameliorates motor disturbance through inhibition of apoptosis in the cerebellum of valproic acid-induced autistic rat pups. Mol Med Rep. 2013, 8 (2): 327-334.PubMedGoogle Scholar
- Pucilowska J, Puzerey PA, Karlo JC, Galan RF, Landreth GE: Disrupted ERK signaling during cortical development leads to abnormal progenitor proliferation, neuronal and network excitability and behavior, modeling human neuro-cardio-facial-cutaneous and related syndromes. J Neurosci. 2012, 32 (25): 8663-8677. 10.1523/JNEUROSCI.1107-12.2012.PubMedView ArticleGoogle Scholar
- Meechan DW, Tucker ES, Maynard TM, LaMantia AS: Diminished dosage of 22q11 genes disrupts neurogenesis and cortical development in a mouse model of 22q11 deletion/DiGeorge syndrome. Proc Natl Acad Sci U S A. 2009, 106 (38): 16434-16445. 10.1073/pnas.0905696106.PubMedPubMed CentralView ArticleGoogle Scholar
- McCawley LJ, Matrisian LM: Matrix metalloproteinases: they’re not just for matrix anymore!. Curr Opin Cell Biol. 2001, 13 (5): 534-540. 10.1016/S0955-0674(00)00248-9.PubMedView ArticleGoogle Scholar
- Vu TH, Werb Z: Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev. 2000, 14 (17): 2123-2133. 10.1101/gad.815400.PubMedView ArticleGoogle Scholar
- Fujioka H, Dairyo Y, Yasunaga K, Emoto K: Neural functions of matrix metalloproteinases: plasticity, neurogenesis, and disease. Biochem Res Int. 2012, 2012: 789083-PubMedPubMed CentralView ArticleGoogle Scholar
- Chambaut-Guerin AM, Herigault S, Rouet-Benzineb P, Rouher C, Lafuma C: Induction of matrix metalloproteinase MMP-9 (92-kDa gelatinase) by retinoic acid in human neuroblastoma SKNBE cells: relevance to neuronal differentiation. J Neurochem. 2000, 74 (2): 508-517.PubMedView ArticleGoogle Scholar
- Ferguson TA, Muir D: MMP-2 and MMP-9 increase the neurite-promoting potential of schwann cell basal laminae and are upregulated in degenerated nerve. Mol Cell Neurosci. 2000, 16 (2): 157-167. 10.1006/mcne.2000.0859.PubMedView ArticleGoogle Scholar
- Shubayev VI, Myers RR: Matrix metalloproteinase-9 promotes nerve growth factor-induced neurite elongation but not new sprout formation in vitro. J Neurosci Res. 2004, 77 (2): 229-239. 10.1002/jnr.20160.PubMedView ArticleGoogle Scholar
- Abdallah M, Michel T: Matrix metalloproteinases in autism spectrum disorders. J Mol Psychiatry. 2013, 1 (1): 1-5.View ArticleGoogle Scholar
- Abdallah MW, Pearce BD, Larsen N, Greaves-Lord K, Norgaard-Pedersen B, Hougaard DM, Mortensen EL, Grove J: Amniotic fluid MMP-9 and neurotrophins in autism spectrum disorders: an exploratory study. Autism Res. 2012, 5 (6): 428-433. 10.1002/aur.1254.PubMedView ArticleGoogle Scholar
- Janusz A, Milek J, Perycz M, Pacini L, Bagni C, Kaczmarek L, Dziembowska M: The Fragile X mental retardation protein regulates matrix metalloproteinase 9 mRNA at synapses. J Neurosci. 2013, 33 (46): 18234-18241. 10.1523/JNEUROSCI.2207-13.2013.PubMedView ArticleGoogle Scholar
- Bilousova TV, Dansie L, Ngo M, Aye J, Charles JR, Ethell DW, Ethell IM: Minocycline promotes dendritic spine maturation and improves behavioural performance in the fragile X mouse model. J Med Genet. 2009, 46 (2): 94-102.PubMedView ArticleGoogle Scholar
- Paribello C, Tao L, Folino A, Berry-Kravis E, Tranfaglia M, Ethell IM, Ethell DW: Open-label add-on treatment trial of minocycline in fragile X syndrome. BMC Neurol. 2010, 10: 91-10.1186/1471-2377-10-91.PubMedPubMed CentralView ArticleGoogle Scholar
- Vo BT, Morton D Jr, Komaragiri S, Millena AC, Leath C, Khan SA: TGF-beta effects on prostate cancer cell migration and invasion are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway. Endocrinology. 2013, 154 (5): 1768-1779. 10.1210/en.2012-2074.PubMedPubMed CentralView ArticleGoogle Scholar
- Landrigan PJ, Lambertini L, Birnbaum LS: A research strategy to discover the environmental causes of autism and neurodevelopmental disabilities. Environ Health Perspect. 2012, 120 (7): a258-a260. 10.1289/ehp.1104285.PubMedPubMed CentralView ArticleGoogle Scholar
- Ecker C, Ronan L, Feng Y, Daly E, Murphy C, Ginestet CE, Brammer M, Fletcher PC, Bullmore ET, Suckling J, Baron-Cohen S, Williams S, Loth E, Consortium MRCAIMS, Murphy DG: Intrinsic gray-matter connectivity of the brain in adults with autism spectrum disorder. Proc Natl Acad Sci U S A. 2013, 110 (32): 13222-13227. 10.1073/pnas.1221880110.PubMedPubMed CentralView ArticleGoogle Scholar
- Wegiel J, Kuchna I, Nowicki K, Imaki H, Wegiel J, Marchi E, Ma SY, Chauhan A, Chauhan V, Bobrowicz TW, de Leon M, Louis LA, Cohen IL, London E, Brown WT, Wisniewski T: The neuropathology of autism: defects of neurogenesis and neuronal migration, and dysplastic changes. Acta Neuropathol. 2010, 119 (6): 755-770. 10.1007/s00401-010-0655-4.PubMedPubMed CentralView ArticleGoogle Scholar
- Abrams DA, Lynch CJ, Cheng KM, Phillips J, Supekar K, Ryali S, Uddin LQ, Menon V: Underconnectivity between voice-selective cortex and reward circuitry in children with autism. Proc Natl Acad Sci U S A. 2013, 110 (29): 12060-12065. 10.1073/pnas.1302982110.PubMedPubMed CentralView ArticleGoogle Scholar
- Aoki Y, Abe O, Nippashi Y, Yamasue H: Comparison of white matter integrity between autism spectrum disorder subjects and typically developing individuals: a meta-analysis of diffusion tensor imaging tractography studies. Mol Autism. 2013, 4 (1): 25-10.1186/2040-2392-4-25.PubMedPubMed CentralView ArticleGoogle Scholar
- Belmonte MK, Allen G, Beckel-Mitchener A, Boulanger LM, Carper RA, Webb SJ: Autism and abnormal development of brain connectivity. J Neurosci. 2004, 24 (42): 9228-9231. 10.1523/JNEUROSCI.3340-04.2004.PubMedView ArticleGoogle Scholar
- Minshew NJ, Williams DL: The new neurobiology of autism: cortex, connectivity, and neuronal organization. Arch Neurol. 2007, 64 (7): 945-950. 10.1001/archneur.64.7.945.PubMedPubMed CentralView ArticleGoogle Scholar
- Hashimoto T, Tayama M, Murakawa K, Yoshimoto T, Miyazaki M, Harada M, Kuroda Y: Development of the brainstem and cerebellum in autistic patients. J Autism Dev Disord. 1995, 25 (1): 1-18. 10.1007/BF02178163.PubMedView ArticleGoogle Scholar
- Penzes P, Buonanno A, Passafaro M, Sala C, Sweet RA: Developmental vulnerability of synapses and circuits associated with neuropsychiatric disorders. J Neurochem. 2013, 126 (2): 165-182. 10.1111/jnc.12261.PubMedPubMed CentralView ArticleGoogle Scholar
- Cauda F, Geda E, Sacco K, D’Agata F, Duca S, Geminiani G, Keller R: Grey matter abnormality in autism spectrum disorder: an activation likelihood estimation meta-analysis study. Neurol Neurosurg Psychiatry. 2011, 82 (12): 1304-1313. 10.1136/jnnp.2010.239111.View ArticleGoogle Scholar
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