Different genes have already been associated with cortical malformation, including FCD type II, directly related to cell proliferation, cell cycle regulation, neuronal migration, late cortical organization and structure and function of the cytoskeleton (9–12). Alterations in these genes can have consequences for CNS, including the place where the cortex can be affected (5).
In this work, we explored an WNT pathway genes array from samples obtained from dysplastic tissue and from the tissue adjacent to the main dysplastic lesion.
Both patients were consistent to FCD type II, with medically refractory seizures. Histopathology presents cortical dyslamination and large, dysplastic neurons, with balloon cells(5).
Both patients are female and with similar ages, so, there may be similarities in responses of cells. We find it more correct to explore and work on the results of Patient 1, Patient 2, and both patients as a group, separately.
The increased relative expression of KREMEN1 gene by 13.45 times may influence the blocking of activation of the WNT pathway. The KREMEN1 gene encodes the dickkopf homolog 1 high-affinity transmembrane receptor that functionally cooperates with DKK1 and 2 to block signaling in the WNT/beta-catenin pathway. The DKK receptor coupled to LPR5/6 acts as a major inhibitor of WNT pathway activation, not allowing LPR5/6 to bind to Frizzled pathway receptors. The KREMEN1 protein is still associated with the mechanism of endocytosis of LRP5/6 receptors, which in dysplastic brain tissue could further contribute to blocking WNT activation. Physiologically, the reduction or absence of DKK1 potentiates WNT signaling by keeping LRP5 or LRP6 in the cell membrane. The consequence of this process may be an increase in intracellular β-catenin and the need for degradation, given the non-activation of WNT pathway and phosphorylation of β-catenin (13).
The dysplastic tissue of patient 1 also showed a 53.74-fold increase in the expression of the AXIN2 gene, which implies that there is an increase in this transcript in the intracellular environment of this tissue. The AXIN2 protein plays an important role in regulating the stability of β-catenin in the WNT signaling pathway. The AXIN2 protein is responsible together with CK1, GSK and APC to phosphorylate β-catenin. Phosphorylated β-catenin undergoes the process of βTrCP-mediated ubiquitination, increasing proteostasis and degrading β-catenin in the cytosol (14). This AXIN2-mediated mechanism is characteristic of the non-activated WNT pathway where the main objective is the degradation of β-catenin not used as a transcription factor. The β-catenin dysregulation is an important event in the genesis of a number of malignant diseases. Control of AXIN2 mediated β-catenin phosphorylation is essentially important for normal remyelination kinetics, thus AXIN2 is being observed as a regulatory and therapeutic target in brain lesions and newborn remyelination (15).
The dysplastic brain tissue of patient 1 still showed a 7.55-fold increase in the expression of the FZD9 gene. This gene is member of the frizzled gene family that encode transmembrane domain proteins that are receptors for WNT signaling proteins. FZD9 is coupled to the β-catenin canonical signaling pathway (WNT2) and its activation promotes the inhibition of the GSK3 kinase, β-catenin nuclear accumulation and activation of WNT target genes (16). The low activation of receptors of the WNT pathway in this tissue, due to alterations in the β-catenin degradation pathway and DKK blockade, can lead the cell to establish a compensatory mechanism seeking to increase the number of FZD receptors in the cell membrane. Activation of FZD9 has a negative regulatory role in the clustering of acetylcholine receptors (AChR) (17).
Furthermore, the 33.13-fold increase in the expression of the PPP2R1A gene, responsible for the negative control of cell growth and division, suggests that dysplastic tissue presents a response compatible with a control in cell division rates by transcribing a gene that blocks cycle and division, thus appearing to be an unregulated compensation referring to possible hyperactivity of this tissue. In another way, the PPP2CA gene showed a 33.12-fold reduction in relative expression, being also a gene responsible for the negative control of the cell cycle(18).
In short, the molecular presentation of patient 1 allows us to suggest possible alterations in the dysplastic tissue compared to the tissue adjacent to the lesion..
Dysplastic brain tissue from patient 2 showed a smaller amount of genes with alterations in their relative expression compared to patient 1. On the other hand, some mechanisms have similar pathways related to non-activation of the WNT pathway. The 319.57-fold increase in CSNK1A1 also represents a possible increase in β-catenin degradation because this gene transcribes the CK1 protein, which is also associated with GSK, AXIN2 and APC, and promotes β-catenin phosphorylation and ubiquitination and its consequent degradation (19). As in patient 1, there was a 12.7-fold increase in FZD9 expression in dysplastic tissue. As already mentioned, FZD9 is coupled to the β-catenin canonical signaling pathway (WNT2) and its activation promotes the inhibition of the GSK3 kinase, β-catenin nuclear accumulation and activation of WNT target genes. In patient 2, there was a 316.26-fold reduction in the expression of CTNNBIP1, a gene involved in the inhibition of transcription factors in cells after activation of the WNT pathway. The protein translated by this gene is a negative regulator of the WNT signaling pathway. CTNNBIP1 prevents the interaction of TCF/LEF proteins that coupled to intranuclear β-catenin after activation of the WNT pathway, promote the transcription of genes responsible for maintaining growth, proliferation, migration, and cell cycle control(20). The reduction in the expression of this gene may lead to lack of control regarding the coupling of non-phosphorylated β-catenin after activation of the WNT pathway, as well as it may be a cellular response to a possible accumulation of phosphorylated β-catenin that tries to be controlled by the increase in CSNK1A1 and non-phosphorylated that tries to be used as a transcription factor together with TCF/LEF due to the increased interaction of these factors mediated by the reduction in the expression of CTNNBIP1.
Analyzing the results of the relative expression when grouping patient 1 with patient 2 and comparing the injured tissue with the adjacent tissue, the results follow suggestive of an increase in cytoplasmic β-catenin degradation by phosphorylation and inactivation of the WNT pathway mediated by an increase in DKK on LPR5/6 receptor. There was a 7.13-fold increase in the expression of AXIN2, involved in the phosphorylation and ubiquitination of β-catenin and its consequent degradation in the non-active form of the WNT pathway. There was also an increase in KREMEN1 and KREMEN2 of 5.03 and 6.38 times, respectively, which induces the blocking of WNT activation by the increase of DKK, causing the non-activation of LPR5/6 receptors and bound to the Frizzled WNT receptors. As in patient 2, analyzing in a grouped way, there was a 33.89-fold reduction in the expression of the inhibitor of the binding between β-catenin and the transcription factor TCF/LEF, also appearing to be a compensation mechanism for transcription of genes related to activation of the WNT pathway which in diseased tissue appears not physiologically activated.
Hypothesis about the WNT pathway in brain tissue with focal cortical dysplasia
Overall, this study revealed some changes that may be suggestive of the refractory epileptogenic phenotype associated with diseased tissue in patients with Focal Cortical Dysplasia type II. Based on the results obtained in this study and in the current literature, the hypothesis of the involvement of the WNT pathway in the mechanisms associated with high excitability of the diseased tissue can be constructed and alternative suggestions for mediators for the control of seizures can be considered based on blockade, silencing or stimulation of transcription of genes involved in the WNT pathway.
The WNT pathway has recently been associated with epilepsies as it participates in the pathway in neurogenesis induced by seizures, as well as in the essential characteristic of the pathology, being observed as a potential therapeutic target for future antiepileptogenic treatments (15).
Specifically, in relation to epilepsy, the focus of WNT signaling involvement has been in relation to seizure-induced neurogenesis and neuronal death in the acute and chronic phases of the seizure. In different experimental models of epilepsy, increases in the expression of WNT signaling components were evidenced and they were associated with an increase in neurogenesis and neuronal death, commonly observed after seizures (21–23) .
Some protective and beneficial mechanisms related to protection against neuronal death are observed after modulation of the WNT pathway in models of cerebral ischemia and induced seizures, with the WNT pathway being a non-aberrant repair control mechanism of neurogenic brain areas (24).
The increase in DKK signaling induced by the increase in the KREMEN 1 and 2 gene transcript can induce inhibition of the WNT pathway by blocking LRP5/5 receptors and their consequent non-binding to Frizzled receptors, thus keeping cells in the dysplastic tissue, in a not activated state of the pathway. It was demonstrated in an experimental model that when using an antagonist of the endogenous DKK1 inhibitor, there was a protection against the development of hippocampal sclerosis, a finding that is closely related to the problems of temporal lobe epilepsy (25). Therefore, by antagonizing the DKK1 inhibitor, there is protection against the formation of hippocampal sclerosis after a seizure. This process using the DKK1 antagonist, which is an inhibitor of the WNT pathway, probably increases the binding of LPR5/6 to Frizzled receptors, leaving the WNT pathway in a more active state in these cells and this activation mediates protection to this tissue. When comparing dysplastic brain tissue with the adjacent one, we suggest that there is an increase in DKK blocking the WNT pathway in this diseased tissue, not allowing an adequate homeostasis necessary to promote a better post-seizure condition. WNT blockade may be related to the worsening of the epileptogenesis process since reduced neurogenesis in the chronic phase of epilepsy can potentially exacerbate tissue hyper excitability over time due to a reduction in the number of inhibitory neurons and their generated connections, consequently keeping the tissue more excitable (26, 27).
Another important finding in the molecular profile of brain tissue with FCD type II was the increased expression of AXIN2 and CSNK1A1 genes, both involved in the mechanism of phosphorylation and ubiquitination of β-catenin and its consequent cytoplasmic degradation. One of the first studies to demonstrate the role of β-catenin in tissue after seizures elucidated that seizures induced by electroconvulsive technique increase the expression of β-catenin and ligand WNT2 in new neurons generated in the hippocampus of rats (21). Therefore, the seizure seems to promote an increase in cytoplasmic β-catenin, to reorganize the neurogenic environment and promote the generation of new neurons mediated by the activation of the WNT pathway and its consequent transcription of genes related to homeostasis and cellular control. It was demonstrated in a study using a murine model of WNT signaling reporter mouse line (Topgal mice) that an increase in β-catenin levels occurs in reactive astrocytes after induced cortical ischemia, with increased β-catenin expression being associated with an increase in susceptibility to seizures in these models of ischemia (28). Here, we observed an increase in AXIN2 and CSNK1A1 genes which may be related to an attempt mechanism of β-catenin degradation increased by the convulsive aspect of dysplastic cortical tissue. Furthermore, the increase in β-catenin degradation may be an attempt at degradation not only due to the increase commonly observed in epileptogenic tissues but also due to the non-activation of the WNT pathway and its consequent need to eliminate cytoplasmic β-catenin since it is not used in unphosphorylated form as a transcription factor coupled to CPB and TCF/LEF.
Furthermore, we observed an increase in the FZD9 transcript, suggesting that cells from tissue with FCD are responding to a feedback mechanism in an attempt to generate a greater number of Frizzled receptors to compensate for the inactivity of the WNT pathway in this tissue. The generation of new receptors may be a response of diseased tissue cells to the accumulation of cytoplasmic β-catenin and the consequent increase in its degradation, in addition to blocking the activation of WNT mediated by the increase in available DKK.
The particular condition of patient 2 was a very significant reduction in the expression of the CTNNBIP1 gene. This gene is related to the interaction between β-catenin and the intranuclear TCF/LEF protein that will serve as a transcription factor in the activation of genes related to the WNT pathway. CTNNBIP1 is an inhibitor of this interaction and in patient 2 we observed an increase of more than 300 times the expression of this gene. The high concentration of cytoplasmic β-catenin can promote the reduction of the CTNNBIP1 inhibitor so that the interaction of nuclear transcription factors occurs. This fact leads to a suggestion that the WNT pathway could be more active in this tissue due to the expressive reduction of the gene that inhibits the interaction of non-phosphorylated β-catenin, which occurs only after the activation of the WNT pathway mediated by FZD receptors. We can also suggest that the expected increase in cytoplasmic β-catenin may alter the degradation mechanisms of the non-active pathway as well as seeking a balance in the active pathway mediated by the reduction in the expression of the negative controller gene of the nuclear interaction of non-phosphorylated β-catenin and its consequent use in the activated WNT pathway.