OutFOXed: FOXJ3 Variants Disrupt PTEN-mTOR Signaling in Focal Epilepsy

Abstract

Epilepsy-Associated FOXJ3 Variants Link a Transcriptional Program of the PTEN-mTOR Pathway to Neuronal Specification and Cortical Lamination

Cheng HY, Liu C, Nien CW, Huang HC, Zhao HJ, Nian FS, Chen C, Custodio HM, Sisodiya SM, Lu C, Chen HH, Hsu CS, Pi WC, Chu CC, Hsu JS, Chen PL, Chang FP, Tung CY, Chou SJ, Alavi S, Houlden H, Chen WY, Liu YT, Hou PS, Tsai JW. Nat Commun. 2026;17(1):1815. doi: 10.1038/s41467-026-69241-2. PMID: 41803108; PMCID: PMC12972129.

Focal cortical dysplasia (FCD), a major cause of drug-resistant epilepsy, involves abnormal neuronal migration and cortical architecture, yet its molecular basis remains poorly defined. Here, we identify FOXJ3 pathogenic variants in patients with autosomal dominant focal epilepsy and FCD. In the developing mouse cortex, FOXJ3 declines sharply in neural progenitors after embryonic day 15.5. In utero electroporation-mediated Foxj3 knockdown in mouse brains impairs neuronal migration, disrupts cortical lamination, and alters neuronal specification, promoting upper-layer neuron production at the expense of deeper-layer neurons. ChIP-seq and scRNA-seq analyses identify Pten as a key FOXJ3 target. Notably, Pten overexpression rescues cortical defects caused by FOXJ3 deficiency. FCD-associated variant fails to upregulate Pten, leading to dysregulated mTOR signaling and enlarged neuronal soma, a hallmark of FCD. These findings suggest that mutations in FOXJ3 may cause epilepsy and FCD and define a transcriptional program that regulates the PTEN-mTOR pathway for neuronal specification and cortical lamination.

Commentary

Focal cortical dysplasia (FCD), which is characterized by disrupted neuronal organization, is highly associated with drug-resistant focal epilepsy. ¹ FCD type II includes both disrupted cortical architecture and distinctive cytological abnormalities, including dysmorphic neurons and/or balloon cells. ² Hyperactivation of the PI3K-PTEN-mTOR pathway is strongly linked to FCD type II, often through pathogenic variants that disrupt PTEN, a critical negative regulator of mTOR signaling.^3,4 In this study, Cheng et al identify FOXJ3, a forkhead family transcription factor not previously implicated in human epilepsy, as a novel contributor to epileptogenesis through its role in coordinating transcriptional programs linked to PTEN–mTOR signaling and cortical development. ⁵ Rather than acting within the canonical mTOR signaling pathway, FOXJ3 appears to function upstream as a regulator of gene expression programs that shape both neuronal specification and cortical organization, thereby linking early developmental decisions to later seizure susceptibility.

The authors initially identified rare, putatively deleterious FOXJ3 variants in several individuals with developmental epilepsy. Although there was phenotypic variability across affected individuals, the convergence on seizure disorders hinted at a shared underlying biological mechanism. This genetic evidence places FOXJ3 among a growing class of epilepsy-associated genes that encode transcriptional regulators rather than components of cell signaling machinery. ⁶ Such findings reinforce the idea that epilepsies, particularly those with early onset, may frequently arise from perturbations in developmental programs that establish the structural and molecular architecture of cortical circuits.

To test the role of FOXJ3 in cortical development, the authors used in utero electroporation in mice to disrupt FOXJ3 expression in neural progenitors at different timepoints during prenatal development. Through a series of elegant studies, they demonstrated that perturbing FOXJ3 induces structural abnormalities in cortical organization consistent with disrupted lamination. Specifically, they demonstrated that FOXJ3 is critical for neuronal migration and the production of deep-layer neurons, with its dysfunction favoring the development of upper-layer callosal projection neurons. This phenotype was tied to changes in progenitor cell fate and cell cycle progression. This suggests that FOXJ3 plays a role in coordinating cell fate decisions during cortical development, ensuring that appropriate neuronal identities are generated in correct proportions. Together, these developmental perturbations provide a plausible substrate for long-term circuit dysfunction, as neurons that are both mis-specified and mispositioned are likely to integrate abnormally into emerging networks.

To interrogate the molecular consequences of FOXJ3 disruption, the authors integrated ChIP-sequencing and transcriptomic analyses in mice. A central observation was that FOXJ3 binds Pten and Tsc1, suggesting that it regulates a gene expression program enriched for components of the PTEN–mTOR pathway. Overexpressing FOXJ3 in mice upregulated Pten gene expression, whereas its loss led to decreased Pten expression and a downstream increase in phosphorylated S6, a sign of mTOR pathway activity. Overexpressing Pten during mouse corticogenesis rescued the neuronal migration deficits associated with FOXJ3 disruption, whereas overexpressing Tsc1 was ineffective, therefore supporting a model in which FOXJ3 directly regulates PTEN to influence cortical development.

To model a patient-specific variant, the authors introduced the FCD-associated FOXJ3 p.N351S genetic variant into mice. Whereas overexpressing wild-type FOXJ3 led to clear increases in Pten gene expression, the mutant form had no effect, suggesting it was unable to act on Pten. A defining feature of FCD type II is the presence of dysmorphic neurons and balloon cells. Indeed, neurons expressing the p.N351S variant had enlarged soma reminiscent of the abnormal cell types observed in FCD type II, thus making a direct link between a FOXJ3 genetic variant and clinically relevant phenotypes.

The authors propose an integrated model in which FOXJ3 coordinates transcriptional programs that modulate components of the PTEN–mTOR pathway to regulate neuronal fate specification. Disruption of this coordination leads to a cascade of developmental abnormalities, including altered neuronal identity, disrupted cortical layering, and reprogrammed mTOR signaling. While the precise contribution of each phenotype to seizure generation remains to be fully delineated, these changes could conceivably increase vulnerability to epileptogenesis by producing cortical circuits that are developmentally miswired and biochemically primed for dysregulated activity. No spontaneous seizures were observed in this study; however, the nature of the mouse model was a very sparse knockdown with few cells affected. Future studies addressing seizures in alternative mouse models or cellular and circuit-level electrophysiology could add to our understanding of this issue.

This study adds to a growing recognition that epilepsy can arise from perturbations in the transcriptional architecture of brain development. In this framework, FOXJ3 represents a key upstream node that integrates developmental patterning with growth signaling pathways, thereby influencing the fundamental determinants of cortical circuit assembly. Genetic variants in FOXJ3 may be more common than currently recognized, especially given the variable penetrance reported here, so the inclusion of this gene in future clinical genetics studies may identify more cases. At a broader level, the findings of this study underscore the importance of considering epileptogenesis as a multilevel process in which transcriptional regulation, cellular identity, and signaling pathways converge to define network stability.