Drinking from the fountain of youth for mTOR-related epilepsy

Abstract

Targeting Pathological Cells With Senolytic Drugs Reduces Seizures in Neurodevelopmental mTOR-Related Epilepsy

Ribierre T, Bacq A, Donneger F, Doladilhe M, Maletic M, Roussel D, Le Roux I, Chassoux F, Devaux B, Adle-Biassette H, Ferrand-Sorbets S, Dorfmuller, G, Chipaux, M, Baldassari, S, Poncer, JC, Baulac, S. Nature Neuroscience. 2024:1–12.

Cortical malformations such as focal cortical dysplasia type II (FCDII) are associated with pediatric drug-resistant epilepsy that necessitates neurosurgery. FCDII results from somatic mosaicism due to post-zygotic mutations in genes of the PI3K-AKT-mTOR pathway, which produce a subset of dysmorphic cells clustered within healthy brain tissue. Here we show a correlation between epileptiform activity in acute cortical slices obtained from human surgical FCDII brain tissues and the density of dysmorphic neurons. We uncovered multiple signatures of cellular senescence in these pathological cells, including p53/p16 expression, senescence-associated secretory phenotype expression, and senescence-associated β-galactosidase activity. We also show that administration of senolytic drugs (dasatinib/quercetin) decreases the load of senescent cells and reduces seizure frequency in an MtorS2215F FCDII preclinical mouse model, providing proof of concept that senotherapy may be a useful approach to control seizures. These findings pave the way for therapeutic strategies selectively targeting mutated senescent cells in FCDII brain tissue.

Commentary

Focal cortical dysplasia (FCD), characterized by cortical malformations, neurodevelopmental delay, and behavioral challenges, ¹ is one of the main causes of pediatric treatment-resistant epilepsy. There are 3 types of FCD, with FCD type II (FCDII) being recognized by the presence of cytomegalic cells, also known as neurofilament-accumulating dysmorphic neurons (DNs), and balloon cells (BCs). ² Pathogenic somatic mutations in the P13K-AKT-mTOR signaling cascade that arise during cortical development are important genetic contributors to FCDII and other mTORopathies, and mutations in genes within this pathway result in a cellular mosaic pattern of mTOR hyperactivation. ³

Current antiseizure treatment for FCDII is based on seizure type (focal seizures vs epileptic spasms) and age of onset, ¹ but available antiseizure drugs are often not sufficient to achieve seizure control. Thus, patients with childhood FCDII onset make up a large percent of epilepsy surgical cases. However, despite advances in neuroimaging and surgical treatments, surgical success rates remain suboptimal, ¹ providing impetus for the identification of alternative treatment options for FCDII and other mTORopathies. Tuberous sclerosis complex (TSC) is a multiple-organ disorder characterized by cortical malformations, and shares histopathological, molecular, and physiological similarities with FCDII. ¹ Preclinical studies and results from the EXIST-3 clinical trial (NCT01713946) showed that everolimus, an mTOR inhibitor, reduced seizure frequency by over 60% in children under 6 years of age with TSC. Given the overlap between TSC and FCDII, there is currently an ongoing single-center study to examine the effect of everolimus on brain mTOR activity and cortical excitability in patients with TSC or FCDII (NCT02451696). Despite the possibility of using mTOR inhibitors for mTORopathies, there remains a need for improved treatments.

In the current study, Ribierre and colleagues utilized a combination of human cortical slices from patients with FCDII and 2 mouse models of mTOR-related epilepsy to identify a potentially targetable biomarker for mTOR-related epilepsy. ⁴ Using acute cortical slices from 2 patients with FCDII, the authors found a positive correlation between epileptiform activity and pS6⁺ (a marker for mTOR activity) in DNs. Importantly, pS6⁺ DNs were not observed in brain regions without epileptiform activity, suggesting that only neurons with increased mTOR activity contribute to the observed epileptiform activity.

Cellular senescence is an irreversible cell cycle arrest state that is involved in multiple processes, including tumor suppression, wound healing, and aging. ⁵ Senescent cells are characterized by distinct morphological changes and increased lysosomal content, as well as their ability to resist apoptosis and to produce and secrete pro-inflammatory cytokines. ⁵ Interestingly, there is evidence for the contribution of the mTOR signaling cascade to cellular senescence and senescence-associated secretory phenotype (SASP) regulation. ⁵ In the current study, the authors also observed evidence of cellular senescence in brain tissues from 22 patients with FCDII but not in adjacent normal-sized cells from the same tissues nor in tissue samples from control patients with epilepsy due to non-mTOR-related cortical malformation. ⁵ All tissue samples were from patients between the ages of 6 months to 16 years. Consistent with their previous observations, ³ the authors also noted an enrichment of somatic mutations in FCDII cytomegalic cells. Together, these findings identify cellular senescence as a potential biomarker for pathogenic FCDII cells.

Loss-of-function mutations in DEPDC5 result in activation of mTOR signaling and are associated with FCDII and genetic focal epilepsies.^6,7 To investigate whether cellular senescence occurs in a mouse model of FCDII, Ribierre et al., examined mTOR activation and markers of cellular senescence in a conditional Depdc5 knockout mouse model (Depdc5^cKO) between 3 and 10 weeks of age, prior to seizure onset. Notably, the authors observed hyperactivation of the mTOR pathway in 3-week-old Depdc5^cKO mice and markers of cellular senescence in 8-week-old mutants; however, these markers were not present in the wild-type (WT) littermates. Furthermore, pro-inflammatory cytokines, including those associated with mTOR-regulated SASP, were upregulated in 10-week-old Depdc5^cKO mice relative to WT, whereas at earlier ages, comparable levels of pro-inflammatory markers were observed between Depdc5^cKO and WT mice, ⁴ suggesting that cellular senescence ensues following mTOR hyperactivation and is independent of spontaneous seizure activity.

To investigate the therapeutic utility of targeting senescent cells in mTOR-related epilepsy, 16-week old Mtor^S2215F mutant mice, which recapitulate many features of human FCDII, were treated with a senolytic cocktail composed of dasatinib and quercetin (DQ) via oral gavage for 9 days. ⁴ One month following cessation of DQ treatment, the authors noted an approximately 70% reduction in the number of pS6⁺ neurons. Furthermore, markers of cellular senescence and mTOR-regulated SASP molecules were significantly reduced in DQ-treated mutant mice. ⁴ In a separate cohort of Mtor^S2215F mutant mice (12 weeks old), EEG electrodes were implanted and spontaneous seizure frequency was recorded for 2 weeks at baseline, at the end of 9 days of DQ treatment, and 1 month after treatment cessation. DQ treatment significantly reduced spontaneous seizure frequency in the mutants at the end of the 9-day treatment period, and the mutants were seizure-free at 1 month following treatment cessation. ⁴ The authors observed no difference in seizure susceptibility in DQ-treated WT mice, ⁴ providing evidence that the antiseizure effects of DQ treatment are specifically mediated by targeting senescent cells.

In summary, Ribierre and colleagues identified cellular senescence as a targetable biomarker in FCDII and demonstrated that treatment with senolytic drugs provided robust and sustained seizure protection. ⁴ Senolytic drugs work by inducing apoptosis in senolytic cells; thus, given that Ribierre and colleagues found that senescent cells themselves are epileptogenic in mTOR-related epilepsy, senolytic drugs effectively reduced the number of senescent cells which in turn, decreased spontaneous seizure activity in mTOR-related epilepsy. ⁴ However, it is unclear whether new senescent cells will develop following treatment cessation. Furthermore, the time course by which senolytic drugs can effectively reduce the number of senescent cells in mTOR-related epilepsy and how soon seizure freedom is achieved remains to be determined. In addition, it would be valuable to establish the duration of seizure freedom following treatment with senolytics and whether a shorter treatment duration would also have a robust effect on seizure frequency.

While senolytic drugs seem to offer long-lasting benefits in a mouse model of mTOR-related epilepsy, further research is warranted before considering treatment with senolytics in children with mTOR-associated disease. Cellular senescence plays an important role in normal neurodevelopment and aging, ⁵ and thus, the long-term consequence of senolytic drugs, which are currently being used for the treatment of age-related phenotypes and chronic disease, ⁸ during a critical neurodevelopmental period is unclear. Another concern is that senolytic drugs can have wide-spread effects throughout the body and might yield unwanted side effects. Despite these trepidations, the findings from the current study highlight the potential of repurposing senolytic drugs to target cellular senescence in FCDII and other mTOR-related diseases. Furthermore, the identification of cellular senescence in multiple mouse models of mTOR-related epilepsy and human cortical tissue from patients with FCDII in this study open the possibility of senolytic treatment as a potential cell-based approach for mTORopathies regardless of the genetic cause.