Targeting pathological cells with senolytic drugs reduces seizures in neurodevelopmental mTOR-related epilepsy.

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, SASP expression and senescence-associated ß-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.

Hippocampal and neocortical BRAF mutant non-expansive lesions in focal epilepsies.

OBJECTIVE: Mesial Temporal Lobe Epilepsy-associated Hippocampal Sclerosis (MTLE-HS) is a syndrome associated with various aetiologies. We previously identified CD34-positive extravascular stellate cells (CD34+ cells) possibly related to BRAFV600E oncogenic variant in a subset of MTLE-HS. We aimed to identify the BRAFV600E oncogenic variants and characterise the CD34+ cells. METHODS: We analysed BRAFV600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE-HS samples (25 with CD34+ cells) and nine non-expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi-electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAFwildtype MTLE-HS and BRAFV600E mutant non-expansive lesion of hippocampus and/or neocortex. RESULTS: We identified a BRAFV600E oncogenic variant in five MTLE-HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAFV600E mutant samples. The co-expression of the oncogene-induced senescence marker p16INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions. INTERPRETATION: BRAFV600E underlies a subset of MTLE-HS and epileptogenic non-expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies.

Epilepsy related to focal neuronal lipofuscinosis: extra-frontal localization, EEG signatures and GABA involvement.

Focal neuronal lipofuscinosis (FNL) is an uncommon epileptic disorder related to an excess of lipofuscin accumulation within dysmorphic-appearing neurons (DANs), whose epileptogenic mechanisms are still poorly understood. It shares some clinical and neuroimaging similarities with focal cortical dysplasia of type IIb (FCDIIb), but it represents a different pathological entity. Here, we identified two patients with FNL among a 10-year cohort of 323 patients who underwent neurosurgery for a focal pharmacoresistant epilepsy. We describe the electroclinical, metabolic and neuropathological features of both patients with FNL who benefited from a comprehensive presurgical investigation. While the previous reports showed frontal lobe localization of the lesion, FNL was identified in the temporal lobe, in one of our patients. EEG investigations in both patients showed striking focal and rich interictal activity resembling that described in FCDIIb. Besides focal intraneuronal lipofuscin accumulation, the neuropathological analysis demonstrated that somata of DANs were surrounded by a large amount of GABAergic presynaptic buttons, suggesting the involvement of interneurons in the epileptogenicity of FNL. To further explore the role of GABAergic transmission in the generation of epileptiform activity in FNL, we performed in vitro multi-electrode array recordings on the post-surgery tissue from one patient. Spontaneous interictal-like discharges (IILDs) were identified only in the restricted area displaying the highest density of lipofuscin-containing DANs, suggesting a close correlation between the density of lipofuscin-containing neurons and epileptogenicity. Moreover, IILDs were blocked by the GABAA receptor antagonist gabazine. All together, these findings showed how GABA signaling may contribute to the generation of interictal-like activity in FNL tissue.