Lapinskas, E. | bioRxiv Science

bioRxiv2026

Activating Ras-MAPK pathway variants drive hippocampal clonal competition in human epilepsy

Mesial (a.k.a., medial) temporal lobe epilepsy (MTLE) is the most common focal epilepsy1,2 and, in drug-resistant cases, is treated by surgical removal of the anterior temporal lobe, which often shows neuronal loss and gliosis consistent with hippocampal sclerosis (HS)2. MTLE with HS has minimal contribution from germline genetic variation3, and is associated with prior precipitating insults such as prolonged childhood seizures and head trauma4-6. Somatic variants in Ras-MAPK pathway genes were recently reported in a few MTLE surgical specimens7,8, but their prevalence, clinical relevance, and underlying biological mechanisms remain unknown. Targeted duplex sequencing of hippocampal DNA from 462 surgical resections revealed significant enrichment of deleterious somatic variants in MTLE versus controls, with >40% of MTLE specimens harboring activating Ras-MAPK variants in PTPN11, NF1, BRAF, KRAS, and twelve genes not previously associated with focal epilepsy. Eight Ras-MAPK genes showed positive clonal selection in MTLE. Increased somatic variant burden predicted worse surgical outcome. Somatic Ras-MAPK variants at ultra-low (<0.5%) variant allele fractions were associated with older seizure onset and HS pathology, supporting a late prenatal or postnatal origin. Ras-MAPK variants in MTLE were enriched in cells derived from hippocampal progenitors--neurons, astrocytes, oligodendrocytes--in line with the known neuronal hyperexcitability and seizures induced by Ras-MAPK overactivation9,10; in contrast, Alzheimer disease hippocampi exhibited microglial enrichment of Ras-MAPK variants, consistent with prior reports11. Single-nucleus RNA sequencing showed increased expression of Ras-MAPK genes in neurons and upregulation of pathways mediating neurogenesis and neural development in MTLE. Functional validation of novel, recurrent PTPN11 variants confirmed gain-of-function, while cellular modeling in induced pluripotent stem cells demonstrated proliferative/survival advantages for mutant cells in mosaic culture. Overall, our data suggest that somatic Ras-MAPK variants and acquired risk factors may converge on clonal competition in the hippocampus to modulate epilepsy risk.

genetics↗

bioRxiv2020

CaMKII binds both substrates and effectors at the active site

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. Once activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A longstanding model invokes two distinct S- and T-sites. If an interactor binds at the T-site, it will preclude autoinhibition and allow substrates to be phosphorylated at the S-site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model that persistent CaMKII activity is facilitated by high affinity binding partners, which kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.

biochemistry↗