Abstracts

Rationale: Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy and drug-resistant in roughly one-third of patients. Our work shows that somatic variants activating Ras-MAPK signaling are present in >40% of hippocampi from patients with MTLE, with over half the variants in the PTPN11 gene. Germline PTPN11 variants activate Ras-MAPK signaling through a dominant gain-of-function mechanism, although it’s unknown how somatic PTPN11 variants confer seizure risk in the hippocampus. The SHP2 protein is a member of the protein tyrosine phosphatase family and naturally auto-inhibited due to the interaction of its two tandem Src homology-2 domains and the PTP domain. Most PTPN11 variants identified in our MTLE cohort cluster to two hotspots in the N-SH2 and PTP domains of the protein, therefore we hypothesized that the PTPN11 variants disrupt the SHP2 auto-inhibited state and increase phosphatase activity. Given the late developmental origins of PTPN11 variants, we next hypothesized that mutant progenitors in the dentate gyrus of the hippocampus have a competitive advantage that results in their greater contribution to hippocampal development and pathogenesis.


Methods: We synthesized and purified twenty-one SHP2 protein variants, the wild-type protein, and the PTP domain alone, through a bacterial expression system followed by High-Performance Liquid Chromatography. We quantified enzymatic activity for each variant using a fluorogenic substrate and compared them against the wild-type protein and PTP. To test the mutants’ competitive advantage, we conducted a mixing experiment involving a mutant SHP2 G503R iPSC line and its isogenic control. We plated the iPSCs at a 50:50 ratio and assessed clone abundance using digital droplet PCR genotyping of bulk DNA. Subsequently, we replicated the experiment with Neural Progenitor Cells (NPCs) to explore the threshold at which the mutant NPCs fail to outcompete the wild-type population.

Results: We discovered that most SHP2 protein variants exhibited increased phosphatase activity relative to the wild-type SHP2. Furthermore, we showed that SHP2 allosteric inhibitors like SHP099 and TNO155 restore normal activity to MTLE-associated SHP2 variants with gain of phosphatase function. From the iPSC mixing experiment, we saw that after the third passage, the mutant clone constituted approximately 80% of the cell population, supporting the competitive advantage of the mutant cells. When replicated in NPCs, we noted interestingly, an initial increase in the mutant population at all tested ratios followed by a decrease with subsequent passages, suggesting that the mutant NPCs potentially differentiated into post-mitotic neurons more rapidly.

Conclusions: In conclusion, most MTLE-associated PTPN11 variants appear to cause a dominant gain of SHP2 function, which may be normalized with clinically available SHP2 drugs. Furthermore, we provide a potential mechanism for how progenitors harboring PTPN11 variants make an asymmetrically greater contribution to hippocampal developmental which is likely key in MTLE pathophysiology.

Funding: NINDS K08-NS128272

NINDS R01-NS035129

Burroughs Wellcome Fund Career Award for Medical Scientists

Howard Hughes Medical Institute