Authors :
Presenting Author: Kyle Stokes, PhD – University of Michigan
Sandra Mojica-Perez, PhD – Research Specialist, Neurology, University of Michigan; Sheethal Jahagirdar, PhD – Research Technician Associate, Neurology, University of Michigan; Wei Niu, PhD – Reseach Assistnat Professor, Neurology, University of Michigan; Peter Crino, MD, PhD – Professor, Neurology, Neurology; Phillip Iffland, PhD – Assistant Professor, Neurology, University of Maryland; Jack Parent, MD – Professor, Neurology, University of Michigan
Rationale:
Hyperactivation of mTOR, a ubiquitous regulator of metabolism, growth, and protein translation, causes malformations of cortical development. Kaptin actin binding protein (KPTN), a KICSTOR complex member, activates the GATOR1 complex to inhibit mTOR. Individuals with KPTN LoF variants develop megacephaly (ME), intellectual disability and epilepsy. mTOR dysregulation is known to affect the transition from radial glia to progenitor cells, but it is unclear how KPTN LoF variants affects this dynamic. Human cortical organoids (hCO) are useful in vitro models for assaying human cortical development. We hypothesize that hCOs with KPTN LoF variants will exhibit mTOR hyperactivation and an expanded cortical progenitor pool that increases hCO size.
Methods:
We reprogrammed blood cells from a KPTN patient with biallelic KPTN variants (KPTN-/-) into induced pluripotent stem cells (iPSCs) using the Yamanaka factors (OCT3/4, SOX2, KLF4, L-MYC). Patient-derived iPSCs and unaffected controls (KPTN+/+) were differentiated into hCOs using a novel protocol, Self-Organizing Single Rosette–Cortical Organoids (SOSR-COs), a high-fidelity model of cortical development. SOSR-COs were evaluated by immunofluorescence, qPCR and size analysis.
Results:
KPTN-/- SOSR-COs show increased levels of pS6 (S235,S236), a marker of mTOR activation. From 30 to 90 days in vitro, KPTN-/- SOSR-COs displayed a significant increase in size compared to controls, although this effect varied by clone. We observed no change in the intermediate progenitor (IP) marker TBR2 but found a significant increase in the outer-radial glial (oRG) marker HOPX in KPTN-/- SOSR-COs.
Conclusions:
Our results show that human SOSR-COs are a viable method for modeling KPTN LoF variants. The increase in size and mTOR hyperactivation are similar to findings in patients and mouse models. Interestingly, KPTN-/- SOSR-COs display an increase in oRG cells hinting at a possible ME mechanism. Future experiments will involve lineage tracing to confirm our observed increase in oRG. Understanding how KPTN LoF variants causes ME and epilepsy will help to develop precision therapies for patients with KPTN LoF and potentially other mTORopathies.
Funding:
AES Postdoctoral Fellowship (KS), NINDS R37NS125632 to PBC.