Authors :
Presenting Author: Kyle Stokes, PhD – University of Michigan
Samantha Jacobs, BSc – University of Michigan
Kaylin Hanna, Undergraduate researcher – University of Michigan
Emmanuel Crespo, PhD – University of Michigan
M. Carmen Varela, BSc Ba – University of Michigan
Miranda Walker, BSc – University of Michigan
Tuo Ji, PhD – University of Michigan
Wei Niu, PhD – University of Toledo
Jack Parent, MD – University of Michigan
Rationale:
Syntaxin binding protein-1 (STXBP1) is a chaperone protein that primarily functions to carry Syntaxin-1 to the synapse where it participates in SNARE mediated exocytosis. However emerging evidence suggests STXBP1 acts as a chaperone for multiple proteins in the cell. De novo loss of function variants in STXBP1 leading to haploinsufficiency (HI) cause cognitive impairment and severe epilepsy. Most studies on STXBP1 dysfunction have focused on synaptic activity, which is altered in excitatory and subplate interneurons, but exocytosis is also needed for proper cell migration during development which may precede synaptic deficits. Many cortical interneurons originate in the medial ganglionic eminence (MGE) and migrate tangentially to the cortex to synapse onto excitatory neurons. Deficits in this critical step could lead to impaired inhibition and altered maturation of both inhibitory and excitatory neurons. Studying developmental and neurological disease in humans are difficult. We recently developed human MGE-like organoids that give rise to somatostatin- and parvalbumin-expressing cortical interneurons. We hypothesize that impaired interneuron development contributes to STXBP1 related epilepsy and intellectual disability which can be modeled using human MGEOs.
Methods:
Using both male and female human pluripotent stem cell lines we performed CRISPR-Cas9 gene editing to generated STXBP1 haploinsufficient, knockout (KO) and isogenic control lines. We also used induced pluripotent stem cells (iPSC) from a patient harboring a p.G544Vif*2 STXBP1 variant and from an unrelated control. These PSC lines were then differentiated into self-organizing single-rosette (SOSR) MGEOs. MGEOs were then plated on a basement membrane-like substrate or fused to excitatory cortical organoids (SOSR-COs) to assess immature interneuron migration using Fiji. qPCR and immunofluorescence staining were also used to assess potential cell type changes.
Results:
We did not find significant differences in interneuron cell type marker expression at early stages of development in STXBP1 HI and KO organoids. Interneurons that migrate out of STXBP1 HI, KO and patient-derived MGEOs show decreased movement and travelled shorter distances than those from control MGEOs.Conclusions:
Timely interneuron migration from the MGE to the cortex is important for proper inhibitory synapse formation and interneuron maturation. The deficits we have found in STXBP1 HI and KO interneurons may contribute to seizures and cognitive impairment in STXBP1 epileptic encephalopathy. Understanding how STXBP1 contributes to interneuron migration may allow for more targeted approaches to novel therapy development for this disorder.
Funding:
Supported by NIH (NINDS) U54NS117170
NSERC Postdoctoral fellowship