Overview of collaborative workflow of the EpiMVP. Synergy of Project 1 with EpiMVP projects, GVCC and HECT

Fig1.: Overview of collaborative workflow of the EpiMVP. Synergy of Project 1 with EpiMVP projects, GVCC and HECT

Milestone 1: Assess in silico and in vitro model systems for VUS functional analyses

  • Computational modeling: with the gene and variant curation core (GVCC), generate and assess In silico tools to improve modeling of VUS pathogenicity
  • Protein functions: use 2-dimentional (2-D) cultures of HEK293T cells in biochemical tests of each studied VUS
  • Cell morphology/intracellular function: test the impact of a VUS on subcellular localization, protein trafficking and/or post translational processing
Computational modeling. Rationale for evaluating VUS and using functional information from projects with GVCC investigators to build and refine the EpiPred tool for variant pathogenesis prediction.

Fig 2. Computational modeling. Rationale for evaluating VUS and using functional information from projects with GVCC investigators to build and refine the EpiPred tool for variant pathogenesis prediction.

Milestone 2: Establish assays of cell autonomous effects in vitro

We’ll use human pluripotent stem cells (hPSCs) engineered by the human epilepsy tool core (HETC) to express doxycycline inducible Neurogenin 2 to differentiate excitatory neurons (iNeurons) or ASCL1/DLX2 to differentiate inhibitory neurons (iGN). They will be made to express WT or VUS containing genes on a null background to evaluate 2-D cultures of these progenitors and induced neurons to:

  • Progenitor proliferation, cell survival
  • Potential for differentiation into iNeurons or iGNs and
  • Cell morphology, protein intracellular localization,
  • Cell motility and
  • Gene expression using RNAseq tools
Neurons differentiated from hPSC expressing patient VUS. Engineered hPSC, iNeurons and iGN reagents from the HETC are used in Project 1 to create 2-D neuronal cultures. Cultures are used to assess morphology, protein expression, synapse formation and turnover.

Fig 3.: Neurons differentiated from hPSC expressing patient VUS. Engineered hPSC, iNeurons and iGN reagents from the HETC are used in Project 1 to create 2-D neuronal cultures. Cultures are used to assess morphology, protein expression, synapse formation and turnover.

Milestone 3: Determine the functional impact of VUS on synapse and network properties

These iNeurons (iN and iGN) expressing wildtype, known pathogenic and VUS mutations will be examined for:

  • Synapse formation
  • Synaptic spine and synaptic specialization turnover (plasticity), 
  • Intracellular protein transport and 
  • Firing properties on multi-electrode arrays (MEA)
Multielectrode Array activity of neurons differentiated from hPSC expressing patient VUS

Fig 4.: Multielectrode Array activity of neurons differentiated from hPSC expressing patient VUS

Participants

M. Elizabeth Ross, MD, PhD

Professor, Feil Family Brain and Mind Research Institute

Department of Neuroscience, Department of Neurology

Weill Cornell Medical College

Jack Parent, MD

William J Herdman Professor, Research Professor

Department of Neurology, Michigan Neuroscience Institute

University of Michigan

Vanessa Aguiar-Pulido, PhD

Assistant Professor (Miami)/Adjunct Assistant Professor (Cornell)

Department of Computer Science (Miami)/Feil Family Brain and Mind Research Institute (Cornell)

University of Miami

Weill Cornell Medical College

Stanislav Kholmanskikh, PhD

Research Assistant Professor

Department of Neuroscience

Weill Cornell Medical College

Chengbing Wang, PhD

Research Associate

Department of Neuroscience

Weill Cornell Medical College

Tuo Ji, PhD

Research Specialist

Parent Lab

University of Michigan

Adil Sabir, MA

Bioinformatician

Department of Neuroscience

Weill Cornell Medical College

Shawn Singh, BS

Senior Technician

Department of Neuroscience

Weill Cornell Medical College