Challenge: Glutamate is the principal excitatory neurotransmitter of the human central nervous system (CNS). Disruption of glutamate homeostasis is a central feature of many neurological diseases, including stroke, glaucoma and Alzheimer’s disease. Glutamate levels in CNS are controlled by glial cells which facilitate its recycling and uptake through Excitatory Amino Acid Transporters (EAATs). Consequently, the EAAT proteins are attractive targets for drug discovery. However, there is currently no tool to identify and monitor the effect of drugs that could alter glutamate levels in cells.
Solution: Cyto-iGluSnFR (pronounced “sight-oh-eye-glue-sniffer”) is an engineered protein, built from a bacterial glutamate receptor domain (Glt1) and a circularly permuted green fluorescent protein (GFP), that senses glutamate, allowing to measure the rate by which glutamate enters cells. This glutamate biosensor can be used to screen thousands of compounds to identify new potential EAAT drugs that can modulate the transport of glutamate into glial cells. The identified molecules can then be tested using the same biosensor in mouse models – where neurons and glial cells function as they do in humans – to assess drug safety and effectiveness for patients.
Achievements/Impact: The team optimized a cell-based assay that allowed screening of compounds affecting glutamate transport through EAAT1 and EAAT2. Mouse embryonic stem cells with targeted integration of Cyto-iGluSnFR variants were also produced to generate two knock-in mouse models of glaucoma and stroke. These preclinical models were used to validate the technology as a tool to monitor glutamate dynamics and evaluate pharmacological modulators of EAATs in vivo. The use of Cyto-iGluSnFR technology displays multiple advantages over existing methodologies as it can be used both in vitro and in vivo, allowing rapid measurements and repeated stimulations in living cells. The technology can be scaled up to 96-well and 384-well assay formats, which are amenable for high-throughput drug screening.
| Principal Investigator: Don Van Meyel The Research Institute of the McGill University Health Centre |
| Co-investigators: Keith K. Murai The Research Institute of the McGill University Health Centre Adriana Di Polo Centre de recherche du CHUM Timothy H. Murphy University of British Columbia |
| Completed Project |
| $1,417,000 / 3 years |
| Supported by CQDM through: – Merck – Pfizer – MEI – BL-NCE |
| And by co-founding partners: – Brain Canada |
