Grin1 p.Tyr647Ser Project
Background. Grin genes across species encode for proteins that make up the different NMDA receptor subunits. GRIN1 mutations (usually heterozygous) are associated with intellectual disability with epilepsy in humans, including the rare p.Tyr647Ser (Y647S) variant. This missense mutation occurs in the M3 helix of the transmembrane domain of GRIN1 which, importantly, is conserved between human GRIN1 and mouse Grin1. Currently, no effective pharmacological intervention or gene therapy exists to treat human patients with GRIN1 disorders. Patients with different variants to the GRIN1gene vary widely in symptom presentation. Thus, it is imperative to independently study each of the variants preclinically when developing treatments.
To do this, we generated Grin1Y647S mice using CRISPR and bred founder mice to develop F1 Grin1+/Y647S progeny. As proof-of-concept for our study, our first aim is to evaluate the behaviour of Grin1+/Y647S mice to ascertain whether there is phenotypic similarity to GRIN1 Y647S patients.
This entry describes pilot results from the Ramsey Laboratory regarding behavioural consequences of heterozygous Y647S mutation in mice.
Behavioural Assays, Results, and Discussion:
Each graph contains data from n= 4 WT and n =4 Grin1+/Y647S mice of >12 weeks of age mixed sexes.
- Open Field Test
To measure locomotor activity.Mice were placed in plexiglass arenas of 20x20x45 cm and their locomotor activity was measured using digital activity monitoring of infrared light beam breaks for 2h in 5min bins:
This data has been removed in anticipation of a research grant outcome. Please email k.intson@utoronto.ca if you are interested in hearing about progress on this project.
- Social Affiliative Assay
To measure social behaviour. Mice were placed in an opaque open-top arena of 62x42x22 cm containing two wire cups. One wire cup contained a novel, same-age, same-sex mouse as a social stimulus, while the other cup was empty. Mice were allowed to freely explore the arena for 8min and their activity was measured using video recording motion software. The time spent in the 3cm zone surrounding each cup was recorded, as well as the track length each mouse accumulated around each cup, and the number of visits they made to each cup.
This data has been removed in anticipation of a research grant outcome. Please email k.intson@utoronto.ca if you are interested in hearing about progress on this project.
- Elevated Plus Maze
To measure anxiety-like behaviour.Mice were placed in a elevated maze (39 cm high) composed of two opposing open arms (30.5×5 cm) and two opposing closed arms (30.5x5x15.2 cm). Mice were placed in an open arm of the maze and allowed to freely explore for 8 min in dim light while being recorded. Motion tracking software measured the amount of time spent in open and closed arms.
This data has been removed in anticipation of a research grant outcome. Please email k.intson@utoronto.ca if you are interested in hearing about progress on this project.
Other observations. I report that 1 male and 1 female Grin1+/Y647S mouse each displayed a seizure when placed on the elevated plus maze. I would describe this seizure as atonic-like in nature: while mice stood on their hind legs, I observed rapid chewing movements, light convusions, and they fell to the floor of the EPM. Seizures lasted ~10s and mice rapidly regained consciousness.
Planned experiments. Future experiments will involve boosting behaviour experimental group numbers, and examining biochemical consequences of Y647S on overall NMDA receptor function in order to determine treatment strategies for GRIN1patients.
Welcome Katheron – and your elegant results! Has anyone tested if Tyr647Ser GRIN1 localises to the membrane or whether it is retained in the ER and/or degraded by ERAD? Are there any other rare-disease associated GRIN1 mutations that produce mutant GRIN1 retaining some of its activity – but again it is retained in the ER and/or degraded by ERAD? I am asking for a friend 🙂
Pietro – thanks for your comment. It’s definitely a pressing question for us to figure out whether this variant localizes to the membrane. There were some initial studies conducted at Emory on this variant, but they were inconclusive. Hope to have an answer/update for you soon 😉