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CFERV 2019 Ramsey

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Grin1 knockdown mice (Grin1KD) have a loss-of-function mutation that is caused by the insertion of foreign DNA into an intron of the Grin1 gene. Although this mouse does not model a
specific patient variant, it is a useful model to understand how brain functions are altered by a global reduction of NMDA receptors. Grin1KD mice display many phenotypes that are similar to patient symptoms, including increased motor activity, decreased muscle tone, increased stereotypic movements,
and impairments in multiple domains of cognition. We asked whether phenotypes of Grin1KD mice could be improved by genetic rescue of the knockdown mutation in adult mice. To do this, we used a tamoxifen inducible Cre recombinase to excise the mutation and restore the Grin1 gene to a wildtype configuration. We discovered that several forms of cognition were improved or even normalized when genetic rescue was performed in adult mice. Our results suggest that the adult brain has sufficient plasticity to overcome developmental insults to NMDA receptor function, which has important implications for patients with GRIN disorders.
While gene therapy may be possible in the future, there are a number of pharmacological interventions that can be tested in this model and quickly translated to patients. As an example, we
tested the ability of a ketogenic diet or dietary ketone ester to improve behavioural phenotypes of Grin1KD mice. We discovered that either the ketogenic diet or beta-hydroxybutyrate improved the Grin1KD phenotypes of hyperactivity, sociability, sensory processing, and spatial memory. Similar studies could be used to test novel pharmacological agents that increase NMDA receptor activity.

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CFERV 2019 Ramsey

  1. 1. Genetic and pharmacological strategies to improve postnatal recovery from GRIN1 hypofunction Amy Ramsey Department of Pharmacology & Toxicology University of Toronto © Amy Ramsey
  2. 2. Grin1KD mice as a general model of loss-of- function mutation in Grin1 • Grin1KD mice have an insertion mutation in an intron • Reduction in the amount of mRNA that is made • Reduction in the amount of GluN1 protein that is made • No change in biophysical properties have been observed © Amy Ramsey
  3. 3. Can we restore cognitive deficits if we rescue NMDA receptor levels in adults? Generate mice with a conditional, floxed mutation that produces low levels of NMDA receptors (Grin1KD mice). Restore Grin1 locus to wildtype with Cre recombinase and recover NMDAR levels in adult mice (Grin1RESCUE mice). Test cognitive behaviours of adult mice. © Amy Ramsey
  4. 4. Catharine MielnikWendy Horsfall Mielnik et al, in submission © Amy Ramsey
  5. 5. Cre reporter line (Rosa26▸stop▸dTomato) used to confirm global Cre activation with tamoxifen regimen Mielnik et al, in submission © Amy Ramsey
  6. 6. Experiment Design WT WTCre Grin1KD Grin1RESCUE tamoxifen chow tamoxifen chow tamoxifen chow tamoxifen chow PD70 PD70 PD70 PD70 regular chow regular chow regular chow regular chow PD98 PD98 PD98 PD98 Measure recovery at molecular cellular & behavioural level © Amy Ramsey
  7. 7. Sonny Chen Emily Johansson Mielnik et al, in submission © Amy Ramsey
  8. 8. Katheron Intson © Amy Ramsey
  9. 9. © Amy Ramsey
  10. 10. NMDA-elicited current in Layer 5 cortical neurons is restored Mary Binko Mielnik et al, in submission © Amy Ramsey
  11. 11. Synaptic levels of GluN1 peptide detected by PSD-95 IP MS Adam Funk Mielnik et al, in submission © Amy Ramsey
  12. 12. Locomotor hyperactivity is improved, and habituation is restored to wildtype level. Mielnik et al, in submission © Amy Ramsey
  13. 13. Executive function assessed by puzzle box test is improved Mielnik et al, in submission © Amy Ramsey
  14. 14. Affiliative social interaction is improved D Mielnik et al, in submission © Amy Ramsey
  15. 15. Summary of genetic rescue in adult mice • Effective rescue in glutamatergic neurons, but gabaergic neurons did not show the same level of rescue. Chromatin structure in the specific region of the gene is more accessible in Vglut+ cells than Gad1+ cells. • Similar levels of recovery at molecular and behavioural level were achieved with earlier intervention (at 6 weeks instead of 10) and with longer recovery (8 weeks instead of 4). • Most substantial behavioural improvements in social interaction, sensorimotor gating, puzzle box executive function. Improvements also seen in locomotor activity, stereotypy, elevated plus maze, and fear conditioning. No improvement in acoustic startle. © Amy Ramsey
  16. 16. Can we improve behavioural phenotypes by improving metabolic phenotype of Grin1KD mice? • Decreased 2-deoxyglucose uptake during exploratory behaviour (Duncan et al 2002). • Reduced expression of glucose transporter genes in brain (Sullivan et al 2019). • Changes in the synaptic proteome indicate upregulation of enzymes for glycolysis & mitochondrial respiration (Sullivan et al 2019). • Increased levels of glucagon in serum (Wesseling et al, 2014). • Reduced glucose tolerance. Grin1KD expression of glucose transporters Glucose Tolerance 0 15 30 60 120 0 5 10 15 20 WT Grin1KD Time (min) Bloodglucose(mmol/L) © Amy Ramsey
  17. 17. Effects of ketogenic diet on NMDAR deficient mice Working Memory in Y-maze 0 20 40 60 80 WT WT KETO GluN1 GluN1 KETO a b 15 15 7 6 %3ArmAlternation Motor Activity (KETO) 5 10 15 20 25 30 0 300 600 900 1200 WT (12) WT-KETO (14) Grin1KD (6) Grin1KD KETO (6) Time (min) Distance(cm) Time in Social Investigation (KETO) SO C IA L N O N SO C IA L N O N SO C IA L N O N SO C IA L N O N 0 5 10 15 WT WT-KETO Grin1KD Grin1KD KETO Time/visit(s) © Amy Ramsey
  18. 18. Effects of BHB supplementation on NMDAR deficient mice Locomotor Activity (BHB) 5 10 15 20 25 30 0 300 600 900 1200 WT (11) WT-BHB (10) Grin1KD (12) Grin1KD BHB (10) * ** * ** Time (min) Distance(cm) Time in Social Investigation (BHB) SO C IA L N O N SO C IA L N O N SO C IA L N O N SO C IA L N O N 0 5 10 15 WT WT-BHB Grin1KD Grin1KD BHB Time/visit(s) © Amy Ramsey
  19. 19. Effects of BHB on blood ketone and blood glucose levels Blood ketone level B A SA L B H B B A SA L B H B 0.0 0.5 1.0 1.5 WT Grin1KD ### ### * Betaketones(mmol/L) Blood glucose level B A SA L B H B B A SA L B H B 0 2 4 6 8 * p=0.03 WT Grin1KD Glucose(mmol/L) © Amy Ramsey
  20. 20. Summary of ketogenic diet results • There are several indications that glucose utilization is impaired in Grin1KD mice. • Ketogenic diet improves several phenotypes of Grin1KD mice (locomotor activity, social interaction, working memory). • Similar effects are obtained with BHB supplementation to a normal diet. • Future studies are planned to investigate EEG patterns in mice and ability of these regimens to normalize EEG and seizure threshold. © Amy Ramsey
  21. 21. Wendy Horsfall Catharine Mielnik Katheron Intson Sonny Chen Emily JohanssonMary Binko Adam Funk Phoebe Yan Tatiana Lipina © Amy Ramsey
  22. 22. Thanks to Catharine Mielnik Katheron Inston Phoebe Yan Sonny Chen Tatiana Lipina Wendy Horsfall Rehnuma Islam Marija Milenkovic Ali Salahpour Chinmaya Sandagi Graham Collingridge John Georgiou University of Toronto University of Toledo Rob McCullumsmith Adam Funk Sinead O’Donovan Courtney Sullivan University of Bordeaux Laurent Groc Emily Johansson Evelyn Lambe Mary Binko © Amy Ramsey

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