Andres Barria, Dominque Muller, Victor Derkach, Leslie C. Griffith, Thomas R. Soderling

1. “Regulatory Phosphorylation of AMPA-Type Glutamate Receptors by CaM-KII During Long Term Potentiation” Andres Barria, Dominque Muller, Victor Derkach, Leslie C. Griffith, Thomas R. Soderling

2. Direct phosphorylation of AMPA-Rs in response to LTP, has not been previously shown • How can this relationship be demonstrated? • Associate the activity of a kinase with increased phosphorylation of AMPA receptors. • CaMKII has been previously shown to increase the responsiveness of AMPA receptors in CA1 pyramidal cells • How can we test for CAMKII activity? • LTP mediated activity is due to autophosphorylation at Thr286 therefore increased phosphorylation at this site should result in an increase in CaMKII activity

3. Detection of Phosphorylation of CaMKII at Thr286 Test for immunoreactivity of an antibody that is specific for CaMKII phosphorylated at Thr286 (AbP-Thr286) Experiment: 1)Induce LTP using theta-burst stimulation 2)Measure the immunoreacitivty for AbP-Thr286 using protein immunoblot test at 5, 15, and 60 minutes Results: The reactivity was increased relative to the control at all three time intervals Conclusion: The measured increase of immunoreactivty of AbP-Thr286 indicates an increase in phosphorylation at the Thr286 site on CaMKII during LTP

4. Immunoreactivity of a general CaMKII antibody was used to confirm that the increase in phosphorylation was not due to upregulation of CaMKII • None of the values vary significantly from the control

5. Does induction of LTP increase 32P labeling of AMPA-Rs? • >90% of GluR1 was immunoprecipitated from the homogenate and treated with GluR antibodies (Figure A). • The amount of 32P-AMPA-R was significantly elevated 15 min and 60 min after induction of LTP compared with the low-frequency stimulated control (Figure B). • The enhanced phosphorylation is not due to LTP-induced total protein phosphorylation (Figure C), so an increase in ATP-specific activity is ruled out.

6. Experiment: inject KN-62 into half of hippocampal slices and visualize changes in EPSP before and after LTP induction as well as use 32P-labelingto detect levels of AMPAR phosphorylation Hypothesis: if CaM-KII is involved in AMPAR phosphorylation, KN-62 should inhibit EPSP after induction Further demonstration of the relationship between CaMKII and AMPAR using CaM-Kinase inhibitor (KN-62)

7. Experimental Graph & Results • Black diamond = (+) KN-62 • White circle = ( - ) KN-62 • Did not affect short-term potentiation (STP) • Blocked expression of LTP • Blocked expression of enhanced phosphorylation of CaM-KII and AMPAR

8. Experimental Graphs & Results • After treatment with KN-62 and induction of LTP, no change in AMPAR phosphorylation was observed

9. Conclusion: • CaM-KII is needed for phosphorylation of AMPARs to occur • Even though PKA and PKC can phosphorylate AMPA-Rs during LTP, they did not cause the phosphorylation shown in these experiments. The phosporylation of AMPA-Rs is solely due to CaM-KII

10. 32P-peptide mapping of AMPA-Rs Identical maps suggest that the same site was phosphorylated in both

11. Enhancement of AMPA-R responsiveness by CaM-KII • Little 32P labeling of GluR1 in control, but 10-fold increase when coexpressed with CaM-KII (Figure C). • CaM-KII enhanced the AMPA-R response in HEK-293 cells (Figures E and F). • The site phosphorylated by CaM-KII in both HEK-293 cells and hippocampal slices after induction of LTP enhanced AMPA-R responsiveness.

12. Conclusions • The evidence for prolonged biochemical postsynaptic changes during LTP is consistent with the current model presented in Trends in Neuroscience (1994; 17:406). • The enhanced phosphorylation of Thr286 was not due to an LTP-mediated increase in CaM-KII protein. • The amount of 32P-AMPA-R was significantly elevated after induction of LTP. • CaM-KII is the catalyst of AMPA-R phosphorylation.

13. “Autophosphorylation at Thr286 of the Calcium-Calmodulin KinaseII in LTP and Learning” Karl Peter Giese, Nikolai B. Ferdorov, Robert K. Filipkowski, Alcino J. Silva

14. Is autophosphorylation of CaMKII at Thr286 required for LTP and learning? • Experiment:Substitute Thr286 for alanine (A) (T286A) • T286A mutant is unable to switch to its CaM-independent state (no autophosphorylation) • Generate the CaMKIIT286A-129B6F2 mutant with the Pointlox procedure • Test if Long-term potentiation is impaired in CaMKIIT286A-129B6F2 mutants

15. Other Variables to Consider • Detect target construct • Confirm point mutations and loxP site did not alter expression of CaMKII gene • Confirm only total CaM-independent CaMKII activity in mutants was decreased

16. Is Long-term potentiation impaired in CaMKIIT286A-129B6F2 mutants?

17. Could other reasons account for the LTP deficit? • Decrease in Synaptic Connectivity • No. Synaptic responses collected were similar in mutant and wild-type mice • Abnormalities in inhibition • No. Blocked GABA(A) receptors in order to rule out abnormal inhibition • Abnormal NMDAR Function • No. In presence of CNQX and PTX, NMDAR currents were normal in the mutants

18. Blocking GABAA

19. NMDAR and GABAA BLOCKED

20. Summary • Substitute Thr286 for alanine (A) (T286A) to test whether autophosphorylation of CaMKII at Thr286 is required for LTP • Long-term potentiation was impaired in CaMKIIT286A-129B6F2 mutants • In conclusion, autophosphorylation of CaMKII at Thr286 is required for LTP

21. Mice were tested for spatial learning in the hidden platform Morris Water Maze (hippocampus-dependent test) 10 mutants 11 wild types (Transfer tests had been done at the end of training) Autophosphorylation of CaMKII and Spatial Learning

22. Data Analysis • (A) The mutants were impaired in locating the hidden platform, but they were normal during the first three training blocks. • (B) During a transfer test after training, the mutants did not search selectively in the target quadrant and showed no swimming speed deficits. • (C) The mutants also did not selectively cross the TQ site.

23. How about other non-hippocampus impairments that might affect the results? • What if the mutants do not have vision at all? • What if they are not motivated to swim around and find the platform? • What if mutation caused problems in motor coordination?

24. Data Analysis (Continued) • (D) To check for visual disabilities, motivational problems, and motor coordination the visible platform test was done. • Mutants were able to learn the task (despite blocks 2, 3) CONCLUSION: Therefore the mutants had the vision, motivation and motor coordination for learning in Morris water maze.

25. Data Analysis (Continued) • (E) The visible platform was replaced by a hidden platform and the mutants were impaired during training. • CONCLUSION: This test following the visible platform test, confirmed the spatial learning impairments of these mutants.

26. Conclusion Autophosphorylation of CaMKII at Thr286 is required for spatial learning.

27. Challenges • What if mice cheat? • They might guess an area and swim a search pattern, getting to the platform quite quickly.

31. Pointlox Procedure

32. Generation of CaMKIIT286A-129B6F2 Mutant

33. Detection of target construct in mutant mice • At first PCR detected the loxP site determining the genotype and a second PCR was used to identify the point mutations. • The electrophoresis gel shows the Hinc II-digested PCR products from homozygotes (/) and wild-type (+/+) mice.

34. Did the point mutations and the loxP site alter the expression of the CaMKII gene? b a