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Role of Autophosphorylation at Thr286 in αCaMKII: Implications for LTP and Learning

This study investigates the significance of autophosphorylation at Thr286 of αCaMKII in long-term potentiation (LTP) and learning processes. The research demonstrates that mutations affecting this site lead to impaired CaMKII function and, consequently, a reduction in LTP capabilities. Utilizing various experiments, including electrophysiology in hippocampal slices from mutant mice, the findings suggest that Thr286 autophosphorylation is crucial for mediating synaptic plasticity and may be essential for effective learning.

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Role of Autophosphorylation at Thr286 in αCaMKII: Implications for LTP and Learning

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  1. Autophosphorylation at Thr286 of the αCalcium-Calmodulin Kinase II in LTP and LearningKarl Peter Giese, Nikolai B. Fedorov, Robert K. Filipkowski, Alcino J. Siilva*Science vol.279, no 5352. pp.870-873. 1998

  2. Previous Experiments • Synaptic Strength Related To Learning/Memory • i.e. LTP • Inhibit CaMKII  Inhibit LTP and Learning • Increasing tCaMKII affects Learning/Memory

  3. More Background • Autophosphorylation at Thr286 CaMKII from CaM-independent to CaM-dependent • LTP  Long-lasting Autophosphorylated form of CaMKII at Thr286

  4. Hypothesis The autophosphorylation at the Thr286 site of CaMKII is required for LTP and learning.

  5. CaMKII TT 305/306 Inhibitory B C

  6. Experiment 1 – T286A

  7. Results from Experiment 1 Result • The αCaMKIIT286A-129B6F2 mutation decreased the total CaM-independent CaMKII activity in the mutants • β-CaMKII?

  8. Verify Experiment 1 Result • The point mutations and the loxP site did not alter the expression of theαCaMKII gene

  9. Verify Experiment 1 Result Result • Total CaMKII (CaM independent + dependent) activity remained approximately equal.

  10. Experiment 2 – Testing Mutants for LTP • Method • Extracellular field recordings in the stratum radiatum of hippocampal slices. • Transverse hippocampal slices (400 μm) from 5-10 month old mice placed in a submerged recording chamber perfused continuously with artificial cerebrospinal fluid (ACSF). • Extracellular fEPSPs recorded with an electrode filled with ACSF in CA1 stratum radiatum. • Schaeffer collaterals were stimulated

  11. Testing for LTP in αCaMKIIT286A-129B6F2 mutants • Protocol • 100Hz tetanus (1s) • Check for potentiation60 minutes • Results • LTP deficient in αCaMKIIT286A-129B6F2 mutants

  12. Testing for LTP in αCaMKIIT286A-129B6F2 mutants  NO OVERLAP LTP impairments in the αCaMKIIT286A-129B6F2 mutants

  13. Verify LTP Deficiency in Mutant • Test that LTP impairments not due to defect in synaptic connectivity in the CA1 region. • Synaptic Transmission during tetanus • Biphasic Change at 10 Hz Stimulus  LTP impairments NOT due to prepotentiation of Synaptic Transmission

  14. Experiment 3 - Pairing Protocol Background • To confirm the LTP deficiency of αCaMKIIT286A-129B6F2 mutants Method • EPSP currents recorded from CA1 pyramidal neurons from 6-12 month old mice with a patch electrode in the whole-cell voltage clamp mode. • Postsynaptic depolarization up to +10mV • 2 Hz synaptic stimulation for 50s

  15. Pairing Protocol Result Pairing Protocol Mutant: 132 ± 8% WT: 277 ± 21% No overlap LTP deficits in the αCaMKIIT286A-129B6F2 mutants

  16. Robust Protocol • γ-aminobutyric acidA (GABAA) receptors blocked with picrotoxin (PTX) during these recordings.  LTP impairments not due to abnormalities in inhibition.

  17. Experiment 4 – NMDAR-dependent LTP • Procedure • 100 Hz Tetanus for 1 s • NMDAR in the presence of AP5 • Results (after 30 min) Mutant Insensitive to AP5  Mutant NMDAR-dependent LTP already deficient

  18. Early Potentiation via NMDAR • 2 Theta Burst Tetanus • 2 high-frequency bursts of four stimuli at 100 Hz, with 200 ms separating the onset of each burst • After 2 seconds • WT + AP5: 113.7 ± 2.0 % Potentiation • Mutant: 108.8 ± 2.6 % Potentiation • After 10 Seconds • WT + AP5: 106.3 ± 2.0 % Potentiation • Mutant: 112.4 ± 3.3 % Potentiation • Mutants without AP5 had very similar potentiation as Wild Type + AP5 • Thus, early mutant potentiation was not NMDAR dependent

  19. Possible Problem • The autophosphorylation of αCaMKII at Thr286 leads to trapping of Calmodulin. • Calmodulin can reduce the opening probability of NMDARs. • Proposed Problematic Model: • T286A  no phosphorylation  more Calmodulin  reduced probability of NMDARs opening  reduced LTP

  20. Check NMDAR opening probability • Results •  Amplitude of NMDAR currents normal in mutants when compared to wild type •  Voltage dependence of NMDAR currents normal in mutants when compared to wild type. • Extracellular Field Recordings – 50 μA Stimulus • Mutants: 0.159 ± 0.053 mV • Wild-Type: 0.200 ± 0.020 mV  LTP impairments in the mutant αCaMKIIT286A-129B6F2 were not due to abnormal NMDAR function.

  21. Original Hypothesis: • The autophosphorylation at the Thr286 site of CaMKII is required for LTP and learning. • Conclusion Thus Far: • Autophosphorylation of αCaMKII is required for LTP. • Next Question: • Is autophosphorylation of αCaMKII required for learning?

  22. Autophosphorylation and Spatial Learning Hidden Platform Version • Hippocampal Dependent • 2-5 Month old mice • 5 days • 12 trials per day • Blocks of 4 trials • Transfer tests at the end of days 3, 5 Visible Platform Version • Hippocampal Independent • Tested for 2 days • 12 trials per day • Transfer test at the end Hidden Platform Version • Performed after Visible Platform Version to verify data Morris Water Maze

  23. Autophosphorylation and Spatial Learning

  24. Visible Platform Test • Visible Platform F. Transfer Test • Hidden Platform G. Transfer Test

  25. Result αCaMKIIT286A-129B6F2 mutants have deficits in spatial learning

  26. Conclusion • Autophosphorylation of aCaMKII is required LTP and Learning

  27. Criticism/Future Experimentation • What about bCaMKIIs? • Visible Platform Test Early Results QUESTIONS?

  28. Powerpoint created by: Wendy Lee Jill Marti Power point presented by: Jason Ly Parth Makker Powerpoint assisted by: Ruby Liu

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