Autophosphorylation at Thr286 of the α Calcium-Calmodulin Kinase II

in LTP and Learning

Karl Peter Giese, Nikolai B. Fedorov, Robert K. Filipkowski, Alcino J. Siilva*

Science vol.279, no 5352. pp.870-873. 1998

Previous Experiments

• Synaptic Strength Related To Learning/Memory– i.e. LTP

• Inhibit CaMKII Inhibit LTP and Learning

• Increasing tCaMKII affects Learning/Memory

More Background

• Autophosphorylation at Thr286 CaMKII from CaM-independent to CaM-dependent

• LTP Long-lasting Autophosphorylated form of CaMKII at Thr286

Hypothesis

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

CaMKII

TT

305/306Inhibitory

B C

Experiment 1 – T286A

Results from Experiment 1

Result• The αCaMKIIT286A-129B6F2 mutation decreased the total

CaM-independent CaMKII activity in the mutants

  CaM-Independent Activity

Mutant 0.40 ± 0.06 pmol-1 min

Wild Type 1.09 ± 0.12 pmol-1 min

• β-CaMKII?

Verify Experiment 1 Result

The point mutations and the loxP site did not alter the expression of theαCaMKII gene

Verify Experiment 1 Result

  Total CaMKII Activity

Mutant 4.74 ± 1.12 pmol-1 min

Wild Type 5.07 ± 0.91 pmol-1 min

Result• Total CaMKII (CaM independent + dependent) activity

remained approximately equal.

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

Testing for LTP in αCaMKIIT286A-129B6F2 mutants

• Protocol– 100Hz tetanus (1s)– Check for potentiation

60 minutes

• Results– LTP deficient in

αCaMKIIT286A-129B6F2 mutants

  Potentiation Sample

Mutant 110.8 ± 6.2% 7 Mice, 7 Slices

Wild Type 153.5 ± 7.5 % 10 Mice, 10 Slices

LTP impairments in the αCaMKIIT286A-129B6F2 mutants

NO OVERLAP

Testing for LTP in αCaMKIIT286A-129B6F2 mutants

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

  Maximum Increase Maximum Decrease

Mutant 146.7 ± 4.4 % 91.6 ± 6.9 %

Wild Type 140.3 ± 3.2 % 79.3 ± 6.9 %

LTP impairments NOT due to prepotentiation of Synaptic Transmission

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

Pairing Protocol Result

Pairing Protocol

Mutant: 132 ± 8%

WT: 277 ± 21%No overlap

LTP deficits in the αCaMKIIT286A-129B6F2 mutants

Robust Protocol

• γ-aminobutyric acidA (GABAA) receptors blocked with picrotoxin (PTX) during these recordings.

LTP impairments not due to abnormalities in inhibition.

Experiment 4 – NMDAR-dependent LTP

• Procedure– 100 Hz Tetanus for 1 s– NMDAR in the presence of AP5

• Results (after 30 min)

  (+) AP5 - Potentiation (-) AP5 - Potentiation

Mutant 108.4 ± 3.5 % 113.9 ± 2.8 %

Wild Type 112.8 ± 3.9 % 158.1 ± 8.7 %

Mutant Insensitive to AP5

Mutant NMDAR-dependent LTP already deficient

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 + AP5Thus, early mutant potentiation was not NMDAR dependent

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

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.

• 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?

Autophosphorylation and Spatial Learning

Morris Water Maze

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

Autophosphorylation and Spatial Learning

D. Visible Platform F. Transfer Test

E. Hidden Platform G. Transfer Test

Visible Platform Test

αCaMKIIT286A-129B6F2 mutants have deficits in spatial learning

Result

Conclusion

•Autophosphorylation of CaMKII is required LTP and Learning

Criticism/Future Experimentation

• What about CaMKIIs?

• Visible Platform Test Early Results

QUESTIONS?

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Power point presented by:

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