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Hyejin Kang, Linus D. Sun, Coleen M. Atkins, Thomas R. Soderling, Matthew A. Wilson,

An Important Role of Neural Activity-Dependent CaMKIV Signaling in the Consolidation of Long-Term Memory. Hyejin Kang, Linus D. Sun, Coleen M. Atkins, Thomas R. Soderling, Matthew A. Wilson, and Susumu Tonegawa. Pathway. Background. Goal: Assess CaMKIV’s role in memory

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Hyejin Kang, Linus D. Sun, Coleen M. Atkins, Thomas R. Soderling, Matthew A. Wilson,

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  1. An Important Role of Neural Activity-Dependent CaMKIV Signaling in the Consolidation of Long-Term Memory Hyejin Kang, Linus D. Sun, Coleen M. Atkins, Thomas R. Soderling, Matthew A. Wilson, and Susumu Tonegawa

  2. Pathway

  3. Background • Goal: Assess CaMKIV’s role in memory • Problems with CREB knockouts • compensatory increases in other CREB isoforms? • Problems with CaMKIV knockouts: • Some knockouts showed significant physiological impairment

  4. Definitions • Dominant Negative: mutant protein that blocks function of the WT protein by binding either to the WT protein or a protein upstream or downstream of the WT protein in a pathway. • C-Fos: An “immediate early gene” dependent on CREB phosphorylation

  5. *Abstract: Biochemical Aspect In mice with dnCaMKIV limited to the postnatal forebrain: • Basic synaptic function and early LTP (E-LTP) were unaffected. • CREB phosphorylation and C-Fos expression were reduced, while other protein levels were unaffected. • The dnCaMKIV resulted in a deficit in hippocampal late LTP (L-LTP), analogous to the effects of a protein synthesis inhibitor.

  6. Dominant Negative CaMKIV • CP: Regulatory domain of CamKII • FLAG: epitope tag • SV40: Viral DNA which can be differentiated from endogenous DNA • dnCaMKIV • HMDT308-311DEDD (autoinhibitory domain mutation) • L71A (ATP binding site) • T196A (phosphorylation site of CAMKK)

  7. Effects of dnCaMKIV • CaMKK enhances CaMKIV activity. • dnCaMKIV significantly reduces caCaMKIV activity • No statistically significant difference between the CREB mutation and dnCaMKIV…but a trend?

  8. Localization of dnCaMKIV to Forebrain • C34: name of animal line expressing dnCaMKIV • In situ hybridization to SV40 probe showed dnCAMKIV localized to the cerebral cortex, hippocampus, lower striatum, amygdala, and olfactory bulb

  9. dnCaMKIV: present and not affecting basal levels of synaptic proteins • Western Blot: • Flag: transgene expression in hippocampal extracts • dnCaMKIV does not affect CaMKII or Actin levels • Basal CREB, MAPK, and pMAPK levels are also unaffected by the transgene (data not shown).

  10. Method for Testing the Effects of Stimulation on Mutants • Hippocampal slices are perfused with saline containing either: • 90mM KCl (depolarizes membrane: VGCC allow Calcium influx into soma) • 100µM Glutamate • 30 minutes later: • Ser133-phosphorylated CREB (pCREB) and C-fos expression measured by immunoreactivity analysis

  11. After Stimulation, PCREB and C-Fos levels are reduced in C34 mutants • Basal levels of pCREB and C-Fos are unaffected by dnCaMKIV • After stimulation (Glu and KCl), C34 hippocampal slices show a deficit in pCREB and c-Fos expression compared to WT slices. • Fosk (which activates the PKA pathway) is not affected by dnCaMKIV: dnCaMKIV effects seem to be limited to the pathway of interest.

  12. Early LTP (E-LTP) is Normal in C34 Hippocampal Slices • Potentiation through theta-burst stimulation • Early synaptic changes (e.g. those mediated by CaMKII) do not seem to be affected.

  13. Long-term LTP (L-LTP) is reduced in dnCaMKIV mutants • Potentiation through 4 x tetanic stimulation • WT maintained LTP up to 200 minutes while potentiation in C34 continually decayed • Decay in C34 potentiation resembles decay in WT potentiation in the presence of anisomycin (a protein synthesis inhibitor)

  14. Points of Concern • CAMKIV is necessary to produce L(Late phase)-LTP in the hippocampus, but what about other brain regions? (Area of future research!) • The loss of L-LTP in C34 cannot be directly linked to the effect of dnCaMKIV on CREB phosphorylation (CaMKIV may regulate other transcription machinery).

  15. Conclusion • dnCaMKIV does not affect basic synaptic function or E-LTP. • CaMKIV plays a role in the protein synthesis-dependent component of L-LTP.

  16. Abstract: Behavioral • Morris water maze and Fear conditioning • Impairment in long-term memory • Specifically, the consolidation/retention was affected • Short-term memory was intact • The acquisition phase appeared unaffected • Short-term retention also unaffected

  17. Morris Water Maze: Hidden Platform • Transgenic mice shows longer escape latencies. • No latencies differences during the first two days of training, so it is not from motor impairment.

  18. Morris Water Maze: Fixed visible platform (in a new location) • Transgenic and wild-type shows similar latency curves • Therefore, latency from hidden platform is not due to swimming speed and fractional periphery occupancy. • Wild type swam to the previous location of the platform instead of the new location • Transgenic mice swam directly to the new platform

  19. Morris Water Maze: Fixed visible platform • Wild type mice employ spatial memory strategy even in visible platform of Morris water maze. • C34 swim directly to the visible platform • Rely on cue-platform association strategy instead of spatial memory strategy

  20. Contextual Fear Conditioning: Context • Since Morris water maze requires training over several days, it is likely that spatial memory can be consolidated. • Therefore, Contextual fear conditioning is used to test CaMKIV’s involvement in latter phases. • dnCaMKIV transgenic • Similar levels of freezing 24 hours after training • Reduction in context dependent freezing after 7 days

  21. Cued Fear Conditioning • dnCaMKIV transgenic • Similar levels of freezing 24 hours after training and 7 days after training • Suggest that transgenic mice have selective deficit in the consolidation/retention phase of context-dependent fear memory.

  22. CREB and Fear Conditioning

  23. Limitation of Experiment • dnCaMKIV was strongly expressed in the hippocampus as well as the cerebral cortex. The deficits in behavior may reflect decreased CaMKIV activity in either or both areas.

  24. Conclusion • The acquisition phase appeared unaffected • Short-term retention also unaffected • Specifically, the consolidation/retention was affected

  25. CREB levels (unphosphorylated) are the same in WT and C34

  26. Input-Output Curves: Synaptic Transmission is Normal in C34 • Fiber Volley Amplitude: Input (presynaptic) • Field EPSP Slope: Output (postsynaptic)

  27. *Open-field and Plus Maze Test • C34 transgenic and Wild-type mice are indistinguishable in … • Percent dwell time in open arm • Percent entries in open arm • Total number of entries in both open and closed arm • Suggest that C34 transgenic mice impairment is due to spatial learning and not general emotional defects (reduce or increase in anxiety).

  28. CaMKIV mutants BACK

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