An Important Role of Neural Activity-Dependent CaMKIV Signaling in the Consolidation of Long-

Term MemoryHyejin 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– Problems with CREB knockouts

• compensatory increases in other CREB isoforms?

– Problems with CaMKIV knockouts:• Some knockouts showed significant

physiological impairment

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

*Abstract: Biochemical Aspect

In mice with dnCaMKIV limited to the postnatal forebrain:1. Basic synaptic function and early LTP (E-LTP)

were unaffected.

2. CREB phosphorylation and C-Fos expression were reduced, while other protein levels were unaffected.

3. The dnCaMKIV resulted in a deficit in hippocampal late LTP (L-LTP), analogous to the effects of a protein synthesis inhibitor.

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)

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?

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

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).

Method for Testing the Effects of Stimulation on Mutants

• Hippocampal slices are perfused with saline containing either:

A. 90mM KCl (depolarizes membrane: VGCC allow Calcium influx into soma)

B. 100µM Glutamate

• 30 minutes later:– Ser133-phosphorylated CREB (pCREB)

and C-fos expression measured by immunoreactivity analysis

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.

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.

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)

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).

Conclusion

1.dnCaMKIV does not affect basic synaptic function or E-LTP.

2.CaMKIV plays a role in the protein synthesis-dependent component of L-LTP.

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

• Transgenic mice shows longer escape latencies.• No latencies differences during the first two days of training, so it is not

from motor impairment.

Morris Water Maze: Hidden Platform

Morris Water Maze: Fixed visible platform (in a new location)

• Wild type swam to the previous location of the platform instead of the new location

• Transgenic mice swam directly to the new platform

• Transgenic and wild-type shows similar latency curves– Therefore, latency from

hidden platform is not due to swimming speed and fractional periphery occupancy.

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

Contextual Fear Conditioning: Context

• dnCaMKIV transgenic– Similar levels of freezing 24 hours after training – Reduction in context dependent freezing after 7 days

• 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.

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.

CREB and Fear Conditioning

• 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.

Limitation of Experiment

Conclusion

1. The acquisition phase appeared unaffected2. Short-term retention also unaffected3. Specifically, the consolidation/retention was

affected

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

Input-Output Curves: Synaptic Transmission is Normal in C34

• Fiber Volley Amplitude: Input (presynaptic)• Field EPSP Slope: Output (postsynaptic)

*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).

CaMKIV mutants

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