An Important Role of Neural Activity-Dependent
CaMKIV Signaling in the Consolidation of LongTerm 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
– 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 thetaburst 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 ELTP.
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
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.
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
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
• 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
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
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.
Conclusion
1. The acquisition phase appeared unaffected
2. Short-term retention also unaffected
3. 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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