an important role of neural activity-dependent camkiv signaling in the consolidation of long- term...
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![Page 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,](https://reader036.vdocuments.mx/reader036/viewer/2022070400/56649f125503460f94c25a35/html5/thumbnails/1.jpg)
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
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Pathway
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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
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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
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*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.
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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)
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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?
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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
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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).
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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
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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.
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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.
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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)
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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).
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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.
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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
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• 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
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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.
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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
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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.
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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.
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CREB and Fear Conditioning
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• 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
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Conclusion
1. The acquisition phase appeared unaffected2. Short-term retention also unaffected3. Specifically, the consolidation/retention was
affected
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CREB levels (unphosphorylated) are the same in WT and C34
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Input-Output Curves: Synaptic Transmission is Normal in C34
• Fiber Volley Amplitude: Input (presynaptic)• Field EPSP Slope: Output (postsynaptic)
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*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).
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CaMKIV mutants
BACK