1) The document discusses the differential localization and functions of PKA regulatory subunits R1b and R2b in the brain. 2) An experiment found that knocking down R1b using shRNA in hippocampal neurons led to decreased levels of pCREB after forskolin treatment, indicating impaired cAMP/PKA/CREB signaling and likely L-LTP impairment. 3) Knocking down R2b did not decrease pCREB levels, suggesting it does not play as important a role in cAMP/PKA/CREB signaling and L-LTP induction.

1. PKAR Subunits Exhibit Differential
Localizations and Functions in the Brain
Lior Schenk
R. Ilouz, S. Taylor, T. Stiles, L. Schenk, J.L. Goldberg
UC San Diego Division of Biological Sciences

2. RCSB Protein Data Bank
(http://www.rcsb.org/pdb/101/motm.do?momID=152)
C C
R R
Inactive PKA
R- Regulatory subunit
C- Catalytic Subunit
C
C
R R
cAMP
cAMP and the PKA holoenzyme
How does the differential
expression of PKAR isoforms
affect LTP induction?

3. PKA Signaling System
PKA: cAMP dependent protein kinase
Hormone
GPCR
Adenylate
Cyclase
ATP
C
C
C C
R R
R R
Inactive PKA
DNA CRE
CREB
R- Regulatory subunit
C- Catalytic Subunit
βγ
α
γ β
α
cAMP
Transcription
Cytosol
R R
Functions:
Memory
NT Metabolism
Glycogen Metabolism
Steroid Metabolism
Muscle Contraction
R. Ilouz

4. Variable expression of R1b
and R2b throughout the brain
Images courtesy R. Ilouz
R1b k/o: LTP and LTD impairment
R2b k/o: “lean” phenotype
Noticeably different localization in
CA3 vs CA1 cells

5. Connecting the dots…
• cAMPPKA
pCREBL-LTP
• Which PKA
isoforms are
responsible?
• Hypothesis: R1b
and R2b, which
differentially
localize in the
hippocampus, play
dissimilar roles in
the induction of
LTP.Kandel; Squire (2000). Memory:
From Mind to Molecules.
Does loss of R1b or R2b function lead to a loss of pCREB, which is necessary for L-LTP?

6. Experimental Procedure
• Hippocampal neuron prep (courtesy R Ilouz)
• GFP-shRNA knockdowns of R1/R2 subunits
• Forskolin treatment  elevated cAMP  PKA
 pCREB  cL-LTP
• Immunofluorescence
– Transfection backbone
– α-CREB / α-pCREB
– DAPI
Irr R1b R2b
CREB
p-CREB

7. RESULTS

8. irr: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI
pCREB levels are visibly decreased in R1b fields.

9. irr: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI
pCREB levels are visibly decreased in R1b fields.

10. R2b: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI
1, 2, 3, 5, 6, 7, 9, 10
pCREB levels are visibly decreased in R1b fields.

11. R2b: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI
1, 2, 3, 5, 6, 7, 9, 10
pCREB levels are visibly decreased in R1b fields.

12. R2b: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI
1, 2, 3, 5, 6, 7, 9, 10
pCREB levels are visibly decreased in R1b fields.

13. R2b: GFP, pCREB, DAPIR1b: GFP, pCREB, DAPI
pCREB levels are visibly decreased in R1b fields.
1, 2, 3, 5, 6, 7, 9, 10

14. Data quantification
Fluorescence Quantification
(in ImageJ)
• Select nuclear area
(freeform selection tool,
DAPI channel)
• Transpose selection onto
pCREB channel
• Analyze  Mean Gray
Value

15. 0
50
100
150
200
250
irr r1b r2b
pCREB mean fluorescence

16. 0
50
100
150
200
250
irr r1b r2b
pCREB mean fluorescence

17. DISCUSSION
pCREB levels are visibly decreased in R1b fields.
Specific interference of R1b may lead to cL-LTP impairment.
cAMP  PKA  pCREB  L-LTP
0
50
100
150
200
250
irr r1b r2b
pCREB mean fluorescence
irr R1b R2b

18. Future Considerations
• Follow-up to reproduce findings
– Implement optimized vector  more transfected
cells, more useful control
• Introduce new stain
– B3-tubulin @ cy5
– Identify add’l neurons + transfection efficiency
• Increase cell density
– More synapses, branching
– Cell-to-cell comparison

19. Thank you!