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. 2013 Apr 24;33(17):7475-87.
doi: 10.1523/JNEUROSCI.4387-12.2013.

dCREB2-mediated Enhancement of Memory Formation

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Free PMC article

dCREB2-mediated Enhancement of Memory Formation

Thomas C Tubon Jr et al. J Neurosci. .
Free PMC article

Abstract

CREB-responsive transcription has an important role in adaptive responses in all cells and tissue. In the nervous system, it has an essential and well established role in long-term memory formation throughout a diverse set of organisms. Activation of this transcription factor correlates with long-term memory formation and disruption of its activity interferes with this process. Most convincingly, augmenting CREB activity in a number of different systems enhances memory formation. In Drosophila, a sequence rearrangement in the original transgene used to enhance memory formation has been a source of confusion. This rearrangement prematurely terminates translation of the full-length protein, leaving the identity of the "enhancing molecule" unclear. In this report, we show that a naturally occurring, downstream, in-frame initiation codon is used to make a dCREB2 protein off of both transgenic and chromosomal substrates. This protein is a transcriptional activator and is responsible for memory enhancement. A number of parameters can affect enhancement, including the short-lived activity of the activator protein, and the time-of-day when induction and behavioral training occur. Our results reaffirm that overexpression of a dCREB2 activator can enhance memory formation and illustrate the complexity of this behavioral enhancement.

Figures

Figure 1.
Figure 1.
The 572 transgene expresses a 28 kDa CRE-binding protein. A, Schematic organization of the dCREB2 locus. Exons 1–7 of the dCREB2 locus are boxed, and the ATG1, ATG2, and TAA (stop) codons are shown. 792 is the previously identified dCREB2-a isoform. 572 is identical to 792 except it contains a frameshift mutation (*) that results in a premature translational stop within exon 1 (572STOP), indicated by an arrowhead. The 807 ORF initiates from the ATG2 located within exon 3. The epitopes of the various antibodies are indicated. B, ATG1 and ATG2 are used to initiate translation in rabbit reticulocyte lysates. Equivalent amounts of RNA transcripts (bottom) are used to program reticulocyte lysates. Translation products are labeled with 35S-Met and resolved by SDS PAGE. ΔATG1, ΔATG2, or ΔATG1/ATG2 indicate base substitutions from ATG to ATT. C, ATG1 and ATG2 are used in transiently transfected cells. Equal amounts of plasmid DNA encoding various ORFs fused to a FLAG epitope are transfected into S2 cells, allowed to express, and equivalent amounts of cell lysate are analyzed using Western blots probed with an αFLAG-specific antibody. D, Induction of the 572 and 807 transgenes make a protein with an apparent mobility of 28 kD. Head extracts from heat-shock-induced (+), or not (−), wild type (WT), 807 or 572 flies analyzed using Western blots probed with the ATG2 antibody (top two panels) or the 657 antibody (bottom panel). The top panel is a short exposure, while the middle panel shows the region around 28 kD upon a longer exposure. The bottom panel shows a window that was stripped and reprobed with the 657 antibody.
Figure 2.
Figure 2.
Induction of 807 and 572 produce a 28 kDa protein with identical activities. A, Time course of 807 inducible expression in head extracts. 807 transgenic flies are heat shock induced (room temperature to 37°C for 30′), or not, and equal amounts of total protein are separated using SDS PAGE. The proteins are transferred and probed using the αC21 antibody. B, The 572 and 807 transgenic flies increase dCREB2-containing CRE-binding activity in an induction-dependent manner. Head extracts are made from wild-type, 572, or 807 transgenic flies that are heat shock induced (HS+), or not (HS−), and equivalent amounts of protein are used in EMSA assays using a radioactive CRE-containing probe. The addition of the α657 (monoclonal) antibody produces supershifted complexes. C, The EMSA protein complexes from 572 and 807 extracts contain the 28 kDa dCREB2 band. The αC21 antibody is used for Western analysis of EMSA complexes that are excised from a preparative gel, eluted, and rerun using SDS PAGE. D, Both the 572 and 807 transgenes transiently activate a CRE-luciferase reporter in vivo. The time course of luciferase activity for progeny flies when wild-type, 572, and 807 flies are crossed with the CRE-luciferase reporter fly, heat-shock induced (INDUCTION), and assayed biochemically is shown. All of the progeny flies contain a single copy of the CRE-luciferase transgene, and either no other transgene (WT), or one copy of 572 or 807. Statistical significance of the dataset was determined via two-way ANOVA, with a p < 0.001.
Figure 3.
Figure 3.
Wild-type flies contain an endogenous 28 kDa protein. A, A cluster of protein bands, including the 28 kDa isoform, are missing in the dCREB2 mutant S162. Western blot analysis of head extracts from wild-type (2U) and the CREB-null mutant, S162, indicates a loss of the 28 kDa CREB isoform (*). For each line, 30 μg of head extracts were fractionated on a 12% SDS-PAGE gel, blotted onto a nitrocellulose membrane, and probed with αCREB657 monoclonal antibody. αProhibitin antibody was used at 1:50 to monitor total protein content in each sample. B, Multiple antibodies recognize a 28 kDa protein band in extracts from wild-type flies. Western blot analysis of equivalent amounts of protein from wild-type flies probed with different antibodies. C, The αC21 antibody immunoprecipitates a 28 kDa protein from wild-type head extracts. Blank (MOCK IP), αC21, or a control (β-galactosidase) antibody is used for immunoprecipitation reactions from equivalent amounts of total adult head protein. Immunoprecipitates are reseparated using SDS PAGE and Western blotted using the α657 antibody.
Figure 4.
Figure 4.
The ATG2 codon is conserved and functional across Drosophila species. A, The ATG2 codon is conserved across Drosophila species. Sequence alignment of dCREB2 genes from different species. B, Evolutionary conservation between Drosophila species (C) The 28 kDa band is detectable in other Drosophila species. Western blot analysis of head extracts from different Drosophila species using the αC21 antibody.
Figure 5.
Figure 5.
A temporal window constrains memory enhancement. A, 807-mediated memory enhancement has a temporal window. Different groups of 807 transgenic flies are heat shock (25°C to 34°C for 30′) induced (+), or not (−), transferred to food-containing vials, and allowed to recover for varying amounts of time (shown on the x-axis), trained with 3 cycles of massed training (3×M), then returned to food-containing vials, and tested for 24 h memory. Error bars represent SEM, and the asterisk indicates statistical significance (**p < 0.01) using Student's t test. N = 8 for all groups, except +/−HS 30′, and +/−HS 90′, for which N = 16. B, 572- and 807-mediated memory enhancement. Transgenic 572 (the original C28 line) or 807 flies are heat-shock induced (+), or not (−), allowed to recover for 90′, trained with a single cycle of training (1×), or 3 cycles of massed training (3×M), and tested for 24 h memory. To equalize induced expression, the 807 flies are induced using a weaker heat-shock regimen (25°C to 34°C for 30′) than the one used for the 572 (C28) line (25°C to 37°C for 30′). Error bars represent SEM, and the asterisk indicates statistical significance (*p < 0.05, **p < 0.01) using Student's t test. N = 8 for all groups.
Figure 6.
Figure 6.
The time of day affects memory enhancement. A, The time of induction and training affect memory enhancement. Transgenic flies carrying either of two independent insertions of 807 (Lines 1 and 2) are entrained on a strict 12/12 h light/dark schedule. Different groups of flies are heat shock induced (+), or not (−) for 30′ (25 to 34°C) beginning at either ZT = 6 or ZT = 11, allowed to recover for 90′, trained with 3 cycles of massed training, returned to circadian cycling conditions, and tested for 4 d memory. Since heat shock lasts 30 min, and the flies are allowed to recover for 90 min, training begins at either ZT = 8 or ZT = 13. Error bars represent SEM, and the asterisk indicates statistical significance (***p < 0.001) using Student's t test. N = 8 for all groups. B, Memory enhancement can result after a single cycle of training. Transgenic 807 flies (Line 2) are entrained on a strict 12/12 h light/dark schedule. Different groups of flies are heat shock induced (+), or not (−), for 30′ (25 to 34°C) beginning at ZT = 11, allowed to recover for 90′, trained with a single cycle of training (1×), and tested for 4 d memory. Error bars represent SEM, and the asterisk indicates statistical significance (***p < 0.001) using Student's t test. N = 8 for all groups. C, Memory enhancement is ATG2-dependent. Transgenic 807 (Line 2) and ΔATG2 flies are entrained on a strict 12/12 h light/dark schedule. Flies are heat shock induced (+) for 30′ (25 to 34°C) beginning at ZT = 11, allowed to recover for 90′, trained with a single cycle of training (1×), and tested for 1 d memory. Error bars represent SEM, and the asterisk indicates statistical significance (*p < 0.05) using Student's t test. N = 8 for all groups. D, The 807 and ΔATG2 transgenic flies induce equivalent amounts of dCREB2 mRNA. Total head RNA is collected from three different flies (807, ΔATG2 or wild-type) that were exposed to one of two treatments [heat shock induction (+) or not (−)]. Each individual sample (of the 12 total) underwent RT-PCR (Lanes 7–12) or PCR only (Lanes 1–6), and the products were separated on an agarose gel and visualized using ethidium bromide staining.
Figure 7.
Figure 7.
Induction of 807 enhances memory of suppression of courtship conditioning. A, 807-mediated memory enhancement is induction-dependent. Mean courtship index 3 d following training of 807 and wild-type (WT) flies heat shock induced (+) or not (−). Left to right: open column represents uninduced 807 flies (N = 25); gray column represents heat shock induced 807 flies (N = 33); stippled column represents uninduced wild-type flies (N = 33); black column represents heat shock induced wild-type flies (N = 35). B, Transgene induction does not affect immediate performance after training (learning). Mean courtship index for 807 flies exposed to different induction and training conditions. Testing occurred 15 min following 5 h of courtship training. Left to right: open column represents uninduced but trained 807 flies (N = 14); gray column represents heat shock induced and trained flies (N = 19); black column represents untrained but heat shock induced 807 flies (N = 27). C, Memory enhancement is induction-, training-, and ATG2-dependent. Mean courtship index 3 d following 5 h of training in both 807 and ATG-2 flies exposed to different HS and training conditions. Left to right: open column represents uninduced and trained 807 flies (N = 16); gray column represents heat shock induced and trained 807 flies (N = 27); black column represents heat shock induced but not trained 807 flies (N = 23), and stippled column represents heat shock induced and trained ΔATG2 flies (N = 26). In all figures, **p < 0.005; ***p < 0.001, and error bars show SEM. Since the mean courtship index was not distributed normally, the data were analyzed using the Mann–Whitney U test in the Sigma Stat statistical software package (Systat Software).

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