Keywords: cortex, memory consolidation, insulin-like signaling, aging, MAPK signaling

Improved Long-Term Memory and Flexibility Learning inS1/2^−/− Mice.

To address the function of SHARP1 and SHARP2 in the context of learning and memory formation, we analyzed wild type,Sharp1 andSharp2 single and double null mutants (24) in the contextual fear conditioning paradigm (27). Because of the prominent expression ofSharp1 and -2 in hippocampal and cortical regions and their coupling to physiologically and pathologically altered neuronal activity in the cortex (23,28,29), we tested recent fear memory formation (1 d after shock) to study hippocampal as well as remote fear memory (28 d after shock) to also assess cortical functions (Fig. 1A andB). ACx regions such as the ACC appear to be critically involved in remote fear memory formation (1).S1^−/− andS2^−/− single mutants performed under all tested condition as WT animals (Fig. S1A andB).S1/2^−/− double mutants were also indistinguishable from WTs in the recent test but displayed significantly increased numbers of freezing rates in the remote test, indicating enhanced anterior cortex-dependent memory consolidation inS1/2^−/− mice (Fig. 1C andD). Freezing was not compromised by sensory perception as assessed by the hot plate assay where no differences between the genotypes were detected (Fig. 1E).

To further substantiate these findings, we applied a modified water maze task including a reversal test that monitors learning flexibility, which is also dependent on the anterior cortex (30). In the initial learning phase, performance remained unaltered during 6 d of training (Fig. 1F), whereas in the probe trial performed at day 7,S1/2^−/− mice stayed significantly longer in the target quadrant but did not display increased numbers of crossings of the exact platform position, and swimming speed was unaltered between genotypes (Fig. 1G–I). In contrast, after changing the platform to a new position (“reversal test”)S1/2^−/− mice relearned the task significantly faster compared with WT controls, which was already apparent on the first day of training (Fig. 1J andK). In the reversal probe trial performed at day 14,S1/2^−/− mice stayed significantly longer in the target quadrant and crossed the exact platform position more often (Fig. 1L andM). Swimming speed was unaltered between genotypes (Fig. 1N). This observation supports the Acx-specific phenotype inS1/2^−/− mice and indicates that improved learning and not impaired forgetting underlies the enhanced memory consolidation seen in the remote fear conditioning (Fig. 1D). The absence of respective phenotypes inS1^−/− andS2^−/− single mutants in the water maze supported the functional redundancy of both genes in cognitive processing (Fig. S1C–E).

Enhanced MAPK and S6 Activity in the Anterior Cortex ofS1/2^−/− Mice.

To further characterize the Acx-specific phenotype inS1/2^−/− mice, we analyzed a set of surrogate markers of several signaling pathways (i.e., phosphorylation/activation states of p44/42-MAPK, S6, PI3K, mTOR, AMPK, GSK3β, and AKT) implicated in neuronal plasticity and hippocampal learning (8–14). We speculated that activated pathways selectively operating in the ACx and not the Hi might be among the underlying processes of the cortex-selective enhancement of learning and memory formation inS1/2^−/− mice. We observed that phosphorylation of p44/42-MAPK as well as S6 inS1/2^−/− mice was higher in the ACx and reduced in the Hi compared with WT controls (Fig. 1A–D). This deregulation of p44/42-MAPK activity was not observed inS1^−/− andS2^−/− single mutants (Fig. S2). In parallel, GSK3β phosphorylation was increased in the ACx and Hi, both at a similar 1.5-fold level and we observed elevated AKT phosphorylation only in the Hi of double mutants (Fig. S3). All other signaling markers remained unaltered between the genotypes and brain regions (Fig. S3). Thus, only increased phospho-MAPK and -S6 were selectively elevated in the ACx but not the Hi ofS1/2^−/−. We previously observed thatS1/2^−/− mice displayed an attenuated sleep–wake amplitude in accordance with an altered 24 activity profile by monitoring voluntary wheel running (20). Therefore, to rule out that differential physical activities and/or sleep–wake profiles might have caused differential MAPK signaling, we determined phospho-MAPK levels in 4-h bins over the complete 24-h period in the ACx and Hi ofS1/2^−/− and WT controls that were housed in cages equipped with running wheels. We again observed elevated phospho-MAPK levels in the ACx ofS1/2^−/− mice throughout the complete circadian cycle (Fig. 2E andF), whereas genotype differences were not detectable in the Hi under these conditions (Fig. 2G andH). We further analyzed signaling in the ACC ofS1/2^−/− mice by applying immunohistochemical (IHC) staining with antibodies directed against phospho-S6 before fear conditioning and directly after remote fear memory test. In agreement with published observations (31), the number of phospho-S6 positive cells increased in the ACC of WT mice upon neuronal activity, but were constitutively elevated in S1/2^−/− mice (Fig. 2I andFig. S4A andB). We did not detect genotype differences in the CA1 region of the Hi (Fig. 2J andFig. S4A andC).

ElevatedIgf2 Expression in the Anterior Cortex ofS1/2^−/− Mice.

Thus far, we observed an elevated level of phospho-MAPK and -S6 in the ACx that correlated with the improved anterior cortex-dependent learning performance inS1/2^−/− mice. Because MAPK and S6K/S6 signaling have been shown to be coordinately activated upon growth factor and insulin signaling, we scanned available microarray data (20) at lowered significance thresholds for growth factor candidates with a differential expression in the cortex ofS1/2^−/− versus WT mice. The only relevant candidates identified wereIgf2 andIgfbp5 as up- and down-regulated inS1/2^−/− mice (Fig. S5). We used qRT-PCR to analyze differential mRNA expression in the ACx ofS1/2^−/− and WT mice over the complete circadian cycle (see above) and validated the up- and down-regulation ofIgf2 andIgfbp5 expression, respectively, in comparison with two housekeeping genes that did not display genotype or time-dependent changes (Rpl3a andAtp5b) (Fig. 3A–D). Moreover, we also validated increasedIgf2 and decreasedIgfbp5 expression in the ACx ofS1/2^−/− versus WT mice, normalized to the housekeeping genes (Rpl13a andCyc1), which were unregulated (Fig. 3E andF). No differences of genotype or time-dependent changes ofIgf2 andIgfbp5 mRNA expression were detected in the Hi (Fig. 3F). Plotting the 24-h kinetics of Igf2 expression and phospho-MAPK levels obtained from WT animals only revealed their circadian profile in the ACx whereby the increase of Igf2 expression precedes the peak of MAPK activation (Fig. S6). Finally, we detected significantly elevated IGF2 protein levels in the ACx ofS1/2^−/− versus WT mice (Fig. 3G).

In consequence, we hypothesized that elevating or reducing IGF2 signaling in the ACC of WT mice should enhance and inhibit remote fear memory formation, respectively. Therefore, we generated AAV2 constructs expressingIgf2 andIgfbp5 under the control of the CAG promoter (32) and injected corresponding viruses including an empty control virus bilaterally into the ACC of WT mice (Fig. 3H). Four weeks after injection and recovery, animals were subjected to fear conditioning and were analyzed for recent and remote memory formation. Mice with virus-mediated overexpression ofIgf2 in the ACC displayed a slight yet significant improvement of remote fear memory formation (P < 0.05) (Fig. 3I). In contrast, animals that were injected with anIgfbp5-encoding virus showed a significantly impaired remote fear memory formation compared with the control (P < 0.05) and to Igf2 injections (P < 0.01) (Fig. 3I). To better reflect the situation inS1/2^−/− mice, whereIgf2 expression is elevated andIgfbp5 expression is reduced in the ACx (Fig. 2A–G), we coinjected a pool of AAV2 constructs coding both for IGF2 and those expressing shRNAs directed againstIgfbp5 bilaterally into the ACC and assessed recent and remote fear memory. The simultaneous increase of IGF2 and decrease of the insulin-signaling inhibitor IGFBP5 resulted in a highly significant enhancement of remote fear memory (P < 0.001) (Fig. 3J). Modulation of IGF2 signaling by virus-mediatedIgf2/Igfbp5 expression in the ACC did not affect hippocampus-dependent recent fear memory (Fig. 3I andJ).

Redundant Function of INSR and IGF1R in the Forebrain Is Associated with Reduced MAPK Signaling in the ACx but Not the Hi.

The canonical receptors that are known to bind insulin-like peptides including IGF2 are the INSR and IGF1R, which both mediate downstream signaling via the ERK/MAPK pathway (33). When inactivated individually, however, deficits in learning and memory formation have not been described so far (34,35). Therefore, we generated forebrain-specific neuronalInsR andIgf1R double null mutant mice by crossingIgf1R/InsR^fl/fl mice (36,37) withCaMKII-cre–expressing mice (38) to study consequences on MAPK signaling and fear memory formation. As expected by the expression pattern of CaMKII, INSR and IGF1R protein levels were highly reduced in the ACx (Fig. 4A andB) and Hi (Fig. 4D andE) inIgf1R/InsR^CaMKII-cre compared withIgf1R/InsR^fl/fl mice. Phosphorylation levels of p44/42-MAPK were, however, significantly reduced only in the ACx (Fig. 4C) but not in the Hi (Fig. 4D) of the double mutants. We subsequently subjectedIgf1R/InsR^fl/fl andIgf1R/InsR^CaMKII-cre mice to a series of standard behavioral tests and did not observe any altered behavior among both groups (Fig. S7). Thus, forebrain restricted deletion of the INSR and IGF1R inCaMKII positive neurons does not alter basic behavior. We next used the contextual fear conditioning paradigm and analyzed recent and remote memory formation (Fig. 4G).Igf1R/InsR^CaMKII-cre mice displayed a highly significant impaired recent and remote fear memory performance compared with the controls (Fig. 4H). As it was shown before for neuronalInsR knockout mice under high fat diet, the double knockout mice displayed a significant gain of weight already under standard diet (Fig. S8), which has been attributed to reduced insulin signaling (39). These data reveal that INSR and IGF1R are at least partially redundant in the forebrain and the loss of both receptors impairs MAPK signaling in the ACx but not the Hi. In addition, our observations indirectly support the implication of the IGF2-IGF1R/INSR-MAPK signaling axis for memory consolidation in the cortex.

Impaired Fear Memory Formation in AgedS1/2^−/− Mutant Mice.

To understand the long-term effects of SHARP1 and SHARP2 deficiency caused by enhancedIgf2 signaling, we studied agingS1/2^−/− mice and observed an age-dependent weight loss and increased mortality rate in agedS1/2^−/− mice (Fig. 5A andB). WhereasIgf2 expression remained elevated in the ACx and unchanged in the Hi, expression levels ofIgfbp5 were similar in the ACx and Hi of agedS1/2^−/− and WT mice (Fig. 5D). The phosphorylation of p44/42-MAPK in the ACx ofS1/2^−/− mice was reduced to WT levels (Fig. 5F andG) and remained reduced in the Hi ofS1/2^−/− mice (Fig. 5H andI). In parallel, the overall performance ofS1/2^−/− mice in contextual fear conditioning was significantly reduced in the recent and reduced to wild-type levels in the remote task (Fig. 5J andK).

Animal Experiments.

All animal experiments were performed in accordance with Regierungspräsidium Braunschweig institutional guidelines and were approved by the government of Lower Saxony, Germany.

Mice were housed at a 12-h light/12-h dark cycle and received food and water available ad libitum. All experiments were performed with cohorts of adult male mice at the same age between 4–5 mo and 12–16 mo for aging studies.S1^−/−,S2^−/−,Igf1R^fl/fl, andInsR^fl/fl single knockout mice as well asCaMKII-cre heterozygous mice were backcrossed to C57BL/6J for more than 10 generations as described previously (24,36–38). WT andSharp1 and -2 double mutant mice (S1/2^−/−) were obtained from double heterozygous breeding pairs (24,58).Igf1R/InsR^fl/fl andIgf1R/InsR^CaMKII-cre mice were obtained fromIgf1R/InsR^fl/fl homozygous andCaMKII-cre heterozygous breeding pairs.Igf1R/InsR^fl/fl homozygous mice were obtained from Igf1R/InsR^fl/+ heterozygous breeding pairs.Igf1R/InsR^fl/+ heterozygous mice were obtained fromIgf1R^fl/fl andInsR^fl/fl breeding pairs. All experiments were performed blinded to genotypes.

Stereotactic Injections.

Surgery was performed under intraperitoneal anesthesia with ketamine/xylazine (100 mg/kg; 10 mg/kg). Viral vectors (AAV2, AAV2-Igf2, AAV2-Igfbp5, or a pool of AAV2-Igf2/Igfbp5shRNA1-3) were injected into the anterior cingulate cortex (1 mm anterior to bregma, 0.5 mm lateral to midline, 1.5 mm ventral to surface). One microliter was administered at each injection site using a 10-μL Hamilton syringe at a rate of 0.5 μL/min with a total of 2–4 × 10¹¹ virus particles per injection site. Immediately after surgery, mice were given buprenorphine (0.2 mg/kg, s.c.) for analgesia.

Western Blot Analyses.

Brain tissues (ACx bregma 1–3 mm and Hi bregma −1 to −3 mm) were isolated using a “rodent brain matrix” 1 mm coronal slicer (ASI Instruments) from adult WT and S1/2^−/− mice harvested at indicated time points. ACx and Hi samples were frozen on dry ice and lysed with a Polytron PT 2000 homogenizer in sucrose buffer. Samples obtained at defined Zeitgeber time points were collected as described above and pooled (n = 3). For preservation of proteins as well as of protein phosphorylation cOmplete (Roche) and PhosSTOP tablets were freshly added to the buffer. After lysis of the tissue, a fraction of the lysate was removed and added to an appropriate volume of RNA lysis buffer (Qiagen) for further RNA extraction. Protein lysates were quantified with a Lowry-based DC Protein Assay Kit II (Bio-Rad) and added to a final concentration of 1 mM DTT and 1× NuPAGE lithium dodecyl sulfate (LDS) sample buffer. Samples were heated for 10 min at 70 °C and separated in 4–12% NuPAGE Bis-Tris gel/1× Mes buffer (Invitrogen). Protein was transferred to a 0.2-μm PVDF membrane (Roche) and blocked overnight in 5% milk in 1× TBS with 0.05% Tween 20. Immunoblots were visualized with HRP-coupled secondary antibodies and an INTAS ECL Imager.

Enzyme Immunoassay.

Detection of IGF2 protein levels in ACx homogenates fromS1/2^−/− and WT mice was essentially performed as described (59) with minor modifications. Protein extracts were concentrated using Amicon Ultra filter units (Millipore) and IGF2 levels were determined following the instructions provided by the manufacturer’s protocol (Abcam) and absorbance was measured using a 96-well microtiter plate reader (Berthold) against a standard curve obtained from serial dilutions of recombinant human IGF2 (R&D Systems).

Plasmids.

ORF-containing expression cassettes encoding mouse IGF2 and IGFBP5 were synthesized using gene blocks (Integrated DNA Systems) and subcloned into an AAV2 plasmid backbone containing a CAG promoter (obtained from M. Klugmann, University of New South Wales, Australia) Three hairpin shRNAs constructs targeting Igfbp5 mRNA (NM_010518.2) were designed using an online tool available atcancan.cshl.edu/RNAi_central using standard settings, synthesized as oligonucleotides, and subcloned into the same AAV2-CAG backbone as follows: shRNA1: TGCTGTTGACAGTGAGCGCAGACTTTATAGGCATATAAAT TAGTGAAGCCACAGATGTAATTTATATGCCTATAAAGTCTATGCCTACTGCCTCGGA; shRNA2: TGCTGTTGACAGTGAGCGATCTCTTGATGGCATGGCATAG TAGTGAAGCCACAGATGTACTATGCCATGCCATCAAGAGAGTGCCTACTGCCTCGGA; and shRNA3: TGCTGTTGACAGTGAGCGCCCTCTGGCTTTCAAGAGAAAG TAGTGAAGCCACAGATGTACTTTCTCTTGAAAGCCAGAGGATGCCTACTGCCTCGGA.

The Igfbp5 targeting sequence is underlined.

Quantification of shRNA constructs was performed with qRT-PCR on cDNA obtained from mouse NIH 3T3 cells nucleofected with AAV2, AAV2-Igfbp2-shRNA1, AAV2-Igfbp2-shRNA2, AAV2-Igfbp2-shRNA3, and a pool of AAV2-Igfbp2-shRNA1–3 plasmid constructs performed in triplicates. Transfection efficiency was determined to be higher than 70%. Knockdown efficiency in reference to the AAV2 control was 32 ± 6% for shRNA1, 43 ± 9% for shRNA2, 39 ± 4% for shRNA2, and 65 ± 7% for a pool of shRNAs1–3. Therefore, a mix of AAV2-Igfbp5-shRNA1–3 plasmids was used for virus generation and injection in a pool with AAV2-Igf2.

Antibodies.

The following antibodies were used in our experiments: AKT (1:2,000; Cell Signaling), phospho-AKT (1:2,000; Cell Signaling), AMPK (1:1,000; Cell Signaling), phospho-AMPK (1:1,000; Cell Signaling), GAPDH (1:500; Stressgen), GSK3β (1:2,000; Cell Signaling), phospho-GSK3β (1:2,000; Cell Signaling), IGF1R (1:1,000; Cell Signaling), INSR (1:1,000; Cell Signaling), p44/42-MAPK (1:2,000; Cell Signaling), phospho-p44/42-MAPK (1:2,000; Cell Signaling), mTOR(1:1,000; Cell Signaling), phospho-mTOR (1:1,000; Cell Signaling), PI3K (1:1,000; Cell Signaling), phospho-PI3K (1:1,000; Cell Signaling), S6 (1:1,000; Cell Signaling), phospho-S6 (1:1,000; Cell Signaling), and α-tubulin (1:10,000; Sigma).

RNA Expression Analyses.

RNA was isolated according to the manufacturer’s manual using RNeasy columns (Qiagen). All isolated RNA samples were tested for quality and quantity with the Bioanalyzer (Agilent Technologies). RNA-integrity (RIN) values were higher than 8. SYBRgreen real-time PCR experiment was performed with a LC480 detection system (Roche). All primers were designed online at the assay design center of the Roche Universal Probe Library:Atp5b (S:GGATCTGCTGGCCCCATAC, AS:CTTTCCAACGCCAGCACCT),Cyc1 (S:CAGAGCATGACCATCGAAAA, AS:CACTTATGCCGCTTCATGG),Igf2 (S:CGCTTCAGTTTGTCTGTTCG, AS:GCAGCACTCTTCCACGATG),Igfbp5 (S:CTACCGCGAGCAAGTCAAG, AS:GTCTCCTCGGCCATCTCA), andRpl13a (S:ATCCCTCCACCCTATGACAA, AS:GCCCCAGGTAAGCAAACTT).

Immunostainings.

Mice were anesthetized with avertin and perfused with gassed ACSF following 4% (wt/vol) PFA in PBS. Then, brains were treated overnight in 4% PFA at 4 °C and embedded in paraffin. Immunohistochemical analysis was performed on 10-μm-thick coronal brain sections. Hematoxylin-eosin (H&E) stain as well as a DAB-based immunostaining Dako-LSAB2 kit was used according to the manufacturer's manual. Primary antibody was used against phospho-S6 (1:100; Cell Signaling). For quantification, cells were counted within a defined identical square, whereas only cells were counted that were completely in the square.

Behavioral Analyses.

Behavioral tests were performed as described (60) with minor modifications. In short, to test anxiety behavior, the light–dark preference test was performed in a box consisting of two equal parts, a dark and a transparent compartment connected by a door. Within 5 min, time spent in the dark was analyzed. The spontaneous locomotor activity of mice was tested in the open field test. Test time for each animal was 10 min. In this time, the total time active, the number of rearing, the total time of rearing, traveled distance, time at the center and the periphery, and corner visits were analyzed. The setup was modified for the hole board test by changing the floor to a plastic plate with 16 symmetrical holes. Within 10 min, the traveled distance, the number of visits at the holes, the total exploration time, as well as the time spent for one exploration was measured and analyzed. To test the pain threshold with the hot plate test, each mouse was placed on a 52 °C metal plate, and the time until the mice licked the hind paw was measured. To test the overall motivation behavior of mice, a tail suspension test was performed. For this purpose, mice hung on their tails for 6 min and the time of struggling to get free was measured. To test the anxiety behavior, mice were tested in an elevated plus maze test. The test setup has two open arms and two closed arms, and preference of mice was tested. Test duration was 5 min, during which total distance traveled, distance traveled in closed arms, time spent in center, running speed, and relative time in closed arms were analyzed. Spatial learning and memory were analyzed in the Morris water maze test. The test started with a 2-d visible platform task to allow the mice to learn the platform position. For initial and reversal learning the flag was removed from the platform. During the following 6 d of spatial learning (initial learning) each mouse was released in the tank four times per day and allowed to swim until it found the hidden platform. After 6 d, a probe trial was performed to access the memory of the mice by removing the platform and measuring the time spent in the target quadrant, number of crossings of the platform position, and swim speed. After a 7-d break, the reversal learning test was performed. The hidden platform was placed in the opposite quadrant and learning was assessed for 6 d. Finally, the reversal probe trial was performed to assess memory formation. Contextual fear memory was assessed in fear conditioning paradigm with several cohorts of mice. All cohorts were tested with TSE Systems equipment, with 0.4-mA shock intensity and 2-s duration. AAV2-injected mice were tested with a Ugo Basile system. Parameters for shock intensity and duration (0.4 mA, 2 s) were identical for AAV2, AAV2-IGF2, and AAV2-IGFBP5 injections. Duration was reduced to 1 s for AAV2 and AAV2-IGF2/IGFBP5-shRNA coinjections to avoid ceiling effects. The freezing rate of the AAV-injected mice was analyzed automatically by Any Maze software (Stoelting). Animals for immunohistochemical analyses were killed before conditioning (baseline) and 28 d after conditioning, respectively. More specific information on behavioral experiments was described previously (60).

Aging Cohort.

Weight was noted every month from 6 to 15 mo of age. Genotype differences in the survival curve until the age of 80 wk were analyzed statistically with the Gehan–Breslow–Wilcoxon test.

Protein and RNA Quantification.

Western blot signal intensities were quantified with ImageJ software. Phosphorylation-specific antibodies were normalized with respective pan antibodies to the total amount of the studied protein. Other protein signals were quantified to tubulin. Real-time PCR signals were normalized to two housekeeping genes out ofAtp5b,Cyc1, andRpl13a, respectively.

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