Conserved age‐related increases in hippocampal PDE11A4 cause unexpected proteinopathies and cognitive decline of social associative memories

Abstract In humans, associative memories are more susceptible to age‐related cognitive decline (ARCD) than are recognition memories. Reduced cAMP/cGMP signaling in the hippocampus may contribute to ARCD. Here, we found that both aging and traumatic brain injury‐associated dementia increased the expression of the cAMP/cGMP‐degrading enzyme phosphodiesterase 11A (PDE11A) in the human hippocampus. Further, age‐related increases in hippocampal PDE11A4 mRNA and protein were conserved in mice, as was the increased vulnerability of associative versus recognition memories to ARCD. Interestingly, mouse PDE11A4 protein in the aged ventral hippocampus (VHIPP) ectopically accumulated in the membrane fraction and filamentous structures we term “ghost axons.” These age‐related increases in expression were driven by reduced exoribonuclease‐mediated degradation of PDE11A mRNA and increased PDE11A4‐pS117/pS124, the latter of which also drove the punctate accumulation of PDE11A4. In contrast, PDE11A4‐pS162 caused dispersal. Importantly, preventing age‐related increases in PDE11 expression via genetic deletion protected mice from ARCD of short‐term and remote long‐term associative memory (aLTM) in the social transmission of food preference assay, albeit at the expense of recent aLTM. Further, mimicking age‐related overexpression of PDE11A4 in CA1 of old KO mice caused aging‐like impairments in CREB function and remote social—but not non‐social—LTMs. RNA sequencing and phosphoproteomic analyses of VHIPP identified cGMP‐PKG—as opposed to cAMP‐PKA—as well as circadian entrainment, glutamatergic/cholinergic synapses, calcium signaling, oxytocin, and retrograde endocannabinoid signaling as mechanisms by which PDE11A deletion protects against ARCD. Together, these data suggest that PDE11A4 proteinopathies acutely impair signaling in the aged brain and contribute to ARCD of social memories.

In situ hybridization. Brains were stored at −80 °C until being cryosectioned at −19 to -17 °C in the sagittal plane at 20 μm, with sections thaw mounted on superfrost slides. As previously described [8,9], slides were pretreated in a series of 4% paraformaldehyde, 1M Triethanolamine/0.9% NaCl, ethanol (70-100%), chloroform, ethanol (100-70%), and sterile water and were then allowed to air dry before storing at -80 °C. In situ hybridization for PDE11A4 was also conducted as previously described [8,9] using an S35-labelled antisense probe (5′-ccaccagttcctgttttccttttcgcatcaagtaatc-3′). The probe was end labeled with 35 S-dATPα using terminal deoxytransferase and extracted using Illustra Microspin G-25 columns (GE Healthcare, North America). Each slide was then hybridized with 600,000 cpms of labeled probe in 125 µl of hybridization buffer and slides incubated overnight in a humid chamber warmed to 37° C. The following day, slides were washed in a series of saline-sodium citrate (SSC) buffers with 0.1 M dithiothreitol (2X SSC room temperature then 0.2X SSC at 55° C). Slides were dehydrated in 70% ethanol and allowed to air dry. Once dry, slides were exposed to Kodak BioMax MR film for 14 days, generating optical densities that were within the linear range of the film. Note, antisense probes yielded consistent expression patterns (see Figure 1) while sense probes yielded no signal as previously published [8,9].
Biochemical Fractionation and Western Blotting. Biochemical fractionation was performed as previously described [2,7,10] (see Figure S1A for validation of our fractionation protocol). In short, cells or tissue were sonicated in ice cold fractionation buffer (20 mM Tris-HCl, pH 7.5; 2 mM MgCl2; Thermo Pierce Scientific phosphatase tablet #A32959 and protease inhibitor 3 #P0044). For those samples processed by biochemical fractionation, successive rounds of differential centrifugation were conducted. Following a low-speed spin (1000 x g) to isolate the nuclear fraction pellet, the supernatant was transferred to a new tube and subjected to a high-speed spin (89,000 x g) to isolate the cytosolic proteins in the supernatant and the membrane proteins in the pellet.
The pellet was then resuspended in fractionation buffer with 0.5% Triton-X 100 to solubilize the protein, with a subsequent high-speed spin (60,000 x g) to isolate the supernatant containing soluble membrane proteins. Note that the nuclear and membrane pellets were "rinsed" prior to the resuspensions described above. That is, these pellets were resuspended in the previous buffer and respun with resulting supernatant discarded. Protein concentrations were determined by DC Protein Assay kit (Bio-Rad; Hercules, CA, USA) according to manufacturer protocol, and were subsequently equalized across samples. Samples were stored at -80 o C until further processing. For western blotting, 10µg of total protein for cells, 30µg of total protein for tissue, or 20µg for membrane, cytosolic, or nuclear fractions from tissue were loaded onto 12% NuPAGE Bis-Tris gels (Invitrogen, Waltham MA) and electrophoresed for one hour at 180 volts. Previously verified hippocampal tissue from a PDE11A KO mouse was included in all PDE11A4 blots as a negative control. Protein was transferred onto a 0.45µm nitrocellulose membrane for two hours at 100 mA. Membranes were then washed twice in tris-buffered saline with 0.1% tween20 (TBS-T) before staining with Ponceau S to determine total protein loading. Note, Ponceau S was chosen over a housekeeping gene as a loading control based on the bestpractice statement of the Journal of Biological Chemistry [11]. Images of the stained membranes were collected to later quantify the optical density of the total protein stain (i.e., spanning ~200kDa to 10kDa), and then the membranes were rinsed in TBS-T to remove the stain. Blots to be probed with the #1 antibody were blocked in 5% milk while all others were blocked in Superblock Blocking Buffer (ThermoFisher, Cat#37515), each with 0.1% Tween 20. Overnight primary antibody incubation at 4°C was completed for the following targets: PDE11A (Aves custom PDE11#1 at 1:10,000; Fabgennix PD11A-112 at 1:500), CREB (Cell Signaling #4820 at 1:10,000) as well as synaptophysin (BD Transduction #611880 at 1:200,000), histone 3 (Millipore 05-928 at 1:5000) and p-AKT (Cell Signaling 9271S at 1:1000). The next day, membranes were washed four times with TBS-T for ten minutes each. Secondary antibody incubation was completed for one hour at room temperature.

Immunofluorescence (IF)/Immunohistochemistry (IHC).
For most experiments, fresh-harvested, flash-frozen brains were embedded in matrix, cryosectioned in the sagittal plane at 20 µM, and thaw-mounted two sections per slide. One exception was that perfused tissue was sectioned with a vibratome at 50 µm for labeling using the AC3 antibodies described below. As previously described [12], 4% paraformaldehyde in PBS for 20 minute was used to fix frozen tissue. After fixation, the tissue was washed three times for ten minutes each in PBS with bovine serum albumin and triton X-100 (PBT). To reduce non-specific labeling, antibodies hosted in rodent species were pretreated with anti-Mouse FabFragments (0.15mg/ml; Jackson Immunoresearch # 715-007-003) in PBS for 2 hours. The FabFragments were then washed off by 3x10 minute washes in PBT prior to adding primary antibody. Overnight incubation with antibodies to the following targets was done at 4°C.
The following day, sections were washed 4x in PBT and incubated for 90 min in secondary antibody, with colabeling studies prioritizing secondaries recognizing the PDE11A antibodies first (Alexafluor 488 AffiniPure Donkey Anti-Chicken, 1:1000, Jackson Immunoresearch #703-545-155 or Alexafluor 488 AffiniPure Donkey Anti-Rabbit, 1:1000, Jackson Immunoresearch # 711-545-152), followed by 3X10 min PBT washes, then the secondaries recognizing the co-labeling marker antibody (Alexafluor 594 AffiniPure species-specific, 1:1000, Jackson Immunoresearch). After the secondary antibody incubation(s), slides were washed in PBT three times for ten minutes each. The slides were then rinsed ten times in PBS. For IF, slides were then mounted with DAPI fluoromount (Southern Biotech, #0100-20). In the case of immunohistochemistry, slides were processed as previously described [13]. Slides were treated with ABC solution (Vector Laboratories, PK-6100) prepared in PBT for 90 minutes. More specifically, after washing 3X10 washes with PBS sections were subjected to a DAB reaction (Vector Laboratories, SK-4100) until fully developed. Slides were placed in PBS once the reaction was complete. After being dried at room temperature, slides were mounted with Permount mounting media (Fisher, SP15-100) and a coverslip. Slides were kept covered and refrigerated until viewed and imaged. For quantification of PDE11A-filled structures and some co-labeling studies, images were taken using a CX9000 camera and Neurolucida imaging software (MBF Bioscience; Williston VT) mounted on a Nikon Eclipse 80i bright-field microscope that is equipped with a dry 20X objective. For co-labeling studies, images were captured using a LASX software and a Leica SP8 confocal microscope with oil immersion objectives 20X-63X.
The number of structures was quantified by an experimenter blind to treatment.
Social Transmission of Food Preference. As previously described [5,12], mice were food restricted such that access to food was limited to 1 hour per day for 2 days prior to the start of training. 24 hours prior to training, all mice were first habituated to a powdered version of their regular food (i.e., their standard Envigo Teklad 8904 food pellets were pulverized using a standard kitchen blender) by being given access to a jar full of it in a clean home cage. The next day, a mouse designated as the "demonstrator" was moved to a clean home cage alone and fed a powdered version of their food laced with a household spice for one hour (e.g. McCormick brand basil vs. ginger, thyme vs turmeric, mint vs cardamom, orange vs anise, or basil vs thyme available in any grocery store). Note that some cohorts of mice were trained and tested at a given retrieval time point with one spice combination and then trained and tested at a different retrieval time point with a different spice combination in order to reduce the total number of mice used herein. For example, data shown in Figure S6 are all from the same cohort of middle-aged mice that were trained/tested at different times, with a different spice combination used at each retrieval time point. We have extensively detailed elsewhere that such an approach does not confound interpretation of our data [5]. The demonstrator is then returned to the home cage and their "observer" cage mates interact with the demonstrator for 15 minutes, during which time the observers make an association between the non-social odor (household spice) and the social pheromones in the breath of the demonstrator. 15 minutes, 24 hours, or 7 days after training, the observer mice are then tested for 60 minutes by being placed in a clean home cage by themselves and given access to 2 powdered foods--one laced with the spice that the demonstrator was given and the other a novel spice. The amount of food eaten and the amount of time spent eating the food (scored for 1 minute out of every 10 minutes) was calculated by an experimenter blind to group. All mice met minimum inclusion criteria of eating at least 0.25 gm of food and being observed to eat for at least 5 seconds. Memory formation/retrieval is defined as the observer mice eating more of the food laced with the familiar spice that was on their demonstrator's breath versus the novel spiced food (preference ratio: familiar-novel/familiar+novel).
Odor Recognition. As previously described [5,12], 1'' round wooden beads (Woodworks) were placed in the subjects' home cages for at least 7 days prior to testing to allow for habituation and saturation of the beads with social odors. Training involved a habituation trial with 3 beads from the subject's home cage, followed by 2 subsequent trials that included 2 home-cage beads and 1 novel-scented bead. For social odor recognition (SOR), the novel-scented bead came from a cage of sex-matched mice of a different strain (e.g., C57BL/6J Jax #000664, BALB/cJ Jax #000651, 129S6/ SvEv Taconic #129SVE, C3H/HeJ #000659, A/J #000646). For non-social odor recognition (NSOR), the novel-scented bead came from a baggie of bedding saturated with a household spice (e.g., basil, ginger, marjoram, cumin, etc.). Memory was then tested 1 hour (short-term memory, STM), 24 hours (recent long-term memory, LTM) or 7 days later (remote LTM). For SOR testing, mice were presented with one home cage bead, one bead from the familiar donor strain used during training, and one bead from a second novel donor strain. For NSOR, mice were presented with 1 bead scented with the training spice and 1 bead with a novel household spice. The donor/spice assigned as "novel" within a given set of scents was counterbalanced across subjects. Mice were allowed to investigate the beads for 2 minutes and time spent investigating beads was manually scored by an experimenter blind to the conditions. All mice met minimum inclusion criteria of spending at least 3 seconds sniffing. Memory is defined as spending more time investigating the novel vs familiar scent (preference ratio: novel-familiar/novel+familiar).
Plasmid generation. Plasmids were generated as previously described [10]. Briefly, Genscript (Piscataway, NJ) generated constructs expressing either EmGFP alone containing an A206Y mutation to prevent EmGFP dimerization [14] or the mouse Pde11a4 (NM_001081033) sequence fused at the N-terminal with EmGFP. These constructs were initially generated on a pUC57 backbone and then subcloned into a pcDNA3.1+ mammalian expression vector (Life Technologies; Walthan, MA). Pde11a4 mutations (i.e., S117A, S124A, S117D, S124D, S162A, S162D) were introduced using the QuickChange procedure and products as per manufacturer's instructions (Agilent Technologies; Santa Clara, CA). Oligonucleotide primers used in the QuickChange procedure were generated by Integrated DNA Technologies (Coralville, IA) and mutated DNA sequences were subsequently verified by Functional Biosciences (Madison, WI).
Cell culture and transfections. Cell culture and transfection were conducted as previously described [10] For most studies, cells were allowed to grow for five hours before sample processing (e.g. fixation for imaging, sonication for Western blot, etc.) and, thus, were harvested ~24 hours following transfection. The one exception was in the case of the COS1 experiment that tracked protein expression changes between the S117A/S124A versus S117D/S124D mutants from 24 hours to 72 hours following transfection. For imaging experiments, cells were fixed with 4% paraformaldehyde in PBS for fifteen minutes and then kept in PBS for imaging using a CoolSNAP EZ CCD camera (Photometrics, Tuscon AZ) that was mounted on an inverted Leica (Wetzlar DE) DMIL microscope with a Fluotar 10X/0.3 ∞/ 1.2 objective. The images were captured using NIS-Elements BR-2.3 (Nikon,Tokyo Japan) software. Images were then systematically hand scored by an experimenter blind to condition. This systematic process involved placing a 4x5 grid over the image in powerpoint to help keep track of position and using the same digital zoom for all images within an experiment, typically between 250% to 400%, depending on the experimenter. The cells within each box of the grid were then classified as exhibiting cytosolic-only labeling versus punctate labeling, and data were then entered for that grid into a grid-shaped template. Once all grids for an image were evaluated, data were typed into an .xls template that calculated the total number of cells that were labelled and the percent of labeled cells that exhibited cytosolic versus punctate labeling (data graphed as the latter).
PDE assay. As previously described [15], cells were harvested in buffer containing 20 mM Tris-HCl and 10 mM Stereotaxic Surgery. As previously described [5], the NeuroStar motorized stereotaxic and drill and injection robot (Tubingen, Germany) was used to conduct stereotaxic surgery. Mice were anesthetized with a mixture of oxygen and isoflurane at an induction rate of 3%. Upon initial administration, lack of reflexes was verified and isoflurane administration was maintained at 1.5-2%. A robotic drill was used to drill small holes at the following coordinates relative to Bregma (dCA1 AP, 1.7, dCA1 ML, +/ 1.6, vCA1 AP, 3.5, vCA1 ML, +/3.0).
Mice were allowed to recover for at least 2 weeks prior to behavioral testing. software. STAR v2.3.1 [17] was used to align sequences to the mouse mm10 genome (http://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/). Reads, which were summarized at exon, transcript, or gene level were, counted using the featureCounts function of the Subreads package [18] in R (https://www.R-project.org/) using Gencode M6 GTF (http://www.gencodegenes.org/mouse_stats/archive.html). Only those reads mapping uniquely to the genome were used. A minimum threshold of 10 was used for mapping quality (MAPQ). The edgeR package within R [19] was used for differential expression analysis. The average read depth for the samples was 64 million reads, and only genes with at least 1 count per million average depth were considered for differential expression analysis. The trimmed mean of m-values (TMM) method was used to normalize raw counts. The estimateGLMRobustDisp function [20] was then used to calculate the dispersion estimates. The function glmFit [21] was then used to fit normalized read counts to a generalized linear model. The function glmLRT was then used to perform gene-wise tests for significant differential expression. Benjamini-Hochburg's FDR [22] was then used to correct the raw P-value for multiple testing. Genes that differed between WT versus KO mice with aLogFC >+/-0.5 and an FDR-P<0.01) were included in pathway analyses using String v11.0 (stringdb.org) [23] (accessed 7/27/21).  Data Analysis. Data were collected blind to treatment and experiments were designed to counterbalance technical variables across biological variables. Data points greater than two standard deviations away from the mean were removed as outliers prior to analyses, as previously described (e.g., [5,8,24,25]). Outliers removed/total n: Figure 1O Figure S7C, 5/108. Data were analyzed for effect of genotype, age, behavioral parameter (e.g., bead, food, etc), and sex for experiments with greater than 6/sex/genotype [4,5,8,12]). Where datasets met assumptions of normality (Shapiro-Wilk test) and equal variance (Levene's test), the following parametric statistical analyses were run on Sigmaplot 11.2 (San Jose, CA, USA): ANOVA (F), Student's t-test (t), one-sample t-test (t; Table 1). In the case of one-sample t-tests (determining whether or not a given group demonstrated memory), a false-rate discovery (FDR) correction was applied to all P-values within an experiment to mitigate the risk of Type I error associated with multiple comparisons. Where normality and/or equal variance assumptions failed, the following non-parametrical statistical analyses were run: Kruskal-Wallis ANOVA (H), Mann-Whitney rank sum test (T), or Wilcoxon Signed Rank Test (Z). Repeated measures analyses were used where appropriate (e.g., in analysis of behavior across multiple trials). Post hoc analyses were performed according to the Student-Newman-Keuls or Dunn's method and significance was defined as P<0.05. Figure S1. PDE11A4 dimers are only found in the nucleus and cytosol. A) Biochemical fractionation of VHIPP yields the expected distribution of fraction markers phospho-AKT (~60 kDa), synaptophysin (~39 kDa) and histone 3 (~17kDa). B) Lysates from COS-1 cells transfected with either GFP-mPDE11A4 (11A4) or GFP alone (GFP) as well as hippocampi from Pde11a WT and KO mice were run on a native gel (different portions of the film shown at different optimal exposures that yield no signal in the negative control samples). Bands corresponding to the molecular weight of a PDE11A4 homodimer (i.e, ~242 kDa for GFP-tagged PDE11A4 and ~200 kDa for endogenous PDE11A4) can be identified in inputs, nuclear fractions and cytosolic fractions but not membrane fractions. Additional specific bands are observed at ~450 kDa and above, pointing to the incorporation of PDE11A4 within macromolecular complexes as has been reported for other PDE families [26]. The fact that PDE11A4 dimers are not detected in the membrane fraction suggests PDE11A4 located in membrane fractions is not there via a direct interaction (i.e,. via palmitoylation, myristolation, etc), but rather via protein-protein interactions. C) Lysates from COS-1 cells were also loaded on a 4-12% Bis Tris denaturing gel to validate antibodies detecting total PDE11A4 (PD11-112AP), PDE11A4-pS117 (PD11-140AP) or PDE11A4-pS117/pS124 (PD11-150AP). Whereas PD11-112 detects wild-type (WT) and phosphoresistant versions of a GFP-PDE11A4 fusion protein around ~121 kDA (note: studies in Pde11a KO vs. WT mice show endogenous PDE11A4 migrates ~95 kDA), PD11-140AP recognizes WT, S124A, and S162A but not S117A nor S117A/S124A. PD11-150AP also recognizes WT and S162A, but shows a somewhat reduced signal with S117A, greatly reduced signal with S124A, and no signal with S117A/S124A. Together, this pattern suggests that PD11-150AP preferentially recognizes pS124 and weakly recognizes pS117. Brightness and/or contrast of images adjusted for graphical clarity. Figure S2. BALB/cJ and 129S6/SvEv mice show age-related increases in PDE11A-filled filamentous structures as observed in C57BL/6J mice. A) Immunofluorescence using an antibody that detects PDE11A4-pS117/pS124 (i.e., PD11-150AP) replicates and extends findings presented in Figure 1L-O by showing an agerelated increase in PDE11A4-pS117/pS124 ghost axons occurs in both C57BL/6J and BALB/cJ mice. B) Quantification replicates the significant increase in PDE11A4-filled ghost axons that occurs in the C57BL/6J (n=3/age/sex) ventral CA1 (vCA1; failed normality; Rank Sum test for effect of age: T(6,6)=26.5, P=0.041) and ventral subiculum (vSub; failed normality; Rank Sum test for effect of age: T(6,6)=25.0, P=0.026) and C) shows this effect extends to vCA1 (t(12)=-4.07, P=0.002) and vSub (fails normality; Rank Sum test for effect of age: T(7,7)=36.0, P=0.038) of BALB/cJ mice (n=3M,4F/age). D) Qualitative assessments with antibodies recognizing total PDE11A4 (i.e, PD-112AP and 11A-#1) were also conducted in 129S6/SvEv mice (n=2M,1F/age), with PDE11A4-filled ghost axons rarely observed in vCA1 or vSub of young mice but plentiful in old mice. Together, these data suggest this phenomena is conserved across mouse strains. *vs. Young, P=0.041-0.002. Data plotted as mean ±SEM. Histogram stretch, brightness, and/or contrast of images adjusted for graphical clarity. Figure S3. Electron microscopy (EM) suggest that age-related increases in PDE11A4 protein expression lead to an accumulation of PDE11A4 in axons that either occludes co-localization of other axonal proteins or possibly leads to the degeneration of the surrounding axon. Immunohistochemistry for PDE11A4 protein was conducted in ventral subiculum of Pde11a WT and KO mice followed by EM as per [27] by the Instrument Resource Facility at the University of South Carolina School of Medicine, except that 0.5% Triton X-100 was used (as in our normal immunoflourescence protocol) instead of the 0.04% triton that is normally used for electron microscopy. Unfortunately, this higher concentration of triton was necessary for the antibody to label the tissue, which led to compromising of the ultrastructure. Brightness and/or contrast of images adjusted for graphical clarity. Figure S4. The fact that associative long-term memory (aLTM)-but not recognition long-term memory (rLTM)-is sensitive to age-related cognitive decline in humans [28][29][30][31][32][33][34] can be modeled in mice. A) 7 days after training, old mice (n=14M,9F) demonstrated worse remote aLTM for social transmission of food preference (STFP) than young mice (Yng; n=11/sex; effect of age: F(1,41)=4.98, P=0.031). Overall, old mice also ate less food than young mice (young 0.82 ±0.05 gm; old 0.69 ±0.1 gm; fails normality; Rank Sum test for effect of age: T(22,23)=616.5, P=0.012), which is consistent with reports of age-related deficits in homeostatic regulation of food intake [35][36][37]. B) In contrast, old mice (n=11M,12F) had equivalent remote rLTM relative to young mice (n=10M,12F) 7 days after training for social odor recognition (SOR; fails equal variance; Rank Sum test for effect of age: T(22, 23)=543.0, P=0.407), with old mice spending somewhat less time investigating the beads during the memory test ( 20.4 ±2.8 sec) relative to young mice (28.6 ±3.3 sec; fails normality; Rank Sum test for effect of age: T(22,23)=585, P=0.074). C) Old mice (n=6M,8F) also had equivalent remote non-social odor rLTM relative to young (n=7/sex) 7 days after training (effect of age: F(1,24)=0.33, P=0.574), with no difference in total time spent investigating the beads during the memory test (young 34.7 ±3.5 sec; old 29.0 ±3.3 sec; F(1,24)=1.62, P=0.215). D) Importantly, young and old mice demonstrated equivalent learning during SOR training, with a reduction in time spent exploring the novel bead from the first to the second training trial. That said, old mice spent less time exploring the novel SOR beads (effect of age x trial: F(1,41)=13.66, P=0.0006; Post hoc: trial 1 vs trial 2, P<0.001 for each age; old vs young trial 1, P=0.0009). E) Young and old mice also showed equivalent learning during NSOR training, with no differences in the total amount of time investigating the novel beads (effect of trial: F(1,24)=65.29, P<0.00001). *vs Young, P=0.031 to <0.001; @vs. Trial 1, P<0.001; m). # significant memory (i.e., greater than 0), FDR-P=0.015 to <0.001. Data plotted as mean ±SEM. Figure S5. Deletion of PDE11A does not affect cycle length or reproductive senescence in female mice. As expected, vaginal smears conducted for a period of 12 days verified that young adult Pde11a WT, HT and KO females all demonstrate cycles of equivalent duration and all old Pde11a WT, HT and KO females similarly experience estropause (i.e., each genotype shows a similar distribution of mice paused in the persistent estrous vs. persistent diestrous phase). Indeed, cycling was unlikely to be a key driver of our PDE11A-related effects since 1) we see equivalent effect sizes and variability in our male and female mice, both in terms of age-related increases in PDE11A4 expression and in terms of the preservation of memory observed in our aged PDE11A mutant mice, and 2) the data shown herein were collected from multiple cohorts of mice tested across multiple years, which strongly decreases the likelihood that all females happen to have been tested at the same point in their cycle. Data in B plotted as mean ±SEM. Brightness and/or contrast of images adjusted for graphical clarity.   Figure 3), there was an effect of age-but not genotype nor sex-on the total amount of time mice spent investigating the novel-scented bead (3-factor with RM ANOVA across trials fails normality; 3 Way ANOVA on trial 1 effect of age: F(1,169)=10.01, P=0.0018) and trial 2 (3 Way ANOVA on trial 2 fails normality and equal variance; Rank Sum effect of age: T(87,90)=8490, P=0.028). That said, all groups learned as indicated by a significant reduction in the amount of time spent exploring the novel scent during trial 2 vs. trial 1 (Wilcoxon Signed Rank Test for effect of trial: Z=-11.45, P<0.0001). B) During NSOR training (combined data for STM, 24hr and 7d test groups), there were no significant differences in the total amount of time each group spent exploring the novel bead during trial 1 or trial 2, and all groups learned as indicated by a significant reduction in the amount of time spent exploring the novel scent during trial 2 vs. trial 1 (3-factor with RM ANOVA fails normality; Wilcoxon Signed Rank test for effect of trial: Z=-9.94, P<0.0001). C) When mice are tested for STM or recent LTM for SOR (from Figure3D-E) and then retested for remote LTM 7 days after SOR training, WT-Young (n=12M,11F), WT-Old (n=14M,15F), KO-Young (n=13M,10F) and KO-Old mice (14M,14F) all show strong memory that does not differ from one another, although overall males demonstrated stronger memory than females (effect of sex: F(1,95)=4.51,P=0.036). This stands in contrast to the enhanced memory that can be observed in KO versus WT mice when mice are tested for the first time on day 7 ( Figure 3F). The ability to detect an enhancement in KOs when tested for the first time on day 7 is due to the decline in memory that is seen in the WT mice in absence of an earlier retrieval test ( Figure 1F), and is consistent with our previous observation in young mice alone [5]. @ main effect of trial (i.e., trial 2 vs trial 1 across groups), P<0.001; & main effect of age (i.e., old versus young across genotypes), P=0.028-0.001; # significant memory (i.e., greater than 0), FDR-P<0.001. Data plotted as mean ±SEM. Figure S8. Recombinant GFP-mPDE11A4 performs similarly to endogenous PDE11A4. A) Mapping of GFP expression observed >2 months following injection shows all mice received a targeted injection of dorsal and ventral CA1, with a minority of mice showing spread outside the targeted subfield. B) Recombinant PDE11A4 is phosphorylated at S117 and S124 in COS-1 cells and the ventral hippocampus (i.e., in vivo). Antibody signals are specific when compared to expression of GFP-alone (in cells and hippocampus) or expression of the S117A or S117AS124A phosphoresistant mutants (COS-1 cells). Molecular weights: GFP-tagged PDE11A4 constructs = ~124 kDa; GFP alone= 27 kDa; Actin = ~45 kDa. C) KO-11A4 mice (n = 10M,12F) and KO-GFP mice (n= 8M,11F) learned equally well during training for SOR (effect of group: F(2,41)=0.008, P=0.93; effect of trial: