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Hippocampal microRNA-132 mediates stress-inducible cognitive deficits through its acetylcholinesterase target G. Shaltiel, M. Hanan, Y. Wolf,
S. Barbash, E. Kovalev, S. Shoham &
Brain Structure and Function

ISSN 1863-2653
Brain Struct Funct
DOI 10.1007/s00429-011-0376-z
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Brain Struct Funct Hippocampal microRNA-132 mediates stress-inducible cognitivedeficits through its acetylcholinesterase target G. Shaltiel • M. Hanan • Y. Wolf • S. Barbash •E. Kovalev • S. Shoham • H. Soreq Received: 7 November 2011 / Accepted: 29 December 2011Ó The Author(s) 2012. This article is published with open access at Springerlink.com Diverse stress stimuli induce long-lasting cog- of the soluble ‘‘readthrough'' AChE-R variant without the nitive deficits, but the underlying molecular mechanisms 30-untranslated region binding site for miR-132. TgR mice are still incompletely understood. Here, we report three different stress models demonstrating that stress-inducible enhanced c-fos labeling and correspondingly intensified increases in microRNA-132 (miR-132) and consequent reaction to the cholinergic agonist pilocarpine. They fur- decreases in its acetylcholinesterase (AChE) target are ther showed excessive hippocampal expression of miR- causally involved. In a mild model of predator scent- 132, accompanied by reduced host AChE-S mRNA and the induced anxiety, we demonstrate long-lasting hippocampal GTPase activator p250GAP target of miR-132. At the elevation of miR-132, accompanied by and associated behavioral level, TgR mice showed abnormal nocturnal with reduced AChE activity. Using lentiviral-mediated locomotion patterns and serial maze mal-performance in suppression of ‘‘synaptic'' AChE-S mRNA, we quantified spite of their reduced AChE-S levels. Our findings attribute footshock stress-inducible changes in miR-132 and AChE stress-inducible cognitive impairments to cholinergic- and its corresponding cognitive damages. Stressed mice mediated induction of miR-132 and consequently sup- showed long-lasting impairments in the Morris water maze.
pressed ACHE-S, opening venues for intercepting these In contrast, pre-stress injected AChE-suppressing lentivi- rus, but not a control virus, reduced hippocampal levels ofboth miR-132 and AChE and maintained similar cognitive Acetylcholinesterase  P250GAP  performance to that of naı¨ve, non-stressed mice. To dis- Cholinergic  Cognition  MicroRNA-132  sociate between miR-132 and synaptic AChE-S as poten- Psychological stress tial causes for stress-inducible cognitive deficits, we furtherused engineered TgR mice with enforced over-expression Shaltiel G and Hanan M contributed equally.
Acute and chronic stress can both induce anxiety (McEwenand Gianaros ) and either impair learning and memory Electronic supplementary material The online version of this performance (Diamond et al. Nijholt et al. or article contains supplementarymaterial, which is available to authorized users.
enhance it (Blank et al. Manipulating cholinergicneurotransmission in the hippocampus, which receives G. Shaltiel  M. Hanan  Y. Wolf  S. Barbash  H. Soreq (&) extensive cholinergic innervations, changes the cognitive The Edmond and Lily Safra Center of Brain Sciences, control over executive function and error monitoring The Silberman Institute of Life Sciences,The Hebrew University of Jerusalem, (Carter et al. suggesting cholinergic involvement in Jerusalem 91904, Israel stress responses. Recent studies associate acute and chronic stress reactions with specific microRNA (miR)-mediatedsilencing of affected transcripts (Meerson et al. E. Kovalev  S. ShohamHerzog Memorial Hospital, Jerusalem 91351, Israel MiRs are short (*22 nucleotides), non-coding RNAs that Brain Struct Funct regulate various molecular pathways (Bartel Krol malfunctioning, then maintaining miR-132 excess even et al. by post-transcriptional gene silencing (Fili- under AChE-S suppression would cause cognitive deficits.
powicz et al. ; Rana ). Each miR may target To investigate this triple hypothesis, we employed three several mRNAs, often in specific locations on their diverse stress paradigms: the acute predator scent test, 30-untranslated region (30-UTR) and can modulate entire acute unpredictable footshocks accompanied by hippo- pathways in a rheostat-like manner (Chen et al. campal AChE knockdown, and engineered mice with an [reviewed in (Soreq and Wolf However, the sig- inherited anxiogenic-like phenotype (Salas et al. due naling pathway(s) controlling the levels of neuronal miRs, to enforced excess of AChE-R, but with a depleted miR- the corresponding miR-target interactions involved in their 132 binding site which ensures continuous suppression of diverse functions and the consequent cognitive effects the host AChE-S, but not the transgenic AChE-R proteins remain incompletely understood.
(Shaked et al. ). In the two latter models, we also MiR-132 is prominently neuronal-enriched, highly evaluated cognitive deficits evolving from the stress induced by the cAMP-response element binding protein (CREB). In cortical neurons, miR-132 enhances neuronalmorphogenesis and neurite outgrowth by decreasing thelevels of the Rho family member GTPase-activating pro- tein, p250GAP (Vo et al. ). Mice engineered to over-express miR-132 in forebrain neurons correspondingly show marked increase in dendrite spine density, accom-panied by deficits in a novel object recognition test Mice were kept in an animal room at a constant tempera- (Wayman et al. However, which neurotransmission ture (22 ± 1°C) and a 12-h light/dark cycle with free signal(s) are responsible for miR-132 changes and which of access to food and water. They were tested in the same its target(s) mediate these effects remained unanswered.
room under dim illumination (30 lux) by the same testers Another target of miR-132 is the acetylcholine hydro- between 10 and 16 h. All experimental procedures were lyzing enzyme acetylcholinesterase (AChE) (Shaked et al.
approved by The Hebrew University's Committee for ; Meshorer and Soreq ). In acute psychological Animal Experimentation (NS-08-11485-4).
stress, activated cholinergic neurons display rapid, yettransient increases in a normally rare soluble AChE-R splice variant (Kaufer et al. that replaces the ‘‘syn-aptic'' AChE variant (Kaufer et al. Meshorer et al.
Intraperitoneal (i.p) injection of 0.1 ml of 25 mg/kg pilo- ; Meshorer and Soreq ). In macrophages, AChE carpine hydrochloride (Sigma, Rehovot, Israel), or saline suppression by miR-132 accentuates cholinergic signaling followed 30 min habituation to a holding cage.
(Shaked et al. In transfected neurons, tetracycline-controlled antisense suppression of AChE leads to Stereotactic surgeries enhanced dendrite extension and causes hyper-locomotionin engineered mice (Sklan et al. ). Taken together, we Group housed C57Bl/6J 9 weeks old male mice underwent surmised that miR-132-mediated suppression of hippo- stereotactic surgery, after which they were singly-housed campal AChE levels may enhance cholinergic signaling, throughout all subsequent testing. Control mice were singly contributing to the neurite extension, cognitive and loco- housed as well. Mice were anesthetized by i.p. injections of motion impairments caused by stressful experiences.
ketamine (50 mg/kg) (Forth Dodge, IA, USA) and domitor To challenge these predictions, we sought appropriate (0.5 mg/kg) (Orion Pharma, Espoo, Finland) mix, and then mouse models and experimental strategies. Our rationale mounted in a stereotactic apparatus for intra-hippocampal was as follows: (1) If indeed stress-inducible changes in injections. Coordinates of the injection sites (in mm) rel- hippocampal miR-132 and AChE cause long-lasting effects, ative to bregma were AP: -2.0, L: 1.8, DV: -1.5. Bilateral miR-132 increases and AChE activity decreases should be injections of 0.5 ul lentiviral suspensions were conducted observed in different stress models and even several days using a 10-l Glenco syringe (Houston, TX, USA). After after the initiation of psychological stress. (2) If the changes each injection, the needle was left in situ for 5 min before in miR-132 and ‘‘synaptic'' AChE-S are inter-related, then being retracted to allow complete diffusion.
preventing one would avoid the other, and if these changesare the cause for stress-induced cognitive deficits, then preventing them both from happening would avoid thesedeficits. (3) Alternatively, if miR-132 elevation by itself is The water maze consisted of a round tank, 1.6 m in sufficient to cause the stress phenotype and cognitive diameter, filled with water. Mice were trained to find the Brain Struct Funct location of a hidden platform (16 cm in diameter), sub- paraformaldehyde containing 4% sucrose (pH 7.4) or merged 1 cm below the water surface, using extra maze removed and kept frozen in -70°C.
visual cues. The training part consisted of 4 trials per day,with a 1-h brake between trials, for 3 days. The escape Predator scent exposure and footshock acute stress latency, i.e., the time required by the mouse to find theplatform and climb on it, was recorded for up to 60 s. Each Group housed C57Bl/6J 9 weeks old male mice were mouse was allowed to remain on the platform for 30 s and placed on well-soiled cat litter for 10 min (in use by the cat was then moved from the maze to its home cage. If the for 2 days, sifted for stools) (Cohen et al. or were mouse did not find the platform within 60 s, it was placed placed in a footshock delivery system (Campden Instru- gently on the platform for 30 s, and then returned to its ments, UK) where they received seven inescapable electric home cage. On the fourth day of the experiment, the footshocks (0.3 mA; 2 ms) at unequal intervals over a total platform was removed and a probe trial was conducted: period of 120 min.
mice were placed in the maze for 60 s, in which thenumber of crosses over the four quadrants of the maze was Elevated plus maze recorded. Increased swimming in the quadrant where theplatform was originally placed was considered as an indi- Anxiety-related behaviors were tested in a Plexiglas plus- cation of spatial acquisition. One day after the end of shaped maze containing two dark and enclosed arms behavioral tests mice were anesthetized by Isoflurane (30 9 5 cm with a 5 9 5 cm center area and 40 cm high inhalation and decapitated. Brains were removed and two walls) and two 30 9 5 cm open and lit arms, all elevated half sagital sections were immediately frozen in liquid 50 cm above ground. Individual mice were placed in the nitrogen. One section was sliced in cryostat for Karnov- center of the maze, tracked for 5 min with a video sky–Roots staining experiments (Sternfeld et al. and camera, and then returned to their home cage. The plus the other was taken for mRNA measurements. In brief, maze was wiped clean between trials with a 10% alcohol AChE activity staining was conducted using the Karnov- sky–Roots staining method (Karnovsky and Roots ;Kaufer et al. 40-um frozen sections were incubatedat room temperature in soft shaking for 5 min. Staining Lentiviral procedures solution contained 2 mM acetylthiocholine iodide (Sigma,Israel), dissolved in PBS, 5 mM sodium citrate, 3 mM The packaged virus was collected at 24 and 48 h post- cupric sulfate, 0.5 mM potassium ferricyanide, 0.1 mM transfection and concentrated using ultracentrifugation butyrylcholinesterase specific inhibitor, ISO-OMPA.
(70,000g, 2 h, 15°C). Dilutions of concentrated virus werefollowed by infection of HEK-293T cells with diluted Two unit serial maze virus. The resulting titer (*1 9 109 infectious particlesper ml) was assessed for shRNA expressing viruses using 3- to 6-months-old female 30 UTR-null AChE and FVB/N puromycin selection and for GFP expressing viruses by cell mice were group housed. Only females were taken for this fluorescence counting. Screening of two putative AChE experiment due to their higher stress responsiveness com- shRNAs, sh799 and sh800, designed to down-regulate pared with males (Goel and Bale ). Modular units mouse/rat AChE levels was first conducted in neuronal-like placed in a series constitute this maze (Quartermain et al.
PC12 cells and compared to a control shRNA (shCON) that ). To complete one ‘‘run'', a 22 h water-deprived does not target any mammalian gene. Both shRNAs mouse must choose between turning right or left to receive, reduced the AChE-S variant and suppressed AChE total at the end of the maze, a reward in the form of 40 ll of 5% activity in these cells, but only Sh799 down-regulated sucrose and one ‘‘run'' is considered complete. Then the overall AChE mRNA levels (Online Resources Fig. 1).
mouse must shuttle back to the other end of the maze whereit gets the same reward. There were 5 runs per session; one Viral infection of primary cortical cell culture session per day. Quantitative measures of performanceinclude number of left/right choice errors and ‘‘retrace Cerebral cortex was separated from the brain of 15th errors''—episodes in which a mouse moves in the wrong day embryos of FVB/N mice, minced, and cells plated direction (toward the end without a reward) (Farchi et al.
on poly-L-ornithine coated 12-well plates in Neurobasal ). Mice were euthanized 80–90 min after the behav- medium, 2% B27 supplement, 20% Glutamax and 20% ioral experiment by an intra-peritoneal injection of Penicillin/streptomycin (Invitrogen, Grand Island, NY, 200 mg/kg sodium pentobarbital (‘‘Pental''). Brains were USA). 72 h post-plating, 250 ul of non-concentrated either fixed by transcardial perfusion with ice-cold 4% medium containing viral particles was added to each well.
Brain Struct Funct to bregma. Images of each hippocampal sub-region wereacquired and analyzed using AnalySIS software (SIS, RNA was extracted (RNeasy lipid tissue kit, Qiagen, Germany). Karnovsky staining of histochemical AChE Valencia, CA, USA), DNase treated and its integrity con- activity was as in (Sternfeld et al. In situ hybrid- firmed by gel electrophoresis.
ization was essential as described (Berson et al. ).
For SYBR green real-time PCR, cDNA synthesis (Pro- mega, Madison, WI, USA) involved 1 lg RNA samples in AChE activity measurement 20 ll reactions. Duplicate real-time reverse transcriptase(RT) PCR tests involved ABI prism 7900HT, SYBR green AChE activity was measured by Ellman's assay (Ellman master mix (Applied biosystems, Foster City, CA, USA) et al. adjusted to multiwell plates and normalized and ROX, and a passive reference dye for signal normali- according to the protein content of the cell lysate (DC zation across the plate. Primer sequences are listed in protein assay, Bio-Rad, Hercules, CA, USA).
Table . 18S rRNA or GAPDH was used as referencetranscripts. Annealing temperature was 60°C for all prim- Statistical analysis ers. Serial dilution of samples served to evaluate primersefficiency and the appropriate cDNA concentration that The following statistical analyses were done using yields linear changes. Melting curve analysis and ampli- STATISTICA9 and GraphPad Prism 5 software: Student's cons sequencing verified the identity of end products.
t test, one-way ANOVA and two-way ANOVA with Tukey MicroRNA quantification using The TaqMan MicroRNA post hoc comparisons and Pearson's correlation test.
Assays (Applied Biosystems, Foster City, CA, USA) wasconducted in two-step RT-PCR kit according to the man-ufacturer's instructions. For miR-132 (Applied Biosystems, Foster City, CA, USA) measurements, a reference tran-script of SNO-135 (Applied Biosystems) was used.
Elevated predator stress-inducible miR-132 associateswith suppression of AChE-S Histochemistry and immunohistochemistry Our first experimental model served to explore the inter- The AChE-R C-terminal peptide (ARP) was labeled by a relationship between a long-lasting stress phenotype, rabbit polyclonal antibody (Berson et al. The com- induced by predator scent, increases in hippocampal miR- mon domain shared by both AChE variants was labeled 132 and decreases in AChE-S. Seven days following a with goat anti-human AChE (Santa Cruz, CA, USA, anti- predator scent stress (Fig. C57BI/6J mice subjected to body N19). c-fos was stained using rabbit anti c-fos an elevated plus maze (EPM) showed sustained anxiety.
(Sigma, Rehovot, Israel). In co-localization studies, we Compatible with previous reports on predator smell-stres- used fluorescein (FITC)-labeled donkey anti-rabbit to sed mice (Cohen et al. they spent less time in the visualize AChE-R and streptavidin-Cy3 to visualize gen- maze open arms and more time in its closed arms and eral AChE. Two to three coronal brain sections were attempted fewer entries to the open arms compared to naı¨ve sampled at an estimated distance of 2.8–3.3 mm posterior mice (Fig. b–d). Given that miR-132 targets the AChE Table 1 Primer sequences for Forward primer 50–30 Reverse primer 50–30 SYBR green real-time PCR

Brain Struct Funct Fig. 2 Neuronal AChE and the GTPase activator p250GAP are bothtargets of miR-132. a The ‘seed' region in miR-132 is complementaryto the targeted 30-UTR in the AChE-S and P250GAP transcripts[created using Targetscan and Pictar websites (Online ResourcesTable 1)]. b Hippocampal mP250GAP mRNA, but not mAChE-Slevels are reduced in stressed compared to naı¨ve mice (Student'st tests: *p 0.05), likely reflecting different balances between therates of transcription and miR-132-mediated destruction of these twotranscripts relationships with its neuronal targets, we quantified themajor AChE-S mRNA and the p250GAP transcript, withpredictably tighter hybridization and higher energy inter- Fig. 1 Predator scent stress leads to inter-related miR-132 elevation action with miR-132 (Fig. Intriguingly, p250GAP but and AChE and p250GAP suppression. a Mice were exposed to the not AChE mRNA levels were suppressed in the predator predator scent test and 7 days later were tested in the elevated plusmaze (EPM). b–d Compared to naı¨ve, stressed mice spent less time in scent-stressed hippocampus (Student's t test: p 0.05, the open EPM arms (b; Student's t test: *p 0.05), more time in the Fig. b), suggesting less robust stress-inducible transcrip- closed EPM arms (c; *p 0.05) and attempted less entries to the tion and/or more efficient destruction of p250GAP mRNA open arms (d; *p 0.05). e AChE-S activation leads to miR-132 under stress.
up-regulation, which in return down-regulates the AChE-S transcript.
f–h 7 days post predator scent exposure, hippocampal miR-132 isup-regulated by *220% (f; Student's t test, t18 = -3.22, *p 0.01), AChE-S knockdown prevents miR-132 up-regulation AChE activity is reduced (g; Student's t test, t23 = 2.00, *p 0.05) and memory impairments and hippocampal miR-132 levels show significant negative correla-tion to AChE activity (h; Pearson's test: r = -0.55; p 0.01) Predicting that the stress-inducible changes in hippocampalmiR-132/AChE-S impair the cholinoceptive capacities of mRNA transcript (Fig. we quantified hippocampal hippocampal neurons (Gray et al. we next set out to miR-132 levels, which we found to be higher by *220% establish an in vivo experimental model where these compared to naı¨ve mice (Student's t test: p 0.01; changes could be avoided. To achieve this goal, we intro- Fig. accompanied by 25% reduction in AChE activity duced AChE knockdown. For this purpose, we used a pre- (t test: p 0.05; Fig. A significant negative correla- calibrated shACHE agent targeted to the exon2 common to tion between hippocampal miR-132 levels and AChE the AChE-S and AChE-R transcripts (Fig. and which activity suggested causal relationship (Pearson's test: efficiently infected cultured primary neurons (Fig. b), r = -0.55; p 0.01; Fig. h), compatible with the reduced AChE activity by over 50% within 12–24 h post- capacity of miR-132 to suppress AChE activity in cultured infection (Fig. c) and suppressed AChE-S and AChE-R primary neurons (Online Resources Fig. 2).
mRNA levels as well as the common domain shared by Our previous findings showed stress-inducible increases these two transcripts (Fig. Bilateral injection of this in neuronal AChE gene expression (Meshorer et al. ; agent to the hippocampus CA1 region of this AChE-tar- Meshorer and Soreq ). Given that mRNA levels reflect geted lentiviral shRNA (shAChE) was then followed by a a steady state between its synthesis and degradation, we 10-days healing period. At this time point, long after the predicted that the observed decline in AChE activity transient stress-inducible increases in hippocampal AChE could potentially mask a balance between increases in the have ceased, histochemical staining showed reduced AChE ‘‘synaptic'' AChE-S mRNA and miR-132-induced decrea- activity in the hippocampus of lentiviral injected mice, ses in its levels. To find more information on miR-132 with predictably larger reduction of AChE activity in

Brain Struct Funct AChE suppression prevents stress-inducible cognitivedamages Lentivirus-injected mice were subjected, 10 days post-injection, to unpredictable footshock stress compared tomice injected with a control virus (shCON) (Fig. Naı¨ve and non-injected mice exposed to the footshockstress were tested in comparison. Similar to predator scent,footshock stress alone led to 63% increase in hippocampalmiR-132, and infection with control lentiviruses extendedthe effect to a twofold increase in miR-132 levels. Theeffects on miR-132 of both stress and the chronic state ofinfection were completely prevented following shAChEsuppression (one-way ANOVA: p 0.003; Fig. sug-gesting that miR-132 elevation under stress is induced bythe reduced cholinergic neurotransmission which is pre-vented by AChE suppression. In rotorod tests, injectedmice showed normal performance reflecting unimpairedmotor coordination (Online Resources. Fig. 3). Neverthe-less, all of the stressed mice, regardless of their treatmentspent less time in the open arms of the maze compared tonaı¨ve mice (one-way ANOVA: p 0.001; Fig. dem-onstrating that preventing miR-132 increases failed toavoid the stress-inducible anxiety.
In the Morris water maze test, both shAChE-injected stressed mice and naı¨ve mice learned to reach the hiddenplatform faster than either non-injected or shCON-injectedstressed mice (post hoc test: p 0.05 for both, Fig. d),which exhibited spatial learning deficits. By the third dayof learning trials, all four groups displayed the same escapelatencies; however, in the probe test, shAChE-injectedmice showed control-like spatial memory, unlike the defi- Fig. 3 Lentiviral mediated AChE-suppression in hippocampal neu- cits shown by the stressed non-injected and shCON-injec- rons in vitro and in vivo. a The sh-799 agent is complementary toexon 2 in the endogenous mouse gene that is included in the two ted groups. Both naı¨ve and shAChE-injected stressed mice alternative transcripts. b Transduction efficiency of our GFP coding crossed more frequently over the missing platform quad- vector. Mouse primary cortical culture were transduced with vector rant, deviating from either non-injected or shCON-injected coding for GFP (MOI = 1). Expression reveals high transduction mice, at least in one time point; demonstrating better efficiency, with more than 90% of cells being transduced. c Primaryneuronal culture total AChE activity is suppressed 18, 24 and 48 h learning capacity to reach the hidden platform and following viral infection with shAChE-S versus shCON. Two-way reflecting better spatial memory (two-way ANOVA: ANOVA: treatment (F1,10 = 18.97, *p 0.01), time (F2,10 = 1.39, p 0.05; Fig. e, f).
p = NS) and interaction (F2,10 = 0.46, p = NS). d AChE splice shAChE-treated mice showed better cognitive perfor- variants mRNA levels in mouse cortical primary culture followingtreatment with sh799 versus shCON: overall AChE transcript was mance that could potentially be due to the enforced downregulated by 80% [n = 4 (sh799 and shCON); **p 0.02].
decreases in AChE-S, the prevented increases in miR-132, AChE-S was down-regulated by 68% [n = 3 (sh799 and shCON); corresponding changes in other transcripts (e.g., p250GAP) Mann–Whitney U test: *p 0.03], while the considerably lower or to all of these reasons combined. To test for probable AChE-R mRNA levels remained unchanged [n = 4 (sh799), n = 3(shCON); p = NS]. e Histochemical staining of AChE activity in correlations between the measured molecular elements and shAChE or shCON treated hippocampal sections. f Reduced AChE the modified cognitive performance of stressed mice, we activity in the shAChE versus shCON CA1 neuronal cell bodies and plotted the route traversed by each mouse in the first axons (relative to dentate gyrus; Student's t test; t13 = 18.5, quadrant as a function of the levels of relevant RNA **p 0.001 and t13 = 5.24, *p 0.002, respectively) transcripts. Impressively, miR-132 levels in both control neuronal cell bodies compared to axons in the injected area and shRNA-injected mice showed a significant correlation (t test: p 0.001 and p 0.002, respectively; Fig. e, f) to the cognitive malfunction reflected in the route traversed compared to hippocampi injected with a control virus.
in the missing platform quadrant during the probe test

Brain Struct Funct Fig. 5 Hippocampal miR-132 levels show inverse association withthe post-stress cognitive performance. a Significant negative corre-lation between hippocampal miR-132 levels and the route traversed inthe missing platform quadrant (Q1) in the probe test (Pearson's test:r = -0.58; p 0.01). b Suppressed hippocampal AChE-S (One-wayANOVA: F3,24 = 7.36, p 0.001; Post hoc: naı¨ve versus shCON**p 0.001; shCON versus shAChE **p 0.01) and inverseelevation p 0.001; Post hoc: naı¨ve versus stress *p 0.05, versus shAChE***p 0.0001; stress versus shAChE ***p 0.0001) mRNA levelsin naı¨ve, stressed, shCON stressed versus shAChE stressed mice.
Note that neither AChE mRNA nor p250GAP mRNA levels showedassociation with the transverse route, attributing the cognitive declineto miR-132 itself Fig. 4 AChE knockdown prevents the cognitive decline following virus-injected mice but not stressed and shRNA injected footshock stress. a Mice subjected to CA1 hippocampal injection oflentivirus-mediated knockdown of AChE (shAChE) or irrelevant mice showed AChE mRNA up-regulation (one-way control virus (shCON) and un-injected stressed and naı¨ve mice were ANOVA: p 0.001, Fig. b). Furthermore, as observed in exposed 3 weeks later to 7 unpredicted and inescapable footshock the predator scent test, p250GAP was down-regulated stress followed by EPM and Morris water maze (MWM) tests.
following footshock stress, and intercepting AChE and b Hippocampal miR-132 mRNA levels increase by 63 and 100% instressed and shCON stressed mice, but remained unchanged in miR-132 up-regulation resulted in enlarged p250GAP shAChE stressed mice. ANOVA test: F3,28 = 6.14, p 0.003. Post increases (one-way ANOVA: p 0.001; Fig. How- hoc LSD test: naı¨ve versus stressed *p 0.05, versus shCON- ever, neither AChE-S mRNA nor p250GAP levels were infected and stressed ***p 0.001; shCON-infected and stressed associated to the cognitive performance.
versus shAChE-infected and stressed mice **p 0.01. c Naı¨ve micespend more time in the EPM open arms than all stressed groups (one-way ANOVA: F3,28 = 8.49, p 0.001). d Naı¨ve and shAChE- Chronic hippocampal miR-132 increases associate infected stressed mice learn to reach the MWM hidden platform faster with AChE-S and p250GAP decreases than stressed or shCON stressed mice. Two-way ANOVA: trialnumber (F11,323 = 12.33, p 0.001), treatment (F3,323 = 10.11,p 0.001), interaction (F33,323 = 0.75, p = NS). Bonferroni post To further challenge the causal association between miR- hoc comparison—trial no. 9: naı¨ve versus stressed *p 0.05, versus 132 and stress-inducible cognitive malfunctioning, we shCON-infected and stressed *p 0.05, versus shAChE-infected and sought an experimental model where miR-132 would be stressed p = NS. e Representative illustrations of mice swimming elevated and AChE-S would be diminished. The AChE-R tracks in the MWM probe test (gray circles: place of the missingplatform in quadrant #1): Bottom shAChE stressed mouse. Top over-expressing TgR mice present such a system. In TgR shCON stressed mouse. f Naı¨ve and shAChE stressed groups display mice, enforced expression of the human hAChE-R tran- more crosses over the previously situated-platform quadrant. Two- script leads to excessive ACh hydrolysis which in turn way ANOVA: quadrant (F3,100 = 9.95, p 0.0001), treatment elevates brain miR-132 levels (Shaked et al. The (F3,100 = 0.002, p = NS), interaction (F9,100 = 3.38, p 0.012).
Bonferroni post hoc comparison for quadrant #1 (where the platform elevated miR-132 may target nascent host AChE-S mRNA was previously situated): Naı¨ve versus stressed *p 0.05, versus but not the transgenic 30UTR-null AChE-R transcripts shCON-infected and stressed *p 0.05, versus shAChE-infected and (Fig. a). Correspondingly, TgR mice show an anxiogenic- stressed p = NS; shCON-infected and stressed versus shAChE- like phenotype accompanied by hyper-reactivity to nicotine infected and stressed *p 0.05 (Salas et al. ). Hippocampal miR-132 levels increased (Pearson's test: r = -0.58; p 0.01; Fig. Normal by *2.7-fold in TgR mice compared to non-transgenic AChE mRNA levels were already retrieved at this time FVB/Ns (Student's t test: p 0.001; Fig. This pre- following footshock stress; however, stressed and control dictably led to suppression of both host AChE-S and yet

Brain Struct Funct Fig. 6 Hippocampal miR-132 elevation and suppressed AChE-S andp250GAP in TgR mice. a The mouse AChE gene in chromosome 5encodes the miR-132-targeted synaptic mAChE-S transcript, whereasthe CMV-regulated transgenic 30 UTR-null human AChE-R is miR-132 refractory. b Hippocampal miR-132 mRNA levels are increasedby 2.7-fold in TgR compared to non-transgenic FVB/N mice(Student's t test, t19 = 3.72, *p 0.001). c Hippocampal AChE-Sand p250GAP mRNA levels, are both reduced in TgR versus FVB/Nmice (Student's t tests: *p 0.05) more so, of p250GAP levels (Student's t test: p 0.05;Fig. Therefore, if AChE-S suppression could by itselfprevent stress-inducible cognitive decline, TgR mice shouldshow no such decline; but if miR-132 increases are thecause, then these mice should present a stress phenotype.
Chronic AChE-S suppression associateswith intensified cholinergic hyper-excitation The AChE-R transgene avoids miR-132 surveillance owingto the lack of its native 30-UTR (Fig. Therefore, the TgRbrain presents continuous excess of AChE-R which cannotbe suppressed, mimicking prolonged conditions of stress(Meshorer and Soreq ). Of note, choline acetyl trans-ferase (ChAT)-expressing cholinergic neurons appeared inthe engineered brains in normal numbers and size, with anormal symmetric distribution between right and left hemi- Fig. 7 miR-132-refractory AChE excess induces cholinergic hyper- spheres. In general, the engineered AChE-R protein accu- reactivity. a Mouse AChE-S, but not transgenic AChE-R with a mulated in cholinoceptive brain regions that tend to express truncated 30-UTR, is recognized by miR-132. b CA1 TgR neurons the primary synaptic AChE-S variant [e.g., the CA1 and display higher than background expression of human AChE-RmRNA. c–d CA1 and dentate gyrus (DG) TgR neurons express dentate gyrus (DG) in the hippocampus and the entorhi- higher than background human AChE-R protein. f–g Schemes of TgR nal cortex]. Typically, the neuronal cytoplasm, nucleus coronal brain sections showing mouse and transgenic-human AChE-R and dendrite(s), but not axons, were stained (Fig. b–e).
labeling patterns in hippocampal regions (e.g., dentate gyrus, DG, Furthermore, TgR mice presented an extreme sensitivity to CA1), subcortical regions [e.g. red nucleus (RN), lateral hypothala-mus (LH), amygdala (Amyg)] and in the entrohinal cortex (Ent Cx).
cholinergic stimulators. Thus, intraperitoneal exposure to Large and small green circles denote intense or faint transgenic 25 mg/kg of the muscarinic agonist pilocarpine (Dickson protein expression, respectively. Green triangles show intracellular and Alonso, induced massive up-regulation of increases (up) or decreases (down) in host and transgenic protein AChE-R in multiple cholinergic brain regions (Fig. g).
expression under pilocarpine treatment, respectively Specifically, the hippocampus and entorhinal cortex showed Brain Struct Funct numerous AChE-expressing cells with further intensified AChE protein increases in the hippocampal CA1 region of labeling under pilocarpine treatment. Under control condi- the transgenic mice, revealing an hyper-excitatory sensitiv- tions, several regions showed intense AChE-R labeling; but, ity (Frankland et al. ) with c-fos significantly correlated pilocarpine treatment caused massive AChE-R elevation to AChE-R labeling (Pearson's test: saline r = 0.86, throughout the hippocampus (Fig. Yet more specifi- p 0.05; pilocarpine r = 0.77, p 0.05; Fig. c).
cally, both TgR and strain-matched FVB/N control miceshowed intensified labeling under pilocarpine administration TgR mice show motion and cognitive malfunctioning compared to saline-injected mice of the Ca2?-responsiveCREB-dependent c-fos mRNA, associated with contextual Nocturnal activity monitoring of TgR mice versus non- fear conditioning (two-way ANOVA: p 0.05; Fig. a).
transgenic controls revealed interchangeable hyper- and The c-fos labeling pattern largely overlapped the observed hypo-locomotion activity of the transgenics during thedark, but not the light phase of the day compared to con-trols (Fig. ANOVA repeated measures: p 0.001).
Given parallel, yet distinct changes in the nocturnal activityof mice over-expressing the synaptic AChE-S variant(Cohen et al. the altered nocturnal activity of theTgR mice likely reflects changes in cholinergic signaling.
At the molecular level, hippocampal host AChE-S proteinamounts were 55% reduced in TgR mice compared toFVB/N controls (t test: p 0.01; Fig. To evaluatelearning capacities and adaptive behavior, we subjected Fig. 9 TgR mice show nocturnal hyper locomotion, suppressedACHE-S and impaired memory. a Nocturnal locomotion patterns ofTgR versus FVB/N mice. One-way ANOVA: Light F1,22 = 0.4,p = NS; Dark F1,22 = 79.02, p 0.001. b Reduced hippocampal Fig. 8 Pilocarpine induces robust hyperactivation of transgene and mAChE-S protein levels in TgR mice compared to FVB/N mice c-fos expression in the TgR brain. a Pilocarpine induces CA1 c-fos (Student's t test: t13 = 2.71, *p 0.01). c A serial maze task requires and AChE-R protein increases in TgR mice. b Pilocarpine-induced a water-deprived mouse to find a sweetened water reward at each end c-fos activation in the CA1 TgR hippocampus: two-way ANOVA of the maze. The mouse must shuffle five times between the two ends shows a significant effect of treatment (F1,19 = 226, p 0.0001) with of the maze to obtain five rewards. d TgR mice display lower serial a significant interaction between strain and treatment (F2,19 = 5.88, maze performance than FVB/Ns. Right/left errors: F1,33 = 9.97; p 0.05). c AChE-R labeling associates with c-fos activation.
Retrace errors: F1,33 = 9.37, *p 0.005. ANOVA with repeated Pearson's test: for saline r = 0.86, p 0.05 and for pilocarpine measures reveals a significant transgene effect in the two measures of Brain Struct Funct these mice to the serial choice maze and measured theanimals' ability to avoid right or left turning and/or retraceerrors (See Fig. c for a scheme of the maze). The cogni-tive test was conducted during the light phase of the daywhere no changes in total locomotion activity wereobserved between the two tested groups. TgR mice dis-played significantly more right/left choice errors andretrace errors compared to control FVB/N mice (two-wayANOVA: p 0.005; Fig. d). Close examination of vid-eotaped maze behavior revealed two major types of error:‘‘trapping behavior''—running in a repeated path severaltimes without correction, and lack of spatial orientationwith respect to reward location, manifested in repeatedvisits at the end of the maze where the last reward wasgiven and hence no reward was to be expected. Our tests ofsensory-motor function did not reveal deficits (OnlineResources: Methods) and thus excluded the possibility thattrapping behavior was due to the secondary effects of thetransgenic intervention.
Stress-inducible total alterations and inter-individualvariability in miR-132 and p250GAP Our working hypothesis predicted that the key processeswhich are activated under stress conditions would be com-mon to different animal strains and stress paradigms.
Therefore, we compared the outcome of the three models westudied integrated together by calculating the percentagechange under stress in specific hippocampal transcripts. Thisanalysis again showed increases in miR-132 in both thepredator scent (t test: p 0.01) and the AChE-R excessmodels (p 0.01), but not in the footshock stress under Fig. 10 Integrated analysis of the 3 stress models. Shown in fold shAChE, which led to miR-132 decreases (p 0.05); an changes from controls are the inter-animal variability values and meanchanges in hippocampal miR-132, AChE and p250GAP transcript accompanying decline in p250GAP in both models levels in the predator scent (a, Student's t test: miR-132 **p 0.01, (p 0.01 and p 0.05 for the predator scent and AChE-R AChE p = NS, p250GAP **p 0.01), footshock (b, Student's t test: excess models, respectively) with miR-132 increases. In miR-132 *p 0.05, AChE *p 0.05, p250GAP *p 0.05) and addition, in all three models, the stress-inducible changes in transgenic AChE (c, Student's t test: miR-132 **p 0.01, AChE*p 0.05, p250GAP *p 0.05) stress models employed in our study.
the hippocampal levels of miR-132 and its p250GAP and d Scheme of the proposed mechanism involved AChE targets showed inverse patterns of individual vari-ability. Thus, both predator-stressed C57BI/6J and the p250GAP. Our analysis thus attributes the observed stress- chronically anxious TgR FVB/N mice showed larger inter- inducible cholinergic hyper-excitation to the feed-forward animal hippocampal miR-132 variability compared to mat- regulation of miR-132 and AChE transcripts, and shows that ched controls. Inversely, they both presented smaller avoiding p250GAP reduction and the corresponding breadth p250GAP variability under stress than in control mice of inter-individual variability in miR-132 associates with (Fig. , Online Resources Fig. 4), suggesting that miR-132 stress-inducible cholinergic hyper excitation and cognitive changes serve to mitigate p250GAP variability under stress impairments (Fig. in these two models. In comparison, C57BI/6J mice sub-jected to shAChE knockdown predictably showed a ten-dency to reduce AChE levels (60–80% of control levels; p 0.05), while p250GAP levels were elevated in thismodel (p 0.5; Fig. ). These findings are compatible Using three different mouse stress models, we found long- with the hypothesis that the inter-individual variability in lasting stress-inducible enhancement of both the levels hippocampal miR-132 is inversely interlocked with that of and the individual variability in hippocampal miR-132 Brain Struct Funct expression. This phenomenon was accompanied by the (Salas et al. hyper-excitation in TgR mice is likely suppression of the levels of hippocampal AChE and the due to the failure of miR-132 in these mice to suppress GTPase activator p250GAP, which are both validated AChE. This hypothesis is reinforced by the co-expression miR-132 targets. Knockdown of AChE production greatly of TgR AChE with the early immediate protein c-fos, limited miR-132 increases in a footshock stress model, which is expressed in neurons whose activity is strongly suggesting continuous surveillance by cholinergic signaling stimulated by synaptic input (Dragunow and Faull ; of miR-132 levels in the hippocampal which is disrupted Frankland et al. ). c-fos is also essential for hippo- under psychological stress due to AChE over-production.
campus-dependent learning and memory (Fleischmann Suppressing AChE further prevented footshock stress- et al. and represents part of the signal transduction inducible damages in cognition, but not anxiety, attributing cascade underlying the molecular basis of long-term to miR-132 a regulatory role over post-stress cognition but potentiation (Miyamoto suggesting relevance for the not anxiety. Corroborating this finding, engineered mice observed cognitive impairments in TgR mice. In stressed with chronic excess of both miR-132 and engineered AChE wild type mice, however, activation of the AChE gluco- showed an anxiogenic-like phenotype, impaired locomo- corticoid-responsive element also takes place (Meshorer tion and cognition, and cholinergic hyper-excitation when and Soreq ). This might reduce ACh levels and con- exposed to pilocarpine. Of note, hippocampal AChE sequently suppress CREB-inducible miR-132 transcription, mRNA levels remained elevated 7 days following predator regaining homeostasis. Hippocampal miR-132 was not scent test and 14 days following footshock stress accom- up-regulated following stress when AChE-S was sup- panied by chronic hippocampal lentiviral infection. In the pressed is compatible with proposed inter-locked regula- pre-frontal cortex, we found weeks-long elevation of AChE tion of these two stress-inducible genes (Fig. d).
following mild stress (Meshorer et al. ). In the hip- Cre-lox mediated deletion of miR-132 in newborn pocampus, we noted such elevation during 1 h and 1 day hippocampal neurons decreases dendrite length and post-stress (Kaufer et al. , Nijholt et al. AChE arborization in adult mice (Magill et al. ), supporting a levels were normal in mice 14 days following footshock long-lasting role for miR-132 in neuronal differentiation, stress alone thus indicates the transient nature of this stress synaptogenesis and maintenance. The GTPase activator in the hippocampus.
p250GAP emerges as an additional target of miR-132 In predator-stressed C57BI/6J mice and in FVB/N mice which is involved in mediating the stress-inducible cogni- with engineered over-expression of AChE, we found ele- tive malfunctioning. Suppressing p250GAP in cultured vated inter-animal variability of hippocampal miR-132 cortical neurons enhances neurite sprouting, similar to the levels which was accompanied by narrower variability of neurological reaction following stress (Kawashima et al.
its p250GAP target, suggesting causal links between miR- ). Both neuronal activity and the GABAA inhibitor 132 and this neuronal protein. Given the in-bred features of bicuculline induce miR-132 transcription, which further these mouse strains, we hypothesized that the stress- down-regulates p250GAP and might enhance neurite inducible inter-animal variability in hippocampal miR-132 growth (Wayman et al. Also, miR-132 is up-regu- reflects life-long differences in individual experience lated during post-natal development, when massive neurite which mediate these changes. Reinforcing this notion, sprouting occurs; miR-132-targeted antisense oligonucle- personal experience determines much of the stress reac- otides attenuate neurite growth (Ponomarev et al. tions in human patients with post-traumatic stress disorder The reported link between p250GAP and the NMDA (Feder et al. ).
receptor (Nakazawa et al. further supports a causal Both miR-132 and AChE transcription are controlled by role in the post-stress cognitive impairments for p250GAP CREB (Shaked et al. which is notably involved in suppression. Likewise, engineered anti-sense suppression learning and memory, neural growth and by the neuronal of AChE modulates neuronal sprouting in the mouse hip- growth factor BDNF associated with cholinergic func- pocampus (Sklan et al. Our findings of miR-132 tioning (Cogswell et al. ; Im et al. ; Wayman excess in the hippocampus of mature TgR mice and in et al. ). Correspondingly, individual differences in footshock-stressed mice over a month after the insult, and response to chronic stress were recently attributed to hip- 7 days post-predator scent stress are compatible with the pocampal BDNF (Taliaz et al. ). Also, contextual fear hypothesis that the long-term dual suppression of AChE conditioning increases pri-miR-132 levels in the mouse and p250GAP in the hippocampus might serve as a com- hippocampus (Ponomarev et al. and in the hippo- pensatory mechanism to balance the damages associated campus of chronically-stressed rats (Meerson et al. with excessive neuronal sprouting.
and the cholinergic agonist pilocarpine leads to a transient AChE suppression prevents both miR-132 increases and up-regulation of pri- and mature-miR-132 (Nudelman et al.
the accompanying cognitive malfunctioning that suggests ). The observed pilocarpine and nicotine-inducible the stress-associated changes in neurite sprouting is a Brain Struct Funct pivotal cause of these damages, and opens new venues for treating trauma patients by mitigating irreversible changesin their neuronal network. Nevertheless, our study did not Alvarez-Saavedra M, Antoun G, Yanagiya A, Oliva-Hernandez R, exclude the involvement of other experimentally validated Cornejo-Palma D, Perez-Iratxeta C, Sonenberg N, Cheng HY miR-132 targets. For example, the miR-132-targeted light- (2011) miRNA-132 orchestrates chromatin remodeling andtranslational control of the circadian clock. Hum Mol Genet induced transcription regulatory factor X 4 (RFX4), 20(4):731–751. doi: abundant in the supra-chiasmatic nucleus (SCN) of the Bartel DP (2009) MicroRNAs: target recognition and regulatory hypothalamus, regulates biological clocks and rhythms functions. Cell 136(2):215–233. (Alvarez-Saavedra et al. Cheng and Obrietan Berson A, Knobloch M, Hanan M, Diamant S, Sharoni M, Schuppli D, Geyer BC, Ravid R, Mor TS, Nitsch RM, Soreq H (2008) In wild type mice, miR-132 levels are lower during the Changes in readthrough acetylcholinesterase expression modu- dark part of the circadian cycle in the SCN (Cheng et al.
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