Genetic manipulation of STEP reverses ... - Wiley Online Library

Genes, Brain and Behavior (2012) 11: 586–600

doi: 10.1111/j.1601-183X.2012.00781.x

Genetic manipulation of STEP reverses behavioral abnormalities in a fragile X syndrome mouse model S. M. Goebel-Goody∗,† , E. D. Wilson-Wallis† , S. Royston‡ , S. M. Tagliatela‡ , J. R. Naegele‡ and P. J. Lombroso† † Child Study Center, Yale University School of Medicine, New Haven, CT, USA, and ‡ Department of Biology and Program in

Neuroscience and Behavior, Wesleyan University, Middletown, CT, USA *Corresponding author: S. M. Goebel-Goody, PhD, Yale University School of Medicine, Child Study Center, 230 South Frontage Road, New Haven, CT 06519, USA. E-mail: [email protected]

Fragile X syndrome (FXS), the most common inherited form of intellectual disability and prevailing known genetic basis of autism, is caused by an expansion in the Fmr1 gene that prevents transcription and translation of fragile X mental retardation protein (FMRP). FMRP binds to and controls translation of mRNAs downstream of metabotropic glutamate receptor (mGluR) activation. Recent work shows that FMRP interacts with the transcript encoding striatal-enriched protein tyrosine phosphatase (STEP; Ptpn5 ). STEP opposes synaptic strengthening and promotes synaptic weakening by dephosphorylating its substrates, including ERK1/2, p38, Fyn and Pyk2, and subunits of N -methyl-D-aspartate (NMDA) and AMPA receptors. Here, we show that basal levels of STEP are elevated and mGluR-dependent STEP synthesis is absent in Fmr1KO mice. We hypothesized that the weakened synaptic strength and behavioral abnormalities reported in FXS may be linked to excess levels of STEP. To test this hypothesis, we reduced or eliminated STEP genetically in Fmr1KO mice and assessed mice in a battery of behavioral tests. In addition to attenuating audiogenic seizures and seizureinduced c-Fos activation in the periaqueductal gray, genetically reducing STEP in Fmr1KO mice reversed characteristic social abnormalities, including approach, investigation and anxiety. Loss of STEP also corrected select nonsocial anxiety-related behaviors in Fmr1KO mice, such as light-side exploration in the light/dark box. Our findings indicate that genetically reducing STEP significantly diminishes seizures and restores select social and nonsocial anxiety-related behaviors in Fmr1KO mice, suggesting that strategies to inhibit STEP activity may be effective for treating patients with FXS. Keywords: Anxiety, audiogenic seizures, behavior, c-Fos, fragile X syndrome, social, STEP, tyrosine phosphatase

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Received 15 December 2011, revised 9 February 2012 and 5 March 2012, accepted 6 March 2012

Individuals with fragile X syndrome (FXS) exhibit behavioral abnormalities, including intellectual disability, anxiety, hyperactivity and seizures (Boyle & Kaufmann 2010). They are also impaired socially, with 30% meeting the criteria for autism (Harris et al . 2008). The majority of cases are attributed to a CGG expansion in the Fmr1 5 untranslated region (Kaufmann & Reiss 1999) that becomes hypermethylated, leading to transcriptional silencing and diminished expression of fragile X mental retardation protein (FMRP) (Chen et al . 1995). FMRP binds select mRNAs and suppresses their translation (Siomi et al . 1993; Darnell et al . 2001, 2011; Laggerbauer et al . 2001; Zalfa et al . 2003). In the absence of FMRP, translation of some of these mRNAs is upregulated (Zalfa et al . 2003; Lu et al . 2004; Hou et al . 2006; Westmark & Malter 2007; Gross et al . 2010). Huber et al . (2002) report that Fmr1 knockout (Fmr1KO ) mice show exaggerated metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD), suggesting that FMRP regulates protein synthesis-dependent synaptic plasticity. These results mark the inception of the mGluR theory of FXS, linking dysregulation of proteins normally suppressed by FMRP to mGluR-LTD. This theory posits that many FXS phenotypes, including elevated protein synthesis, increased glutamate receptor endocytosis, immature spine development and behavioral abnormalities, originate in exaggerated mGluR signaling (Bear et al . 2004; Krueger & Bear 2011). Consequently, genetic and pharmacological approaches that decrease mGluR signaling attenuate several abnormalities in Fmr1KO mice (Chuang et al . 2005; Yan et al . 2005; Dolen et al . 2007; de Vrij et al . 2008; Osterweil et al . 2010; Hays et al . 2011; Thomas et al . 2011). Another focus of FXS research has been to discover alternative therapeutic targets by identifying mRNAs regulated by FMRP and/or mGluR-dependent signaling (Krueger & Bear 2011). One excellent candidate is striatalenriched protein tyrosine phosphatase (STEP), a brainenriched phosphatase that regulates proteins that are important for synaptic plasticity and are associated with the postsynaptic density (PSD), including N -methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) receptors (AMPARs), Fyn, Pyk2 and the mitogen-activated protein kinase (MAPK) family of proteins ERK1/2 and p38 (Goebel-Goody et al . 2012). STEP has been coined an ‘LTD protein’ because it is translates in response to mGluR activation and promotes mGluR-stimulated AMPAR endocytosis (Zhang et al . 2008; Luscher & Huber 2010). Additionally, STEP opposes synaptic

© 2012 The Authors Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society

Manipulation of STEP reverses FXS behaviors

strengthening by promoting NMDAR internalization and inactivating ERK1/2, Fyn and Pyk2 (Nguyen et al . 2002; Pelkey et al . 2002; Paul et al . 2003; Snyder et al . 2005; Venkitaramani et al . 2011). Cross-linking studies on FMRP-RNA complexes show that the transcript encoding STEP (Ptpn5 ) interacts with FMRP (Darnell et al . 2011). We hypothesized that mGluRdependent STEP translation would be dysregulated in the absence of FMRP and that excess basal levels of STEP might contribute to behavioral phenotypes characteristic of Fmr1KO mice (Musumeci et al . 2000; Peier et al . 2000; Spencer et al . 2005, 2011; Yan et al . 2005; Liu & Smith 2009; Mines et al . 2010). We show that genetically modulating STEP levels corrects select behavioral abnormalities in Fmr1KO mice, indicating STEP as viable target in FXS.

Materials and methods Subjects All protocols were approved by the Yale University Institutional Animal Care and Use Committee and strictly adhered to the NIH Guide for the Care and Use of Laboratory Animals. Mice carrying a targeted null mutation of the Ptpn5 gene (B6N.129Ptpn5tm1Pjlo , referred to as STEPKO mice, C57BL/6N background backcrossed for 9–12 generations; Venkitaramani et al . 2009, 2011) were crossed with mice carrying a targeted null mutation of the Fmr1 gene (B6N.129-Fmr1tm1Cgr , referred to as Fmr1KO mice, C57BL/6J background backcrossed for 10–11 generations, courtesy of Dr William Greenough, University of Illinois Urbana-Champaign). These crossings generated F1 STEP heterozygous (HT )/Fmr1HT female breeders, as well as STEPHT /Fmr1KO male breeders (Fig. 2a). Breeders from different litters were mated to produce F2 progeny with a selective reduction in STEP or Fmr1. Mice used in the current study were generated from F2 STEPHT /Fmr1HT female and STEPHT /Fmr1KO male mice further crossed for at least five generations to ensure that progeny were on a genetic background that was between C57BL/6N and C57BL/6J. Unless otherwise noted, mice were weaned and housed in groups of 2–5 in standard ventedrack cages in a 12:12 h light:dark cycle with food and water available ad libitum. Mice weaned to solitary housing were excluded from the study.

Genotyping Primers used to detect the presence or absence of the WT or KO (neomycin) allele on the Fmr1 gene were as follows: 5 WT primerGGTTAAAAGTCATCCGTGGCTA-3 , 5 KO primer-CTGAGCCCAGAA AGCGAA-3 , 5 common primer-ACCACCACTGCCCTTCTGAT-3 . Fmr1 primers were combined and the following multiprimer polymerase chain reaction (PCR) was used: 94◦ C for 5 min; 35 cycles of 94◦ C for 45 seconds, 60◦ C for 1 min, 72◦ C for 1 min; final extension at 72◦ C for 10 min. Fmr1 products were electrophoresed on a 2% agarose gel to detect bands at 500 bp (KO), 350 bp (WT) or both (HT; Fig. 2b). Primers used to detect the presence or absence of the WT or KO (neomycin) allele on the Ptpn5 gene were as follows: 5 -CCCTACTCTCATTCCTCCCTTCCC-3 , 5 KO primer-CCACCAAAGAACGGAGCC-3 , 5 common primerGGCAGCAGATGCTGGTGGC-3 . Ptpn5 primers were combined and the following multiprimer PCR was used: 94◦ C for 5 min; 36 cycles of 95◦ C for 45 seconds, 59◦ C for 1 min, 72◦ C for 1 min; final extension at 72◦ C for 10 min. Ptpn5 products were electrophoresed on a 2% agarose gel to detect bands at 400 bp (WT), 200 bp (KO) or both (HT; Fig. 2b).

Antibodies and reagents Antibodies, dilutions used and sources are listed in Table 1. Normal horse serum, avidin: biotinylated enzyme complex (ABC) Vectastain Genes, Brain and Behavior (2012) 11: 586–600

Table 1: List of primary and secondary antibodies used in immunoblotting and immunohistochemistry Antibody Anti-STEP (23E5) Anti-PSD-95 Anti-GAPDH Anti-rabbit Anti-mouse Anti-c-Fos Biotinylated anti-goat

Host

Dilution

Mouse

1:1000

Rabbit Mouse Goat Goat Goat

1:5000 1:40 000 1:10 000 1:5000 1:500

Horse

1:300

Source Boulanger et al . (1995) Millipore Millipore Bio-Rad Laboratories Bio-Rad Laboratories Santa Cruz Biotechnology, Inc. Vector Laboratories

Elite Kit and diaminobenzidine (DAB) Peroxidase Substrate Kit were purchased from Vector Laboratories (Burglingame, CA, USA). Heparin was purchased from Hospira (Lake Forest, IL, USA) and Histoclear was obtained from National Diagnostics (Atlanta, GA, USA). (S )-3,5Dihydroxyphenylglycine hydrate (DHPG) was obtained from SigmaAldrich (St. Louis, MO, USA).

Synaptoneurosome preparation and DHPG treatments Synaptoneurosomes (SNs) were prepared from hippocampi from 4- to 8-month-old STEPWT /Fmr1WT or STEPWT /Fmr1KO mice (three mice pooled for n = 1) using a modified protocol (Hollingsworth et al . 1985) as previously described (Zhang et al . 2008). SNs were first incubated for 10 min at 37◦ C and then either treated with 50 μM DHPG or remained as untreated, for 10 min at 37◦ C. SNs were lysed by adding sodium dodecyl sulfate (SDS) to a final concentration of 1%. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as a standard.

Immunoblotting

Proteins (10–40 μg) were resolved by SDS-polyacrylamide gel electrophoresis, transferred, blocked and incubated appropriately with primary and secondary antibodies as described (GoebelGoody et al . 2009). Immunodetection was accomplished using chemiluminescence (SuperSignal Kits, Pierce) and visualized with a G:BOX imaging station using GeneSnap software (Syngene, Frederick, MD, USA). Image J software (version 1.43u; NIH) was used for quantification. Protein signals were normalized to the average signal of all lanes on the same blot (Osterweil et al . 2010). This value was divided by the normalized glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal in the same lane. All biochemical results refer to STEP61 , which is the membrane-associated and only isoform of STEP present in the hippocampus (Boulanger et al . 1995; Oyama et al . 1995; Bult et al . 1996).

Behavioral tests Two cohorts of age-matched, sexually nave mice were used for testing: Cohort 1 for audiogenic seizures (AGS) and c-Fos analysis and Cohort 2 for the remainder of the behavioral battery. All mice in Cohort 2 were tested in the following order with a minimum of a 1 week intertest interval: open field, marble burying, elevated plus maze (EPM), social choice test, social dominance tube test and light/dark box. The original number of mice tested in each cohort is listed in Table S1. Given the large number of tasks conducted and the low probability of obtaining the genotypes of interest (Fig. 2a), multiple rounds of the behavioral battery were performed over several months. On each test day, at least one mouse of each genotype was represented, and they were all of similar age. As a result, the variability of age was equally distributed across genotypes.

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Goebel-Goody et al. Unless otherwise noted, the room was brightly lit with constant background noise (60 dB). With the exception of AGS, marble burying and social dominance tube test, ANY-maze video tracking software (Stoelting, Wood Dale, IL, USA) was utilized to automate behavioral testing. Investigators remained blind to the genotypes during all testing. All apparatuses were wiped thoroughly with MB-10 (200 ppm) and 70% ethanol between mice to disinfect and eliminate odor trails. Tests were conducted between 0800 and 1700 h.

Audiogenic seizures For AGS, mice were born and reared in static cages in a quiet room. Mice were weaned at least 24 h before testing. At postnatal day (PND) 20–24 (average age PND 23), mice were first habituated for 30 seconds to the chamber (40-cm diameter × 60-cm-high plexiglass cylinder encased in sound-proof foam). The 130-dB stimulus (PAL1, Lane Self Defense, Roseville, MN, USA) was powered by a direct current (DC) converter and mounted on the lid. Mice were graded by two observers as having no response, wild running, clonic/tonic seizures or respiratory arrest. The stimulus lasted until seizure onset or 5 min (whichever was first). No differences were observed between male and female mice in the incidence of AGS (data not shown); therefore, the data were pooled together for statistical analysis.

mouse ≤1-inch margin around the cup) was recorded during each phase. During habituation, the test mouse freely explored the three chambers, with the sides containing only cups. During socialization, a stranger mouse was placed under a cup in one side, while the other remained empty, and the test mouse freely explored the apparatus. Socialization index was calculated as the time spent in close proximity to the mouse divided by total time in close proximity to the mouse and cup. During social novelty, a novel stranger mouse was positioned under the remaining empty cup, while the stranger introduced during the socialization phase remained (familiar), and the test mouse freely explored the apparatus. Novelty index was determined as the time spent in close proximity to the novel mouse divided by the total time in close proximity to the novel and familiar mice. All stranger mice were nonlittermate, age-matched mice of varying genotypes, which had been previously habituated to being enclosed under the cup for 10 min on the day of testing.

Social dominance tube test

Total activity and center square exploration of male mice (average age 2.5 months, range 2–5 months) were assessed in an empty open-field box constructed of white, opaque plexiglass (50-cm long × 50-cm wide × 30-cm tall) for 5 min. The center square was designated as the central 25% of the box.

Male mice (average age 8.6 months, range 5–10 months) were tested in a social dominance tube test and evaluated for their number of ‘wins’ against STEPWT /Fmr1WT mice. Immediately before the match, mice were individually habituated to a clear plastic tube (30.5cm long × 3.8-cm inner diameter) contained within the open-field box for 5 min. Matches began when two opposing mice were placed head first at either end of the tube and released simultaneously. Matches concluded when one mouse completely backed out and retreated from the tube (designated as the loser). Matches lasting longer than 2 min or where mice crawled over each other were excluded. Each mouse performed three matches against nonlittermate, age-matched STEPWT /Fmr1WT mice.

Marble burying

Light/dark box exploration

Open field

Male mice (average age 2.8 months, range 2–5 months) were singly housed in static cages containing Alpha-Dri bedding (2.5 cm) for 48 h before testing. On the test day, the mouse was briefly removed from the home cage and 20 black glass marbles (15-mm diameter) were evenly arranged in a 4 × 5 pattern. The mouse was returned to the cage and allowed to freely explore for 20 min. On completion, an overhead picture was captured. The percentage of each visible marble was quantified using ImageJ (version 1.43u; NIH). Images were converted to grayscale, thresholded and a constant cutoff value established whereby anything darker than that value was considered to be a visible marble. Scales were normalized to the width of the cage. The area of each marble was measured and compared with the area of a completely visible marble (control = 100% visible). Marbles with a visible area of ≤60% of the control marble (i.e. buried at least 40%) were considered buried.

Elevated plus maze Male mice (average age 2.8 months, range 2–5 months) were rehoused with their littermates in groups of 2–5 in vented-rack cages for at least 48 h before conducting EPM. The apparatus comprised four arms (30-cm long × 7-cm wide) configured in a plus manner situated 40-cm high, with two opposing arms constructed with 15-cm-tall walls. Mice were placed in the center square and allowed to freely explore for 5 min under dim light (6–7 lux), while the investigator left the room. Any mice that fell were excluded. Entries were defined as four paws (or 75% of its body) in the zone.

Social choice test Male mice (average age 4.8 months, range 2–8 months) were tested in an opaque rectangular plexiglass box (64-cm long × 43-cm wide × 38-cm tall) divided into three compartments (20-cm long × 43-cm wide) by clear plexiglass walls with small, retractable entryways. Each side chamber contained an inverted wire cup (1-cm bar spacing). The task consisted of three 10-min phases: (1) habituation, (2) socialization and (3) social novelty. Activity in each chamber and in close proximity to either the mouse or cup (front 25% of the

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Male mice (average age 9.6 months, range 6–11 months) were examined in a partitioned light/dark box (38-cm long × 38-cm wide × 36-cm tall) composed of two equally sized plexiglass chambers joined by a small opening, where one side was clear and the other was black and covered with a black lid. The room was brightly lit so that the clear side was fully illuminated. Mice were individually placed in the illuminated chamber and allowed to freely explore for 10 min. Entries were defined as four paws (or 75% of its body) in the zone.

c-Fos immunohistochemistry Thirty minutes after the AGS stimulus ended, mice were perfused transcardially with 0.1 M sodium phosphate buffer (PB; pH 7.4) containing 5% sucrose and 1 unit/ml heparin followed by 4% paraformaldehyde in PB. Brains were postfixed in the same solution for 12 h, then cryoprotected by equilibration in 20% and 30% sucrose in PB, embedded in a 0.9% saline solution containing 20% BSA and 0.5% gelatin, polymerized with 4.6% glutaraldehyde and frozen. Fiftyμm-thick coronal free-floating sections were obtained and stored at −20◦ C in cryoprotectant solution (30% glycerol and 30% ethylene glycol in 0.1 M PB). Every sixth section from the anterior hippocampus to the posterior inferior colliculus was selected, resulting in a stained series at 300-μm intervals. Sections were rinsed in phosphate buffered saline (PBS), submerged in 3% H2 O2 for 20 min, blocked in 3% normal horse serum with 0.3% Triton X-100 (blocking buffer) for 30 min and incubated for 72 h in blocking buffer containing antic-Fos antibody (4◦ C). Sections were washed with PBS plus 0.3% Triton X-100 and incubated for 1 h in blocking buffer containing the biotinylated secondary antibody. Sections were then incubated in ABC Vectastain Elite Kit for 60 min, washed and reacted for 3 min in Tris buffer (pH 7.5) containing DAB, nickel substrate and hydrogen peroxide according to the manufacturer’s recommendations (DAB Peroxidase Substrate Kit). Nuclei were stained with methyl green, and sections were dehydrated by ethanol, defatted in Histoclear and mounted in DPX Mounting Media (Leica Microsystems Inc., Buffalo Grove, IL, USA). Genes, Brain and Behavior (2012) 11: 586–600


Quantification of c-Fos staining Three sections containing the periaqueductal gray (PAG) were projected at a 200× magnification onto paper using a microprojector (Ken-a-Vision 1000, Ken-A-Vision Manufacturing Company, Inc., Kansas City, MO, USA) and the locations of all c-Fos+ neurons were plotted. An investigator who was blind to genotype and AGS response quantified the total c-Fos+ neurons in the PAG from three sections per mouse.

Statistical analysis

All data were presented as means ± SEM (with the exception of AGS and social dominance tube test) and significance set at P ≤ 0.05 as determined by SPSS software version 19 (IBM, New York, NY, USA). For biochemical assessments, a two-way analysis of variance (ANOVA) was used with genotype and drug treatment as factors. Pairwise comparisons were accomplished using one-tailed, unpaired two-sample t tests to calculate differences in response to DHPG treatment in SNs compared with STEPWT /Fmr1WT mice because changes in one direction only were expected based on previous results (Zhang et al . 2008). Two-tailed X 2 tests were conducted to determine significant differences in AGS and the social dominance tube test. For c-Fos expression and all other behavioral tasks, significant differences between groups were determined by one-way ANOVA with LSD post hoc tests following confirmation of a significant main effect of group. Where indicated, pairwise comparisons in the social choice task were accomplished using two-tailed, unpaired two-sample t tests. Within a given behavioral task, if a subject was

(a)

Figure 1: Steady-state levels of STEP are upregulated and mGluR-induced STEP synthesis is absent in Fmr1KO mice. (a) Characterization of SN and cytosolic (Cyto) fractions. Representative blots with equal amounts of protein showing that PSD-95 is expressed almost exclusively in SNs, and, while expressed in both fractions, STEP is enriched in SNs. (b) Expression of STEP and PSD-95 is significantly higher in SNs from STEPWT /Fmr1KO mice, regardless of treatment (main effect of genotype ∗ P ≤ 0.05, twoway ANOVA). Pairwise comparisons indicate that DHPG treatment significantly increases STEP levels in STEPWT /Fmr1WT SNs but not STEPWT /Fmr1KO SNs (# P ≤ 0.05; two-sample t test). (c) Expression of STEP in unstimulated Cyto fractions does not differ with genotype, suggesting that the basal increase in STEP expression in STEPWT /Fmr1KO mice occurs selectively in SNs. Data represent the mean normalized protein signal divided by the normalized GAPDH signal ± SEM for all values (n = 3).

found to be an outlier (two standard deviations away from the mean) for any parameter of that task, it was excluded from analysis for the entire task. Each task was analyzed independently for outliers.

Results Loss of functional FMRP in mice increased basal STEP protein levels and prevented rapid mGluR-dependent STEP synthesis in SNs A recent study utilized high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation to rigorously identify STEP mRNA as a target of FMRP (Darnell et al . 2011). Accordingly, we predicted that baseline STEP expression would be upregulated in STEPWT /Fmr1KO mice. We also assessed whether mGluR-stimulated translation of STEP was disrupted. Hippocampal SNs from STEPWT /Fmr1WT and STEPWT /Fmr1KO mice were prepared and stimulated with the mGluR agonist DHPG (50 μM, 10 min; Fig. 1). We first verified enrichment of PSD-95 in the SN fraction and confirmed that, while expressed in both fractions, STEP was most enriched in the SN fraction (Fig. 1a). Main effect analysis showed that STEP expression was significantly greater in SNs from STEPWT /Fmr1KO mice than (b)

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STEPWT /Fmr1WT mice (F1,8 = 6.177, P ≤ 0.05; Fig. 1b). As reported previously (Zhang et al . 2008), pairwise comparisons indicated that DHPG treatment significantly increased STEP expression in STEPWT /Fmr1WT SNs (t4 = −2.15, P ≤ 0.05); however, this increase was absent in STEPWT / Fmr1KO SNs (t4 = −0.12, P > 0.05; Fig. 1b). No significant differences were observed in unstimulated cytosolic between genotypes (t4 = 0.91, P > 0.05; Fig. 1c). The baseline increase in STEP levels in STEPWT /Fmr1KO mice was not limited to the hippocampus, because STEP levels were also significantly higher in whole brain extracts from STEPWT /Fmr1KO as compared with STEPWT /Fmr1WT mice (t4 = −2.33, P ≤ 0.05; data not shown). As a positive control, we found a significant main effect of genotype on PSD-95 expression, where PSD-95 was significantly higher in SNs from STEPWT /Fmr1KO compared with STEPWT /Fmr1WT mice (F1,8 = 20.72, P ≤ 0.05; Fig. 1b), consistent with increased steady-state levels of PSD-95. Previous studies reported that DHPG stimulation significantly increased PSD-95 levels in STEPWT /Fmr1WT , but not STEPWT /Fmr1KO , neurons (Todd et al . 2003; Muddashetty et al . 2007). Similarly, pairwise comparisons indicated a trend toward a DHPG-induced increase in PSD-95 expression in STEPWT /Fmr1WT SNs (t4 = −1.63, P = 0.09), which was absent in STEPWT /Fmr1KO SNs (t4 = −0.87, P > 0.05). No significant main effects of genotype or drug treatment were observed in GAPDH expression (Fig. S1).

Genetic reduction of STEP decreased AGS and reduced seizure-induced c-Fos activation in the PAG of Fmr1KO mice We hypothesized that excessive STEP levels contribute to abnormal behavioral phenotypes normally present in Fmr1KO mice. To test this prediction, we genetically reduced or eliminated STEP in Fmr1KO mice (Fig. 2a). Examination of both STEPHT /Fmr1KO and STEPKO /Fmr1KO increased therapeutic relevance by testing whether a 50% or 100% reduction in STEP protein (Venkitaramani et al . 2009) corrected behavioral phenotypes. STEPHT /Fmr1WT and STEPKO /Fmr1WT mice were also examined because their performance in the tasks used in this study had not yet been examined. (a)

Table 2: Breakdown of AGS responses following genetic reduction in Fmr1 and/or STEP Genotype STEPWT /Fmr1WT STEPHT /Fmr1WT STEPKO /Fmr1WT STEPWT /Fmr1KO STEPHT /Fmr1KO STEPKO /Fmr1KO

Wild running

Seizure (clonic/tonic)

0/12 0/13 0/5 9/21 2/30 3/23

0/12 0/13 0/5 7/21 14/30 7/23

We first tested whether genetically reducing STEP decreased AGS in Fmr1KO mice. None of the STEPWT /Fmr1WT mice showed seizure-related activity, and neither did STEPHT /Fmr1WT nor STEPKO /Fmr1WT mice (Fig. 3a and Table 2). Consistent with other reports, 76.2% of STEPWT /Fmr1KO mice showed seizure activity, which differed significantly from STEPWT /Fmr1WT mice (X12 = 17.75, P ≤ 0.0001). This value dropped to 53.3% in STEPHT /Fmr1KO mice and 43.5% in STEPKO /Fmr1KO mice (Fig. 3a and Table 2). The incidence of AGS was significantly lower in STEPKO /Fmr1KO mice compared with STEPWT /Fmr1KO mice (X12 = 4.86, P ≤ 0.03), showing that loss of STEP significantly reduced AGS in Fmr1KO mice. AGSs are mediated through brainstem circuits involving the PAG (Faingold 1999; Faingold & Randall 1999; N’Gouemo & Faingold 1999). To evaluate possible genotypic differences in neuronal activity induced by the audio stimulus, we examined expression of c-Fos in the PAG shortly after stimulation (Fig. 3b–h). A significant main effect of group was observed (F4,21 = 35.72, P ≤ 0.005). Although no genotypic differences were found in the number of c-Fos+ PAG cells in the absence of seizures (Fig. 3h), the number of c-Fos+ cells was significantly greater following tonic/clonic seizures in both STEPWT /Fmr1KO and STEPKO /Fmr1KO mice compared with all groups without seizure (P s ≤ 0.0005; LSD post hoc test; Fig. 3b–h). Even so, STEPKO /Fmr1KO mice that experienced a seizure consistently showed fewer PAG c-Fos+ cells than STEPWT /Fmr1KO mice (P ≤0.01; LSD post hoc test; Fig. 3c, d and f–h).

(b)

Figure 2: Generation of Fmr1KO mice with a selective reduction in STEP. (a) Breeding strategy employed in the study. Fmr1KO mice were cross-bred with STEP KO mice to produce breeder progeny that were STEPHT /Fmr1HT (females) and STEPHT /Fmr1KO (males). Breeders from different litters were then mated to produce offspring with a selective reduction or elimination of STEP and Fmr1. The six offspring analyzed in this study are listed. (b) Representative gels of PCR screening for the presence or absence of the WT and KO (neomycin) Ptpn5 (STEP) and Fmr1 alleles.

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Social abnormalities of Fmr1KO mice in the social choice task were altered following genetic reduction in STEP Mice are typically social beings that prefer to spend time sniffing and interacting with other mice (Murcia et al . 2005). We evaluated sociability using a three-chambered social choice task (Crawley 2007; Moy et al . 2009). During the socialization phase, pairwise comparisons indicated that all

genotypes spent significantly more time in close proximity to the mouse compared with the cup (Fig. S2a). There were no significant genotypic differences shown in the socialization index (F5,46 = 0.88, P > 0.05), or in the time spent in close proximity to the mouse (F5,46 = 1.07, P > 0.05) or cup (F5,46 = 0.24, P > 0.05; Figs. 4c and S2a and b), suggesting that all mice had appropriate sociability. No significant differences were found between groups in the total distance

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Figure 4: Abnormalities in social approach are reversed in Fmr1KO mice following genetic reduction in STEP. (a) Schematic of apparatus during the socialization phase of the social choice task. No significant differences among genotypes are detected in total distance traveled (b) or time in close proximity to the stranger mouse (c). (d) STEPWT /Fmr1KO mice enter the area in close proximity to the stranger mouse more times than all genotypes that are Fmr1WT , and this phenotype is reversed in STEPHT /Fmr1KO mice (main effect of group P ≤ 0.005, one-way ANOVA; # P s ≤ 0.005, LSD post hoc ). (e) The duration of each entry in close proximity to the stranger mouse is also significantly different between genotypes (main effect of group P ≤ 0.05 , one-way ANOVA; ∗ P s ≤ 0.02, LSD post hoc ). Data represent the mean for each measure ± SEM [STEPWT /Fmr1WT (n = 8), STEPHT / Fmr1WT (n = 11), STEPKO /Fmr1WT (n = 6), STEPWT /Fmr1KO (n = 13), STEPHT / Fmr1KO (n = 8) and STEPKO /Fmr1KO (n = 6)].

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traveled (F5,46 = 1.37, P > 0.05), showing similar locomotion (Fig. 4b). Social approach was determined by the number of entries in close proximity to the stranger mouse in the socialization phase, a method providing similar measurements to number of nose contacts or sniffs (Mines et al . 2010). A significant main effect of group was observed in the number of entries in close proximity to the mouse (F5,46 = 5.42, P ≤ 0.005), where STEPWT /Fmr1KO mice had significantly more entries than all genotypes that were Fmr1WT (P s ≤ 0.005; LSD post hoc test; Fig. 4d). Moreover, STEPHT /Fmr1KO mice had significantly fewer visits in close proximity to the stranger mouse than STEPWT /Fmr1KO mice (P ≤ 0.005; LSD post

hoc test; Fig. 4d), indicating that genetically reducing STEP significantly decreased the abnormalities in social approach, which were observed in Fmr1KO mice. We also found a significant main effect of group on the duration of each visit in close proximity to the mouse (F5,46 = 2.70, P ≤ 0.05; Fig. 4e). The time per entry was significantly greater for STEPHT /Fmr1WT , STEPKO /Fmr1WT and STEPHT /Fmr1KO mice compared with STEPWT /Fmr1KO mice (P s ≤ 0.02; LSD post hoc test). These results are consistent with an increase in the length of each visit following genetic reduction in STEP. No significant genotypic differences were observed for the number of entries (F5,46 = 1.45, P > 0.05) or duration of each entry (F5,46 = 0.92,

Figure 3: Genetically reducing STEP decreases AGS and reduces seizure-induced c-Fos activation in the PAG of Fmr1KO mice. (a) None of the STEPWT /Fmr1WT (n = 12), STEPHT /Fmr1WT (n = 13) or STEPKO /Fmr1WT (n = 5) mice show seizure activity in response to a 130-dB stimulus, while 76.2% of STEPWT /Fmr1KO (n = 21) have a response. Genetically reducing STEP decreases AGS because only 53.3% of STEPHT /Fmr1KO (n = 30) and 43.5% of STEPKO /Fmr1KO (n = 23) mice have a seizure-related response (∗ P ≤ 0.01 vs. STEPWT /Fmr1WT , # P ≤ 0.03 vs. STEPWT /Fmr1KO ; X 2 test). Data represent the mean percentage of mice showing seizure incidence (wild running and tonic/clonic seizure). (b–g) Comparison of c-Fos immunoreactivity in the PAG among different genotypes and responses 30 min after exposure to the audio stimulus. Scale equals 200 μm in (b–d) and 10 μm in (e–g). Boxes in (b–d) reflect locations for higher magnification images in (e–g). (b and e) Only a sparse number of c-Fos+ neurons are found in the PAG of STEPWT /Fmr1WT mice when no seizure response is observed. (c and f) STEPWT /Fmr1KO mice that have seizures show robust c-Fos staining in the PAG. (d and g) The number of seizure-induced c-Fos+ neurons is consistently lower in STEPKO /Fmr1KO mice compared with STEPWT /Fmr1KO mice. (h) In the absence of a seizure, there are no significant differences in c-Fos expression between STEPWT /Fmr1WT (n = 5), STEPWT /Fmr1KO (n = 4) and STEPKO /Fmr1KO (n = 6) mice. Following seizures, the number of c-Fos+ PAG neurons is significantly increased regardless of genotype (main effect of group P ≤ 0.005, one-way ANOVA; ∗ P s ≤ 0.0005 vs. both seizure groups, LSD post hoc ); however, this number is significantly less in STEPKO /Fmr1KO mice (n = 6) compared with STEPWT /Fmr1KO mice (n = 5; # P ≤ 0.01 vs. STEPWT /Fmr1KO seizure group). Data represent the mean c-Fos+ cells in the PAG ± SEM.

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Figure 5: Significant differences are observed between genotypes in overall locomotor activity during the social novelty phase. (a) Schematic of apparatus during the social novelty phase of the social choice task. (b) Total distance traveled is significantly different between genotypes (main effect of group P ≤ 0.005, one-way ANOVA). STEPKO /Fmr1WT mice tend to be hypoactive, whereas STEPWT /Fmr1KO tend to be hyperactive (∗ P s ≤ 0.05, # P s ≤ 0.02, LSD post hoc ). No significant differences among genotypes are detected the time (c), number of entries (d) or duration of each entry (e) in close proximity to the novel mouse. Data represent the mean for each measure ± SEM [STEPWT / Fmr1WT (n = 8), STEPHT /Fmr1WT (n = 11), STEPKO /Fmr1WT (n = 6), STEPWT / Fmr1KO (n = 13), STEPHT /Fmr1KO (n = 8) and STEPKO /Fmr1KO (n = 6)].

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P > 0.05) in close proximity to the cup (data not shown), indicating that the observed differences in the socialization phase were specific for the social aspect of the task. Mice were subsequently evaluated during the social novelty phase, where a novel mouse was introduced to the task (Figs. 5a and 2c and d). There was a striking main effect of group in locomotor activity during this phase for mice lacking both STEP and Fmr1 (F5,46 = 4.35, P ≤ 0.005; Fig. 5b). STEPWT /Fmr1KO mice traveled significantly greater distance than STEPHT /Fmr1WT , STEPKO /Fmr1WT and STEPKO /Fmr1KO (P s ≤ 0.02; LSD post hoc ), and there was also a strong trend for STEPWT /Fmr1KO mice to travel more than STEPWT /Fmr1WT (P = 0.075; LSD post hoc ), consistent with novelty-induced hyperactivity in STEPWT /Fmr1KO mice. In contrast, STEPKO /Fmr1WT traveled significantly less distance than STEPWT /Fmr1WT (P ≤ 0.05; LSD post hoc ), suggesting hypoactivity in STEP-deficient mice. The net result was that genetically eliminating STEP in Fmr1KO mice reduced overall locomotor activity, mimicking levels seen in STEPWT /Fmr1WT mice. Previous reports have indicated that STEPWT /Fmr1WT mice on the C57BL/6 background should have a preference for the novel mouse in this phase (Moy et al . 2009); however, pairwise comparisons indicated none of the genotypes spent more time in close proximity to the novel mouse compared with the familiar one (Fig. S2c). Moreover, no significant genotypic differences between groups were observed in the novelty index (F5,46 = 0.663, P > 0.05), in the raw time spent in close proximity to the novel mouse (F5,46 = 2.38, P > 0.05) or familiar mouse (F5,46 = 0.54, P > 0.05; Figs. 5c Genes, Brain and Behavior (2012) 11: 586–600

and 2a and b). Unlike the socialization phase, there were no significant differences detected between groups in the number of entries (F5,46 = 1.89, P > 0.05) or duration of each entry (F5,46 = 1.64, P > 0.05) in close proximity to the novel mouse (Fig. 5d and e).

Genetically reducing STEP mice decreased social anxiety in Fmr1KO in a social dominance tube test We utilized the social dominance tube test (Lindzey et al . 1961; Spencer et al . 2005) as an additional test of social anxiety (Fig. 6). As published previously (Spencer et al . 2005), STEPWT /Fmr1KO mice won only 34.5% of the time (10/29 matches; X12 = 5.59, P ≤ 0.02 vs. STEPWT /Fmr1WT ). This percentage increased to 56.6% in STEPHT /Fmr1KO mice (13/23 matches; X12 = 0.78, P = 0.38 vs. STEPWT /Fmr1WT ), showing that STEPHT /Fmr1KO and STEPWT /Fmr1WT mice retreat from the tube at a similar frequency. Taken further, STEPKO /Fmr1KO mice won 76.9% of the time (10/13 matches; X12 = 7.54, P ≤ 0.02 vs. STEPWT /Fmr1WT ). These findings showed that genetically reducing STEP in Fmr1KO mice increased the number of wins in the social dominance tube test. Given that the number of wins was significantly greater for STEPKO /Fmr1KO mice than STEPWT /Fmr1WT mice, we next investigated whether loss of STEP alone caused the mice to win more often. We found that STEPKO /Fmr1WT mice did not win significantly more than STEPWT /Fmr1WT mice (61.1% wins; 11/18 matches; X12 = 1.78, P = 0.18), showing that loss of STEP alone was not causing STEPKO /Fmr1KO mice to win more often.

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Figure 6: Social anxiety in Fmr1KO mice is reversed following genetic reduction in STEP in a social dominance tube test. The percentage of wins by STEPWT /Fmr1KO mice (n = 10), STEPHT /Fmr1KO mice (n = 8) and STEPKO /Fmr1KO mice (n = 6) against STEPWT /Fmr1WT mice is presented. Each mouse was tested against three STEPWT /Fmr1WT mice. STEPWT /Fmr1KO mice win significantly fewer times than STEPWT /Fmr1WT mice, suggesting social anxiety. Genetically reducing STEP in Fmr1KO mice increases the number of wins against STEPWT /Fmr1WT mice, indicating reversal of social anxiety (∗ P ≤ 0.02 vs. STEPWT /Fmr1WT ; X 2 test). Data represent the mean percentage of wins against STEPWT /Fmr1WT mice.

Loss of STEP normalized select nonsocial anxiety-related phenotypes in Fmr1KO mice To address whether genetically reducing STEP also reversed nonsocial anxiety-related phenotypes in Fmr1KO mice, we evaluated mice for their exploration in a light/dark box test (Fig. 7). Significant differences between groups were observed for both time (F5,49 = 3.39, P ≤ 0.02) and total distance traveled (F5,49 = 2.55, P ≤ 0.05) in the illuminated side (Fig. 7a and b). STEPWT /Fmr1KO mice spent more time (P s ≤ 0.05; LSD post hoc test; Fig. 7a) and traveled greater distance (P s ≤ 0.05; LSD post hoc test; Fig. 7b) in the light side than all genotypes that were Fmr1WT (Fig. 7a), indicating decreased anxiety in Fmr1KO mice. Genetically reducing STEP in Fmr1KO mice corrected this phenotype, because the time and distance traveled was significantly less in STEPHT /Fmr1KO mice compared with STEPWT /Fmr1KO mice (P s ≤ 0.05; LSD post hoc test; Fig. 7a and b). Significant genotypic differences were also detected in the number of total light and dark entries (i.e. transitions between zones; F5,49 = 3.07, P ≤ 0.05; Fig. 7c). Mice with reduced or eliminated levels of STEP had significantly less total entries than those that were STEPWT regardless of Fmr1 genotype (with the exception of STEPKO /Fmr1KO mice; P s ≤ 0.05; LSD post hoc test). These findings are consistent with increased anxiety following genetic reduction in STEP. Although light-side exploration and total number of light/dark transitions are often highly correlated (Crawley & Goodwin 1980), STEPWT /Fmr1WT and STEPWT /Fmr1KO mice did not differ in their number of total entries (P = 0.47; LSD post hoc test; Fig. 7c).

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Mice were also evaluated for their exploration in the EPM. A significant main effect of group was observed for the time spent in the closed arms (F5,53 = 2.38, P ≤ 0.05), where STEPWT /Fmr1KO mice spent less time than STEPWT /Fmr1WT and STEPKO /Fmr1WT mice (P s ≤ 0.05; LSD post hoc test; Fig. 8a). Concomitantly, STEPWT /Fmr1KO mice spent a greater amount of time in more anxiogenic zones, including the center square (F5,53 = 2.49, P ≤ 0.05; P s ≤ 0.05; LSD post hoc test; Fig. 8e) and open arms; however, no significant main effect was detected for open arm time (F5,53 = 1.89, P = 0.11; Fig. 8c). The number of open arm entries was also affected by genotype (F5,53 = 2.74, P ≤ 0.03; Fig. 8d), where STEPWT /Fmr1KO mice had significantly more entries than STEPKO /Fmr1WT and STEPKO /Fmr1KO mice (P s ≤ 0.05; LSD post hoc test) and a strong trend was observed for STEPWT /Fmr1KO mice entering the open arms more frequently than STEPWT /Fmr1WT mice (P = 0.09; LSD post hoc test; Fig. 8d). We observed no significant effects of genotype on the number of closed arm entries (F5,53 = 0.57, P > 0.05; Fig. 8b) or total distance traveled (F5,53 = 0.54, P > 0.05; Fig. 8f), indicating that the observed effects were not accounted for by differences in overall locomotion. In all parameters evaluated from the EPM (Fig. 8), genetically reducing STEP did not appear to significantly affect domains where STEPWT /Fmr1KO mice differed from STEPWT /Fmr1WT mice. We did not observe main effects of genotype for the total distance traveled (F5,73 = 0.61, P > 0.05) or the ratio of center/total distance traveled (F5,73 = 0.93, P > 0.05) in the open-field task (Fig. S3a and b). Discrepancies between our findings and those showing hyperactivity in Fmr1KO mice (Peier et al . 2000; Spencer et al . 2011) could be accounted for based on methodological differences, as we assessed open-field activity for only 5 min.

Perseverative-related behaviors did not explain the observed behavioral abnormalities associated with Fmr1KO mice To determine if the abnormalities we observed in Fmr1KO mice were due to increased perseverative and/or repetitive behaviors, we examined marble burying behaviors. Although marble burying was initially characterized as a measure of anxiety (Njung’e & Handley 1991), more recent studies indicate that it is a reflection of repetitive and/or perseverative behaviors (Thomas et al . 2009). In an effort to reduce interinvestigator variability and subjectivity, we devised a novel method to quantitate the percentage of each marble buried. Nonetheless, we observed no significant differences between genotypes on the number of marbles buried (F5,68 = 1.59, P > 0.05; Fig. S3c) or the home cage total distance traveled in the presence (F5,68 = 1.12, P > 0.05) or absence (F5,68 = 0.20, P > 0.05) of marbles (data not shown).

Discussion Much FXS research aims to discover new target proteins whose translation is controlled by mGluRs and FMRP Genes, Brain and Behavior (2012) 11: 586–600


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Figure 7: Genetically reducing STEP in Fmr1KO mice reverses abnormal exploration in the illuminated side of the light/dark box. STEPWT /Fmr1KO mice spend significantly more time (a; main effect of group P ≤ 0.02, one-way ANOVA) and travel significantly greater distance (b; main effect of group P ≤ 0.05, one-way ANOVA) in the illuminated side of the light/dark box than all genotypes that are Fmr1WT mice (∗ P s ≤ 0.05, LSD post hoc ). Both the time (a) and distance traveled (b) in the light side is significantly less in STEPHT /Fmr1KO mice compared with STEPWT /Fmr1KO mice (∗ P s ≤ 0.05, LSD post hoc ). (c) Total number of light/dark side entries is significantly different between groups (main effect of group P ≤ 0.05, one-way ANOVA), where mice with reduced or eliminated levels of STEP had significantly less total entries than those that were STEPWT , regardless of Fmr1 genotype (with the exception of STEPKO /Fmr1KO mice; ∗ P s ≤ 0.05; LSD post hoc test). Data represent the mean for each measure ± SEM [STEPWT /Fmr1WT (n = 8), STEPHT /Fmr1WT (n = 10), STEPKO /Fmr1WT (n = 7), STEPWT /Fmr1KO (n = 11), STEPHT /Fmr1KO (n = 10) and STEPKO /Fmr1KO (n = 9)].

and is dysregulated in Fmr1KO mice (Bear et al . 2004; Krueger & Bear 2011). Here, we describe and validate STEP as a novel target. We establish increased basal STEP expression and loss of mGluR-induced STEP synthesis in Fmr1KO mice. Genetic modulation of STEP corrects select behavioral abnormalities normally present in Fmr1KO mice. Fmr1KO mice null for STEP exhibit fewer AGS and show a corresponding reduction in PAG neuronal activity following seizures. Moreover, most social and nonsocial anxietyrelated abnormalities associated with Fmr1KO mice are reversed by genetically reducing STEP. Altogether, these findings highlight the benefit of reducing STEP in Fmr1KO mice and suggest that developing STEP inhibitors may be worthy of clinical consideration for FXS. FMRP suppresses mRNA translation by stalling ribosomal translocation (Muddashetty et al . 2007; Darnell et al . 2011). Accordingly, the expression of some proteins encoded by FMRP target mRNAs is upregulated in Fmr1KO mice (Zalfa et al . 2003; Lu et al . 2004; Hou et al . 2006; Westmark & Malter 2007; Gross et al . 2010). Given that STEP is an FMRP target (Darnell et al . 2011) and that mGluR stimulation normally upregulates STEP in a translationdependent manner (Zhang et al . 2008), we predicted that steady-state levels of STEP would be increased in Fmr1KO mice. We confirm that STEP levels are basally elevated in synaptoneurosomal, but not cytosolic, fractions and that mGluR-stimulated synthesis of STEP is absent in Fmr1KO Genes, Brain and Behavior (2012) 11: 586–600

mice, consistent with impaired mGluR-induced dendritic translation of FMRP targets (Zalfa et al . 2003; Lu et al . 2004; Hou et al . 2006; Muddashetty et al . 2007; Westmark & Malter 2007; Park et al . 2008). Improper recruitment of mRNAs to active polyribosomes likely causes this disruption in Fmr1KO mice (Weiler et al . 2004; Muddashetty et al . 2007); however, decreased degradation could also be playing a role. Additional assays will be needed to determine the mechanism(s) behind STEP upregulation in Fmr1KO mice. STEP is found in dendrites and has emerged as critical modulator of synaptic plasticity and key player in several neuropsychiatric disorders (Boulanger et al . 1995; Pelkey et al . 2002; Zhang et al . 2008, 2010; Goebel-Goody et al . 2012). STEP-mediated dephosphorylation of Fyn, Pyk2, ERK1/2 and p38 inactivates these enzymes, whereas dephosphorylation of surface NMDARs and AMPARs promotes their endocytosis (Nguyen et al . 2002; Pelkey et al . 2002; Munoz et al . 2003; Paul et al . 2003; Snyder et al . 2005; Zhang et al . 2008; Venkitaramani et al . 2011). Thus, STEP appears to oppose synaptic strengthening and promote synaptic weakening. Given that STEP is among a handful of ‘LTD proteins’ dysregulated in Fmr1KO mice (Zalfa et al . 2003; Lu et al . 2004; Hou et al . 2006; Westmark & Malter 2007; Park et al . 2008), we propose that it is the complex interaction of STEP with other affected proteins in FXS that leads to behavioral abnormalities.

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Figure 8: Effects of genetically modulating STEP in Fmr1KO mice in the EPM. (a) STEPWT /Fmr1KO mice spend less time in the closed arms than both STEPWT /Fmr1WT and STEPKO /Fmr1WT mice (main effect of group P ≤ 0.05, one-way ANOVA; ∗ P s ≤ 0.05, LSD post hoc ) indicating decreased anxiety. No significant differences among genotypes are detected in the number of closed arm entries (b) or total distance traveled (f), indicating similar locomotion. (c) No significant differences are detected among genotypes in open arm time; however, (e) STEPWT /Fmr1KO mice spend significantly more time in the center square compared with all mice that are Fmr1WT (main effect of group P ≤ 0.05, one-way ANOVA; ∗ P s ≤ 0.05, LSD post hoc ). (d) Genotype also affects the number of open arm entries (main effect of group P ≤ 0.03, one-way ANOVA; ∗ P s ≤ 0.05, LSD post hoc ); however, only modest difference between STEPWT /Fmr1WT and STEPWT /Fmr1KO mice are detected. Data represent the mean for each measure ± SEM [STEPWT /Fmr1WT (n = 10), STEPHT /Fmr1WT (n = 11), STEPKO /Fmr1WT (n = 10), STEPWT /Fmr1KO (n = 13), STEPHT /Fmr1KO (n = 8) and STEPKO /Fmr1KO (n = 7)].

Using a comprehensive behavioral battery, we evaluated whether genetically reducing STEP corrects abnormalities in Fmr1KO mice. One robust phenotype of Fmr1KO mice is their increased AGS susceptibility (Musumeci et al . 2000; Yan et al . 2005). Motivation for assessing AGS stems from prior work showing a striking seizure resistance in STEP KO mice (Briggs et al . 2011). We establish that loss of STEP in Fmr1KO mice significantly reduces AGS. Although the basis for this reduced susceptibility in STEPKO /Fmr1KO mice is still unknown, studies point to altered excitatory/inhibitory balance. Fmr1KO mice exhibit decreased neocortical inhibition, leading to hyperexcitability (Gibson et al . 2008; Olmos-Serrano et al . 2010; Paluszkiewicz et al . 2011) and increased seizure susceptibility (Musumeci et al . 2000; Yan

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et al . 2005). In contrast, loss of STEP increases hippocampal inhibitory excitability and decreases excitation, thereby reducing seizure susceptibility (Briggs et al . 2011). Seizure activity and neuronal activation can be mapped by examining c-Fos expression (Morgan et al . 1987; Herrera & Robertson 1996). We found that AGS significantly increases c-Fos activation in the PAG, a modulatory structure in the AGS efferent pathway (Faingold 1999; Faingold & Randall 1999; N’Gouemo & Faingold 1999; Ross & Coleman 2000). Even so, AGS-induced c-Fos staining is significantly less in STEPKO /Fmr1KO mice compared with STEPWT /Fmr1KO mice, suggesting attenuated seizure-induced PAG activation. Changes in c-Fos number and/or intensity are associated with varying degrees of severity and stages (Morgan et al . 1987), indicating that loss of STEP may reduce the severity or Genes, Brain and Behavior (2012) 11: 586–600


progression of AGS in Fmr1KO mice. Although it is likely that other subcortical auditory nuclei are affected (Le Gal La Salle & Naquet 1990), altogether, our findings show that loss of STEP significantly reduces AGS frequency and PAG activation in Fmr1KO mice. In this study, we focus on social anxiety and autismlike symptoms, which are observed in FXS (Cornish et al . 2008). Individuals with FXS exhibit socially anxious behaviors with abnormal approach and interaction (Wolff et al . 1989; Cornish et al . 2008; Harris et al . 2008). These impairments are attributed to anxiety and hyperarousal to social stimuli, because the motivation for the interaction is present but is too overwhelming to maintain (Cornish et al . 2008). Aberrant social behaviors are also found in Fmr1KO mice (Spencer et al . 2005; Mineur et al . 2006; McNaughton et al . 2008; Liu & Smith 2009; Moy et al . 2009; Mines et al . 2010; Liu et al . 2011). Using the social choice task and social dominance tube test, we show increased social anxiety alongside heightened interest and hyperactivity in Fmr1KO mice that is reversed by genetically manipulating STEP. Our results are consistent with Fmr1KO showing greater active behaviors, but decreased nose contact time, with stranger mice (Spencer et al . 2005; McNaughton et al . 2008; Mines et al . 2010). One speculation is that these abnormalities reflect hyperarousal to social stimuli concomitantly with social anxiety, as seen in FXS humans. Disparities between our work and those finding diminished social approach and novelty preference in Fmr1KO mice may be explained by differences in age or methodology used to quantify nose contacts (Liu & Smith 2009; Moy et al . 2009; Liu et al . 2011). We were somewhat surprised by the observed finding that all genotypes lacked preference for social novelty in the choice task; however, this phase is designed to test for social recognition or memory and is therefore less relevant for assessing social anxiety (Yang et al . 2011). Although we do not make any claims regarding social novelty preference or social memory, our findings are consistent with a recent report showing only subtle differences in novelty preference scores using C57BL/6 mice (Pietropaolo et al . 2011). Another well-characterized phenotype of Fmr1KO mice is reduced anxiety in nonsocial paradigms (Peier et al . 2000; Qin et al . 2002; Spencer et al . 2005, 2011; Qin & Smith 2008; Mines et al . 2010; Yuskaitis et al . 2010; Liu et al . 2011). We found that the light/dark box and most aspects of the EPM corroborate this phenotype. An alternative explanation might be that Fmr1KO mice show increased exploration in novel environments, because EPM center square time is enhanced in Fmr1KO mice and this relies heavily on exploratory behavior (Rodgers & Johnson 1995). Whether the abnormality associated with Fmr1KO mice is due to reduced anxiety and/or increased exploration, loss of STEP in Fmr1KO mice reverses light-side exploration in the light/dark box, thereby normalizing this phenotype. In our study, findings from other nonsocial anxiety and repetitive tests are not as clear in showing a robust phenotype of Fmr1KO mice; therefore, the impact of STEP is difficult to interpret in the absence of a significant deficit. We also report novel social and nonsocial anxiety-related phenotypes of STEP HT and STEP KO mice. First, each social interaction is longer in the socialization phase of Genes, Brain and Behavior (2012) 11: 586–600

the choice task, so it is interesting to consider that the quality of each interaction is enhanced. Because STEP KO mice exhibit more dominance behaviors in some tasks (Venkitaramani et al . 2011), another explanation might be that increased aggression leads to longer visits. This possibility seems unlikely, because STEPKO /Fmr1WT and STEPWT /Fmr1WT mice retreat from the social dominance tube test at a similar frequency. Second, STEP-deficient mice tend to be hypoactive in the social novelty phase and the light/dark box, consistent with increased anxiety and/or decreased exploration. Although the basis for these phenotypes remain unknown, altered social, anxiety and depressive-like behaviors are observed following genetic modulation of STEP substrates, including NMDARs and ERK2 (Boyce-Rustay & Holmes 2006; Satoh et al . 2011). In conclusion, we establish that genetically reducing STEP in Fmr1KO mice, either fully or partially, reduces AGS susceptibility and normalizes select social and nonsocial anxiety-related behaviors. Thus, our results suggest a potential therapeutic role of STEP in reversing characteristic phenotypes of FXS. Further investigation is required to uncover the molecular basis and/or downstream pathways responsible for attenuating Fmr1KO behaviors when STEP is reduced. Moreover, because genetically manipulating STEP may elicit unknown secondary or developmental modifications, it remains unknown whether acute or chronic inhibition of STEP will have the same effect in adult Fmr1KO mice. Future work utilizing selective inhibitors of STEP will address this question.

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Acknowledgments We thank Dr Pradeep Kurup, Dr Niki Carty and other lab members for helpful discussions and advice on biochemical experiments;

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Goebel-Goody et al. Deborah Hall, Jeesun (Iris) Park, Carole Nasrallah, Faten Sayed, Linda Li and Matthew Baum for assistance with behavioral experiments and genotyping of mice and Nicholas Woods and Elizabeth Litvina for performing c-Fos quantification. We also wish to thank Dr Michael Tranfaglia, Dr Peter Olausson and Dr Natalina Salmaso for advice on conducting and analyzing the behavioral experiments. This research was supported by a FRAXA Research Foundation Fellowship and NIH Grant 5T32MH018268 to S.M.G.-G. and by NIH Grants MH52711 and MH091037 to P.J.L. Support for J.R.N. was provided by the McKnight Foundation Brain Disorders Award. The authors declare no conflict of interest.

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1: Expression of GAPDH is not significantly altered by genotype or DHPG treatment. A two-way ANOVA indicated no significant main effect of genotype (F1,8 = 2.55, P > 0.05), treatment (F1,8 = 3.05, P > 0.05), or genotype × treatment interaction (F1,8 = 0.14, P > 0.05). Moreover, pairwise comparisons within each genotype failed to detect any significant differences in GAPDH levels after DHPG treatment (Fmr1WT : t4 = 1.22, P > 0.05; Fmr1KO : t4 = 1.38, P > 0.05; two-sample t test). For these reasons, we normalized all protein signals in Fig. 1 to GAPDH. Similar results were obtained when normalizing to ERK1/2 and tubulin (data not shown); however, we had concerns using either of these in this study. Specifically, ERK1/2 is a STEP substrate and phosphorylation of ERK1/2 is dysregulated in Fmr1KO mice (Paul et al . 2003; Kim et al . 2008; Osterweil et al . 2010). Moreover, because abnormal dendritic spine morphology is a hallmark of Fmr1KO mice and in FXS humans (Comery et al ., 1997), we felt that use of tubulin for normalization was inappropriate because of its role in regulating cytoskeletal dynamics. Figure S2: No significant differences are detected among genotypes in sociability or preference for social novelty in the social choice task. (a) Pairwise comparisons indicate that each genotype spends significantly more time in close proximity to the mouse compared with the cup [∗ P s ≤ 0.05, two-sample t test: STEPWT /Fmr1WT (t14 = 2.41), STEPHT /Fmr1WT (t20 = 5.09), STEPKO /Fmr1WT (t10 = 5.43),

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STEPWT /Fmr1KO (t24 = 6.72), STEPHT /Fmr1KO (t14 = 5.68) and STEPKO /Fmr1KO (t10 = 5.35)], indicating appropriate sociability. (b) No significant genotypic differences are found in the socialization index (% of time spent in close proximity to the mouse relative to the total time spent in close proximity to the mouse and cup). (c) Pairwise comparisons indicate that none of the genotypes spend more time with the novel mouse than the familiar one [P s > 0.05, two-sample t test: STEPWT /Fmr1WT (t14 = −0.31), STEPHT /Fmr1WT (t20 = 04), STEPKO /Fmr1WT (t10 = 0.73), STEPWT /Fmr1KO (t24 = 1.96), STEPHT /Fmr1KO (t14 = 1.18) and STEPKO /Fmr1KO (t10 = 0.23)], suggesting lack of social novelty preference. (d) No significant genotypic differences are found in the social novelty index (% of time spent in close proximity to the novel mouse relative to the total time spent in close proximity to the novel and familiar mice). Data represent the mean for each measure ± SEM. Figure S3: No significant differences are detected among genotypes in open-field activity or marble burying. (a) Total distance traveled in the open-field arena (5 min) is not significantly different between groups. (b) Genotype also does not affect the ratio of center to total distance traveled in our study. (c) The number of marbles buried >40% does not differ significantly between genotypes. Data represent the mean for each measure ± SEM [openfield activity: STEPWT /Fmr1WT (n = 12), STEPHT /Fmr1WT (n = 14), STEPKO /Fmr1WT (n = 9), STEPWT /Fmr1KO (n = 20), STEPHT /Fmr1KO (n = 17) and STEPKO /Fmr1KO (n = 7); marble burying: STEPWT /Fmr1WT (n = 11), STEPHT /Fmr1WT (n = 14), STEPKO /Fmr1WT (n = 11), STEPWT /Fmr1KO (n = 17), STEPHT /Fmr1KO (n = 11) and STEPKO /Fmr1KO (n = 10)]. Table S1: Original numbers of mice included in each cohort for behavioral analysis. A total of eight mice (three STEPWT /Fmr1KO and five STEPHT /Fmr1KO ) were assessed in open field but did not complete the remainder of the behavioral battery because they died from natural causes before the experiment. (SZ, seizure, n.e., not examined). As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
