1. NF1 mutation selectively disrupts social learning/memory which is rescued by p21-activated kinase 1 (Pak1) J.P. Spence 1, A. Dietrich 1, E.G. Michels 2, D.W. Clapp 2, A. Shekhar 1. Dept. of Psychiatry 1 and Pediatrics 2 Indiana University School of Medicine, Indianapolis, IN 46202 inactivation: Implications for the pathophysiology and treatment of autism spectrum disorders IndianaCTSI INTRODUCTION Neurofibromatosis type 1 (NF1) is a common neurocutaneous disorder that results from the deletion of one copy of the NF1 gene. In addition to various physical features, NF1 patients often exhibit problems with cognition, learning, attention and social function. Studies suggest that the NF1 gene deletion is also associated with some forms of autism. The incidence of NF1 is dramatically increased in patients with autism by roughly 150-190 fold, and the NF1 locus has been significantly associated with autism using linkage analysis. The NF1 gene encodes neurofibromin, a GAP-like protein that negatively regulates p21 ras activity. Mutation in the NF1 gene up- regulates p21 ras activity, thereby, propagating both the MAPK and PI3K signaling cascades. These biochemical pathways mediate important functions involved in cellular proliferation and synaptic plasticity and have been implicated in learning and memory. Recently, it was shown that the p21-activated kinase type 1 (Pak1) positively regulates MAPK activation, and the co-deletion of Pak1 in Nf1+/- mast cells restores the hyperactivation of Ras- MAPK pathway (McDaniel et al, 2008). The purpose of this study was to determine whether the Nf1 gene deletion (Nf1+/-; Jacks et al, 1994) in mice affects social behaviors, social versus avoidance learning, and anxiety-like or depression-like responses; and (2) to determine whether the co-deletion of Pak1 gene in Nf1+/- mice rescues the resulting deficits. METHODS Animals. Wild-type (Wt), Nf1+/-, and Nf1+/- / Pak1 -/- mice bred on a C57BL/6J background were tested at approximately 10 weeks of age. Wt (n=12) and Nf1+/- (n=12) mice were first tested, and then Nf1+/- (n=12) and Nf1+/- / Pak1-/- (n=11) mice were tested in subsequent experiments. In addition, age- matched adult male C57BL/6J mice (n=20) were used as “stimulus” mice for the social behavior tests. All mice were singly housed for 6 days before testing began. Social Behavior Tests. The social behavior tests included four 10 min sessions that were performed sequentially: (1) habituation, (2) social interaction, (3) preference for social novelty, and (4) social learning (performed 24 hr. later). The apparatus consisted of three adjoining chambers constructed of black Plexiglass (Sankoorikal et al, 2006). The chambers on opposite ends of the apparatus both contained wire-mesh cylinders that were used to house “stimulus” mice. Elevated Plus Maze (EPM). The EPM apparatus consisted of 2 open arms and 2 enclosed arms that emanate from a central platform raised above the floor. Entries into and time spent in the open arm were measured during two 5 min. trials (separated by 24 hrs.) Open Field Test. The open field consisted of a rectangular box with 9 predefined areas. Entries into and time spent in the center region as well as total entries were measured during a 10 min. trial. Forced Swim Test. The forced swim apparatus consisted of a clear Plexiglas cylinder containing 6 in. of water (25 ± 1.0°C). Immobility was determined during the 2-6 min. interval of two trials (separated by 24 hr.). The first trial was 15 min, while second was 6 min. Olfactory Habituation Test. 6 WT, 10 Nf1+/- and 4 Nf1+/- / Pak1- /- mice were presented with cotton swabs scented with either water (3x), almond extract (3x) and novel mouse (3x). Each cotton swab was presented for 2 min., and the total duration of each test was18 min. SOCIAL BEHAVIORS Figure 1. Social Behaviors in Wt, Nf1+/- and Nf1+/- / Pak1 -/- Mice. The mean (± SEM) represents social interaction (SI) or the percent time spent sniffing each respective cylinder divided by the total time (300 sec). For analyses, only the first 5 min of each ten minute test was utilized. A)Social Interaction. To measure general SI, the “test” mice were exposed to a novel “stimulus” mouse during a 10 min. session. All three strains exhibited similar levels of SI during the 0-5 min. interval. B)Preference for Social Novelty. To measure preference for social novelty, the “test” mice were presented with the same “familiar” mouse from the previous SI session and a novel “stimulus” mouse. All three strains displayed significant preference for the novel mouse during the 0-5 min. interval. C)Social Learning. As an indicator of long-term social learning, preference for social novelty was measured 24 hours later using the same “familiar” mouse from the previous day and a novel “stimulus” mouse. Both Wt and Nf1+/- / Pak1-/- mice showed significant preference for the novel mouse during the 0-5 min. interval, while the Nf1+/- strain showed no preference for social novelty. * AVOIDANCE LEARNING / ANXIETY-LIKE BEHAVIORS Figure 2. Avoidance Learning and Anxiety-like Behavior in Wt, Nf1+/- and Nf1 +/- / Pak1 -/- Mice. Open arm duration/entries (day 1) and center duration/entries represent an inverse measure of anxiety-like behavior in rodents. Avoidance learning was defined as the change in open arm duration / entries comparing day 2 relative to day 1. A)Elevated Plus Maze. a) The mean (± SEM) for open arm duration was defined as the percent time (sec) spent in the open arm divided by the total time (300 sec). No differences were detected in open arm duration between strains on day 1 or day 2, and all three strains showed a reduction in open arm duration on day 2 relative to day 1. b) An entry was defined as having all four paws into the open arm of the EPM. Similar to open arm duration, no differences were detected between strains in open arm entries on day 1 or day 2. All three strains exhibited a reduction in open arm entries on day 2. B)Open Field Test. a) The mean (± SEM) for center duration was defined as the percent time (sec) spent in the center region divided by the total time (600 sec), No differences were detected between the Wt, Nf1+/- and Nf1+/- / Pak1-/- strains in center duration. The three strains also displayed similar (b) entries into the center region as well as (c) total entries, a measure of general locomotor activity. DEPRESSION / IMMOBILITY Figure 3. Depression-associated Immobility. The mean (± SEM) represent the percent time (sec) spent immobile divided by the total time (240 sec) during the 2-6 min. interval of each trial. Immobility was defined as the cessation of limb movements, except minor involuntary hind limb movements. Relative to the Wt and Nf1+/- strains, the Nf1+/- / Pak1 strain exhibited significantly decreased immobility on day 1. None of the three strains showed a significant increase in immobility on day 2 relative to day 1. OLFACTORY HABITUATION Figure 4. The Olfactory Habituation Test. The mean (± SEM) represents the time (sec) spent sniffing cotton swabs dipped in each respective scent during a 2 min. trial. The three strains showed similar habituation to each scent. CONCLUSIONS 1)The Nf1 gene deletion is significantly correlated with selective deficits in long-term social learning / memory. 2)The co-deletion of Pak1 in Nf1+/- mice rescues the deficits in long-term social learning / memory. 3)Nf1+/- / Pak1-/- mice exhibit decreases in depression-associated immobility as compared to the Wt and Nf1+/- strains. 4)The Nf1-related hyperactivation of the Ras-MAPK pathway represents a probable mechanism for the deficits in long-term social learning / memory seen in the Nf1+/- strain. 5)Pak1 inhibitors could potentially provide novel treatments for the secondary prevention of NF1- related social disorders as well as some forms of autism spectrum disorders. Supported by CTSI TL1 pre-doctoral award & RO1 MH 065702 to AS * * * C) SOCIAL LEARNING * P<0.05 vs. Mouse-1 Wt Nf1+/- Nf1+/- Nf1+/- / Pak1-/- %time sniffing cylinder/total B) PREFERENCE FOR SOCIAL NOVELTY * P<0.05 vs. Mouse-1 Wt Nf1+/- Nf1+/- Nf1+/- / Pak1-/- %time sniffing cylinder/total A) SOCIAL INTERACTION * P<0.001 vs. Empty Wt Nf1+/- Nf1+/- Nf1+/- / Pak1-/- %time sniffing cylinder/total * * * * * * Entries b) OPEN ARM ENTRIES * P<0.05 vs. Day 1 * Wt Nf1+/- Nf1+/- Nf1+/- / Pak1-/- * * A) ELEVATED PLUS MAZE Wt Nf1+/- Nf1+/- Nf1+/- / Pak1-/- %open arm/total a) OPEN ARM DURATION * P<0.05 vs. Day 1 * * Entries c) TOTAL ENTRIES Wt Nf1+/- Nf1+/- Nf1+/- / Pak1-/- Entries b) CENTER ENTRIES Wt Nf1+/- Nf1+/- Nf1+/- / Pak1-/- B) OPEN FIELD TEST %center/total a) CENTER DURATION Wt Nf1+/- Nf1+/- Nf1+/- / Pak1-/- FORCED SWIM TEST † P<0.05 vs. Wt & Nf1+/- %immobility/total duration † Time (sec) Water Almond Novel Extract Mouse