Mechanism and treatment for the learning and memory deficits associated with mouse models of Noonan syndrome

In Noonan Syndrome (NS) 30% to 50% of subjects show cognitive deficits of unknown etiology and with no known treatment. Here, we report that knock-in mice expressing either of two NS-associated Ptpn11 mutations show hippocampal-dependent spatial learning impairments and deficits in hippocampal long-term potentiation (LTP). In addition, viral overexpression of the PTPN11D61G in adult hippocampus results in increased baseline excitatory synaptic function, deficits in LTP and spatial learning, which can all be reversed by a MEK inhibitor. Furthermore, brief treatment with lovastatin reduces Ras-Erk activation in the brain, and normalizes the LTP and learning deficits in adult Ptpn11D61G/+ mice. Our results demonstrate that increased basal Erk activity and corresponding baseline increases in excitatory synaptic function are responsible for the LTP impairments and, consequently, the learning deficits in mouse models of NS. These data also suggest that lovastatin or MEK inhibitors may be useful for treating the cognitive deficits in NS.


Introduction
Noonan syndrome (NS) is an autosomal dominant genetic disorder with an incidence of ~1 in 2,500 live births characterized by facial abnormalities, short stature, motor delay and cardiac defects 1,2 . Importantly, 30% to 50% of NS patients show cognitive deficits [3][4][5][6] . NS patients also show clumsiness, motor delay, hearing loss, deficits in spatial knowledge, planning, and social/emotional problems 3,4,7 . Recent studies showed that NS patients show impairments in hippocampal-dependent memory tasks 4, 8, 9 . Germ line mutations in genes involved in Ras-Erk signaling such as PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, SHOC2, MEK1 and CBL have been reported to cause NS 1,10 . Among those, mutations in the PTPN11 gene, which encodes the non-receptor protein tyrosine phosphatase SHP2, account for ~ 50% of NS cases 1 . SHP2 is a positive regulator for Ras-Erk signaling 11 which is critically involved in many cellular processes including learning and memory 12 . The PTPN11 mutations found in NS patients result in gain-of-function alleles that up-regulate this signaling cascade 11,[13][14][15] . Cognitive problems, such as learning disabilities and memory impairments, are common in NS 3,5,6 . However, little is known about the role of PTPN11 in synaptic plasticity and learning and memory in the mammalian brain. Furthermore, there is no available treatment for cognitive deficits associated with this common genetic disorder.
Previous studies, that used NS mouse models derived by knocking-in mutations in the NSassociated Ptpn11 gene, demonstrated that the heterozygous knock-in mice show phenotypes similar to those found in NS patients. These include short stature, craniofacial abnormalities, myeloproliferative disease and multiple cardiac defects 14,16 . In the present study, we first tested whether NS mutant mice have deficits in learning and memory and synaptic plasticity. Then, we asked whether increasing SHP2 activity in adult brain affects synaptic function, LTP and learning and memory. Finally, we examined whether it is possible to rescue the LTP and learning deficits of NS mutant mice in adults.

NS mutant mice show deficits in spatial learning and memory
To investigate the underlying mechanism of the learning and memory deficits associated with NS, we studied two lines of heterozygous knock-in mice harboring gain-of-function mutations found in NS patients 14,16 : Ptpn11 D61G/+ and Ptpn11 N308D/+ . Previous studies showed that the Ptpn11 D61G/+ mutation causes more severe phenotypes than the Ptpn11 N308D/+ mutation 14,16 . As NS patients show deficits in spatial function and in memory tasks dependent on the hippocampus 4,8,9 , we tested both Ptpn11 mutants in the hidden platform-version of the Morris water maze 17 . In this task, mice learn to use spatial cues around a pool to find an escape platform hidden beneath the water surface. Following training, memory is assessed in probe trials wherein the mice search for 60 seconds with the platform removed from the pool. Ptpn11 N308D/+ mutants showed comparable latencies to find the hidden platform to their wild-type (WT) controls during training ( Fig. 1a; Repeated measures ANOVA, F 1, 18 = 2.078, P = 0.167) and showed normal swimming speeds in probe trials (WT, 17.33 ± 1.55 cm/s, n = 11 mice; Ptpn11 N308D/+ , 18.37 ± 0.82 cm/s, n = 9 mice; unpaired two-tailed t-test, t = 0.554, P = 0.586). However, in probe trials Ptpn11 N308D/+ mutants spent significantly less time than WT mice in the target quadrant where the platform was located during training ( Fig. 1b; WT, 57.87 ± 4.83 %; Ptpn11 N308D/+ , 41.85 ± 4.30 %; unpaired two-tailed t-test, t = 2.421, * P < 0.05). Also, the searches of WT mice during the probe trials were closer to the target platform than those of the mutants (WT, 32.53 ± 2.26 cm; Ptpn11 N308D/+ , 40.18 ± 2.05 cm; unpaired two-tailed ttest, t = 2.450, * P < 0.05). In contrast, Ptpn11 N308D/+ mutants performed normally in the visible-platform version of the water maze ( Supplementary Fig. 1), suggesting that the Ptpn11 N308D/+ mutation does not impair either visuomotor function or motivation. After extended training, the Ptpn11 N308D/+ mutants reached a level of performance comparable to WT mice in probe trials, demonstrating that they can acquire spatial information, albeit at a slower rate than WT mice ( Supplementary Fig. 2). In addition, Ptpn11 N308D/+ mutants also showed deficits in contextual fear conditioning, another hippocampus-dependent task ( Supplementary Fig. 3).

Adult expression of PTPN11 D61G impairs LTP and memory
The mutations in Ptpn11 mice are present throughout development, affect the entire body and could disrupt the function of brain structures other than the hippocampus. Similarly, NS is a systemic developmental disorder, and it has been assumed that developmental defects are responsible for the cognitive deficits in these patients 20 . Viral vectors provide spatial and temporal regulation of gene expression critical for testing the specific role of Ptpn11 mutations in the adult brain. Moreover, NS alleles severely compromise the viability of mutant mice 14 , thus making it very difficult to obtain sufficient number of mutant mice for all studies envisioned (Supplementary table 1). To test whether altered Shp2 signaling in the adult hippocampus can cause LTP and, consequently, learning deficits, we overexpressed mutant PTPN11 D61G using recombinant adeno-associated virus (AAV-PTPN11 D61G ) in the CA fields (CA1, CA2 and CA3) of the hippocampus of adult WT mice. PTPN11 D61G overexpression in the hippocampus ( Fig. 3a and Supplementary Fig. 6) resulted in increased Erk activation as assessed by immunoblotting p-Erk, confirming that AAV-expressed PTPN11 D61G is functional ( Fig. 3b; n = 5 hippocampi for each group, unpaired two-tailed ttest, t = 2.452, * P < 0.05). Consistently, AAV-PTPN11 D61G expression impaired performance in probe trials of the water maze (Fig. 3c). AAV-PTPN11 D61G -expressing mice spent significantly less time in the target quadrant than did AAV-GFP/vehicle-injected control mice ( Fig. 3c; PTPN11 D61G /veh, 25.89 ± 3.38 %, n=10; GFP/veh, 35.88 ± 2.95 %, n=13; unpaired two-tailed t-test, t = 2.231, * P < 0.05). Unlike the Ptpn11 D61G/+ mutation in mice, AAV-PTPN11 D61G expression did not affect swimming speed or other performance variables during the acquisition phase of the water maze ( Supplementary Fig. 7), suggesting that the acute expression of PTPN11 D61G in the hippocampus only affects learning and memory, whereas deregulation of Shp2-Erk signaling during development in the Ptpn11 D61G/+ mice might affect other functions including motor coordination. Notably, overexpressing WT PTPN11 did not affect basal p-Erk levels or spatial learning and memory ( Supplementary Fig. 8), demonstrating that the adverse impact on Erk signaling and learning and memory is specific to the NS-related PTPN11 mutation.
AAV-PTPN11 D61G -expressing mice were also tested in another hippocampus-dependent task (object-place recognition) 24-hours after training; WT AAV-PTPN11-expressing mice were used as controls (Fig. 3d). Consistent with the water maze results, PTPN11 D61G expression in the CA fields of the hippocampus caused memory deficits in this task: the control mice spent significantly more time exploring the object at the new location ( Fig. 3d; n = 15 mice, 59.79 ± 3.72 % for new place, one-sample paired t-test compared to 50 %, t = 2.633, * P < 0.05), but the PTPN11 D61G mice did not ( Fig. 3d; n = 15 mice, 52.61 ± 4.10 % for new place, one-sample paired t-test compared to 50 %, t = 0.636, P = 0.535). Importantly, AAV-PTPN11 D61G -expressing mice showed comparable total exploration time to that of WT AAV-PTPN11-expressing mice during training (WT AAV-PTPN11, 43.70 ± 3.98 s, n = 15; AAV-PTPN11 D61G , 39.29 ± 4.94 s, n = 15; unpaired t-test, t = 0.695 P = 0.493). All together, these data show that expressing PTPN11 D61G in the adult CA fields of the hippocampus is sufficient to disrupt memory, and demonstrate that PTPN11 plays a critical role in adult brain function, in addition to its effects on development 20 .
To test whether reducing Erk activity could reverse the memory deficits in AAV-PTPN11 D61G -expressing mice, we treated these mice with the MEK inhibitor SL327 or vehicle daily, 30 min before training. SL327 treatment (32 mg/kg, intraperitoneal injection) decreased Erk activation in the hippocampus of control and AAV-PTPN11 D61G mice ( Supplementary Fig. 9). We choose a sub-threshold dose of the drug that does not impair spatial learning in WT mice and only decreases hippocampal Erk activation in WT mice by ~ 25% (Supplementary Fig. 9). Importantly, this SL327 treatment rescued the spatial learning deficits of the AAV-PTPN11 D61G mice without affecting the performance of the AAV-GFP group ( Fig. 3c; PTPN11 D61G /SL327, one-way ANOVA, F 3, 36 = 10.44, P < 0.001; target vs. other quadrants, Dunnett's Multiple Comparison Test, * P < 0.05). Consistent with the water maze results, the same SL327 treatment also rescued the memory deficits in the object-place recognition task ( Fig. 3e; WT PTPN11, n = 5, 58.83 ± 2.01 % for new place, two-tailed paired t-test compared to 50 %, t = 4.395, * P < 0.05; PTPN11 D61G , n = 8, 59.90 ± 3.41 % for new place, two-tailed paired t-test compared to 50 %, t = 2.904, * P < 0.05). These results demonstrate that increased Ras-Erk signaling in adult CA fields of the hippocampus contribute to the memory deficits in AAV-PTPN11 D61G -expressing mice. Remarkably, SL327 also reversed the memory deficits of the adult Ptpn11 D61G/+ mutant mice in the Morris water maze, showing that normalizing Erk activity in adults can reverse the behavioral deficits even in mutant mice with germ line mutations ( Supplementary Fig.  10).
Consistent with the hypothesis that increased Ras-Erk activity leads to the LTP deficits responsible for spatial learning impairment in Ptpn11 mutant mice, the levels of 5 TBSinduced LTP in lovastatin-treated Ptpn11 D61G/+ mice were significantly higher than those in the vehicle-treated mutants and indistinguishable from those in WT control animals (Fig. 6f, g; two-way ANOVA with genotype as between-subjects factor, F 1, 25 = 5.936, * P < 0.05, Bonferroni post-test reveals a significant effect of lovastatin treatment only on Ptpn11 D61G/+ group, ** P < 0.01). By contrast, lovastatin treatment had no effect on LTP in hippocampal slices from WT mice (Fig. 6f, g). Thus, lovastatin treatment can normalize LTP deficits and spatial learning impairments even in adult Ptpn11 D61G/+ mice. Although we cannot exclude the possibility that lovastatin may affect other biological processes 24 , our data suggest that lovastatin reverses the spatial learning deficits of Ptpn11 D61G/+ mice, by reducing Erk activation and consequently correcting LTP deficits.

Discussion
Our study provides compelling evidence that the spatial learning and memory deficits in mouse models of NS are caused by enhanced Ras-Erk activation, which disrupts the balance between excitation and inhibition (E/I) and impairs hippocampal long-term potentiation. Furthermore, our experiments with viral vectors demonstrate that Ptpn11 plays critical roles not only in regulating development 20,25 , but also in adult brain functions. Consistent with our findings, expression of the fly ortholog of SHP2 (Csw) bearing gain-of-function mutations impaired long-term memory in Drosophila 26 .
In the present study, we used two knock-in mutant mice harboring a D61G or a N308D mutation in PTPN11. The D61G mutation is associated with both NS and leukemia and shows higher enzymatic activity than N308D, which is only associated with NS 15 . Consistently, Ptpn11 D61G/+ mice showed more severe deficits in LTP and learning than Ptpn11 N308D/+ mice. Although basal-level of p-Erk was significantly higher in the hippocampus of Ptpn11 D61G/+ mice compared to WT littermates (Fig. 6), we could not detect significant increases in basal p-Erk levels in the hippocampus of Ptpn11 N308D/+ mice, perhaps because these mice showed an overall milder phenotype (Supplementary Fig. 9c).
The activation of Ras-Erk signaling facilitates AMPA receptor trafficking during LTP 21 and abnormal hyperactivation of postsynaptic Erk signaling impairs hippocampal LTP and learning 27,28 . Our findings suggest that the PTPN11 D61G mutation increases the number of synapses with postsynaptic AMPA receptors, thus occluding LTP and therefore impairing learning. In agreement with the hypothesis that there are more synapses with AMPA receptors, we found that PTPN11 D61G expression increases mEPSC frequency, but does not affect PPF ratio (Fig. 4c), a form of plasticity very sensitive to changes in pre-synaptic function. Additionally, PTPN11 D61G expression increased the evoked AMPA:NMDA ratios (Fig. 4a, b), another observation consistent with the hypothesis that the PTPN11 D61G expression resulted in more synapses with AMPA receptors. Importantly, these observations were reproduced in both AAV-PTPN11 D61G -transfected mice and in the germ line mutants. Importantly, PTPN11 D61G expression enhanced the surface expression of GluA1 and increased the number of surface GluA1 clusters in cultured hippocampal neurons, a finding consistent with the hypothesis that the enhancement in excitatory synaptic function driven by PTPN11 D61G expression is caused by postsynaptic mechanisms. Interestingly, deletion of a Ras-Erk regulator (SynGAP) was reported to increase ERK signaling, enhance the levels of AMPA receptors, increase mEPSC frequency and impair LTP 27 .
Deregulation of Ras-Erk signaling has been associated with other genetic disorders including neurofibromatosis type I (NF1), Costello syndrome, LEOPARD syndrome, CFC syndrome, and Legius syndrome 10,29 . Among these, studies with the Nf1 +/− mutant mouse, which is a model of NF1, demonstrated that increased Ras signaling results in increased GABA release (excitation is normal in these mice) that leads to deficits in LTP and, consequently, learning and memory impairments [30][31][32][33] . Altogether these findings demonstrate that similar behavioral (e.g., spatial learning deficits) and even electrophysiological phenotypes (i.e., LTP deficits) can be caused by different cellular mechanisms: increases in AMPARs in NS mice and increases in GABA release in NF1 mice. Homozygous deletion of the NF1 gene in mouse post-natal excitatory neurons does not affect either synaptic transmission or learning 32 , whereas expression of the NS-mutation PTPN11 D61G in post-natal excitatory neurons does disrupt both synaptic transmission and learning, a direct demonstration of the distinct roles of these two Ras signaling modulators.
In this study, we show that postnatal treatment with an FDA-approved drug, lovastatin, can reverse learning and memory as well as LTP deficits in an adult NS mouse model. A previous study showed that lovastatin treatment can rescue spatial learning problems, attention deficits and pre-pulse inhibition deficits in Nf1 +/− mutant mice 22 . Thus, our studies suggest that this FDA-approved drug with a strong safety profile may also be useful for treatment of cognitive deficits associated with NS.

Methods
Mice-Ptpn11 D61G/+ mice were backcrossed to 129S6/SvEv and Ptpn11 N308D/+ mice were backcrossed to C57Bl/6J mice at least 6 times before experiments. Three to six month-old male and female mice were used. For AAV experiments, 3 -4 month-old male C57Bl/6J mice (Jackson Laboratory) were used. Mice were randomly assigned to treatment and experimental condition. All experiments used littermates as controls and were carried and analyzed with the experimenters blinded to genotype and treatment. Animals were group housed (2 -4) on a 12 h light/dark cycle in vivarium at UCLA and CAU. All studies were approved by the Animal Research Committee at UCLA and CAU.
Drugs-SL327 (Tocris) was dissolved in DMSO (16 mg/ml) and was injected intraperitoneally once daily, 30 min before the water maze experiment at a dose of 32mg/kg. The volume of a single injection was under 80 μl. Lovastatin (Mevinolin, Sigma) was prepared as previously described 22 . Briefly, lovastatin was dissolved in ethanol (final concentration of 8%) and 1N NaOH was added to convert mevinolin to the sodium salt. The pH of the final solution (4 mg/ml) was adjusted to 7.5 with HCl. The Vehicle solution was prepared with the same procedure. Lovastatin was administered daily (subcutaneous injection, 10 mg/kg) for 3 days before the first training day of the water maze and 6 h before training every day thereafter.
AAV-The coding sequence of human PTPN11 with or without the D61G mutation was subcloned into the HindIII -NsiI site of the AAV expression vector pSOFF. The resultant vector expresses mutant PTPN11 under the synthetic CBA promoter (CMV enhancer and chicken beta-actin promoter). Recombinant virus (rAAV5) was purified as previously described 34 . Briefly, an iodixanol gradient purification was performed followed by an ion exchange chromatography step which results in a 99 % pure vector preparation as judged by silver stained-SDS acrylamide gel fractionation. After the chromatography, the buffer was exchanged and the virus was concentrated in Ringer's solution using a Biomax 100 K concentrator (Millipore). Vector titers were determined by Real Time PCR. Typical titers were 3.09 × 10 12 genome copies/ml. rAAV5-GFP expressing only GFP was used as a control. Virus was infused into two sites per hemisphere (1 μl per injection, AP=−2.5, Lat= +/−2, DV=−1.7; AP=−1.8, Lat=+/−1, DV=−1.6) over 5 min through a 30-gauge Hamilton microsyringe. Viruses (GFP, WT PTPN11 or PTPN11 D61G ) were randomly assigned for infusion. After completion of infusion, the syringe was left in place for an additional 5 min. All the experiments were done three weeks after the infusion.
Behavior-Behavioral experiments were performed during the light cycle. In the hidden platform-version of Morris water maze, mice were trained with two blocks of 2 trials (ITI = 1min) spaced about 45 min apart. In each training trial, mice were released from a different starting position and then were allowed to search for the escape platform for 60 s. The platform was submerged 1 cm under the surface of the water. Once a mouse found the platform, it was left there for 15 s. If a mouse did not find the platform within 60 s, it was guided to the platform and remained on the platform for 15 s before being removed from the pool. Mice were trained for 5 -7 consecutive days. Memory was assessed in probe trials that were given after completion of training as described in the main text. During the probe trials, the platform was removed and the mice were allowed to search for it for 60 s. One mouse was excluded from further analysis because of floating (no voluntary movement for more than 10 s in more than 2 trials). The same group of mice was tested in the visible platform-version of Morris water maze. Data were acquired and analyzed using WaterMaze software (Actimetrics).
The object-place recognition task included a training and a test session. Before training, mice were handled 5 min per day for 4 days, and then habituated in a square box (27.5 cm × 27.5 cm × 25 cm) for 15 min for another 2 days. One side of the experimental box included a prominent cue. During the 10-min training session, mice were placed in the box, exposed to two identical objects and allowed to explore these objects. During the test session (24h after training), mice were placed back into the experimental box with the same two objects for 5 min: one object (Old location) stayed in the same location as during training, while the other object (New location) was moved to a new location. For the rescue experiment, SL327 (32 mg/kg, i.p.) was injected 30 min before the training session. The objects changed during the test sessions were randomly counterbalanced between mice. Experiments were videotaped and the exploration times were manually analyzed.
Electrophysiology-For extracellular recordings of field excitatory postsynaptic potentials (fEPSP), sagittal slices (400 μm) were prepared with a vibratome (VT1000S, Leica) in ice-cold artificial cerebrospinal fluid (ACSF). Slices recovered at room temperature for at least 90 min before recording in ACSF saturated with 95 % O 2 and 5 % CO 2 containing the following (in mM): 120 NaCl, 3.5 KCl, 2.5 CaCl 2 , 1.3 MgSO 4 , 1.25 NaH 2 PO 4 , 20 NaHCO 3 and 10 D-glucose. Recording was performed in a submerged chamber perfused with ACSF (32 °C). fEPSPs were recorded with platinum-iridium electrodes placed in the CA1 stratum radiatum. Bipolar platinum stimulating electrodes were placed in Schaffer collaterals. Baseline responses were measured with stimulation (0.017 Hz, 0.1 ms pulse duration) at an intensity (typically 20 -30 μA) that evoked a response that was approximately one third of the maximum evoked response. LTP was induced with theta-burst stimulation (2 or 5 bursts, each burst consisting of four pulses at 100 Hz with a 200 ms inter-burst interval). Initial fEPSP slopes were measured and normalized to the average of baseline (with Clampfit 10.2).
Western blot and immunohistochemistry-Dissected hippocampi were homogenized in protein lysis buffer (10mM Tris-Cl pH 6.8, 1.6 % SDS) containing protease and phosphatase inhibitor cocktails (Sigma). Supernatants were collected after centrifugation and the protein concentration was determined using a BCA assay kit (Thermo). Equal amounts of proteins (5 μg) were separated by electrophoresis on a 4 % -12 % SDS-PAGE (Invitrogen), and then transferred to nitrocellulose membranes. After blocking with 5% BSA in TBS-T (Tris-buffer saline containing 0.1% Tween-20) for 1 hr at room temperature, membranes were hybridized with a primary antibody overnight at 4°C. After washing with TBS-T, membranes were incubated with a secondary antibody in 5% non-fat milk/TBS-T for 1 hr at room temperature. Signals were visualized by ECL (Thermo) and exposure time was adjusted so that the signals measured were in a linear range. After detecting phospho-Erk, the membranes were stripped and re-probed with a total Erk antibody. The total Erk levels were used to normalize each sample. The following primary antibodies were used: anti-phospho-Erk (#9101S, Cell Signaling, 1:6000), anti-total Erk (#9102S, Cell Signaling, 1:5000) and anti-SHP2 (sc-280, Santa Cruz, 1:3000).
Sindbis viral vector construction and immunocytochemistry-The coding sequence of human PTPN11 with or without the D61G mutation was subcloned into a Sindbis viral expression vector (pSinRep5; Invitrogen) and GFP was inserted into the 3′ region of the coding sequence along with an additional subgenomic promoter for bicistronic expression. Sindbis viruses were produced according to the manufacturer's protocol (Invitrogen) and directly added to the medium of cultured rat hippocampal neurons (DIV21). Twelve hours after infection, immunocytochemistry was performed with or without permeabilization by using anti-GluA1-N (#AGC-004, Alomone labs) antibody and Cy3conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Lab). Images were acquired by using confocal microscope (Zeiss LSM 710) and analyzed by using ImageJ (ver. 1.42q). Statistics-For water maze data, we used ANOVAs to analyze quadrant occupancy (% time spent in quadrant). After initial ANOVA analyses, searching specificity for each genotype was determined by comparing target quadrant to other quadrants using Dunnett's Multiple Comparison Test. We also used two-way ANOVAs to analyze the interaction between genotypes and pool quadrants. In addition, we compared target quadrant occupancy among different groups by using the unpaired two-tailed t-test. Proximity measures between two genotypes also were analyzed by the unpaired two-tailed t-test. Effects of drug treatments on different genotypes were analyzed by using two-way ANOVA followed by appropriate post-hoc tests. LTP data were analyzed by using repeated-measures ANOVA followed by Bonferroni test on the responses after LTP induction and unpaired two-tailed ttest on the average of the last 10 min of recording. For other experiments, we used Student's t-test to compare two groups and ANOVA to compare three or more groups. We did not use statistical methods to predetermine the sample sizes, but our sample sizes are similar to those reported in previously published papers [31][32][33] . Data distribution was assumed to be normal but this was not formally tested. All the data are represented as mean ± s.e.m.