Mechanism and treatment for learning and memory deficits in mouse models of Noonan syndrome

Abstract

In Noonan syndrome (NS) 30-50% 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 mutations in Ptpn11, which encodes the nonreceptor protein tyrosine phosphatase Shp2, show hippocampal-dependent impairments in spatial learning and deficits in hippocampal long-term potentiation (LTP). In addition, viral overexpression of an NS-associated allele PTPN11(D61G) in adult mouse hippocampus results in increased baseline excitatory synaptic function and deficits in LTP and spatial learning, which can be reversed by a mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, brief treatment with lovastatin reduces activation of the GTPase Ras-extracellular signal-related kinase (Erk) pathway in the brain and normalizes deficits in LTP and learning in adult Ptpn11(D61G/+) 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.

Figure 1. NS mice show spatial memory deficits

a. Escape latencies of Ptpn11^N308D/+ (n = 9) and WT littermates (n = 11) were not different in the hidden platform version of the water maze. b. Ptpn11^N308D/+ and WT littermates selectively searched in the target quadrant in a probe trial given after 3 days of training (Ptpn11^N308D/+, n = 9 mice, One-way ANOVA, F_{3, 32} = 13.82, *** P < 0.001; WT, n = 11 mice, One-way ANOVA, F_{3, 40} = 48.48, *** P < 0.001). However, Ptpn11^N308D/+ mice spent significantly less time in the target quadrant than WT mice. Two-way ANOVA for quadrant occupancy with genotype as between-subjects factor and pool quadrant as within-subjects factor, genotype x pool quadrant interaction: F_3,54 = 4.091, * P < 0.05. Pool quadrants; target (T), adjacent right (AR), opposite (O), and adjacent left (AL) quadrant. c. Ptpn11^D61G/+ mutants (n = 10) showed significantly longer latency to the platform during training compared with WT controls (n = 15) in the hidden–platform version of the water maze. d. Quadrant occupancy for the probe trial conducted after 3 days of training reveals that Ptpn11^D61G/+ mice (n=10) did not show preference for the target quadrant, but their WT littermates (n=15) did. In addition, Ptpn11^D61G/+ mice spent significantly less time in the target quadrant than did WT mice (Ptpn11^D61G/+, 27.44 ± 2.04 %; WT, 37.14 ± 2.09, ** P < 0.01; unpaired two-tailed t-test). Two-way ANOVA for quadrant occupancy with genotype as between-subjects factor and pool quadrant as within-subjects factor, genotype x pool quadrant interaction: F_{3, 69} = 2.884, * P < 0.05. n.s., not significant (P > 0.05).

Figure 2. NS mice show LTP deficits

a. LTP induced by a 5 TBS was reduced significantly in hippocampal slices from Ptpn11^N308D/+ mice compared with their WT littermates (WT, n = 6 slices from 6 mice; Ptpn11^N308D/+, n = 6 slices from 6 mice; Repeated-measures ANOVA: F_{1, 10} = 7.893, P < 0.05). b. LTP induced by a 5 TBS protocol was reduced in hippocampal slices from Ptpn11^D61G/+ mice compared with those from WT mice (WT, n = 7 slices from 7 mice; Ptpn11^D61G/+, n = 7 slices from 6 mice; Repeated-measures ANOVA: F_1,12 = 5.828, P < 0.05). fEPSP slopes normalized to the average baseline response before LTP induction (at time 0) are plotted in 2-min blocks. Sample traces show responses during baseline (gray) and the last 10 min (black) of the recording (average of ten recording traces). Scale: vertical bar, 0.5 mV; horizontal bar, 4 ms. Error bars represent s.e.m.

Figure 3. PTPN11^D61G overexpression induces learning and memory and LTP deficits that can be reversed by MEK inhibition

a. AAV–PTPN11^D61G infection results in overexpression of SHP2^D61G. Anti–SHP2 immunohistochemistry shows robust overexpression of SHP2 in the hippocampus of AAV–PTPN11^D61G–infused brains (left) compared with AAV–GFP infused brains (right). Full-length blots/gels are presented in Supplementary Figure 11. b. PTPN11^D61G overexpression increases basal Erk activity (phospho–Erk level) and prevents further Erk activation in response to TBS. Left, Representative immunoblot showing p–Erk (upper) and total Erk (lower) in PTPN11^D61G–expressing slices and GFP–expressing slices. Slices were prepared 1 h after TBS. Right, Bar graph displays normalized p–Erk levels (mean ± s.e.m.). CTL, control without TBS. c. MEK inhibitor SL327 reverses spatial memory deficits in PTPN11^D61G–overexpressing mice in the Morris water maze. Quadrant occupancy analysis for the probe trial reveals that PTPN11^D61G/veh mice showed no preference for the target quadrant (target vs. other quadrants, Dunnett’s Multiple Comparison Test after one-way ANOVA, P > 0.05). PTPN11^D61G/veh mice also spent significantly less time in the target quadrant compared with GFP/veh mice. SL327 treatment significantly increased the time spent in the target quadrant in PTPN11^D61G-expressing mice compared with vehicle-treated PTPN11^D61G mice (PTPN11^D61G/SL327, 37.25 ± 3.50 %, n=10, unpaired two-tailed t-test, t = 2.335, * P < 0.05). d. PTPN11^D61G overexpression in the hippocampus impairs memory in the object–place recognition test. Control mice expressing WT PTPN11 spent significantly more time exploring the object in the new place than exploring the object in the old place during the test session 24 h after training. However, PTPN11^D61G–overexpressing mice did not show preference for the object in the new place. e. MEK inhibitor SL327 rescues memory deficits in object–place recognition test caused by PTPN11^D61G overexpression. When SL327 (32 mg/kg) was injected 30 min before training in the object-place recognition test, both PTPN11– and PTPN11^D61G–expressing mice spent significantly more time exploring the object in the new place than exploring the object in the old place during the test session 24 h after training. f. and g. MEK inhibitor SL327 reverses LTP deficits caused by PTPN11^D61G overexpression. f. PTPN11^D61G overexpression significantly impaired 5 TBS–induced LTP, and bath application of SL327 reversed the deficit (Repeated-measures ANOVA, F_{3, 72} = 140.2, P < 0.0001). SL327 (1 μM) was applied for 1 h before LTP induction, and then maintained in the bath throughout recording. g. Average % fEPSP changes (last 10 minutes of recording) shows a significant LTP deficit in the vehicle-treated PTPN11^D61G group compared with the vehicle-treated GFP group (GFP/veh, 154.8 ± 4.18 %, n=7; PTPN11^D61G/veh, 131.9 ± 4.38, n=10; unpaired two-tailed t-test, t = 3.625, ** P < 0.01) and significant reversal by SL327 treatment (PTPN11^D61G/SL327, 146.1 ± 4.36 %, n=10; unpaired two-tailed t-test, t = 2.309, * P < 0.05). SL327 did not affect LTP in the GFP group (GFP/SL327, 146.2 ± 4.37 %, n=7; unpaired two-tailed t-test, t = 1.414, P = 0.183).

Figure 4. PTPN11^D61G overexpression enhances excitatory synaptic function through increased Ras-Erk signaling

a. AMPA receptor-mediated currents were measured at the peak of the currents at − 65 mV, and NMDA currents were measured 50 ms after onset at + 40 mV. The average of 15 traces is shown. Scale, 100 pA and 40 ms. b. Group data showing the increased AMPA:NMDA current ratio in AAV–PTPN11^D61G mice compared with AAV-GFP mice. SL327 treatment (1 μM, 1 h) significantly reversed the AMPA:NMDA current ratio in the PTPN11^D61G group without affecting GFP–expressing mice. Two-way ANOVA, interaction between viral treatment and drug, F_{1, 31} = 10.53, ** P < 0.01. Bonferroni post-test reveals significant effect of SL327 treatment only on PTPN11^D61G group (** P < 0.01). c. Paired-pulse facilitation ratio is unaffected by PTPN11^D61G. There was no significant difference at 25 ms or 50 ms intervals between the two groups. d. PTPN11^D61G overexpression increases excitatory synaptic function. Left, Representative traces of mEPSC recordings from GFP or PTPN11^D61G–expressing hippocampus. Middle, mEPSC frequency was increased in AAV–PTPN11^D61G–transfected mice compared with AAV–GFP mice, and was reversed by SL327 (1 μM) treatment without affecting on the AAV–GFP group. Two-way ANOVA with viral treatment as between-subjects factor, F_{1, 30} = 10.31, ** P < 0.01. Right, mEPSC amplitudes were not significantly different among groups. Two-way ANOVA with viral treatment as between-subjects factor, F_{1, 30} = 0.470, P = 0.498. Scale, 20 pA and 200 ms. e. Excitatory synaptic function is increased in Ptpn11^D61G/+ mutant mice and reversed by SL327 treatment. Left, Representative traces of mEPSC recordings from WT or Ptpn11^D61G/+ mice. Middle, mEPSC frequency was increased in Ptpn11^D61G/+ mice compared with WT littermates, and was reversed by SL327 (1 μM) treatment. Two-way ANOVA with genotype as between-subjects factor, F_{1, 33} = 5.914, * P < 0.05. Right, mEPSC amplitudes were not significantly different among groups. Two-way ANOVA with genotype as between-subjects factor, F_{1, 33} = 0.418, P = 0.839. Scale, 20 pA and 200 ms. f. and g. mIPSC was not changed in either AAV–PTPN11^D61G-transfected mice or Ptpn11^D61G/+ mutants. f. Representative traces of mIPSC recordings from GFP or PTPN11^D61G–expressing hippocampus. g. Representative traces of mIPSC recordings from Ptpn11^D61G/+ mutant mice or WT littermates. Scale, 20 pA and 1 s.

Figure 5. PTPN11^D61G overexpression increases surface AMPA receptor expression

a. and b. Representative images of surface GluA1 staining in cultured neurons. GFP alone (a) or PTPN11^D61G and GFP (b) were co–expressed using a bicistronic Sindbis viral vector in cultured hippocampal neurons (DIV21). Scale, 20μm. c. Representative images of western blotting of total and biotinylated surface proteins. Cadherin and Rab-4 were used as markers for surface and cytosol expression, respectively. Full-length blots/gels are presented in Supplementary Figure 11. d. Surface expression of GluA1 was significantly increased in PTPN11^D61G expressing neurons compared to WT PTPN11 expressing neurons, while the total expression level of GluA1 did not differ between WT PTPN11 and PTPN11^D61G transfected neurons.

Figure 6. Lovastatin treatment reverses spatial learning and memory and LTP deficits in Ptpn11^D61G/+ mice

a. Lovastatin treatment reverses increased Erk activation in hippocampi from Ptpn11^D61G/+ mice. Left, Representative immunoblot showing p-Erk (upper) and total Erk (lower) levels in WT and Ptpn11^D61G/+ mutant mice. Hippocampi were dissected 6 h after the 4^th day of lovastatin injection (subcutaneous (s.c.) injections, 10 mg/kg). Full-length blots/gels are presented in Supplementary Figure 11. Right, Bar graph displaying normalized p-Erk levels (mean ± s.e.m.). b. Vehicle-treated Ptpn11^D61G/+ mutant mice showed significantly longer latency to the hidden platform during training sessions compared with vehicle-treated WT mice. Lovastatin-treated Ptpn11^D61G/+ mice showed comparable latency to WT mice. c. Lovastatin treatment did not improve swimming speed. d and e. Lovastatin treatment (10 mg/kg) reverses spatial memory deficits in Ptpn11^D61G/+ mice at a concentration that does not affect WT controls. d. Quadrant occupancy analysis for the probe trail reveals that Ptpn11^D61G/+ mice with vehicle treatment (Ptpn11^D61G/+/veh) showed no preference for the target quadrant (target vs. other quadrants, Dunnett’s Multiple Comparison Test after one-way ANOVA, P > 0.05). By contrast, the Ptpn11^D61G/+/Lova group selectively searched for the target quadrant, suggesting that lovastatin treatment reversed the spatial memory deficit in Ptpn11^D61G/+ mice (target vs. other quadrants, Dunnett’s Multiple Comparison Test after one-way ANOVA, *** P < 0.0001). The Ptpn11^D61G/+/Lova group also spent significantly more time in the target quadrant compared with Ptpn11^D61G/+/veh mice. e. Proximity analysis reveals that the spatial memory deficit in Ptpn11^D61G/+ mice can be reversed by lovastatin treatment. f ang g. Lovastatin treatment reverses LTP deficits in Ptpn11^D61G/+ mice at concentrations that do not affect WT littermates. f. Ptpn11^D61G/+ mice showed a deficit in 5 TBS-induced LTP that was reversed by systemic administration of lovastatin (Repeated-measures ANOVA, F_{3, 96} = 14.38, P < 0.0001). g. Average % fEPSP changes (last 10 minutes of recordings) show that lovastatin treatment significantly rescued the LTP deficit in Ptpn11^D61G/+ mice (WT/veh, 159.6 ± 5.33 %, n=7; WT/Lova, 150.7 ± 5.49 %, n=6; Ptpn11^D61G/+/veh, 131.7 ± 2.31 %, n=9; Ptpn11^D61G/+/Lova, 154.2 ± 6.88 %, n=7; unpaired two-tailed t-test, ** P < 0.01, *** P < 0.001).