Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase

Abstract

Children with neurofibromatosis type 1 (NF1) are increasingly recognized as having a high prevalence of social difficulties and autism spectrum disorders (ASDs). We demonstrated a selective social learning deficit in mice with deletion of a single Nf1 allele (Nf1(+/-)), along with greater activation of the mitogen-activated protein kinase pathway in neurons from the amygdala and frontal cortex, structures that are relevant to social behaviors. The Nf1(+/-) mice showed aberrant amygdala glutamate and GABA neurotransmission, deficits in long-term potentiation and specific disruptions in the expression of two proteins that are associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (Adam22) and heat shock protein 70 (Hsp70), respectively. All of these amygdala disruptions were normalized by the additional deletion of the p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1(+/-) mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide insights and therapeutic targets for patients with NF1 and ASDs.

Figure 1. Nf1^+/− mice show selective deficits in long-term social learning and increased MAPK activation in the amygdala compared to wild-type (WT) mice

Figure 1a–c depicts the time spent sniffing wire cages containing “stimulus” mice in a three-chambered apparatus (refer to insets that illustrate the orientation of cages and when cages were empty or contained a novel or familiar mouse). No differences were detected between strains in (a) preference for social interaction or (b) short-term social learning (1–3 min. following exposure to “test” mouse). c–d) Unlike WT, Nf1^+/− mice show deficits in long-term social learning, as measured by preference for social novelty (24 hrs. following exposure to “test” mouse). (d) represents comprehensive heat maps of time spent in each region. No differences were observed in learning in the (e) novel object recognition test or (f) passive avoidance test; anxiety states in the (g–h) elevated plus maze; depression/despair in the (i) forced swim test; or olfaction in an (j) olfactory habituation. (k) The Nf1^+/− genotype is associated with MAPK hyperexcitation in neuronal cultures from the prefrontal cortex (PFC, left graph. Western blotting was used to quantify pERK1/2 in cultured cortical neurons at basal levels and 2 min following stimulation with SCF (10 ng/ml), and total Erk1/2 was used as loading controls. Compared to WT mice, MAPK hyperexcitation was not apparent in the basolateral amydala (BLA) region of Nf1^+/− mice in (k, right graph and cropped western blots; full-length blots/gels are presented in Suppl. Fig. 3), but it (l) was apparent in cells immunoreactive for pERK within the BLA, but not medial amygdala (MEA) of Nf1^+/− mice exposed to a novel mouse. Graphs depict mean ± SEM for WT (white) and Nf1^+/− (red) mice. *P < 0.05. High magnification photographs (I, right) show pERK immunoreactive cells in lateral BLA (lBLA) and medial BLA/amygdaloid intermedullary grey (mBLA/IMG) of WT and Nf1^+/− mice.

Figure 2. Deficits in long-term social learning in Nf1^+/− mice are rescued in Nf1^+/−/Pak1^−/− mice

Figure 2a–d depicts the time spent sniffing wire cages containing “stimulus” mice in a three-chambered apparatus (refer to insets that illustrate the orientation of cages and when cages were empty or contained a novel or familiar mouse; Nf1^+/−; Nf1^+/−/Pak1^−/−; Pak1^−/−). Similar to our previous findings, no differences were detected between strains in (a) preference for social interaction or (b) short-term social learning. (c) Unlike Nf1^+/− mice, Nf1^+/−/Pak1^−/− showed significant preference for social novelty or restored long-term social learning (24 hrs. following exposure to “test” mouse). (d) represents comprehensive heat maps of time spent in each region. No differences were observed in (e) anxiety as measured by elevated plus maze, (f) depression-associated behavior, or (g) olfactory habituation. Graphs depict mean ± SEM for Nf1^+/− (red), Nf1^+/−/Pak1^−/− (purple), and Pak1^−/− (blue) mice. *P < 0.05.

Figure 3. Nf1^+/− mice exhibit elevated GABA and glutamate tones and significant LTP deficits in the BLA. Co-deletion of the Pak1 gene restores GABA neurotransmission and LTP in the BLA

(a) Representative electrophysiological recordings of sIPSC at holding potential of −55 mV from pyramidal neurons of the BLA of WT, Nf1^+/−, Nf1^+/−/Pak1^+/+ and Pak1^+/+ mice. (b,c) Neurons from Nf1^+/− mice exhibit no differences in sIPSC and mIPSC amplitude, but have significantly higher sIPSC and mIPSC frequency in the BLA compared to WT (n = 11,11,14,14). (d) The representative recordings of isolated sEPSC and mEPSC from BLA pyramidal neurons at a holding potential of −60 mV in WT, Nf1^+/−, Nf1^+/−/Pak1^−/−, Pak1^−/− mice. (e,f) Compared to WT, Nf1^+/− mice showed significant increases in frequency of sEPSCs (n = 17,14,16,19) and mEPSCs (n = 17,15,13,15), but not amplitude of sEPSCs and mEPSCs. Nf1^+/−/Pak1^−/− and Pak1^−/− mice demonstrated significant increase of sEPSC and mEPSC amplitude (e) and no significant changes in sEPSC and mEPSC frequency (f) compared to WT mice. *represents P ≤ 0.05 compared to control. (g) Representative traces of EPSPs before (1) and 60 min after (2) TBS stimulation of thalamic inputs to the BLA at holding potential of −70 mV. (h) Time course of averaged evoked EPSCs in response to stimulation of thalamic afferents in all neurons recorded from WT (white circle), Nf1^+/− (red), Nf1^+/−/Pak1^−/− (purple) and Pak1^−/− (blue) mice, respectively. TBS-LTP amplitude was significantly lower in Nf1^+/−, compared to WT mice. The significant depression of LTP was restored by co-deletion of Pak1. Data represent the mean ± SEM for each group; *represents P < 0.05 for comparison within groups. ^#(black) represents P < 0.05 Nf1^+/− vs WT mice, ^#(purple) - Nf1^+/− vs Nf1^+/−/Pak1^−/− mice. (i) The summary graph showing the group data for the effects of tetanic stimulation protocol on EPSP amplitude before (Control) and 60 min after stimulation in WT (white, n = 7), Nf1^+/− (red, n = 12), Nf1^+/−/Pak1^−/− (purple, n = 8), and Pak1^−/− (blue, n = 5) mice. Graphs depict mean ± SEM for each group. *represents P < 0.05 compared to control..

Figure 4. Reductions in ADAM22 levels and number of immunopositive cells seen in the BLA of Nf1^+/− mice were rescued by the co-deletion of Pak1 gene

Graphs in (a–b) represents normalized protein expression of ADAM22, CAP1 and HSP70 in the BLA of WT, Nf1^+/−, Nf1^+/−/Pak1^−/−, and Pak1^−/− mice. Graphs in (d–f) respectively represents the number of ADAM22, CAP1 and HSP70 immunoreactive (ir) cells in the BLA of WT and Nf1^+/− mice (and additional Nf1^+/−/Pak1^−/−, and Pak1^−/− mice for only ADAM22). Bars represent the mean and error bars represent the standard error of the mean. * and ^# in a–c respectively indicate P < 0.05. (f) contains low (left) and high (right) photomicrographs of ADAM22-immunoreactive cells in the amygdala of WT, Nf1^+/−, Nf1^+/−/Pak1^−/−, and Pak1^−/− mice. CeA and BLA are indicated with arrows in left row. Dashed lined box in left photomicrographs indicated where cells were counted and also where high magnification photomicrographs to right are taken. Scale bar for photomicrographs in left and right rows and inset are respectively, 25, 75 and 375 μm. See Suppl. Fig. 1b for a hypothetical illustration depicting glutamate and GABA regulation of normal EPSCs, LTP and IPSCs activity in WT mice, and how in Nf1^+/− mice disruption of ADAM22 interrupts anchoring of AMPA receptors to the post-synaptic membrane and leads to non-sustainable LTP as demonstrated in Figure 3h. Similarly, increased expression of HSP70 could contribute to increases in pre-synaptic GABA release and increased frequency of IPSCs seen in Figure 3c. Abbreviations: BLA, basolateral amygdala; CeA, central amygdala.

Figure 5. Bilateral injections of a PAK1 inhibitor IPA3 (and not the inactive enantiomer PIR3.5) into the BLA of Nf1^+/− mice rescues their social learning deficits

(a) depicts the time spent in close proximity to either the novel or familiar mouse cage (≤ 2.5cm from wire cage) [* indicates a novel mouse preference, ^# indicates an enhanced social preference in WT mice, and a rescue of social preference in Nf1^+/− mice, P < 0.05]. Graphs depict mean ± SEM. (b) is an illustration of a coronal mouse brain section (taken from Paxinos & Franklin stereotaxic atlas of the mouse brain, 3^rd edition) indicating the location of the injection sites (indicated by red circles) within the BLA from −1.46 to −1.94 mm bregma with a 0.5 mm scale bar at bottom right. Abbreviations: CeA, central amygdala; ec, external capsule; LA, lateral amygdala; opt, optic tract. (c) representative heat maps of time spent in each region with IPA3 groups spending more time near novel mouse cage. (d) Bar graph represents the number of ADAM22-immunoreactive (ir) cells in the BLA of WT and Nf1^+/− mice treated with vehicle or IPA3 from crossover experiment from Fig. 5a–c. Bars represent the mean and error bars represent the standard error of the mean. *indicates a P < 0.05 versus all other groups. (e) contains photomicrographs of ADAM22-immunoreactive cells in the BLA of WT, Nf1^+/− mice treated with vehicle or IPA3. Scale bar for photomicrograph is 25 μm.