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

Nature Neuroscience volume 17, pages 1583–1590 (2014)Cite this article

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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.

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Figure 1: Nf1+/− mice show selective deficits in long-term social learning and increased Mapk activation in the amygdala.
Figure 2: Deletion of the Pak1 gene restores long-term social deficits in Nf1+/− mice.
Figure 3: Nf1+/− mice have elevated GABA-glutamate neurotransmission and disrupted LTP in the amygdala, which was restored by deletion of Pak1.
Figure 4: Nf1+/− mice have altered Adam22, Cap1 and Hsp70 protein expression levels in the amygdala that are partially rescued by deletion of Pak1.
Figure 5: Amygdalar preinjection of Pak1 inhibitor restores long-term social behavioral deficits and normalizes local Adam22 expression in Nf1+/− mice.

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Acknowledgements

This work was supported by grants from the National Center for Advanced Translational Sciences, US National Institutes of Health UL1RR025761/TR000006, R01 MH52619 and MH065702 (to A.S.), a predoctoral fellowship to J.P.S. (TL1 RR 025759), K01AG044466 (to P.L.J.) and R01 CA74177-06 (to D.W.C.). We thank T. Jacks (Massachusetts Institute of Technology) for providing Nf1+/− mice and J. Chernoff (Fox Chase Cancer Center) for providing Pak1−/− mice.

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Author notes

  1. Andrei I Molosh and Philip L Johnson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Andrei I Molosh, Philip L Johnson, John P Spence, David Arendt, Steven P Janasik & Anantha Shekhar

  2. Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Andrei I Molosh, Philip L Johnson, Lauren M Federici, Rajesh Khanna, Weiguo Zhu & Anantha Shekhar

  3. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Philip L Johnson, Lauren M Federici & Cristian Bernabe

  4. Program in Medical Neurosciences, Indiana University School of Medicine, Indianapolis, Indiana, USA

    John P Spence & Lauren M Federici

  5. Department of Chemistry, METACyt Biochemical Analysis Center, Indiana University, Bloomington, Indiana, USA

    Zaneer M Segu & Yehia S Mechref

  6. Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Rajesh Khanna, Weiguo Zhu & Anantha Shekhar

  7. Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Chirayu Goswami & Lang Li

  8. Department of Pediatrics, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Su-Jung Park & D Wade Clapp

  9. Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA

    D Wade Clapp

  10. Indiana Clinical and Translational Sciences Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Anantha Shekhar

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Contributions

A.S., along with A.I.M., P.L.J., J.P.S. and D.W.C., formulated the hypotheses and designed the studies. S.-J.P. and D.W.C. maintained the mouse colony and genotyped all mice. J.P.S. and S.P.J. performed the behavioral assays shown in Figure 1, and J.P.S. performed those shown in Figure 2. For Figure 1 and Supplementary Figure 3, western blots were performed by R.K. and W.Z., and immunohistochemistry was performed by L.M.F. and P.L.J. A.I.M. performed all electrophysiology experiments shown in Figure 3. For Table 1 and Figure 4a,b and Supplementary Table 1, A.I.M. micropunched the BLA, Y.S.M. and Z.M.S. conducted the proteomics assay, and C.G., L.L. and P.L.J. analyzed the data. The immunohistochemistry shown in Figure 4c–f was performed by L.M.F. and P.L.J. The stereotaxic surgeries and behavioral assays shown in Figure 5 were performed by D.A. and C.B. The stereotaxic surgeries and immunohistochemistry shown in Supplementary Figure 2 were done by D.A. and P.L.J. A.I.M. and P.L.J. analyzed data and created figures. A.I.M., P.L.J., J.P.S., D.W.C. and A.S. interpreted the data and collectively wrote the main draft of the article, with all other authors contributing to the revisions, and all authors approved of the final version.

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Correspondence to Anantha Shekhar.

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Integrated supplementary information

Supplementary Figure 1 Hypothetical graphical illustrations of Nf1 and Pak1 gene pathways and glutamate and GABA regulation of electrophysiological activity in the basolateral amygdala of WT and Nf1+/– mice.

(a) The hypothetical graphical illustration, pathways adapted from Le and Parada (2007)1, showing the interaction of Nf1 and Pak1 gene products in growth factor signal transduction. Neurofibromin, a cytoplasmic GAP-like protein, negatively regulates RAS activation by accelerating the conversion of RAS-GTP to RAS-GDP and increasing RAS–RAF-MEK signal transduction, whereas Pak1 has an activating effect on this pathway. (b) is a hypothetical illustration depicting glutamate and GABA regulation of normal EPSCs, LTP and IPSCs activity in WT mice (top illustration), and how in Nf1+/- mice (bottom illustration) 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

Supplementary Figure 2 Effects of a Pak1 inhibitor on DMSO-induced increases in pErk expression within the basolateral amygdala

(a)Bilateral injections of 100% DMSO + the inactive enantiomer PIR3.5 (a pharmacological control for IPA3, 100 μM/100 nl) into the BLA of WT mice induced a robust increase in the no. of local pERK-ir cells, which was attenuated in WT mice that were injected with 100% DMSO + the PAK1 inhibitor IPA3 (100 μM/100 nl: F2,11 = 22.6, p < 0.001, n = 5,3,4). (b) Bilateral injections of 100% DMSO + the inactive enantiomer PIR3.5 (a pharmacological control for IPA3, 100 μM/100 nl) into the BLA of WT mice also reduced in the no. of local ADAM22-ir cells, which was attenuated in WT mice that were injected with 100% DMSO + the PAK1 inhibitor IPA3 (100 μM/100 nl: F2,11 = 22.6, p < 0.001, n = 5,3,4). (c) is a plotted linear regression that illustrates that the no. of ADAM22-ir cells in the BLA is inversely correlated with the no. of pERK-ir cells in the BLA in WT mice from experiment in SFig- 1a-b. (d) contains photomicrographs of pERK and ADAM22-immunoreactive cells in the amygdala from each treatment group. Scale bar is 25 μm. (e) is an illustration of a coronal mouse brain section (taken from Franklin & Paxinos stereotaxic atlas of the mouse brain, 3rd edition) indicating the location of the injection sites for each treatment group within the BLA from -1.46 to -1.94 mm bregma with a 0.5mm scale bar at bottom right. Abbreviations: CeA, central amygdala; ec, external capsule; LA, lateral amygdala; opt, optic tract.

Supplementary Figure 3 Full-length blots for figure 1k

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Molosh, A., Johnson, P., Spence, J. et al. Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase. Nat Neurosci 17, 1583–1590 (2014). https://doi.org/10.1038/nn.3822

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