Molecular Neurobiology

, Volume 54, Issue 10, pp 8090–8109 | Cite as

Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice

  • Joan A. Lenihan
  • Orthis Saha
  • Victoria Heimer-McGinn
  • John F. Cryan
  • Guoping Feng
  • Paul W. Young
Article
  • 440 Downloads

Abstract

NUMB is a key regulator of neurogenesis and neuronal differentiation that can be ubiquitinated and targeted for proteasomal degradation by ligand of numb protein-X (LNX) family E3 ubiquitin ligases. However, our understanding of LNX protein function in vivo is very limited. To examine the role of LNX proteins in regulating NUMB function in vivo, we generated mice lacking both LNX1 and LNX2 expression in the brain. Surprisingly, these mice are healthy, exhibit unaltered levels of NUMB protein and do not display any neuroanatomical defects indicative of aberrant NUMB function. Behavioural analysis of LNX1/LNX2 double knockout mice revealed decreased anxiety-related behaviour, as assessed in the open field and elevated plus maze paradigms. By contrast, no major defects in learning, motor or sensory function were observed. Given the apparent absence of major NUMB dysfunction in LNX null animals, we performed a proteomic analysis to identify neuronal LNX-interacting proteins other than NUMB that might contribute to the anxiolytic phenotype observed. We identified and/or confirmed interactions of LNX1 and LNX2 with proteins known to have presynaptic and neuronal signalling functions, including the presynaptic active zone constituents ERC1, ERC2 and LIPRIN-αs (PPFIA1, PPFIA3), as well as the F-BAR domain proteins FCHSD2 (nervous wreck homologue) and SRGAP2. These and other novel LNX-interacting proteins identified are promising candidates to mediate LNX functions in the central nervous system, including their role in modulating anxiety-related behaviour.

Keywords

LNX1 LNX2 LIPRIN/PPFIA ERC1/ERC2 NUMB Anxiety 

Abbreviations

PDZ

PSD-95

DlgA

ZO-1

RING

Really Interesting New Gene

LNX

Ligand of numb protein X

CNS

Central nervous system

ERC

ELKS/Rab6-interacting/CAST

SVZ

Subventricular zone

PBK

PDZ-binding kinase

DKO

Lnx1exon3−/−;Lnx2 −/− double knockout

DHET

Lnx1 exon3+/− ;Lnx2 +/− double heterozygous knockout

GFP

Green fluorescent protein

GST

Glutathione S-transferase

PCR

Polymerase chain reaction

PTB

Phosphotyrosine-binding domain

Notes

Acknowledgements

We are extremely grateful to Pat Fitzgerald and James O’Leary for their generous assistance and advice regarding behavioural experiments. We thank Mary McCaffrey (University College Cork) for access to her Science Foundation Ireland-funded confocal microscope and Jane McGlade (University of Toronto) for providing the rabbit anti-LNX1/2-RING/NPAY antibody. This work was supported by a Research Frontiers Programme grant from Science Foundation Ireland (08/RFP/NSC1382) and an Irish Research Council EMBARK Postgraduate Research Scholarship to Joan Lenihan. We thank Tom Moore for access to the EVOS FL Cell Imaging System.

Compliance with Ethical Standards

All animal experiments were approved by the Animal Experimentation Ethics Committee of University College Cork (No: 2013/028) and were conducted under licence (No: AE19130/P013) issued by the Health Products Regulatory Authority of Ireland, in accordance with the European Union Directive 2010/63/EU for animals used for scientific purposes.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2016_261_MOESM1_ESM.pdf (156 kb)
ESM 1 (PDF 155 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Joan A. Lenihan
    • 1
  • Orthis Saha
    • 1
    • 2
  • Victoria Heimer-McGinn
    • 1
    • 3
  • John F. Cryan
    • 4
    • 5
    • 6
  • Guoping Feng
    • 7
    • 8
    • 9
  • Paul W. Young
    • 1
    • 6
  1. 1.School of Biochemistry and Cell BiologyUniversity College CorkCorkIreland
  2. 2.Institut de Biologie de l’ENS (IBENS), INSERM, CNRS, École Normale SupérieurePSL Research UniversityParisFrance
  3. 3.Department of Cognitive, Linguistic and Psychological SciencesBrown UniversityProvidenceUSA
  4. 4.Department of Anatomy and NeuroscienceUniversity College CorkCorkIreland
  5. 5.Alimentary Pharmabiotic CentreUniversity College CorkCorkIreland
  6. 6.Cork Neuroscience CentreUniversity College CorkCorkIreland
  7. 7.McGovern Institute for Brain Research, Department of Brain and Cognitive SciencesMassachusetts Institute of TechnologyCambridgeUSA
  8. 8.Key Laboratory of Brain Functional Genomics (Ministry of Education and Science and Technology Commission of Shanghai Municipality), Institute of Cognitive Neuroscience, School of Psychology and Cognitive ScienceEast China Normal UniversityShanghaiChina
  9. 9.Stanley Center for Psychiatric ResearchBroad Institute of Massachusetts Institute of Technology and HarvardCambridgeUSA

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