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

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.

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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

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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.

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Correspondence to Paul W. Young.

Ethics declarations

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.

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The authors declare that they have no conflict of interest.

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Lenihan, J.A., Saha, O., Heimer-McGinn, V. et al. Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice. Mol Neurobiol 54, 8090–8109 (2017). https://doi.org/10.1007/s12035-016-0261-0

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Keywords

  • LNX1
  • LNX2
  • LIPRIN/PPFIA
  • ERC1/ERC2
  • NUMB
  • Anxiety