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

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Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice Mol Neurobiol DOI 10.1007/s12035-016-0261-0 Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice Joan A. Lenihan1 & Orthis Saha 1,2 & Victoria Heimer-McGinn1,3 & John F. Cryan4,5,6 & Guoping Feng7,8,9 & Paul W. Young 1,6 Received: 23 August 2016 /Accepted: 25 October 2016 # Springer Science+Business Media New York 2016 Abstract NUMB is a key regulator of neurogenesis and neu- analysis to identify neuronal LNX-interacting proteins other ronal differentiation that can be ubiquitinated and targeted for than NUMB that might contribute to the anxiolytic phenotype proteasomal degradation by ligand of numb protein-X (LNX) observed. We identified and/or confirmed interactions of family E3 ubiquitin ligases. However, our understanding of LNX1 and LNX2 with proteins known to have presynaptic LNX protein function in vivo is very limited. To examine the and neuronal signalling functions, including the presynaptic role of LNX proteins in regulating NUMB function in vivo, active zone constituents ERC1, ERC2 and LIPRIN-αs we generated mice lacking both LNX1 and LNX2 expression (PPFIA1, PPFIA3), as well as the F-BAR domain proteins in the brain. Surprisingly, these mice are healthy, exhibit un- FCHSD2 (nervous wreck homologue) and SRGAP2. These altered levels of NUMB protein and do not display any neu- and other novel LNX-interacting proteins identified are prom- roanatomical defects indicative of aberrant NUMB function. ising candidates to mediate LNX functions in the central ner- Behavioural analysis of LNX1/LNX2 double knockout mice vous system, including their role in modulating anxiety- revealed decreased anxiety-related behaviour, as assessed in related behaviour. 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 dys- Keywords LNX1 . LNX2 . LIPRIN/PPFIA . ERC1/ERC2 . function in LNX null animals, we performed a proteomic NUMB . Anxiety Electronic supplementary material The online version of this article (doi:10.1007/s12035-016-0261-0) contains supplementary material, which is available to authorized users. * Paul W. Young 6 Cork Neuroscience Centre, University College Cork, Cork, Ireland [email protected] 7 McGovern Institute for Brain Research, Department of Brain and 1 School of Biochemistry and Cell Biology, University College Cork, Cognitive Sciences, Massachusetts Institute of Technology, Cork, Ireland Cambridge, MA 02139, USA 2 Present address: Institut de Biologie de l’ENS (IBENS), INSERM, CNRS, École Normale Supérieure, PSL Research University, 8 Key Laboratory of Brain Functional Genomics (Ministry of 75005 Paris, France Education and Science and Technology Commission of Shanghai Municipality), Institute of Cognitive Neuroscience, School of 3 Present address: Department of Cognitive, Linguistic and Psychology and Cognitive Science, East China Normal University, Psychological Sciences, Brown University, Providence, RI, USA Shanghai 200062, China 4 Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland 9 Stanley Center for Psychiatric Research, Broad Institute of 5 Alimentary Pharmabiotic Centre, University College Cork, Massachusetts Institute of Technology and Harvard, Cork, Ireland Cambridge, MA 02142, USA Mol Neurobiol Abbreviations LNX1 and LNX2 have the same domain structure, com- PDZ PSD-95 prising an amino-terminal Really Interesting New Gene DlgA ZO-1 (RING) domain, a NUMB-binding motif (NPAY or NPAF) RING Really Interesting New Gene and four carboxyl-terminal PSD-95, DlgA and ZO-1 (PDZ) LNX Ligand of numb protein X domains (Fig. 1). The RING domain harbours the catalytic E3 CNS Central nervous system ubiquitin ligase activity, but notably, the shorter LNX1 p70 ERC ELKS/Rab6-interacting/CAST and p62 isoforms that are expressed in the brain lack the SVZ Subventricular zone RING domain, suggesting that they might have functions that PBK PDZ-binding kinase are independent of ubiquitination [1, 11]. No such alternative DKO Lnx1exon3−/−;Lnx2−/− double knockout splicing of Lnx2 has been reported. The combination of a DHET Lnx1exon3+/−;Lnx2+/− double heterozygous knockout RING and one or more PDZ domains is unique to the LNX GFP Green fluorescent protein family [12]. PDZ domains function as protein–protein inter- GST Glutathione S-transferase action modules, most commonly binding to the carboxyl- PCR Polymerase chain reaction termini of other proteins. Wolting et al. [13] catalogued around PTB Phosphotyrosine-binding domain 220 LNX-interacting proteins both from their own work and the published literature, while a subsequent study by Guo et al. [14] added approximately 30 additional proteins to this list. Introduction Most of these interactions are PDZ domain-mediated and were identified using either yeast two-hybrid assays or arrays Ligand of NUMB protein X (LNX) proteins were first charac- of PDZ domains and PDZ-binding motifs. To date, only a terized based on their ability to bind to NUMB and small number of the described LNX-interacting proteins have NUMBLIKE [1, 2]. LNX1 and LNX2 are closely related E3 been shown to be substrates for ubiquitination by LNX. For ubiquitin ligases that can ubiquitinate specific isoforms of example, ubiquitination of c-Src and PDZ-binding kinase NUMB, and LNX-mediated ubiquitination, at least in the case (PBK) by LNX1 targets them for proteasomal degradation of LNX1, has been shown to target NUMB for proteasomal [8, 14], while LNX-mediated ubiquitination of CLAUDINS degradation [3–5]. NUMB is a negative regulator of Notch sig- and CD-8α appears to cause their internalization from the cell nalling, and degradation of NUMB upon LNX1 overexpression surface, via endocytic pathways [15, 16]. Nevertheless, these was shown to moderately enhance Notch signalling in cultured examples indicate that the ubiquitin ligase activity of LNX cells [4]. However, LNX2 knockdown in colorectal cancer cell proteins can be targeted to specific substrates via PDZ- lines caused a decrease in NUMB levels, a result that does not fit mediated interactions. Thus, we need to consider interacting with the notion of LNX2 targeting NUMB for degradation [6]. proteins beyond NUMB and NUMBLIKE that may be sub- Developmentally, expression of both Lnx1andLnx2 messenger strates for ubiquitination by LNX proteins or that may mediate RNA (mRNA) is prominent in the embryonic and adult central E3 ligase-independent LNX functions. Given the low and nervous system (CNS) [1, 2]. This observation suggests a pos- potentially cell type-restricted expression patterns of LNX sible role for LNX1 and LNX2 in modulating neural develop- proteins [8, 11, 17], the identification of physiologically rele- ment through their interaction with NUMB and/or its paralogue vant interacting proteins and substrates will be key to eluci- NUMBLIKE—key regulators of mammalian neurogenesis and dating the in vivo functions of LNX proteins. neuronal differentiation [7]. However, LNX proteins are present To explore the neuronal functions of LNX proteins in vivo, at very low levels in the brain, despite the presence of Lnx we generated double knockout mice that lack LNX protein mRNAs [8], and the regulation of NUMB by endogenous levels expression in the CNS. These mice exhibit decreased of LNX proteins has not been definitively demonstrated. One of anxiety-related behaviours, in the apparent absence of any many aspects of neural development regulated by sensory, motor or learning deficits. However, we do not find NUMB/NUMBLIKE is the development of the neurogenic evidence to support the hypothesis that LNX proteins are ma- subventricular zone (SVZ) [9]. A recent study has reported an jor regulators of NUMB/NUMBLIKE function during CNS upregulation of LNX2 within the SVZ of mice lacking the Gli3 development. To identify other proteins that may mediate transcriptional repressor and demonstrated that these mice have LNX functions in the CNS, we characterized brain proteins lower levels of NUMB protein [10]. However, a causal relation- that bind LNX1 and LNX2 PDZ domains using affinity puri- ship between these two observations was not proven and the fication and mass spectrometry. This approach revealed inter- question of whether NUMB is modulated by endogenous levels actions of LNX1 and LNX2 with proteins that have of LNX proteins in the SVZ has not been addressed. There have established synaptic or neuronal functions, including not been any in vivo Lnx loss-of-function studies in a mamma- ERC1/ERC2, LIPRIN-αs, FCHSD2 and SRGAP2—provid- lian context, and hence, the physiological significance of the ing candidates, in addition to NUMB, that may play a role in LNX–NUMB interaction remains unclear. the altered anxiety-related behaviour in LNX-deficient mice. Mol Neurobiol A B Lnx1 - wild type Lnx2 - wild type ATG p80 p70 p62 F1 R1 E x o n 1 2 s 0 n 1 n 5 E x o n 3 x o n n 2 n 4 1 s 5 - E x o n x o n 1-6 3 o o o E x o E x E x E x H H E E Lnx2 - floxed ATG Lnx1 - exon3 knockout 1 F2 F1 R1 E x o n 2 n s 1 0 p80 E x o n - p70p770 p6p6262 R o 3 Neo E x H H E x o n 1 5 E x o n 3 E x o n 2 E x o n 4 n s 6 - 1 5 R n E x o Neo o x E Lnx2 - knockout ATG Frt E x o n 1 F2 R1 n s 3 - 1 0 o LoxP C RING NPAY PDZ domains E x LNX1 p80 D LNX1 p70 RING NPAF PDZ domains LNX2 LNX1 p62 (75kDa) E F 1 2 3 C WT DHET DKO –400 8kb- WT LNX1 –200 5kb- Floxed –400 3kb- LNX2 –200 (bp) 2kb- T PCR WT PCRKO PCRWT PCRKO PCRW KO PCR G Lysate IP: αLNX1/2 E14.5 P1 P18 P18 Blot: * -75 anti- LNX2 -75 LNX2 p70 * LNX1/2 LNX1p70 LNX1 p62 LNX1p62 -58 LNX1 -58 T T O T WT W W W DKO DKO DK DKO H Lysate IP: αLNX1/2 E14.5 P1 P7 P18 Ad P1 P18 LNX2 -75 -75 Blot: LNX2 T T T anti- WT W KO W W WT DKO D DKO DKO DKO T O O LNX2 W K WT K D D P1: Olf B Fore Br Mid Br Br Stm Cereb Sp Cord LNX2 -75 O T T O WT WT W KO W WT WT K DK DKO D DKO DKO D Materials and Methods of the transcripts that codes for the p70 and p62 neuronal isoforms of LNX1, is replaced by a neomycin resistance gene Animals (Fig.
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