Deletion of Dlk2 increases the vulnerability to anxiety-like behaviors and impairs the anxiolytic action of alprazolam
Introduction
The NOTCH system is an evolutionary conserved signaling pathway identified as a critical regulator of neurogenesis in the adult brain (Imayoshi and Kageyama, 2011). In mammals, NOTCH signaling is triggered by the interaction of at least one of the four membrane receptors (NOTCH1–4) with one or more of the five JAGGED of DELTA canonical ligands (JAGGED1, JAGGED2, DLL1, DLL3, and DLL4) (Kageyama and Ohtsuka, 1999, D'Souza et al., 2010). NOTCH receptors and their ligands are transmembrane proteins belonging to the epidermal growth factor (EGF)-like family. The interaction induces metalloproteinase-mediated and γ-secretase-mediated cleavage of the NOTCH receptor releasing an active NOTCH intracellular domain (NICD) that translocates to the nucleus and forms a complex with CBF-1/RBP-J, mediating the transcription of target genes, for instance, the expression of the basic loop helix transcription factors Hes1, Hes5 and Hey1 (D'Souza et al., 2008). Recently, two EGF-like proteins highly related to the NOTCH ligand family, named Delta-like 1 (DLK1) and Delta-like 2 (DLK2), were identified as non-canonical NOTCH ligands, based on the fact that they interact with NOTCH receptors, despite lacking the DSL domain characteristic of the canonical ligands. Although the precise function of these non-canonical ligands still remains unclear, DLK1 and DLK2 act as NOTCH receptor antagonists (Baladron et al., 2005, Sanchez-Solana et al., 2011, Traustadottir et al., 2016).
Among the different functional implications of the NOTCH signaling pathway, numerous evidences emphasize its crucial involvement in cell differentiation and proliferation processes (Falix et al., 2012), playing a key role in tumorigenesis, carrying important therapeutic implications (Shao et al., 2012, Espinoza and Miele, 2013, Gu et al., 2016, Liu et al., 2016). In addition, in the central nervous system (CNS), the NOTCH pathway contributes to the maintenance of an adequate balance between neurogenesis and gliogenesis (Snyder et al., 2012), to the regulation of synaptic plasticity (Alberi et al., 2011), and to neuron survival (Hitoshi et al., 2004, Mizutani et al., 2007). Indeed, recent studies described how the NOTCH pathway is involved in the regulation of different brain functions, such as learning, memory (Costa et al., 2003, Conboy et al., 2007, Yoon et al., 2012, Sargin et al., 2013, Ding et al., 2016) or emotional behavior (Guo et al., 2009, Wang et al., 2012, Dias et al., 2014, Monsalve et al., 2014, Sun et al., 2016). However, additional studies are still needed to elucidate the precise neurobiological mechanisms underlying NOTCH-mediated actions.
DLK2 is a transmembrane protein highly homologous to the previously known DLK1, characterized by a widespread expression in different mouse tissues (Nueda et al., 2007). Besides its role as a non-canonical NOTCH ligand inhibiting NOTCH signaling, there is very scarce information about the functional role of DLK2. Interestingly, DLK2 is widely distributed in different brain areas such as the arcuate nucleus, the hypophysis, the dorsal raphe, the amygdala, the hippocampus and the prefrontal cortex (Gregg et al., 2010, Monsalve et al., 2014, Bonthuis et al., 2015, Carithers and Moore, 2015, Lizio et al., 2015). Since all these regions are involved in the regulation of stress, emotional response, learning, or memory, it is tempting to suggest that DLK2 may play a relevant role in the regulation of these behaviors.
Mice deficient for the Dlk2 gene (Dlk2−/− mice) were generated and used to evaluate the relevance of this gene in emotional behavior. Basic behavioral aspects related with motor activity (open field test), anxiety-like (light-dark box test, elevated plus maze test) and depressive-like behavior (novelty suppressed feeding test) were performed in WT and Dlk2–/– mice. The neurobiological changes induced by challenging animals to an acute stressful stimulus (restraint stress) and the pharmacological response to the anxiolytic benzodiazepine (alprazolam) were evaluated. Analyses of the relative gene expression levels of corticotropin releasing hormone (Crh) in the paraventricular nucleus (PVN), glucocorticoid receptor (Nr3c1), FK506 binding protein 5 (Fkbp5) in the hippocampus (HIPP), and the transcription factors Hes1, Hes5 and Hey1 in PVN, HIPP and amygdala (AMY) were carried out in Dlk2–/– and WT mice under basal conditions and after exposure to restraint stress. In addition, gamma-aminobutyric acid (GABA) A receptor, subunit alpha-2 (Gabra2) and subunit gamma-2 (Gabrg2), gene expression levels were also evaluated in the amygdala (AMY) of Dlk2−/− and WT mice under basal conditions and after exposure to restraint stress.
Section snippets
Generation of Dlk2 knockout mice
To generate Dlk2 knockout mice we designed a targeting strategy consisting in the insertion in this locus of a single distal loxP site within the intron 1 and a loxP site together with a flippase recognition target (FRT) flanked neomycin selection cassette within the intron 4 (Fig. S1). Three of the confirmed properly recombined embryonic stem (ES) cell clones (from S129P2/Ola background) were then injected into blastocysts (C57BL6/J background), aiming to generate Dlk2 constitutive knockout
Motor activity-open field
No significant differences between Dlk2−/− mice and their corresponding control group were observed in any of the parameters analyzed (Fig. 1) (periphery distance travelled Student’s t-test, t = 0.570, 28df, p = 0.573; center distance travelled Student’s t-test, t = −1.795, 28df, p = 0.083; total distance travelled Student’s t-test, t = −0.782, 28df, p = 0.441; periphery speed Student’s t-test, t = −1.452, 28df, p = 0.158; center speed Student’s t-test, t = −1.452, 28df, p = 0.158; total speed Student’s t-test, t =
Discussion
The results of the present study point to DLK2 as playing a major role in the regulation of emotional behaviors. This assumption is based on the following facts: 1) Deletion of Dlk2 increased vulnerability to anxiety-like (light-dark box and elevated plus maze paradigms) and depressive-like behaviors (novelty suppressed feeding test); 2) Crh, Nr3c1 and Fkbp5 gene expression levels were altered in Dlk2–/– mice under both basal and restraint stress conditions compared with WT mice; 3) Hes1, Hes5
Author contribution
JL and JM were responsible for the study concept and design. FN and MSGG contributed to the acquisition of animal data. FN, MSGG, JL and JM assisted with data analysis and interpretation of findings. JL designed and directed the generation of the animals. FN drafted the manuscript. JL and JM provided critical revision of the manuscript for important intellectual content. All authors critically reviewed content and approved the final version for publication.
Conflict of interest
All authors state that they have no biomedical financial interest or potential conflicts of interest.
Acknowledgments
This research was supported by “Instituto de Salud Carlos III” (RTA, RD16/0017/0014, Fondos FEDER), “Plan Nacional sobre Drogas” (PNSD, 2016/016), ‘Ministerio de Economía y Competitividad’ (FIS, PI14/00438), and ‘Junta de Comunidades de Castilla-La Mancha’ (PCI08-0134-6712, PEI-1-2014-033, with FEDER funds).
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Both authors share equal credit for the direction of this work.