International Journal of Molecular Sciences Article Dual Mkk4 and Mkk7 Gene Deletion in Adult Mouse Causes an Impairment of Hippocampal Immature Granule Cells Rubén Darío Castro-Torres 1,2 , Jordi Olloquequi 3 , Miren Etchetto 4,5,6 , Pablo Caruana 1 , Luke Steele 1 , Kyra-Mae Leighton 1 , Jesús Ureña 1,5,6 , Carlos Beas-Zarate 2 , Antoni Camins 4,5,6 , Ester Verdaguer 1,5,6,† and Carme Auladell 1,5,6, *,† 1 Department of Cell Biology, Physiology and Immunology, Biology Faculty, Universitat de Barcelona, 08028 Barcelona, Spain; dario.castro@cucba.udg.mx (R.D.C.-T.); pcaruanas@gmail.com (P.C.); luketaylorsteele@gmail.com (L.S.); kyramael34@gmail.com (K.-M.L.); jurena@ub.edu (J.U.); everdaguer@ub.edu (E.V.) 2 Laboratory of Neurobiotechnology CUCBA, Department of Cell and Molecular Biology, Universidad de Guadalajara, Jalisco 45200, Mexico; carlos.beas@academicos.udg.mx 3 Laboratory of Cellular and Molecular Pathology, Health Sciences Faculty, Biomedical Sciences Institute, Universidad Autónoma de Chile, Talca 3460000, Chile; jordiig82@gmail.com 4 Department of Pharmacology, Toxicology and Therapeutic Chemistry, Pharmacy and Food Sciences Faculty, Universitat de Barcelona, 08028 Barcelona, Spain; mirenettcheto@ub.edu (M.E.); camins@ub.edu (A.C.) 5 Centre for Biomedical Research of Neurodegenerative Diseases (CIBERNED), Instituto de Salud Carlos III, 28029 Madrid, Spain  6 Institut de Neurociències, Universitat de Barcelona, 08035 Barcelona, Spain  * Correspondence: cauladell@ub.edu Citation: Castro-Torres, R.D.; † These two authors contributed equally. Olloquequi, J.; Etchetto, M.; Caruana, P.; Steele, L.; Leighton, K.-M.; Ureña, Abstract: (1) Background: The c-Jun-NH2-terminal protein kinase (JNK) is a mitogen-activated J.; Beas-Zarate, C.; Camins, A.; protein kinase involved in regulating physiological processes in the central nervous system. However, Verdaguer, E.; et al. Dual Mkk4 and the dual genetic deletion of Mkk4 and Mkk7 (upstream activators of JNK) in adult mice is not reported. Mkk7 Gene Deletion in Adult Mouse The aim of this study was to induce the genetic deletion of Mkk4/Mkk7 in adult mice and analyze Causes an Impairment of their effect in hippocampal neurogenesis. (2) Methods: To achieve this goal, Actin-CreERT2 (Cre+/ − ), Hippocampal Immature Granule Mkk4flox/flox , Mkk7flox/flox mice were created. The administration of tamoxifen in these 2-month-old Cells. Int. J. Mol. Sci. 2021, 22, 9545. mice induced the gene deletion (Actin-CreERT2 (Cre+/− ), Mkk4∆/∆ , Mkk7∆/∆ genotype), which was https://doi.org/10.3390/ijms 22179545 verified by PCR, Western blot, and immunohistochemistry techniques. (3) Results: The levels of MKK4/MKK7 at 7 and 14 days after tamoxifen administration were not eliminated totally in CNS, Academic Editor: Yusaku Nakabeppu unlike what happens in the liver and heart. These data could be correlated with the high levels of these proteins in CNS. In the hippocampus, the deletion of Mkk4/Mkk7 induced a misalignment Received: 18 July 2021 position of immature hippocampal neurons together with alterations in their dendritic architecture Accepted: 31 August 2021 pattern and maturation process jointly to the diminution of JNK phosphorylation. (4) Conclusion: Published: 2 September 2021 All these data supported that the MKK4/MKK7–JNK pathway has a role in adult neurogenic activity. Publisher’s Note: MDPI stays neutral Keywords: Cre-LoxP; MKK4; MKK7; pJNK; DCX; hippocampus with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction The c-Jun NH2-terminal kinases (JNKs) are members of the Mitogen-Activated Protein Kinases (MAPKs) super-family. JNK activity regulates several cellular functions, such Copyright: © 2021 by the authors. as cell growth, differentiation, survival, and apoptosis through their impact on gene Licensee MDPI, Basel, Switzerland. expression, cytoskeletal protein dynamics, and cell death/survival pathways. In fact, the This article is an open access article best-described mechanism linked to the JNK pathway signaling is its pro-apoptotic action distributed under the terms and following sustained or intense exposure to cellular stress (including oxidative, genotoxic, conditions of the Creative Commons and osmotic stress) or pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ and interleukin (IL)-1β. This allows regulating physiological and pathological processes 4.0/). Int. J. Mol. Sci. 2021, 22, 9545. https://doi.org/10.3390/ijms22179545 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9545 2 of 18 in autoimmune diseases, diabetes, cancer, cardiac hypertrophy, and neurodegenerative diseases [1–5]. Two MAP Kinase Kinases (MAPKKs)–MKK4 and MKK7–activate JNK proteins through dual phosphorylation at threonine and tyrosine residues (TPY-motif). In turn, MKK4 and MKK7 are activated by multiple MAP Kinase Kinase Kinase (MAPKKK) and MAPKKKKs, including the Mixed-Lineage Kinase (MLK), Transforming Growth Factor β-Activated Ki- nase (TAK), Apoptosis Signal-regulating Kinase (ASK), MAPK/RK Kinase Kinase (MEKKs), Dual Leucine Zipper Kinase (DLK), and their different isoforms in response to different stimuli. The scaffold JNK-Interacting Proteins (JIP) facilitate this sequential phospho- rylation cascade. Their inactivation is regulated by Mitogen-Activated Protein Kinase Phosphatase (MKP)-1 and-7 [6]. MKK4 and MKK7 are crucial in the central and peripheral nervous systems’ (CNS and PNS) developmental processes, such as commissural fibers development, cell migration, and correct positioning of neuronal cells [7,8]. Indeed, it has been evidenced that MKK4 has dynamic changes during embryogenesis and postnatal development not only in the brain but also in other organs, such as liver and thymus [9]. In this way, Mkk4 knock-out (KO) mice display altered hepathogenesis and die early in embryonic development, specifically between days 10.5 and 12.5, as also occurs to the c-jun−/− mice. Moreover, Mkk4 has been identified as a tumor-suppressor gene [9]. In turn, Mkk4 gene deletion has revealed a compensatory effect of Mkk7 [3,10,11]. MKK4 and MKK7 proteins are differentially located in neurons: while MKK4 is present in the cell body and their processes (dendrites and axons), MKK7 is mainly detected in the nucleus. Therefore, whereas both MKK4 and MKK7 can stimulate JNK in the nuclear fraction, MKK4 activity is likely to take part in maintaining the high basal activity on neurites. Consequently, MKK4 seems to be the kinase that mediates JNK dendritic outgrowth and establishes neural circuits in the brain [7]. Studies using KO mice with a single genetic mutation or mutation combinations of JNK1, JNK2, and JNK3 isoforms reported valuable data about their function [12]. Accord- ingly, jnk3-null mice show a reduction of c-JUN phosphorylation in ischemia–hypoxia [13] and in excitotoxicity [14], while Jnk1−/− mice display progressive degeneration of long nerve fibers together with alterations in microtubule stability, evidencing a role of JNK1 in axonal growth and dendritic architecture maintenance [15,16]. In addition, the use of Jnk1−/− , Jnk2−/ − , and Jnk3−/− mice supported the fact that the JNK signaling pathway controls adult neurogenesis [17,18]. However, the specific mechanisms by which the molecular effectors of the JNK pathway are involved in all these processes remain obscure. Moreover, KO conventional murine models have significant limitations since the deletion key members of the pathway such as Mkk4 or Mkk7 and the double deletion Jnk1/Jnk2 affect embryonic development and induce lethality [7,8,19,20]. In this sense, the conditional KO mice for Mkk4 and Mkk7 created with the Cre-LoxP system and Nestin promoter surpass the embryonic lethality, but the animals die soon after birth [7,8]. To overcome these limitations, the present study aimed to assess the role of MKK4 and MKK7 in immature hippocampal neurons and dendritic architecture maintenance, using a new conditional-induced KO mice, the hemizygous Actin-CreERT2 (Cre+/− ), Mkk4flox/flox , Mkk7flox/flox . These mice allow a controlled deletion of Mkk4 and Mkk7 genes in postnatal stages or adults. The results obtained in the present work demonstrate that the Mkk4∆/∆ Mkk7∆/∆ geno- type mice can be induced in adulthood, showing a decrease in MKK4 and MKK7 proteins in different areas of the CNS (hippocampus, cortex, and cerebellum) as well as in heart and liver. This protein depletion was correlated with a reduction of JNK phosphorylation. Moreover, the analyses of immature neurons in the subgranular zone (SGZ) of the hip- pocampus in these KO Mkk4∆/∆ Mkk7∆/∆ mice evidenced modifications in their distribution and dendritic pattern when compared with wild-type (WT) mice. Finally, alterations in the dendritic distribution pattern of cortical neurons were also detected. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 18 Int. J. Mol. Sci. 2021, 22, 9545 3 ofin and dendritic pattern when compared with wild-type (WT) mice. Finally, alterations 18 the dendritic distribution pattern of cortical neurons were also detected. 2. Results 2. Results 2.1. 2.1. Characterization Characterization of of Mkk4 Mkk4 and and Mkk7 Mkk7 Gene Gene Deletion Deletion and and Protein Protein Elimination Elimination in in the the Δ/Δ Δ/Δ Hippocampus of Actin-Cre Hippocampus of Actin-Cre ERT2 ERT2 (Cre +/− +/),−Mkk4 (Cre ), Mkk4 , Mkk7 ∆/∆ , Mkk7 Adult ∆/∆ Mice Adult Mice The The relative relative quantification quantificationof of MKK4 MKK4 and and MKK7 MKK7 proteins proteins was was evaluated evaluated in in the the CNS, CNS, heart, and liver in two-month-old WT mice by immunoblot assays. heart, and liver in two-month-old WT mice by immunoblot assays. The results showed The results showed that that the the amounts amounts of of MKK4 MKK4 and and MKK7 MKK7 werewere higher higher inin the the CNS CNS than than in in heart heart andand liver liver (Figure (Figure 1A–C). In addition, the lower protein abundance of MKK7 was found in the 1A–C). In addition, the lower protein abundance of MKK7 was found in the heart heart and and liver liver (Figure (Figure 1A,C). 1A,C). Figure 1. Levels of MKK4 and MKK7 proteins in different CNS areas, heart, and liver in mice adulthood. (A) The Figure 1. Levels of MKK4 and MKK7 proteins in different CNS areas, heart, and liver in mice adulthood. (A) The im- immunoblot munoblot shows shows MKK4 MKK4 andand MKK7 MKK7 protein protein levels levels inindifferent differenttissues WTadult tissuesofofWT adultmice. mice.(B) (B)The Thebar bargraph graphrepresents represents the the MKK4 protein MKK4 protein levels levels in in the the CNS CNS (cortex, (cortex, cerebellum, cerebellum, andand hippocampus), hippocampus), heart, heart, and and liver. liver. (C) (C) The The bar bar graph graph represents represents thethe MKK7 protein MKK7 protein levels in the CNS (cortex, cerebellum, and hippocampus), heart, and liver. Abbreviations. MKK4, MKK4, Mitogen Mitogen Kinase Kinase Kinase 4; 4; MKK7, MKK7,Mitogen MitogenKinase KinaseKinase Kinase7; 7; GAPDH, GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; glyceraldehyde-3-phosphate-dehydrogenase; CTX, CTX, cortex; cortex; CB, CB, cerebellum; cerebellum; HP, HP, hippocampus. hippocampus. One-way One-way ANOVAANOVA and hoc and post postFisher’s hoc Fisher’s LeastLeast Significant Significant Difference Difference (LSD)(LSD) (n = 3)(nwere = 3) were used used in theinstatistical the statistical analysis. analysis. * p