Norbin ablation results in defective adult hippocampal neurogenesis and depressive-like behavior in mice

Adult neurogenesis in the hippocampus subgranular zone is associated with the etiology and treatment efficiency of depression. Factors that affect adult hippocampal neurogenesis have been shown to contribute to the neuropathology of depression. Glutamate, the major excitatory neurotransmitter, plays a critical role in different aspects of neurogenesis. Of the eight metabotropic glutamate receptors (mGluRs), mGluR5 is the most highly expressed in neural stem cells. We previously identified Norbin as a positive regulator of mGluR5 and showed that its expression promotes neurite outgrowth. In this study, we investigated the role of Norbin in adult neurogenesis and depressive-like behaviors using Norbin-deficient mice. We found that Norbin deletion significantly reduced hippocampal neurogenesis; specifically, the loss of Norbin impaired the proliferation and maturation of newborn neurons without affecting cell-fate specification of neural stem cells/neural progenitor cells (NSCs/NPCs). Norbin is highly expressed in the granular neurons in the dentate gyrus of the hippocampus, but it is undetectable in NSCs/NPCs or immature neurons, suggesting that the effect of Norbin on neurogenesis is likely caused by a nonautonomous niche effect. In support of this hypothesis, we found that the expression of a cell–cell contact gene, Desmoplakin, is greatly reduced in Norbin-deletion mice. Moreover, Norbin-KO mice show an increased immobility in the forced-swim test and the tail-suspension test and reduced sucrose preference compared with wild-type controls. Taken together, these results show that Norbin is a regulator of adult hippocampal neurogenesis and that its deletion causes depressive-like behaviors.Major depressive disorder (MDD) is one of the most common and debilitating psychiatric illnesses with a lifetime prevalence of ∼17% (1). Clinical symptoms of MDD include anhedonia, depressed mood, helplessness, and cognitive disruption. Because of the clinical and etiological heterogeneity of MDD, the pathophysiology of MDD remains elusive (2), and the biological underpinnings remain unclear. Decades of research have clearly established that various neurotransmitters, especially monoamine neurotransmitters, as well as neurotrophic factors, contribute to MDD (3). More recently glutamatergic signaling also has been linked to MDD. With the confirmation of continuous neurogenesis in adult brains of most mammals, including humans, a neurogenic hypothesis of MDD has been put forward and continues to drive research (4).The neurogenic hypothesis of MDD is based on several correlative studies. Brain imaging and postmortem studies of MDD patients suggest that reduction in hippocampal volume could be reflective of reduced neurogenesis in addition to mature neuronal cell loss (5, 6). Antidepressant treatment in animals increases hippocampal neurogenesis with a lag time that is reminiscent of the delayed onset of antidepressant efficacy typically observed in humans (7). In addition, stress, a common risk factor for depression, inhibits neurogenesis in nonhuman primates, and this phenomenon can be corrected by antidepressant treatment (8). In rodents, ablation of hippocampal neurogenesis causes a depressive phenotype and abolishes the effect of certain antidepressants (9). Therefore, it is hypothesized that reduced adult hippocampal neurogenesis may underlie the pathological mechanism of MDD and that an up-regulation of neurogenesis would oppose the action of stress and/or depression, having a potential therapeutic benefit for MDD.In the hippocampus, adult neurogenesis involves the five following steps: (i) proliferation of neural stem/progenitor cells (NSCs/NPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG); (ii) fate specification (neurons versus astrocytes); (iii) massive loss of progenitor cells; (iv) neuronal differentiation and maturation; and (v) ultimate integration to the existing neuronal circuitry of the DG (10). Each step is modulated by both physiological stimuli and pathophysiological conditions. Two important determinants, cell-autonomous factors and neurogenic niche, play a key role in determining the ultimate outcome of adult neurogenesis. Cell-autonomous factors are intrinsic NSC/NPC genetic traits that dictate their fate, and the neurogenic niche corresponds to their local microenvironment defined by various parameters (e.g., neuronal inputs, vasculature, and glia composition) that interact with NSCs/NPCs and also influence their fate (11).Previous studies indicated that neurotransmitter glutamate regulates DG neurogenesis (12, 13). Of the eight metabotropic glutamate receptors (mGluRs), mGluR5 is most highly expressed in NSCs (14, 15). In vivo activation of mGluR5 receptors benefits the proliferation and/or survival of NSCs in the hippocampus. In vitro, pharmacological blockade of mGluR5 reduced NSC proliferation and survival, whereas activation of mGluR5 receptors substantially enhanced cell proliferation, suggesting that mGluR5 has a cell-autonomous role of in neurogenesis (16, 17).We previously identified Norbin as a positive regulator of mGluR5 (18). Forebrain-specific Norbin-KO mice display phenotypic characteristics that resemble phenotypes observed in cases of reduced mGluR5 activity, including impaired synaptic plasticity and sensitivity to psychostimulants. In the CNS, Norbin is a neuronal gene whose expression is up-regulated concomitantly with long-term potentiation, the electrophysiological mechanism underlying learning and memory. Norbin expression correlates with neurite outgrowth and also was found to have an impact on both the quantity and the length of neurites in cultured neuroblastoma N2a cells. In summary, Norbin (i) regulates mGluR5, whose activity affects DG neurogenesis; (ii) promotes neurite outgrowth; (iii) is highly expressed in the DG of the hippocampus, the niche for adult DG neurogenesis. Norbin-KO mice also display impaired cognitive functions. For all these reasons, we investigated the role of Norbin in adult hippocampal neurogenesis and whether the loss of Norbin could contribute to depression-like behaviors.We found here that neuron-specific Norbin ablation in mice, using two distinct Cre systems, results in a significant reduction in hippocampal neurogenesis. Both the proliferation and survival of newborn neurons are impaired. This impairment is likely the result of a nonautonomous niche effect because Norbin expression is limited to mature granular neurons in the DG. In addition, gene-expression analysis of Norbin-KO mice compared with wild-type controls indicated that Norbin deletion results in a significant reduction in desmoplakin (DSP) expression. In light of the observed reduction in adult hippocampal neurogenesis in Norbin-KO mice, we used three well-established behavioral paradigms to determine if there also was a depressive-like consequence of Norbin KO. Results from the forced-swim test (FST), the tail-suspension test (TST), and the sucrose preference test indicated that Norbin-KO mice display depressive-like characteristics.