br Introduction Diacylglycerol kinase DGK
Introduction Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol (DG) to produce phosphatidic Iloperidone (PA) [, , , ]. DG and PA act as lipid second messengers in a wide variety of biological processes. Ten DGK isozymes (α, β, γ, δ, η, κ, ε, ζ, ι, and θ) have been identified and classified into five subtypes based on their structural features [, , , ]. Type II DGKs  include δ-, η-, and κ-isozymes [, , ]. Moreover, several alternative splicing products of DGKδ (δ1 and δ2)  and DGKη (η1, η2, η3 and η4) [, , ] have been identified. Previously, we reported that DGKδ isozyme was abundantly expressed in the mouse brain . Because conventional DGKδ-knockout (KO) mice die within 24 h of birth , we generated brain-specific DGKδ-KO mice and performed behavioral analysis on the mice . Intriguingly, the brain-specific DGKδ-KO mice exhibited obsessive-compulsive disorder (OCD)-like behaviors. For example, in the novel object recognition test, the mice showed irrational object contact, such as compulsive checking. Moreover, OCD-like phenotypes were evaluated using the marble burying test where the DGKδ-KO mice buried more marbles than the control mice. Furthermore, these phenotypes were significantly alleviated by the administration of fluoxetine, a selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitor (SSRI) used as an OCD therapy [16,17]. SSRIs target the serotonin (5-HT) transporter (SERT), which reuptakes 5-HT from the synaptic cleft into the presynaptic neuron for recycling or metabolic degradation. OCD is a psychiatric affliction with a lifetime prevalence of 1–3% [18,19]. The essential features of OCD are recurrent obsessions and compulsions (e.g., intrusive obsessive thinking, doubting, checking, washing/cleaning and arranging) that are time consuming and cause marked affliction. Although several neural systems have been implicated in the pathology of OCD, the nature of the OCD-related neurotransmitter system dysfunction remains unknown. Knowing that fluoxetine attenuated the OCD-like behaviors in the DGKδ-KO mice, we here performed biochemical analysis to reveal the molecular mechanisms underlying OCD-like abnormal behaviors in brain-specific DGKδ-KO mice and the abnormalities of serotonergic system in the brain.
Materials and methods
Discussion We have previously reported that the brain-specific DGKδ-KO mice exhibited OCD-like behaviors . However, the molecular mechanisms causing those OCD-like abnormal behaviors have been unclear. As SSRIs are the main therapies for OCD, depletion of 5-HT in the synaptic clefts is thought to be the main cause of OCD [26,27]. In the present study, we revealed that DGKδ deficiency increased the protein levels of SERT, a target of SSRIs, in the mouse cerebral cortex (Fig. 1). Moreover, DGKδ-KO mice showed a decreased 5-HT amount in the cerebral cortex (Fig. 4), which was likely caused by decreased TPH-2 expression and increased MAO-A expression (Fig. 4). Therefore, in the DGKδ-KO mouse brain, it is possible that decreased TPH-2 and increased MAO-A levels caused 5-HT reduction in pre-synaptic neurons. Moreover, increased SERT levels also result in further depletion of 5-HT in the synaptic clefts. These data strongly suggest that OCD-like behaviors in the DGKδ-KO mice are caused by comprehensive and composite serotonergic hypofunction. Therefore, it is likely that SERT inhibitors (SSRI) alleviated abnormal mouse behaviors  through attenuation of the SERT activity increased by DGKδ-deficiency. SERT is known to interact with many proteins such as calcineurin , Hic-5 (hydrogen peroxide-inducible clone 5) , nNOS (neuronal nitric oxide synthase) , PICK1 (protein interacting with C kinase) , α-synuclein  and SCAMP2 (secretory carrier membrane protein 2) . In the present study, we found that DGKδ also interacted with SERT (Fig. 2, Fig. 3). Therefore, DGKδ was added to the list of SERT-interacting proteins. Among them, only DGKδ controls SERT expression. Moreover, DGKδ uniquely regulates THP-2 and MAO-A protein levels. Intriguingly, DGKδ oligomerizes through its sterile α-motif domain [, , ]. Therefore, it is possible that DGKδ forms a substantially large complex with SERT and those other SERT-interacting proteins. Interestingly, we reported that PA, the product of DGK, enhanced α-helix formation and aggregation of α-synuclein . Thus, it is possible that, in addition to SERT, DGKδ may also regulate α-synuclein function in a large complex.