Full-length in meso structure and mechanism of rat kynurenine 3-monooxygenase inhibition.

The structural mechanisms of single-pass transmembrane enzymes remain elusive. Kynurenine 3-monooxygenase (KMO) is a mitochondrial protein involved in the eukaryotic tryptophan catabolic pathway and is linked to various diseases. Here, we report the mammalian full-length structure of KMO in its membrane-embedded form, complexed with compound 3 (identified internally) and compound 4 (identified via DNA-encoded chemical library screening) at 3.0 Å resolution. Despite predictions suggesting that KMO has two transmembrane domains, we show that KMO is actually a single-pass transmembrane protein, with the other transmembrane domain lying laterally along the membrane, where it forms part of the ligand-binding pocket. Further exploration of compound 3 led to identification of the brain-penetrant compound, 5. We show that KMO is dimeric, and that mutations at the dimeric interface abolish its activity. These results will provide insight for the drug discovery of additional blood-brain-barrier molecules, and help illuminate the complex biology behind single-pass transmembrane enzymes.

Memantine, an NMDA receptor antagonist, improves working memory deficits in DGKß knockout mice.

Diacylglycerol kinase (DGK) ß is a type 1 isozyme of the DGK family. We previously reported that DGKß was deeply involved in neurite spine formation, and DGKß knockout (KO) mice exhibited behavioral abnormalities concerning spine formation, such as cognitive, emotional, and attentional impairment. Moreover, some of these abnormalities were ameliorated by the administration of a mood stabilizer. However, there is no data about how memory-improving drugs used in the treatment of Alzheimer's disease affect DGKß KO mice. In the present study, we evaluated the effect of an anti-Alzheimer's drug, memantine on the working memory deficit observed in DGKß KO mice. In the Y-maze test, the administration of memantine significantly improved working memory of DGKß KO mice. We also found that the expression levels of the NR2A and NR2B N-methyl-d-aspartate (NMDA) receptor subunits were increased in the prefrontal cortex, but decreased in the hippocampus of DGKß KO mice. These altered expression levels of NR2 subunits might be related to the effect of an NMDA receptor antagonist, memantine. Taken together, these findings may support the hypothesis that DGKß has a pivotal role in cognitive function.

Involvement of diacylglycerol kinase ß in the spine formation at distal dendrites of striatal medium spiny neurons.

Spine formation, a salient feature underlying neuronal plasticity to adapt to a changing environment, is regulated by complex machinery involving membrane signal transduction. The diacylglycerol kinase (DGK) family, which is involved in membrane lipid metabolism, catalyzes the phosphorylation of a lipid second messenger, diacylglycerol (DG). Of the DGKs, DGKß is characterized by predominant expression in a specific brain region: the striatum. We previously demonstrated that DGKß is expressed selectively in medium spiny neurons (MSNs) and that it is highly enriched in the perisynaptic membrane on dendritic spines contacted with excitatory terminals. Moreover, DGKß regulates spinogenesis through actin-based remodeling in an activity-dependent manner. However, the detailed mechanisms of spinogenesis regulation and its functional significance remain unclear. To address these issues, we performed Golgi-Cox staining to examine morphological aspects of MSNs in the striatum of DGKß-knockout (KO) mice. Results show that striatal MSNs of DGKß-KO mice exhibited lower dendritic spine density at distal dendrites than wild-type mice did. We also sought protein targets that interact with DGKß and identified the GluA2 AMPA receptor subunit as a novel DGKß binding partner. In addition, DGKß-deficient brain exhibits significant reduction of TARP γ-8, which represents a transmembrane AMPA receptor regulatory protein. These findings suggest that DGKß regulates the spine formation at distal dendrites in MSNs.

Diacylglycerol kinase ß knockout mice exhibit attention-deficit behavior and an abnormal response on methylphenidate-induced hyperactivity.

BACKGROUND: Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. DGKß is one of the subtypes of the DGK family and regulates many intracellular signaling pathways in the central nervous system. Previously, we demonstrated that DGKß knockout (KO) mice showed various dysfunctions of higher brain function, such as cognitive impairment (with lower spine density), hyperactivity, reduced anxiety, and careless behavior. In the present study, we conducted further tests on DGKß KO mice in order to investigate the function of DGKß in the central nervous system, especially in the pathophysiology of attention deficit hyperactivity disorder (ADHD). METHODOLOGY/PRINCIPAL FINDINGS: DGKß KO mice showed attention-deficit behavior in the object-based attention test and it was ameliorated by methylphenidate (MPH, 30 mg/kg, i.p.). In the open field test, DGKß KO mice displayed a decreased response to the locomotor stimulating effects of MPH (30 mg/kg, i.p.), but showed a similar response to an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801 (0.3 mg/kg, i.p.), when compared to WT mice. Examination of the phosphorylation of extracellular signal-regulated kinase (ERK), which is involved in regulation of locomotor activity, indicated that ERK1/2 activation induced by MPH treatment was defective in the striatum of DGKß KO mice. CONCLUSIONS/SIGNIFICANCE: These findings suggest that DGKß KO mice showed attention-deficit and hyperactive phenotype, similar to ADHD. Furthermore, the hyporesponsiveness of DGKß KO mice to MPH was due to dysregulation of ERK phosphorylation, and that DGKß has a pivotal involvement in ERK regulation in the striatum.

Luteolin shows an antidepressant-like effect via suppressing endoplasmic reticulum stress.

Depression is a significant public health problem and some reports indicate an association between depression and endoplasmic reticulum stress. Luteolin is a flavonoid contained in many plants and with a variety of known pharmacological properties such as anti-inflammatory, anti-anxiety, and memory-improving effects, suggesting that luteolin penetrates into the brain. In the present study, we investigated the effects of luteolin on endoplasmic reticulum stress-induced neuronal cell death. Luteolin significantly suppressed tunicamycin-induced cell death at 1 to 10 µM in human neuroblastoma cells. Luteolin increased in the expression of the 78 kDa glucose-regulated protein and 94 kDa glucose-regulated protein and decreased in the cleavage activation of caspase-3. Additionally, to investigate whether chronic luteolin treatment has an antidepression effect, we performed some behavioral tests. Chronic luteolin treatment showed antidepressant-like effects in behavioral tests and, luteolin attenuated the expression of endoplasmic reticulum stress-related proteins in the hippocampus of corticosterone-treated depression model mice. These findings indicate that luteolin has antidepressant-like effects, partly due to the suppression of endoplasmic reticulum stress.

Diacylglycerol kinase ß knockout mice exhibit lithium-sensitive behavioral abnormalities.

BACKGROUND: Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). DGKß is widely distributed in the central nervous system, such as the olfactory bulb, cerebral cortex, striatum, and hippocampus. Recent studies reported that the splice variant at the COOH-terminal of DGKß was related to bipolar disorder, but its detailed mechanism is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we performed behavioral tests using DGKß knockout (KO) mice to investigate the effects of DGKß deficits on psychomotor behavior. DGKß KO mice exhibited some behavioral abnormalities, such as hyperactivity, reduced anxiety, and reduced depression. Additionally, hyperactivity and reduced anxiety were attenuated by the administration of the mood stabilizer, lithium, but not haloperidol, diazepam, or imipramine. Moreover, DGKß KO mice showed impairment in Akt-glycogen synthesis kinase (GSK) 3ß signaling and cortical spine formation. CONCLUSIONS/SIGNIFICANCE: These findings suggest that DGKß KO mice exhibit lithium-sensitive behavioral abnormalities that are, at least in part, due to the impairment of Akt-GSK3ß signaling and cortical spine formation.

Essential role of neuron-enriched diacylglycerol kinase (DGK), DGKbeta in neurite spine formation, contributing to cognitive function.

BACKGROUND: Diacylglycerol (DG) kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Of the 10 subtypes of mammalian DGKs, DGKbeta is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We, therefore, developed DGKbeta KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKbeta. In addition, overexpression of DGKbeta in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKbeta, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKbeta but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that membrane-localized DGKbeta regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory.

Metallothionein-3 deficient mice exhibit abnormalities of psychological behaviors.

Metallothioneins (MTs) are metal binding proteins and have four isoforms. MT-3, known as growth inhibitory factor (GIF), exists mainly in the central nervous system. It regulates zinc levels and exhibits a neuroprotective effect in the various types of brain diseases. However, the reports demonstrate that the relation between MT-3 and psychiatric disorder is still unknown. In the present study, the authors carried out behavioral tests on MT-3 knock-out (KO) mice. The duration of the MT-3 KO mice's social interactions were significantly shorter than that of the wild-type (WT) mice. The acoustic startle response of the MT-3 KO mice showed diminished prepulse inhibition (PPI) at all prepulse intensities. However, the locomotor activity tests of the MT-3 KO mice displayed normal circadian rhythm, activity, and habituation to a novel environment. In the novel object recognition test, the MT-3 KO mice exhibited normal memory. These findings indicate that abnormalities of psychological behavior were observed in the MT-3 KO mice. Further experiments will be needed to clarify the involvement of MT-3 in higher brain function.

Generation and characterization of conditional heparin-binding EGF-like growth factor knockout mice.

Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a) behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b) altered dopamine and serotonin levels in the brain, (c) decreases in spine density in neurons of the prefrontal cortex, (d) reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor and post-synaptic protein-95 (PSD-95), (e) decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II) signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.

Sirtuin 1 overexpression mice show a reference memory deficit, but not neuroprotection.

Sirtuin 1 (SIRT1) is the closest mammalian homologue of yeast silent information regulator 2 (Sir2) and has a role in lifespan modulation. Reportedly, SIRT1 is also linked to neurodegenerative diseases. However, there are limited studies that report the relation between SIRT1 and neurodegenerative diseases using in vivo transgenic (Tg) methods. In the present study, we generated neuron-specific enolase (NSE) SIRT1 Tg mice that overexpress human SIRT1 in neurons. We examined possible neuroprotective effects of SIRT1 overexpression and compared their higher brain functions with those of wild-type (WT) mice. Overexpression of SIRT1 did not have any neuroprotective effects against the neuronal damage induced by ischemia or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, SIRT1 Tg mice exhibited a reference memory deficit. These findings suggest that an excessive expression of SIRT1 might induce the memory deficit in mice, but not neuroprotective effects.