RAGE in circulating immune cells is fundamental for hippocampal inflammation and cognitive decline in a mouse model of latent chronic inflammation.

BACKGROUND: Latent chronic inflammation has been proposed as a key mediator of multiple derangements in metabolic syndrome (MetS), which are increasingly becoming recognized as risk factors for age-related cognitive decline. However, the question remains whether latent chronic inflammation indeed induces brain inflammation and cognitive decline. METHODS: A mouse model of latent chronic inflammation was constructed by a chronic subcutaneous infusion of low dose lipopolysaccharide (LPS) for four weeks. A receptor for advanced glycation end products (RAGE) knockout mouse, a chimeric myeloid cell specific RAGE-deficient mouse established by bone marrow transplantation and a human endogenous secretory RAGE (esRAGE) overexpressing adenovirus system were utilized to examine the role of RAGE in vivo. The cognitive function was examined by a Y-maze test, and the expression level of genes was determined by quantitative RT-PCR, western blot, immunohistochemical staining, or ELISA assays. RESULTS: Latent chronic inflammation induced MetS features in C57BL/6J mice, which were associated with cognitive decline and brain inflammation characterized by microgliosis, monocyte infiltration and endothelial inflammation, without significant changes in circulating cytokines including TNF-α and IL-1ß. These changes as well as cognitive impairment were rescued in RAGE knockout mice or chimeric mice lacking RAGE in bone marrow cells. P-selectin glycoprotein ligand-1 (PSGL-1), a critical adhesion molecule, was induced in circulating mononuclear cells in latent chronic inflammation in wild-type but not RAGE knockout mice. These inflammatory changes and cognitive decline induced in the wild-type mice were ameliorated by an adenoviral increase in circulating esRAGE. Meanwhile, chimeric RAGE knockout mice possessing RAGE in myeloid cells were still resistant to cognitive decline and brain inflammation. CONCLUSIONS: These findings indicate that RAGE in inflammatory cells is necessary to mediate stimuli of latent chronic inflammation that cause brain inflammation and cognitive decline, potentially by orchestrating monocyte activation via regulation of PSGL-1 expression. Our results also suggest esRAGE-mediated inflammatory regulation as a potential therapeutic option for cognitive dysfunction in MetS with latent chronic inflammation.

Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

Relationship between lamellar sensory corpuscles distributed along the upper arm's deep arteries and pulsating sensation of blood vessels.

Vibration is detected by mechanoreceptors, including Pacinian corpuscles (PCs), which are widely distributed in the human body including the adventitia of large blood vessels. Although the distribution of PCs around large limb vessels has been previously reported, there remains no consensus on their distribution in the adventitia of the human deep blood vessels in the upper arm. In addition, the physiological functions of PCs located around the deep limb blood vessels remain largely unknown. This study aimed to elucidate detailed anatomical features and physiological function of lamellar sensory corpuscles structurally identified as PCs using the immunohistochemical methods around the deep vessels in the upper arm. We identified PCs in the connective tissue adjacent to the deep vessels in the upper arm using histological analysis and confirmed that PCs are located in the vascular sheath of the artery and its accompanying vein as well as in the connective tissue surrounding the vascular sheath and nerves. PCs were densely distributed on the distal side of deep vessels near the elbow. We also examined the relationship between vascular sound and pulsating sensation to evaluate the PCs functions around deep arteries and veins and found that the vascular sound made by pressing the brachial arteries in the upper arm was associated with the pulsating sensation of the examinee. Our results suggest that PCs, around deep vessels, function as bathyesthesia sensors by detecting vibration from blood vessels.

Platelet-derived growth factor receptor α gene is regulated by multiple first exons.

Transcription of the platelet-derived growth factor receptor α (PDGFRA/Pdgfra) gene is considered to be precisely regulated. We have previously reported that the PDGFRA/Pdgfra gene is regulated by a dual promoter system in human and mouse, in which a novel PDGFRA/Pdgfra transcript has a first exon (exon 1ß) different from that of the canonical PDGFRA/Pdgfra transcript (exon 1α). To elucidate the function of each transcript, we first investigated the contribution of different PDGFRA transcripts to final protein levels. Notably, knockdown experiments suggested the existence of other PDGFRA transcripts, and we identified five additional first exons (exons 1γ, 1δ, 1ε, 1ζ, and 1η) in intron 1 in both the human and mouse genes. The first exons of the mouse Pdgfra gene showed unique expression patterns: exon 1α was broadly expressed; exon 1ß was highly expressed in embryos; exon 1γ was observed at relatively high levels in the adult central nervous system (CNS); and exon 1δ was expressed at relatively high levels in the developing CNS. Furthermore, in silico analysis of common putative transcription factor binding sites in the upstream regions of the first exons of both human and mouse PDGFRA/Pdgfra genes predicted common (such as Sry, Mzf1, and Cdx) and unique (such as Sox5, Lmo2, and GATA) transcription factors. Our findings show the diversity of the transcriptional regulation of the PDGFRA/Pdgfra gene.

DBZ regulates cortical cell positioning and neurite development by sustaining the anterograde transport of Lis1 and DISC1 through control of Ndel1 dual-phosphorylation.

Cell positioning and neuronal network formation are crucial for proper brain function. Disrupted-in-Schizophrenia 1 (DISC1) is anterogradely transported to the neurite tips, together with Lis1, and functions in neurite extension via suppression of GSK3ß activity. Then, transported Lis1 is retrogradely transported and functions in cell migration. Here, we show that DISC1-binding zinc finger protein (DBZ), together with DISC1, regulates mouse cortical cell positioning and neurite development in vivo. DBZ hindered Ndel1 phosphorylation at threonine 219 and serine 251. DBZ depletion or expression of a double-phosphorylated mimetic form of Ndel1 impaired the transport of Lis1 and DISC1 to the neurite tips and hampered microtubule elongation. Moreover, application of DISC1 or a GSK3ß inhibitor rescued the impairments caused by DBZ insufficiency or double-phosphorylated Ndel1 expression. We concluded that DBZ controls cell positioning and neurite development by interfering with Ndel1 from disproportionate phosphorylation, which is critical for appropriate anterograde transport of the DISC1-complex.

Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke.

BACKGROUND: Microglia are the resident macrophage population of the central nervous system (CNS) and play essential roles, particularly in inflammation-mediated pathological conditions such as ischemic stroke. Increasing evidence shows that the population of vascular cells located around the blood vessels, rather than circulating cells, harbor stem cells and that these resident vascular stem cells (VSCs) are the likely source of some microglia. However, the precise traits and origins of these cells under pathological CNS conditions remain unclear. METHODS: In this study, we used a mouse model of cerebral infarction to investigate whether reactive pericytes (PCs) acquire microglia-producing VSC activity following ischemia. RESULTS: We demonstrated the localization of ionized calcium-binding adaptor molecule 1 (Iba1)-expressing microglia to perivascular regions within ischemic areas. These cells expressed platelet-derived growth factor receptor-ß (PDGFRß), a hallmark of vascular PCs. PDGFRß(+) PCs isolated from ischemic, but not non-ischemic, areas expressed stem/undifferentiated cell markers and subsequently differentiated into various cell types, including microglia-like cells with phagocytic capacity. CONCLUSIONS: The study results suggest that vascular PCs acquire multipotent VSC activity under pathological conditions and may thus be a novel source of microglia.

RhoA/ROCK pathway mediates p38 MAPK activation and morphological changes downstream of P2Y12/13 receptors in spinal microglia in neuropathic pain.

Recent studies have indicated an important role of ATP receptors in spinal microglia, such as P2Y12 or P2Y13, in the development of chronic pain. However, intracellular signaling cascade of these receptors have not been clearly elucidated. We found that intrathecal injection of 2-(methylthio)adenosine 5'-diphosphate (2Me-SADP) induced mechanical hypersensitivity and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the spinal cord. Intrathecal administration of P2Y12/P2Y13 antagonists and Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor H1152 suppressed not only p38 MAPK phosphorylation, but also mechanical hypersensitivity induced by 2Me-SADP. In the rat peripheral nerve injury model, intrathecal administration of antagonists for the P2Y12/P2Y13 receptor suppressed activation of p38 MAPK in the spinal cord. In addition, subarachnoidal injection of H1152 also attenuated nerve injury-induced spinal p38 MAPK phosphorylation and neuropathic pain behavior, suggesting an essential role of ROCK in nerve injury-induced p38 MAPK activation. We also found that the antagonists of the P2Y12/P2Y13 receptor and H1152 had inhibitory effects on the morphological changes of microglia such as retraction of processes in both 2Me-SADP and nerve injured rats. In contrast these treatments had no effect on the number of Iba1-positive cells in the nerve injury model. Collectively, our results have demonstrated roles of ROCK in the spinal microglia that is involved in p38 MAPK activation and the morphological changes. Inhibition of ROCK signaling may offer a novel target for the development of a neuropathic pain treatment.

Peripherally increased artemin is a key regulator of TRPA1/V1 expression in primary afferent neurons.

BACKGROUND: Artemin, a member of the glial cell line-derived neurotrophic factor family, is known to have a variety of neuronal functions, and has been the subject of attention because it has interesting effects, including bi-directional results in modulation in neuropathic and inflammatory pain. It has been shown that the overexpression of artemin is associated with an increase in the expression of TRP family channels in primary afferents and subsequent hyperalgesia, and an increase in neuronal activity. The purpose of this study was to examine the peripheral synthesis of artemin in inflammatory and neuropathic pain models, and to demonstrate the effects of long-term or repeated application of artemin in vivo on pain behaviors and on the expression of TRP family channels. Further, the regulatory mechanisms of artemin on TRPV1/A1 were examined using cultured DRG neurons. RESULTS: We have demonstrated that artemin is locally elevated in skin over long periods of time, that artemin signals significantly increase in deep layers of the epidermis, and also that it is distributed over a broad area of the dermis. In contrast, NGF showed transient increases after peripheral inflammation. It was confirmed that the co-localization of TRPV1/A1 and GFRα3 was higher than that between TRPV1/A1 and TrkA. In the peripheral sciatic nerve trunk, the synthesis of artemin was found by RT-PCR and in situ hybridization to increase at a site distal to a nerve injury. We demonstrated that in vivo repeated artemin injections into the periphery changed the gene expression of TRPV1/A1 in DRG neurons without affecting GFRα3 expression. Repeated artemin injections also induced mechanical and heat hyperalgesia. Using primary cultured DRG neurons, we found that artemin application significantly increased TRPV1/A1 expression and Ca(2+) influx. Artemin-induced p38 MAPK pathway regulated the TRPV1 channel expression, however TRPA1 upregulation by artemin is not mediated through p38 MAPK. CONCLUSIONS: These data indicate the important roles of peripherally-derived artemin on the regulation of TRPV1/A1 in DRG neurons in pathological conditions such as inflammatory and neuropathic pain.

WAVE2-Abi2 complex controls growth cone activity and regulates the multipolar-bipolar transition as well as the initiation of glia-guided migration.

Glia-guided migration (glia-guided locomotion) during radial migration is a characteristic yet unique mode of migration. In this process, the directionality of migration is predetermined by glial processes and not by growth cones. Prior to the initiation of glia-guided migration, migrating neurons transform from multipolar to bipolar, but the molecular mechanisms underlying this multipolar-bipolar transition and the commencement of glia-guided migration are not fully understood. Here, we demonstrate that the multipolar-bipolar transition is not solely a cell autonomous event; instead, the interaction of growth cones with glial processes plays an essential role. Time-lapse imaging with lattice assays reveals the importance of vigorously active growth cones in searching for appropriate glial scaffolds, completing the transition, and initiating glia-guided migration. These growth cone activities are regulated by Abl kinase and Cdk5 via WAVE2-Abi2 through the phosphorylation of tyrosine 150 and serine 137 of WAVE2. Neurons that do not display such growth cone activities are mispositioned in a more superficial location in the neocortex, suggesting the significance of growth cones for the final location of the neurons. This process occurs in spite of the "inside-out" principle in which later-born neurons are situated more superficially.

Phosphorylation of ezrin/radixin/moesin (ERM) protein in spinal microglia following peripheral nerve injury and lysophosphatidic acid administration.

Peripheral nerve injury activates spinal glial cells, which may contribute to the development of pain behavioral hypersensitivity. There is growing evidence that activated microglia show dynamic changes in cell morphology; however, the molecular mechanisms that underlie the modification of the membrane and cytoskeleton of microglia are not known. Here, we investigated the phosphorylation of ezrin, radixin, and moesin (ERM) proteins in the spinal cord after peripheral nerve injury. ERM is known to function as membrane-cytoskeletal linkers and be localized at filopodia- and microvilli-like structures. ERM proteins must be phosphorylated at a specific C-terminal threonine residue to be in the active state. The nature of ERM proteins in the spinal cord of animals in a neuropathic pain model has not been investigated and characterized. In the present study, we observed an increase in the phosphorylated ERM in the spinal microglia following spared nerve injury. The intrathecal administration of lysophosphatidic acid induced the phosphorylation of ERM proteins in microglia along with the development of mechanical pain hypersensitivity. Intrathecal administration of ERM antisense locked nucleic acid suppressed nerve injury-induced tactile allodynia and decreased the phosphorylation of ERM, but not the Iba1 staining pattern, in spinal glial cells. These findings suggest that lysophosphatidic acid induced the phosphorylation of ERM proteins in spinal microglia and may be involved in the emergence of neuropathic pain. These findings may underlie the pathological mechanisms of nerve injury-induced neuropathic pain.

A Bone Morphogenetic Protein Signaling Inhibitor, LDN193189, Converts Ischemia-Induced Multipotent Stem Cells into Neural Stem/Progenitor Cell-Like Cells.

Stem cell therapy is used to restore neurological function in stroke patients. We have previously reported that ischemia-induced multipotent stem cells (iSCs), which are likely derived from brain pericytes, develop in poststroke human and mouse brains. Although we have demonstrated that iSCs can differentiate into neural lineage cells, the factors responsible for inducing this differentiation remain unclear. In this study, we found that LDN193189, a bone morphogenetic protein (BMP) inhibitor, caused irreversible changes in the shape of iSCs. In addition, compared with iSCs incubated without LDN193189, the iSCs incubated with LDN193189 (LDN-iSCs) showed upregulated expression of neural lineage-related genes and proteins, including those expressed in neural stem/progenitor cells (NSPCs), and downregulated expression of mesenchymal and pericytic-related genes and proteins. Moreover, microarray analysis revealed that LDN-iSCs and NSPCs had similar gene expression profiles. Furthermore, LDN-iSCs differentiated into electrophysiologically functional neurons. These results indicate that LDN193189 induces NSPC-like cells from iSCs, suggesting that bioactive molecules regulating BMP signaling are potential targets for promoting neurogenesis from iSCs in the pathological brain, such as during ischemic stroke. We believe that our findings will bring us one step closer to the clinical application of iSCs.

Morphology of Schwann Cell Processes Supports Renal Sympathetic Nerve Terminals With Local Distribution of Adrenoceptors.

Nerves in the renal parenchyma comprise sympathetic nerves that act on renal arteries and tubules to decrease blood flow and increase primary urine reabsorption, respectively. Synaptic vesicles release neurotransmitters that activate their effector tissues. However, the mechanisms by which neurotransmitters exert individual responses to renal effector cells remain unknown. Here, we investigated the spatial and molecular compositional associations of renal Schwann cells (SC) supporting the nerve terminals in male rats. The nerve terminals of vascular smooth muscle cells (SMCs) enclosed by renal SC processes were exposed through windows facing the effectors with presynaptic specializations. We found that the adrenergic receptors (ARs) α2A, α2C, and ß2 were localized in the SMC and the basal side of the tubules, where the nerve terminals were attached, whereas the other subtypes of ARs were distributed in the glomerular and luminal side, where the norepinephrine released from nerve endings may have indirect access to ARs. In addition, integrins α4 and ß1 were coexpressed in the nerve terminals. Thus, renal nerve terminals could contact their effectors via integrins and may have a structure, covered by SC processes, suitable for intensive and directional release of neurotransmitters into the blood, rather than specialized structures in the postsynaptic region.

Phldb2 is essential for regulating hippocampal dendritic spine morphology through drebrin in an adult-type isoform-specific manner.

Morphologically dynamic dendritic spines are the major sites of neuronal plasticity in the brain; however, the molecular mechanisms underlying their morphological dynamics have not been fully elucidated. Phldb2 is a protein that contains two predicted coiled-coil domains and the pleckstrin homology domain, whose binding is highly sensitive to PIP3. We have previously demonstrated that Phldb2 regulates synaptic plasticity, glutamate receptor trafficking, and PSD-95 turnover. Drebrin is one of the most abundant neuron-specific F-actin-binding proteins that are pivotal for synaptic morphology and plasticity. We observed that Phldb2 bound to drebrin A (adult-type drebrin), but not to drebrin E (embryonic-type drebrin). In the absence of Phldb2, the subcellular localization of drebrin A in the hippocampal spines and its distribution in the hippocampus were altered. Immature spines, such as the filopodium type, increased relatively in the CA1 regions of the hippocampus, whereas mushroom spines, a typical mature type, decreased in Phldb2-/- mice. Phldb2 suppressed the formation of an abnormal filopodium structure induced by drebrin A overexpression. Taken together, these findings demonstrate that Phldb2 is pivotal for dendritic spine morphology and possibly for synaptic plasticity in mature animals by regulating drebrin A localization.

Acute stress induces severe neural inflammation and overactivation of glucocorticoid signaling in interleukin-18-deficient mice.

Interleukin-18 (IL18) is an inflammatory cytokine that is related to psychiatric disorders such as depression and cognitive impairment. We previously found that IL18 deficiency may cause hippocampal impairment, resulting in depression-like behavioral changes. However, the potential role of IL18 in stressful conditions remains uncertain. In the present study, we examined the effect of IL18 on neural inflammation and stress tolerance during acute stress. Littermate Il18+/+ and Il18-/- mice were exposed to a single restraint stress for 6 h, and all assessments were performed 18 h after the mice were released from the restraint. In Il18-/- mice exposed to acute stress, the immobility times in both the forced swim test and tail suspension test were decreased, although no difference was observed in Il18+/+ mice. Il1ß, Il6, and Tnfα expression levels in the hippocampus of stressed Il18-/- mice were significantly higher than those in the other groups. Moreover, the numbers of astrocytes and microglia, including those in the active form, were also increased compared with those in other groups. Regarding the molecular mechanism, the HSF5 and TTR genes were specifically expressed in stressed Il18-/- mice. As a potential treatment, intracerebral administration of IL18 to Il18-/- mice resulted in partial recovery of changes in behavioral assessments. Our results revealed that IL18-deficient mice were more sensitive and had a longer response to acute stress than that in normal mice. In addition, neural inflammation and augmentation of glucocorticoid signals caused by stress were more intense and remained longer in Il18-/- mice, resulting in behavioral changes. In conclusion, IL18 might be an indispensable factor that modulates the stress response and maintains balance between neural inflammation and glucocorticoid signaling.

LL5beta directs the translocation of filamin A and SHIP2 to sites of phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) accumulation, and PtdIns(3,4,5)P3 localization is mutually modified by co-recruited SHIP2.

Phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)) accumulates at the leading edge of migrating cells and works, at least partially, as both a compass to indicate directionality and a hub for subsequent intracellular events. However, how PtdIns(3,4,5)P(3) regulates the migratory machinery has not been fully elucidated. Here, we demonstrate a novel mechanism for efficient lamellipodium formation that depends on PtdIns(3,4,5)P(3) and the reciprocal regulation of PtdIns(3,4,5)P(3) itself. LL5beta, whose subcellular localization is directed by membrane PtdIns(3,4,5)P(3), recruits the actin-cross-linking protein Filamin A to the plasma membrane, where PtdIns(3,4,5)P(3) accumulates, with the Filamin A-binding Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2). A large and dynamic lamellipodium was formed in the presence of Filamin A and LL5beta by the application of epidermal growth factor. Conversely, depletion of either Filamin A or LL5beta or the overexpression of either an F-actin-cross-linking mutant of Filamin A or a mutant of LL5beta without its PtdIns(3,4,5)P(3)-interacting region inhibited such events in COS-7 cells. Because F-actin initially polymerizes near the plasma membrane, it is likely that membrane-recruited Filamin A efficiently cross-links newly polymerized F-actin, leading to enhanced lamellipodium formation at the site of PtdIns(3,4,5)P(3) accumulation. Moreover, we demonstrate that co-recruited SHIP2 dephosphorylates PtdIns(3,4,5)P(3) at the same location.

Histone deacetylase SIRT1 modulates neuronal differentiation by its nuclear translocation.

Neural precursor cells (NPCs) differentiate into neurons, astrocytes, and oligodendrocytes in response to intrinsic and extrinsic changes. Notch signals maintain undifferentiated NPCs, but the mechanisms underlying the neuronal differentiation are largely unknown. We show that SIRT1, an NAD(+)-dependent histone deacetylase, modulates neuronal differentiation. SIRT1 was found in the cytoplasm of embryonic and adult NPCs and was transiently localized in the nucleus in response to differentiation stimulus. SIRT1 started to translocate into the nucleus within 10 min after the transfer of NPCs into differentiation conditions, stayed in the nucleus, and then gradually retranslocated to the cytoplasm after several hours. The number of neurospheres that generated Tuj1(+) neurons was significantly decreased by pharmacological inhibitors of SIRT1, dominant-negative SIRT1 and SIRT1-siRNA, whereas overexpression of SIRT1, but not that of cytoplasm-localized mutant SIRT1, enhanced neuronal differentiation and decreased Hes1 expression. Expression of SIRT1-siRNA impaired neuronal differentiation and migration of NPCs into the cortical plate in the embryonic brain. Nuclear receptor corepressor (N-CoR), which has been reported to bind SIRT1, promoted neuronal differentiation and synergistically increased the number of Tuj1(+) neurons with SIRT1, and both bound the Hes1 promoter region in differentiating NPCs. Hes1 transactivation by Notch1 was inhibited by SIRT1 and/or N-CoR. Our study indicated that SIRT1 is a player of repressing Notch1-Hes1 signaling pathway, and its transient translocation into the nucleus may have a role in the differentiation of NPCs.

Exploring Molecular Mechanisms Involved in the Development of the Depression-Like Phenotype in Interleukin-18-Deficient Mice.

Interleukin-18 (IL-18) is an inflammatory cytokine that has been linked to energy homeostasis and psychiatric symptoms such as depression and cognitive impairment. We previously revealed that deficiency in IL-18 led to hippocampal abnormalities and resulted in depression-like symptoms. However, the impact of IL-18 deficiency on other brain regions remains to be clarified. In this study, we first sought to confirm that IL-18 expression in neural cells can be found in human brain tissue. Subsequently, we examined the expression of genes in the prefrontal cortex of Il18 -/- mice and compared it with gene expression in mice subjected to a chronic mild stress model of depression. Extracted genes were further analyzed using Ingenuity® Pathway Analysis, in which 18 genes common to both the chronic mild stressed model and Il18 -/- mice were identified. Of those, 16 were significantly differentially expressed between Il18+/+ and Il18 -/- mice. We additionally measured protein expression of α-2-HS-glycoprotein (AHSG) and transthyretin (TTR) in serum and the brain. In the prefrontal cortex of Il18 -/- mice, TTR but not AHSG was significantly decreased. Conversely, in the serum of Il18 -/- mice, AHSG was significantly increased but not TTR. Therefore, our results suggest that in IL-18-deficit conditions, TTR in the brain is one of the mediators causally related to depression, and AHSG in peripheral organs is one of the regulators inducing energy imbalance. Moreover, this study suggests a possible "signpost" to clarify the molecular mechanisms commonly underlying the immune system, energy metabolism, neural function, and depressive disorders.

PIP3-Phldb2 is crucial for LTP regulating synaptic NMDA and AMPA receptor density and PSD95 turnover.

The essential involvement of phosphoinositides in synaptic plasticity is well-established, but incomplete knowledge of the downstream molecular entities prevents us from understanding their signalling cascades completely. Here, we determined that Phldb2, of which pleckstrin-homology domain is highly sensitive to PIP3, functions as a phosphoinositide-signalling mediator for synaptic plasticity. BDNF application caused Phldb2 recruitment toward postsynaptic membrane in dendritic spines, whereas PI3K inhibition resulted in its reduced accumulation. Phldb2 bound to postsynaptic scaffolding molecule PSD-95 and was crucial for localization and turnover of PSD-95 in the spine. Phldb2 also bound to GluA1 and GluA2. Phldb2 was indispensable for the interaction between NMDA receptors and CaMKII, and the synaptic density of AMPA receptors. Therefore, PIP3-responsive Phldb2 is pivotal for induction and maintenance of LTP. Memory formation was impaired in our Phldb2-/- mice.

Interleukin-18-deficient mice develop hippocampal abnormalities related to possible depressive-like behaviors.

Interleukin-18 (IL-18) is an inflammatory cytokine linked to major depressive disorder (MDD). MDD is closely related to metabolic disorders, such as diabetes mellitus (DM) and obesity. Moreover, DM is associated with cognitive impairment and promotes apoptosis of hippocampal cells by activating pro-apoptotic and inhibiting anti-apoptotic factors. IL-18-deficient (Il18-/-) mice are obese and have DM. Therefore, we hypothesized a close relationship between IL-18 and death of hippocampal cells, affecting neurogenesis related to behavioral changes such as MDD. Il18-/- male mice were generated on the C57Bl/6 background and Il18+/+ mice were used as controls. Behavioral, histopathological, and molecular responses, as well as responses to intracerebral recombinant IL-18 administration, were examined. Compared with Il18+/+ mice, Il18-/- mice had impaired learning and memory and exhibited lower motivation. In the Il18-/- mice, degenerated mitochondria were detected in synaptic terminals in the molecular layer, the polymorphic layer, and in mossy fibers in the dentate gyrus, suggesting mitochondrial abnormalities. Because of the degeneration of mitochondria in the dentate gyrus, in which pro-apoptotic molecules were upregulated and anti-apoptotic factors were decreased, apoptosis inducers were not cleaved, indicating inhibition of apoptosis. In addition, neurogenesis in the dentate gyrus and the maturity of neuronal cells were decreased in the Il18-/- mice, while intracerebral administration of recombinant IL-18 promoted significant recovery of neurogenesis. Our findings suggested that IL-18 was indispensable for mitochondrial homeostasis, sustaining clearance of degenerative neural cells, and supporting neurogenesis, normal neuronal maturation and hippocampal function.

Molecular characterization of the ankle-link complex in cochlear hair cells and its role in the hair bundle functioning.

Several lines of evidence indicate that very large G-protein-coupled receptor 1 (Vlgr1) makes up the ankle links that connect the stereocilia of hair cells at their base. Here, we show that the transmembrane protein usherin, the putative transmembrane protein vezatin, and the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing submembrane protein whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells and are absent in Vlgr1-/- mice that lack the ankle links. Direct in vitro interactions between these four proteins further support their involvement in a molecular complex associated with the ankle links and scaffolded by whirlin. In addition, the delocalization of these proteins in myosin VIIa defective mutant mice as well as the myosin VIIa tail direct interactions with vezatin, whirlin, and, we show, Vlgr1 and usherin, suggest that myosin VIIa conveys proteins of the ankle-link complex to the stereocilia. Adenylyl cyclase 6, which was found at the base of stereocilia, was both overexpressed and mislocated in Vlgr1-/- mice. In postnatal day 7 Vlgr1-/- mice, mechanoelectrical transduction currents evoked by displacements of the hair bundle toward the tallest stereocilia (i.e., in the excitatory direction) were reduced in outer but not inner hair cells. In both cell types, stimulation of the hair bundle in the opposite direction paradoxically resulted in significant transduction currents. The absence of ankle-link-mediated cohesive forces within hair bundles lacking Vlgr1 may account for the electrophysiological results. However, because some long cadherin-23 isoforms could no longer be detected in Vlgr1-/- mice shortly after birth, the loss of some apical links could be involved too. The premature disappearance of these cadherin isoforms in the Vlgr1-/- mutant argues in favor of a signaling function of the ankle links in hair bundle differentiation.