Adult neurogenesis modulates the hippocampus-dependent period of associative fear memory.

Acquired memory initially depends on the hippocampus (HPC) for the process of cortical permanent memory formation. The mechanisms through which memory becomes progressively independent from the HPC remain unknown. In the HPC, adult neurogenesis has been described in many mammalian species, even at old ages. Using two mouse models in which hippocampal neurogenesis is physically or genetically suppressed, we show that decreased neurogenesis is accompanied by a prolonged HPC-dependent period of associative fear memory. Inversely, enhanced neurogenesis by voluntary exercise sped up the decay rate of HPC dependency of memory, without loss of memory. Consistently, decreased neurogenesis facilitated the long-lasting maintenance of rat hippocampal long-term potentiation in vivo. These independent lines of evidence strongly suggest that the level of hippocampal neurogenesis play a role in determination of the HPC-dependent period of memory in adult rodents. These observations provide a framework for understanding the mechanisms of the hippocampal-cortical complementary learning systems.

Usefulness of gallium-67 scintigraphy for evaluating the histopathological activity in interstitial nephritis.

BACKGROUND: Interstitial nephritis is a common cause of renal failure. Gallium-67 scintigraphy is reportedly useful for diagnosing interstitial nephritis; however, its ability to assess disease activity remains unknown. We aimed to analyze the relationship between the renal uptake of gallium-67 and the disease activity in interstitial nephritis. METHODS: We retrospectively analyzed the data of patients who underwent gallium-67 scintigraphy at a hospital in Tokyo. The renal uptake adjusted for the soft tissues beneath the kidneys was semi-quantitatively evaluated. We compared the renal uptake levels between patients clinically diagnosed with and without interstitial nephritis. Among those undergoing renal biopsy, we evaluated the predictive ability of gallium-67 scintigraphy and analyzed the renal uptake levels regarding the disease activity through a histopathological analysis. RESULTS: We included 143 patients; among them, 30, 17, and 96 patients were clinically diagnosed with interstitial nephritis, other kidney diseases, and non-kidney diseases, respectively. The renal uptake of gallium-67 was the highest among patients with interstitial nephritis. Among the 25 patients who underwent renal biopsy, 15 were pathologically diagnosed with interstitial nephritis. The renal uptake levels showed a high discriminative ability (C-statistic: 0.83). Furthermore, net reclassification improvement with the addition of gallium-67 scintigraphy to N-acetyl-ß-D-glucosaminidase for the prediction of interstitial nephritis was 1.14. Histopathological analysis revealed a positive correlation between renal uptake and inflammation in the cortex and peritubular capillaries. CONCLUSIONS: This study confirmed the diagnostic value and potential usefulness of gallium-67 scintigraphy for evaluating interstitial nephritis.

Generation and analysis of novel Reln-deleted mouse model corresponding to exonic Reln deletion in schizophrenia.

AIM: A Japanese individual with schizophrenia harboring a novel exonic deletion in RELN was recently identified by genome-wide copy-number variation analysis. Thus, the present study aimed to generate and analyze a model mouse to clarify whether Reln deficiency is associated with the pathogenesis of schizophrenia. METHODS: A mouse line with a novel RELN exonic deletion (Reln-del) was established using the CRISPR/Cas9 method to elucidate the underlying molecular mechanism. Subsequently, general behavioral tests and histopathological examinations of the model mice were conducted and phenotypic analysis of the cerebellar granule cell migration was performed. RESULTS: The phenotype of homozygous Reln-del mice was similar to that of reeler mice with cerebellar atrophy, dysplasia of the cerebral layers, and abrogated protein levels of cerebral reelin. The expression of reelin in heterozygous Reln-del mice was approximately half of that in wild-type mice. Conversely, behavioral analyses in heterozygous Reln-del mice without cerebellar atrophy or dysplasia showed abnormal social novelty in the three-chamber social interaction test. In vitro reaggregation formation and neuronal migration were severely altered in the cerebellar cultures of homozygous Reln-del mice. CONCLUSION: The present results in novel Reln-del mice modeled after our patient with a novel exonic deletion in RELN are expected to contribute to the development of reelin-based therapies for schizophrenia.

Low-dose vitamin K2 (MK-4) supplementation for 12 months improves bone metabolism and prevents forearm bone loss in postmenopausal Japanese women.

Menaquinone-4 (MK-4) administered at a pharmacological dosage of 45 mg/day has been used for the treatment of osteoporosis in Japan. However, it is not known whether a lower dose of MK-4 supplementation is beneficial for bone health in healthy postmenopausal women. The aim of this study was to examine the long-term effects of 1.5-mg daily supplementation of MK-4 on the various markers of bone turnover and bone mineral density (BMD). The study was performed as a randomized, double-blind, placebo-controlled trial. The participants (aged 50-65 years) were randomly assigned to one of two groups according to the MK-4 dose received: the placebo-control group (n = 24) and the 1.5-mg MK-4 group (n = 24). The baseline concentrations of undercarboxylated osteocalcin (ucOC) were high in both groups (>5.1 ng/ml). After 6 and 12 months, the serum ucOC concentrations were significantly lower in the MK-4 group than in the control group. In the control group, there was no significant change in serum pentosidine concentrations. However, in the MK-4 group, the concentration of pentosidine at 6 and 12 months was significantly lower than that at baseline. The forearm BMD was significantly lower after 12 months than at 6 months in the control group. However, there was no significant decrease in BMD in the MK-4 group during the study period. These results suggest that low-dose MK-4 supplementation for 6-12 months improved bone quality in the postmenopausal Japanese women by decreasing the serum ucOC and pentosidine concentrations, without any substantial adverse effects.

Role of nuclear localization of PSMB1 in transcriptional activation.

The production of retinol binding protein 4 (RBP4) is higher in adipose tissue in type 2 diabetes. We have found that proteasome subunit beta type 1 (PSMB1) is a transcriptional activator of Rbp4. In the present study, the putative tyrosine phosphorylation site in PSMB1 was mutated to phenylalanine. The mutated form of PSMB1 displayed increased nuclear translocation, resulting in activation of transcription in adipocytes.

Case report: A case of fetal umbilical vein varix presenting disseminated intravascular coagulation, polycythemia, and neonatal hepatitis in an extremely low birth weight infant.

Reports on the clinical course of fetal umbilical vein varix in premature infants are limited. We report a case of an extremely low body weight infant with intra-abdominal umbilical vein varix who developed disseminated intravascular coagulation, polycythemia, and hyperbilirubinemia after birth; late-onset neonatal hepatitis; and fetal thrombotic vasculopathy confirmed by placental histopathology. Ultrasonography after birth showed a dilated portion of the umbilical vein at the hepatic hilum with thrombi inside. We speculate that the umbilical vein varix caused the fetal thrombotic vasculopathy, and the presence of umbilical vein varix and fetal thrombotic vasculopathy in combination with prematurity caused coagulopathy, polycythemia, hyperbilirubinemia, and hepatitis. Despite the favorable outcomes reported in the literature, premature infants with umbilical vein varix may require careful observation and management for coagulopathy and late-onset hepatitis. Furthermore, placental histopathology could aid in the understanding of various clinical outcomes in infants with umbilical vein varices.

Vitamin D receptor is not essential for extracellular signal-related kinase phosphorylation by vitamin D(3) in human Caco-2/TC7 cells.

Vitamin D(3) initiated rapid extracellular signal-related kinase (ERK) phosphorylation, but the contribution of vitamin D receptor (VDR) to this event is unclear. We investigated the use of RNA interference (RNAi) to knockdown VDR. RNAi downregulated VDR as well as its targeted gene expression, but vitamin D(3) dependent ERK phosphorylation remained. Thus VDR might not be involved in ERK phosphorylation by vitamin D(3).

Identification of glucose transporter 4 knockdown-dependent transcriptional activation element on the retinol binding protein 4 gene promoter and requirement of the 20 S proteasome subunit for transcriptional activity.

Retinol binding protein 4 (RBP4) is the transport protein that carries retinol in blood. RBP4 was described recently as a new adipokine that reduced insulin sensitivity. Mice lacking glucose transporter 4 (GLUT4) in adipocytes have enhanced Rbp4 gene expression; however, the molecular mechanism is unknown. We found a G4KA (GLUT4 knockdown-dependent transcriptional activation) element located approximately 1.3 kb upstream of the Rbp4 promoter. Mutations within the G4KA sequence significantly reduced expression of the Rbp4 promoter-reporter construct in G4KD-L1 (GLUT4 knockdown 3T3-L1) adipocyte cells. In a yeast one-hybrid screen of a G4KD-L1 cell cDNA library, using the G4KA element as bait, we identified subunits of the 20 S proteasome, PSMB1 and PSMA4, as binding partners. In chromatin immunoprecipitation assays, both subunits bound to the G4KA element; however, only PSMB1 was tightly bound in the GLUT4 knockdown model. PSMB1 RNA interference, but not PSMA4, significantly inhibited Rbp4 transcription. Nuclear transportation of PSMB1 was increased in G4KD-L1 cells. These results provide evidence for an exclusive proteasome subunit-related mechanism for transcriptional activation of RBP4 within a GLUT4 knockdown model.

Anti-myelin oligodendrocyte glycoprotein antibody-positive acute disseminated encephalomyelitis mimicking limbic encephalitis: A case report.

Anti-myelin oligodendrocyte glycoprotein antibodies (MOG-ab) have been detected in various disorders of the central nervous system including acute disseminated encephalomyelitis (ADEM), neuromyelitis optica spectrum disorders (NMOSD), optic neuritis, myelitis, and cortical encephalitis. We report an atypical case of MOG-ab-associated encephalomyelitis with part of the clinical manifestations resembling limbic encephalitis. Multifocal, hyperintense, bilateral lesions predominantly affecting the white matter on brain magnetic resonance imaging and marked response to steroid therapy were compatible with a MOG-ab-associated disease. This case illustrates that MOG-ab-associated disease should be considered in encephalomyelitis involving the bilateral limbic system.

ARHGAP10, which encodes Rho GTPase-activating protein 10, is a novel gene for schizophrenia risk.

Schizophrenia (SCZ) is known to be a heritable disorder; however, its multifactorial nature has significantly hampered attempts to establish its pathogenesis. Therefore, in this study, we performed genome-wide copy-number variation (CNV) analysis of 2940 patients with SCZ and 2402 control subjects and identified a statistically significant association between SCZ and exonic CNVs in the ARHGAP10 gene. ARHGAP10 encodes a member of the RhoGAP superfamily of proteins that is involved in small GTPase signaling. This signaling pathway is one of the SCZ-associated pathways and may contribute to neural development and function. However, the ARHGAP10 gene is often confused with ARHGAP21, thus, the significance of ARHGAP10 in the molecular pathology of SCZ, including the expression profile of the ARHGAP10 protein, remains poorly understood. To address this issue, we focused on one patient identified to have both an exonic deletion and a missense variant (p.S490P) in ARHGAP10. The missense variant was found to be located in the RhoGAP domain and was determined to be relevant to the association between ARHGAP10 and the active form of RhoA. We evaluated ARHGAP10 protein expression in the brains of reporter mice and generated a mouse model to mimic the patient case. The model exhibited abnormal emotional behaviors, along with reduced spine density in the medial prefrontal cortex (mPFC). In addition, primary cultured neurons prepared from the mouse model brain exhibited immature neurites in vitro. Furthermore, we established induced pluripotent stem cells (iPSCs) from this patient, and differentiated them into tyrosine hydroxylase (TH)-positive neurons in order to analyze their morphological phenotypes. TH-positive neurons differentiated from the patient-derived iPSCs exhibited severe defects in both neurite length and branch number; these defects were restored by the addition of the Rho-kinase inhibitor, Y-27632. Collectively, our findings suggest that rare ARHGAP10 variants may be genetically and biologically associated with SCZ and indicate that Rho signaling represents a promising drug discovery target for SCZ treatment.

Identification of a minimal segment of complexin II essential for preferential distribution in axons.

Complexin II (CPLX2) is a soluble pre-synaptic protein believed to regulate neurotransmitter release from pre-synaptic terminals. CPLX2 is localized in pre-synaptic terminals in mature brain, but the mechanism of selective localization remains unclear. Here we identified an essential segment of CPLX2 for preferential axonal distribution. Myc-tagged CPLX2 was expressed in cultured rat hippocampal neurons and its distribution between axons and dendrites was compared by immunocytochemistry and image analysis. Fluorescence signals were detected in both axons and dendrites; however, their respective distribution varied significantly. Despite the fact that signal intensity decreased almost linearly from the base to the tip of the dendrite, a substantial level was sustained along the axon, even at a position near the tip. Image analyses using a series of mutants indicated that the deletion of 19 amino acid residues, G71-P89, within the 'central core' for binding to soluble N-ethylmaleimide sensitive factor attachment protein receptor proteins resulted in the loss of preferential axonal distribution. The enhanced green fluorescent protein derivative fused with the G71-P89 fragment exhibited a similar localization to that of wild type CPLX2, indicating that the G71-P89 region of CPLX2 is essential and sufficient for preferential axonal distribution.

Synaptopodin maintains the neural activity-dependent enlargement of dendritic spines in hippocampal neurons.

Synaptopodin (SYNPO) is an F-actin interacting protein expressed in dendritic spines and upregulated during the late-phase of long-term potentiation. Here, we investigated whether SYNPO regulates spine morphology through interactions with F-actin, the major cytoskeletal element of spines. In primary hippocampal neuron cultures, both endogenous and exogenous SYNPO localized preferentially in large spines under basal conditions. SYNPO overexpression did not affect the number or volume of spines in unstimulated neurons. Pharmacological activation of synaptic NMDA receptors transiently increased spine volume in control neurons, while the increase was persistent in neurons overexpressing SYNPO. In addition, exogenous SYNPO in PtK2 cells suppressed staurosporine-dependent disruption of F-actin stress fibers, suggesting that SYNPO protected F-actin from disruption. These results suggest that SYNPO stabilized activity-dependent increases in spine volume and imply that late-phase changes in spine morphology involve SYNPO.

Development- and activity-dependent regulation of SNAP-25 phosphorylation in rat brain.

Synaptosomal-associated protein of 25kDa (SNAP-25), a member of the SNARE proteins essential for neurotransmitter release, is phosphorylated at Ser(187) in PC12 cells and in the rat brain in a protein kinase C-dependent manner. It remains unclear how the phosphorylation of SNAP-25 is regulated during development and by neuronal activity. We studied the mode of SNAP-25 phosphorylation at Ser(187) in the rat brain using an anti-phosphorylated SNAP-25 antibody. Both the expression and phosphorylation of SNAP-25 increased remarkably during the early postnatal period, but their onsets were quite different. SNAP-25 expression was detected as early as embryonic Day 18, whereas the phosphorylation of SNAP-25 could not be detected until postnatal Day 4. A delay in the onset of phosphorylation was also observed in cultured rat hippocampal neurons. The phosphorylation of SNAP-25 was regulated in a neuronal activity-dependent manner and, in the rat hippocampus, decreased by introducing seizures with kainic acid. These results clearly indicated that the phosphorylation of SNAP-25 at Ser(187) is regulated in development- and neuronal activity-dependent manners, and is likely to play important roles in higher brain functions.

Mutation screening of GRIN2B in schizophrenia and autism spectrum disorder in a Japanese population.

N-methyl-d-aspartate receptors (NMDARs) play a critical role in excitatory synaptic transmission and plasticity in the central nervous systems. Recent genetics studies in schizophrenia (SCZ) show that SCZ is susceptible to NMDARs and the NMDAR signaling complex. In autism spectrum disorder (ASD), several studies report dysregulation of NMDARs as a risk factor for ASD. To further examine the association between NMDARs and SCZ/ASD development, we conducted a mutation screening study of GRIN2B which encodes NR2B subunit of NMDARs, to identify rare mutations that potentially cause diseases, in SCZ and ASD patients (n = 574 and 152, respectively). This was followed by an association study in a large sample set of SCZ, ASD, and normal healthy controls (n = 4145, 381, and 4432, respectively). We identified five rare missense mutations through the mutation screening of GRIN2B. Although no statistically significant association between any single mutation and SCZ or ASD was found, one of its variant, K1292R, is found only in the patient group. To further examine the association between mutations in GRIN2B and SCZ/ASD development, a larger sample size and functional experiments are needed.

Generation of reelin-positive marginal zone cells from the caudomedial wall of telencephalic vesicles.

An early and fundamental step of the laminar organization of developing neocortex is controlled by the developmental programs that critically depend on the activities of reelin-positive cells in the marginal zone. However, the ontogeny of reelin-positive cells remained elusive. To gain insights into the spatial and temporal regulation of reelin-positive marginal zone cell development, we used a transgenic mouse line in which we defined the green fluorescent protein (GFP) transgene as a novel reliable molecular marker of reelin-positive marginal zone cells from the early stages of their development. We further used exo utero electroporation-mediated gene transfer that allows us to mark progenitor cells and monitor the descendants in the telencephalon in vivo. We show here the generation of reelin-positive marginal zone cells from the caudomedial wall of telencephalic vesicles, including the cortical hem, where the prominent expression of GFP is initially detected. These neurons tangentially migrate at the cortical marginal zone and are distributed throughout the entire neocortex in a caudomedial-high to rostrolateral-low gradient during the dynamic developmental period of corticogenesis. Therefore, our findings on reelin-positive marginal zone cells, in addition to the cortical interneurons, add to the emerging view that the neocortex consists of neuronal subtypes that originate from a focal source extrinsic to the neocortex, migrate tangentially into the neocortex, and thereby underlie neural organization of the neocortex.

Neuron type-selective effects of activin on development of the hippocampus.

Activin is a member of the transforming growth factor-beta superfamily and affects the viability of hippocampal neurons during postnatal neurogenesis. We used primary hippocampal neuron to study the actions of activin on developing neurons. Continuous treatment of hippocampal cultures with activin suppressed the emergence of GAD67(+) neurons, which are a subtype of GABAergic interneurons, and increased the percentage of Prox1(+) neurons, which are dentate granule cells. The effects of activin were abolished by co-treatment with follistatin, which is a direct inhibitor of activin. In contrast, follistatin treatment alone increased the percentage of GAD67(+) neurons and decreased the percentage of Prox1(+) neurons. These results indicate that changes in activin signaling during postnatal neural development alter the composition of the neural circuitry and suggest that alterations in the ratio of excitatory to inhibitory neurons may be responsible for changes in the spontaneous and evoked-reactivity of these neurons to other neural inputs.

Two distinct regulatory mechanisms of neurotransmitter release by phosphatidylinositol 3-kinase.

Recent studies have indicated that various growth factors are involved in synaptic functions; however, the precise mechanisms remain unclear. In order to elucidate the molecular mechanisms of the growth factor-mediated regulation of presynaptic functions, the effects of epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) on neurotransmitter release were studied in rat PC12 cells. Brief treatment with EGF and IGF-1 enhanced Ca2+-dependent dopamine release in a concentration-dependent manner. EGF activated both mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase) pathways, and the EGF-dependent enhancement of DA release was suppressed by a MAPK kinase inhibitor as well as by PI3-kinase inhibitors. In striking contrast, IGF-1 activated the PI3-kinase pathway but not the MAPK pathway, and IGF-1-dependent enhancement was suppressed by a PI3-kinase inhibitor but not by a MAPK kinase inhibitor. The enhanced green fluorescent protein-tagged pleckstrin homology (PH) domain of protein kinase B, which selectively binds to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate, was translocated to the plasma membrane after treatment with either EGF or NGF. By contrast, no significant redistribution was induced by IGF-1. These results indicate that PI3-kinase participates in the enhancement of neurotransmitter release by two distinct mechanisms: EGF and NGF activate PI3-kinase in the plasma membrane, whereas IGF-1 activates PI3-kinase possibly in the intracellular membrane, leading to enhancement of neurotransmitter release in a MAPK-dependent and -independent manner respectively.

Distinct ontogenic and regional expressions of newly identified Cajal-Retzius cell-specific genes during neocorticogenesis.

Cajal-Retzius (CR) cells are early-generated transient neurons and are important in the regulation of cortical neuronal migration and cortical laminar formation. Molecular entities characterizing the CR cell identity, however, remain largely elusive. We purified mouse cortical CR cells expressing GFP to homogeneity by fluorescence-activated cell sorting and examined a genome-wide expression profile of cortical CR cells at embryonic and postnatal periods. We identified 49 genes that exceeded hybridization signals by >10-fold in CR cells compared with non-CR cells at embryonic day 13.5, postnatal day 2, or both. Among these CR cell-specific genes, 25 genes, including the CR cell marker genes such as the reelin and calretinin genes, are selectively and highly expressed in both embryonic and postnatal CR cells. These genes, which encode generic properties of CR cell specificity, are eminently characterized as modulatory composites of voltage-dependent calcium channels and sets of functionally related cellular components involved in cell migration, adhesion, and neurite extension. Five genes are highly expressed in CR cells at the early embryonic period and are rapidly down-regulated thereafter. Furthermore, some of these genes have been shown to mark two distinctly different focal regions corresponding to the CR cell origins. At the late prenatal and postnatal periods, 19 genes are selectively up-regulated in CR cells. These genes include functional molecules implicated in synaptic transmission and modulation. CR cells thus strikingly change their cellular phenotypes during cortical development and play a pivotal role in both corticogenesis and cortical circuit maturation.