Hisatake Kondo

Comprehensive Behavioral Analysis of Calcium/Calmodulin-Dependent Protein Kinase IV Knockout Mice

Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO) mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency.

Epidermal FABP (FABP5) Regulates Keratinocyte Differentiation by 13(S)-HODE-Mediated Activation of the NF-κB Signaling Pathway

Fatty acid-binding proteins (FABPs) are postulated to serve as lipid shuttles that solubilize hydrophobic fatty acids and deliver them to appropriate intracellular sites. Epidermal FABP (E-FABP/FABP5) is predominantly expressed in keratinocytes and is overexpressed in the actively proliferating tissue characteristic of psoriasis and wound healing. In this study, we found decreased expression of the differentiation-specific proteins keratin 1, involucrin, and loricrin in E-FABP(-/-) keratinocytes relative to E-FABP(+/+) keratinocytes. We also determined that incorporation of linoleic acid was significantly reduced in E-FABP(-/-) keratinocytes. Although linoleic acid did not directly affect keratinocyte differentiation, keratin 1 expression was induced by the linoleic acid derivative 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE), and this induction was concomitant with increased NF-κB activity. In E-FABP(-/-) keratinocytes, the expression of 13(S)-HODE and the subsequent induction of NF-κB activity was lower than in wild-type keratinocytes. The reduction of linoleic acid in E-FABP(-/-) keratinocytes led to decreased cellular 13(S)-HODE content, resulting in decreased keratin 1 expression through downregulation of NF-κB activity. The regulation of fatty acid metabolism by E-FABP during keratinocyte differentiation suggests that E-FABP may have a role in the pathogenesis of psoriasis.

Diacylglycerol kinase β accumulates on the perisynaptic site of medium spiny neurons in the striatum

Following activation of Gq protein‐coupled receptors, phospholipase C yields a pair of second messengers, i.e. diacylglycerol (DAG) and inositol 1,4,5‐trisphosphate. The former activates protein kinase C and the latter mobilizes Ca2+ from intracellular store. DAG kinase (DGK) then phosphorylates DAG to produce another second messenger (phosphatidic acid). Of 10 mammalian DGK isozymes, DGKβ is expressed in dopaminergic projection fields with the highest level in the striatum and its particular splice variant is differentially expressed in patients with bipolar disorder. To gain molecular anatomical evidence for its signaling role, we investigated the cellular expression and subcellular localization of DGKβ in the striatum of rat brain. DGKβ was expressed in medium spiny neurons constituting the striatonigral and striatopallidal pathways, whereas striatal interneurons were below the detection threshold. DGKβ was distributed in somatodendritic elements of medium spiny neurons and localized in association with the smooth endoplasmic reticulum and plasma membrane or in the narrow cytoplasmic space between them. In particular, DGKβ exhibited dense accumulation at perisynaptic sites on dendritic spines forming asymmetrical synapses. The characteristic anatomical localization was consistent with exclusive enrichment of DGKβ in the microsomal and postsynaptic density fractions. Intriguingly, DGKβ was very similar in immunohistochemical and immunochemical distribution to Gq‐coupled receptors, such as metabotropic glutamate receptors 1 and 5, and also to other downstream molecules involving DAG metabolism, such as phospholipase C β and DAG lipase. These findings suggest that abundant DGKβ is provided to perisynaptic sites of medium spiny neurons so that it can effectively produce phosphatidic acid upon activation of Gq‐coupled receptors and modulate the cellular state of striatal output neurons.

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP+ astrocytes (21% of FABP7+ cells) and NG2+ oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7+/NG2+ cells, while there was a significant increase in FABP7+/GFAP+ cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU+ astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.

The postsynaptic density protein, IQ-ArfGEF/BRAG1, can interact with IRSp53 through its proline-rich sequence

IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor for Arf1 and Arf6, is localized at the postsynaptic density (PSD) and interacts with PSD-95. In this study, we identified a novel interaction of IQ-ArfGEF/BRAG1 with insulin receptor tyrosine kinase substrate of 53 kDa (IRSp53), also known as brain-specific angiogenesis inhibitor 1-associated protein 2. The interaction was mediated by the binding of the C-terminal proline-rich sequence of IQ-ArfGEF/BRAG1 to the SH3 domain of IRSp53. IQ-ArfGEF/BRAG1 and IRSp53 were colocalized at the PSD of excitatory synapses of certain neuronal populations. Our present findings suggest that IQ-ArfGEF/BRAG1 may play roles downstream of NMDA receptors through the interaction with multivalent PSD proteins such as IRSp53 and PSD-95.

Vezatin, a potential target for ADP-ribosylation factor 6, regulates the dendritic formation of hippocampal neurons.

ADP-ribosylation factor 6 (ARF6) is a small GTPase that regulates neuronal morphogenesis processes such as axonal, dendritic, and spine formation possibly through the actin cytoskeleton and membrane trafficking. In an attempt to define the molecular mechanisms that regulate neuronal morphogenesis by ARF6, we identified vezatin as a novel binding partner of active GTP-bound ARF6 using yeast two-hybrid screening. Vezatin was able to bind specifically to GTP-ARF6 among the ARF family. In the adult mouse brain, vezatin exhibited widespread gene expression with high levels in the hippocampus and medial habenular nucleus. In hippocampal neurons, vezatin was localized at dendrites as well as cell bodies. Knockdown of endogenous vezatin significantly reduced total dendritic length and arborization of cultured hippocampal neurons, while overexpression of vezatin increased dendritic length. Our present study suggests that vezatin may regulate dendritic formation as a downstream effector of ARF6.

Identification of FABP7 in fibroblastic reticular cells of mouse lymph nodes

Fatty acids and their metabolites regulate immune cell function. The present study was undertaken to examine the detailed distribution of fatty acid binding proteins (FABPs), the cytosolic chaperones of fatty acids, in mouse peripheral immune organs. Using immunohistochemistry, FABP7 was localized to the alpha-smooth muscle actin (SMA)+ fibroblastic reticular cells, which construct the stromal reticula in the T cell areas of the peripheral lymph nodes and spleen. Immunoelectron microscopy showed that FABP7+ cells enclosed the collagen fibers, forming a conduit system, which transport lymph and associated low-molecular-mass proteins. In contrast, FABP5+ cells were distributed throughout the lymph node and contained well-developed lysosome and phagocytic materials within the cytoplasm. The mesenteric lymph nodes of FABP7 knockout mice showed normal histological features, but the percentage of CD4+ cells was significantly increased compared with that in wild-type mice. These data indicate that FABP7 may be involved in T cell homeostasis, possibly by modulating lipid metabolism in fibroblastic reticular cells within the peripheral lymph nodes.

Localization of fatty acid binding proteins (FABPs) in the cochlea of mice.

Various fatty acids (FAs) are involved in many different functions in the organism as a source of energy, as essential ingredients of membranous lipids as well as intracellular signaling molecules. Intracellular fatty acid binding proteins (FABPs) comprise a family of soluble lipid binding proteins with low molecular masses and which can make long chain FAs soluble to allow intracellular translocation in the aqueous cytosol. To clarify the possible involvement of FAs and FABPs in hearing function, the present study investigated the localization of FABPs in the cochlea of adult mice using immunohistochemical procedures. Among various FABP species, H (heart-type)-FABP was localized in inner and outer pillar cells and outer phalangeal cells, while B (brain-type)-FABP was localized in border cells and cells of Hensen, and fibrocytes in the spiral limbus and spiral prominence. In the spiral ganglion, moderate to low H-FABP immunoreactivity was observed in almost all neurons, while B-FABP immunoreactivity was found in satellite cells. The discrete localization of the two FABPs in different non-receptor cells in the Organ of Corti suggests that the FABP species and/or their ligands, FAs, play important roles in the regulation of the hearing function.

Expression of epidermal fatty acid binding protein (E-FABP) in septoclasts in the growth plate cartilage of mice

Abstractn-3 Polyunsaturated fatty acids play a role in regulating the growth of the long bones. Fatty acid-binding proteins (FABPs) bind and transport hydrophobic long-chain fatty acids intracellularly, and epidermal-type FABP (E-FABP) has an affinity for n-3 fatty acids. This study aimed to clarify the localization of E-FABP in the growth plate of the mouse tibia. At the chondro-osseous junction (COJ) of the growth plate, E-FABP-immunoreactivity was exclusively localized in mononuclear, spindle-shaped cells with several long processes. These E-FABP-immunoreactive cells were identified as being septoclasts, i.e., cells that resorb uncalcified transverse septa. The processes of these immunoreactive septoclasts terminated between the longitudinal and transverse septa. E-FABP-immunoreactivity was found in the entire cytoplasm and on the mitochondrial outer membrane. In ontogeny, immunoreactive septoclasts were observed immediately after emergence of the primary ossifying center and were distributed not only at the COJ but also in the metaphysis near the COJ. The number of septoclasts increased at the postnatal age of 1xa0week (P1w)-P2w, and thereafter gradually decreased; and the cells became concentrated at the COJ after P3w-P4w. The immunoreactivity for peroxisome proliferator-activated receptor (PPAR)β/δ was detected in these E-FABP-immunoreactive septoclasts. The present results suggest that fatty acids, preferably n-3 ones, are intracellularly transported by E-FABP to various targets, including mitochondria and nucleus, in which PPARβ/δ may play functional roles in the transcriptional regulation of genes involved in the endochondral ossification.

The sensitivity of male rat reproductive organs to monosodium glutamate.

OBJECTIVEnThis study aimed to investigate the sensitivity of the testis, epididymis, seminal vesicle, and sperm acrosome reaction (AR) to monosodium L- glutamate (MSG) in rats.nnnMATERIALS AND METHODSnRats were divided into four groups and fed with non-acidic MSG at 0.25, 3 or 6 g/kg body weight for 30 days or without MSG. The morphological changes in the reproductive organs were studied. The plasma testosterone level, epididymal sperm concentration, and sperm AR status were assayed.nnnRESULTSnCompared to the control, no significant changes were discerned in the morphology and weight of the testes, or the histological structures of epididymis, vas deferens and seminal vesicle. In contrast, significant decreases were detected in the weight of the epididymis, testosterone levels, and sperm concentration of rats treated with 6 g/kg body weight of MSG. The weight loss was evident in the seminal vesicle in MSG-administered rats. Moreover, rats treated with MSG 3 and 6 g/kg exhibited partial testicular damage, characterized by sloughing of spermatogenic cells into the seminiferous tubular lumen, and their plasma testosterone levels were significantly decreased. In the 6 g/kg MSG group, the sperm concentration was significantly decreased compared with the control or two lower dose MSG groups. In AR assays, there was no statistically significant difference between MSG-rats and normal rats.nnnCONCLUSIONnTesticular morphological changes, testosterone level, and sperm concentration were sensitive to high doses of MSG while the rate of AR was not affected. Therefore, the consumption of high dose MSG must be avoided because it may cause partial infertility in male.