Yasukazu Hozumi

Mutant SOD1 linked to familial amyotrophic lateral sclerosis, but not wild-type SOD1, induces ER stress in COS7 cells and transgenic mice

Mutations in a Cu, Zn-superoxide dismutase (SOD1) cause motor neuron death in human familial amyotrophic lateral sclerosis (FALS) and its mouse model, suggesting that mutant SOD1 has a toxic effect on motor neurons. However, the question of how the toxic function is gained has not been answered. Here, we report that the mutant SOD1s linked to FALS, but not wild-type SOD1, aggregated in association with the endoplasmic reticulum (ER) and induced ER stress in the cDNA-transfected COS7 cells. These cells showed an aberrant intracellular localization of mitochondria and microtubules, which might lead to a functional disturbance of the cells. Motor neurons of the spinal cord in transgenic mice with a FALS-linked mutant SOD1 also showed a marked increase of GRP78/BiP, an ER-resident chaperone, just before the onset of motor symptoms. These data suggest that ER stress is involved in the pathogenesis of FALS with an SOD1 mutation.

Nuclear localization of diacylglycerol kinase ζ in neurons

Diacylglycerol kinase (DGK) is involved in intracellular signal transduction as a regulator of levels of diacylglycerol which leads to protein kinase C activation. Previous studies have revealed that DGK consists of a family of isozymes in mammalian species and that most if not all of them show abundant expression in the central nervous system, suggesting the importance of this enzyme in neuronal function. Among the isozymes, DGKζ (previously also known as DGK‐IV for the rat clone) has unique structural features, such as four ankyrin‐like repeats and a nuclear localization signal (NLS), and shows intense mRNA expression in neurons of the olfactory bulb, hippocampus and cerebral and cerebellar cortices (Goto, K. & Kondo, H. (1996), Proc. Natl Acad. Sci. USA, 93, 11196–11201). However, previous studies have given conflicting results about whether or not DGKζ localizes to the nucleus in these cells. In this study, we have used immunohistochemistry with specific antibodies in brain tissues and cDNA transfection into primary cultured neurons to address this question. We have shown that, while DGKζ is primarily a nuclear protein in neurons, it can also be cytoplasmic in some conditions, and the subcellular location depends not only on the cell type but also on the developmental state or growth conditions of the cell. In addition, we have used deletion mutants to show that nuclear transport of DGKζ depends on a cooperative interaction between the NLS and the C‐terminal region including ankyrin repeats in a manner which suggests that the NLS is a cryptic site whose exposure is regulated by the C‐terminal region. Together, these results support the hypothesis that the localization of DGKζ may be regulated by differential expression of these various proteins which interact with its C‐terminal region.

Modulatory effects of oligodendrocytes on the conduction velocity of action potentials along axons in the alveus of the rat hippocampal CA1 region.

Like neurons and astrocytes, oligodendrocytes have a variety of neurotransmitter receptors and ion channels. However, except for facilitating the rapid conduction of action potentials by forming myelin and buffering extracellular K(+), little is known about the direct involvement of oligodendrocytes in neuronal activities. To investigate their physiological roles, we focused on oligodendrocytes in the alveus of the rat hippocampal CA1 region. These cells were found to respond to exogenously applied glutamate by depolarization through N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors. Electrical stimulation of the border between the alveus and stratum oriens evoked inward currents through several routes involving glutamate receptors and inward rectifier K(+) channels. Moreover, electrical stimulation resembling in vivo activity evoked long-lasting depolarization. To examine the modulatory effects of oligodendrocytes on neuronal activities, we performed dual, whole-cell recording on CA1 pyramidal neurons and oligodendrocytes. Direct depolarization of oligodendrocytes shortened the latencies of action potentials evoked by antidromic stimulation. These results indicate that oligodendrocytes increase the conduction velocity of action potentials by a mechanism additional to saltatory conduction, and that they have active roles in information processing in the brain.

Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation

Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1. We earlier established a monoclonal antibody (mAb), IMab-1, which is specific for R132H-containing IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-specific mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using Sendai virus envelope, IDH1-R132S-specific mAbs were screened in ELISA. One mAb, SMab-1, reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-specific. Furthermore, SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not react with IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1-R132S-specific mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.

Cell Biology and Pathophysiology of the Diacylglycerol Kinase Family: Morphological Aspects in Tissues and Organs

Diacylglycerol kinase phosphorylates diacylglycerol to produce phosphatidic acid. These lipids serve not only as intermediate products in the synthesis of several lipids but also as bioactive molecules. Therefore diacylglycerol kinase is thought to play one of the central roles in lipid signal transduction via the metabolism of two messenger molecules. Molecular and cellular studies have revealed that diacylglycerol kinase consists of a family of isozymes and each has a unique character in terms of regulatory mechanism, binding partner, and subcellular localization. This review focuses on pathophysiological findings of the enzyme family, principally from a morphological point of view in tissues and organs in animal studies, which helps us to develop a picture of how diacylglycerol kinase works in our body.

Selective translocation of diacylglycerol kinase ζ in hippocampal neurons under transient forebrain ischemia

The molecular mechanisms responsible for differential neuronal vulnerability to ischemic injury are incompletely understood. Previous studies have reported that the expression and activity of protein kinase C (PKC), some subtypes of which are activated by Ca(2+) and diacylglycerol (DG), are altered after ischemic insults. Therefore, DG kinase (DGK), which is responsible for controlling PKC activity through DG metabolism, may also be involved in this process. DGKzeta, which is abundantly expressed in the brain, contains a nuclear localization signal (NLS), suggesting its involvement in some nuclear processes in neuronal cells. To elucidate the functional implications of DGKzeta in ischemia, we examined detailed localization of DGKzeta in rat brain after ischemic insults. We used an ischemic model of global cerebral ischemia for 20 min by bilateral common carotid artery occlusion combined with hypotension and followed time-points of reperfusion. DGKzeta expression was evaluated by immunohistochemistry using affinity-purified anti-DGKzeta antibody. In sham-operated rats, a strong DGKzeta-immunoreactivity was observed in the nucleus of neurons in various parts of the brain. In the global ischemic model DGKzeta-immunoreactivity was reduced in intensity in the hippocampal formation and detected in the cytoplasm of CA1 pyramidal neurons throughout reperfusion time courses. Change in the subcellular localization was restricted to the pyramidal cells in CA1 and later in CA3, but not observed in other areas of hippocampus. No change was observed in the cerebral and cerebellar cortices. The present study suggests that DGKzeta might be involved in the process of selective vulnerability of hippocampal pyramidal neurons in postischemic brain.

Nuclear diacylglycerol kinase-zeta is a negative regulator of cell cycle progression in C2C12 mouse myoblasts.

The nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting diacylglycerol (DG) into phosphatidic acid, terminate DG‐dependent events. It has been demonstrated that nuclear DGK‐ζ interferes with cell cycle progression. We previously reported that nuclear DGK‐ζ expression increased during myogenic differentiation, whereas its down‐regulation impaired differentiation. Here, we evaluated the possible involvement of nuclear DGK‐ζ in cell cycle progression of C2C12 myoblasts. Overexpression of a wild‐type DGK‐ζ, which mainly localized to the nucleus (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked the cells in the G1 phase of the cell cycle, as demonstrated by in situ analysis of biotinylated‐16‐dUTP incorporated into newly synthesized DNA and by flow cytometry. In contrast, down‐regulation of endogenous DGK‐ζ by short interfering RNA (siRNA) increased the number of cells in both the S and G2/M phases of the cell cycle. Cell cycle arrest of cells overexpressing wild‐type DGK‐ζ was accompanied by decreased levels of retino‐blastoma protein phosphorylated on Ser‐807/811. Down‐regulation of endogenous DGK‐ζ, using siRNA, prevented the cell cycle block characterizing C2C12 cell myogenic differentiation. Overall, our results identify nuclear DGK‐ζ as a key determinant of cell cycle progression and differentiation of C2C12 cells.—Evan‐gelisti, C., Tazzari, P. L., Riccio, M., Fiume, R., Ho‐zumi, Y., Fala, F., Goto, K., Manzoli, L., Cocco, L., Martelli, A. M. Nuclear diacylglycerol kinase‐ζ is a negative regulator of cell cycle progression in C2C12 mouse myoblasts. FASEB J. 21, 3297–3307 (2007)

Diacylglycerol kinase β promotes dendritic outgrowth and spine maturation in developing hippocampal neurons

BackgroundDiacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to phosphatidic acid and comprises multiple isozymes of distinct properties. Of DGKs, mRNA signal for DGKβ is strongly detected in the striatum, and one of the transcripts derived from the human DGKβ locus is annotated in GenBank as being differentially expressed in bipolar disorder patients. Recently, we have reported that DGKβ is expressed in medium spiny neurons of the striatum and is highly concentrated at the perisynapse of dendritic spines. However, it remains elusive how DGKβ is implicated in pathophysiological role in neurons at the cellular level.ResultsIn the present study, we investigated the expression and subcellular localization of DGKβ in the hippocampus, together with its functional implication using transfected hippocampal neurons. DGKβ is expressed not only in projection neurons but also in interneurons and is concentrated at perisynaptic sites of asymmetrical synapses. Overexpression of wild-type DGKβ promotes dendrite outgrowth at 7 d in vitro (DIV) and spine maturation at 14 DIV in transfected hippocampal neurons, although its kinase-dead mutant has no effect.ConclusionIn the hippocampus, DGKβ is expressed in both projection neurons and interneurons and is accumulated at the perisynapse of dendritic spines in asymmetrical synapses. Transfection experiments suggest that DGKβ may be involved in the molecular machineries of dendrite outgrowth and spinogenesis through its kinase activity.

Subnuclear localization and differentiation-dependent increased expression of DGK-ζ in C2C12 mouse myoblasts

Diacylglycerol kinases (DGKs) catalyze phosphorylation of diacylglycerol (DG) to yield phosphatidic acid (PA). Previous evidence has shown that the nucleus contains several DGK isoforms. In this study, we have analyzed the expression and subnuclear localization of DGK‐ζ employing C2C12 mouse myoblasts. Immunocytochemistry coupled to confocal laser scanning microscopy showed that both endogenous and green fluorescent protein‐tagged overexpressed DGK‐ζ localized mostly to the nucleus. In contrast, overexpressed DGK‐α, ‐β, ‐δ, and ‐ι did not migrate to the nucleus. DGK‐ζ was present in the nuclear speckle domains, as also revealed by immuno‐electron microscopy analysis. Moreover, DGK‐ζ co‐localized and interacted with phosphoinositide‐specific phospholipase Cβ1 (PLCβ1), that is involved in inositide‐dependent signaling pathways important for the regulation of cell proliferation and differentiation. Furthermore, we report that DGK‐ζ associated with nuclear matrix, the fundamental organizing principle of the nucleus where many cell functions take place, including DNA replication, gene expression, and protein phosphorylation. Nuclear DGK‐ζ increased during myogenic differentiation of C2C12 cells, while DGK‐ζ down‐regulation by siRNA markedly impaired differentiation. Overall, our findings further support the importance of speckles and nuclear matrix in lipid‐dependent signaling and suggest that nuclear DGK‐ζ might play some fundamental role during myogenic differentiation of C2C12 cells. J. Cell. Physiol. 209: 370–378, 2006.

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.