Junya Mitoma

Ceramide and its interconvertible metabolite sphingosine function as indispensable lipid factors involved in survival and dendritic differentiation of cerebellar Purkinje cells

Abstract: Ceramide generated from sphingomyelin has emerged as a new but conserved type of biologically active lipid. We previously found that endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and that sphingomyelin is present abundantly in the somatodendritic region of these cells. To gain further insight into a potential role of the sphingomyelin/ceramide pathway, we investigated the effects of depletion of sphingolipids on the phenotypic growth and survival of immature Purkinje cells and the ability of ceramide or other sphingolipids to antagonize these effects. Inhibition of ceramide synthesis by ISP‐1, a specific inhibitor of serine palmitoyltransferase, decreased cellular levels of sphingolipids. This treatment resulted in a decrease in cell survival accompanied by an induction of apoptotic cell death and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons. Cell‐permeable ceramides, sphingosine, or sphingomyelin overcame these abnormalities more effectively than other sphingolipids when added simultaneously with ISP‐1. Exposure to bacterial sphingomyelinase in turn enhanced cell survival and dendritic branching complexity of Purkinje cells at different optimal concentrations. Furthermore, cell‐permeable ceramide acted synergistically with the neurotrophin family, which has been previously shown to support Purkinje cell survival. These observations suggest that ceramide is a requisite for the survival and the dendritic differentiation of Purkinje cells.

Charged amino acids at the carboxyl-terminal portions determine the intracellular locations of two isoforms of cytochrome b5.

Outer mitochondrial membrane cytochromeb 5 (OMb), which is an isoform of cytochromeb 5 (cyt b 5) in the endoplasmic reticulum, is a typical tail-anchored protein of the outer mitochondrial membrane. We cloned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitochondrial targeting signal at the amino terminus. To identify the region responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The deletion of more than 11 amino acid residues from the carboxyl-terminal end of OMb abolished the targeting of the protein to the mitochondria. When the carboxyl-terminal 10 amino acids of OMb were fused to the cytb 5 that was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the carboxyl-terminal 10 amino acid residues of OMb have sufficient information to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 amino acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b 5, in which the acidic amino acid in its carboxyl-terminal end was replaced by basic amino acid, could be transported to the mitochondria. It would thus seem that charged amino acids in the carboxyl-terminal portion of these proteins determine their locations in the cell.

The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum.

Cytochrome b5 is an integral membrane protein located on the outer surface of the endoplasmic reticulum (ER). This cytochrome is considered to be synthesized on free ribosomes and to be inserted post‐translationally into the ER membrane, without participation of a signal recognition particle. To elucidate the signal responsible for targeting of cytochrome b5 to the ER membrane in vivo, DNAs encoding various derivatives of the cytochrome were constructed and introduced into cultured mammalian COS cells, and the subcellular distributions of the derivatives expressed in the cells were then analyzed. The deletion of more than 11 amino acid residues at the carboxy‐terminal end of cytochrome b5 abolished the targeting and anchoring of the cytochrome to the ER membrane. Fusion proteins consisting of the carboxy‐terminal 10 amino acid residues of cytochrome b5 and passenger proteins with the hydrophobic portion could be localized in the ER membrane. Thus, the last 10 amino acid residues of cytochrome b5 carry information necessary for the cytochrome to be targeted to the ER membrane.

Bipotential roles of ceramide in the growth of hippocampal neurons: promotion of cell survival and dendritic outgrowth in dose- and developmental stage-dependent manners.

Ceramide is now regarded as a lipid messenger molecule involved in a variety of cellular processes, including growth, differentiation, and cell death. Previously, we demonstrated that ceramide is required for cell survival and dendritic growth of cerebellar Purkinje neurons (Furuya et al.: J Neurochem 65:1551–1561, 1995). Here, we show that ceramide plays growth‐supportive roles in hippocampal neurons at immature stages of development. Application of cell‐permeable N‐hexanoyl‐D‐erythro‐sphingosine (C6‐ceramide) at a concentration of 3 μM promoted cell survival and dendritic outgrowth of the immature neurons. A structurally related compound, N‐hexanoyl‐D‐erythro‐dihydrosphingosine (C6‐dihydroceramide), was ineffective, showing a requirement of 4‐5 double bonds in the sphingosine moiety for activity. Incorporation of 5‐bromo‐2′‐deoxyuridine into neurons was not altered by the treatment with C6‐ceramide, indicating that C6‐ceramide did not facilitate neuronal proliferation but protected hippocampal neurons against basal cell death. The survival‐promoting activity of C6‐ceramide, however, appeared to be biphasic; C6‐ceramide at a concentration of 10 μM caused retraction of the dendrites and detachment of the neurons from the culture plate followed by cell death. In contrast to the immature neurons, the treatment of mature hippocampal neurons with C6‐ceramide did not support cell survival but caused nonnecrotic cell death, even at a concentration of 3 μM. These results suggest strongly that ceramide regulates the fate of hippocampal neurons, depending on its concentration and on the developmental stage. J. Neurosci. Res. 51:712–722, 1998.

A novel metabolic communication between neurons and astrocytes: non-essential amino acid L-serine released from astrocytes is essential for developing hippocampal neurons

A hippocampal astrocyte conditioned medium (HACM) supported the survival of hippocampal neurons under a serum-, glia-free culture setting. The neurotrophic activity in HACM was mostly recovered in low molecular weight fractions (Mr < 3000), which contained high levels of L-serine and L-alanine. However, L-serine alone significantly improved the neuronal survival and neurite growth in a stereo-specific manner. Other non-essential amino acids had no effect. These results strongly suggest that L-serine, released by astrocytes, is essential for the survival and phenotypic growth of hippocampal neurons.

RETENTION OF CYTOCHROME B5 IN THE ENDOPLASMIC RETICULUM IS TRANSMEMBRANE AND LUMINAL DOMAIN-DEPENDENT

Cytochrome b 5 (b5), a typical tail-anchored protein of the endoplasmic reticulum (ER) membrane, is composed of three functionally different domains: amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxyl-terminal ER-targeting domains (Mitoma, J., and Ito, A. (1992) EMBO J. 11, 4197–4203). To analyze the potential retention signal of b5, mutant proteins were prepared to replace each domain with natural or artificial sequences, and subcellular localizations were examined using immunofluorescence microscopy and cell fractionation. The transmembrane domain functioned to retain the cytochrome in the ER, and the mutation of all or part of the transmembrane domain with an artificial hydrophobic sequence had practically no effect on intracellular distribution of the cytochrome. However, when the transmembrane domain was extended systematically, a substantial portion of the protein with the domain of over 22 amino acid residues leaked from the organelle. Thus, the transmembrane length functions as the retention signal. When cytochromes with mutations at the carboxyl-terminal end were overexpressed in cells, a substantial portion of the protein was transported to the plasma membrane, indicating that the carboxyl-terminal luminal domain also has a role in retention of b5 in the ER. Carbohydrate moiety of the glycosylatably-mutated b5 was sensitive to endoglycosidase H but resistant to endoglycosidase D. Therefore, both transmembrane and carboxyl-terminal portions seems to function as the static retention signal.

Occurrence of an Unusual Phospholipid, Phosphatidyl-l-threonine, in Cultured Hippocampal Neurons EXOGENOUS l-SERINE IS REQUIRED FOR THE SYNTHESIS OF NEURONAL PHOSPHATIDYL-l-SERINE AND SPHINGOLIPIDS

We have recently reported thatl-serine released from astroglial cells supports the survival and neuritogenesis of hippocampal neurons under a serum- and glia-free culture condition (Mitoma, J., Furuya, S., and Hirabayashi, Y. (1998) Neurosci. Res. 30, 195–199). In this study, we show that exogenous l-serine is required for the synthesis of phosphatidyl-l-serine (PS) and sphingolipids in hippocampal neurons. When hippocampal neurons were maintained under an astroglial cell-free condition, the levels of sphingolipids and phosphatidyl-l-serine in the neurons were greatly reduced in the absence of external l-serine or glycine. Instead, a novel phospholipid appeared just ahead of PS on TLC. This novel lipid was determined to be phosphatidyl-l-threonine by TLC blotting/negative secondary ion mass spectrometry and amino acid analysis. Biochemical studies on rat brain microsomes have indicated that phosphatidyl-l-threonine is synthesized by the base exchange enzyme that is involved in PS synthesis with much lower affinity, that is, approximately 1 150 of l-serine. Addition of l-serine or glycine to the culture medium restored the synthesis of PS and sphingolipids in the neurons. These observations show that hippocampal neurons require exogenousl-serine for the synthesis of PS and sphingolipids in the absence of astroglial cells and suggested that astroglial cells contribute to neuronal lipid synthesis through the supply ofl-serine.

Sialidase NEU4 Hydrolyzes Polysialic Acids of Neural Cell Adhesion Molecules and Negatively Regulates Neurite Formation by Hippocampal Neurons

Background: Despite crucial roles of polysialic acid (polySia) in neural functions, the enzyme involved in degradation of polysialic acid in its physiological turnover remains uncertain. Results: Sialidase NEU4 catalytically degrades polySia and negatively regulates neurite outgrowth of hippocampal neurons. Conclusion: Sialidase NEU4 is probably the major degradation enzyme for polySia in vertebrate. Significance: The findings contribute to elucidation of the physiological turnover of polySia. Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.

Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation

Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.

L-serine released from astrocytes is essential for survival and neurite growth of cultured hippocampal neurons

To invesigate the physiological significance of microglia-derived plasminogen (PGn) in the central nervous system, we determined the effects of PGn on the production of plasminogen activator system in cultured astrocytes. By addition of PGn, plasminogen activator inhibitor (PAI) released into the medium was found to be increased in doseand timedependent manner. The reverse zymography and Western blotting revealed that the increasing PA1 is PAI1 type. In addition, PGn caused the activation of p38 MAP kinase in cultured astrocytes. Thus, it might be suggested that the signaling cascade including p38 MAP kinase is associated with the enhancement of PAIrelease from astrocytes taken. Together, these results suggest that microglia-derived plasminogen is a regulatory factor for plasminogen activator activity from astrocytes.