3-Phosphoglycerate dehydrogenase, a key enzyme for l-serine biosynthesis, is preferentially expressed in the radial glia/astrocyte lineage and olfactory ensheathing glia in the mouse brain.

l-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids d-serine and glycine. We have recently shown that l-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo l-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to l-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss.

Mitochondrial targeting signal of rat liver monoamine oxidase B is located at its carboxy terminus.

Monoamine oxidase B, a typical intrinsic protein of the outer mitochondrial membrane, has an uncleavable targeting signal and is inserted into the membrane without proteolytic maturation. To investigate the region responsible for targeting the enzyme to the outer mitochondrial membrane, various mutated proteins were expressed in cultured mammalian cells, and the distributions of the expressed proteins were analyzed by immunofluorescence microscopy and subcellular fractionation. Deletion of the carboxy-terminal 28 amino acids of monoamine oxidase B abolished the transfer of the enzyme to mitochondria, while the deletion of the amino-terminal 55 amino acids had no effect on the transfer to mitochondria. The existence of the targeting signal at the carboxy-terminal portion of the enzyme was confirmed by using hybrid proteins in which the amino- or carboxy-terminal portion of the enzyme was fused to the hydrophilic portion of cytochrome b5. The fused protein with the carboxy-terminal 29 amino acid residues of monoamine oxidase B was localized in mitochondria, whereas that with 10 amino acids remained in the cytoplasm. These results indicate that the targeting signal of monoamine oxidase B is present within its carboxy-terminal 29 amino acid residues.

In situ topology of cytochrome b5 in the endoplasmic reticulum membrane.

Cytochrome b5 is tail-anchored in the ER membrane and is composed of three functionally different portions; amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxy-terminal ER-targeting portions [Mitoma, J. and Ito, A. (1992) EMBO J. 11, 4197-4203]. In situ topology of cytochrome b5 in the ER-membrane was studied using immunofluorescence microscopy. Antibodies were raised against the hydrophilic portion (anti-b5) and the carboxy-terminal seven amino acid residues (anti-peptide) of cytochrome b5 and used for detection of the cytochrome in COS cells which expressed the rat cytochrome. Anti-b5 antibody detected the cytochrome in a reticular staining pattern characteristic of the ER, even when the cell plasma membrane was permeabilized with Streptolysin O. The anti-peptide displayed a fluorescence signal only with Triton-permeabilized cells in which the antibody was able to penetrate into the ER lumen. In a double immuno-staining of the cell using the antipeptide antibody and the antibody against protein disulfide isomerase, both antibodies showed the same staining pattern in the presence of either Triton X-100 or Streptolysin O. The results indicate that the carboxy-terminal hydrophilic stretch is exposed to the luminal side. Cytochrome b5 was tagged with c-myc peptide at the carboxy-terminal end and the topology of the c-myc peptide was analyzed by the same method. Anti c-myc monoclonal IgG detected the tagged cytochrome b5 only after Triton treatment of the fixed cells, suggesting that the addition of c-myc peptide to the carboxy-terminal end does not affect insertion or orientation of the cytochrome in the ER membrane.

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.

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.

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

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.

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 microM 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 microM 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 microM. These results suggest strongly that ceramide regulates the fate of hippocampal neurons, depending on its concentration and on the developmental stage.

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 that L-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 exogenous L-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 of L-serine.

Retention of cytochrome b5 in the endoplasmic reticulum is transmembrane and luminal domain-dependent.

Cytochrome b5 (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.

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

Outer mitochondrial membrane cytochrome b5 (OMb), which is an isoform of cytochrome b5 (cyt b5) 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 cyt b5 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 b5, 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.

Synthetic cationic amphiphiles for liposome-mediated DNA transfection.

The compounds with efficient DNA transfection ability into eukaryotic cells were searched from various synthetic amphiphiles which have cationic heads and long saturated hydrocarbon tails. The efficiency of amphiphiles in gene transfer was examined by the transient expression of cytochrome b5 from its cDNA in COS cells. Among various synthetic amphiphiles, including N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride which is commercially available lipid, O,O'-didodecyl-N-[p-(2-trimethylammonioethyloxy)benzoyl]-(L) -glutamate bromide was highest in efficiency. The optimum condition for the amount of the amphiphile and DNA, and the incubation time were established to be 7.5-15 micrograms/22 mm dish and 1-10 micrograms/22 mm dish, and 48-72 h, respectively.

L-Serine regulates the activities of microglial cells that express very low level of 3-phosphoglycerate dehydrogenase, an enzyme for L-Serine biosynthesis.

Microglia are well known to become activated during various kinds of neuropathological events. The factors that are responsible for the activation, however, are not fully determined. In the present study, L-Ser was shown to enhance production of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) by lipopolysaccharide (LPS)-stimulated cultured rat microglial cells. L-Ser, however, did not enhance the expression of mRNAs encoding inducible NO synthase, IL-6 and TNF alpha. On the other hand, astrocytes did not depend on L-Ser for release of IL-6 and TNF alpha. The expression of an enzyme 3-phosphoglycerate dehydrogenase (3PGDH), which is essential for L-Ser biosynthesis from a glycolytic intermediate 3-phosphoglycerate, was investigated. As revealed by Western blotting and immunocytochemical staining, 3PGDH-protein expression in vitro was the highest in astrocytes, intermediate in neurons and the lowest in microglial cells. Semiquantitative RT-PCR showed that microglial cells expressed 3PGDH-mRNA at a lower level than astrocytes. In frozen sections from rat forebrain, only astrocytes were immunoreactive for 3PGDH. The present study suggested that L-Ser is able to modulate microglial function mainly at the translation level because microglial cells cannot synthesize sufficient amount of L-Ser due to the scarce expression of 3PGDH.

L-serine and glycine serve as major astroglia-derived trophic factors for cerebellar Purkinje neurons.

Glial cells support the survival and development of central neurons through the supply of trophic factors. Here we demonstrate that l-serine (l-Ser) and glycine (Gly) also are glia-derived trophic factors. These amino acids are released by astroglial cells and promote the survival, dendritogenesis, and electrophysiological development of cultured cerebellar Purkinje neurons. Although l-Ser and Gly are generally classified as nonessential amino acids, 3-phosphoglycerate dehydrogenase (3PGDH), a key enzyme for their biosynthesis, is not expressed in Purkinje neurons. By contrast, the Bergman glia, a native astroglia in the cerebellar cortex, highly expresses 3PGDH. These data suggest that l-Ser and Gly mediate the trophic actions of glial cells on Purkinje neurons.