Katsura Takano

Protection by exogenous pyruvate through a mechanism related to monocarboxylate transporters against cell death induced by hydrogen peroxide in cultured rat cortical neurons.

In cortical neurons cultured for 3 or 9u2003days inu2003vitro (DIV), exposure to hydrogen peroxide (H2O2) led to a marked decrease in cell viability in a concentration‐dependent manner at a concentration range of 10u2003µm to 1u2003mm irrespective of the duration between 6 and 24u2003h. However, H2O2 was more potent in decreasing cellular viability in cortical neurons cultured for 9u2003DIV than in those for 3u2003DIV. Pyruvate was effective in preventing the neuronal cell death at 1u2003mm even when added 1–3u2003h after the addition of H2O2. Semi‐quantitative RT–PCR and western blotting analyses revealed significantly higher expression of both mRNA and protein for a particular monocarboxylate transporter (MCT) in neurons cultured for 9u2003DIV than in those for 3u2003DIV. A specific inhibitor of MCT significantly attenuated the neuroprotection by pyruvate in neurons cultured for 9u2003DIV, without markedly affecting that in neurons cultured for 3u2003DIV. These results suggest that vulnerability to H2O2 may at least in part involve expression of particular MCT isoforms responsible for the bi‐directional transport of pyruvate across cell surfaces in cultured rat cortical neurons.

Deletion of SERP1/RAMP4, a Component of the Endoplasmic Reticulum (ER) Translocation Sites, Leads to ER Stress

ABSTRACT Stress-associated endoplasmic reticulum (ER) protein 1 (SERP1), also known as ribosome-associated membrane protein 4 (RAMP4), is a Sec61-associated polypeptide that is induced by ER stress. SERP1−/− mice, made by targeted gene disruption, demonstrated growth retardation, increased mortality, and impaired glucose tolerance. Consistent with high levels of SERP1 expression in pancreas, pancreatic islets from SERP1−/− mice failed to rapidly synthesize proinsulin in response to a glucose load. In addition, reduced size and enhanced ER stress were observed in the anterior pituitary of SERP1−/− mice, and growth hormone production was slowed in SERP1−/− pituitary after insulin stimulation. Experiments using pancreatic microsomes revealed aberrant association of ribosomes and the Sec61 complex and enhanced ER stress in SERP1−/− pancreas. In basal conditions, the Sec61 complex in SERP1−/− microsomes was more cofractionated with ribosomes, compared with SERP1+/+ counterparts, in high-salt conditions. In contrast, after glucose stimulation, the complex showed less cofractionation at an early phase (45 min) but more at a later phase (120 min). Although intracellular insulin/proinsulin levels were not significantly changed in both genotypes, these results suggest that subtle changes in translocation efficiency play an important role in the regulation of ER stress and rapid polypeptide synthesis.

Oxidative metabolites are involved in polyamine-induced microglial cell death

Pathological activation of microglia, which reside quiescently in physiological CNS, is associated with various neurodegenerative diseases. Endogenous polyamines, spermidine and spermine, are known to be activators of cell proliferation and differentiation. We previously reported that both spermidine and spermine induce dose-dependent cell death in cultured rat brain microglia at a submicromolar concentration range via apoptotic process, whereas cultured astrocytes were less sensitive to these polyamines [Neuroscience 120 (2003) 961]. These polyamine effects were observed only in the presence of fetal bovine serum. In the present study we examined further the mechanism of polyamine-induced microglial cell death. Amine oxidase in fetal bovine serum produces hydrogen peroxide and an aminoaldehyde from spermine, and the latter generates acrolein spontaneously. Acrolein was found to be much more toxic to microglia than to astrocytes and the effective concentration of acrolein was similar to that of spermine, whereas hydrogen peroxide was marginally toxic. Aminoguanidine, an inhibitor of amine oxidase, blocked the toxic effects of spermine on microglia. Spermine cytotoxicity was also prevented by antioxidant reagents; glutathione (reduced form), cysteine, and N-acetylcysteine. These results suggest that polyamine-induced apoptotic cell death of microglia is triggered by an oxidative stress with acrolein, which is produced by amine oxidase from polyamine. The different toxicities of polyamine between two glial cells may regulate the balance of glial activation in some pathological conditions of CNS.

Microglial cell death induced by a low concentration of polyamines

Pathological activation of microglia, which reside quiescently in physiological CNS, contributes various neurodegenerative diseases. Endogenous polyamines, spermidine (SPD) and spermine (SPM) are known to be activators of cell proliferation and differentiation. We examined the effect of polyamines on microglial activation in culture. Cultured microglia prepared from the whole brains of newborn rats produced nitric oxide (NO) by the stimulation with lipopolysaccharide (LPS). LPS-induced NO production was markedly inhibited by SPD and SPM; half effective concentrations (EC(50)) of SPD and SPM were about 3 and 1 microM, respectively. Cell viability assessed by total mitochondrial activity decreased by the incubation with SPD and SPM for 24 h at similar concentration ranges. After the treatment with SPM for 24 h, the cells changed into small round morphology, and were strongly stained with propidium iodide. By the staining with bis-benzimide trihydrochloride, condensation and fragmentation of the nucleus were often observed. Semiquantitative analysis of fragmented DNA with enzyme-linked immunosorbent assay technique revealed that a large amount of fragmented DNA appeared in cytosol prior to disruption of the cell membrane. Fragmented DNA in the cytosol increased dose dependently with SPM; EC(50) was less than 10 microM. Furthermore, most of the cells after 24 h incubation with 10 microM SPD and SPM were positive for terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end labeling. These results suggest that microglial cell death is induced by a low concentration of polyamines via an apoptotic process rather than necrotic one.

Uptake of [3H]L-serine in rat brain synaptosomal fractions.

Accumulation of [3H]L-serine in crude synaptosomal fractions freshly prepared from rat brain has been found to be temperature-sensitive and to consist of both Na(+)-dependent and Na(+)-independent components. The accumulation of [3H]L-serine measured at submicromolar concentrations had a distinct substrate selectivity, different from the uptake of [3H]L-proline, [3H]L-glutamate and [3H]GABA. It was fully inhibited by L-glutamine, L-asparagine, L-cysteine, L-alanine, L-leucine, L-isoleucine, L-tyrosine, L-phenylalanine, L-threonine and by the synthetic marker for the large neutral amino acid transport systems 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, but not influenced by beta-alanine, taurine, glycine nor was it inhibited by the marker for the A system, L-2-methylamino isobutyric acid. D-Serine at 1 mM concentration produced no significant inhibition of the accumulation of 10 nM [3H]L-serine. We conclude that L-serine uptake observed in the present study is mediated by at least two distinct transport systems: a Na(+)-dependent one of lower affinity (K(m) in mM range) and a Na(+)-independent system of higher affinity (K(m) approximately 20-100 micro M). Characteristics of [3H]L-serine accumulation displayed at low substrate concentrations suggest that it was mediated neither by the typical A, nor by the large neutral, amino acid transport systems but predominantly by transporters belonging to the recently identified LAT (L-amino acid transporter) family.

Functional expression of A glutamine transporter responsive to down-regulation by lipopolysaccharide through reduced promoter activity in cultured rat neocortical astrocytes.

The prevailing view is that the glutamine (Gln) transporter (GlnT/ATA1/SAT1/SNAT1) is a member of the system A transporter superfamily with the ability to fuel the glutamate/Gln cycle at nerve terminals in glutamatergic neurons. Semiquantitative reverse transcription‐polymerase chain reaction revealed similarly high expression of mRNA for GlnT by rat brain neocortical astrocytes as well as neurons, with progressively lower expression by cerebellar astrocytes, hippocampal astrocytes, and whole‐brain microglia in culture. [3H]Gln was accumulated in a temperature‐dependent manner with a saturable profile in both cultured neocortical neurons and astrocytes, whereas biochemical and pharmacological analyses on [3H]Gln accumulation revealed the expression of both system A and system L transporters by cultured neocortical neurons and astrocytes. Exposure to lipopolysaccharide (LPS) for 24 hr resulted in a significant decrease in both GlnT mRNA expression and [3H]Gln accumulation, with a concomitant drastic increase in nitrite formation in cultured neocortical astrocytes. Moreover, LPS significantly inhibited the promoter activity of GlnT in the astrocytic cell line C6 glioma cells as well as primary rat neocortical astrocytes in culture. These results suggest that activation by LPS would lead to down‐regulation of the expression of GlnT responsible for the incorporation of extracellular Gln into intracellular spaces across plasma membranes through the inhibition of its promoter activity in cultured rat neocortical astrocytes.

Modulation of DNA binding of nuclear transcription factors with leucine-zipper motifs by particular endogenous polyamines in murine central and peripheral excitable tissues

Transcriptional regulation is one of the most important functions of polyamines in the nucleus of eukaryotic cells. The addition of the endogenous polyamines spermine and spermidine markedly increased DNA binding activity of the transcription factor activator protein-1 (AP1) in a concentration-dependent manner at a concentration range of 50 to 500 microM in nuclear extracts of murine whole brain when determined in the absence of added MgCl(2) on gel retardation electrophoresis. Similar but less potent potentiation was seen with DNA binding of cAMP responsive element binding protein (CREB), while both polyamines were ineffective in affecting that of c-Myc irrespective of the addition of MgCl(2). Unlabeled AP1 probe was invariably more potent in competing for AP1 binding than unlabeled CREB probe in either the presence or absence of spermine and spermidine. In addition to whole brain, both polyamines significantly increased AP1 binding in retina, adrenal and pituitary, without significantly affecting that in spleen. Moreover, ultraviolet and circular dichroism spectra analyses revealed that these two polyamines induced DNA topological transition of AP1 probe under the conditions favorable for the increase in AP1 binding. These results suggest that both spermine and spermidine may modulate gene transcription through cis- and trans-actions on AP1 binding in the nucleus in murine central and peripheral structures with high excitability.

A possible novel mechanism underlying temperature-dependent uptake of [3H]spermidine in nuclear fractions of murine brain.

[3H]Spermidine (SPD) was accumulated in subcellular fractions enriched of the nucleus in a temperature-dependent manner with a saturable profile in murine brain. The accumulation reached a plateau within 30 min at 2 degrees C and 30 degrees C, while excess unlabeled SPD significantly inhibited the accumulation at 2 degrees C without markedly affecting that at 30 degrees C when added after equilibrium. The temperature-dependent accumulation of [3H]SPD was significantly inhibited by the triamine SPD and the tetraamine spermine, but not by the diamine putrescine. Phospholipases were invariably effective in significantly inhibiting the accumulation at 30 degrees C in a concentration-dependent manner. Amongst different discrete murine central structures examined, the temperature-dependent [3H]SPD accumulation was highest in neocortex with progressively lower activities in striatum, hypothalamus, spinal cord, medulla-pons, hippocampus, midbrain and cerebellum. These results suggest the possible presence of a hitherto unidentified nuclear transport system for particular polyamines in murine brain.

Possible correlation between abilities of a variety of polyamines to increase activator protein-1 DNA binding and to inhibit [3H]spermidine transport in nuclear fractions of murine brain

The addition of a number of natural and synthetic polyamines significantly increased activator protein (AP1) DNA binding in nuclear extracts of murine whole brain, which occurred in a manner positively correlated with their potencies to inhibit temperature-dependent transport of [3H]spermidine in brain nuclear fractions. These results suggest that polyamines may affect gene transcription by AP1 complex after incorporation into the nucleus in rodent brain.