Yutaka Inaguma

Riluzole enhances expression of brain-derived neurotrophic factor with consequent proliferation of granule precursor cells in the rat hippocampus

The dentate gyrus of the hippocampus, generating new cells throughout life, is essential for normal recognition memory performance. Reduction of brain‐derived neurotrophic factor (BDNF) in this structure impairs its functions. To elucidate the association between BDNF levels and hippocampal neurogenesis, we first conducted a search for compounds that stimulate endogenous BDNF production in hippocampal granule neurons. Among ion channel modulators tested, riluzole, a neuroprotective agent with anticonvulsant properties that is approved for treatment of amyotrophic lateral sclerosis, was highly effective as a single dose by an intraperitoneal injection, causing a rise in BDNF localized in dentate granule neurons, the hilus, and the stratum radiatum of the CA3 region. Repeated, but not single, injections resulted in prolonged elevation of hippocampal BDNF and were associated with increased numbers of newly generated cells in the granule cell layer. This appeared due to promoted proliferation rather than survival of precursor cells, many of which differentiated into neurons. Intraventricular administration of BDNF‐specific antibodies blocked such riluzole effects, suggesting that BDNF increase is necessary for the promotion of precursor proliferation. Our results suggest the basis for a new strategy for treatment of memory dysfunction.

Modulation of the Stress‐Induced Synthesis of hsp27 and αB‐Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract: The possible participation of cyclic AMP in the stress‐induced synthesis of two small stress proteins, hsp27 and αB‐crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB‐crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β‐adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite‐ or heat‐induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB‐crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB‐crystallin showed that the modulation of the stress‐induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB‐crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB‐crystallin is suppressed.

αB crystallin and HSP28 are enhanced in the cerebral cortex of patients with Alzheimer's disease

Abstract The localization of two small heat shock proteins, αB crystallin and 28-kDa heat shock protein (HSP28), was studied immunochemically and immunohistochemically in cerebral cortex of patients with Alzheimers disease (AD) and control groups. By specific immunoassays for αB crystallin and HSP28, we found that the concentrations of αB crystallin are elevated in the temporal and frontal lobes, while those of HSP28 are elevated in the temporal, frontal, and parietal lobes in the AD brains. Immunohistochemically, αB crystallin was localized in astrocytes and oligodendrocytes, while HSP28 was present in degenerating neurons in the cerebral cortex of both AD and control brains. However, αB crystallin-immunoreactive astrocytes and HSP28-immunoreactive degenerating neurons were more frequently observed in AD brains. The immunoreactivity for HSP28 was also found in the senile plaques in the AD brains. These findings suggest that the increased accumulations of these small heat shock proteins appear to be part of reactive processes of glial cells and neurons under pathologic conditions.

Brain-derived neurotrophic factor, nerve growth factor and neurotrophin-3 in selected regions of the rat brain following kainic acid-induced seizure activity

Abstract Changes in levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) in various regions of the rat brain following kainic acid-induced seizure activity were investigated. BDNF protein, as measured by a two-site enzyme immunoassay, increased transiently 12–24 h after the intraperitoneal administration of kainic acid to 61.6 ng/g wet weight in the hippocampus (∼10-fold increase), 19.5 ng/g in the piriform plus entorhinal cortex (∼10-fold) and 8.2 ng/g in the olfactory bulb (∼16-fold), and then rapidly decreased. Increases of 2- to 4-fold in levels of BDNF were also detected in the septum, cerebral cortex, striatum and hypothalamus, but not in the cerebellum. In contrast, levels of NGF and NT-3 decreased 24 h after the administration of kainic acid. Western and Northern blotting analyses of hippocampal tissues, respectively, revealed increase in levels of a 14-kDa protein corresponding to BDNF and its mRNA at both 4.2 and 1.4 kb. Hippocampal mRNAs for NGF and NT-3 increased and decreased, respectively, in kainic acid-treated rats. Immunohistological investigations showed that, in the hippocampus, the administration of kainic acid enhanced a homogeneous immunoreactivity of BDNF in the polymorph inner layer (the stratum radiatum of the CA3/CA4 regions and the hilar region) and in granule cells of the dentate gyrus. BDNF protein was found in neurons, but not at all in glial cells or in blood vessels, and was localized in the cytoplasm, the nucleoplasm and the primary dendrites of neurons as well as in perisynaptic extracellular spaces, but hardly in their axons. Our results show that kainic acid treatment increases levels of BDNF, but not NGF or NT-3, in various regions of the rat brain, other than the cerebellum. Also, the majority of BDNF newly synthesized by hippocampal granule neurons is secreted into the perisynaptic extracellular space in the polymorph inner layer of the dentate gyrus, supporting an autocrine-like role for the factor in synaptic functions.

Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27

Abstract We previously showed that the aggregated form of Hsp27 in cultured cells becomes dissociated as a result of phosphorylation with various types of stress. In order to clarify the signal transduction cascade involved, the effects of various inhibitors of protein kinases and dithiothreitol on the dissociation of Hsp27 were here examined by means of an immunoassay after fractionation of cell extracts by sucrose density gradient centrifugation. The dissociation of Hsp27 induced by exposure of U251 MG human glioma cells to metals (NaAsO2 and CdCl2), hypertonic stress (sorbitol and NaCl), or anisomycin, an activator of p38 mitogen–activated protein (MAP) kinase, was completely suppressed by the presence of SB 203580 or PD 169316, inhibitors of p38 MAP kinase, but not by PD 98059 and Uo 126, inhibitors of MAP kinase kinase (MEK), nor by staurosporine, Go 6983, and bisindolylmaleimide I, inhibitors of protein kinase C. Phorbol ester (PMA)–induced dissociation of Hsp27 was completely suppressed by staurosporine, Go 6983, or bisindolylmaleimide I and partially suppressed by SB 203580, or PD 169316 but not by PD 98059 or Uo 126, indicating mediation by 2 cascades. The presence of 1 mM dithiothreitol in the culture medium during exposure to chemicals suppressed the dissociation of Hsp27 induced by arsenite and CdCl2 but not by other chemicals. These results suggest that the phosphorylation of Hsp27 is catalyzed by 2 protein kinases, p38 MAP kinase–activated protein (MAPKAP) kinase- 2/3 and protein kinase C. In addition, metal-induced signals are sensitive to reducing power.

Developmental and Age-Dependent Changes of 28-kDa Calbindin-D in the Central Nervous Tissue Determined with a Sensitive Immunoassay Method

Abstract: For the quantitative analysis of vitamin D‐dependent 28‐kDa calcium‐binding protein (calbindin‐D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin‐D from rat kidneys, and the antibodies were purified with a calbindin‐D‐coupled Sepharose column. The purified antibodies were specific for calbindin‐D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich‐type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin‐D, which was sufficiently sensitive for the measurement of calbindin‐D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin‐D in a single Purkinje cell was 0.05 pg. Calbindin‐D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin‐D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4–5 weeks of postnatal age. In contrast, kidney calbindin‐D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin‐D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain. However, the concentrations in various regions of cerebral cortex from patients with Alzheimers disease showed values similar to those in the relevant regions of the age‐matched control patients.

Induction of brain-derived neurotrophic factor by convulsant drugs in the rat brain: involvement of region-specific voltage-dependent calcium channels

A high level of hippocampal brain‐derived neurotrophic factor (BDNF) in normally aged as compared with young rats suggests that it is important to maintain a considerable level of hippocampal BDNF during aging in order to keep normal hippocampal functions. To elucidate possible mechanisms of endogenous BDNF increase, changes in levels of BDNF were studied in the rat brain following systemic administration of various convulsant agents; excitotoxic glutamate agonists, NMDA, kainic acid and (+/–)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA); GABA receptor antagonists, picrotoxin, pentylenetetrazole (PTZ) and lindane (γ‐hexachlorocyclohexane); and l‐type voltage‐dependent calcium channel agonist, BAY‐K 8644. Kainic acid and AMPA, but not NMDA, caused remarkable increases in BDNF protein in the rat hippocampus and entorhinal cortex. Picrotoxin, PTZ and lindane stimulated BDNF production in the entorhinal cortex and also in the hippocampus of rats showing very severe convulsions. On the other hand, BAY‐K 8644 treatment increased BDNF levels in the neocortex and entorhinal cortex. Maximal levels of BDNF protein were observed at 12–24 h, 8–16 h and 6 h following administration of kainic acid, PTZ and BAY‐K 8644, respectively. Kainic acid stimulated BDNF synthesis in presynaptic hippocampal granule neurons, but not in postsynaptic neurons with its receptors, while PTZ and BAY‐K 8644 produced the same effects in postsynaptic neurons in the entorhinal cortex (in granule neurons in the hippocampus) and in the whole cortex, respectively. Nifedipine inhibited almost completely BAY‐K 8644, but not PTZ, effects. ω‐Conotoxin GVIA and DCG‐IV partially blocked kainic acid‐induced enhancement of BDNF, indicating involvement of l‐type and N‐type voltage‐dependent calcium channels, respectively. In addition, BDNF levels in the hippocampus of mice deficient in d‐myo‐inositol‐1,4,5‐triphosphate receptor gene were scarcely different from those in the same region of controls, suggesting little involvement of intracellular calcium increase through this receptor. BAY‐K 8644, but not kainic acid or PTZ, stimulated the phosphorylation of cyclic AMP responsive element binding protein. Our results indicate convulsant‐dependent stimulation of BDNF production and involvement of region‐specific voltage‐dependent calcium channels.

cDNA cloning of a 20-kDa protein (p20) highly homologous to small heat shock proteins: developmental and physiological changes in rat hindlimb muscles

A cDNA clone encoding p20, a novel member of the small heat-shock protein family in mammals, was isolated from a rat soleus cDNA library. The clone contained an insert of 1.3 kb with an open reading frame specifying a polypeptide of 162 amino-acid residues. Southern blot analysis suggested that the p20 gene is a single gene in rat genome. Developmental changes and a sciatic nerve denervation experiment suggested that the expression of p20 in rat hindlimb muscle is related to muscle contraction, and specifically in slow-twitch muscles.

Translocation and induction of αB crystallin by heat shock in rat glioma (GA-1) cells

Abstract Response to heat shock of αB crystallin expressed in rat astrocytoma GA-1 cells was analysed quantitatively using an immunoassay method. GA-1 cells contained a considerable amount of αB crystallin at growing phase. When the growing cells were heated at 45°C, concentrations of αB crystallin in cell extracts were decreased to less than one-fifth of the original level within 15 min, with an increase in the insoluble fraction which was detected by immunoblotting. The low level of αB crystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the control level within several hours. At 10 h following heat shock (45°C for 15 min), the concentration of αB crystallin in the soluble extract was about twice that of the control level, with little detectable amounts in the insoluble fraction. These results are additional evidence that suggest that αB crystallin is a small heat shock protein.

Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease.

The phosphorylation state of αB‐crystallin accumulated in the brains of two patients with Alexanders disease (one infantile and one juvenile type) was determined by means of SDS‐PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against αB‐crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25–28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of αB‐crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser‐59 in both cases. A significant level of phosphorylation at Ser‐45 but not Ser‐19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. αB‐crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. αB‐crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimers disease and aged controls. The phosphorylation site was mostly at Ser‐59 in all cases. Immunohistochemically, αB‐crystallin was stained in Rosenthal fibers in brains of patients with Alexanders disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser‐59. These results indicate that the major phosphorylation site in αB‐crystallin in brains of patients with Alexanders disease or Alzheimers disease as well as in aged controls is Ser‐59.