Tokiko Hama

Interleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week-old rats in cultures

Interleukin-6 (human recombinant) supported the survival of cultured mesencephalic, catecholaminergic and septal cholinergic neurons from postnatal, two-week-old (P13-P15) rats. Significantly, more catecholaminergic neurons, stained by monoclonal anti-tyrosine hydroxylase antibody, were found in cultures supplemented with interleukin-6 at a concentration of 5 ng/ml than in cultures not treated with interleukin-6. The optimal dose used was 50 ng/ml. The survival effect of interleukin-6 on postnatal rat, tyrosine hydroxylase-positive neurons was observed both in cultures using serum-containing and serum-free medium. Contents of dopamine and noradrenaline in cultures with interleukin-6 were also larger than in control cultures. Interleukin-6 also increased the survival of cultured embryonic (E17) rat midbrain tyrosine hydroxylase-positive neurons. The effect on these neurons was, however, smaller, and the optimal dose of interleukin-6 was nearly 5 ng/ml. Interleukin-6 also supported the survival of cultured postnatal (P13) rat septal cholinergic neurons, visualized by acetylcholinesterase staining. The concomitant addition of mouse nerve growth factor (100 ng/ml) and interleukin-6 (50 ng/ml) had a synergetic effect on the survival of acetylcholinesterase-positive neurons in culture. Our data suggest that the survival of cultured tyrosine hydroxylase-positive, mesencephalic, and acetylcholinesterase-positive, septal neurons from postnatal two-week-old rats was supported by interleukin-6, just as there was a different dose dependency of interleukin-6 on the cultured postnatal neurons compared with embryonic neurons.

Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats

Human recombinant interleukin-6 (IL-6, B-cell stimulating factor-2) was capable of supporting neuronal survival in cholinergic neuron culture, prepared from 10-day-old rat brain septal region. Cell survival of the cultured cholinergic neurons was estimated by measuring the remaining choline acetyltransferase (ChAT) activities after 6 days of culture. IL-6 at a concentration of 5 ng/ml maintained a more than 3-fold higher ChAT activity in the culture as compared with that in cultures without IL-6. The maximal dose of IL-6 was near 50 ng/ml. The concomitant addition of mouse nerve growth factor (NGF) and IL-6, both at maximal doses, had a synergistic effect on cholinergic cell survival. These results indicate that IL-6 can act as a neurotrophic agent, independent of the action of NGF, supporting neuronal survival of cultured postnatal rat septal cholinergic neurons. On the other hand, IL-6 did not affect the differentiation of the cultured embryonic rat septal cholinergic neurons, differently from the differentiation action by NGF.

Interleukin-6 as a neurotrophic factor for promoting the survival of cultured catecholaminergic neurons in a chemically defined medium from fetal and postnatal rat midbrains

Interleukin-6 (IL-6, human recombinant) promoted the survival of catecholaminergic neurons from fetal and postnatal rat midbrains as assessed by an immunohistochemical staining for tyrosine hydroxylase (TH) in culture using a chemically defined medium. The maximal dose of IL-6 for the cell survival of postnatal P15 rat mesencephalic TH-positive neurons in culture for 7 days was 50 ng/ml. The survival-promoting effects on P15 cultures were observed both in high- and low-density cultures. The survival effect of IL-6 on the cultured P15 TH-positive neurons was significant for only 4-15 days in vitro. However, the viable number of TH-positive neurons with IL-6 was less than that of the control at early points in the culture process (1-2 days in vitro). Continuous presentation of IL-6 was required for promoting survival. The optimal dose of IL-6 for the survival of fetal E16 midbrain TH-positive neurons was 5 ng/ml, and the survival promoting effect was less than that for the P15 cultures. The maximal dose of IL-6 for the survival of P2 TH-positive neurons was 5 ng/ml and that of P8 was 50 ng/ml, indicating that the response of rat mesencephalic TH-positive neurons to IL-6 changes during the first postnatal week.

Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14).

In the present paper, the distribution of a neuron-specific phosphoneuroprotein 14 (PNP 14) in cell and tissue was investigated in detail by the immunoblot method using affinity-purified antibody against this protein. The immunoblot of the supernatant fractions of various tissue homogenates of rat clearly demonstrated that PNP 14 was enormously rich in the brain. The content in rat brain was as much as 0.1% of the homogenate. The immunocytochemical study showed that the protein was localized at nerve endings in the cerebellum. Existence of the protein was also confirmed in cultured neuronal cells from postnatal rat midbrain, but not in glial cells. Examination of subcellular localization of PNP 14 indicates that the protein was present in synaptic plasma membranes and synaptic supernatant fractions, but not in synaptic vesicles. During the development of rat brain, PNP 14 came into existence after birth and its amount linearly increased to a maximum at 21-28 days after birth. The content of the protein then remained at the same level for more than 10 months. We concluded that this protein is neuron specific and supposed that it may be involved in neuronal formation and function.

Protein kinase C and Ca2+/calmodulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles

A monoclonal antibody was made using the spleen cells of a mouse immunized with chick synaptic membranes and designated as mAb 1D12. It immunoprecipitated 25% of the omega-conotoxin binding protein but no dihydropyridine binding protein solubilized from chick brain membranes. By immunoblotting, a polypeptide of 58-kDa was identified as the antigen of this antibody in chick, rat, rabbit and guinea pig brain. Immunohistochemical observation indicated the immunoreactivity of mAb 1D12 to be localized in the synaptic regions of central and peripheral neurons. In peripheral organs, there was additional staining in the distal portions of nerve fibers. Immunoelectron microscopy showed immunoreactivity to be located in synaptic vesicle and presynaptic plasma membranes. In the subcellular fractionation of rat brain, 58-kDa protein was recovered in the fractions of synaptic vesicles and plasma membranes but not soluble proteins. This protein could be extracted from membranes by Triton X-100 but treatment with EDTA, acid, base or high salt failed to have such effect. Solubilized 58-kDa protein of rat brain was purified by immunoaffinity chromatography using mAb 1D12. Both protein kinase C and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated purified 58-kDa protein, and maxima of 0.47 and 0.94 mol of phosphates, respectively, were incorporated per mol of 58-kDa protein. 58-kDa protein was not phosphorylated by either cAMP-dependent or cGMP-dependent protein kinase. When present in membranes, it was also phosphorylated by protein kinase C and CaM kinase II. Possible involvement of 58-kDa protein in the protein kinase C and CaM kinase II-mediated regulation of synaptic transmission in central and peripheral neurons is discussed.

Characteristics of brain injury-derived neurotrophic peptide-binding sites on rat brain synaptosomes and neurons in culture.

Brain injury-derived neurotrophic peptide is the fragmental 13-mer peptide of the novel neurotrophic factor which was extracted and purified from Sponge Gelform made of gelatin implanted at the mechanically-induced injury site in neonatal rat brains. Brain injury-derived neurotrophic peptide supports survival of septal cholinergic and mesencephalic dopaminergic neurons in culture, and rescues hippocampal neurons in culture from glutamate neurotoxicity. Here we studied the binding characteristics of brain injury-derived neurotrophic peptide to synaptosomes from normal adult rat brains and neurons in culture from neonatal rat brains. [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide binding to rat brain synaptosomes was specific and saturable. Equilibrium binding studies revealed that [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide bound to 1.1 pmol/mg protein with a Kd (dissociation constant) of 0.17 microM in hippocampal synaptosomes and to 2.0 pmol/mg protein with a Kd of 0.38 microM in septal synaptosomes. [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide could bind to a subpopulation of hippocampal neurons in culture from embryonic rat brains. Affinity cross-linking with the carboxyl-reactive cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-HCl and [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide produced radiolabeled bands corresponding to 100,000, 50,000 and 40,000 mol. wt molecules on hippocampal neurons in culture. These results suggest that the 13-mer sequence of brain injury-derived neurotrophic peptide plays a crucial role in expressing the neurotrophic properties of the factor.

Amphiphilic helix is essential for the activity of brain injury-derived neurotrophic peptide (BINP)

To study the structure‐activity relationships of brain injury‐derived neurotrophic peptide (BINP), 12 analogs were synthesized by replacing each amino acid residue with Gly. BINP showed CD spectra typical of an α‐helical conformation in TFE solution which mimics the membrane environment. In the α‐helical conformation, BINP showed an amphiphilic profile. Neurotrophic activities of BINP and its analogs were estimated from the effects on supporting septal cholinergic neurons and on rescuing hippocampal neurons from injury caused by glutamate. Both assays showed that the residues on the hydrophobic side of the amphiphilic helix were essential for the neurotrophic activity.

Development of an antibody against a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein and identification of a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein in hippocampal neurons.

Brain injury-derived neurotrophic peptide is a 13-amino acid peptide derived from a 15,000 mol. wt neurotrophic factor released from sites of mechanical injury in neonatal rat brain. This peptide promotes survival of septal cholinergic neurons and mesencephalic dopaminergic neurons, and protects hippocampal neurons from glutamate-induced neurotoxicity. In this study, we have developed a monoclonal antibody against a brain injury-derived neurotrophic peptide-binding protein by immunizing mice with septal synaptosomes from five-week-old rat brain. Monoclonal antibodies were screened for inhibition of the binding of a 125I-labeled analogue of brain injury-derived neurotrophic peptide to rat brain synaptosomes. The monoclonal antibody 6A22 suppressed the biological activity of brain injury-derived neurotrophic peptide and abolished the protective effect of the neurotrophic peptide against glutamate-induced neurotoxicity. This monoclonal antibody recognized a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein, which was also identified by cross-linking experiments. Immunohistochemical studies showed that the 6A22 antibody bound to the cell surfaces of a subpopulation (about 60%) of hippocampal neurons in culture. These results are consistent with the possibility that the 40,000 mol. wt protein belongs to brain injury-derived neurotrophic peptide receptors.

A new system for studying neuronal cell death using NG-108 cell and BINP

SHIGEKI FURUYA’, TOSHIHIDE TABATA 2.3,4, JUNYA MITOMA’, ASAMI MAKINO’, MASANOBU KANO**‘, 4. YOSHIO HIRABAYASHI’ ‘Lab. for Cellular Glycobiol., Frontier Res. Prog.. RIKEN, Wako, Saitama 35 I-0198, 2CREST. Japan Sci. & Tech. Corp. 3Lab. for Cellular Neurophysiol., Brain Sci. Inst., RIKEN, Wake, Saitama 35 I-019X. 4Dept. of Physiol., Kanazawa Univ. Schl. of Med.. Kanazawa, lshikawa 920-X640 Purkinje cell survival and dendritic development appear to be regulated by granule cell afferents. It has been demonstrated that purified Purkinje cells survive poorly and fail to extend dendrites in the absence of granule cells under a monolayer culture condition. Despite these observations, the molecular mechanism underlying the survival and dendritic development of Purkinje cells remains largely unknown. We observed poor Purkinje cell survival with undeveloped dendrites under a certain serum-free culture condition, even in the presence of granule cells. This impaired growth was overcome by the addition of a conditioned medium prepared from cerebellar astrocyte cultures (CACM), but not neurotrophins and other known neurotrophic factors. The addition of CACM produced a 3.0 to 3.5-fold increase in the number of Purkinje cells at 14 days in vitro. A conditioned medium prepared from hippocampal astrocytes also improved Purkinje cell survival and dendritic development to a similar extent. The growth-promoting activity in CACM is heat stable and recovered in the low molecular weight fraction (Mr < -3000). These results strongly suggest that Purkinje cells require an additional factor that is supplied by astrocytes for their survival and dendritic development. The molecular nature of the astrocyte-derived Purkinje cell growth-promoting factor is currently being investigated.

1316 Characterization of BINP (brain injury-derived neurotrophic peptide) binding protein in synaptosomes and cultures from rat brain

Sheng-Tian Li’ , Kunio Kato3, Katsuhiko Mikoshiba’ Long-term depression (LTD) in the CA1 area of rat hippocampal slices was studied. We employed a special perfusion solution composed of variable concentrations of Ca 2+ Mg2+ and K+ to substitute for low frequency , stimulation in order to induce LTD without electrical stimulation. LTD was successfully induced with this conditioning solution (Ca 2f: 4 mM; Mg2+: 0.1 mM; Kf: 5mM) applied for 20min (-28.5h8.8’30; N=12). The advantage of this protocol is that the whole slice is exposed to conditioning solution, and therefore LTD should be induced in all of the synapses, possibly yielding clearer results in biochemical studies than electrical stimulation-induced LTD. We tested the effect of protein phosphatase inhibitors, such as FK-506 and cyclosporine A on the LTD induced by the conditioning solution. We also investigated phosphatase activity during LTD induction by biochemical methods, and compared protein phosphatase activity before and after the induction of LTD.