Kayoko Yamashita

Contribution of mitogen-activated protein kinases to NMDA-induced neurotoxicity in the rat retina

We examined the contributions of the mitogen-activated protein kinases (MAPKs) family [extracellular signal-regulated kinase (ERK), p38 kinase (p38), and c-Jun N-terminal kinase (JNK)] to N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. Detection of apoptotic cell death in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining began 6 h after intravitreal NMDA (100 nmol) injection and continued to increase thereafter. Western blot analysis showed that phosphorylated MAPKs (p-MAPKs) were expressed in the retina following a temporal manner: maximal expression of phosphorylated ERK (p-ERK) at 1 h, maximal expression of phosphorylated p38 (p-p38) at 6 h, and beginning of phosphorylated JNK (p-JNK) significant increase at 6 h after injection. An immunohistochemical/TUNEL co-localization study showed that p-JNK- and p-p38-positive cells in the RGCL were frequently TUNEL-positive, whereas few p-ERK-positive cells were TUNEL-positive. Moreover, co-injection of inhibitors for JNK (0.2 nmol SP600125) and/or p38 (2.0 nmol SB203580) with NMDA was effective in ameliorating NMDA-induced apoptotic cell loss in the RGCL 12 h after injection, as shown by TUNEL-positive cell counts. These inhibitors also protected the inner retina as shown by morphometric studies such as cell counts in the RGCL and measurement of the IPL thickness 7 days after injection. On the other hand, an ERK inhibitor (2.0 nmol U0126) did not suppress NMDA-induced cell death in the RGCL nor thinning of the IPL. These findings suggest that JNK and p38 are proapoptotic in NMDA-induced cell death in the RGCL, but not ERK.

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

SummarySnell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.

Developmental changes in the localization of activated C-JUN N-terminal kinase (JNK/SAPK) in the chick spinal cord.

To examine the role of c‐Jun N‐terminal kinase (JNK/SAPK) in the developing nervous system of vertebrates, the localization of an active form of JNK, phosphorylated JNK (p‐JNK), was studied in the lumbosacral spinal cord of the chick embryo. We also examined the localization of phosphorylated neurofilaments (NFs, potential targets of p‐JNK) and cyclin‐dependent kinase 5 (Cdk5), which is known to phosphorylate cytoskeletal proteins, including NFs, and compared their expression with that of p‐JNK. Additionally, the localization of phosphorylated forms of c‐Jun and ATF‐2 was compared with that of p‐JNK. On embryonic day 3 (E3), the expression of p‐JNK was observed in regions containing early‐projecting axons. Axons in these regions also expressed phosphorylated NFs. Subsequently, on E5 and E8, the expression of both p‐JNK and phosphorylated NFs increased concomitantly in the axonal tracts in the spinal white matter. Thus, white matter expressed both p‐JNK and phosphorylated NFs, whereas there was only weak expression of Cdk5. By E13, the spinal cord expression pattern of p‐JNK and phosphorylated NFs had changed compared to earlier ages. Although phosphorylated NFs were still expressed in the white matter, the expression of p‐JNK was decreased in axons in the white matter, whereas strong p‐JNK expression appeared in cell nuclei in the gray matter. In summary, the present study revealed that the localization of p‐JNK in the spinal cord changes dramatically from axons to cell nuclei during development, suggesting multiple roles of p‐JNK, depending on the developmental age. J. Comp. Neurol. 426:622–631, 2000.

Mechanisms of neurite elongation induced by proteasome inhibitor

RITSLJKO OHTANI-KANEKO’: MAKOTO YOKOSUKA’, MASAYUKl IIGO’, MASAYUKI HARA’, KAYOKO YAMASHITA’. KOJI I‘AKADA’. SEIICHI KAWASHIMA’. & KAZUAKI HIRATA’ ‘Dept. of Anatomy, St. Marianna Univ. Sch. of Med., Miyamae-ku. Kawasaki 2 1 h-851 1. ‘Dep. of-Biochem.( I )_ Jikei Univ. Sch. of Med., Minato-ku. l‘okyo 105-8461, “Dept. of Mol. Biol.. Rinsho-ken. Hunkyo-ku, l‘olqo 113-8613 While proteasome inhibitors have been known to Induce neurite elongation of many neuroblastoma cells, its mechamsms still remain unclear. In the present study. vve investigated the contribution of protein synthesis as vvell as phosphonlation and polymerization of cytoskeletal proteins to neurite elongation caused by proteasome inhibitors. Lactacystin or benzyloxycarbonyl-Leu-Leu-Ieucinal (%l.I.I.al) was used as a proteasome inhibitor m this study. PC12h cells were treated with actinomycin D (ActD) or cycloheximide (CHX) to inhibit protein systhesis in combination with a proteasome inhibitor. CHX inhibited neurite elongation nearly to the control level. though the cells could elongated their neurites after ActI)treatment. Studies on phosphorylation of neurofilament proteins re\,ealed that phosphoi-ylated neurofilament Hand Msubunits were increased by. proteasome inhibitors. In order to examine the involvement of phosphoFlation of neurofilaments and polymerization of microtubules m neurite elongation. effects of a cdc2 protein kinase inhibitor (olomoucine). vvhrch also inhibits ~33”‘“’ ~25 kinase. and colchicine vvere also studied