Jun Kosaka

Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury

Retinal ganglion cells (RGCs) die by apoptosis after optic nerve injury. A number of reports have separately shown changes in pro-apoptotic proteins such as the Bcl-2 family members following optic nerve injury. However, induction time of these apoptotic signals has not been identified due to different treatments of the optic nerve, and insufficient time intervals for measurements. Therefore, the stream of cell death signals is not well understood. In the present study, we systematically reinvestigated a detailed time course of these cell death/survival signals in the rat retina after optic nerve crush, to determine the signal cascade leading to RGC apoptosis. The most conspicuous changes detected in the retina were the rapid inactivation of phospho-Akt and phospho-Bad proteins 2-3 days after optic nerve damage, and the subsequent gradual activation of Bax protein and caspase-3 activity accompanied by cell loss of RGCs 6 days after nerve injury. Cellular localization of these molecular changes was limited to RGCs. Furthermore, amount of insulin-like growth factor-I (IGF-I), an activator of the phosphatidyl inositol-3-kinase (PI3K)/Akt system, was initially decreased from RGCs 1-2 days just prior to the inactivation of phospho-Akt by optic nerve crush. Conversely, supplementation with IGF-I into the rat retina induced upregulation of phospho-Akt expression and cell survival of RGCs both in vitro and in vivo. Thus, injury to the optic nerve might induce early changes in cellular homeostasis with a plausible loss of trophic support for injured RGCs. Actually, IGF-I drastically enhanced neurite outgrowth from adult rat RGCs via a wortmannin-dependent mechanism in a retinal explant culture. Our data strongly indicate that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stage of optic nerve injury.

Up-regulation of Hrk, a regulator of cell death, in retinal ganglion cells of axotomized rat retina.

Hrk, a regulator of cell death, belongs to the family of pro-apoptotic BH3-only proteins and is known to induce apoptosis in nerve tissue. We examined Hrk gene expression to clarify the role of BH3-only proteins in apoptosis of retinal ganglion cells (RGCs) after optic nerve transection in the adult rat. Reverse transcription-polymerase chain reaction showed that Hrk was up-regulated in retina from 12 h after axotomy, and continued to be elevated for 1 week. In situ hybridization histochemistry revealed that Hrk was expressed in a sub-population of axotomized RGCs. These results suggest that Hrk is involved in the induction of apoptosis in RGCs after optic nerve transection.

Doublecortin expression continues into adulthood in horizontal cells in the rat retina.

The doublecortin (DCX) protein is associated with microtubules, and is essential for neuronal migration, differentiation, and plasticity. In mammals, it is expressed in developing neurons and new immature neuroblasts in the adult brain, but not generally in mature neurons. In the retina, doublecortin is detectable as early as embryonic day 15 (E15), is highly expressed between E18 and E20, and is poorly expressed postnatally. In this study, we investigated immunohistochemically the expression and cellular localization of doublecortin in the adult rat retina. Doublecortin was expressed in the outer plexiform layer (OPL), and in cells in the outer border of the inner nuclear layer (INL). No other layers were labeled by anti-doublecortin antibodies. In double-labeling experiments, doublecortin expression co-localized with the expression of the marker for horizontal cells, calbindin D. By contrast, the marker for immature neuroblasts, polysialylated neural cell-adhesion molecule, was not expressed in horizontal cells. These results suggest that either horizontal cells have the capacity to continuously remodel their neurites or doublecortin has a different function in horizontal cells from the control of neuronal plasticity that it is known to modulate other neurites. In addition, doublecortin might be an alternative molecular marker for horizontal cells in the adult rat retina.

The influence of the timing of administration of thiopentone sodium on nitric oxide-mediated neurotoxicity in vitro

Thiopentone sodium is a highly useful pharmacological agent that provides a neuroprotection against cerebral ischaemia. Since not all patients can receive thiopentone sodium before cerebral ischaemia occurs, we investigated the influence of timing of thiopentone sodium administration on the neurotoxicity induced by nitric oxide (NO) using Shibutas established model of primary brain cultures. Cortical neurones prepared from 16-day gestational rat foetuses were used after 13-14 days in culture. The cells were exposed to an NO-donor, NOC-5 at 30 microM. Thiopentone sodium administered at 30 and 10 min before or 5, 10 and 15 min after exposure to NOC-5, but not thereafter, significantly attenuated NO-induced neurotoxicity compared with controls. The survival rate of the neurones in which thiopentone sodium was administered at 15 min after exposure to NOC-5 was 55.7+/-2.4%, compared to a 10.0+/-1.6% survival rate in neurones when thiopentone sodium was administered at 30 min after exposure to NOC-5. These findings demonstrate that thiopentone sodium, which protects cerebral cortical neurones against NO-mediated cytotoxicity, should be given as soon as possible in case ischaemic or hypoxic neuronal damage is predicted.

JNK inhibitory kinase is up-regulated in retinal ganglion cells after axotomy and enhances BimEL expression level in neuronal cells

Optic nerve transection results in retinal ganglion cell (RGC) death in adult mammals, after the alteration of gene expression of RGCs. To elucidate the molecular mechanism by which axotomy induces RGC death, we isolated the molecules up‐regulated after optic nerve transection. One of these, axomtomy‐related gene (ARG)357, an 895‐amino‐acid protein containing a complete serine–threonine kinase domain, was isolated from a subtraction library of the rat retina. The sequence showed that this gene was a rat homolog of human c‐Jun N‐terminal kinase (JNK) inhibitory kinase and so belonged to the germinal center kinase‐VIII subfamily of Sterile20s protein kinase. We designated ARG357 as rat JNK inhibitory kinase (JIK). Rat JIK was expressed ubiquitously in various tissues and was highly expressed in the retina, with selective expression in RGCs. After axotomy, BimEL and Hrk, which are BH3‐only proteins, and rat JIK were up‐regulated in RGCs. Overexpression of rat JIK in neuronal cells up‐regulated the expression of BimEL, but not that of Hrk. These results indicate that JIK may contribute to axotomy‐induced RGC death by up‐regulating the expression of BH3‐only protein.

Loss of Ahi1 Affects Early Development by Impairing BM88/Cend1-Mediated Neuronal Differentiation

Mutations in the Abelson helper integration site-1 (AHI1) gene result in N-terminal Ahi1 fragments and cause Joubert syndrome, an autosomal recessive brain malformation disorder associated with delayed development. How AHI1 mutations lead to delayed development remains unclear. Here we report that full-length, but not N-terminal, Ahi1 binds Hap1, a huntingtin-associated protein that is essential for the postnatal survival of mice and that this binding is regulated during neuronal differentiation by nerve growth factor. Nerve growth factor induces dephosphorylation of Hap1A and decreases its association with Ahi1, correlating with increased Hap1A distribution in neurite tips. Consistently, Ahi1 associates with phosphorylated Hap1A in cytosolic, but not in synaptosomal, fractions isolated from mouse brain, suggesting that Ahi1 functions mainly in the soma of neurons. Mass spectrometry analysis of cytosolic Ahi1 immunoprecipitates reveals that Ahi1 also binds Cend1 (cell cycle exit and neuronal differentiation protein 1)/BM88, a neuronal protein that mediates neuronal differentiation and is highly expressed in postnatal mouse brain. Loss of Ahi1 reduces the levels of Cend1 in the hypothalamus of Ahi1 KO mice, which show retarded growth during postnatal days. Overexpressed Ahi1 can stabilize Cend1 in cultured cells. Furthermore, overexpression of Cend1 can rescue the neurite extension defects of hypothalamic neurons from Ahi1 KO mice. Our findings suggest that Cend1 is involved in Ahi1-associated hypothalamic neuronal differentiation in early development, giving us fresh insight into the mechanism behind the delayed development in Joubert syndrome.

C38, equivalent to BM88, is developmentally expressed in maturing retinal neurons and enhances neuronal maturation

J. Neurochem. (2010) 112, 1235–1248.

Quantification of In Situ Hybridization Signals in Rat Testes

We performed basic research into quantifying in situ hybridization (ISH) signals in rat testis, a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA in paraffin sections. Specimens fixed with Bouins fixative and hybridized with digoxygenin-labeled probes could easily be analyzed quantitatively through “posterization” of the images. The amount of rRNA hybridized with the probe was greatest in early primary spermatocytes, followed by pachytene primary spermatocytes, then diplotene spermatocytes, and finally by secondary spermatocytes and spermatids. The amounts reached low levels in metaphase, anaphase, and telophase of meiotic division and early step 1 spermatids, and then slightly increased during spermiogenesis. ISH rRNA staining was a useful parameter for evaluation of the quantitative analysis of mRNA and the levels of hybridizable RNA in tissue sections. (J Histochem Cytochem 52:813–820, 2004)

Comparative morphology and development of extra-ocular muscles in the lamprey and gnathostomes reveal the ancestral state and developmental patterns of the vertebrate head

The ancestral configuration of the vertebrate head has long been an intriguing topic in comparative morphology and evolutionary biology. One peculiar component of the vertebrate head is the presence of extra-ocular muscles (EOMs), the developmental mechanism and evolution of which remain to be determined. The head mesoderm of elasmobranchs undergoes local epithelialization into three head cavities, precursors of the EOMs. In contrast, in avians, these muscles appear to develop mainly from the mesenchymal head mesoderm. Importantly, in the basal vertebrate lamprey, the head mesoderm does not show overt head cavities or signs of segmental boundaries, and the development of the EOMs is not well described. Furthermore, the disposition of the lamprey EOMs differs from those the rest of vertebrates, in which the morphological pattern of EOMs is strongly conserved. To better understand the evolution and developmental origins of the vertebrate EOMs, we explored the development of the head mesoderm and EOMs of the lamprey in detail. We found that the disposition of lamprey EOM primordia differed from that in gnathostomes, even during the earliest period of development. We also found that three components of the paraxial head mesoderm could be distinguished genetically (premandibular mesoderm: Gsc+/TbxA–; mandibular mesoderm: Gsc–/TbxA–; hyoid mesoderm: Gsc–/TbxA+), indicating that the genetic mechanisms of EOMs are conserved in all vertebrates. We conclude that the tripartite developmental origin of the EOMs is likely to have been possessed by the latest common ancestor of the vertebrates. This ancestor’s EOM developmental pattern was also suggested to have resembled more that of the lamprey, and the gnathostome EOMs’ disposition is likely to have been established by a secondary modification that took place in the common ancestor of crown gnathostomes.

Only extra-high dose of ketamine affects l-glutamate-induced intracellular Ca2+ elevation and neurotoxicity ☆

The neurotoxic effects of anesthetics on the developing brain are a concern. Although most of the anesthetics are GABAA agonists or NMDA antagonists, the differences in these effects on prospective glutamate-neurotoxicity in the brain is not fully understood. We examined the degree of L-glutamate-induced intracellular calcium ([Ca(2+)]i) elevation and neurotoxicity in neurons exposed to anesthetics. Primary cortical neurons from E17 rats were preincubated with 1-100 μM of ketamine or thiopental sodium (TPS) for the first 72 h of culturing. Two weeks later, the neurons were exposed to L-glutamate. The extent of glutamate toxicity was evaluated using Ca(2+)-imaging and morphological experiments. Preincubation with 100 μM ketamine but not with other concentrations of ketamine and TPS for the first 72 h in culture significantly enhanced L-glutamate-induced [Ca(2+)]i elevation 2 weeks later. Morphology experiments showed that vulnerability to L-glutamate-mediated neurotoxicity was only altered in neurons preincubated with 100 μM ketamine but not with TPS. Although preincubation with high concentration of ketamine showed enhancement of L-glutamate-induced [Ca(2+)]i elevation 2 weeks later, long-term exposure to TPS or ketamine at clinical doses during developmental periods may not result in a dose-related potentiation of exogenous glutamate-induced neurotoxicity, once the intravenous anesthetics are discontinued.