Akinori Akaike

Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell–based transplantation therapies for retinal diseases.

α7 Nicotinic Receptor Transduces Signals to Phosphatidylinositol 3-Kinase to Block A β-Amyloid-induced Neurotoxicity

Multiple lines of evidence, from molecular and cellular to epidemiological, have implicated nicotinic transmission in the pathogenesis of Alzheimers disease (AD). Here we show the signal transduction mechanism involved in nicotinic receptor-mediated protection against β-amyloid-enhanced glutamate neurotoxicity. Nicotine-induced protection was suppressed by an α7 nicotinic receptor antagonist (α-bungarotoxin), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2). Levels of phosphorylated Akt, an effector of PI3K, and Bcl-2 were increased by nicotine. The α7 nicotinic receptor was physically associated with the PI3K p85 subunit and Fyn. These findings indicate that the α7 nicotinic receptor transduces signals to PI3K in a cascade, which ultimately contributes to a neuroprotective effect. This might form the basis of a new treatment for AD.

Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia?

Localization of the mRNAs for fractalkine, a CX3C chemokine, and for its receptor CX3CR1 was investigated in the rat brain. In situ hybridization study revealed that fractalkine mRNA was dominantly expressed in neuronal cells particularly in the olfactory bulb, cerebral cortex, hippocampus, caudate putamen and nucleus accumbens. In vitro study using enriched neuronal or glial culture supported the dominant expression of fractalkine mRNA in neurons. On the other hand, CX3CR1 mRNA was dominantly expressed in glial cells throughout the whole brain. The in vitro study suggested the cells expressing CX3CR1 mRNA are microglia, not astrocytes or neurons. Fractalkine appears to function as a signal molecule from neuron to microglia.

Phosphatidylinositol 3-kinase mediates neuroprotection by estrogen in cultured cortical neurons

It has been shown that estrogen replacement in menopausal women is effective in slowing down the progression of cognitive impairment in Alzheimers disease. Although recent studies have demonstrated the neuroprotective effects of estrogen, the precise mechanism of neuroprotection has not been elucidated. In the present study, we show that the phosphatidylinositol 3‐kinase (PI3‐K) cascade is involved in the neuroprotective mechanism stimulated by estrogen. Exposure to glutamate reduced the viability of rat primary cortical neurons. Pretreatment with 10 nM 17β‐estradiol significantly attenuated the glutamate‐induced toxicity. This neuroprotective effect of 17β‐estradiol was blocked by co‐administration with LY294002, a selective PI3‐K inhibitor, but not by co‐administration with PD98059, a selective mitogen activated protein kinase kinase inhibitor. Pretreatment with ICI182780, a specific estrogen receptor antagonist, also blocked the neuroprotection. Immunoblotting assay revealed that treatment with 17β‐estradiol induced the phosphorylation of Akt/PKB, an effector immediately downstream of PI3‐K. These results suggest that PI3‐K mediates the neuroprotective effect of 17β‐estradiol against glutamate‐induced neurotoxicity. J. Neurosci. Res. 60:321–327, 2000

Wnt Signaling Promotes Regeneration in the Retina of Adult Mammals

Regeneration in the mammalian CNS is severely limited. Unlike in the chick, current models hold that retinal neurons are never regenerated. Previously we demonstrated that, in the adult mammalian retina, Müller glia dedifferentiate and produce retinal cells, including photoreceptors, after acute neurotoxic injury in vivo. However, the number of newly generated retinal neurons is very limited. Here we demonstrate that Wnt (wingless-type MMTV integration site family)/β-catenin signaling promotes proliferation of Müller glia-derived retinal progenitors and neural regeneration after damage or during degeneration. Wnt3a treatment increases proliferation of dedifferentiated Müller glia >20-fold in the photoreceptor-damaged retina. Supplementation with retinoic acid or valproic acid induces differentiation of these cells primarily into Crx (cone rod homeobox)-positive and rhodopsin-positive photoreceptors. Notably, injury induces nuclear accumulation of β-catenin, cyclin D1 upregulation, and Wnt/β-catenin reporter activity. Activation of Wnt signaling by glycogen synthase kinase-3β inhibitors promotes retinal regeneration, and, conversely, inhibition of the signaling attenuates regeneration. This Wnt3a-mediated regeneration of retinal cells also occurs in rd mice, a model of retinal degeneration. These results provide evidence that Wnt/β-catenin signaling contributes to CNS regeneration in the adult mammal.

Estradiol protects mesencephalic dopaminergic neurons from oxidative stress-induced neuronal death.

Oxidative stress is important in the process of dopaminergic neuronal degeneration in Parkinsons disease. Recent studies suggest that estrogens have neuroprotective effects in neurodegenerative disorders, including Alzheimers disease. In the present study, we investigated neuroprotection against oxidative stress afforded by estradiol using primary neuronal culture of the rat ventral mesencephalon. Oxidative stress induced by glutamate, superoxide anions, and hydrogen peroxide caused significant neuronal death. Although simultaneous administration of 17β‐estradiol and glutamate did not show any significant effects, preincubation with 17β‐estradiol provided significant neuroprotection against glutamate‐induced neurotoxicity (ED50 was 50 μM for dopaminergic and 15 μM for nondopaminergic neurons). Neuroprotection occurred even after a brief preincubation with 17β‐estradiol and was not significantly blocked by either an estrogen receptor antagonist or a protein synthesis inhibitor. These findings indicate that the neuroprotection against glutamate neurotoxicity is mediated by neither estrogen receptors nor activation of genome transcription. Other steroids (corticosterone, testosterone, and cholesterol) did not provide significant neuroprotection against glutamate‐induced neurotoxicity. Furthermore, preincubation with 17β‐estradiol provided neuroprotection against neuronal death induced by both superoxide anions and hydrogen peroxide. Dichlorofluorescin diacetate, a marker of oxygen radicals, revealed that preincubation with 17β‐estradiol suppressed intracellular oxygen radicals induced by hydrogen peroxide. The biologically inactive stereoisomer of estradiol, 17α‐estradiol, provided neuroprotection against glutamate‐induced toxicity in dopaminergic neurons, as well as the17β isoform. 17α‐estradiol may be a potential therapeutic agent used to prevent dopaminergic neuronal death induced by oxidative stress in Parkinsons disease. J. Neurosci. Res. 54:707–719, 1998.

Microglia‐derived interleukin‐6 and leukaemia inhibitory factor promote astrocytic differentiation of neural stem/progenitor cells

Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. It has been recognized that astrocytes promote neurogenic differentiation of NSPCs, suggesting the importance of cell–cell interactions between glial cells and NSPCs. Recent studies have demonstrated that microglia, one type of glial cells, play an important role in neurogenesis. However, little is known about how activated microglia control the proliferation and differentiation of NSPCs. In this study, we investigated the possibility that microglia‐derived soluble factors regulate the behaviour of NSPCs. To this end, NSPCs and microglial cultures were obtained from rat embryonic day 16 subventricular zone (SVZ) and rat postnatal 1 day cortex, respectively, and the conditioned medium from microglia was prepared. Microglial‐conditioned medium had no significant effect on the proliferation of NSPCs. In contrast, it increased the percentage of cells positive for a marker of astrocytes, glial fibrillary acidic protein (GFAP) during differentiation. The induction of astrocytic differentiation by microglial‐conditioned medium was reduced by the inhibition of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) and mitogen‐activated protein kinase (MAPK) pathways. Furthermore, microglia‐derived interleukin (IL)‐6 and leukaemia inhibitory factor (LIF) were identified as essential molecules for this astrocytic differentiation using neutralizing antibodies and recombinant cytokines. Our results suggest that microglia as well as astrocytes contribute to the integrity of the local environment of NSPCs, and at least IL‐6 and LIF released by activated microglia promote astrocytic differentiation of NSPCs via the activation of the JAK/STAT and MAPK pathways.

Nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via α7-neuronal receptors and neuronal CNS receptors

We examined the effects of nicotine on glutamate-induced cytotoxicity using primary cultures of rat cortical neurons. The cell viability decreased significantly when cultures were exposed to glutamate for 10 min and then incubated with glutamate-free medium for 1 h. The exposure of cultures to nicotine (10 microM) for 8-24 h prior to glutamate application ameliorated the glutamate-induced cytotoxicity, with no significant effect of nicotine alone on the cell viability. Neuroprotection by nicotine was dependent on the incubation period. alpha-bungarotoxin (alpha-BTX) and methyllycaconitine (MLA), both of which are alpha7-neuronal receptor antagonists, and dihydro-beta-erythroidine (DHbetaE), a neuronal central nervous system (CNS) receptor antagonist, each significantly antagonized the protection by nicotine against glutamate-induced cytotoxicity. Ionomycin, a calcium ionophore, and S-nitrosocysteine (SNOC), a nitric oxide (NO) donor, also induced cytotoxicity in a manner similar to glutamate. Nicotine protected cultures against ionomycin-induced cytotoxicity, but not against SNOC-induced cytotoxicity. These results suggest that nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via alpha7-neuronal receptors and neuronal CNS receptors by reducing NO-formation triggered by Ca2+ influx.

Stimulation of α4β2 nicotinic acetylcholine receptors inhibits β-amyloid toxicity

In a regenerative air preheater of the stationary cylindrical regenerator chamber type, a rotatable heat-exchange element carrier is provided at the cold end of the regenerator chamber. The chamber wall has an aperture opposite the carrier through which access can be had to the heat-exchange elements in the carrier for their radial installation or removal.

Analgesia induced by microinjection of morphine into, and electrical stimulation of, the nucleus reticularis paragigantocellularis of rat medulla oblongata.

A dose-related analgesia was produced by microinjections of morphine into the nucleus reticularis paragigantocellularis (NRPG) of the medulla oblongata of the rat in doses of 0.15 – 15 nanograms of which the minimum dose approximates concentrations of morphine distributed in the medulla oblongata after systemic administration of analgesic doses of the opiate. The analgesia was antagonized by naloxone pretreatment. Focal electrical stimulation of the NRPG elicited an analgesia. It would thus appear that the NRPG is a highly sensitive site to morphine and electrical stimulation.