Geranylgeranylacetone Suppresses N-Methyl-N-nitrosourea-Induced Photoreceptor Cell Loss in Mice.

Retinitis pigmentosa is a disease characterized by the loss of photoreceptor cells. The N-methyl-N-nitrosourea (MNU)-induced retinal degeneration model is widely used to study the mechanism of these retinal degenerative disorders because of its selective photoreceptor cell death. As for the cell death mechanism of MNU, calcium-calpain activation and lipid peroxidation processes are involved in the initiation of this cell death. Although such molecular mechanisms of the MNU-induced cell death have been described, the total image of the cell death is still obscure. Heat shock protein 70 (HSP70) has been shown to function as a chaperon molecule to protect cells against environmental and physiological stresses. In this study, we investigated the effect of geranylgeranylacetone (GGA), an accylic polyisoprenoid, on MNU-induced photoreceptor cell loss. HSP70 induction by GGA was effective against MNU-induced photoreceptor cell loss as a result of its ability to prevent HSP70 degradation. The data indicate that GGA may help to suppress the onset and progression of retinitis pigmentosa.

Nitric Oxide Synthase Activation as a Trigger of N-methyl-N-nitrosourea-Induced Photoreceptor Cell Death.

Retinal degeneration (RD) such as retinitis pigmentosa and age-related macular degeneration are major causes of blindness in adulthood. As one of the model for RD, intraperitoneal injection of N-methyl-N-nitrosourea (MNU) is widely used because of its selective photoreceptor cell death. It has been reported that MNU increases intracellular calcium ions in the retina and induces photoreceptor cell death. Although calcium ion influx triggers the neuronal nitric oxide synthase (nNOS) activation, the role of nNOS on photoreceptor cell death by MNU has not been reported yet. In this study, we investigated the contribution of nNOS on photoreceptor cell death induced by MNU in mice. MNU significantly increased NOS activation at 3 day after treatment. Then, we evaluated the effect of nNOS specific inhibitor, ethyl[4-(trifluoromethyl) phenyl]carbamimidothioate (ETPI) on the MNU-induced photoreceptor cell death. At 3 days, ETPI clearly inhibited the MNU-induced cell death in the ONL. These data indicate that nNOS is a key molecule for pathogenesis of MNU-induced photoreceptor cell death.

Cell Fate of Müller Cells During Photoreceptor Regeneration in an N-Methyl-N-nitrosourea-Induced Retinal Degeneration Model of Zebrafish.

Zebrafish can regenerate several organs such as the tail fin, heart, central nervous system, and photoreceptors. Very recently, a study has demonstrated the photoreceptor regeneration in the alkylating agent N-methyl-N-nitrosourea (MNU)-induced retinal degeneration (RD) zebrafish model, in which whole photoreceptors are lost within a week after MNU treatment and then regenerated within a month. The research has also shown massive proliferation of Müller cells within a week. To address the question of whether proliferating Müller cells are the source of regenerating photoreceptors, which remains unknown in the MNU-induced zebrafish RD model, we employed a BrdU pulse-chase technique to label the proliferating cells within a week after MNU treatment. As a result of the BrdU pulse-chase technique, a number of BrdU(+) cells were observed in the outer nuclear layer as well as the inner nuclear layer. This implies that regenerating photoreceptors are derived from proliferating Müller cells in the zebrafish MNU-induced RD model.

Involvement of neuronal nitric oxide synthase in N-methyl-N-nitrosourea-induced retinal degeneration in mice.

N-methyl-N-nitrosourea (MNU) is widely used to study the mechanism of retinal degenerative diseases (RDs) because of its selectivity of photoreceptor cell death. Many reports suggest that excessive nitric oxide (NO) plays a crucial role in neuronal cell death. We hypothesized that nitric oxide synthase (NOS)/NO are involved in photoreceptor cell death by MNU. We found that the levels of NO increased after MNU treatment. Furthermore, we demonstrated that neuronal NOS specific inhibitor attenuated photoreceptor cell death by MNU in mice. We believe that our findings might be a new target for the treatment of RDs.

Upregulation of leukemia inhibitory factor (LIF) during the early stage of optic nerve regeneration in zebrafish.

Fish retinal ganglion cells (RGCs) can regenerate their axons after optic nerve injury, whereas mammalian RGCs normally fail to do so. Interleukin 6 (IL-6)-type cytokines are involved in cell differentiation, proliferation, survival, and axon regrowth; thus, they may play a role in the regeneration of zebrafish RGCs after injury. In this study, we assessed the expression of IL-6-type cytokines and found that one of them, leukemia inhibitory factor (LIF), is upregulated in zebrafish RGCs at 3 days post-injury (dpi). We then demonstrated the activation of signal transducer and activator of transcription 3 (STAT3), a downstream target of LIF, at 3-5 dpi. To determine the function of LIF, we performed a LIF knockdown experiment using LIF-specific antisense morpholino oligonucleotides (LIF MOs). LIF MOs, which were introduced into zebrafish RGCs via a severed optic nerve, reduced the expression of LIF and abrogated the activation of STAT3 in RGCs after injury. These results suggest that upregulated LIF drives Janus kinase (Jak)/STAT3 signaling in zebrafish RGCs after nerve injury. In addition, the LIF knockdown impaired axon sprouting in retinal explant culture in vitro; reduced the expression of a regeneration-associated molecule, growth-associated protein 43 (GAP-43); and delayed functional recovery after optic nerve injury in vivo. In this study, we comprehensively demonstrate the beneficial role of LIF in optic nerve regeneration and functional recovery in adult zebrafish.

Function of Sox2 in ependymal cells of lesioned spinal cords in adult zebrafish.

The sex-determining region Y-box 2 (Sox2) is related not only to pluripotency, but also to cell proliferation. Zebrafish can regain their motor function after spinal cord injury (SCI). Following SCI, new motor neurons are produced from proliferating ependymal cells. Here, we investigated the expression and function of Sox2 after SCI in zebrafish. Sox2 was upregulated as early as 1 day post-lesion (dpl) in ependymal cells, which was followed by cell proliferation. Sox2 knockdown significantly decreased the number of proliferating cells at 5dpl. The results of this study suggest a role of Sox2 as one of the proliferation initiators in ependymal cells after SCI.

Upregulation of anti-apoptotic factors in upper motor neurons after spinal cord injury in adult zebrafish.

Unlike mammals, fish motor function can recover within 6-8weeks after spinal cord injury (SCI). The motor function of zebrafish is regulated by dual control; the upper motor neurons of the brainstem and motor neurons of the spinal cord. In this study, we aimed to investigate the framework behind the regeneration of upper motor neurons in adult zebrafish after SCI. In particular, we investigated the cell survival of axotomized upper motor neurons and its molecular machinery in zebrafish brain. As representative nuclei of upper motor neurons, we retrogradely labeled neurons in the nucleus of medial longitudinal fasciculus (NMLF) and the intermediate reticular formation (IMRF) using a tracer injected into the lesion site of the spinal cord. Four to eight neurons in each thin sections of the area of NMLF and IMRF were successfully traced at least 1-15days after SCI. TUNEL staining and BrdU labeling assay revealed that there was no apoptosis or cell proliferation in the axotomized neurons of the brainstem at various time points after SCI. In contrast, axotomized neurons labeled with a neurotracer showed increased expression of anti-apoptotic factors, such as Bcl-2 and phospho-Akt (p-Akt), at 1-6days after SCI. Such a rapid increase of Bcl-2 and p-Akt protein levels after SCI was quantitatively confirmed by western blot analysis. These data strongly indicate that upper motor neurons in the NMLF and IMRF can survive and regrow their axons into the spinal cord through the rapid activation of anti-apoptotic molecules after SCI. The regrowing axons from upper motor neurons reached the lesion site at 10-15days and then crossed at 4-6weeks after SCI. These long-distance descending axons from originally axotomized neurons have a major role in restoration of motor function after SCI.