Hitomi Isago

Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa

Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.

Age-Dependent Differences in Recovered Visual Responses in Royal College of Surgeons Rats Transduced with the Channelrhodopsin-2 Gene

The objective of this study is to investigate age-related differences in recovered visual function in Royal College of Surgeons (RCS) rats transduced with the Channelrhodopsin-2 (ChR2) gene. An adeno-associated virus vector that contained ChR2 was injected intravitreously into young or aged RCS rats. After 4xa0months, visual evoked potentials were recorded. To estimate the transduction efficiencies, ChR2V-expressing cells and retrograde labeled retinal ganglion cells (RGCs) were counted. After photoreceptor degradation, immunohistochemistry was used to detect glial fibrillary acidic protein (GFAP) in the retinas. The amplitudes and latencies from young RCS rats were higher and shorter, respectively, than those from aged RCS rats. ChR2V was expressed in the RGCs of both groups of rats; there was no significant difference in the transduction efficiency of either group. However, the number of RGCs in aged RCS rats was significantly less than that in young RCS rats. In addition, strong GFAP immunoreactivity was observed after photoreceptor degeneration, whereas it was weaker in ChR2V-expressing RGCs. ChR2 transduction produced photosensitive RGCs in both young and aged rats. However, the degree of recovery depended on the age at the time of transduction.

Differentiation of neuronal cells from NIH/3T3 fibroblasts under defined conditions

We attempted to test whether the differentiated NIH/3T3 fibroblasts could be differentiated into neuronal cells without any epigenetic modification. First, a neurosphere assay was carried out, and we successfully generated neurosphere‐like cells by floating cultures of NIH/3T3 fibroblasts in neural stem cell medium. These spheres have the ability to form sub‐spheres after three passages, and express the neural progenitor markers Nestin, Sox2, Pax6, and Musashi‐1. Second, after shifting to a differentiating medium and culturing for an additional 8u2003days, cells in these spheres expressed the neuronal markers β‐tubulin and neurofilament 200 and the astrocytic marker glial fibrillary acidic protein (GFAP). Finally, after treating the spheres with all‐trans retinoic acid and taurine, the expression of β‐tubulin was increased and the staining of photoreceptor markers rhodopsin and recoverin was observed. The present study shows that NIH/3T3 fibroblasts can generate neurosphere‐like, neuron‐like, and even photoreceptor‐like cells under defined conditions, suggesting that the differentiated non‐neuronal cells NIH/3T3 fibroblasts, but not pluripotent cells such as embryonic stem cells or induced pluripotent stem cells, may have the potential to be transdifferentiated into neuronal cells without adding any epigenetic modifier. This transdifferentiation may be due to the possible neural progenitor potential of NIH/3T3 fibroblasts that remains dormant under normal conditions.

Notch signaling pathway regulates proliferation and differentiation of immortalized Müller cells under hypoxic conditions in vitro.

Previous studies have indicated that Müller glia in chick and fish retinas can re-enter the cell cycle, express progenitor genes, and regenerate neurons via the Notch signaling pathway in response to retinal damage or growth factors. Here, we investigated the role of Notch signaling and the effect of hypoxia, as a means to induce retinal damage, on the proliferation of an immortalized Müller cell line (rMC-1 cells). Our data showed that rMC-1 cells expressed Müller glia and neural and retinal progenitor markers but did not express neuronal or retinal markers. Hypoxia increased rMC-1 cell proliferation by activating the positive cell-cycle regulators, cyclins A and D1, as well as the neural and retinal progenitor markers, Notch1, Hes1, nestin, Sox2, Msi1, Pax6, and NeuroD1. However, hypoxia did not significantly influence the expression of Müller glial markers GS, CRALBP, and cyclin D3 or the death of the rMC-1 cells. The increase in cell proliferation induced by hypoxia was greatly attenuated by blocking Notch signaling with the inhibitor DAPT, resulting in the reduced expression of positive cell-cycle regulators (cyclins A and D1) and neural and retinal progenitor markers (Notch1, Hes1, Sox2, Pax6, and NeuroD1). Blockade of the Notch signaling pathway by DAPT after hypoxia promoted the differentiation of rMC-1 cells to neurons, as demonstrated by the induction of neural marker (Tuj1), retinal amacrine (Syntaxin1), and retinal ganglion cell (Brn3b) markers, although the expression of the latter marker was low. Taken together, our data indicate that Notch signaling is required for proliferation under hypoxic conditions either by activating the positive cell-cycle regulators or by skewing their de-differentiation towards a neural progenitor lineage. These findings indicate that the Notch signaling pathway regulates hypoxia-induced proliferation and differentiation of Müller glia.

Establishment of monocular-limited photoreceptor degeneration models in rabbits

BackgroundNumerous rodent models of photoreceptor degeneration have been developed for the study of visual function. However, no viable model has been established in a species that is more closely related to Homo sapiens. Here, we present a rabbit model of monocular photoreceptor degeneration.MethodsWe tested 2 chemicals, verteporfin and sodium nitroprusside (SNP), for developing a 1-eye limited photoreceptor degeneration model in pigmented rabbits. After the intravenous injection of verteporfin, the retina was exposed to light from a halogen lamp for 0, 10, 30, or 60 min. Alternately, 100 μL of various concentrations of sodium nitroprusside (0.1 mM, 0.5 mM, and 1 mM) were intravitreously injected into the rabbit eye. Retinal degeneration was evaluated by fundus photography, electroretinogram (ERG), and histological examinations.ResultsFundus photographs of animals in the verteporfin- or SNP-treated groups showed evidence of retinal degeneration. The severity of this degradation depended on the duration of light exposure and the concentration of SNP administered. The degeneration was clearly limited to the light-exposed areas in the verteporfin-treated groups. Extensive retinal atrophy was observed in the SNP-treated groups. The a- and b-wave amplitudes were dramatically decreased on the ERGs from SNP-treated groups. Histological examination revealed that either verteporfin or SNP induced severe photoreceptor degeneration. High-dose SNP treatment (1 mM) was also associated with inner retinal layer degeneration.ConclusionsBoth SNP and verteporfin clearly caused photoreceptor degeneration without any effect on the contralateral eye. These compounds therefore represent valuable tools for the empirical investigation of visual function recovery. The findings will inform guidelines for clinical applications such as retinal prostheses, cell-based therapy, and gene therapy.

Gene Therapy for Retinitis Pigmentosa

The retina comprises diverse differentiated neurons that have specific functions. Photoreceptor cells, the first-order neurons in the retina, have photopigments (rhodopsin and opsin) that absorb photons. Signals produced by the photoreceptor cells are transmitted to second-order neurons. Finally, visual signals are transmitted to the brain from the third-order neurons, the retinal ganglion cells (RGCs). Major diseases that cause blindness in advanced countries include glaucoma, diabetic retinopathy, retinitis pigmentosa (RP), and age-related retinop‐ athy. Loss of vision due to these diseases is irreversible. However, with regard to glaucoma, eye drops that have the effect of reducing intraocular pressure have been developed. In diabetic retinopathy, effective surgical treatments such as vitrectomy and photocoagulation have been established. Blindness due to glaucoma and diabetic retinopathy can be prevented by admin‐ istering these treatments in the early phase. On the other hand, in diseases caused by gene mutations, such as RP, effective treatments for delaying photoreceptor degeneration have not yet been established. Degeneration of photoreceptor cells results in loss of vision, even if other retinal neurons are intact [1-3].