Evaluation of Local Retinal Function in Light-Damaged Rats Using Multifocal Electroretinograms and Multifocal Visual Evoked Potentials.

Electroretinograms (ERGs) are often used to evaluate retinal function. However, assessing local retinal function can be challenging; therefore, photopic and scotopic ERGs are used to record whole-retinal function. This study evaluated focal retinal function in rats exposed to continuous light using a multifocal ERG (mfERG) system. The rats were exposed to 1000 lux of fluorescent light for 24 h to induce photoreceptor degeneration. After light exposure, the rats were reared under cyclic light conditions (12 h: 5 lux, 12 h: dark). Photopic and multifocal ERGs and single-flash and multifocal visual evoked potentials (mfVEPs) were recorded 7 days after light exposure. Fourteen days following light exposure, paraffin-embedded sections were prepared from the eyes for histological evaluation. The ERG and VEP responses dramatically decreased after 24 h of light exposure, and retinal area-dependent decreases were observed in mfERGs and mfVEPs. Histological assessment revealed severe damage to the superior retina and less damage to the inferior retina. Considering the recorded visual angles of mfERGs and mfVEPs, the degenerated area shown on the histological examinations correlates well with the responses from multifocal recordings.

Properties of a Single Amino Acid Residue in the Third Transmembrane Domain Determine the Kinetics of Ambient Light-Sensitive Channelrhodopsin.

Channelrhodopsins have been utilized in gene therapy to restore vision in patients with retinitis pigmentosa and their channel kinetics are an important factor to consider in such applications. We investigated the channel kinetics of ComV1 variants with different amino acid residues at the 172nd position. Patch clamp methods were used to record the photocurrents induced by stimuli from diodes in HEK293 cells transfected with plasmid vectors. The channel kinetics (τon and τoff) were considerably altered by the replacement of the 172nd amino acid and was dependent on the amino acid characteristics. The size of amino acids at this position correlated with τon and decay, whereas the solubility correlated with τon and τoff. Molecular dynamic simulation indicated that the ion tunnel constructed by H172, E121, and R306 widened due to H172A variant, whereas the interaction between A172 and the surrounding amino acids weakened compared with H172. The bottleneck radius of the ion gate constructed with the 172nd amino acid affected the photocurrent and channel kinetics. The 172nd amino acid in ComV1 is a key residue for determining channel kinetics as its properties alter the radius of the ion gate. Our findings can be used to improve the channel kinetics of channelrhodopsins.

SIG-1451, a Novel, Non-Steroidal Anti-Inflammatory Compound, Attenuates Light-Induced Photoreceptor Degeneration by Affecting the Inflammatory Process.

Age-related macular degeneration is a progressive retinal disease that is associated with factors such as oxidative stress and inflammation. In this study, we evaluated the protective effects of SIG-1451, a non-steroidal anti-inflammatory compound developed for treating atopic dermatitis and known to inhibit Toll-like receptor 4, in light-induced photoreceptor degeneration. SIG-1451 was intraperitoneally injected into rats once per day before exposure to 1000 lx light for 24 h; one day later, optical coherence tomography showed a decrease in retinal thickness, and electroretinogram (ERG) amplitude was also found to have decreased 3 d after light exposure. Moreover, SIG-1451 partially protected against this decrease in retinal thickness and increase in ERG amplitude. One day after light exposure, upregulation of inflammatory response-related genes was observed, and SIG-1451 was found to inhibit this upregulation. Iba-1, a microglial marker, was suppressed in SIG-1451-injected rats. To investigate the molecular mechanism underlying these effects, we used lipopolysaccharide (LPS)-stimulated rat immortalised Müller cells. The upregulation of C-C motif chemokine 2 by LPS stimulation was significantly inhibited by SIG-1451 treatment, and Western blot analysis revealed a decrease in phosphorylated I-κB levels. These results indicate that SIG-1451 indirectly protects photoreceptor cells by attenuating light damage progression, by affecting the inflammatory responses.

Development of an optogenetic gene sensitive to daylight and its implications in vision restoration.

Optogenetic gene-mediated therapy for restoring vision is thought to be a useful treatment for blind patients. However, light sensitivity achieved using this gene therapy is inferior to that of daylight vision. To increase light sensitivity, we designed three mutants using a bioinformatics approach. Nucleotide sequences encoding two sites in the extracellular loops (ex1, ex3) of mVChR1 close to simulated ion-conducting pathways were replaced by homologous amino acid-encoding sequences of ChR1 or ChR2. The light sensitivity of ex3mV1 was higher than that of mVChR1 at 405-617 nm. Visual responses were restored in Royal College of Surgeons rats with genetically degenerating photoreceptor cells transfected with ex3mV1Co, wherein transmembrane of sixth (TM6) in ex3mV1 was additionally replaced with the corresponding domain of CoChR; these rats responded to light in the order of µW/mm2. Thus, ex3mV1Co might be useful for the restoration of advanced visual function.

Phototoxicities Caused by Continuous Light Exposure Were Not Induced in Retinal Ganglion Cells Transduced by an Optogenetic Gene.

The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.

Geranylgeranyl acetone prevents glutamate-induced cell death in HT-22 cells by increasing mitochondrial membrane potential.

Geranylgeranyl acetone (GGA) protects against various types of cell damages by upregulating heat shock proteins. We investigated whether GGA protects neuronal cells from cell death induced by oxidative stress. Glutamate exposure was lethal to HT-22 cells which comprise a neuronal line derived from mouse hippocampus. This configuration is often used as a model for hippocampus neurodegeneration in vitro. In the present study, GGA protected HT-22 cells from glutamate-induced oxidative stress. GGA pretreatment did not induce heat shock proteins (Hsps). Moreover, reactive oxygen species increased to the same extent in both GGA-pretreated and untreated cells exposed to glutamate. In contrast, glutamate exposure and GGA pretreatment increased mitochondrial membrane potential. However, increases in intracellular Ca2+ concentration were inhibited by GGA pretreatment. In addition, the increase of phosphorylated ERKs by the glutamate exposure was inhibited by GGA pretreatment. These findings suggest that GGA protects HT-22 cells from glutamate-provoked cell death without Hsp induction and that the mitochondrial calcium buffering capacity plays an important role in this protective effect.

Neuroprotective effect of a dietary supplement against glutamate-induced excitotoxicity in retina.

AIM: To evaluate the neuroprotective effect of a dietary supplement (ClearVision EX®; CV) against glutamate-induced excitotoxicity in retina. METHODS: We evaluated the protective effects CV on glutamate-induced cell toxicity of an immortalized mouse hippocampal cell line (HT-22) in vitro and N-methyl-D-aspartate (NMDA) induced retinal injury in vivo. Once-daily oral administration of CV or vehicle (5% Arabic gum) was started the day before the NMDA injection and continued until the end of the study. Electroretinograms (ERGs) were recorded to evaluate the retinal function at 2d after NMDA injection. Furthermore, a histological evaluation, Western blot analysis, and immunohistochemistry were performed for assessing the signal transduction pathway. RESULTS: HT-22 cell death was induced by the addition of glutamate and co-incubation with CV protected against it. Oral administration of CV inhibited the decrease in scotopic threshold response amplitudes induced by the intravitreal injection of NMDA and those of the thickness of the inner retinal layer in the histological evaluation. The increased phosphorylated levels of extracellular signal-regulated kinase (ERK) but not cAMP response element binding protein (CREB) or Akt were observed 1h after NMDA injection in both the vehicle- and CV-treated rats; however, pERK activation was no more upregulated at 3h after NMDA injection. pERK upregulation was observed in Müller cells. CONCLUSION: CV shows a protective effect against both glutamate-induced HT-22 cell death and NMDA-induced retinal damage. pERK upregulation in the Müller cells plays a key role in the protective effect of CV against glutamate-induced retinal toxicity.

N-Methyl-N-Nitrosourea-Induced Photoreceptor Degeneration Is Inhibited by Nicotinamide via the Blockade of Upstream Events before the Phosphorylation of Signalling Proteins.

N-methyl-N-nitrosourea (MNU), a known carcinogen, is generally used in animal models to chemically induce photoreceptor degeneration. It has been reported that nicotinamide (NAM) exerts a protective effect on MNU-induced photoreceptor degeneration. We investigated the molecular mechanisms on MNU-induced photoreceptor degeneration. Intraperitoneal MNU injection (75 mg/kg) in rats induced selective photoreceptor degeneration in 7 days. NAM administration completely inhibited photoreceptor degeneration. Photoreceptor layer abnormality was observed within 6 hours after MNU injection, whereas it was restored in the NAM-treated retina, as detected by optical coherence tomography. One day following MNU administration, phosphorylation of the cell death-associated signalling proteins c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) increased, while the apoptosis-related proteins, full-length poly(ADP-ribose) polymerase (PARP) and apoptosis-inducing factor (AIF), were depleted. These changes were not observed in the NAM-treated retinas. Cell survival signalling, such as extracellular signal-regulated kinase (ERK), Akt, and cAMP response element binding protein (CREB) phosphorylation, increased in the MNU- but not in the NAM-treated rat retinas. Increased phosphorylated ERK (p-ERK) levels were observed within 6 hours after MNU administration, suggestive of cell survival signalling activation. This did not occur in NAM-treated retinas. These results indicate that NAM regulates upstream cellular events prior to the activation of cell death-related signalling events, such as JNK and p38 phosphorylation.

Natronomonas pharaonis halorhodopsin Ser81 plays a role in maintaining chloride ions near the Schiff base.

Optogenetic technologies have often been used as tools for neuronal activation or silencing by light. Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven chloride ion pump. Upon light absorption, a chloride ion passes through the cell membrane, which is accompanied by the temporary binding of a chloride ion with Thr126 at binding site-1 (BS1) near the protonated Schiff base in NpHR. However, the mechanism of stabilization of the binding state between a chloride ion and BS1 has not been investigated. Therefore, to identify a key component of the chloride ion transport pathway as well as to acquire dynamic information about the chloride ion-BS1 binding state, we performed a rough analysis of the chloride ion pathway shape followed by molecular dynamics (MD) simulations for both wild-type and mutant NpHR structures. The MD simulations showed that the hydrogen bond between Thr126 and the chloride ion was retained in the wild-type protein, while the chloride ion could not be retained at and tended to leave BS1 in the S81A mutant. We found that the direction of the Thr126 side chain was fixed by a hydroxyl group of Ser81 through a hydrogen bond and that Thr126 bound to a chloride ion in the wild-type protein, while this interaction was lost in the S81A mutant, resulting in rotation of the Thr126 side chain and reduction in the interaction between Thr126 and a chloride ion. To confirm the role of S81, patch clamp recordings were performed using cells expressing NpHR S81A mutant protein. Considered together with the results that the NpHR S81A-expressing cells did not undergo hyperpolarization under light stimulation, our results indicate that Ser81 plays a key role in chloride migration. Our findings might be relevant to ongoing clinical trials using optogenetic gene therapy in blind patients.

Kinetic profiles of photocurrents in cells expressing two types of channelrhodopsin genes.

Channelrhodopsin-2 (ChR2), a light-activated cation-selective ion channel, has been widely used as a tool in optogenetic research. ChR2 is specifically sensitive to wavelengths less than 550 nm. One of the methods to expand the sensitivity of a channelrhodopsin to a wider range of wavelengths is to express another channelrhodopsin in the cells by the transduction of an additional gene. Here, we report the characteristic features of cells expressing two types of channelrhodopsins, each having different wavelength sensitivities. In HEK293 cells stably expressing ChR2, photocurrents were elicited at stimuli of 400-550 nm, and the wavelength sensitivity range was expanded by the additional transduction of the modified Volvox channelrhodopsin-1 (mVChR1) gene, which has broad wavelength sensitivities, ranging from 400 to 600 nm. However, the photocurrent at 550 nm was lower than that of the mVChR1-expressing cell; moreover, the turning-on and turning-off constants were delayed, and the deactivation rates were decreased. Meanwhile, the response to lower light intensity was improved by the additional gene. Thus, the transduction of an additional gene is a useful method to improve the light and wavelength sensitivities, as well as photocurrent kinetic profiles, of channelrhodopsins.

Visual Responses of Photoreceptor-Degenerated Rats Expressing Two Different Types of Channelrhodopsin Genes.

Optogenetic technologies are expected to be applicable for clinical use in restoring vision. However, the degree of recovered visual function is highly dependent on the function of the chosen optogenetic gene. To investigate the effect on visual function of dual expression of genes with different wavelength sensitivities, we transduced a modified Volvox-derived channelrhodopsin gene (mVChR1) via an adeno-associated virus vector into transgenic rats harbouring the ChR2 gene in retinal ganglion cells. These transgenic rats were given an intraperitoneal injection of N-methyl-N-nitrosourea to induce the degeneration of native photoreceptor cells prior to transduction of mVChR1. Optical coherence tomography images indicated the degeneration of the native photoreceptor cells after the N-methyl-N-nitrosourea injection. Complete loss of function of the native photoreceptor cells was confirmed using electroretinograms. In the ChR2 transgenic rats, visually evoked potentials were clearly detectable in spite of native photoreceptor function abolishment; however the responses were limited to within blue wavelengths. In contrast, the limited wavelength sensitivities were improved by the additional transduction of mVChR1, which exhibited sensitivities to green and red. Thus, the transductions of dual genes encoding channelrhodopsins that exhibit different wavelength sensitivities represents a promising candidate method to expand and to enhance rescued wavelength sensitivities in blind subjects.

Improved transduction efficiencies of adeno-associated virus vectors by synthetic cell-permeable peptides.

Various serotypes of adeno-associated virus (AAV) vectors have been used for gene therapy and as research tools. Among these serotypes, the AAV type 2 vector has been used successfully in human gene therapies. However, the transduction efficiency of AAV2 depends on the cell type, and this poses a problem in the efficacy of gene therapy. To improve the transduction efficiency of AAV2, we designed a small peptide consisting of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor peptide and the HIV-Tat sequence Tat-Y1068. Pre- or co-treatment of CYNOM-K1 cells from cynomolgus monkey embryo skin with Tat-Y1068 increased the transduction efficiencies in a dose-dependent manner and caused p38 phosphorylation. The transduction efficiency of AAV2 into the rat fibroblast cell line RAT-1 highly expressing EGFR was less than the transduction efficiency of AAV2 into CYNOM-K1 cells. Tat-Y1068 increased the transduction efficiency in RAT-1 cells in the same manner as in CYNOM-K1 cells. In conclusion, cell-permeable peptides possessing the EGFR tyrosine kinase inhibitor function might serve as a useful ingredient of AAV2 vector solution for increasing the transduction efficiency of gene therapies.

Measurement of Electroretinograms and Visually Evoked Potentials in Awake Moving Mice.

The development of new treatments for intractable retinal diseases requires reliable functional assessment tools for animal models. In vivo measurements of neural activity within visual pathways, including electroretinogram (ERG) and visually evoked potential (VEP) recordings, are commonly used for such purposes. In mice, the ERG and VEPs are usually recorded under general anesthesia, a state that may alter sensory transduction and neurotransmission, but seldom in awake freely moving mice. Therefore, it remains unknown whether the electrophysiological assessment of anesthetized mice accurately reflects the physiological function of the visual pathway. Herein, we describe a novel method to record the ERG and VEPs simultaneously in freely moving mice by immobilizing the head using a custom-built restraining device and placing a rotatable cylinder underneath to allow free running or walking during recording. Injection of the commonly used anesthetic mixture xylazine plus ketamine increased and delayed ERG oscillatory potentials by an average of 67.5% and 36.3%, respectively, compared to unanesthetized mice, while having minimal effects on the a-wave and b-wave. Similarly, components of the VEP were enhanced and delayed by up to 300.2% and 39.3%, respectively, in anesthetized mice. Our method for electrophysiological recording in conscious mice is a sensitive and robust means to assess visual function. It uses a conventional electrophysiological recording system and a simple platform that can be built in any laboratory at low cost. Measurements using this method provide objective indices of mouse visual function with high precision and stability, unaffected by anesthetics.

Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina.

The transcription factor nuclear factor kappaB (NF-κB) plays various roles in cell survival, apoptosis, and inflammation. In the rat retina, NF-κB activity increases after exposure to damaging light, resulting in degeneration of photoreceptors. Here, we report that in dark-adapted rats exposed for 6 h to bright white light, the p65 subunit of retinal NF-κB translocates to the mitochondria, an event associated with a decrease in expression of cytochrome c oxidase subunit III (COX III). However, sustained exposure for 12 h depleted p65 from the mitochondria, and enhanced COX III expression. Treatment with the protective antioxidant PBN prior to light exposure prevents p65 depletion in the mitochondria and COX III upregulation during prolonged exposure, and apoptosis in photoreceptor cells. These results indicate that COX III expression is sensitive to the abundance of NF-κB p65 in the mitochondria, which, in turn, is affected by exposure to damaging light.

Restoration of the majority of the visual spectrum by using modified Volvox channelrhodopsin-1.

We previously showed that blind rats whose vision was restored by gene transfer of Chlamydomonas channelrhodopsin-2 (ChR2) could only detect wavelengths less than 540 nm because of the action spectrum of the transgene product. Volvox-derived channelrhodopsin-1, VChR1, has a broader spectrum than ChR2. However, the VChR1 protein was mainly localized in the cytoplasm and showed weak ion channel properties when the VChR1 gene was transfected into HEK293 cells. We generated modified Volvox channelrhodopsin-1 (mVChR1), which is a chimera of Volvox channelrhodopsin-1 and Chlamydomonas channelrhodopsin-1 and demonstrated increased plasma membrane integration and dramatic improvement in its channel properties. Under whole-cell patch clamp, mVChR1-expressing cells showed a photo-induced current upon stimulation at 468-640 nm. The evoked currents in mVChR1-expressing cells were ~30 times larger than those in VChR1-expressing cells. Genetically, blind rats expressing mVChR1 via an adeno-associated virus vector regained their visual responses to light with wavelengths between 468 and 640 nm and their recovered visual responses were maintained for a year. Thus, mVChR1 is a candidate gene for gene therapy for restoring vision, and gene delivery of mVChR1 may provide blind patients access to the majority of the visible light spectrum.

Essential role of thioredoxin 2 in mitigating oxidative stress in retinal epithelial cells.

The retina is constantly subjected to oxidative stress, which is countered by potent antioxidative systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems leads to the development of age-related macular degeneration. Thioredoxin 2 (Trx2) is a potent antioxidant, which acts directly on mitochondria. In the present study, oxidative stress was induced in the human RPE cell line (ARPE-19) using 4-hydroxynonenal (4-HNE) or C2-ceramide. The protective effect of Trx2 against oxidative stress was investigated by assessing cell viability, the kinetics of cell death, mitochondrial metabolic activity, and expression of heat shock proteins (Hsps) in Trx2-overexpressing cell lines generated by transfecting ARPE cells with an adeno-associated virus vector encoding Trx2. We show that overexpression of Trx2 reduced cell death induced by both agents when they were present in low concentrations. Moreover, early after the induction of oxidative stress Trx2 played a key role in the maintenance of the cell viability through upregulation of mitochondrial metabolic activity and inhibition of Hsp70 expression.