Increased lentivirus titer using ultra expression vectors.

Lentivirus is an efficient gene transfer system that is widely used in basic science. We aimed to improve viral titer by applying an ultra-expression vectors to lentiviral packaging. Application of the ultra-expression vectors increased biological titer 4 times for standard preparation. We also evaluated the efficacy of the ultra-expression vectors to relatively longer insert fragments, such as CSII-CMV-CNROE containing 5 genes in multiple cloning sites. Packaging of the ultra-expression vectors showed 3.5 times higher biological titer compared with the original method. Our improved packaging system could be applied to lentivirus to produce higher titers.

Sheep-derived cell immortalization through the expression of cell cycle regulators and biological characterization using transcriptomes.

Sheep are important domestic animals for the production of wool and meat. Although numerous cultured cell lines from humans and mice have been established, the number of cell lines derived from sheep is limited. To overcome this issue, the efficient establishment of a sheep-derived cell line and its biological characterization is reported. Mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase were introduced into sheep muscle-derived cells in an attempt to immortalize primary cells using the K4DT method. Furthermore, the SV40 large T oncogene was introduced into the cells. The successful immortalization of sheep muscle-derived fibroblasts was shown using the K4DT method or SV40 large T antigen. Furthermore, the expression profile of established cells showed close biological characteristics of ear-derived fibroblasts. This study provides a useful cellular resource for veterinary medicine and cell biology.

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.

Optogenetics-Mediated Gene Therapy for Retinal Diseases.

The visual system consists of various types of neurons and a single-nucleotide mutation can sometimes lead to blindness. The phototransduction pathway in the retina starts from the first-order neurons, the photoreceptor cells, and transmits the signals to second-order neurons. Finally, the output signal from third-order neurons, the ganglion cells, is carried to the brain. The photoreceptor cells are the only neurons in the retina that can respond to a light signal; they are hyperpolarised when they receive light, and the ganglion cells carry the signals to the brain following the depolarization. Recently, various types of channelrhodopsins have been found and developed. It is expected that the gene therapies using the cation channel as well as anion channelrhodopsin genes would be effective against diseases that cause severe destruction of visual function, such as the retinitis pigmentosa and age-related macular degeneration. In this review, we mainly describe mVChR1-mediated gene therapy for retinitis pigmentosa and the future application of optogenetic genes in retinal diseases.

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.

Presence of ES1 homolog in the mitochondrial intermembrane space of porcine retinal cells.

ES1 homologs are conserved among prokaryotes and eukaryotes, and the gene expression of ES1 homologs has been confirmed in diverse mammalian tissues. However, the localization and function of mammalian ES1 proteins remain poorly understood. ES1 protein was found specifically expressed in the cone cells of zebrafish and was proposed to contribute to the formation of mega mitochondria. We also observed mega mitochondria in the cone cells of porcine retinas, which raised the question regarding the localization of the porcine ES1. Therefore, in the present study, we aimed to determine the localization of ES1 in porcine retinas. We prepared a rabbit polyclonal antibody against the ES1 C-terminal and performed western blotting analysis and immunoelectron microscopy. The ES1 was found to be localized mainly in the mitochondrial intermembrane space of the porcine retinal cells. Immunopositive signals for ES1 were observed in the mitochondria of almost all retinal cells, and not specifically in cone cells. Our results and the ES1 sequences indicated that the glyoxalase III activity of ES1 might contribute to the stable functionality of the active mitochondria in a protective manner.

Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress.

Over 11 million people in the United States alone have some form of age-related macular degeneration (AMD). Oxidative stress, cell death, and the degeneration of retinal pigment epithelial (RPE) cells contribute to AMD pathology. Recent evidence suggests that ceramide (Cer), a cellular sphingolipid mediator that acts as a second messenger to induce apoptosis, might play a role in RPE cell death. The lysosomal breakdown of Cer by acid ceramidase [N-acylsphingosine amidohydrolase (ASAH)1] into sphingosine (Sph) is the major source for Sph 1-phosphate production, which has an opposing role to Cer and provides cytoprotection. Here, we investigated the role of Cer in human RPE-derived ARPE19 cells under hydrogen peroxide-induced oxidative stress, and show that Cer and hexosyl-Cer levels increase in the oxidatively stressed ARPE19 cells, which can be prevented by overexpression of lysosomal ASAH1. This study demonstrates that oxidative stress generates sphingolipid death mediators in retinal cells and that induction of ASAH1 could rescue retinal cells from oxidative stress by hydrolyzing excess Cers.

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.

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.

The poly-cistronic expression of four transcriptional factors (CRX, RAX, NEURO-D, OTX2) in fibroblasts via retro- or lentivirus causes partial reprogramming into photoreceptor cells.

The introduction of four key transcriptional factors (CRX, RAX, NEURO-D, OTX2) allows the direct differentiation of fibroblasts to retinal photoreceptor cells. This reprogramming was achieved with a combination of mono-cistronic viruses. Although the combination of mono-cistronic viruses was useful, a relatively high titer of recombinant viruses was necessary because co-infections are required. To overcome this issue, we established a poly-cistronic expression system for direct reprogramming and analyzed the biological characteristics of introduced cells after the exogenous introduction. The coding region of four reprogramming factors and EGFP (CRX, RAX, NEURO-D, OTX2, and EGFP; CNROE) was inserted into multiple sites of the pMYs-IP retrovirus or CSII-CMV lentivirus vector. The recombinant viruses were exposed to HE16 human embryonic fibroblasts. The expression levels of cone related genes were detected with real-time PCR. We detected the activation of two of the photoreceptor-related genes after the poly-cistronic expression of CRX, RAX, NEURO-D, and OTX2, but the rest of the genes did not exhibit transcriptional elevation. We concluded that the poly-cistronic expression of CNROE induced partial reprogramming into photoreceptor cells. We hypothesize that the direct reprogramming into photoreceptor cells might require relatively high protein expression levels of transcriptional factors.

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.

The protection of rat retinal ganglion cells from ischemia/reperfusion injury by the inhibitory peptide of mitochondrial µ-calpain.

Intracellular Ca(2+)-dependent cysteine proteases such as calpains have been suggested as critical factors in retinal ganglion cell (RGC) death. However, it is unknown whether mitochondrial calpains are involved in RGC death. The purpose of the present study was to determine whether the inhibition of mitochondrial µ-calpain activity protects against RGC death during ischemia/reperfusion (I/R) injury. This study used a well-established rat model of experimental acute glaucoma involving I/R injury. A specific peptide inhibitor of mitochondrial µ-calpain, Tat-µCL, was topically applied to rats via eye drops three times a day for 5 days after I/R. RGC death was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The truncation of apoptosis-inducing factor (AIF) was determined by western blot analyses. Retinal morphology was determined after staining with hematoxyline and eosin. In addition, the number of Fluoro Gold-labeled RGCs in flat-mounted retinas was used to determine the percentage of surviving RGCs after I/R injury. After 1 day of I/R, RGC death was observed in the ganglion cell layer. Treatment with Tat-µCL eye drops significantly prevented the death of RGCs and the truncation of AIF. After 5 days of I/R, RGC death decreased by approximately 40%. However, Tat-µCL significantly inhibited the decrease in the retinal sections and flat-mounted retinas. The results suggested that mitochondrial µ-calpain is associated with RGC death during I/R injury via truncation of AIF. In addition, the inhibition of mitochondrial µ-calpain activity by Tat-µCL had a neuroprotective effect against I/R-induced RGC death.

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.

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.

Proteolysis of mitochondrial calpain-13 in cerebral ischemia-reperfusion injury.

Calpains are calcium-dependent cysteine proteases activated by intracellular Ca2+. Although calpains mainly exist in the cytosol, calpain-13 is present in the mitochondria in mouse brains; however, the enzymatic properties and physiological functions of calpain-13 remain unknown. Hence, in this study, we predicted and evaluated the enzymatic properties of calpain-13. Based on our bioinformatic approaches, calpain-13 possessed a catalytic triad and EF-hand domain, similar to calpain-1, a well-studied calpain. Therefore, we hypothesized that calpain-13 had calpain-1-like enzymatic properties; however, calpain-13 was not proteolyzed in C57BL/6J mouse brains. Subsequently, cerebral ischemia/reperfusion (I/R) injury caused proteolysis of mitochondrial calpain-13. Thus, our study showed that mitochondrial calpain-13 was proteolyzed in the mitochondria of the I/R injured mouse brain. This finding could be valuable in further research elucidating the involvement of calpain-13 in cell survival or death in brain diseases, such as cerebral infarction.

Establishment of immortalized Egyptian Rousettus bat cell lines.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin-dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung-derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart-derived SV40 cell lines had aberrant karyotypes and the young bat-derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

Role of calpain-5 in cerebral ischemia and reperfusion injury.

BACKGROUND: Ischemia and reperfusion (I/R) injury exacerbate the prognosis of ischemic diseases. The cause of this exacerbation is partly a mitochondrial cell death pathway. Mitochondrial calpain-5 is proteolyzed/autolyzed under endoplasmic reticulum stress, resulting in inflammatory caspase-4 activation. However, the role of calpain-5 in I/R injury remains unclear. We hypothesized that calpain-5 is involved in ischemic brain disease. METHODS: Mitochondria from C57BL/6J mice were extracted via centrifugation with/without proteinase K treatment. The expression and proteolysis/autolysis of calpain-5 were determined using western blotting. The mouse and human brains with I/R injury were analyzed using hematoxylin and eosin staining and immunohistochemistry. HT22 cells were treated with tunicamycin and CAPN5 siRNA. RESULTS: Calpain-5 was expressed in the mitochondria of mouse tissues. Mitochondrial calpain-5 in mouse brains was responsive to calcium earlier than cytosolic calpain-5 in vitro calcium assays and in vivo bilateral common carotid artery occlusion model mice. Immunohistochemistry revealed that neurons were positive for calpain-5 in the normal brains of mice and humans. The expression of calpain-5 was increased in reactive astrocytes at human infarction sites. The knockdown of calpain-5 suppressed of cleaved caspase-11. CONCLUSIONS: The neurons of human and mouse brains express calpain-5, which is proteolyzed/autolyzed in the mitochondria in the early stage of I/R injury and upregulated in reactive astrocytes in the end-stage. GENERAL SIGNIFICANCE: Our results provide a comprehensive understanding of the mechanisms underlying I/R injury. Targeting the expression or activity of mitochondrial calpain-5 may suppress the inflammation during I/R injuries such as cerebrovascular diseases.

Characterization of Common Minke Whale (Balaenoptera Acutorostrata) Cell Lines Immortalized with the Expression of Cell Cycle Regulators.

Primary cultured cells cannot proliferate infinite. The overcoming of this limit can be classified as immortalization. Bypass of p16 senescence protein induces efficient immortalization various types of mammalians is previously reported. However, the Cetacea species is not known. Here, that common minke whale-derived cells can be immortalized with a combination of human genes, mutant cyclin-dependent kinase 4 (CDK4R24C ), cyclin D1, and Telomerase Reverse Transcriptase (TERT) is reported. These results indicate that the function of cell cycle regulators in premature senescence is evolutionarily conserved. This study describes the conserved roles of cell cycle regulators in the immortalization of cells from humans to Cetacea species. Furthermore, using RNA-seq based on next-generation sequencing, the gene expression profiles of immortalized cells are compared with parental cells as well as those immortalized with SV40 large T antigen, which is once a popular method for cellular immortalization. The profiling results show that newly established common minke-whale-derived immortaliozed cells have completely different profiles from SV40 cells. This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables to establish immortalized cell lines with different biological nature from SV40 expressing cells.