Oral Supplementation with AHCC®, a Standardized Extract of Cultured Lentinula edodes Mycelia, Enhances Host Resistance against SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic has significantly impacted global public health safety and the economy. Multiple antiviral drugs have been developed, and some have received regulatory approval and/or authorization. The use of nutraceuticals can be beneficial for preventing and treating COVID-19 complications. AHCC is a standardized, cultured extract of an edible mushroom Lentinula edodes of the Basidiomycete family of fungi that is enriched in acylated α-1,4-glucans. Here, we evaluated the effects of the oral administration of AHCC on the host response to SARS-CoV-2 infection in two murine models, K18-hACE2 transgenic mice and immunocompetent BALB/c mice. Oral administration of AHCC every other day for one week before and one day post SARS-CoV-2 infection in both strains of mice decreased the viral load and attenuated inflammation in the lungs. AHCC treatment also significantly reduced SARS-CoV-2-induced lethality in the K18-hACE2 mice. AHCC administration enhanced the expansion of γδ T cells in the spleen and lungs before and after viral infection and promoted T helper 1-prone mucosal and systemic T cell responses in both models. In AHCC-fed BALB/c mice, SARS-CoV-2 specific IgG responses were also enhanced. In summary, AHCC supplementation enhances host resistance against mild and severe COVID-19 infection primarily via the promotion of innate and adaptive T cell immune responses in mice.

Synergistic effect of standardized extract of Asparagus officinalis stem and heat shock on progesterone synthesis with lipid droplets and mitochondrial function in bovine granulosa cells.

Progesterone (P4) is a well-known steroid hormone that plays a key role in oocyte growth and the maintenance of pregnancy in mammals, including cattle. Heat stress (HS) has an adverse effect on P4 synthesis through an imbalance in the cellular redox status. We have recently revealed that a standardized extract of Asparagus officinalis stem (EAS) increases P4 through non-HS induction of heat shock protein 70 (HSP70) and a synergistic increase of HSP70 by enhancing the intracellular redox balance, which was adversely affected by HS in bovine granulosa cells (GCs). Bovine GCs collected from bovine ovarian follicles were cultured at 38.5 °C and 41 °C for 12 h with or without 5 mg/mL EAS. After treatment, cells and culture suppernatant were collected for the analysis. Enzyme-linked immunosorbent assay (ELISA) was performed to detect in P4 levels. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to detect expression of steroidogenesis related genes. Fluorescence staining was used to detect mitochondrial activity and lipid droplet. P4 level was increased by EAS treatment in association with increase in steroidogenic acute regulatory protein (STAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), mitochondrial membrane activity and lipid droplet both under non-HS and HS conditions. Notably, synergistic effect of EAS with HS co-treatment was observed to show a greater increase in P4 synthesis when comparison with EAS treatment under non-HS condition. Furthermore, inhibition of HSP70 significantly reduced EAS-induced P4 synthesis, mitochondrial activity and synthesis of lipid droplets. These results suggest that P4 synthesis by EAS is mediated by the steroidogenesis pathway via HSP70-regulated activation of STAR and 3ß-HSD, together with improved mitochondrial activity and lipid metabolism in bovine GCs. Moreover, effect of EAS has a synergistic effect of with HSP70-regulated steroidogenesis pathway.

Effects of Epigenetic Modification of PGC-1α by a Chemical Chaperon on Mitochondria Biogenesis and Visual Function in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary blinding disease characterized by gradual photoreceptor death, which lacks a definitive treatment. Here, we demonstrated the effect of 4-phenylbutyric acid (PBA), a chemical chaperon that can suppress endoplasmic reticulum (ER) stress, in P23H mutant rhodopsin knock-in RP models. In the RP models, constant PBA treatment led to the retention of a greater number of photoreceptors, preserving the inner segment (IS), a mitochondrial- and ER-rich part of the photoreceptors. Electroretinography showed that PBA treatment preserved photoreceptor function. At the early point, ER-associated degradation markers, xbp1s, vcp, and derl1, mitochondrial kinetic-related markers, fis1, lc3, and mfn1 and mfn2, as well as key mitochondrial regulators, pgc-1α and tfam, were upregulated in the retina of the models treated with PBA. In vitro analyses showed that PBA upregulated pgc-1α and tfam transcription, leading to an increase in the mitochondrial membrane potential, cytochrome c oxidase activity, and ATP levels. Histone acetylation of the PGC-1α promoter was increased by PBA, indicating that PBA affected the mitochondrial condition through epigenetic changes. Our findings constituted proof of concept for the treatment of ER stress-related RP using PBA and revealed PBA's neuroprotective effects, paving the way for its future clinical application.

AHCC®, a Standardized Extract of Cultured Lentinula Edodes Mycelia, Promotes the Anti-Tumor Effect of Dual Immune Checkpoint Blockade Effect in Murine Colon Cancer.

Treatment strategies combining immune checkpoint blockade (ICB) with other agents have emerged as a promising approach in the treatment of cancers. AHCC®, a standardized extract of cultured Lentinula edodes mycelia, has been reported to inhibit tumor growth and enhance immune cell function. Here we investigated whether AHCC® promotes the therapeutic effect of immunotherapy in cancers. A combination of oral AHCC® and dual immune checkpoint blockade (DICB), including PD-1/CTLA-4 blockade, had reduced tumor growth and increased granzyme B and Ki-67 expression by tumor-infiltrating CD8+ T cells in MC38 colon cancer bearing mice compared to a combination of water and DICB. In the same tumor bearing mice, AHCC® and DICB treatment also altered the composition of the gut microbiome with the increased abundance of the species of Ruminococcaceae family which is associated with increased therapeutic efficacy of immunotherapy. The anti-tumor effect of AHCC® and DICB was not found in MC38 tumor-bearing mice treated with antibiotics. These data suggest that AHCC® increases the anti-tumor effect of DICB by enhancing T cell function and affecting the gut microbiome.

Lentinula edodes extract increases goblet cell number and Muc2 expression in an intestinal inflammatory model of Trichinella spiralis infection.

AHCC® is a standardized extract of cultured mushroom (Lentinula edodes) mycelia with a wide variety of therapeutic effects including anti-inflammatory, antitumor and antiviral effects. Trichinellosis, a food-borne parasitic zoonosis is caused by the nematode Trichinella spp. Infection with Trichinella is characterized by the induction of a Th1-type response at the beginning of the intestinal phase, followed by a dominant Th2-type response which is essential for parasite expulsion. The aim of this study was to evaluate the immunomodulatory effect of AHCC® in a murine model of Trichinella spiralis infection. Swiss CD1 mice were infected with T. spiralis larvae and treated with AHCC®. Standard treatment with albendazole (ABZ) was used as control in the assessment of parasite burden. The small intestine was taken out and the proximal segment was evaluated for several parameters: gene expression of immune and stress-reticulum mediators, histological damage score, goblet cell count and Mucin 2 (Muc2) gene expression. AHCC® modulated expression levels of both Th1 and Th2 cytokines and reduced histological damage score. In addition, AHCC® diminished the number of adults of T. spiralis in treated animals. AHCC® treatment anticipates T. spiralis expulsion and increases goblet cell number and Muc2 gene expression.

A standardized extract of Asparagus officinalis stem improves HSP70-mediated redox balance and cell functions in bovine cumulus-granulosa cells.

Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.

Neuroprotective Effect of 4-Phenylbutyric Acid against Photo-Stress in the Retina.

Exposure to excessive visible light causes retinal degeneration and may influence the progression of retinal blinding diseases. However, there are currently no applied treatments. Here, we focused on endoplasmic reticulum (ER) stress, which can cause cellular degeneration and apoptosis in response to stress. We analyzed functional, histological, and molecular changes in the light-exposed retina and the effects of administering an ER-stress inhibitor, 4-phenylbutyric acid (4-PBA), in mice. We found that light-induced visual function impairment related to photoreceptor cell loss and outer segment degeneration were substantially suppressed by 4-PBA administration, following attenuated photoreceptor apoptosis. Induction of retinal ER stress soon after light exposure, represented by upregulation of the immunoglobulin heavy chain binding protein (BiP) and C/EBP-Homologous Protein (CHOP), were suppressed by 4-PBA. Concurrently, light-induced oxidative stress markers, Nuclear factor erythroid 2-related factor 2 (Nrf2) and Heme Oxygenase 1 (HO-1), and mitochondrial apoptotic markers, B-cell lymphoma 2 apoptosis regulator (Bcl-2)-associated death promoter (Bad), and Bcl-2-associated X protein (Bax), were suppressed by 4-PBA administration. Increased expression of glial fibrillary acidic protein denoted retinal neuroinflammation, and inflammatory cytokines were induced after light exposure; however, 4-PBA acted as an anti-inflammatory. Suppression of ER stress by 4-PBA may be a new therapeutic approach to suppress the progression of retinal neurodegeneration and protect visual function against photo-stress.

Neuroprotective Effects of Asparagus officinalis Stem Extract in Transgenic Mice Overexpressing Amyloid Precursor Protein.

To mimic Alzheimer's disease, transgenic mice overexpressing the amyloid precursor protein (APP) were used in this study. We hypothesize that the neuroprotective effects of ETAS®50, a standardized extract of Asparagus officinalis stem produced by Amino Up Co., Ltd. (Sapporo, Japan), are linked to the inhibition of the apoptosis cascade through an enhancement of the stress-response proteins: heat shock proteins (HSPs). APP-overexpressing mice (double-transgenic APP and PS1 mouse strains with a 129s6 background), ages 6-8 weeks old, and weighing 20-24 grams were successfully bred in our laboratory. The animals were divided into 5 groups. APP-overexpressing mice and wild-type (WT) mice were pretreated with ETAS®50 powder (50% elemental ETAS and 50% destrin) at 200 mg/kg and 1000 mg/kg body weight. Saline, the vehicle for ETAS®50, was administered in APP-overexpressing mice and WT mice. ETAS®50 and saline were administered by gavage daily for 1 month. Cognitive assessments, using the Morris Water Maze, demonstrated that memory was recovered following ETAS®50 treatment as compared to nontreated APP mice. At euthanization, the brain was removed and HSPs, amyloid ß, tau proteins, and caspase-3 were evaluated through immunofluorescence staining with the appropriate antibodies. Our data indicate that APP mice have cognitive impairment along with elevated amyloid ß, tau proteins, and caspase-3. ETAS®50 restored cognitive function in these transgenic mice, increased both HSP70 and HSP27, and attenuated pathogenic level of amyloid ß, tau proteins, and caspsase-3 leading to neuroprotection. Our results were confirmed with a significant increase in HSP70 gene expression in the hippocampus.

ADIPOR1 deficiency-induced suppression of retinal ELOVL2 and docosahexaenoic acid levels during photoreceptor degeneration and visual loss.

Lipid metabolism-related gene mutations can cause retinitis pigmentosa, a currently untreatable blinding disease resulting from progressive neurodegeneration of the retina. Here, we demonstrated the influence of adiponectin receptor 1 (ADIPOR1) deficiency in retinal neurodegeneration using Adipor1 knockout (KO) mice. Adipor1 mRNA was observed to be expressed in photoreceptors, predominately within the photoreceptor inner segment (PIS), and increased after birth during the development of the photoreceptor outer segments (POSs) where photons are received by the visual pigment, rhodopsin. At 3 weeks of age, visual function impairment, specifically photoreceptor dysfunction, as recorded by electroretinography (ERG), was evident in homozygous, but not heterozygous, Adipor1 KO mice. However, although photoreceptor loss was evident at 3 weeks of age and progressed until 10 weeks, the level of visual dysfunction was already substantial by 3 weeks, after which it was retained until 10 weeks of age. The rhodopsin mRNA levels had already decreased at 3 weeks, suggesting that reduced rhodopsin may have contributed to early visual loss. Moreover, inflammation and oxidative stress were induced in homozygous KO retinas. Prior to observation of photoreceptor loss via optical microscopy, electron microscopy revealed that POSs were present; however, they were misaligned and their lipid composition, including docosahexaenoic acid (DHA), which is critical in forming POSs, was impaired in the retina. Importantly, the expression of Elovl2, an elongase of very long chain fatty acids expressed in the PIS, was significantly reduced, and lipogenic genes, which are induced under conditions of reduced endogenous DHA synthesis, were increased in homozygous KO mice. The causal relationship between ADIPOR1 deficiency and Elovl2 repression, together with upregulation of lipogenic genes, was confirmed in vitro. Therefore, ADIPOR1 in the retina appears to be indispensable for ELOVL2 induction, which is likely required to supply sufficient DHA for appropriate photoreceptor function and survival.

Taurine rescues mitochondria-related metabolic impairments in the patient-derived induced pluripotent stem cells and epithelial-mesenchymal transition in the retinal pigment epithelium.

Mitochondria participate in various metabolic pathways, and their dysregulation results in multiple disorders, including aging-related diseases. However, the metabolic changes and mechanisms of mitochondrial disorders are not fully understood. Here, we found that induced pluripotent stem cells (iPSCs) from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) showed attenuated proliferation and survival when glycolysis was inhibited. These deficits were rescued by taurine administration. Metabolomic analyses showed that the ratio of the reduced (GSH) to oxidized glutathione (GSSG) was decreased; whereas the levels of cysteine, a substrate of GSH, and oxidative stress markers were upregulated in MELAS iPSCs. Taurine normalized these changes, suggesting that MELAS iPSCs were affected by the oxidative stress and taurine reduced its influence. We also analyzed the retinal pigment epithelium (RPE) differentiated from MELAS iPSCs by using a three-dimensional culture system and found that it showed epithelial mesenchymal transition (EMT), which was suppressed by taurine. Therefore, mitochondrial dysfunction caused metabolic changes, accumulation of oxidative stress that depleted GSH, and EMT in the RPE that could be involved in retinal pathogenesis. Because all these phenomena were sensitive to taurine treatment, we conclude that administration of taurine may be a potential new therapeutic approach for mitochondria-related retinal diseases.

Renin-angiotensin system impairs macrophage lipid metabolism to promote age-related macular degeneration in mouse models.

Metabolic syndrome, a condition involving obesity and hypertension, increases the risk of aging-associated diseases such as age-related macular degeneration (AMD). Here, we demonstrated that high-fat diet (HFD)-fed mice accumulated oxidized low-density lipoprotein (ox-LDL) in macrophages through the renin-angiotensin system (RAS). The ox-LDL-loaded macrophages were responsible for visual impairment in HFD mice along with a disorder of the retinal pigment epithelium (RPE), which is required for photoreceptor outer segment renewal. RAS repressed ELAVL1, which reduced PPARγ, impeding ABCA1 induction to levels that are sufficient to excrete overloaded cholesterol within the macrophages. The ox-LDL-loaded macrophages expressed inflammatory cytokines and attacked the RPE. An antihypertensive drug, angiotensin II type 1 receptor (AT1R) blocker, resolved the decompensation of lipid metabolism in the macrophages and reversed the RPE condition and visual function in HFD mice. AT1R signaling could be a future therapeutic target for macrophage-associated aging diseases, such as AMD.

Neuroprotective and vision-protective effect of preserving ATP levels by AMPK activator.

Progression of blinding diseases, such as age-related macular degeneration, is accelerated by light exposure. However, no particular intervention is applied to the photostress. Here, we report neuroprotective effects of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) activator, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), on light-induced visual function impairment, photoreceptor disorders and death in mice. Increase in retinal ATP levels in response to photostress was transient, because oxygen consumption rate (OCR) and cytochrome c oxidase (CcO) activity were reduced under photostress. However, AICAR treatment preserved OCR, CcO activity, and high levels of retinal ATP after light exposure. AMPK knockdown in the photoreceptor-derived cell line revealed that AMPK targeted CcO activity. Further, our data indicated that photostress reduced mitochondrial respiratory function and ATP levels, while AICAR treatment promoted neuronal survival and retained visual function, stabilizing ATP levels through preserved CcO activity. The current study has provided proof of concept for providing cells with sufficient energy to promote cell survival in the presence of cellular stress. This is in contrast to the previous reports which primarily investigated therapeutic approaches to suppress stress signals. Hence, stabilization of the ATP supply may serve as a novel therapeutic approach to support tissue survival under stress and prevent neurodegeneration.

Spatiotemporal imaging documented the maturation of the cardiomyocytes from human induced pluripotent stem cells.

OBJECTIVES: Cardiomyocytes derived from human induced pluripotent stem cells are a promising source of cells for regenerative medicine. However, contractions in such derived cardiomyocytes are often irregular and asynchronous, especially at early stages of differentiation. This study aimed to determine the differentiation stage of initiation of synchronized and regular contractions, using spatiotemporal imaging and physiological and genetic analyses. METHODS: Knock-in human induced pluripotent stem cell lines were established with clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 to analyze cardiac and pacemaker cell maturation. Time-frequency analysis and Ca2+ imaging were performed, and the expression of related proteins and specific cardiac/pacemaker mRNAs in contracting embryoid bodies was analyzed at various differentiation stages. RESULTS: Time-frequency analysis and Ca2+ imaging revealed irregular, asynchronous contractions at the early stage of differentiation with altered electrophysiological properties upon differentiation. Genes associated with electrophysiological properties were upregulated after 70 days of culturing in differentiation media, whereas pacemaker genes were initially upregulated during the early stage and downregulated at the later stage. CONCLUSIONS: A differentiation period >70 days is required for adequate development of cardiac elements including ion channels and gap junctions and for sarcomere maturation.

Dietary Spirulina Supplementation Protects Visual Function From Photostress by Suppressing Retinal Neurodegeneration in Mice.

PURPOSE: We investigated whether daily consumption of Spirulina, an antioxidant generating cyanobacterial nutritional supplement, would suppress photostress-induced retinal damage and prevent vision loss in mice. METHODS: Six-week-old male BALB/cAJcl mice were allowed constant access to either a standard or Spirulina-supplemented diet (20% Spirulina) that included the antioxidants, ß-carotene and zeaxanthin, and proteins for 4 weeks. Following dark adaptation, mice were exposed to 3000-lux white light for 1 hour and returned to their cages. Visual function was analyzed by electroretinogram, and retinal histology by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated, deoxyuridine triphosphate nick-end labeling (TUNEL) assay, and immunohistochemistry. Retinal expression of proteins, reactive oxygen species (ROS), and mRNAs were measured using immunoblot analysis, enzyme-linked immunosorbent assay (ELISA), 2',7'-dichlorofluorescein-diacetate, or ROS Brite 700 Dyes, and real-time reverse-transcription polymerase chain reaction, respectively. RESULTS: Light-induced visual function impairment was suppressed by constant Spirulina intake. Thinning of the photoreceptor layer and outer segments, photoreceptor cell death, decreased rhodopsin protein, and induction of glial fibrillary acidic protein were ameliorated in the Spirulina-intake group. Increased retinal ROS levels after light exposure were reduced by Spirulina supplementation. Light-induced superoxide dismutase 2 and heme oxygenase-1 mRNAs in the retina, and Nrf2 activation in the photoreceptor cells, were preserved with Spirulina supplementation, despite reduced ROS levels, suggesting two pathways for suppressing ROS, scavenging and induction of endogenous antioxidative enzymes. Light-induced MCP-1 retinal mRNA and proteins were also suppressed by Spirulina. CONCLUSIONS: Spirulina ingestion protected retinal photoreceptors from photostress in the retina. TRANSLATIONAL RELEVANCE: Spirulina has potential as a nutrient supplement to prevent vision loss related to oxidative damage in the future.

Aquaporin 4 Suppresses Neural Hyperactivity and Synaptic Fatigue and Fine-Tunes Neurotransmission to Regulate Visual Function in the Mouse Retina.

The bidirectional water channel aquaporin 4 (AQP4) is abundantly expressed in the neural tissue. The advantages and disadvantages of AQP4 neural tissue deficiency under pathological conditions, such as inflammation, and relationship with neural diseases, such as Alzheimer's disease, have been previously reported. However, the physiological functions of AQP4 are not fully understood. Here, we evaluated the role of AQP4 in the mouse retina using Aqp4 knockout (KO) mice. Aqp4 was expressed in Müller glial cells surrounding the synaptic area between photoreceptors and bipolar cells. Both scotopic and photopic electroretinograms showed hyperactive visual responses in KO mice, gradually progressing with age. Moreover, the amplitude reduction after frequent stimuli and synaptic fatigue was more severe in KO mice. Glutamine synthetase, glutamate aspartate transporter, synaptophysin, and the inward potassium channel Kir2.1, but not Kir4.1, were downregulated in KO retinas. KIR2.1 colocalized with AQP4 in Müller glial cells at the synaptic area, and its expression was affected by Aqp4 levels in primary Müller glial cell cultures. Intraocular injection of potassium in wild-type mice led to visual function hyperactivity, as observed in Aqp4 KO mice. Mitochondria molecules, such as Pgc1α and CoxIV, were downregulated, while apoptotic markers were upregulated in KO retinas. AQP4 may fine-tune synaptic activity, most likely by regulating potassium metabolism, at least in part, via collaborating with KIR2.1, and possibly indirectly regulating glutamate kinetics, to inhibit neural hyperactivity and synaptic fatigue which finally affect mitochondria and cause neurodegeneration.

MicroRNA signature in the chemoprevention of functionally-enriched stem and progenitor pools (FESPP) by Active Hexose Correlated Compound (AHCC).

PURPOSE: Many breast cancer patients use natural compounds in their battle against breast cancer. Active Hexose Correlated Compound (AHCC®) is a cultured mushroom mycelium extract shown to favorably modulate the immune system and alleviate cancer burden. Cancer Stem cells (CSCs) are a subset of highly tumorigenic cancer cells that are thought to be responsible for recurrence. CSCs can be epigenetically regulated by microRNAs (miRNAs). We hypothesized that AHCC may influence CSCs by modulating tumor-suppressor or oncogenic miRNAs. METHODS: Functionally-enriched stem and progenitor pools (FESPP) were isolated in the form of mammospheres from MDA-MB-231, MCF-7, and 4T1 cells, exposed to AHCC in both regular and primary culture from Balb/c mice, and analyzed by visual counting and flow cytometry. Cell motility was also observed in MDA-MB-231 cells. Profiling and RT-qPCR were performed to determine AHCC influence on miRNAs in MDA-MB-231 mammospheres. Additionally, Balb/c mice were orally gavaged with AHCC, and tumor growth parameters and miR-335 expression were analyzed. MDA-MB-231 cells were transfected with miR-335 and analyzed by western blot. RESULTS: We demonstrated that AHCC reduced mammosphere growth in three cell lines and in primary culture, prevented cell migration, and upregulated miR-335 expression in MDA-MB-231 cells and mouse tumor samples. Among the differentially regulated miRNAs in CSCs, we focused on tumor suppressor miR-335, known to target extracellular matrix protein Tenascin C (TNC). TNC is involved in CSC immune evasion pathways. In MDA-MB-231, inhibition of miR-335 increased TNC protein expression. CONCLUSIONS: These results support that AHCC limits FESPP growth, partly by targeting miRNA pathways.

Novel channel-mediated choline transport in cholinergic neurons of the mouse retina.

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X2 purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X2 purinoceptors acquire permeability to large cations, such as N-methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,ß-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X2 purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X2 purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X2 purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X2 purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

Knock-in strategy at 3'-end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation.

Fluorescent reporter gene knock-in induced pluripotent stem cell (iPSC) lines have been used to evaluate the efficiency of differentiation into specific cell lineages. Here, we report a knock-in strategy for the generation of human iPSC reporter lines in which a 2A peptide sequence and a red fluorescent protein (E2-Crimson) gene were inserted at the termination codon of the cone-rod homeobox (Crx) gene, a photoreceptor-specific transcriptional factor gene. The knock-in iPSC lines were differentiated into fluorescence-expressing cells in 3D retinal differentiation culture, and the fluorescent cells also expressed Crx specifically in the nucleus. We found that the fluorescence intensity was positively correlated with the expression levels of Crx mRNA and that fluorescent cells expressed rod photoreceptor-specific genes in the later stage of differentiation. Finally, we treated the fluorescent cells with DAPT, a Notch inhibitor, and found that DAPT-enhanced retinal differentiation was associated with up-regulation of Crx, Otx2 and NeuroD1, and down-regulation of Hes5 and Ngn2. These suggest that this knock-in strategy at the 3'-end of the target gene, combined with the 2A peptide linked to fluorescent proteins, offers a useful tool for labeling specific cell lineages or monitoring expression of any marker genes without affecting the function of the target gene.

Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.

We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

Active Hexose Correlated Compound (AHCC) promotes an intestinal immune response in BALB/c mice and in primary intestinal epithelial cell culture involving toll-like receptors TLR-2 and TLR-4.

PURPOSE: Active Hexose Correlated Compound (AHCC(®)) is a cultured mushroom extract that is commercially available and promoted for immune support. Available data suggest that AHCC supplementation affects immune cell populations and immune outcomes, including natural killer cell response to infection. The mechanism by which AHCC exerts its effects is not well understood. The present work aimed to characterize the immunomodulatory activity of AHCC in the gut and to study the effects of AHCC on toll-like receptor (TLR) signaling in intestinal epithelial cells (IECs). METHODS: BALB/c mice were fed AHCC by gavage. In vivo activities were assessed by immunohistochemistry and cytokine production. The effects of AHCC on ex vivo primary cell culture from IECs were examined after challenge with LPS or E. coli alone or in the presence of anti-TLR-2 and TLR-4 blocking antibodies. RESULTS: Feeding AHCC resulted in increased IgA+ cells in the intestine and increased sIgA, IL-10, and IFN-γ in the intestinal fluid. In IECs, contact with AHCC increased IL-6 production but not to the pro-inflammatory level of positive controls, LPS and E. coli. Blocking TLR-2 and TLR-4 reduced the induction of IL-6 by AHCC, suggesting that these innate receptors are involved in generating the immune response of IECs to AHCC. CONCLUSIONS: AHCC may play a role in the orchestration of immune response and the maintenance of immune homeostasis in part by priming the TLR-2 and TLR-4 gate at the intestinal epithelium. Such a response is likely due to the recognition of non-pathogenic food-associated molecular patterns (FAMPs) such as those found associated with other mushroom or yeast-derived compounds.