||Jun Funatsu, Yusuke Murakami, Shotaro Shimokawa, Shunji Nakatake, Kohta Fujiwara, Ayako Okita, Masatoshi Fukushima, Kensuke Shibata, Noriko Yoshida, Yoshito Koyanagi, Masato Akiyama, Shoji Notomi, Shintaro Nakao, Toshio Hisatomi, Atsunobu Takeda, Eleftherios I Paschalis, Demetrios G Vavvas, Yasuhiro Ikeda, Koh-Hei Sonoda, Karen E Nelson, Circulating inflammatory monocytes oppose microglia and contribute to cone cell death in retinitis pigmentosa, PNAS Nexus, 10.1093/pnasnexus/pgac003, 1, 1, 2022.03, Retinitis pigmentosa (RP) is an intractable inherited disease that primarily affects the rods through gene mutations followed by secondary cone degeneration. This cone-related dysfunction can lead to impairment of daily life activities, and ultimately blindness in patients with RP. Paradoxically, microglial neuroinflammation contributes to both protection against and progression of RP, but it is unclear which population(s) - tissue-resident microglia and/or peripheral monocyte-derived macrophages (mφ) - are implicated in the progression of the disease. Here we show that circulating blood inflammatory monocytes (IMo) are key effector cells that mediate cone cell death in RP. Attenuation of IMo and peripherally engrafted mφ by Ccl2 deficiency or immune modulation via intravenous nano-particle treatment suppressed cone cell death in rd10 mice, an animal model of RP. In contrast, the depletion of resident microglia by a colony-stimulating factor 1 receptor inhibitor exacerbated cone cell death in the same model. In human patients with RP, IMo was increased and correlated with disease progression. These results suggest that peripheral IMo is a potential target to delay cone cell death and prevent blindness in RP..
||Murakami Y, Koyanagi Y, Fukushima M, Yoshimura M, Fujiwara K, Akiyama M, Momozawa Y, Ueno S, Terasaki H, Oishi A, Miyata M, Ikeda H, Tsujikawa A, Mizobuchi K, Hayashi T, Fujinami K, Tsunoda K, Park JY, Han J, Kim M, Lee CS, Kim SJ, Park TK, Joo K, Woo SJ, Ikeda Y, Sonoda KH, Genotype and Long-term Clinical Course of Bietti Crystalline Dystrophy in Korean and Japanese Patients, Ophthalmology Retina., 10.1016/j.oret.2021.02.009, 5, 12, 1269-1279, 2021.02, Objective: To investigate the genotype and long-term clinical phenotype of patients with Bietti crystalline dystrophy (BCD) in Korea and Japan.
Design: Retrospective case series.
Subjects: We analyzed the cases of 62 patients with clinical features of BCD who harbor pathogenic biallelic CYP4V2 variants in their homozygote or compound heterozygote.
Methods: Data were collected from patient charts including, age, best-corrected visual acuity (BCVA), Goldmann perimetry, fundus photography, optical coherence tomography, fundus autofluorescence, and electroretinogram. We compared the clinical course of the patients with homozygous c.802-8_810del17insGC [Exon7del], the most common mutation in the East Asian population, with those of the patients with other genotypes.
Main Outcome Measures: BCVA, visual field (VF), and their changes during follow-up.
Results: The mean age at the first visit was 55.2 years, with the mean follow-up of 7.1 years. The mean BCVAs at the first and last visits were 0.28 logarithm of the minimum angle of resolution (logMAR) and 0.89 logMAR, respectively. In genetic testing, c.802-8_810del17insGC was detected in 86 of 124 alleles of the subjects, and 36 patients were homozygous for this mutation. The age, BCVA, VF area, central foveal thickness, and abnormal hypoautofluorescent area at either the first visit or the last visit were not different between the Exon7del homozygotes and the others. The mean BCVA changes per year were 0.089 logMAR in the Exon7del homozygotes and 0.089 logMAR in the others. An age- and sex-adjusted linear regression analysis showed no association between the Exon7del homozygote status and the rate of vision loss. Characteristic crystalline deposits in the posterior pole were generally observed in younger patients and disappeared over time along with progressive retinochoroidal atrophy.
Conclusions: BCD patients with a homozygote for c.802-8_810del17insGC accounted for >50% of this cohort of Korean and Japanese patients, and the clinical effect of this deleterious variant was not severe in the spectrum of CYP4V2 retinopathy..
||Okita A, Murakami Y, Shimokawa S, Funatsu J, Fujiwara K, Nakatake S, Koyanagi Y, Akiyama M, Takeda A, Hisatomi T, Ikeda Y, Sonoda KH, Changes of Serum Inflammatory Molecules and Their Relationships with Visual Function in Retinitis Pigmentosa, Invest Ophthalmol Vis Sci., 10.1167/iovs.61.11.30, 61, 30, 30-30, 2020.09, Purpose: Retinal degeneration involves neuroinflammation, and pro-inflammatory cytokines/chemokines are markedly increased in the eyes of patients with retinitis pigmentosa (RP). In this study, we investigated the changes of serum cytokines/chemokines in RP, and their relationships with visual parameters. Methods: Forty-five consecutive patients with typical RP aged 20 to -39 years and 28 age-matched and gender-matched controls were included. Fifteen cytokines (interleukin [IL]-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, 1L-15, IL-17, IL-23, interferon [IFN]-γ, and tumor necrosis factor [TNF]-α, TNF-β) and 9 chemokines (eotaxin, growth-related oncogene [GRO]-α, I-309, IL-8, IFN-γ-inducible protein [IP]-10, monocyte chemotactic protein [MCP]-1, MCP-2, regulated activation normal T-cell expressed and secreted [RANTES], and thymus and activated regulated chemokine [TARC]) in the serum were simultaneously measured by a multiplexed immunoarray (Q-Plex). Relationships between these cytokines/chemokines and indices of central vision, such as visual acuity (VA), the values of static perimetry tests (Humphrey Field analyzer, the central 10-2 program), and optical coherence tomography measures were analyzed in the patients with RP. Results: Among the 15 cytokines and 9 chemokines, serum IL-8 and RANTES levels were significantly increased in patients with RP compared with controls (IL-8: P < 0.0001; RANTES: P < 0.0001). Among the elevated cytokines/chemokines, the levels of IL-8 were negatively correlated with VA (ρ = 0.3596 and P = 0.0165), and the average retinal sensitivity of four central points (ρ = -0.3691 and P = 0.0291), and 12 central points (ρ = -0.3491 and P = 0.0398), as well as the central subfield thickness (ρ = -0.3961 and P = 0.0094), and ellipsoid zone width (ρ = -0.3841 and P = 0.0120). Conclusions: Peripheral inflammatory response may be activated and serum IL-8 levels are associated with central vision in patients with RP..
||Yoshito Koyanagi, Masato Akiyama, Koji M. Nishiguchi, Yukihide Momozawa, Yoichiro Kamatani, Sadaaki Takata, Chihiro Inai, Yusuke Iwasaki, Mikako Kumano, Yusuke Murakami, Kazuko Omodaka, Toshiaki Abe, Shiori Komori, Dan Gao, Toshiaki Hirakata, Kentaro Kurata, Katsuhiro Hosono, Shinji Ueno, Yoshihiro Hotta, Akira Murakami, Hiroko Terasaki, Yuko Wada, Toru Nakazawa, Tatsuro Ishibashi, Yasuhiro Ikeda, Michiaki Kubo, Koh Hei Sonoda, Genetic characteristics of retinitis pigmentosa in 1204 Japanese patients, Journal of medical genetics, 10.1136/jmedgenet-2018-105691, 56, 10, 662-670, 2019.10, Background The genetic profile of retinitis pigmentosa (RP) in East Asian populations has not been well characterised. Therefore, we conducted a large-scale sequencing study to investigate the genes and variants causing RP in a Japanese population. Methods A total of 1209 Japanese patients diagnosed with typical RP were enrolled. We performed deep resequencing of 83 known causative genes of RP using next-generation sequencing. We defined pathogenic variants as those that were putatively deleterious or registered as pathogenic in the Human Gene Mutation Database or ClinVar database and had a minor allele frequency in any ethnic population of ≤0.5% for recessive genes or ≤0.01% for dominant genes as determined using population-based databases. Results We successfully sequenced 1204 patients with RP and determined 200 pathogenic variants in 38 genes as the cause of RP in 356 patients (29.6%). Variants in six genes (EYS, USH2A, RP1L1, RHO, RP1 and RPGR) caused RP in 65.4% (233/356) of those patients. Among autosomal recessive genes, two known founder variants in EYS [p.(Ser1653fs) and p.(Tyr2935∗)] and four East Asian-specific variants [p.(Gly2752Arg) in USH2A, p.(Arg658∗) in RP1L1, p.(Gly2186Glu) in EYS and p.(Ile535Asn) in PDE6B] and p.(Cys934Trp) in USH2A were found in ≥10 patients. Among autosomal dominant genes, four pathogenic variants [p.(Pro347Leu) in RHO, p.(Arg872fs) in RP1, p.(Arg41Trp) in CRX and p.(Gly381fs) in PRPF31] were found in ≥4 patients, while these variants were unreported or extremely rare in both East Asian and non-East Asian population-based databases. Conclusions East Asian-specific variants in causative genes were the major causes of RP in the Japanese population..
||Shoji Notomi, Kenji Ishihara, Nikolaos E. Efstathiou, Jong Jer Lee, Toshio Hisatomi, Takashi Tachibana, Eleni K. Konstantinou, Takashi Ueta, Yusuke Murakami, Daniel E. Maidana, Yasuhiro Ikeda, Shinji Kume, Hiroto Terasaki, Shozo Sonoda, Judith Blanz, Lucy Young, Taiji Sakamoto, Koh Hei Sonoda, Paul Saftig, Tatsuro Ishibashi, Joan W. Miller, Guido Kroemer, Demetrios G. Vavvas, Genetic LAMP2 deficiency accelerates the age-associated formation of basal laminar deposits in the retina, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1906643116, 116, 47, 23724-23734, 2019.01, The early stages of age-related macular degeneration (AMD) are characterized by the accumulation of basal laminar deposits (BLamDs). The mechanism for BLamDs accumulating between the retinal pigment epithelium (RPE) and its basal lamina remains elusive. Here we examined the role in AMD of lysosome-associated membrane protein-2 (LAMP2), a glycoprotein that plays a critical role in lysosomal biogenesis and maturation of autophagosomes/ phagosomes. LAMP2 was preferentially expressed by RPE cells, and its expression declined with age. Deletion of the Lamp2 gene in mice resulted in age-dependent autofluorescence abnormalities of the fundus, thickening of Bruch’s membrane, and the formation of BLamDs, resembling histopathological changes occurring in AMD. Moreover, LAMP2-deficient mice developed molecular signatures similar to those found in human AMD—namely, the accumulation of APOE, APOA1, clusterin, and vitronectin—adjacent to BLamDs. In contrast, collagen 4, laminin, and fibronectin, which are extracellular matrix proteins constituting RPE basal lamina and Bruch’s membrane were reduced in Lamp2 knockout (KO) mice. Mechanistically, retarded phagocytic degradation of photoreceptor outer segments compromised lysosomal degradation and increased exocytosis in LAMP2-deficient RPE cells. The accumulation of BLamDs observed in LAMP2-deficient mice was eventually followed by loss of the RPE and photoreceptors. Finally, we observed loss of LAMP2 expression along with ultramicroscopic features of abnormal phagocytosis and exocytosis in eyes from AMD patients but not from control individuals. Taken together, these results indicate an important role for LAMP2 in RPE function in health and disease, suggesting that LAMP2 reduction may contribute to the formation of BLamDs in AMD..
||Takashi Ueta, Kenji Ishihara, Shoji Notomi, Jong Jer Lee, Daniel E. Maidana, Nikolaos E. Efstathiou, Yusuke Murakami, Eiichi Hasegawa, Kunihiro Azuma, Tetsuya Toyono, Eleftherios I. Paschalis, Makoto Aihara, Joan W. Miller, Demetrios G. Vavvas, RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1908355116, 116, 47, 23705-23713, 2019.01, Inflammation plays an important role in pathological angiogenesis. Receptor-interacting protein 1 (RIP1) is highly expressed in inflammatory cells and is known to play an important role in the regulation of apoptosis, necroptosis, and inflammation; however, a comprehensive description of its role in angiogenesis remains elusive. Here, we show that RIP1 is abundantly expressed in infiltrating macrophages during angiogenesis, and genetic or pharmacological inhibition of RIP1 kinase activity using kinase-inactive RIP1K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization, Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis is mediated by caspase activation through a kinase-independent function of RIP1 and RIP3. Mechanistically, infiltrating macrophages are the key target of RIP1 kinase inhibition to attenuate pathological angiogenesis. Inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of proangiogenic M2-like markers but not M1-like markers. Similarly, in vitro, catalytic inhibition of RIP1 down-regulates the expression of M2-like markers in interleukin-4–activated bone marrow-derived macrophages, and this effect is blocked by simultaneous caspase inhibition. Collectively, these results demonstrate a nonnecrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may be a therapeutic target of pathological angiogenesis..
||Shunji Nakatake, Yusuke Murakami, Yasuhiro Ikeda, Noriko Morioka, Takashi Tachibana, Kohta Fujiwara, Noriko Yoshida, Shoji Notomi, Toshio Hisatomi, Shigeo Yoshida, Tatsuro Ishibashi, Yusaku Nakabeppu, Koh-Hei Sonoda, MUTYH promotes oxidative microglial activation and inherited retinal degeneration, JCI Insight, 10.1172/jci.insight.87781, 1, 15, e87781, 2016.09, Oxidative stress is implicated in various neurodegenerative disorders, including retinitis pigmentosa (RP), an inherited disease that causes blindness. The biological and cellular mechanisms by which oxidative stress mediates neuronal cell death are largely unknown. In a mouse model of RP (rd10 mice), we show that oxidative DNA damage activates microglia through MutY homolog-mediated (MUYTH-mediated) base excision repair (BER), thereby exacerbating retinal inflammation and degeneration. In the early stage of retinal degeneration, oxidative DNA damage accumulated in the microglia and caused single-strand breaks (SSBs) and poly(ADP-ribose) polymerase activation. In contrast, Mutyh deficiency in rd10 mice prevented SSB formation in microglia, which in turn suppressed microglial activation and photoreceptor cell death. Moreover, Mutyh-deficient primary microglial cells attenuated the polarization to the inflammatory and cytotoxic phenotype under oxidative stress. Thus, MUTYH-mediated BER in oxidative microglial activation may be a novel target to dampen the disease progression in RP and other neurodegenerative disorders that are associated with oxidative stress..
||Yusuke Murakami, Noriko Yoshida, Yasuhiro Ikeda, Shunji Nakatake, Kota Fujiwara, Shoji Notomi, Takahiro Nabeshima, Shintaro Nakao, Toshio Hisatomi, Hiroshi Enaida, Tatsuro Ishibashi, Relationship Between Aqueous Flare and Visual Function in Retinitis Pigmentosa, American Journal of Ophthalmology, 10.1016/j.ajo.2015.02.001, 159, 5, 958-963.e1, 2015.05, PURPOSE: To investigate the correlation between aqueous flare values and central visual function in patients with retinitis pigmentosa (RP). DESIGN: Retrospective, observational case series. METHODS: We retrospectively studied 160 patients diagnosed with typical RP and 59 control subjects. Aqueous flare values were measured by laser flare cell meter. The relationships between aqueous flare and best-corrected visual acuity (VA) and mean deviation (MD) of static perimetry tests were analyzed in RP patients. RESULTS: The aqueous flare values were significantly higher in the RP patients compared to the control subjects (10.6 ± 7.9 vs 5.0 ± 2.1 photon counts per millisecond [pc/ms], P < .0001). In the RP patients, the aqueous flare values were negatively correlated with VA (r = 0.359, P < .0001) and MD (r = -0.330, P < .0001). Age-subgroup analysis showed a significant correlation between aqueous flare and VA in the RP patients' 40s, 50s, and 60s and between aqueous flare and MD in the 30s, 40s, 50s, and 60s. The RP patients with MD values ≥-15 decibels (dB) showed significantly higher levels of aqueous flare than those with MD values <-15 dB (12.0 ± 6.2 vs 8.7 ± 5.8, P = .0001). CONCLUSIONS: Aqueous flare is increased in RP patients and negatively correlates with central visual function. These results suggest a close relationship between inflammation and central vision loss in RP..
||Yusuke Murakami, Yasuhiro Ikeda, Noriko Yoshida, Shoji Notomi, Toshio Hisatomi, Sugako Oka, Gabriele De Luca, Yoshikazu Yonemitsu, Margherita Bignami, Yusaku Nakabeppu, Tatsuro Ishibashi, MutT Homolog-1 Attenuates Oxidative DNA Damage and Delays Photoreceptor Cell Death in Inherited Retinal Degeneration, The American Journal of Pathology, 10.1016/j.ajpath.2012.06.026, 181, 4, 1378-1386, 2012.10, Retinitis pigmentosa (RP) is a genetically heterogenous group of inherited retinal degenerative diseases resulting from photoreceptor cell death and affecting >1 million persons globally. Although oxidative stress has been implicated in the pathogenesis of RP, the mechanisms by which oxidative stress mediates photoreceptor cell death are largely unknown. Here, we show that oxidation of nucleic acids is a key component in the initiation of death-signaling pathways in rd10 mice, a model of RP. Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) increased in photoreceptor cells, and especially within their nuclei, in rd10 mice as well as in Royal College of Surgeons rats, another model of RP caused by different genetic mutations. Vitreous samples from humans with RP contained higher levels of 8-oxo-dG excreted than samples from nondegenerative controls. Transgenic overexpression of human MutT homolog-1, which hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, significantly attenuated 8-oxo-dG accumulation in nuclear DNA and photoreceptor cell death in rd10 mice, in addition to suppressing DNA single-strand break formation, poly(ADP-ribose) polymerase activation, and nuclear translocation of apoptosis-inducing factor. These findings indicate that oxidative DNA damage is an important process for the triggering of photoreceptor cell death in rd10 mice and suggest that stimulation of DNA repair enzymes may be a novel therapeutic approach to attenuate photoreceptor cell loss in RP..
||Yusuke Murakami, Hidetaka Matsumoto, Miin Roh, Jun Suzuki, Toshio Hisatomi, Yasuhiro Ikeda, Joan W. Miller, Demetrios G. Vavvas, Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration, Proceedings of the National Academy of Sciences, 10.1073/pnas.1206937109, 109, 36, 14598-14603, 2012.09, Retinitis pigmentosa comprises a group of inherited retinal photoreceptor degenerations that lead to progressive loss of vision. Although in most cases rods, but not cones, harbor the deleterious gene mutations, cones do die in this disease, usually after the main phase of rod cell loss. Rod photoreceptor death is characterized by apoptotic features. In contrast, the mechanisms and features of subsequent nonautonomous cone cell death remain largely unknown. In this study, we show that receptor-interacting protein (RIP) kinase mediates necrotic cone cell death in rd10 mice, a mouse model of retinitis pigmentosa caused by a mutation in a rod-specific gene. The expression of RIP3, a key regulator of programmed necrosis, was elevated in rd10 mouse retinas in the phase of cone but not rod degeneration. Although rd10 mice lacking Rip3 developed comparable rod degeneration to control rd10 mice, they displayed a significant preservation of cone cells. Ultrastructural analysis of rd10 mouse retinas revealed that a substantial fraction of dying cones exhibited necrotic morphology, which was rescued by Rip3 deficiency. Additionally, pharmacologic treatment with a RIP kinase inhibitor attenuated histological and functional deficits of cones in rd10 mice. Thus, necrotic mechanisms involving RIP kinase are crucial in cone cell death in inherited retinal degeneration, suggesting the RIP kinase pathway as a potential target to protect cone-mediated central and peripheral vision loss in patients with retinitis pigementosa..
||George Trichonas, Yusuke Murakami, Aristomenis Thanos, Yuki Morizane, Maki Kayama, Christine M. Debouck, Toshio Hisatomi, Joan W. Miller, Demetrios G. Vavvas, Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis, Proceedings of the National Academy of Sciences, 10.1073/pnas.1009179107, 107, 50, 21695-21700, 2010.12, Apoptosis has been shown to be a significant form of cell loss in many diseases. Detachment of photoreceptors from the retinal pigment epithelium, as seen in various retinal disorders, causes photoreceptor loss and subsequent vision decline. Although caspase-dependent apoptotic pathways are activated after retinal detachment, caspase inhibition by the pan-caspase inhibitor Z-VAD fails to prevent photoreceptor death; thus, we investigated other pathways leading to cell loss. Here, we show that receptor interacting protein (RIP) kinase-mediated necrosis is a significant mode of photoreceptor cell loss in an experimental model of retinal detachment and when caspases are inhibited, RIP-mediated necrosis becomes the predominant form of death. RIP3 expression, a key activator of RIP1 kinase, increased more than 10-fold after retinal detachment. Morphological assessment of detached retinas treated with Z-VAD showed decreased apoptosis but significantly increased necrotic photoreceptor death. RIP1 kinase inhibitor necrostatin-1 or Rip3 deficiency substantially prevented those necrotic changes and reduced oxidative stress and mitochondrial release of apoptosis-inducing factor. Thus, RIP kinase-mediated programmed necrosis is a redundant mechanism of photoreceptor death in addition to apoptosis, and simultaneous inhibition of RIP kinases and caspases is essential for effective neuroprotection and may be a novel therapeutic strategy for treatment of retinal disorders..
||Yusuke Murakami, Yasuhiro Ikeda, Yoshikazu Yonemitsu, Mitsuho Onimaru, Kazunori Nakagawa, Ri-ichiro Kohno, Masanori Miyazaki, Toshio Hisatomi, Makoto Nakamura, Takeshi Yabe, Mamoru Hasegawa, Tatsuro Ishibashi, Katsuo Sueishi, Inhibition of Nuclear Translocation of Apoptosis-Inducing Factor Is an Essential Mechanism of the Neuroprotective Activity of Pigment Epithelium-Derived Factor in a Rat Model of Retinal Degeneration, The American Journal of Pathology, 10.2353/ajpath.2008.080466, 173, 5, 1326-1338, 2008.11, Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP..