SIRT4 Protects Müller Glial Cells Against Apoptosis by Mediating Mitochondrial Dynamics and Oxidative Stress.

SIRT4 is a member of the sirtuin family, which is related to mitochondrial function and possesses antioxidant and regulatory redox effects. Currently, the roles of SIRT4 in retinal Müller glial cells, oxidative stress, and mitochondrial function are still unclear. We confirmed, by immunofluorescence staining, that SIRT4 is located mainly in the mitochondria of retinal Müller glial cells. Using flow cytometry and Western blotting, we analyzed cell apoptosis, intracellular reactive oxygen species (ROS) levels, apoptotic and proapoptotic proteins, mitochondrial dynamics-related proteins, and mitochondrial morphology and number after the overexpression and downregulation of SIRT4 in rMC-1 cells. Neither the upregulation nor the downregulation of SIRT4 alone affected apoptosis. SIRT4 overexpression reduced intracellular ROS, reduced the BAX/BCL2 protein ratio, and increased the L-OPA/S-OPA1 ratio and the levels of the mitochondrial fusion protein MFN2 and the mitochondrial cleavage protein FIS1, increasing mitochondrial fusion. SIRT4 downregulation had the opposite effect. Mitochondria tend to divide after serum starvation for 24 h, and SIRT4 downregulation increases mitochondrial fragmentation and oxidative stress, leading to aggravated cell damage. The mitochondrial division inhibitor Mdivi-1 reduced oxidative stress levels and thus reduced cell damage caused by serum starvation. The overexpression of SIRT4 in rMC-1 cells reduced mitochondrial fragmentation caused by serum starvation, leading to mitochondrial fusion and reduced expression of cleaved caspase-3, thus alleviating the cellular damage caused by oxidative stress. Thus, we speculate that SIRT4 may protect retinal Müller glial cells against apoptosis by mediating mitochondrial dynamics and oxidative stress.

Noninvasive Light Flicker Stimulation Promotes Optic Nerve Regeneration by Activating Microglia and Enhancing Neural Plasticity in Zebrafish.

Purpose: Forty-hertz light flicker stimulation has been proven to reduce neurodegeneration, but its effect on optic nerve regeneration is unclear. This study explores the effect of 40-Hz light flicker in promoting optic nerve regeneration in zebrafish and investigates the underlying mechanisms. Methods: Wild-type and mpeg1:EGFP zebrafish were used to establish a model of optic nerve crush. Biocytin tracing and hematoxylin and eosin staining were employed to observe whether 40-Hz light flicker promotes regeneration of retinal ganglion cell axons and dendrites. Optomotor and optokinetic responses were evaluated to assess recovery of visual function. Immunofluorescence staining of mpeg1:EGFP zebrafish was performed to observe changes in microglia. Differentially expressed genes that promote optic nerve regeneration following 40-Hz light flicker stimulation were identified and validated through RNA-sequencing analysis and quantitative real-time PCR (qRT-PCR). Results: Zebrafish exhibited spontaneous optic nerve regeneration after optic nerve injury and restored visual function. We observed that 40-Hz light flicker significantly activated microglia following optic nerve injury and promoted regeneration of retinal ganglion cell axons and dendrites, as well as recovery of visual function. Transcriptomics and qRT-PCR analyses revealed that 40-Hz light flicker increased the expression of genes associated with neuronal plasticity, including bdnf, npas4a, fosab, fosb, egr4, and ier2a. Conclusions: To our knowledge, this study is the first to demonstrate that 40-Hz light flicker stimulation promotes regeneration of retinal ganglion cell axons and dendrites and recovery of visual function in zebrafish, which is associated with microglial activation and enhancement of neural plasticity.

Values of macular ganglion cell-inner plexiform layer and 10-2 visual field measurements in detecting and evaluating glaucoma.

AIM: To assess the performance of macular ganglion cell-inner plexiform layer thickness (mGCIPLT) and 10-2 visual field (VF) parameters in detecting early glaucoma and evaluating the severity of advanced glaucoma. METHODS: Totally 127 eyes from 89 participants (36 eyes of 19 healthy participants, 45 eyes of 31 early glaucoma patients and 46 eyes of 39 advanced glaucoma patients) were included. The relationships between the optical coherence tomography (OCT)-derived parameters and VF sensitivity were determined. Patients with early glaucoma were divided into eyes with or without central 10° of the VF damages (CVFDs), and the diagnostic performances of OCT-derived parameters were assessed. RESULTS: In early glaucoma, the mGCIPLT was significantly correlated with 10-2 VF pattern standard deviation (PSD; with average mGCIPLT: ß=-0.046, 95%CI, -0.067 to -0.024, P<0.001). In advanced glaucoma, the mGCIPLT was related to the 24-2 VF mean deviation (MD; with average mGCIPLT: ß=0.397, 95%CI, 0.199 to 0.595, P<0.001), 10-2 VF MD (with average mGCIPLT: ß=0.762, 95%CI, 0.485 to 1.038, P<0.001) and 24-2 VF PSD (with average mGCIPLT: ß=0.244, 95%CI, 0.124 to 0.364, P<0.001). Except for the minimum and superotemporal mGCIPLT, the decrease of mGCIPLT in early glaucomatous eyes with CVFDs was more severe than that of early glaucomatous eyes without CVFDs. The area under the curve (AUC) of the average mGCIPLT (AUC=0.949, 95%CI, 0.868 to 0.982) was greater than that of the average circumpapillary retinal nerve fiber layer thickness (cpRNFLT; AUC=0.827, 95%CI, 0.674 to 0.918) and rim area (AUC=0.799, 95%CI, 0.610 to 0.907) in early glaucomatous eyes with CVFDs versus normal eyes. CONCLUSION: The 10-2 VF and mGCIPLT parameters are complementary to 24-2 VF, cpRNFLT and ONH parameters, especially in detecting early glaucoma with CVFDs and evaluating the severity of advanced glaucoma in group level.

CD38 Deficiency Protects Mouse Retinal Ganglion Cells Through Activating the NAD+/Sirt1 Pathway in Ischemia-Reperfusion and Optic Nerve Crush Models.

Purpose: The purpose of this study was to investigate the protective effect of CD38 deletion on retinal ganglion cells (RGCs) in a mouse retinal ischemia/reperfusion (I/R) model and an optic nerve crush (ONC) model, and to elucidate the underlying molecular mechanisms. Methods: Retinal I/R and ONC models were constructed in mice. PCR was used to identify the deletion of CD38 gene in mice, hematoxylin and eosin (H&E) staining was used to evaluate the changes in retinal morphology, and electroretinogram (ERG) was used to evaluate the changes in retinal function. The survival of RGCs and activation of retinal macroglia were evaluated by immunofluorescence staining. The expression of Sirt1, CD38, Ac-p65, Ac-p53, TNF-α, IL-1ß, and Caspase3 proteins in the retina was further evaluated by protein imprinting. Results: In retinal I/R and ONC models, CD38 deficiency reduced the loss of RGCs and activation of macroglia and protected the retinal function. CD38 deficiency increased the concentration of NAD+, reduced the degree of acetylation of NF-κB p65 and p53, and reduced expression of the downstream inflammatory cytokines TNFα, IL-1ß, and apoptotic protein Caspase3 in the retina in the ONC model. Intraperitoneal injection of the Sirt1 inhibitor EX-527 partially counteracted the effects of CD38 deficiency, suggesting that CD38 deficiency acts at least in part through the NAD+/Sirt1 pathway. Conclusions: CD38 plays an important role in the pathogenesis of retinal I/R and ONC injury. CD38 deletion protects RGCs by attenuating inflammatory responses and apoptosis through the NAD+/Sirt1 pathway.

Resveratrol Alleviates Retinal Ischemia-Reperfusion Injury by Inhibiting the NLRP3/Gasdermin D/Caspase-1/Interleukin-1ß Pyroptosis Pathway.

Purpose: The purpose of this study is to investigate the anti-pyroptotic effect of resveratrol in the context of ischemia-reperfusion (I/R)-induced retinal injury, with a particular focus on Müller glial cells (MGCs) and to elucidate the underlying molecular mechanisms. Methods: The retinal I/R model was constructed in mice and pyroptotic markers were measured at six, 12, 24, 48, and 72 hours after I/R injury to determine the peak of pyroptotic activity. The effects of resveratrol on pyroptosis, inflammasomes, and the activation of MGCs after I/R injury were observed on the retina of mice. Moreover, induction of pyroptosis in rat Müller glial cells (r-MC) via lipopolysaccharide was used to explore the effects of resveratrol on pyroptosis of r-MC in vitro. Results: After the induction of retinal I/R injury in mice, the intricate involvement of pyroptosis in the progressive degeneration of the retina was observed, reaching its zenith at the onset of 24 hours after I/R injury. Resveratrol treatment alleviated I/R injury on the retina, relieved retinal ganglion cells death. In addition, resveratrol inhibited Caspase-1 activation, gasdermin D (GSDMD-N) cleavage, the inflammasome assembly, and the release of inflammatory cytokines, simultaneously relieving the MGCs activation. Furthermore, resveratrol inhibited the pyroptosis-related NLRP3/GSDMD-N/TMS1/ASC/Caspase-1/IL-1ß pathway in r-MC cells, and mitigated cells death in vitro. Conclusions: Pyroptosis plays an important role in the pathogenesis of retinal I/R injury. Resveratrol can attenuate pyroptotic-driven damage in the retina and MGC by inhibiting the NLRP3/GSDMD-N/TMS1/ASC/Caspase-1/IL-1ß pyroptosis pathway.

A pedigree with retinitis pigmentosa and its concomitant ophthalmic diseases.

AIM: To characterize the ophthalmic clinical phenotype of a family with retinitis pigmentosa (RP) and closed-angle glaucoma and to detect pathogenic genes and mutation sites causing RP in this family. METHODS: Ophthalmic clinic performance was examined in detail in 8 enrolled family members. Genomic DNA was extracted from the peripheral blood of 4 family members for whole-exome sequencing (WES) to select potential genetic mutations whose structures were identified by bioinformatics analysis. Then, Sanger sequencing was used in 12 family members and control group members to validate and confirm the disease-causing mutation loci, and we analyzed the genotype-phenotype relationships. RESULTS: The known c.512C>T (p.P171L) mutation in the rhodopsin (RHO) gene was only found in afflicted family members and was confirmed by WES and Sanger sequencing as the pathogenic mutation in this family. In addition to being diagnosed with RP, family member III:4 was found to have bilateral closed-angle glaucoma, high myopia, and concurrent cataracts, and family members II:2 and II:4 had pathological changes of anterior chamber angle narrowing. Family members IV:3 and IV:4 were found to have retinoschisis. CONCLUSION: Glaucoma and related pathological changes, such as retinoschisis, in family members are preliminarily considered RP complications caused by RHO mutation.

Rapamycin suppresses neuroinflammation and protects retinal ganglion cell loss after optic nerve crush.

Pyroptosis, an inflammasome-mediated mode of death, plays an important role in glaucoma. It has been shown that regulating the mTOR pathway can inhibit pyroptosis. Unfortunately, whether rapamycin (RAPA), a specific inhibitor of the mTOR pathway, can inhibit optic nerve crush (ONC)-induced pyroptosis to protect retinal ganglion cells (RGCs) has not been investigated. Our research aimed to confirm the effect of intravitreal injection of RAPA on RGCs. Furthermore, we used the ONC model to explore the underlying mechanisms. First, we observed that intravitreal injection of RAPA alleviated RGC damage induced by various types of injury. We then used the ONC model to further explore the potential mechanism of RAPA. Mechanistically, RAPA not only reduced the activation of glial cells in the retina but also inhibited retinal pyroptosis-induced expression of inflammatory factors such as nucleotide-binding oligomeric domain-like receptor 3 (NLRP3), apoptosis-associated speckle-like protein containing a CARD (ASC), N-terminal of gasdermin-D (GSDMD-N), IL-18 and IL-1ß. Moreover, RAPA exerted protective effects on RGC axons, possibly by inhibiting glial activation and regulating the mTOR/ROCK pathway. Therefore, this study demonstrates a novel mechanism by which RAPA protects against glaucoma and provides further evidence for its application in preclinical studies.

Role of the PGE2 receptor in ischemia-reperfusion injury of the rat retina.

Purpose: To investigate the function and expression of the PGE2 receptors EP1-4 in rat retinal ischemia-reperfusion (I/R) injury and to determine the regulatory role of resveratrol (RES) in this process. Methods: In vitro, we stimulated primary astrocytes extracted from the optic disc of rats with epidermal growth factor (EGF) and RES, and detected the location of EP1-4 expression with immunofluorescence. The expression of antiglial fibrillary acidic protein (GFAP), EGF receptor (EGFR), inducible NOS (iNOS), and EP1-4 in astrocytes was detected with western blotting. In vivo, we established an I/R injury model and RES treatment model with Sprague-Dawley rats. Changes in the thickness of the inner retina were observed with hematoxylin and eosin (H&E) staining. EP1-4 localization in the retina was observed with immunohistochemistry. The expression of COX-2, iNOS, and EP1-4 in the control and model groups was detected with western blotting. Results: In this study, immunofluorescence and immunohistochemistry showed that EP1-4 are expressed in astrocytes and the rat retina. EGF stimulation increased the expression of EGFR, iNOS, EP1, EP2, and EP4 in astrocytes. The expression of EP1-4 was statistically significantly increased on the third day after model induction, and EP1-4 expression decreased to normal levels on day 7. EGF and RES mediated the decrease in the expression of EP2. RES treatment significantly reduced retinal damage and RGC loss, as demonstrated by the relatively intact tissue structure on day 7 observed with H&E staining. Moreover, inflammation was associated with this I/R injury model, as demonstrated by the early induction of proinflammatory mediators, and this inflammation was significantly attenuated after RES treatment. Conclusions: These results indicate that the COX-2/PGE2/EPs pathway is involved in retinal damage and astrocyte inflammation. In addition, the results suggest that the neuroprotective effects of RES may be associated with decreased production of inflammatory mediators. These results suggest that the PGE2 receptor may be a key factor in the treatment of neurodegenerative diseases, and that RES may be used as a possible therapeutic strategy for glaucoma.

Resveratrol Delays Retinal Ganglion Cell Loss and Attenuates Gliosis-Related Inflammation From Ischemia-Reperfusion Injury.

Purpose: Resveratrol has been shown to enhance the survival of retinal ganglion cells (RGCs) following ischemia-reperfusion (I/R) injury for glaucoma. However, the precise mechanisms for resveratrol's protective effects are still unclear. The aim of this study is to determine whether resveratrol can inhibit RGC apoptosis, retinal gliosis, and inflammation, all of which are critical events in retinal degeneration following I/R injury. Methods: Right retinal ischemia was induced in adult male Sprague Dawley rats by increasing intraocular pressure to 110 mm Hg for 60 minutes, and the left eyes maintained at normal pressure serve as the control. Intraperitoneal injection of resveratrol or control buffer was performed continuously for 3 days from pre- to post-I/R injury and the protective effects were evaluated and compared. RGCs were retrogradely labeled with Fluoro-Gold by injection into superior colliculi. Apoptosis was detected by TUNEL staining. Western blotting and immunostaining for Bax, Bcl-2, and Caspase-3 were used to explore the Bax-associated apoptotic pathway. Gliosis was assessed by western blotting and immunostaining of retinal cross sections with anti-glial fibrillary acidic protein (GFAP) antibodies. Results: In this study, resveratrol treatment significantly reduced retinal damage and RGC loss as demonstrated by the relatively intact tissue structure in hematoxylin and eosin staining at day 7 and increased Fluoro-Gold labeling of RGCs at day 14, respectively. We found that resveratrol exhibited an anti-apoptotic effect as assessed by reduced TUNEL staining, inhibition of the early upregulated expression of the apoptosis-related protein Bax, and decreased subsequently cleaved caspase-3. However, it did not affect Bcl-2 levels. Moreover, in our I/R injury model, the combined response of reactive gliosis and related inflammation, which were demonstrated by an early induction of pro-inflammatory mediators and subsequently increased GFAP level, were significantly attenuated after resveratrol treatment. Conclusions: These results demonstrate that resveratrol can prevent RGC death by blocking the Bax-caspase-3-dependent apoptotic pathway and suppressed gliosis-related inflammation in the retina after I/R injury. Together these results support the use of resveratrol as a possible therapeutic strategy for glaucoma.

Expression of Sirtuins in the Retinal Neurons of Mice, Rats, and Humans.

Sirtuins are a class of histone deacetylases (HDACs) that have been shown to regulate a range of pathophysiological processes such as cellular aging, inflammation, metabolism, and cell proliferation. There are seven mammalian Sirtuins (SIRT1-7) that play important roles in stress response, aging, and neurodegenerative diseases. However, the location and function of Sirtuins in neurons are not well defined. This study assessed the retinal expression of Sirtuins in mice, rats, and humans and measured the expression of Sirtuins in aged and injured retinas. Expression of all 7 Sirtuins was confirmed by Western blot and Real-Time PCR analysis in all three species. SIRT1 is highly expressed in mouse, rat, and human retinas, whereas SIRT2-7 expression was relatively lower in human retinas. Immunofluorescence was also used to examine the expression and localization of Sirtuins in rat retinal neurons. Importantly, we demonstrate a marked reduction of SIRT1 expression in aged retinal neurons as well as retinas injured by acute ischemia-reperfusion. On the other hand, none of the other Sirtuins exhibit any significant age-related changes in expression except for SIRT5, which was significantly higher in the retinas of adults compared to both young and aged rats. Our work presents the first composite analysis of Sirtuins in the retinal neurons of mice, rats, and humans, and suggests that increasing the expression and activity of SIRT1 may be beneficial for the treatment of glaucoma and other age-related eye dysfunction.

FkbN and Tcs7 are pathway-specific regulators of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis L19.

FK506 (tacrolimus), which is produced by many Streptomyces strains, is clinically used as an immunosuppressive agent and for treatment of inflammatory skin diseases. Here, we identified that the FK506 biosynthetic gene cluster in an industrial FK506-producing strain Streptomyces tsukubaensis L19 is organized as eight transcription units. Two pathway-specific regulators, FkbN and Tcs7, involved in FK506 biosynthesis from S. tsukubaensis L19 were characterized in vivo and in vitro. FkbN activates the transcription of six transcription units in FK506 biosynthetic gene cluster, and Tcs7 activates the transcription of fkbN. In addition, the DNA-binding specificity of FkbN was determined. Finally, a high FK506-producing strain was constructed by overexpression of both fkbN and tcs7 in S. tsukubaensis L19, which improved FK506 production by 89 % compared to the parental strain.

Functions of Type II Thioesterases in Bacterial Polyketide Biosynthesis.

Many polyketides show biological activities and have thus been applied in clinics, as food additives, and in agriculture. Type II thioesterases (TEIIs) play an important role in polyketide biosynthesis. Most TEIIs belong to α/ß-hydrolase family and usually contain a catalytic triad Ser-His-Asp. In polyketide biosynthesis, TEIIs can play an editing role by removal of aberrant non-extendable acyl units in elongation steps, a starter unit selection role by removal of unfavored starter acyl units in initiation steps, and a releasing role by removal of final product in termination steps. Complementation of TEIIs has been observed and applied.

Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network.

Phosphopantetheinyl transferases (PPTases) play essential roles in both primary metabolisms and secondary metabolisms via post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). In this study, an industrial FK506 producing strain Streptomyces tsukubaensis L19, together with Streptomyces avermitilis, was identified to contain the highest number (five) of discrete PPTases known among any species thus far examined. Characterization of the five PPTases in S. tsukubaensis L19 unveiled that stw ACP, an ACP in a type II PKS, was phosphopantetheinylated by three PPTases FKPPT1, FKPPT3, and FKACPS; sts FAS ACP, the ACP in fatty acid synthase (FAS), was phosphopantetheinylated by three PPTases FKPPT2, FKPPT3, and FKACPS; TcsA-ACP, an ACP involved in FK506 biosynthesis, was phosphopantetheinylated by two PPTases FKPPT3 and FKACPS; FkbP-PCP, an PCP involved in FK506 biosynthesis, was phosphopantetheinylated by all of these five PPTases FKPPT1-4 and FKACPS. Our results here indicate that the functions of these PPTases complement each other for ACPs/PCPs substrates, suggesting a complicate phosphopantetheinylation network in S. tsukubaensis L19. Engineering of these PPTases in S. tsukubaensis L19 resulted in a mutant strain that can improve FK506 production.

The substrate promiscuity of a phosphopantetheinyl transferase SchPPT for coenzyme A derivatives and acyl carrier proteins.

Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of acyl carrier proteins (ACPs) in fatty acid synthases (FASs), ACPs in polyketide synthases, and peptidyl carrier proteins (PCPs) in nonribosomal peptide synthetases (NRPSs) in all organisms. Some bacterial PPTases have broad substrate specificities for ACPs/PCPs and/or coenzyme A (CoA)/CoA analogs, facilitating their application in metabolite production in hosts and/or labeling of ACPs/PCPs, respectively. Here, a group II PPTase SchPPT from Streptomyces chattanoogensis L10 was characterized to accept a heterologous ACP and acetyl-CoA. Thus, SchPPT is a promiscuous PPTase and may be used on polyketide production in heterologous bacterial host and labeling of ACPs.

An acyltransferase domain of FK506 polyketide synthase recognizing both an acyl carrier protein and coenzyme A as acyl donors to transfer allylmalonyl and ethylmalonyl units.

UNLABELLED: Acyltransferase (AT) domains of polyketide synthases (PKSs) usually use coenzyme A (CoA) as an acyl donor to transfer common acyl units to acyl carrier protein (ACP) domains, initiating incorporation of acyl units into polyketides. Two clinical immunosuppressive agents, FK506 and FK520, are biosynthesized by the same PKSs in several Streptomyces strains. In this study, characterization of AT4FkbB (the AT domain of the fourth module of FK506 PKS) in transacylation reactions showed that AT4FkbB recognizes both an ACP domain (ACPT csA) and CoA as acyl donors for transfer of a unique allylmalonyl (AM) unit to an acyl acceptor ACP domain (ACP4FkbB), resulting in FK506 production. In addition, AT4FkbB uses CoA as an acyl donor to transfer an unusual ethylmalonyl (EM) unit to ACP4FkbB, resulting in FK520 production, and transfers AM units to non-native ACP acceptors. Characterization of AT4FkbB in self-acylation reactions suggests that AT4FkbB controls acyl unit specificity in transacylation reactions but not in self-acylation reactions. Generally, AT domains of PKSs only recognize one acyl donor; however, we report here that AT4FkbB recognizes two acyl donors for the transfer of different acyl units. DATABASE: Nucleotide sequence data have been submitted to the GenBank database under accession numbers KJ000382 and KJ000383.