Regulation of Diseases-Associated Microglia in the Optic Nerve by Lipoxin B4 and Ocular Hypertension.

Background: The resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B4 produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB4 neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap. Methods: Cellular targets and signaling of LXB4 in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of the mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB4 in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups was assessed by Student's t-test and one-way ANOVA. Results: Single-cell transcriptomics identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB4 treatment. Conclusion: We identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB4 target in the retina and optic nerve. LXB4 maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB4 pathway.

Dysregulation of neuroprotective lipoxin pathway in astrocytes in response to cytokines and ocular hypertension​.

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte reactivity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension. By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4's neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.

Lipoxins A4 and B4 inhibit glial cell activation via CXCR3 signaling in acute retinal neuroinflammation.

Lipoxins are small lipids that are potent endogenous mediators of systemic inflammation resolution in a variety of diseases. We previously reported that Lipoxins A4 and B4 (LXA4 and LXB4) have protective activities against neurodegenerative injury. Yet, lipoxin activities and downstream signaling in neuroinflammatory processes are not well understood. Here, we utilized a model of posterior uveitis induced by lipopolysaccharide endotoxin (LPS), which results in rapid retinal neuroinflammation primarily characterized by activation of resident macroglia (astrocytes and Müller glia), and microglia. Using this model, we observed that each lipoxin reduces acute inner retinal inflammation by affecting endogenous glial responses in a cascading sequence beginning with astrocytes and then microglia, depending on the timing of exposure; prophylactic or therapeutic. Subsequent analyses of retinal cytokines and chemokines revealed inhibition of both CXCL9 (MIG) and CXCL10 (IP10) by each lipoxin, compared to controls, following LPS injection. CXCL9 and CXCL10 are common ligands for the CXCR3 chemokine receptor, which is prominently expressed in inner retinal astrocytes and ganglion cells. We found that CXCR3 inhibition reduces LPS-induced neuroinflammation, while CXCR3 agonism alone induces astrocyte reactivity. Together, these data uncover a novel lipoxin-CXCR3 pathway to promote distinct anti-inflammatory and proresolution cascades in endogenous retinal glia.

Characterization of bioactive and fruit quality compounds of promising mango genotypes grown in Himalayan plain region.

Twenty mango genotypes grown in the plains of the Himalayas were characterized by their physical, physiological, biochemical, mineral and organoleptic attributes: fruit firmness, weight, peel thickness, shape, dry seed weight, respiration rate, weight loss, and shelf life. Biochemical attributes such as soluble solids, total carotenoids, total phenolic content, antioxidant activity, titratable acidity, ascorbic acid and total sugars were also determined. In addition, mineral content and fruit-softening enzymes were measured, and an organoleptic evaluation was performed. Polygalactouronase (PG), pectin methylesterase (PME) and lipoxygenase (LOX) were measured from the pulp adjacent to the peel. Similarly, biochemical attributes and mineral content were evaluated using fruit pulp, while organoleptic evaluation included fruit pulp characters and the fruit's external appearance. The results of the study showed that the 'Malda' genotype exhibited the highest total phenolic content (560.60 µg/100 g), total antioxidant (5.79 µmol TE/g), and titratable acidity (0.37%) among the tested genotypes. 'Amrapali' had the highest soluble solid content (25.20 °B), 'Jawahar' had the highest ascorbic acid content (44.20 mg/100 g pulp), 'Mallika' had the highest total flavonoid content (700.00 µg/g) and 'Amrapali' had the highest total carotenoid content (9.10 mg/100 g). Moreover, the genotypes 'Malda', 'Safed Malda'and 'Suvarnarekha' had a shelf life of 4-5 days longer than other tested genotypes. The genotypes with high biochemical attributes have practical utility for researchers for quality improvement programmes and processing industries as functional ingredients in industrial products. This study provides valuable information on the nutritional and functional properties of different mango genotypes, which can aid in developing improved varieties with enhanced health benefits and greater practical utility for processing industries.

Dysregulation of Neuroprotective Lipoxin Pathway in Astrocytes in Response to Cytokines and Ocular Hypertension.

Glaucoma leads to vision loss due to retinal ganglion cell death. Astrocyte reactivity contributes to neurodegeneration. Our recent study found that lipoxin B4 (LXB4), produced by retinal astrocytes, has direct neuroprotective actions on retinal ganglion cells. In this study, we aimed to investigate how the autacoid LXB4 influences astrocyte activity in the retina under inflammatory cytokine-induced activation and during ocular hypertension. The protective activity of LXB4 was investigated in vivo using the mouse silicone-oil model of chronic ocular hypertension (n=40). By employing a range of analytical techniques, including bulk RNA-seq, RNAscope in-situ hybridization, qPCR, and lipidomic analyses, we discovered the formation of lipoxins and expression of the lipoxin pathway in rodents (including the retina and optic nerve), primates (optic nerve), and human brain astrocytes, indicating the presence of this neuroprotective pathway across various species. Findings in the mouse retina identified significant dysregulation of the lipoxin pathway in response to chronic ocular hypertension, leading to an increase in 5-lipoxygenase (5-LOX) activity and a decrease in 15-LOX activity. This dysregulation was coincident with a marked upregulation of astrocyte reactivity. Reactive human brain astrocytes also showed a significant increase in 5-LOX. Treatment with LXB4 amplified the lipoxin biosynthetic pathway by restoring and amplifying the generation of another member of the lipoxin family, LXA4, and mitigated astrocyte reactivity in mouse retinas and human brain astrocytes. In conclusion, the lipoxin pathway is functionally expressed in rodents, primates, and human astrocytes, and is a resident neuroprotective pathway that is downregulated in reactive astrocytes. Novel cellular targets for LXB4's neuroprotective action are inhibition of astrocyte reactivity and restoration of lipoxin generation. Amplifying the lipoxin pathway is a potential target to disrupt or prevent astrocyte reactivity in neurodegenerative diseases, including retinal ganglion cell death in glaucoma.

Gene Therapy: Contest between Adeno-Associated Virus and Host Cells and the Impact of UFMylation.

Adeno-associated virus (AAV)-based gene therapy is currently limited by (1) decline in therapeutic gene expression over time, (2) immune cell activation and (3) neutralization by pre-existing antibodies. Hence, studying the interaction of AAV vectors with various cellular pathways during the production and transduction process is necessary to overcome such barriers. Post-translational modifications (PTM) of AAV vectors during the production and transduction process is known to limit its transduction efficiency and further evoke the immune response. Further, AAV vectors are known to trigger cellular stress, resulting in an upregulation of distinct arms of the unfolded protein response (UPR) pathway. Recognition of the AAV genome by Toll-like receptor-9 triggers the myeloid differentiation primary response signaling cascade for innate (IL-6, IFN-α, IFN-ß) and adaptive (CD8+ T-cell, B-cell) immune response against the viral capsid and the transgene product. Herein, we highlight a potential intersection of the UPR, PTMs, and intracellular trafficking pathways, which could be fine-tuned to augment the outcome of AAV-based gene delivery.

AAV6 Vexosomes Mediate Robust Suicide Gene Delivery in a Murine Model of Hepatocellular Carcinoma.

During recombinant Adeno-associated virus (AAV) production, a proportionately large amount of vectors is released in the culture supernatant, which is often discarded. It has been shown that these vectors often associate with vesiculated structures, such as exosomes. Exosome-associated AAV (vexosomes) represent an additional gene-delivery platform. The efficiency of such vexosomes in suicide gene therapy is unexplored. In the present study, we have generated AAV serotype 6 vexosomes containing an inducible caspase 9 (iCasp9) suicide gene by a differential ultracentrifugation-based protocol. We further tested the cytotoxic potential of these vexosomes in a human hepatocellular carcinoma (HCC) model in vitro and in vivo. The AAV6-iCasp9 containing vexosomes, when primed with a pro-drug (AP20187), demonstrated a significant loss in cell viability (57% ± 8% versus 100% ± 4.8%, p < 0.001) in comparison to mock-treated Huh7 cells. An intratumoral administration of AAV6-iCasp9 vexosomes and AP20187 in a murine xenograft model revealed a 2.3-fold increase in tumor regression in comparison to untreated animals. These findings were further corroborated by histological analysis and apoptosis assays. In conclusion, our data demonstrate the therapeutic potential of AAV6 vexosomes in a xenotransplantation model of HCC. Furthermore, the simplicity in production and isolation of vexosomes should further facilitate its application in other malignancies.

Exosome-associated SUMOylation mutant AAV demonstrates improved ocular gene transfer efficiency in vivo.

Exosome associated Adeno-associated virus (AAV) vectors have emerged as a promising tool in gene therapy. Recently, we elucidated the role of SUMOylation post-translational modification in AAV2 capsid and demonstrated that capsid modifications at SUMOylation sites, enhance vector transduction. The present study was designed to study the combinatorial effect of exosome delivery of a SUMOylation site modified AAV2, during ocular gene therapy. In the first set of experiments, we investigated the in vitro gene transfer potential of exosome-associated SUMOylation mutant AAV2 (Exo-K105Q-EGFP) in human retinal pigmental epithelial (ARPE19) cells. Our data showed that, Exo-K105Q vectors had a significantly higher transduction potential in ARPE19 cells when compared to exosomes derived from wildtype AAV2 (Exo-AAV2-EGFP) vector packaging. Subsequently, an intravitreal administration of exosome associated mutant AAV2 vectors in C57BL6/J mice, demonstrated a significant increase reporter gene (EFGP) expression 4 weeks after gene transfer. Further immunostaining, revealed that these exosome-based vectors also had a better permeation across the retinal layers. These data highlight the translational potential of exosome associated SUMOylation mutant AAV for ocular gene therapy.

Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration.

Recombinant adeno-associated virus (AAV)-based gene therapy has been promising, but several host-related transduction or immune challenges remain. For this mode of therapy to be widely applicable, it is crucial to develop high transduction and permeating vectors that infect the target at significantly low doses. Because glycosylation of capsid proteins is known to be rate limiting in the life cycle of many viruses, we reasoned that perturbation of glycosylation sites in AAV2 capsid will enhance gene delivery. In our first set experiments, pharmacological modulation of the glycosylation status in host cells, modestly decreased (1-fold) AAV2 packaging efficacy while it improved their gene expression (∼74%) in vitro. We then generated 24 mutant AAV2 vectors modified to potentially create or disrupt a glycosylation site in its capsid. Three of them demonstrated a 1.3-2.5-fold increase in transgene expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic gene transfer of these vectors in hemophilia B mice, resulted in a 2-fold increase in human coagulation factor (F)IX levels, while its T/B-cell immunogenic response was unaltered. Subsequently, intravitreal gene transfer of glycosylation site-modified vectors in C57BL6/J mice demonstrated an increase in green fluorescence protein expression (∼2- to 4-fold) and enhanced permeation across retina. Subretinal administration of these modified vectors containing RPE65 gene further rescued the photoreceptor response in a murine model of Leber congenital amarousis. Our studies highlight the translational potential of glycosylation site-modified AAV2 vectors for hepatic and ocular gene therapy applications.

Rational Engineering and Preclinical Evaluation of Neddylation and SUMOylation Site Modified Adeno-Associated Virus Vectors in Murine Models of Hemophilia B and Leber Congenital Amaurosis.

Synthetic engineering of viral vectors such as adeno-associated virus (AAV) is crucial to overcome host transduction barriers observed during clinical gene therapy. We reasoned that exploring the role of cellular ubiquitin-like modifiers (UBLs) such as Neddylation or SUMOylation during AAV transduction could be beneficial. Using a combination of in silico biochemical and molecular engineering strategies, we have studied the impact of these UBLs during AAV2 infection and further developed Neddylation or SUMOylation site-modified AAV vectors and validated them in multiple disease models in vitro and in vivo. Hepatic gene transfer of two novel vectors developed, K105Q (SUMOylation-site mutant) and K665Q (Neddylation-site mutant), demonstrated a significantly improved human coagulation factor (F) IX expression (up to two-fold) in a murine model of hemophilia B. Furthermore, subretinal gene transfer of AAV2-K105Q vector expressing RPE65 gene demonstrated visual correction in a murine model of a retinal degenerative disease (rd12 mice). These vectors did not have any adverse immunogenic events in vivo. Taken together, we demonstrate that gene delivery vectors specifically engineered at UBLs can improve the therapeutic outcome during AAV-mediated ocular or hepatic gene therapy.

Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV) 1-rh10 serotypes.

Post-translational modifications in viral capsids are known to fine-tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post-translational modifications during intra-cellular maturation of its capsid proteins. Data on such post-translational modifications in the capsid of recombinant adeno-associated virus serotypes (AAV1-rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post-translational modifications in AAV1-rh10 capsid protein. Our analysis revealed a total of 52 post-translational modifications in AAV2-AAVrh10 capsids, including ubiquitination (17%), glycosylation (36%), phosphorylation (21%), SUMOylation (13%) and acetylation (11%). While AAV1 had no detectable post-translational modification, at least four AAV serotypes had >7 post-translational modifications in their capsid protein. About 82% of these post-translational modifications are novel. A limited validation of AAV2 capsids by MALDI-TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV2 capsid (AAV2-N253Q), which severely compromised its packaging efficiency (~ 100-fold vs. AAV2 wild-type vectors). In order to confirm other post-translational modifications detected such as SUMOylation, mutagenesis of a SUMOylation site(K258Q) in AAV2 was performed. This mutant vector demonstrated reduced levels of SUMO-1/2/3 proteins and negligible transduction, 2 weeks after ocular gene transfer. Our study underscores the heterogeneity of post-translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post-translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. ENZYMES: Trypsin, EC 3.4.21.4.