Mitophagy in Astrocytes Is Required for the Health of Optic Nerve.

Mitochondrial dysfunction in astrocytes has been implicated in the development of various neurological disorders. Mitophagy, mitochondrial autophagy, is required for proper mitochondrial function by preventing the accumulation of damaged mitochondria. The importance of mitophagy, specifically in the astrocytes of the optic nerve (ON), has been little studied. We introduce an animal model in which two separate mutations act synergistically to produce severe ON degeneration. The first mutation is in Cryba1, which encodes ßA3/A1-crystallin, a lens protein also expressed in astrocytes, where it regulates lysosomal pH. The second mutation is in Bckdk, which encodes branched-chain ketoacid dehydrogenase kinase, which is ubiquitously expressed in the mitochondrial matrix and involved in the catabolism of the branched-chain amino acids. BCKDK is essential for mitochondrial function and the amelioration of oxidative stress. Neither of the mutations in isolation has a significant effect on the ON, but animals homozygous for both mutations (DM) exhibit very serious ON degeneration. ON astrocytes from these double-mutant (DM) animals have lysosomal defects, including impaired mitophagy, and dysfunctional mitochondria. Urolithin A can rescue the mitophagy impairment in DM astrocytes and reduce ON degeneration. These data demonstrate that efficient mitophagy in astrocytes is required for ON health and functional integrity.

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD.

Age-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [ CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1α, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.

Aberrant Akt2 signaling in the RPE may contribute to retinal fibrosis process in diabetic retinopathy.

Diabetic Retinopathy (DR) is a complication of diabetes that causes blindness in adults. Retinal fibrosis is closely associated with developing proliferative diabetic retinopathy (PDR). Clinical studies have shown that fibrotic membranes exhibit uncontrolled growth in PDR and contribute to retinal detachment from RPE cells, ultimately leading to vision loss. While anti-VEGF agents and invasive laser treatments are the primary treatments for PDR, retinal fibrosis has received minimal attention as a potential target for therapeutic intervention. Therefore, to investigate the potential role of Akt2 in the diabetes-induced retinal fibrosis process, we generated RPE-specific Akt2 conditional knockout (cKO) mice and induced diabetes in these mice and Akt2fl/fl control mice by intraperitoneal injection of streptozotocin. After an 8-month duration of diabetes (10 months of age), the mice were euthanized and expression of tight junction proteins, epithelial-mesenchymal transition (EMT), and fibrosis markers were examined in the RPE. Diabetes induction in the floxed control mice decreased levels of the RPE tight junction protein ZO-1 and adherens junction proteins occludin and E-cadherin; these decreases were rescued in Akt2 cKO diabetic mice. Loss of Akt2 also inhibited diabetes-induced elevation of RNA and protein levels of the EMT markers Snail/Slug and Twist1 in the RPE as compared to Akt2fl/fl diabetic mice. We also found that in Akt2 cKO mice diabetes-induced increase of fibrosis markers, including collagen IV, Connective tissue growth factor (CTGF), fibronectin, and alpha-SMA was attenuated. Furthermore, we observed that high glucose-induced alterations in EMT and fibrosis markers in wild-type (WT) RPE explants were rescued in the presence of PI3K and ERK inhibitors, indicating diabetes-induced retinal fibrosis may be mediated via the PI3K/Akt2/ERK signaling, which could provide a novel target for DR therapy.

Activated cGAS/STING signaling elicits endothelial cell senescence in early diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of blindness in working-age adults and remains an important public health issue worldwide. Here we demonstrate that the expression of stimulator of interferon genes (STING) is increased in patients with DR and animal models of diabetic eye disease. STING has been previously shown to regulate cell senescence and inflammation, key contributors to the development and progression of DR. To investigate the mechanism whereby STING contributes to the pathogenesis of DR, diabetes was induced in STING-KO mice and STINGGT (loss-of-function mutation) mice, and molecular alterations and pathological changes in the retina were characterized. We report that retinal endothelial cell senescence, inflammation, and capillary degeneration were all inhibited in STING-KO diabetic mice; these observations were independently corroborated in STINGGT mice. These protective effects resulted from the reduction in TBK1, IRF3, and NF-κB phosphorylation in the absence of STING. Collectively, our results suggest that targeting STING may be an effective therapy for the early prevention and treatment of DR.

Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD.

In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1ß: interleukin 1 beta; IS: Inner segment; ITGB1/integrin ß1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.

Microglia-Neutrophil Interactions Drive Dry AMD-like Pathology in a Mouse Model.

In dry age-related macular degeneration (AMD), inflammation plays a key role in disease pathogenesis. Innate immune cells such as microglia and neutrophils infiltrate the sub-retinal space (SRS) to induce chronic inflammation and AMD progression. But a major gap in our understanding is how these cells interact with each other in AMD. Here, we report a novel concept of how dynamic interactions between microglia and neutrophils contribute to AMD pathology. Using well-characterized genetically engineered mouse models as tools, we show that in the diseased state, retinal pigmented epithelial (RPE) cells trigger pro-inflammatory (M1) transition in microglia with diminished expression of the homeostatic marker, CX3CR1. Activated microglia localize to the SRS and regulate local neutrophil function, triggering their activation and thereby inducing early RPE changes. Ligand receptor (LR)-loop analysis and cell culture studies revealed that M1 microglia also induce the expression of neutrophil adhesion mediators (integrin ß1/α4) through their interaction with CD14 on microglia. Furthermore, microglia-induced neutrophil activation and subsequent neutrophil-mediated RPE alterations were mitigated by inhibiting Akt2 in microglia. These results suggest that the Akt2 pathway in microglia drives M1 microglia-mediated neutrophil activation, thereby triggering early RPE degeneration and is a novel therapeutic target for early AMD, a stage without treatment options.

Reducing Akt2 in retinal pigment epithelial cells causes a compensatory increase in Akt1 and attenuates diabetic retinopathy.

The retinal pigment epithelium (RPE) plays an important role in the development of diabetic retinopathy (DR), a leading cause of blindness worldwide. Here we set out to explore the role of Akt2 signaling-integral to both RPE homeostasis and glucose metabolism-to DR. Using human tissue and genetically manipulated mice (including RPE-specific conditional knockout (cKO) and knock-in (KI) mice), we investigate whether Akts in the RPE influences DR in models of diabetic eye disease. We found that Akt1 and Akt2 activities were reciprocally regulated in the RPE of DR donor tissue and diabetic mice. Akt2 cKO attenuated diabetes-induced retinal abnormalities through a compensatory upregulation of phospho-Akt1 leading to an inhibition of vascular injury, inflammatory cytokine release, and infiltration of immune cells mediated by the GSK3ß/NF-κB signaling pathway; overexpression of Akt2 has no effect. We propose that targeting Akt1 activity in the RPE may be a novel therapy for treating DR.

A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.

ßA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity.

ßA3/A1-crystallin, a lens protein that is also expressed in astrocytes, is produced as ßA3 and ßA1-crystallin isoforms by leaky ribosomal scanning. In a previous human proteome high-throughput array, we found that ßA3/A1-crystallin interacts with protein tyrosine phosphatase 1B (PTP1B), a key regulator of glucose metabolism. This prompted us to explore possible roles of ßA3/A1-crystallin in metabolism of retinal astrocytes. We found that ßA1-crystallin acts as an uncompetitive inhibitor of PTP1B, but ßA3-crystallin does not. Loss of ßA1-crystallin in astrocytes triggers metabolic abnormalities and inflammation. In CRISPR/cas9 gene-edited ßA1-knockdown (KD) mice, but not in ßA3-knockout (KO) mice, the streptozotocin (STZ)-induced diabetic retinopathy (DR)-like phenotype is exacerbated. Here, we have identified ßA1-crystallin as a regulator of PTP1B; loss of this regulation may be a new mechanism by which astrocytes contribute to DR. Interestingly, proliferative diabetic retinopathy (PDR) patients showed reduced ßA1-crystallin and higher levels of PTP1B in the vitreous humor.

BNIP3L-mediated mitophagy is required for mitochondrial remodeling during the differentiation of optic nerve oligodendrocytes.

Retinal ganglion cell axons are heavily myelinated (98%) and myelin damage in the optic nerve (ON) severely affects vision. Understanding the molecular mechanism of oligodendrocyte progenitor cell (OPC) differentiation into mature oligodendrocytes will be essential for developing new therapeutic approaches for ON demyelinating diseases. To this end, we developed a new method for isolation and culture of ON-derived oligodendrocyte lineage cells and used it to study OPC differentiation. A critical aspect of cellular differentiation is macroautophagy/autophagy, a catabolic process that allows for cell remodeling by degradation of excess or damaged cellular molecules and organelles. Knockdown of ATG9A and BECN1 (pro-autophagic proteins involved in the early stages of autophagosome formation) led to a significant reduction in proliferation and survival of OPCs. We also found that autophagy flux (a measure of autophagic degradation activity) is significantly increased during progression of oligodendrocyte differentiation. Additionally, we demonstrate a significant change in mitochondrial dynamics during oligodendrocyte differentiation, which is associated with a significant increase in programmed mitophagy (selective autophagic clearance of mitochondria). This process is mediated by the mitophagy receptor BNIP3L (BCL2/adenovirus E1B interacting protein 3-like). BNIP3L-mediated mitophagy plays a crucial role in the regulation of mitochondrial network formation, mitochondrial function and the viability of newly differentiated oligodendrocytes. Our studies provide novel evidence that proper mitochondrial dynamics is required for establishment of functional mitochondria in mature oligodendrocytes. These findings are significant because targeting BNIP3L-mediated programmed mitophagy may provide a novel therapeutic approach for stimulating myelin repair in ON demyelinating diseases.Abbreviations: A2B5: a surface antigen of oligodendrocytes precursor cells, A2B5 clone 105; ACTB: actin, beta; APC: an antibody to label mature oligodendrocytes, anti-adenomatous polyposis coli clone CC1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9A: autophagy related 9A; AU: arbitrary units; BafA1: bafilomycin A1; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CNP: 2',3'-cyclic nucleotide 3'-phosphodiesterase; Ctl: control; COX8: cytochrome c oxidase subunit; CSPG4/NG2: chondroitin sulfate proteoglycan 4; DAPI: 4'6-diamino-2-phenylindole; DNM1L: dynamin 1-like; EGFP: enhanced green fluorescent protein; FACS: fluorescence-activated cell sorting; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; GFP: green fluorescent protein; HsESC: human embryonic stem cell; IEM: immunoelectron microscopy; LAMP1: lysosomal-associated membrane protein 1; LC3B: microtubule-associated protein 1 light chain 3; MBP: myelin basic protein; MFN2: mitofusin 2; Mito-Keima: mitochondria-targeted monomeric keima-red; Mito-GFP: mitochondria-green fluorescent protein; Mito-RFP: mitochondria-red fluorescent protein; MitoSOX: red mitochondrial superoxide probe; MKI67: antigen identified by monoclonal antibody Ki 67; MMP: mitochondrial membrane potential; O4: oligodendrocyte marker O4; OLIG2: oligodendrocyte transcription factor 2; ON: optic nerve; OPA1: OPA1, mitochondrial dynamin like GTPase; OPC: oligodendrocyte progenitor cell; PDL: poly-D-lysine; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; RGC: retinal ganglion cell; ROS: reactive oxygen species; RT-PCR: real time polymerase chain reaction; SEM: standard error of the mean; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; TUBB3: tubulin, beta 3 class III.

Role of glia in optic nerve.

Glial cells are critically important for maintenance of neuronal activity in the central nervous system (CNS), including the optic nerve (ON). However, the ON has several unique characteristics, such as an extremely high myelination level of retinal ganglion cell (RGC) axons throughout the length of the nerve (with virtually all fibers myelinated by 7 months of age in humans), lack of synapses and very narrow geometry. Moreover, the optic nerve head (ONH) - a region where the RGC axons exit the eye - represents an interesting area that is morphologically distinct in different species. In many cases of multiple sclerosis (demyelinating disease of the CNS) vision problems are the first manifestation of the disease, suggesting that RGCs and/or glia in the ON are more sensitive to pathological conditions than cells in other parts of the CNS. Here, we summarize current knowledge on glial organization and function in the ON, focusing on glial support of RGCs. We cover both well-established concepts on the important role of glial cells in ON health and new findings, including novel insights into mechanisms of remyelination, microglia/NG2 cell-cell interaction, astrocyte reactivity and the regulation of reactive astrogliosis by mitochondrial fragmentation in microglia.

The role of lipocalin-2 in age-related macular degeneration (AMD).

Lipocalins are a family of secreted adipokines which play important roles in various biological processes. Lipocalin-2 (LCN-2) has been shown to be involved in acute and chronic inflammation. This particular protein is critical in the pathogenesis of several diseases including cancer, diabetes, obesity, and multiple sclerosis. Herein, we discuss the general molecular basis for the involvement of LCN-2 in acute infections and chronic disease progression and also ascertain the probable role of LCN-2 in ocular diseases, particularly in age-related macular degeneration (AMD). We elaborate on the signaling cascades which trigger LCN-2 upregulation in AMD and suggest therapeutic strategies for targeting such pathways.

Modulating EGFR-MTORC1-autophagy as a potential therapy for persistent fetal vasculature (PFV) disease.

Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress normally. The regulatory mechanisms responsible for fetal vascular regression remain obscure, as does the underlying cause of regression failure. However, there are a few animal models that mimic the clinical manifestations of human PFV, which can be used to study different aspects of the disease. One such model is the Nuc1 rat model that arose from a spontaneous mutation in the Cryba1 (crystallin, beta 1) gene and exhibits complete failure of the hyaloid vasculature to regress. Our studies with the Nuc1 rat indicate that macroautophagy/autophagy, a process in eukaryotic cells for degrading dysfunctional components to ensure cellular homeostasis, is severely impaired in Nuc1 ocular astrocytes. Further, we show that CRYBA1 interacts with EGFR (epidermal growth factor receptor) and that loss of this interaction in Nuc1 astrocytes increases EGFR levels. Moreover, our data also show a reduction in EGFR degradation in Nuc1 astrocytes compared to control cells that leads to over-activation of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway. The impaired EGFR-MTORC1-autophagy signaling in Nuc1 astrocytes triggers abnormal proliferation and migration. The abnormally migrating astrocytes ensheath the hyaloid artery, contributing to the pathogenesis of PFV in Nuc1, by adversely affecting the vascular remodeling processes essential to regression of the fetal vasculature. Herein, we demonstrate in vivo that gefitinib (EGFR inhibitor) can rescue the PFV phenotype in Nuc1 and may serve as a novel therapy for PFV disease by modulating the EGFR-MTORC1-autophagy pathway. ABBREVIATIONS: ACTB: actin, beta; CCND3: cyclin 3; CDK6: cyclin-dependent kinase 6; CHQ: chloroquine; COL4A1: collagen, type IV, alpha 1; CRYBA1: crystallin, beta A1; DAPI: 4'6-diamino-2-phenylindole; EGFR: epidermal growth factor receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; KDR: kinase insert domain protein receptor; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MKI67: antigen identified by monoclonal antibody Ki 67; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP: poly (ADP-ribose) polymerase family; PCNA: proliferating cell nuclear antigen; PFV: persistent fetal vasculature; PHPV: persistent hyperplastic primary vitreous; RPE: retinal pigmented epithelium; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SQSTM1/p62: sequestome 1; TUBB: tubulin, beta; VCL: vinculin; VEGFA: vascular endothelial growth factor A; WT: wild type.

Melatonin as the Possible Link Between Age-Related Retinal Regeneration and the Disrupted Circadian Rhythm in Elderly.

The association between age-related macular degeneration (AMD) and biological rhythms has been insufficiently studied; however there are several reasons to believe that impairment in circadian rhythm may affect incidence and pathogenesis of AMD. The current understanding of AMD pathology is based on age-related, cumulative oxidative damage to the retinal pigmented epithelium (RPE) partially due to impaired clearance of phagocytosed photoreceptor outer segments. In higher vertebrates, phagocytosis of the outer segments is synchronized by circadian rhythms and occurs shortly after dawn, followed by lysosomal-mediated clearance. Aging has been shown to be associated with the changes in circadian rhythmicity of melatonin production, which can be a major factor contributing to the impaired balance between phagocytosis and clearance and increased levels of reactive oxygen species resulting in degenerative changes in the retina. This minireview summarizes studies linking AMD with melatonin production and discusses challenges and perspectives of this area of research.

Neutrophils homing into the retina trigger pathology in early age-related macular degeneration.

Age-related macular degeneration (AMD) is an expanding problem as longevity increases worldwide. While inflammation clearly contributes to vision loss in AMD, the mechanism remains controversial. Here we show that neutrophils are important in this inflammatory process. In the retinas of both early AMD patients and in a mouse model with an early AMD-like phenotype, we show neutrophil infiltration. Such infiltration was confirmed experimentally using ribbon-scanning confocal microscopy (RSCM) and IFNλ- activated dye labeled normal neutrophils. With neutrophils lacking lipocalin-2 (LCN-2), infiltration was greatly reduced. Further, increased levels of IFNλ in early AMD trigger neutrophil activation and LCN-2 upregulation. LCN-2 promotes inflammation by modulating integrin ß1 levels to stimulate adhesion and transmigration of activated neutrophils into the retina. We show that in the mouse model, inhibiting AKT2 neutralizes IFNλ inflammatory signals, reduces LCN-2-mediated neutrophil infiltration, and reverses early AMD-like phenotype changes. Thus, AKT2 inhibitors may have therapeutic potential in early, dry AMD.

A Role for ßA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration.

Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that ßA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells. Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used. Spatial localization of proteins was demonstrated with immunofluorescence, gene expression levels were determined by quantitative PCR (qPCR), and protein levels by Western blotting. Cell movement was evaluated using wound healing and cell migration assays. Co-immunoprecipitation was used to identify binding partners of ßA3/A1-crystallin. Results: ßA3/A1-crystallin is upregulated in polarized RPE cells compared to undifferentiated cells. Loss of ßA3/A1-crystallin in murine and human RPE cells resulted in upregulation of Snail and vimentin, downregulation of E-cadherin, and increased cell migration. ßA3/A1-crystallin binds to cortactin, and loss of ßA3/A1-crystallin resulted in increased P-cortactinY421. The RPE from AMD samples had increased Snail and vimentin, and decreased E-cadherin, compared to age-matched controls. Conclusions: We introduced a novel concept of dry AMD initiation induced by lysosomal clearance defects in the RPE and subsequent attempts by RPE cells to avoid the resulting stress by undergoing EMT. We demonstrate that ßA3/A1-crystallin is a potential therapeutic target for AMD through rejuvenation of lysosomal dysfunction and potentially, reversal of EMT.

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.

The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H+ -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding ßA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression.

Activating the AKT2-nuclear factor-κB-lipocalin-2 axis elicits an inflammatory response in age-related macular degeneration.

Age-related macular degeneration (AMD) is a complex and progressive degenerative eye disease resulting in severe loss of central vision. Recent evidence indicates that immune system dysregulation could contribute to the development of AMD. We hypothesize that defective lysosome-mediated clearance causes accumulation of waste products in the retinal pigmented epithelium (RPE), activating the immune system and leading to retinal tissue injury and AMD. We have generated unique genetically engineered mice in which lysosome-mediated clearance (both by phagocytosis and autophagy) in RPE cells is compromised, causing the development of features of early AMD. Our recent data indicate a link between lipocalin-2 (LCN-2) and the inflammatory responses induced in this mouse model. We show that nuclear factor-κB (NF-κB) and STAT-1 may function as a complex in our animal model system, together controlling the upregulation of LCN-2 expression in the retina and stimulating an inflammatory response. This study revealed increased infiltration of LCN-2-positive neutrophils in the choroid and retina of early AMD patients as compared with age-matched controls. Our results demonstrate that, both in our animal model and in human AMD, the AKT2-NF-κB-LCN-2 signalling axis is involved in activating the inflammatory response, making this pathway a potential target for AMD treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems.

A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70-80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs.