Ocular Amyloid, Condensates, and Aggregates - Higher-Order Protein Assemblies Participate in Both Retinal Degeneration and Function.

The formation of higher-order protein assemblies (commonly called protein aggregates) has long been associated with disease states, particularly in neurodegenerative disorders. Within the eye, protein aggregation has also been implicated in various retinal degenerative diseases ranging from retinitis pigmentosa (RP) to Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) to age-related macular degeneration (AMD). Yet, many essential cellular processes including transcription, translation, and the formation of non-membrane bound organelles require the formation of functional, non-pathologic protein aggregates to maintain cellular homeostasis. Thus, functional protein aggregates, also called condensates, likely play essential roles in maintaining normal retina function. However, currently, there is a critical gap in our knowledge: What proteins form higher-order assemblies under normal conditions within the retina and what function do these structures serve? Herein, we present data suggesting that protein aggregation is identifiable in multiple retinal layers of normal, healthy murine retina, and briefly discuss the potential contributions of aggregated proteins to normal retinal function, with a focus on the photoreceptor inner and outer segment.

A loss-of-function cysteine mutant in fibulin-3 (EFEMP1) forms aberrant extracellular disulfide-linked homodimers and alters extracellular matrix composition.

Fibulin-3 (F3 or EFEMP1) is a disulfide-rich, secreted glycoprotein necessary for maintaining extracellular matrix (ECM) and connective tissue integrity. Three studies have identified distinct autosomal recessive F3 mutations in individuals with Marfan Syndrome-like phenotypes. Herein, we characterize how one of these mutations, c.163T>C; p.Cys55Arg (C55R), disrupts F3 secretion, quaternary structure, and function by forming unique extracellular disulfide-linked homodimers. Dual cysteine mutants suggest that the C55R-induced disulfide species forms because of the new availability of Cys70 on adjacent F3 monomers. Surprisingly, mutation of single cysteines located near Cys55 (i.e., Cys29, Cys42, Cys48, Cys61, Cys70, Cys159, and Cys171) also produced similar extracellular disulfide-linked dimers, suggesting that this is not a phenomenon isolated to the C55R mutant. To assess C55R functionality, F3 knockout (KO) retinal pigmented epithelial (RPE) cells were generated, followed by reintroduction of wild-type (WT) or C55R F3. F3 KO cells produced lower levels of the ECM remodeling enzyme, matrix metalloproteinase 2, and reduced formation of collagen VI ECM filaments, both of which were partially rescued by WT F3 overexpression. However, C55R F3 was unable to compensate for these same ECM-related defects. Our results highlight the unique behavior of particular cysteine mutations in F3 and uncover potential routes to restore C55R F3 loss-of-function.

Specifications of the ACMG/AMP variant curation guidelines for myocilin: Recommendations from the clingen glaucoma expert panel.

The standardization of variant curation criteria is essential for accurate interpretation of genetic results and clinical care of patients. The variant curation guidelines developed by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015 are widely used but are not gene specific. To address this issue, the Clinical Genome Resource (ClinGen) Variant Curation Expert Panels (VCEP) have been tasked with developing gene-specific variant curation guidelines. The Glaucoma VCEP was created to develop rule specifications for genes associated with primary glaucoma, including myocilin (MYOC), the most common cause of Mendelian glaucoma. Of the 28 ACMG/AMP criteria, the Glaucoma VCEP adapted 15 rules to MYOC and determined 13 rules not applicable. Key specifications included determining minor allele frequency thresholds, developing an approach to counting probands and segregations, and reviewing functional assays. The rules were piloted on 81 variants and led to a change in classification in 40% of those that were classified in ClinVar, with functional evidence influencing the classification of 18 variants. The standardized variant curation guidelines for MYOC provide a framework for the consistent application of the rules between laboratories, to improve MYOC genetic testing in the management of glaucoma.

A novel homozygous missense mutation p.P388S in TULP1 causes protein instability and retinitis pigmentosa.

Purpose: Retinitis pigmentosa (RP) is an inherited retinal disorder that results in the degeneration of photoreceptor cells, ultimately leading to severe visual impairment. We characterized a consanguineous family from Southern India wherein a 25 year old individual presented with night blindness since childhood. The purpose of this study was to identify the causative mutation for RP in this individual as well as characterize how the mutation may ultimately affect protein function. Methods: We performed a complete ophthalmologic examination of the proband followed by exome sequencing. The likely causative mutation was identified and modeled in cultured cells, evaluating its expression, solubility (both with western blotting), subcellular distribution, (confocal microscopy), and testing whether this variant induced endoplasmic reticulum (ER) stress (quantitative PCR [qPCR] and western blotting). Results: The proband presented with generalized and parafoveal retinal pigmented epithelium (RPE) atrophy with bone spicule-like pigmentation in the midperiphery and arteriolar attenuation. Optical coherence tomography scans through the macula of both eyes showed atrophy of the outer retinal layers with loss of the ellipsoid zone, whereas the systemic examination of this individual was normal. The proband's parents and sibling were asymptomatic and had normal funduscopic examinations. We discovered a novel homozygous p.Pro388Ser mutation in the tubby-like protein 1 (TULP1) gene in the individual with RP. In cultured cells, the P388S mutation does not alter the subcellular distribution of TULP1 or induce ER stress when compared to wild-type TULP1, but instead significantly lowers protein stability as indicated with steady-state and cycloheximide-chase experiments. Conclusions: These results add to the list of known mutations in TULP1 identified in individuals with RP and suggest a possible unique pathogenic mechanism in TULP1-induced RP, which may be shared among select mutations in TULP1.

Small Molecule-Based Inducible Gene Therapies for Retinal Degeneration.

The eye is an excellent target organ for gene therapy. It is physically isolated, easily accessible, immune-privileged, and postmitotic. Furthermore, potential gene therapies introduced into the eye can be evaluated by noninvasive methods such as fundoscopy, electroretinography, and optical coherence tomography. In the last two decades, great advances have been made in understanding the molecular underpinnings of retinal degenerative diseases. Building upon the development of modern techniques for gene delivery, many gene-based therapies have been effectively used to treat loss-of-function retinal diseases in mice and men. Significant effort has been invested into making gene delivery vehicles more efficient, less toxic, and non-immunogenic. However, one challenge for the treatment of more complex gain-of-function diseases, many of which might be benefited by the regulation of cellular stress-responsive signaling pathways, is the ability to control the strategy in a physiological (conditional) manner. This review is focused on promising retinal gene therapy strategies that rely on small molecule-based conditional regulation and the inherent limitations and challenges of these strategies that need to be addressed prior to their extensive use.

Prospective Application of Activity-Based Proteomic Profiling in Vision Research-Potential Unique Insights into Ocular Protease Biology and Pathology.

Activity-based proteomic profiling (ABPP) is a powerful tool to specifically target and measure the activity of a family of enzymes with the same function and reactivity, which provides a significant advantage over conventional proteomic strategies that simply provide abundance information. A number of inherited and age-related eye diseases are caused by polymorphisms/mutations or abnormal expression of proteases including serine proteases, cysteine proteases, and matrix metalloproteinases, amongst others. However, neither conventional genomic, transcriptomic, nor traditional proteomic profiling directly interrogate protease activities. Thus, leveraging ABPP to probe the activity of these enzyme classes as they relate to normal function and pathophysiology of the eye represents a unique potential opportunity for disease interrogation and possibly intervention.

A High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production.

Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion.

An inducible form of Nrf2 confers enhanced protection against acute oxidative stresses in RPE cells.

Increasing evidence suggests that overt oxidative stress within the retina plays an important role in the progression of age-related retinal decline, and in particular, in the disease age-related macular degeneration (AMD). Nuclear factor erythroid 2-like 2 (Nrf2) is a master transcription factor that upregulates numerous of antioxidant/detoxification genes. Nrf2-/- mice develop progressive retinal degeneration that includes the formation of drusen-like deposits, lipofuscin, and sub-retinal pigment epithelium (RPE) deposition of inflammatory proteins. Furthermore, strategies that promote Nrf2 activation have shown promise for the treatment of cone/rod dystrophies and other forms of retinal degeneration. Herein we explored whether utilizing a small molecule-inducible version of Nrf2 confers additional protection against oxidative stresses when compared to a constitutively expressed version of Nrf2. Stable populations of human ARPE-19 cells were generated that express either constitutive FLAG-tagged (FT) Nrf2 (FT cNrf2) or doxycycline (dox)-inducible FT Nrf2 (FT iNrf2) at low levels (∼4.5 fold vs. endogenous). Expression of either FT cNRF2 or FT iNrf2 upregulated canonical antioxidant genes (e.g., NQO1, GCLC). Both FT cNrf2 and FT iNrf2 ARPE-19 cells were protected from cigarette smoke extract-induced nitric oxide generation to similar extents. However, only FT iNrf2 cells demonstrated enhanced resistance to doxorubicin and cumene hydroperoxide-mediated increases in mitochondrial superoxide and lipid peroxidation, respectively, and did so in a dox-dependent manner. These results suggest that therapeutic approaches which conditionally control Nrf2 activity may provide additional protection against acute oxidative stresses when compared to constitutively expressed Nrf2 strategies.

Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain.

Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼ 70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation.

A novel H395R mutation in MKKS/BBS6 causes retinitis pigmentosa and polydactyly without other findings of Bardet-Biedl or McKusick-Kaufman syndrome.

PURPOSE: To identify the causative mutation in two siblings from a consanguineous family in India with retinitis pigmentosa (RP) and polydactyly without other findings of Bardet-Biedl syndrome (BBS). We also performed functional characterization of the mutant protein to explore its role in this limited form of BBS. METHODS: The siblings underwent a thorough ophthalmological examination, including retinal optical coherence tomography (OCT) imaging, and an extensive physical examination with abdominal ultrasonography to characterize the disease phenotype. Next-generation sequencing (NGS) using a panel targeting retinal degeneration genes was performed on genomic DNA samples from the siblings and parents. Upon identification of the causative mutation, functional characterization was accomplished by performing protein-protein interaction studies in human embryonic kidney (HEK-293T) and human adult retinal pigmented epithelium (ARPE-19) cells. RESULTS: The two siblings showed signs of RP and polydactyly. The patients did not have truncal obesity, renal anomalies, hydrometrocolpos, congenital heart disease, or overt cognitive defects. NGS identified a homozygous c.1184A>G mutation in the MKKS/BBS6 gene in both patients resulting in a p.H395R substitution in the MKKS/BBS6 protein. This mutant protein decreased the interaction of MKKS/BBS6 with BBS12 but did so to a different extent in the HEK-293T versus ARPE-19 cells. Nonetheless, the effect of the H395R variant on disrupting interactions with BBS12 was not as profound as other reported MKKS/BBS6 mutations associated with syndromic RP. CONCLUSIONS: We identified a novel H395R substitution in MKKS/BBS6 that results in a unique phenotype of only RP and polydactyly. Our observations reaffirm the notion that mutations in MKKS/BBS6 cause phenotypic heterogeneity and do not always result in classic MKKS or BBS findings.

Mapping wild-type and R345W fibulin-3 intracellular interactomes.

Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate 'heavy' and 'light' isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.

Genetic ablation of N-linked glycosylation reveals two key folding pathways for R345W fibulin-3, a secreted protein associated with retinal degeneration.

An R345W mutation in the N-glycoprotein, fibulin-3 (F3), results in inefficient F3 folding/secretion and higher intracellular F3 levels. Inheritance of this mutation causes the retinal dystrophy malattia leventinese. N-Linked glycosylation is a common cotranslational protein modification that can regulate protein folding efficiency and energetics. Therefore, we explored how N-glycosylation alters the protein homeostasis or proteostasis of wild-type (WT) and R345W F3 in ARPE-19 cells. Enzymatic and lectin binding assays confirmed that WT and R345W F3 are both primarily N-glycosylated at Asn249. Tunicamycin treatment selectively reduced R345W F3 secretion by 87% (vs. WT F3). Genetic elimination of F3 N-glycosylation (via an N249Q mutation) caused R345W F3 to aggregate intracellularly and adopt an altered secreted conformation. The endoplasmic reticulum (ER) chaperones GRP78 (glucose-regulated protein 78) and GRP94 (glucose-regulated protein 94), and the ER lectins calnexin and calreticulin were identified as F3 binding partners by immunoprecipitation. Significantly more N249Q and N249Q/R345W F3 interacted with GRP94, while substantially less N249Q and N249Q/R345W interacted with the ER lectins than their N-glycosylated counterparts. Inhibition of GRP94 ATPase activity reduced only N249Q/R345W F3 secretion (by 62%), demonstrating this variant's unique reliance on GRP94 for secretion. These observations suggest that R345W F3, but not WT F3, requires N-glycosylation to acquire a stable, native-like structure.

Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy: Similarities to Age-Related Macular Degeneration and Potential Therapies.

Fibulin-3 (F3) is a secreted, disulfide-rich glycoprotein which is expressed in a variety of tissues within the body, including the retina. An Arg345Trp (R345W) mutation in F3 was identified as the cause of a rare retinal dystrophy, Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD). ML/DHRD shares many phenotypic similarities with age-related macular degeneration (AMD). The most prominent feature of ML/DHRD is the development of radial or honeycomb patterns of drusen which can develop as early as adolescence. Two independent mouse models of ML/DHRD show evidence of complement activation as well as retinal pigment epithelium (RPE) atrophy, strengthening the phenotypic connection with AMD. Because of its similarities with AMD, ML/DHRD is receiving increasing interest as a potential surrogate disease to study the underpinnings of AMD. This mini-review summarizes the current knowledge of F3 and points toward potential therapeutic strategies which directly or indirectly target cellular dysfunction associated with R345W F3.

Differential tolerance of 'pseudo-pathogenic' tryptophan residues in calcium-binding EGF domains of short fibulin proteins.

An Arg345Trp (R345W) mutation in the last canonical calcium-binding epidermal growth factor (cbEGF) domain of fibulin-3 (F3) causes the rare macular dystrophy, Malattia Leventinese (ML). In cell culture studies, this mutation leads to inefficient F3 secretion and higher intracellular steady state levels, likely due to F3 disulfide bonding and/or protein folding problems. However, how the R345W mutation actually causes ML is still largely unknown. Herein we tested whether the introduction of analogous, 'pseudo-pathogenic' tryptophan mutations immediately after the bn cysteine (bn+1) in other cbEGF domains also caused protein folding/secretion challenges. We found that introduction of tryptophan mutations into each of the four other F3 canonical cbEGF domains caused a significant reduction in protein secretion ranging from 2.7 to 56% of wild-type (WT) F3 levels. Surprisingly, an R185W mutation in the first canonical cbEGF domain of F3 yielded the highest amount of secretion among the F3 tryptophan mutants, and its secretion defect could be rescued to near WT levels (95%) after growth temperature reduction. Interestingly, when similarly positioned tryptophan mutations were introduced into any of the canonical cbEGF domains of the highly homologous protein, fibulin-5 (F5), there was no effect on secretion. In an attempt to make F3 tolerant of tryptophan residues (like F5), we genetically engineered F3 to have a higher sequence homology with F5 by deleting three insert regions present in F3, but not F5. However, deletion of one or more of these regions did not have a beneficial effect on R345W F3 secretion. Overall, these results demonstrate that the introduction of tryptophan residues at the bn+1 position does not universally disrupt cbEGF domain folding and secretion, but that their effect is context dependent, and in this case, uniquely disrupt the folding of canonical cbEGF domains of F3, but not F5.

Effect of single amino acid substitution on oxidative modifications of the Parkinson's disease-related protein, DJ-1.

Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.

Exploring ocular fibulin-3 (EFEMP1): Anatomical, age-related, and species perspectives.

Fibulin-3 (FBLN3, aka EFEMP1) is a secreted extracellular matrix (ECM) glycoprotein implicated in ocular diseases including glaucoma and age-related macular degeneration. Yet surprisingly, little is known about its native biology, expression patterns, and localization in the eye. To overcome these shortcomings, we conducted gene expression analysis and immunohistochemistry for FBLN3 in ocular tissues from mice, pigs, non-human primates, and humans. Moreover, we evaluated age-related changes in FBLN3 and FBLN3-related ECM remodeling enzymes/inhibitors in aging mice. We found that FBLN3 displayed distinct staining patterns consistent across the mouse retina, particularly in the ganglion cell layer and inner nuclear layer (INL). In contrast, human retinas exhibited a unique staining pattern, with enrichment of FBLN3 in the retinal pigment epithelium (RPE), INL, and outer nuclear layer (ONL) in the peripheral retina. This staining transitioned to the outer plexiform layer (OPL) in the central retina/macula, and was accompanied by reduced RPE immunoreactivity approaching the fovea. Surprisingly, we found significant age-related increases in FBLN3 expression and protein abundance in the mouse retina which was paralleled by reduced transcript levels of FBLN3-degrading enzymes (i.e., Mmp2 and Htra1). Our findings highlight important species-dependent, retinal region-specific, and age-related expression and localization patterns of FBLN3 which favor its accumulation during aging. These findings contribute to a better understanding of FBLN3's role in ocular pathology and provide valuable insights for future FBLN3 research.

Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing.

We present a method of in vitro/in vivo protein detection by pairing CRISPR-Cas9 genome editing with the NanoBiT system. We describe steps for cell culturing, in vitro CRISPR-Cas9 ribonucleoprotein delivery, cell monitoring, efficiency assessments, and edit analysis through HiBiT assays. We then detail procedures to determine edit specificity through genomic DNA analysis, small interfering RNA reverse transfection, and HiBiT blotting. This protocol is simple to execute and multifunctional, and it enables high-throughput screens on endogenous proteins to be conducted with ease.

The Role of Vimentin in Human Corneal Fibroblast Spreading and Myofibroblast Transformation.

Vimentin has been reported to play diverse roles in cell processes such as spreading, migration, cell-matrix adhesion, and fibrotic transformation. Here, we assess how vimentin impacts cell spreading, morphology, and myofibroblast transformation of human corneal fibroblasts. Overall, although knockout (KO) of vimentin did not dramatically impact corneal fibroblast spreading and mechanical activity (traction force), cell elongation in response to PDGF was reduced in vimentin KO cells as compared to controls. Blocking vimentin polymerization using Withaferin had even more pronounced effects on cell spreading and also inhibited cell-induced matrix contraction. Furthermore, although absence of vimentin did not completely block TGFß-induced myofibroblast transformation, the degree of transformation and amount of αSMA protein expression was reduced. Proteomics showed that vimentin KO cells cultured in TGFß had a similar pattern of protein expression as controls. One exception included periostin, an ECM protein associated with wound healing and fibrosis in other cell types, which was highly expressed only in Vim KO cells. We also demonstrate for the first time that LRRC15, a protein previously associated with myofibroblast transformation of cancer-associated fibroblasts, is also expressed by corneal myofibroblasts. Interestingly, proteins associated with LRRC15 in other cell types, such as collagen, fibronectin, ß1 integrin and α11 integrin, were also upregulated. Overall, our data show that vimentin impacts both corneal fibroblast spreading and myofibroblast transformation. We also identified novel proteins that may regulate corneal myofibroblast transformation in the presence and/or absence of vimentin.

E3 ubiquitin ligase Herc3 deficiency leads to accumulation of subretinal microglia and retinal neurodegeneration.

Activated microglia have been implicated in the pathogenesis of age-related macular degeneration (AMD), diabetic retinopathy, and other neurodegenerative and neuroinflammatory disorders, but our understanding of the mechanisms behind their activation is in infant stages. With the goal of identifying novel genes associated with microglial activation in the retina, we applied a semiquantitative fundus spot scoring scale to an unbiased, state-of-the-science mouse forward genetics pipeline. A mutation in the gene encoding the E3 ubiquitin ligase Herc3 led to prominent accumulation of fundus spots. CRISPR mutagenesis was used to generate Herc3-/- mice, which developed prominent accumulation of fundus spots and corresponding activated Iba1 + /CD16 + subretinal microglia, retinal thinning on OCT and histology, and functional deficits by Optomotory and electrophysiology. Bulk RNA sequencing identified activation of inflammatory pathways and differentially expressed genes involved in the modulation of microglial activation. Thus, despite the known expression of multiple E3 ubiquitin ligases in the retina, we identified a non-redundant role for Herc3 in retinal homeostasis. Our findings are significant given that a dysregulated ubiquitin-proteasome system (UPS) is important in prevalent retinal diseases, in which activated microglia appear to play a role. This association between Herc3 deficiency, retinal microglial activation and retinal degeneration merits further study.

Translational attenuation differentially alters the fate of disease-associated fibulin proteins.

Mutations in fibulin proteins that cause cellular secretion deficiencies are linked to a variety of diseases, ranging from retinopathies to cutis laxa (CL). One secretion-deficient fibulin mutant, R345W fibulin-3, causes the macular dystrophy malattia leventinese by increased endoplasmic reticulum retention and/or extracellular misfolding. Herein, we report that small-molecule activation of the PERK arm of the unfolded protein response partially rescues R345W secretion deficiencies through translational attenuation mediated by eIF2α phosphorylation. Enhanced mutant fibulin-3 secretion can also be achieved by activation of a PERK-independent eIF2α kinase through arsenite treatment and is independent of activating transcription factor 4 signaling and protein translation. However, this translational attenuation strategy was unsuccessful for enhancing the secretion deficiencies of fibulin-5 mutants associated with age-related macular degeneration or CL. While lowered growth temperature enhanced the secretion of mutants associated with CL (C217R and S227P), these effects were not mediated through translational attenuation. In stark contrast to the situation with fibulin-3, protein translation was required for efficient wild-type and mutant fibulin-5 secretion. These data suggest that alteration of specific cellular signaling pathways and proteostasis network components can differentially influence fibulin fate, a hypothesis that could be exploited as a therapy for fibulin-related diseases.