Many genes involved in inherited diseases produce alternate mRNA isoforms that remain poorly characterized. Functional assessment of these isoforms could therefore unlock new insights into disease pathobiology or treatment. Here we investigated the function of the newly discovered B isoform of CRB1, a gene implicated in inherited retinal degenerations. CRB1-B is the most abundant retinal isoform, and differs from the canonical CRB1-A isoform in several important ways. Crucially, CRB1-B is the only isoform expressed by photoreceptors -- the cells vulnerable to degeneration. Using a mouse model of CRB1-associated degeneration with degraded visual acuity, we find that restoring CRB1-B to photoreceptors prevents degeneration and completely rescues visual function, despite the absence of other isoforms. The therapeutic mechanism involves preservation of adherens junctions between photoreceptors and supporting glia: We establish that progressive loss of these junctions is a key pathobiological mechanism underlying photoreceptor death, and that junction loss is prevented by restoring photoreceptor expression of CRB1-B. Our findings nominate CRB1-B replacement as a promising gene therapy strategy. More broadly, they suggest that exploring disease gene isoform diversity offers an untapped opportunity to devise novel therapeutics.

Purpose: The Vglut2-Cre;ndufs4(loxP/loxP) mouse strain with retinal ganglion cell (RGC)-specific mitochondrial complex I dysfunction develops severe RGC degeneration by postnatal day 90 (P90), with accompanying retinal mononuclear phagocyte (MNP) accumulation. We have reported that continuous exposure to hypoxia partially rescues RGC death in these mice, with minimal effect on MNP abundance. We hypothesized that pharmacological depletion of MNPs with the colony-stimulating factor-1 receptor inhibitor pexidartinib would enhance RGC neuroprotection by hypoxia. Methods: Iba1(+) retinal MNP depletion was assessed in C57Bl/6J mice fed control or pexidartinib-infused chow beginning at P25. Subsequently, Vglut2-Cre;ndufs4(loxP/loxP) mice and control littermates were raised under normoxia or hypoxia and fed control or pexidartinib chow from P25 to P90. The neuroprotective effect of pexidartinib and hypoxia alone and in combination was assessed by quantifying RGC soma and axon survival in retinal flat mounts and optic nerve cross-sections. Results: Pexidartinib completely depleted retinal MNPs within 1 week of treatment. Untreated Vglut2-Cre;ndufs4(loxP/loxP) mice exhibited the expected approximately 50% reduction of RGC soma and axon survival at P90 (P < 0.0001 for both). Hypoxia or pexidartinib monotherapy each reduced RGC degeneration by more than one-half, whereas their combination resulted in complete RGC neuroprotection (P < 0.01 for all 3 treatments). Normal myelination patterns were restored in mice receiving dual therapy. Conclusions: Pexidartinib effectively depletes retinal MNPs and is neuroprotective in the setting of severe RGC mitochondrial dysfunction. This therapeutic effect is additive to that of hypoxia. Combating retinal neuro-inflammation may therefore be a useful adjunct therapy in mitochondrial optic neuropathies like Leber hereditary optic neuropathy (LHON).

Mitochondria differ depending on their location within a neuron. Morphological heterogeneity between somatic, dendritic, and axonal mitochondria is well established. Emerging evidence suggests that further specialization is needed to meet the unique demands of different neuronal compartments. However, the molecular and functional diversity of mitochondria within a neuron remains poorly understood. Here, we utilized proteomics in MitoTag mice to profile somatodendritic and axonal mitochondria across four distinct neuron types, thereby generating a compendium of intracellular mitochondrial diversity. Combining proteomics, functional, and immunofluorescence analyses, we demonstrated that axonal mitochondria are not defined by the presence of unique proteins, but rather by the selective loss or preservation of specific pathways compared to their somatodendritic counterparts. This results in "impoverished" axonal mitochondria, which are characterized by diminished mtDNA expression and impaired oxidative phosphorylation yet retain other pathways, such as fatty acid metabolism. Bioinformatic analyses of multiomic data identified local translation as one mechanism underlying compartment-specific diversity. Together, these findings provide a comprehensive in vivo framework for understanding mitochondrial specialization across neuronal compartments.

Missing wire thread inserts in mechanical components pose critical safety risks, as undetected defects may lead to mechanical failures and downtime. High-resolution (HR) imaging captures defects but faces challenges: complex metallic backgrounds, low-contrast substrates, and small defect sizes impede reliable detection. Due to limited contextual reasoning, traditional convolutional networks often misclassify textural noise or overlook genuine anomalies. This paper proposes Defect Detection Yolov5 (Ded-YOLOv5), an efficient defect detection framework, to address these issues. The model integrates a Defect Detection Attenion (Ded-Att) module into YOLOv5, adaptively enhancing defect features by modeling channel-spatial dependencies and suppressing background interference. A hierarchical fusion mechanism correlates multi-scale cues, improving localization accuracy for minute defects. Experiments demonstrate that Ded-YOLOv5 reduces false positives from metallic textures and minimizes missed low-visibility defects. The method exhibits superior anti-interference capability against artifacts like uneven illumination and reflective glares compared to conventional approaches. These advancements establish Ded-YOLOv5 as a practical solution for mechanical manufacturing quality assurance.

The retina is light-sensitive neuronal tissue in the back of the eye. The phospholipid composition of the retina is unique and highly enriched in polyunsaturated fatty acids, including docosahexaenoic fatty acid (DHA). While it is generally accepted that a high DHA content is important for vision, surprisingly little is known about the mechanisms of DHA enrichment in the retina. Furthermore, the biological processes controlled by DHA in the eye remain poorly defined as well. Here, we combined genetic manipulations with lipidomic analysis in mice to demonstrate that acyl-CoA synthetase 6 (Acsl6) serves as a regulator of the unique composition of retinal membranes. Inactivation of Acsl6 reduced the levels of DHA-containing phospholipids, led to progressive loss of light-sensitive rod photoreceptor neurons, attenuated the light responses of these cells, and evoked distinct transcriptional response in the retina involving the Srebf1/2 (sterol regulatory element binding transcription factors 1/2) pathway. This study identifies one of the major enzymes responsible for DHA enrichment in the retinal membranes and introduces a model allowing an evaluation of rod functioning and pathology caused by impaired DHA incorporation/retention in the retina.

The first steps of vision take place in the ciliary outer segment compartment of photoreceptor cells. The protein composition of outer segments is uniquely suited to perform this function. The most abundant among these proteins is the visual pigment, rhodopsin, whose outer segment trafficking involves intraflagellar transport (IFT). Here, we report three major findings from the analysis of mice in which ciliary transport was acutely impaired by conditional knockouts of IFT-B subunits. First, we demonstrate the existence of a sorting mechanism whereby mislocalized rhodopsin is recruited to and concentrated in extracellular vesicles prior to their release, presumably to protect the cell from adverse effects of protein mislocalization. Second, reducing rhodopsin expression significantly delays photoreceptor degeneration caused by IFT disruption, suggesting that controlling rhodopsin levels may be an effective therapy for some cases of retinal degenerative disease. Last, the loss of IFT-B subunits does not recapitulate a phenotype observed in mutants of the BBSome (another ciliary transport protein complex relying on IFT) in which non-ciliary proteins accumulate in the outer segment. Whereas it is widely thought that the role of the BBSome is to primarily participate in ciliary transport, our data suggest that the BBSome has another major function independent of IFT and possibly related to maintaining the diffusion barrier of the ciliary transition zone.

Among neurons, retinal ganglion cells (RGCs) are uniquely sensitive to mitochondrial dysfunction. The RGC is highly polarized, with a somatodendritic compartment in the inner retina and an axonal compartment projecting to targets in the brain. The drastically dissimilar functions of these compartments implies that mitochondria face different bioenergetic and other physiological demands. We hypothesized that compartmental differences in mitochondrial biology would be reflected by disparities in mitochondrial protein composition. Here, we describe a protocol to isolate intact mitochondria separately from mouse RGC somatodendritic and axonal compartments by immunoprecipitating labeled mitochondria from RGC MitoTag mice. Using mass spectrometry, 471 and 357 proteins were identified in RGC somatodendritic and axonal mitochondrial immunoprecipitates, respectively. We identified 10 mitochondrial proteins exclusively in the somatodendritic compartment and 19 enriched ≥2-fold there, while 3 proteins were exclusively identified and 18 enriched in the axonal compartment. Our observation of compartment-specific enrichment of mitochondrial proteins was validated through immunofluorescence analysis of the localization and relative abundance of superoxide dismutase ( SOD2 ), sideroflexin-3 ( SFXN3 ) and trifunctional enzyme subunit alpha ( HADHA ) in retina and optic nerve specimens. The identified compartmental differences in RGC mitochondrial composition may provide promising leads for uncovering physiologically relevant pathways amenable to therapeutic intervention for optic neuropathies.

Continuous exposure to environmental hypoxia (11% O2) has been shown to markedly slow the progressive degeneration of retinal ganglion cells (RGCs) in a mouse model of mitochondrial optic neuropathy with RGC-specific deletion of the key mitochondrial complex I accessory subunit ndufs4. As a first step toward identifying the therapeutic mechanism of hypoxia in this model, we conducted a series of experiments to investigate the role of the hypoxia-inducible factor (HIF) regulatory pathway in RGC neuroprotection. Vglut2-Cre; ndufs4loxP/loxP mice were crossed with strains bearing floxed alleles of the negative HIF regulatory vhl or of the two major HIF α-subunit isoforms, Hif1α and Hif2α. Deletion of vhl within ndufs4-deficient RGCs failed to prevent RGC degeneration under normoxia, indicating that HIF activation is not sufficient to achieve RGC rescue. Furthermore, the rescue of ndufs4-deficient RGCs by hypoxia remained robust despite genetic inactivation of Hif1α and Hif2α. Our findings demonstrate that the HIF pathway is entirely dispensable to the rescue of RGCs by hypoxia. Future efforts to uncover key HIF-independent molecular pathways induced by hypoxia in this mouse model may be of therapeutic relevance to mitochondrial optic neuropathies such as Leber hereditary optic neuropathy.

Visual signal transduction takes place within a stack of flattened membranous ‘discs’ enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or “discs”, located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called “incisures”. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

PURPOSE. To test whether continuous hypoxia is neuroprotective to retinal ganglion cells (RGCs) in a mouse model of mitochondrial optic neuropathy. METHODS. RGC degeneration was assessed in genetically modified mice in which the floxed gene for the complex I subunit NDUFS4 is deleted from RGCs using Vlgut2driven Cre recombinase. Beginning at postnatal day 25 (P25), Vglut2-Cre;ndufs4loxP/loxP mice and control littermates were housed under hypoxia (11% oxygen) or kept under normoxia (21% oxygen). Survival of RGC somas and axons was assessed at P60 and P90 via histological analysis of retinal flatmounts and optic nerve cross-sections, respectively. Retinal tissue was also assessed for gliosis and neuroinflammation using western blot and immunofluorescence. RESULTS. Consistent with our previous characterization of this model, at least one-third of RGCs had degenerated by P60 in Vglut2-Cre;ndufs4loxP/loxP mice remaining under normoxia. However, continuous hypoxia resulted in complete rescue of RGC somas and axons at this time point, with normal axonal myelination observed on electron microscopy. Though only partial, hypoxia-mediated rescue of complex I-deficient RGC somas and axons remained significant at P90. Hypoxia prevented reactive gliosis at P60, but the retinal accumulation of Iba1+ mononuclear phagocytic cells was not substantially reduced. CONCLUSIONS. Continuous hypoxia achieved dramatic rescue of early RGC degeneration in mice with severe mitochondrial dysfunction. Although complete rescue was not durable to P90, our observations suggest that investigating the mechanisms underlying hypoxiamediated neuroprotection of RGCs may identify useful therapeutic strategies for optic neuropathies resulting from less profound mitochondrial impairment, such as Leber hereditary optic neuropathy.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1 , was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.

Retinitis pigmentosa is a retinal degenerative disease that leads to blindness through photoreceptor loss. Rhodopsin is the most frequently mutated protein in this disease. While many rhodopsin mutations have well-understood consequences that lead to cell death, the disease association of several rhodopsin mutations identified in retinitis pigmentosa patients, including F220C and F45L, has been disputed. In this study, we generated two knockin mouse lines bearing each of these mutations. We did not observe any photoreceptor degeneration in either heterozygous or homozygous animals of either line. F220C mice exhibited minor disruptions of photoreceptor outer segment dimensions without any mislocalization of outer segment proteins, whereas photoreceptors of F45L mice were normal. Suction electrode recordings from individual photoreceptors of both mutant lines showed normal flash sensitivity and photoresponse kinetics. Taken together, these data suggest that neither the F220C nor F45L mutation has pathological consequences in mice and, therefore, may not be causative of retinitis pigmentosa in humans.

Optic atrophy resulting from retinal ganglion cell (RGC) degeneration is a prominent ocular manifestation of mitochondrial dysfunction. Although transgenic mice lacking the mitochondrial complex I accessory subunit NDUFS4 develop early-onset optic atrophy, severe systemic mitochondrial dysfunction leads to very early death and makes this mouse line impractical for studying the pathobiology of mitochondrial optic neuropathies. Theoretically, RGC-specific inactivation of ndufs4 would allow characterization of RGC degeneration over a longer time course, provided that RGC death from mitochondrial dysfunction is a cell-autonomous process. We demonstrate that the vesicular glutamate transporter VGLUT2 may be exploited to drive robust Cre recombinase expression in RGCs without any expression observed in directly neighboring retinal cell types. Deletion of ndufs4 in RGCs resulted in reduced expression of NDUFS4 protein within the optic nerves of Vglut2-Cre;ndufs4loxP/loxP mice. RGC degeneration in Vglut2-Cre;ndufs4loxP/loxP retinas commenced around postnatal day 45 (P45) and progressed to loss of two-thirds of RGCs by P90, confirming that intrinsic complex I dysfunction is sufficient to induce RGC death. The rapidly-developing optic atrophy makes the Vglut2-Cre;ndufs4loxP/loxP mouse line a promising preclinical model for testing therapies for currently untreatable mitochondrial optic neuropathies such as Leber Hereditary Optic Neuropathy.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood-retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using the Mfsd2a knock-out mouse as animal model. Our lipidome analyses of Mfsd2a-/- retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated in Mfsd2a-/- retinas. Mfsd2a-/- retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructure Mfsd2a-/- photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENT Phospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood-retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin–proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness.