Ophthalmology
Abstract
Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multi-organ ciliopathies or as retina-specific diseases. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used superresolution fluorescence microscopy and electron microscopy (TEM) to localize CEP290 in the CC and in primary cilia of cultured cells with sub-diffraction resolution, and to determine effects of CEP290 deficiency in three mutant models. Radially, CEP290 localizes in close proximity to the microtubule doublets in the region between the doublets and the ciliary membrane. Longitudinally, it is distributed throughout the length of the CC whereas it is confined to the very base of primary cilia in hRPE-1 cells. We found Y-shaped links, ciliary sub-structures between microtubules and membrane, throughout the length of the CC. Severe CEP290 deficiencies in mouse models did not prevent assembly of cilia or cause obvious mislocalization of ciliary components in early stages of degeneration. There were fewer cilia and no normal outer segments in the mutants, but the Y-shaped links were clearly present. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis.
Authors
Valencia L. Potter, Abigail R. Moye, Michael A. Robichaux, Theodore G. Wensel
Abstract
Dry eye disease affects over 16 million adults in the U.S. and the majority of cases are due to Meibomian gland dysfunction. Unfortunately, the identity of the stem cells involved in Meibomian gland development and homeostasis are not well-elucidated. Here, we report that loss of Krox20, a zinc-finger transcription factor involved in development of ectoderm-derived tissues, or deletion of KROX20-expressing epithelial cells disrupts Meibomian gland formation and homeostasis, leading to dry eye disease secondary to Meibomian gland dysfunction. Ablation of Krox20-lineage cells in adult mice also resulted in dry eye disease, implicating Krox20 in homeostasis of the mature Meibomian gland. Lineage tracing and expression analyses revealed a restricted KROX20 expression pattern in the ductal areas of the Meibomian gland, although Krox20-lineage cells generate the full, mature Meibomian gland. This suggests that KROX20 marks a stem/progenitor cell population that differentiates to generate the entire Meibomian gland. Our Krox20 mouse models provide a powerful system that delineated the identity of stem cells required for Meibomian gland development and homeostasis, and can be used to investigate the factors underlying these processes. They are also robust models of Meibomian gland dysfunction-related dry eye disease with a potential for use in pre-clinical therapeutic screening.
Authors
Edem Tchegnon, Chung-Ping Liao, Elnaz Ghotbi, Tracey Shipman, Yong Wang, Renee M. McKay, Lu Q. Le
Abstract
BACKGROUND. This study systematically investigated circulating and retinal tissue lipid determinants of human diabetic retinopathy (DR) to identify underlying lipid alterations associated with severity of DR. METHODS. Retinal tissues were retrieved from postmortem human eyes including 19 individuals without diabetes, 20 with diabetes but without DR, and 20 with diabetes and DR for lipidomic study. In a parallel study, serum samples from 28 American Indians with type 2 diabetes from the Glia River Indian Community including 12 without DR, 7 with mild non-proliferative DR (NPDR), and 9 with moderate NPDR were selected. A mass-spectrometry-based lipidomic platform was used to measure serum and tissue lipids. RESULTS. In the postmortem retinas, we found a graded decrease of long chain acylcarnitines, longer chain fatty acid ester of hydroxyl fatty acids, diacylglycerols, triacylglycerols, phosphatidylcholines, and Ceramide[NS] in central retina from no diabetes to diabetes with DR. The American Indians’ sera also exhibited a graded decrease in circulating long-chain acylcarnitines and a graded increase in the intermediate length saturated and monounsaturated triacylglycerols from no DR to moderate NPDR. CONCLUSION. These findings suggest diminished synthesis of complex lipids and impaired mitochondrial β-oxidation of fatty acids in retinal DR with parallel changes in circulating lipids. TRIALREGISTRION. ClinicalTrials.gov NCT00340678 FUNDING. Supported by NIH grants K08DK106523, R03DK121941, P30DK089503, P30DK081943, P30DK020572, P30 EY007003, The Thomas Beatson Foundation, and JDRF Center for Excellence (5-COE-2019-861-S-B).
Authors
Patrice E. Fort, Thekkelnaycke M. Rajendiran, Tanu Soni, Jaeman Byun, Yang Shan, Helen C. Looker, Robert G. Nelson, Matthias Kretzler, George Michailidis, Jerome E. Roger, Thomas W. Gardner, Steven F. Abcouwer, Subramaniam Pennathur, Farsad Afshinnia
Abstract
The metabolic environment is important for neuronal cells, such as photoreceptors. When photoreceptors undergo degeneration, as occurs during retinitis pigmentosa (RP), patients have progressive loss of vision that proceeds to full blindness. Currently, there are no available treatments for the majority of RP diseases. We performed metabolic profiling of the neural retina in a preclinical model of RP and found that tricarboxylic acid (TCA) cycle intermediates were reduced during disease. We then determined that, 1) promoting citrate production within the TCA cycle in retinal neurons during disease progression protects the photoreceptors from cell death and prolongs visual function, 2) that supplementation with single metabolites within the TCA cycle can provide this therapeutic effect in vivo over time, and, 3) that this therapeutic effect is not specific to a particular genetic mutation but has broad applicability for patients with RP and other retinal degenerative diseases. Overall, targeting TCA cycle activity in the neural retina promotes photoreceptor survival and visual function during neurodegenerative disease.
Authors
Ashley A. Rowe, Pinkal D. Patel, Ruth Gordillo, Katherine J. Wert
Abstract
Inherited retinal diseases such as retinitis pigmentosa (RP) can be caused by thousands of different mutations, a small number of which have been successfully treated with gene replacement. However, this approach has yet to scale and may not be feasible in many cases, highlighting the need for interventions that could benefit more patients. Here, we found that microglial phagocytosis is upregulated during cone degeneration in RP, suggesting that expression of “don’t eat me” signals such as CD47 might confer protection to cones. To test this, we delivered an adeno-associated viral (AAV) vector expressing CD47 on cones, which promoted cone survival in three mouse models of RP and preserved visual function. Cone rescue with CD47 required a known interacting protein, signal regulatory protein alpha (SIRPα), but not an alternative interacting protein, thrombospondin-1 (TSP1). Despite the correlation between increased microglial phagocytosis and cone death, microglia were dispensable for the pro-survival activity of CD47, suggesting that CD47 interacts with SIRPα on non-microglial cells to alleviate degeneration. These findings establish augmentation of CD47-SIRPα signaling as a potential treatment strategy for RP and possibly other forms of neurodegeneration.
Authors
Sean K. Wang, Yunlu Xue, Constance L. Cepko
Abstract
Aniridia is most commonly caused by haploinsufficiency of the PAX6 gene, characterised by variable iris and foveal hypoplasia, nystagmus, cataracts, glaucoma and aniridia related keratopathy (ARK). Genotype-phenotype correlations have previously been described, however detailed longitudinal studies of aniridia are less commonly reported. We identified eighty-six patients from sixty-two unrelated families with molecularly confirmed heterozygous PAX6 variants from a United Kingdom (UK)-based single-centre ocular genetics service. They were categorised into mutation groups and retrospective review of baseline to most recent clinical characteristics (ocular and systemic) were recorded. One hundred and seventy-two eyes were evaluated, with a mean follow up period of 16.3 ± 12.7 years. Nystagmus was recorded in 87.2%, and foveal hypoplasia in 75%. Cataracts were diagnosed in 70.3%, glaucoma in 20.6% and ARK in 68.6% of eyes. Prevalence, age of diagnosis and surgical intervention varied amongst mutation groups. Overall, the missense mutation sub-group had the mildest phenotype, and surgically naïve eyes maintained better visual acuity. Systemic evaluation identified type 2 diabetes in 12.8%, which is twice the UK prevalence. This is the largest longitudinal study of aniridia in the United Kingdom, providing insights into prognostic indicators for patients and guiding clinical management of both ocular and systemic features.
Authors
Vivienne Kit, Dulce Lima Cunha, Ahmed M. Hagag, Mariya Moosajee
Abstract
It remains unresolved how retinal pigment epithelial cell metabolism is regulated following immune activation to maintain retinal homeostasis and retinal function. We exposed retinal pigment epithelium (RPE) to several stress signals, particularly Toll-like receptor stimulation, and uncovered an ability of RPE to adapt their metabolic preference on aerobic glycolysis or oxidative glucose metabolism in response to different immune stimuli. We have identified interleukin-33 (IL-33) as a key metabolic checkpoint that antagonizes the Warburg effect to ensure the functional stability of the RPE. The identification of IL-33 as a key regulator of mitochondrial metabolism suggests roles for the cytokine that go beyond its extracellular “alarmin” activities. IL-33 exerts control over mitochondrial respiration in RPE by facilitating oxidative pyruvate catabolism. We have also revealed that in the absence of IL-33, mitochondrial function declined and resultant bioenergetic switching was aligned with altered mitochondrial morphology. Our data not only shed new light on the molecular pathway of activation of mitochondrial respiration in RPE in response to immune stressors but also uncover a potentially novel role of nuclear intrinsic IL-33 as a metabolic checkpoint regulator.
Authors
Louis M. Scott, Emma E. Vincent, Natalie Hudson, Chris Neal, Nicholas Jones, Ed C. Lavelle, Matthew Campbell, Andrew P. Halestrap, Andrew D. Dick, Sofia Theodoropoulou
Abstract
BACKGROUND. Idiopathic intracranial hypertension (IIH) is a condition predominantly affecting obese women of reproductive age. Recent evidence suggests that IIH is a disease of metabolic dysregulation, androgen excess and an increased risk of cardiovascular morbidity. Here we evaluate systemic and adipose specific metabolic determinants of the IIH phenotype. METHODS. In fasted, matched IIH (N=97) and control (N=43) patients, we assessed: glucose and insulin homeostasis and leptin levels. Body composition was assessed along with an interrogation of adipose tissue function via nuclear magnetic resonance metabolomics and RNA sequencing in paired omental and subcutaneous biopsies in a case control study. RESULTS. We demonstrate an insulin and leptin resistant phenotype in IIH in excess to that driven by obesity. Adiposity in IIH is preferentially centripetal and is associated with increased disease activity and insulin resistance. IIH adipocytes appear transcriptionally and metabolically primed towards depot-specific lipogenesis. CONCLUSIONS. These data show that IIH is a metabolic disorder in which adipose tissue dysfunction is a feature of the disease. Managing IIH as a metabolic disease could reduce disease morbidity and improving cardiovascular outcomes. FUNDING. This study was supported by the National Institute of Health Research UK (NIHR-CS-011-028), the Medical Research Council UK (MR/K015184/1) and the Midlands Neuroscience Teaching and Research Fund.
Authors
Connar S.J. Westgate, Hannah F. Botfield, Zerin Alimajstorovic, Andreas Yiangou, Mark Walsh, Gabrielle Smith, Rishi Singhal, James L. Mitchell, Olivia Grech, Keira A. Markey, Daniel Hebenstreit, Daniel A. Tennant, Jeremy W. Tomlinson, Susan P. Mollan, Christian Ludwig, Ildem Akerman, Gareth G. Lavery, Alexandra J. Sinclair
Abstract
Age-related macular degeneration (AMD) damages the retinal pigment epithelium (RPE), the tissue that safeguards photoreceptor health, leading to irreversible vision loss. Polymorphisms in cholesterol and complement genes are implicated in AMD, yet mechanisms linking risk variants to RPE injury remain unclear. We sought to determine how allelic variants in the apolipoprotein E cholesterol transporter modulate RPE homeostasis and function. Using live-cell imaging, we show that inefficient cholesterol transport by the AMD risk-associated ApoE2 increases RPE ceramide, leading to autophagic defects and complement-mediated mitochondrial damage. Mitochondrial injury drives redox state-sensitive cysteine-mediated phase separation of ApoE2, forming biomolecular condensates that could nucleate drusen. The protective ApoE4 isoform lacks these cysteines and is resistant to phase separation and condensate formation. In Abca4-/- Stargardt macular degeneration mice, mitochondrial dysfunction induces liquid-liquid phase separation of p62/SQSTM1, a multifunctional protein that regulates autophagy. Drugs that decrease RPE cholesterol or ceramide prevent mitochondrial injury and phase separation in vitro and in vivo. In AMD donor RPE, mitochondrial fragmentation correlates with ApoE and p62 condensates. Our studies demonstrate that major AMD genetic and biological risk pathways converge upon RPE mitochondria, and identify mitochondrial stress-mediated protein phase separation as an important pathogenic mechanism and promising therapeutic target in AMD.
Authors
Nilsa La Cunza, Li Xuan Tan, Thushara Thamban, Colin J Germer, Gurugirijha Rathnasamy, Kimberly Toops, Aparna Lakkaraju
Abstract
The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and also is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for co-translational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with >99% efficiency and absolute RPE specificity upon tamoxifen induction at both post-natal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knock-in allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well-suited for studies of gene function and pathophysiology in the RPE.
Authors
Elliot H. Choi, Susie Suh, David E. Einstein, Henri Leinonen, Zhiqian Dong, Sriganesh Ramachandra Rao, Steven J. Fliesler, Seth Blackshaw, Minzhong Yu, Neal S. Peachey, Krzysztof Palczewski, Philip D. Kiser
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