In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype–phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells.

Increased oxidative stress and mitochondrial damage are observed in protein aggregation diseases, such as age-related macular degeneration (AMD). We have recently reported elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in the retinal pigment epithelial cells (RPE) of the dry AMD-resembling NFE2L2/PGC1α double knockout (dKO) mouse model. Here, we provide evidence of a disturbance in the autolysosomal machinery handling mitochondrial clearance in the RPE cells of one-year-old NFE2L2/PGC1α-deficient mice. Confocal immunohistochemical analysis revealed an upregulation of autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as numerous mitophagy markers, such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN) together with damaged mitochondria. However, we detected no evidence of increased autolysosome formation in transmission electron micrographs or of colocalization of lysosomal marker LAMP2 (lysosome-associated membrane protein 2) and the mitochondrial marker ATP synthase β in confocal micrographs. Interestingly, we observed an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells together with autofluorescence aggregates. Our results reveal that there is at least a relative decrease of mitophagy in the RPE cells of NFE2L2/PGC1α dKO mice. This further supports the hypothesis that mitophagy is a putative therapy target in AMD-like pathology.

Glia-neuron partnership is important for inner retinal homeostasis and any disturbances may result in retinal ganglion cell (RGC) death. Müller cells support RGCs with essential functions such as removing excess glutamate and providing energy sources. The aim was to explore the impact of Müller cells on RGC survival. To investigate the Müller cell/RGC interactions we developed a coculture model, in which primary Müller cells were grown in inserts on top of pure primary RGC cultures. The impact of starvation and mitochondrial inhibition on the Müller cell ability to protect RGCs was studied. Moreover, the ability of Müller cells to remove glutamate from the extracellular space was investigated. RGC survival was evaluated by cell viability assays and glutamate uptake was assessed by kinetic uptake assays. We demonstrated a significantly increased RGC survival in presence of untreated and prestarved Müller cells. Additionally, prestarved Müller cells significantly increased RGC survival after mitochondrial inhibition. Finally, we revealed a significantly increased ability to take up glutamate in starved Müller cells. Overall, our study confirms essential roles of Müller cells in RGC survival. We suggest that targeting Müller cell function could have potential for future treatment strategies to prevent blinding neurodegenerative retinal diseases.

Purpose: Müller cells support retinal neurons with essential functions. Here, we aim to examine the impact of starvation and oxidative stress on glutamate uptake and mitochondrial function in Müller cells. Methods: Cultured human retinal Müller cells (MIO-M1) were exposed to H₂O₂ and additional starvation for 24 hours. Effects of starvation and H₂O₂ on glutamate uptake and mitochondrial function were assessed by kinetic glutamate uptake assays and Seahorse assays, respectively. Cell survival was evaluated by cell viability assays. mRNA and protein expressions were assessed by quantitative PCR and Western blot. Results: Starvation of Müller cells increased the glutamate uptake capacity as well as the expression of the most abundant glutamate transporter, EAAT1. Mitochondrial and glycolytic activity were diminished in starved Müller cells despite unaffected cell viability. Simultaneous starvation and exposure to oxidative stress resulted in a reduced glutamate uptake and a collapsed mitochondrial function. In Müller cells with intact energy supply, the glutamate uptake and mitochondrial function were unaffected after exposure to oxidative stress. Conclusions: Here, we identify an increased susceptibility toward oxidative stress in starved Müller cells in spite of unaffected viability and an apparent decreased ability to transport glutamate. Solely exposure to oxidative stress did not affect Müller cell functions. Thus, our study suggests an increased susceptibility of Müller cells in case of more than one cellular stressor. Extrapolating these findings, age-related neurodegenerative retinal diseases may be the result of impaired Müller cell function.

Age‐related macular degeneration (AMD) is a devastating eye disease that causes permanent vision loss in the central part of the retina, known as the macula. Patients with such severe visual loss face a reduced quality of life and are at a 1.5 times greater risk of death compared to the general population. Currently, there is no cure for or effective treatment for dry AMD. There are several mechanisms thought to underlie the disease, for example, ageing‐associated chronic oxidative stress, mitochondrial damage, harmful protein aggregation and inflammation. As a way of gaining a better understanding of the molecular mechanisms behind AMD and thus developing new therapies, we have created a peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha and nuclear factor erythroid 2‐related factor 2 (PGC1α/NFE2L2) double‐knockout (dKO) mouse model that mimics many of the clinical features of dry AMD, including elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen‐like structures in retinal pigment epithelial cells (RPE). In addition, a human RPE cell‐based model was established to examine the impact of non‐functional intracellular clearance systems on inflammasome activation. In this study, we found that there was a disturbance in the autolysosomal machinery responsible for clearing mitochondria in the RPE cells of one‐year‐old PGC1α/NFE2L2‐deficient mice. The confocal immunohistochemical analysis revealed an increase in autophagosome marker microtubule‐associated proteins 1A/1B light chain 3B (LC3B) as well as multiple mitophagy markers such as PTE‐induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN), along with signs of damaged mitochondria. However, no increase in autolysosome formation was detected, nor was there a colocalization of the lysosomal marker LAMP2 or the mitochondrial marker, ATP synthase β. There was an upregulation of late autolysosomal fusion Ras‐related protein (Rab7) in the perinuclear space of RPE cells, together with autofluorescent aggregates. Additionally, we observed an increase in the numbers of Toll‐like receptors 3 and 9, while those of NOD‐like receptor 3 were decreased in PGC1α/NFE2L2 dKO retinal specimens compared to wild‐type animals. There was a trend towards increased complement component C5a and increased involvement of the serine protease enzyme, thrombin, in enhancing the terminal pathway producing C5a, independent of C3. The levels of primary acute phase C‐reactive protein and receptor for advanced glycation end products were also increased in the PGC1α/NFE2L2 dKO retina. Furthermore, selective proteasome inhibition with epoxomicin promoted both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial‐mediated oxidative stress, leading to the release of mitochondrial DNA to the cytosol, resulting in potassium efflux‐dependent activation of the absent in melanoma 2 (AIM2) inflammasome and the subsequent secretion of interleukin‐1β in ARPE‐19 cells. In conclusion, the data suggest that there is at least a relative decrease in mitophagy, increases in the amounts of C5 and thrombin and decreased C3 levels in this dry AMD‐like model. Moreover, selective proteasome inhibition evoked mitochondrial damage and AIM2 inflammasome activation in ARPE‐19 cells.