primary-cell-cultures-from-the-mouse-retinal-pigment-epithelium

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 ...

University of Pittsburgh School of Medicine

The Johns Hopkins University School of Medicine

We have discovered a spontaneous mutation in the Sprague-Dawley rat with a novel eye phenotype that we have named Nuc1. The Nuc1 mutation behaves as a single semi-dominant locus with an intermediate phenotype in the heterozygotes. Heterozygotes exhibit nuclear cataracts. Homozygous Nuc1 rats are fully viable and have microphthalmia, retinal abnormalities and disruption of lens structure shortly before birth. The homozygous mutant shows no obvious pathology outside of the eye, indicating that the mutation is highly eye specific in its effects. An unusual feature of the mutation is that it prevents the normal programmed loss of nuclei from lens fiber cells, but does not affect the loss of other organelles. TUNEL, light, and electron microscopic studies show normal intact nuclei in lens fibers, in contrast to many other models with degenerate nuclei and unlike normal lenses where no such nuclei remain. The beaded filament protein, filensin, is down-regulated in fibers of Nuc1, while heat shock cognate 70 is up-regulated. Homozygous retinas are thicker than normal, and TUNEL labeling indicates roughly half the number of apoptotic cells compared to a wild-type retina. The transient layer of Chievitz persists in adult Nuc1 retina, indicative of delayed development. Hence, Nuc1 is a novel mutation that could be an eye-specific regulator of apoptosis.

A spontaneous mutation affects programmed cell death during development of the rat eye.

Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process. Nuc1 is a spontaneous mutation in the Sprague-Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the betaA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate betaA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.

betaA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development.

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant βA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that βA3/A1-crystallin is essential for normal astrocyte function in the retina.

βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina.

Crystallins, the highly abundant proteins of the ocular lens, are essential determinants of the transparency and refractivity required for lens function. Initially thought to be lens-specific and to have evolved as lens proteins, it is now clear that crystallins were recruited to the lens from proteins that existed before lenses evolved. Crystallins are expressed outside of the lens and most have been shown to have cellular functions distinct from their roles as structural elements in the lens. For one major crystallin group, the β/γ-crystallin superfamily, no such functions have yet been established. We have explored possible functions for the polypeptides (βA3-and βA1-crystallins) encoded by Cryba1, one of the 6 β-crystallin genes, using a spontaneous rat mutant and genetically engineered mouse models. βA3-and βA1-crystallins are expressed in retinal astrocytes and retinal pigment epithelial (RPE) cells. In both cell types, these proteins appear to be required for the proper acidification of the lysosomes. In RPE cells, elevated pH in the lysosomes is shown to impair the critical processes of phagocytosis and autophagy, leading to accumulation of undigested cargo in (auto) phagolysosomes. We postulate that this accumulation may cause pathological changes in the cells resembling some of those characteristic of age-related macular degeneration (AMD). Our studies suggest an important regulatory function of βA3/A1-crystallin in astrocytes. We provide evidence that the cellular function of βA3/A1-crystallin involves its interaction with V-ATPase, the proton pump responsible for acidification of the endolysosomal system.

βA3/A1-crystallin: more than a lens protein.

Persistent fetal vasculature (PFV) is a human disease in which the fetal vasculature of the eye fails to regress normally. The fetal, or hyaloid, vasculature nourishes the lens and retina during ocular development, subsequently regressing after formation of the retinal vessels. PFV causes serious congenital pathologies and is responsible for as much as 5% of blindness in the United States.

βA3/A1-crystallin and persistent fetal vasculature (PFV) disease of the eye.

The retinal pigmented epithelium (RPE) is critically important to retinal homeostasis, in part due to its very active processes of phagocytosis and autophagy. Both of these processes depend upon the normal functioning of lysosomes, organelles which must fuse with (auto)phagosomes to deliver the hydrolases that effect degradation of cargo. It has become clear that signaling through mTOR complex 1 (mTORC1), is very important in the regulation of lysosomal function. This signaling pathway is becoming a target for therapeutic intervention in diseases, including age-related macular degeneration (AMD), where lysosomal function is defective. In addition, our laboratory has been studying animal models in which the gene (Cryba1) for βA3/A1-crystallin is deficient. These animals exhibit impaired lysosomal clearance in the RPE and pathological signs that are similar to some of those seen in AMD patients. The data demonstrate that βA3/A1-crystallin localizes to lysosomes in the RPE and that it is a binding partner of V-ATPase, the proton pump that acidifies the lysosomal lumen. This suggests that βA3/A1-crystallin may also be a potential target for therapeutic intervention in AMD. In this review, we focus on effector molecules that impact the lysosomal-autophagic pathway in RPE cells.

Lysosomes: Regulators of autophagy in the retinal pigmented epithelium.

Maintenance of protein homeostasis, also referred to as "Proteostasis", integrates multiple pathways that regulate protein synthesis, folding, translocation, and degradation. Failure in proteostasis may be one of the underlying mechanisms responsible for the cascade of events leading to age-related macular degeneration (AMD). This review covers the major degradative pathways (ubiquitin-proteasome and lysosomal involvement in phagocytosis and autophagy) in the retinal pigment epithelium (RPE) and summarizes evidence of their involvement in AMD. Degradation of damaged and misfolded proteins via the proteasome occurs in coordination with heat shock proteins. Evidence of increased content of proteasome and heat shock proteins in retinas from human donors with AMD is consistent with increased oxidative stress and extensive protein damage with AMD. Phagocytosis and autophagy share key molecules in phagosome maturation as well as degradation of their cargo following fusion with lysosomes. Phagocytosis and degradation of photoreceptor outer segments ensures functional integrity of the neural retina. Autophagy rids the cell of toxic protein aggregates and defective mitochondria. Evidence suggesting a decline in autophagic flux includes the accumulation of autophagic substrates and damaged mitochondria in RPE from AMD donors. An age-related decrease in lysosomal enzymatic activity inhibits autophagic clearance of outer segments, mitochondria, and protein aggregates, thereby accelerating the accumulation of lipofuscin. This cumulative damage over a person's lifetime tips the balance in RPE from a state of para-inflammation, which strives to restore cell homeostasis, to the chronic inflammation associated with AMD.

Defects in retinal pigment epithelial cell proteolysis and the pathology associated with age-related macular degeneration.

We have previously demonstrated that βA3/A1-crystallin, a member of the β/γ-crystallin superfamily, is expressed in the astrocytes and retinal pigment epithelial (RPE) cells of the eye. In order to understand the physiological functions of βA3/A1-crystallin in RPE cells, we generated conditional knockout (cKO) mice where Cryba1, the gene encoding βA3/A1-crystallin, is deleted specifically from the RPE using the Cre-loxP system. By utilizing the cKO model, we have shown that this protein is required by RPE cells for proper lysosomal degradation of photoreceptor outer segments (OS) that have been internalized in phagosomes and also for the proper functioning of the autophagy process. We also reported that βA3/A1-crystallin is trafficked to lysosomes, where it regulates endolysosomal acidification by modulating the activity of the lysosomal V-ATPase complex. Our results show that the V-ATPase activity in cKO RPE is significantly lower than WT RPE. Since, V-ATPase is important for regulating lysosomal pH, we noticed that endolysosomal pH was higher in the cKO cells compared to the WT cells. Increased lysosomal pH in cKO RPE is also associated with reduced Cathepsin D activity. Cathepsin D is a major lysosomal aspartic protease involved in the degradation of the OS and hence we believe that reduced proteolytic activity contributes to impaired degradation of OS in the cKO RPE. Reduced lysosomal activity in the cKO RPE also contributes to the incomplete degradation of the autophagosomes. Our results also suggest that βA3/A1-crystallin regulates V-ATPase activity by binding to the V0 subunit of the V-ATPase complex. Taken together, these results suggest a novel mechanism by which βA3/A1-crystallin regulates lysosomal function by modulating the activity of V-ATPase.

Modulation of V-ATPase by βA3/A1-Crystallin in Retinal Pigment Epithelial Cells.

Impaired autophagic and proteasomal cleansing have been documented in aged retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD). Omega-3 fatty acids and resveratrol have many positive homeostatic effects in RPE cells. In this work, ARPE-19 cells were treated with 288 ng of Resvega, containing 30 mg of trans resveratrol and 665 mg of omega-3 fatty acids, among other nutrients, with proteasome inhibitor MG-132 or autophagy inhibitor bafilomycin A1 up to 48 h. Autophagy markers p62/SQSTM1 (p62) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting. Fluorescence microscopy with mCherry-GFP-LC3 plasmid was applied to study the autophagy flux, and cytoprotective effects were investigated with colorimetric MTT and LDH assays. Resvega induced autophagy by showing increased autolysosome formation and autophagy flux, and the change in the p62 and LC3 protein levels further confirmed the fluorescent microscopy results. Moreover, Resvega provided a clear cytoprotection under proteasome inhibition. These findings highlight the potential of the nutraceuticals containing resveratrol, omega-3 fatty acids and other nutrients in the prevention of ARPE-19 cell damage.

Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells.

The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.

Debasish Sinha

Department of Ophthalmology

University of Pittsburgh School of Medicine

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Debasish Sinha

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ophthalmology

University of Pittsburgh School of Medicine

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Department of Ophthalmology