Caveolin-1 associates with the endo/lysosomal machinery of cells in culture, suggesting that it functions at these organelles independently of its contribution to cell surface caveolae. Here we explored mice lacking caveolin-1 specifically in the retinal pigment epithelium (RPE Support of the neural retina generally and of adjacent photoreceptor neurons specifically by the retinal pigment epithelium (RPE) 5 is essential for vision (1). A major function of the RPE is its contribution to photoreceptor outer segment renewal, a continuous and life-long rejuvenation process that involves the formation of new membrane disks at the proximal end of the outer segment and diurnal shedding of distal spent outer segment tips (2). Outer segment renewal is critical for photoreceptor function and survival, and any abnormality is thought to impair vision. RPE cells participate in outer segment renewal by clearing shed photoreceptor outer segment fragments (POS) by receptor-mediated phagocytosis (3).Mechanistically, RPE phagocytosis belongs to a family of conserved non-inflammatory clearance phagocytosis pathways that other cell types use to remove apoptotic cells and debris. These pathways have in common that their failure to efficiently clear debris contributes to human disease. However, unlike other forms of phagocytosis, RPE clearance of POS occurs in a strict diurnal rhythm that is regulated by light and circadian mechanisms (4). This is a unique advantage for RPE phagocytosis studies because all steps of the synchronized phagocytic process may be quantified precisely in situ in the intact, undisturbed retinas of experimental animals. Content in the RPE of engulfed rod POS phagosomes peaks shortly after light onset and declines characteristically within several hours as RPE cells complete digestion of their phagocytic load before the next burst of intake (5).Like other phagocytic pathways, ingested phagosomes in the RPE fuse with lysosomal vesicles to form phagolysosomes. In POS phagolysosomes, degradation of opsin, which constitutes ϳ85% of POS protein, requires the aspartic protease cathepsin D and phagosomal acidification (6, 7). Because RPE cells are post-mitotic in the mammalian eye and ingest numerous POS daily, prompt and complete POS engulfment is essential to prevent gradual buildup of undigested debris in the RPE (8). Inefficient RPE lysosomal function causes accumulation of debris in human and experimental animal RPE that can be toxic and
Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling—a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to ‘nucleotide empty’ or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.
Alström syndrome (OMIM #203800) is an autosomal recessive obesity ciliopathy caused by loss-of-function mutations in the ALMS1 gene. In addition to multi-organ dysfunction, such as cardiomyopathy, retinal degeneration and renal dysfunction, the disorder is characterized by high rates of obesity, insulin resistance and early-onset type 2 diabetes mellitus (T2DM). To investigate the underlying mechanisms of T2DM phenotypes, we generated a loss-of-function deletion of alms1 in the zebrafish. We demonstrate conservation of hallmark clinical characteristics alongside metabolic syndrome phenotypes, including a propensity for obesity and fatty livers, hyperinsulinemia and glucose response defects. Gene expression changes in β-cells isolated from alms1 −/− mutants revealed changes consistent with insulin hypersecretion and glucose sensing failure, which were corroborated in cultured murine β-cells lacking Alms1. We also found evidence of defects in peripheral glucose uptake and concomitant hyperinsulinemia in the alms1 −/− animals. We propose a model in which hyperinsulinemia is the primary and causative defect underlying generation of T2DM associated with alms1 deficiency. These observations support the alms1 loss-of-function zebrafish mutant as a monogenic model for mechanistic interrogation of T2DM phenotypes.
Aging is a complex process best characterized as the chronic dysregulation of cellular processes leading to deteriorated tissue and organ function. While aging cannot currently be prevented, its impact on lifespan and healthspan in the elderly can potentially be minimized by interventions that aim to return these cellular processes to optimal function. Recent studies have demonstrated that partial reprogramming using the Yamanaka factors (or a subset; OCT4, SOX2, and KLF4; OSK) can reverse age-related changes in vitro and in vivo. However, it is still unknown whether the Yamanaka factors (or a subset) are capable of extending the lifespan of aged wild type mice. Here, we show that systemically delivered AAVs, encoding an inducible OSK system, in 124-week-old mice extends the median remaining lifespan by 109% over wild-type controls and enhances several health parameters. Importantly, we observed a significant improvement in frailty scores indicating that we were able to improve the healthspan along with increasing the lifespan. Furthermore, in human keratinocytes expressing exogenous OSK, we observed significant epigenetic markers of age-reversal, suggesting a potential reregulation of genetic networks to a younger, potentially healthier state. Together, these results may have important implications for the development of partial reprogramming interventions to reverse age-associated diseases in the elderly
Counting rhodopsin-positive phagosomes residing in the retinal pigment epithelium (RPE) in the eye at different times of day allows a quantitative assessment of engulfment and digestion phases of diurnal RPE phagocytosis, which efficiently clears shed photoreceptor outer segment fragments (POS) from the neural retina. Comparing such activities among age- and background-matched experimental wild-type and mutant mice or rats serves to identify roles for specific proteins in the phagocytic process. Here, we describe experimental procedures for mouse eye harvest, embedding, sectioning, immunofluorescence labeling of rod POS phagosomes in RPE cells in sagittal eye sections, imaging of POS phagosomes in the RPE by laser scanning confocal microscopy, and POS quantification.
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