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Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis.
The degradation of phagosomes, derived from the ingestion of photoreceptor outer segment (POS) disk membranes, is a major role of the retinal pigment epithelium (RPE). Here, POS phagosomes were observed to associate with myosin-7a, and then kinesin-1, as they moved from the apical region of the RPE. Live-cell imaging showed that the phagosomes moved bidirectionally along microtubules in RPE cells, with kinesin-1 light chain 1 (KLC1) remaining associated in both directions and during pauses. Lack of KLC1 did not inhibit phagosome speed, but run length was decreased, and phagosome localization and degradation were impaired. In old mice, lack of KLC1 resulted in RPE pathogenesis that was strikingly comparable to aspects of age-related macular degeneration (AMD), with an excessive accumulation of RPE and sub-RPE deposits, as well as oxidative and inflammatory stress responses. These results elucidate mechanisms of POS phagosome transport in relation to degradation, and demonstrate that defective microtubule motor transport in the RPE leads to phenotypes associated with AMD
Loss of Melanoregulin (MREG) Enhances Cathepsin-D Secretion by the Retinal Pigment Epithelium
Cathepsin-D (Cat-D) is a major proteolytic enzyme in phagocytic cells. In the retinal pigment epithelium (RPE), it is responsible for the daily degradation of photoreceptor outer segments (POSs) to maintain retinal homeostasis. Melanoregulin (MREG)-mediated loss of phagocytic capacity has been linked to diminished intracellular Cat-D activity. Here, we demonstrate that loss of MREG enhances the secretion of intermediate Cat-D (48 kDa), resulting in a net enhancement of extracellular Cat-D activity. These results suggest that MREG is required to maintain Cat-D homeostasis in the RPE and likely plays a protective role in retinal health. In this regard, in the Mreg dsu/dsu mouse, we observe increased basal laminin. Loss of the Mreg dsu allele is not lethal and therefore leads to slow age-dependent changes in the RPE. Thus, we propose that this model will allow us to study potential dysregulatory functions of Cat-D in retinal disease
Loss of Melanoregulin (MREG) Enhances Cathepsin-D Secretion by the Retinal Pigment Epithelium
Abstract Cathepsin-D (Cat-D) is a major proteolytic enzyme in phagocytic cells. In the retinal pigment epithelium (RPE), it is responsible for the daily degradation of photoreceptor outer segments (POSs) to maintain retinal homeostasis. Melanoregulin (MREG)-mediated loss of phagocytic capacity has been linked to diminished intracellular Cat-D activity. Here, we demonstrate that loss of MREG enhances the secretion of intermediate Cat-D (48 kDa), resulting in a net enhancement of extracellular Cat-D activity. These results suggest that MREG is required to maintain Cat-D homeostasis in the RPE and likely plays a protective role in retinal health. In this regard, in the Mreg dsu/dsu mouse, we observe increased basal laminin. Loss of the Mreg dsu allele is not lethal and therefore leads to slow age-dependent changes in the RPE. Thus, we propose that this model will allow us to study potential dysregulatory functions of Cat-D in retinal disease. Copyright © Cambridge University Press, 2013
The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium
A main requisite in the phagocytosis of ingested material is a coordinated series of maturation steps which lead to the degradation of ingested cargo. Photoreceptor outer segment (POS) renewal involves phagocytosis of the distal disk membranes by the retinal pigment epithelium (RPE). Previously, we identified melanoregulin (MREG) as an intracellular cargo-sorting protein required for the degradation of POS disks. Here, we provide evidence that MREG-dependent processing links both autophagic and phagocytic processes in LC3-associated phagocytosis (LAP). Ingested POS phagosomes are associated with endogenous LC3 and MREG. The LC3 association with POSs exhibited properties of LAP; it was independent of rapamycin pretreatment, but dependent on Atg5. Loss of MREG resulted in a decrease in the extent of LC3-POS association. Studies using DQ™-BSA suggest that loss of MREG does not compromise the association and fusion of LC3-positive phagosomes with lysosomes. Furthermore, the mechanism of MREG action is likely through a protein complex that includes LC3, as determined by colocalization and immunoprecipitation in both RPE cells and macrophages. We posit that MREG participates in coordinating the association of phagosomes with LC3 for content degradation with the loss of MREG leading to phagosome accumulation. © 2014, Springer Science+Business Media New York
The Contribution of Melanoregulin to Microtubule-Associated Protein 1 Light Chain 3 (LC3) Associated Phagocytosis in Retinal Pigment Epithelium
A main requisite in the phagocytosis of ingestedmaterial is a coordinated series of maturation steps which leadto the degradation of ingested cargo. Photoreceptor outersegment (POS) renewal involves phagocytosis of the distaldisk membranes by the retinal pigment epithelium (RPE).Previously, we identified melanoregulin (MREG) as an intra-cellular cargo-sorting protein required for the degradation ofPOS disks. Here, we provide evidence that MREG-dependentprocessing links both autophagic and phagocytic processes inLC3-associated phagocytosis (LAP). Ingested POSphagosomes are associated with endogenous LC3 andMREG. The LC3 association with POSs exhibited propertiesof LAP; it was independent of rapamycin pretreatment, butdependent on Atg5. Loss of MREG resulted in a decrease inthe extent of LC3-POS association. Studies using DQâ„¢-BSAsuggest that loss of MREG does not compromise the associ-ation and fusion of LC3-positive phagosomes with lysosomes.Furthermore, the mechanism of MREG action is likelythrough a protein complex that includes LC3, as determinedby colocalization and immunoprecipitation in both RPE cellsand macrophages. We posit that MREG participates incoordinating the association of phagosomes with LC3 forcontent degradation with the loss of MREG leading tophagosome accumulation
Melanoregulin (MREG) Modulates Lysosome Function in Pigment Epithelial Cells
Melanoregulin (MREG), the product of the Mregdsu gene, is a small highly charged protein, hypothesized to play a role in organelle biogenesis due to its effect on pigmentation in dilute, ashen, and leaden mutant mice. Here we provide evidence that MREG is required in lysosome-dependent phagosome degradation. In the Mreg-/- mouse, we show that loss of MREG function results in phagosome accumulation due to delayed degradation of engulfed material. Over time, the Mreg-/- mouse retinal pigment epithelial cells accumulate the lipofuscin component, A2E. MREG-deficient human and mouse retinal pigment epithelial cells exhibit diminished activity of the lysosomal hydrolase, cathepsin D, due to defective processing. Moreover, MREG localizes to small intracellular vesicles and associates with the endosomal phosphoinositide, phosphatidylinositol 3,5-biphosphate. Collectively, these studies suggest that MREG is required for lysosome maturation and support a role for MREG in intracellular trafficking
Elevated PGC-1α Activity Sustains Mitochondrial Biogenesis and Muscle Function without Extending Survival in a Mouse Model of Inherited ALS
SummaryThe transcriptional coactivator PGC-1α induces multiple effects on muscle, including increased mitochondrial mass and activity. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, adult-onset neurodegenerative disorder characterized by selective loss of motor neurons and skeletal muscle degeneration. An early event is thought to be denervation-induced muscle atrophy accompanied by alterations in mitochondrial activity and morphology within muscle. We now report that elevation of PGC-1α levels in muscles of mice that develop fatal paralysis from an ALS-causing SOD1 mutant elevates PGC-1α-dependent pathways throughout disease course. Mitochondrial biogenesis and activity are maintained through end-stage disease, accompanied by retention of muscle function, delayed muscle atrophy, and significantly improved muscle endurance even at late disease stages. However, survival was not extended. Therefore, muscle is not a primary target of mutant SOD1-mediated toxicity, but drugs increasing PGC-1α activity in muscle represent an attractive therapy for maintaining muscle function during progression of ALS
Correction to: Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.
The original article [1] contains an error in the legend of Fig 5 whereby the descriptions for panels 5d and 5e are incorrect; as such, the corrected legend can be viewed below with its respective figure images
The Ternary Rab27a–Myrip–Myosin VIIa Complex Regulates Melanosome Motility in the Retinal Pigment Epithelium
The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light-dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a- and MyoVIIa-interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co-immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live-cell imaging of mouse RPE primary cultures. Wild-type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen- (Rab27a defective) and shaker-1 (MyoVIIa mutant)-derived RPE cells, and in wild-type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a–Myrip–MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery
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