Kimberly A. Toops

Cholesterol-mediated activation of acid sphingomyelinase disrupts autophagy in the retinal pigment epithelium

How autophagy is regulated in the postmitotic retinal pigment epithelium (RPE) is unclear. Visual cycle metabolites and cholesterol that accumulate in the RPE inhibit autophagic flux by activating acid sphingomyelinase (ASMase). Increased ceramide promotes tubulin acetylation, which prevents autophagosome traffic. ASMase inhibition restores RPE autophagy.

A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers

The retinal pigment epithelium (RPE) performs numerous functions that are indispensable for photoreceptor health and vision. This monolayer of cells is also a major site of insult in inherited and age-related macular degenerations. In vitro models of primary RPE such as human fetal and adult RPE cultures have been invaluable for dissecting disease pathways at the cellular and molecular level. However, numerous studies show that it takes over four weeks for human RPE cell monolayers to become fully polarized after plating on semipermeable membrane supports. Poor persistence of transgene expression over this time period critically limits the applicability of human RPE cultures for live imaging studies required to follow dynamic processes like intracellular trafficking and organelle transport that occur over timescales of milliseconds. Here, we provide a detailed three-step protocol for live imaging of polarized primary RPE using high-speed spinning disk confocal microscopy. Step 1: establish porcine RPE monolayers that undergo differentiation within one week after plating on semipermeable membrane supports; step 2: transfect or transduce RPE using either of two different protocols that result in prolonged transgene expression; and step 3: perform multicolor high-speed live imaging of organelle transport in polarized RPE monolayers. Porcine RPE cells and photoreceptor outer segments were isolated from freshly harvested eyes and plated on collagen-coated Transwell® filters to generate polarized monolayers. After seven days, RPE monolayers were highly pigmented, had TER values ≥ 200 Ω.cm2 and cleared outer segments within 5 hours after phagocytosis. These cells expressed RPE65, localized ZO-1 to the tight junction, Na+,K+-ATPase to the apical membrane and acetylated tubulin to the primary cilium. There was an inverse relationship between initial plating density and the time to differentiation. We used nucleofection to express fluorescently tagged genes in RPE cells prior to plating on filters or baculovirus fusion constructs to transfect polarized monolayers. Both these methods resulted in transfection efficiencies over 40% and transgene expression lasted up to 8 days after plating. These filters were imaged by high-speed spinning disk microscopy to follow tubulovesicular trafficking of lysosomes and actin dynamics in the RPE. Four-dimensional image analysis performed using commercially available software was used to analyze live imaging data. In conclusion, this 3-step protocol describes a powerful method to investigate organelle trafficking and function in real time in the RPE that can be used for answering fundamental questions of RPE cell biology and pathobiology.

Protective responses to sublytic complement in the retinal pigment epithelium

Significance The complement system regulates immune defense and inflammation. Abnormal complement activation in the retina is associated with blinding macular degenerations, which have limited treatment options. The retinal pigment epithelium (RPE) is an initial site of injury in macular degenerations, but mechanisms that protect the RPE from complement-mediated damage are unclear. Here, we identify two critical responses to complement in the RPE: accelerated recycling of the complement inhibitor CD59 and lysosome-mediated membrane repair. We show that in models of macular degeneration, excess cholesterol impairs both defense mechanisms, resulting in mitochondrial damage. Drugs that decrease RPE cholesterol restore these mechanisms and help the RPE combat complement attack. Our studies identify promising drug targets to preserve RPE health and function in macular degenerations. The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4−/− Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4−/− mice, indicating that they could be effective therapeutic approaches for macular degenerations.

Should I stay or should I go? Trafficking of sub-lytic MAC in the retinal pigment epithelium.

Assembly of sub-lytic C5b-9 membrane attack complexes (MAC) on the plasma membrane of retinal pigment epithelial cells contributes to the pathogenesis of age-related macular degeneration. C5b-9 pores induce calcium influx, which activates signaling pathways that compromise cell function. Mechanisms that limit sub-lytic MAC activity include: cell surface complement regulatory proteins CD46, CD55, and CD59 that inhibit specific steps of MAC formation; elimination of assembled MAC by exocytosis of membrane vesicles or by endocytosis and subsequent lysosomal degradation; and rapid resealing of pores by the exocytosis of lysosomes. Aging in the post-mitotic retinal pigment epithelium is characterized by the accumulation of cellular debris called lipofuscin, which has also been associated with retinal diseases such as age-related macular degeneration. Lipofuscin has been shown to activate complement components both in vitro and in vivo, suggesting that it could contribute complement-mediated dysfunction in the retinal pigment epithelium. Here, we discuss emerging evidence that vesicular trafficking in the retinal pigment epithelium is critical for efficient removal of MAC from the cell surface and for limiting inflammation in the outer retina.

Apolipoprotein E Isoforms and AMD

The cholesterol transporting protein apolipoprotein E (ApoE) occurs in three allelic variants in humans unlike in other species. The resulting protein isoforms E2, E3 and E4 exhibit differences in lipid binding, integrating into lipoprotein particles and affinity for lipoprotein receptors. ApoE isoforms confer genetic risk for several diseases of aging including atherosclerosis, Alzheimers disease, and age-related macular degeneration (AMD). A single E4 allele increases the risk of developing Alzheimers disease, whereas the E2 allele is protective. Intriguingly, the E4 allele is protective in AMD. Current thinking about different functions of ApoE isoforms comes largely from studies on Alzheimers disease. These data cannot be directly extrapolated to AMD since the primary cells affected in these diseases (neurons vs. retinal pigment epithelium) are so different. Here, we propose that ApoE serves a fundamentally different purpose in regulating cholesterol homeostasis in the retinal pigment epithelium and this could explain why allelic risk factors are flipped for AMD compared to Alzheimers disease.

Novel roles for the radial spoke head protein 9 in neural and neurosensory cilia

Cilia are cell surface organelles with key roles in a range of cellular processes, including generation of fluid flow by motile cilia. The axonemes of motile cilia and immotile kinocilia contain 9 peripheral microtubule doublets, a central microtubule pair, and 9 connecting radial spokes. Aberrant radial spoke components RSPH1, 3, 4a and 9 have been linked with primary ciliary dyskinesia (PCD), a disorder characterized by ciliary dysmotility; yet, radial spoke functions remain unclear. Here we show that zebrafish Rsph9 is expressed in cells bearing motile cilia and kinocilia, and localizes to both 9 + 2 and 9 + 0 ciliary axonemes. Using CRISPR mutagenesis, we show that rsph9 is required for motility of presumptive 9 + 2 olfactory cilia and, unexpectedly, 9 + 0 neural cilia. rsph9 is also required for the structural integrity of 9 + 2 and 9 + 0 ciliary axonemes. rsph9 mutant larvae exhibit reduced initiation of the acoustic startle response consistent with hearing impairment, suggesting a novel role for Rsph9 in the kinocilia of the inner ear and/or lateral line neuromasts. These data identify novel roles for Rsph9 in 9 + 0 motile cilia and in sensory kinocilia, and establish a useful zebrafish PCD model.

Let's play a game of chutes and ladders: Lysosome fusion with the epithelial plasma membrane.

In non-polarized cells, calcium-induced exocytosis of “conventional” lysosomes is important in diverse processes like membrane repair after exposure to pore-forming toxins and clearance of cellular debris. Resealing of torn membranes is especially critical for barrier epithelia that directly interact with pathogens and toxins, which can result in membrane microdisruptions and lesions. However, whether lysosomes participate in membrane repair in polarized epithelia has been an open question. We recently reported that in polarized Madin-Darby canine kidney (MDCK) cells, localized influx of calcium induces lysosomes to fuse with the basolateral membrane. This spatial segregation of exocytosis depends on an intact actin cytoskeleton, membrane cholesterol and restricted distribution of fusion machinery such as the t-SNARE syntaxin 4. Our data show that the polarity of syntaxin 4 (which is regulated by the clathrin adaptor protein AP-1) dictates whether lysosomes parachute down to the basolateral membrane or take a ladder up to the apical membrane. Here, we speculate about additional machinery (such as the lysosomal calcium sensor synaptotagmin VII and the v-SNARE VAMP7) that could be involved in polarized fusion of lysosomes with the epithelial membrane. We also discuss the potential importance of lysosome exocytosis in maintaining membrane integrity in the retinal pigment epithelium, the primary tissue affected in blinding diseases such as age-related macular degeneration.

The effect of glial fibrillary acidic protein expression on neurite outgrowth from retinal explants in a permissive environment

BackgroundIncreased expression of glial fibrillary acidic protein (GFAP) within macroglia is commonly seen as a hallmark of glial activation after damage within the central nervous system, including the retina. The increased expression of GFAP in glia is also considered part of the pathologically inhibitory environment for regeneration of axons from damaged neurons. Recent studies have raised the possibility that reactive gliosis and increased GFAP cannot automatically be assumed to be negative events for the surrounding neurons and that the context of the reactive gliosis is critical to whether neurons benefit or suffer. We utilized transgenic mice expressing a range of Gfap to titrate the amount of GFAP in retinal explants to investigate the relationship between GFAP concentration and the regenerative potential of retinal ganglion cells.FindingsExplants from Gfap-/- and Gfap+/- mice did not have increased neurite outgrowth compared with Gfap+/+ or Gfap over-expressing mice as would be expected if GFAP was detrimental to axon regeneration. In fact, Gfap over-expressing explants had the most neurite outgrowth when treated with a neurite stimulatory media. Transmission electron microscopy revealed that neurites formed bundles, which were surrounded by larger cellular processes that were GFAP positive indicating a close association between growing axons and glial cells in this regeneration paradigm.ConclusionsWe postulate that glial cells with increased Gfap expression support the elongation of new neurites from retinal ganglion cells possibly by providing a scaffold for outgrowth.

Aberrant early endosome biogenesis mediates complement activation in the retinal pigment epithelium in models of macular degeneration

Significance The first lines of communication between cells and the environment are early endosomes, which sort incoming cargo to regulate cell health. Endosomal abnormalities are seen in neurodegenerative diseases, yet the molecular mechanisms remain obscure. Here, using human donor cells and disease models, we demonstrate that excess ceramide promotes expansion of early endosomes in the retinal pigment epithelium (RPE), a primary site of injury in Stargardt and age-related maculopathies. Complement C3 uptake into enlarged endosomes and subsequent cleavage is associated with abnormal mechanistic target of rapamycin activity. Decreasing ceramide using Food and Drug Administration-approved drugs corrects endosomal defects and prevents C3 activation. Our studies establish how organelles modulate RPE complement activity, and identify ceramide as a drug target for macular degenerations. Abnormally enlarged early endosomes (EEs) are pathological features of neurodegenerative diseases, yet insight into the mechanisms and consequences of EE expansion remains elusive. Here, we report swollen apical EEs in the retinal pigment epithelium (RPE) of aged human donors and in the pigmented Abca4−/− mouse model of Stargardt early-onset macular degeneration. Using high-resolution live-cell imaging, we show that age-related and pathological accumulation of lipofuscin bisretinoids increases ceramide at the apical surface of the RPE, which promotes inward budding and homotypic fusion of EEs. These enlarged endosomes internalize the complement protein C3 into the RPE, resulting in the intracellular generation of C3a fragments. Increased C3a in turn activates the mechanistic target of rapamycin (mTOR), a regulator of critical metabolic processes such as autophagy. The antidepressant desipramine, which decreases ceramide levels by inhibiting acid sphingomyelinase, corrects EE defects in the RPE of Abca4−/− mice. This prevents C3 internalization and limits the formation of C3a fragments within the RPE. Although uncontrolled complement activation is associated with macular degenerations, how complement contributes to pathology in a progressive disease is not well understood. Our studies link expansion of the EE compartment with intracellular complement generation and aberrant mTOR activation, which could set the stage for chronic metabolic reprogramming in the RPE as a prelude to disease. The pivotal role of ceramide in driving EE biogenesis and fusion in the Abca4−/− mice RPE suggests that therapeutic targeting of ceramide could be effective in Stargardt disease and other macular degenerations.