Mitochondria-dependent phase separation of disease-relevant proteins drives pathological features of age-related macular degeneration.

Age-related macular degeneration (AMD) damages the retinal pigment epithelium (RPE), the tissue that safeguards photoreceptor health, leading to irreversible vision loss. Polymorphisms in cholesterol and complement genes are implicated in AMD, yet mechanisms linking risk variants to RPE injury remain unclear. We sought to determine how allelic variants in the apolipoprotein E cholesterol transporter modulate RPE homeostasis and function. Using live-cell imaging, we show that inefficient cholesterol transport by the AMD risk-associated ApoE2 increases RPE ceramide, leading to autophagic defects and complement-mediated mitochondrial damage. Mitochondrial injury drives redox state-sensitive cysteine-mediated phase separation of ApoE2, forming biomolecular condensates that could nucleate drusen. The protective ApoE4 isoform lacks these cysteines and is resistant to phase separation and condensate formation. In Abca-/- Stargardt macular degeneration mice, mitochondrial dysfunction induces liquid-liquid phase separation of p62/SQSTM1, a multifunctional protein that regulates autophagy. Drugs that decrease RPE cholesterol or ceramide prevent mitochondrial injury and phase separation in vitro and in vivo. In AMD donor RPE, mitochondrial fragmentation correlates with ApoE and p62 condensates. Our studies demonstrate that major AMD genetic and biological risk pathways converge upon RPE mitochondria, and identify mitochondrial stress-mediated protein phase separation as an important pathogenic mechanism and promising therapeutic target in AMD.

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

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.

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.

Protective responses to sublytic complement in the retinal pigment epithelium.

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.

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, Alzheimer's disease, and age-related macular degeneration (AMD). A single E4 allele increases the risk of developing Alzheimer's 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 Alzheimer's 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 Alzheimer's disease.

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

Autophagy is an essential mechanism for clearing damaged organelles and proteins within the cell. As with neurodegenerative diseases, dysfunctional autophagy could contribute to blinding diseases such as macular degeneration. However, precisely how inefficient autophagy promotes retinal damage is unclear. In this study, we investigate innate mechanisms that modulate autophagy in the retinal pigment epithelium (RPE), a key site of insult in macular degeneration. High-speed live imaging of polarized adult primary RPE cells and data from a mouse model of early-onset macular degeneration identify a mechanism by which lipofuscin bisretinoids, visual cycle metabolites that progressively accumulate in the RPE, disrupt autophagy. We demonstrate that bisretinoids trap cholesterol and bis(monoacylglycero)phosphate, an acid sphingomyelinase (ASMase) cofactor, within the RPE. ASMase activation increases cellular ceramide, which promotes tubulin acetylation on stabilized microtubules. Live-imaging data show that autophagosome traffic and autophagic flux are inhibited in RPE with acetylated microtubules. Drugs that remove excess cholesterol or inhibit ASMase reverse this cascade of events and restore autophagosome motility and autophagic flux in the RPE. Because accumulation of lipofuscin bisretinoids and abnormal cholesterol homeostasis are implicated in macular degeneration, our studies suggest that ASMase could be a potential therapeutic target to ensure the efficient autophagy that maintains RPE health.

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.

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.

BACKGROUND: Increased 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. FINDINGS: Explants 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. CONCLUSIONS: We 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.

Mechanism of polarized lysosome exocytosis in epithelial cells.

Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigate the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain. In cells lacking either the ubiquitous AP-1A or the epithelial-specific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia and suggest that AP-1 is a crucial regulator of this process.

Hydrocortisone stimulates neurite outgrowth from mouse retinal explants by modulating macroglial activity.

PURPOSE: There is mounting evidence that retinal ganglion cells (RGCs) require a complex milieu of trophic factors to enhance cell survival and axon regeneration after optic nerve injury. The authors' goal was to examine the contribution of components of a combination of hormones, growth factors, steroids, and small molecules to creating a regenerative environment and to determine if any of these components modulated macroglial behavior to aid in regeneration. METHODS: Postnatal day 7 mouse retinal explants embedded in collagen were used as an in vitro model of neurite regeneration. Explants were treated with the culture supplements fetal bovine serum, N2, and G5 and a mixture of G5 and N2 components, designated enhanced N2 (EN2). Explants were evaluated for neurite outgrowth over 7 days in culture. The effects of each treatment were also evaluated on cultured RGCs purified by Thy1 immunopanning. Immunohistochemistry and qPCR analysis were used to evaluate differences in gene expression in the explants due to different treatments. RESULTS: EN2 stimulated significant neurite outgrowth from explants but not from purified RGCs. Elimination of hydrocortisone (HC) from EN2 reduced the mean neurites per explant by 37%. EN2-treated explants demonstrated increased expression of Gfap, Glul, Glt1, Cntf, Pedf, and VegfA compared with explants treated with EN2 without HC. Subsequent experiments showed that increased expression of Cntf and Glul was critical to the trophic effect of HC. CONCLUSIONS: These data suggest that the HC in EN2 indirectly contributed to neurite outgrowth by activating macroglia to produce neurotrophic and neuroprotective molecules.

Modulation of diabetes-induced palate defects by maternal immune stimulation.

Maternal diabetes can induce a number of developmental abnormalities in both laboratory animals and humans, including deformities of the face and palate. The incidence of birth defects in newborns of women with diabetes is approximately 3 to 5 times higher than among nondiabetics. In mice, nonspecific activation of the maternal immune system can reduce fetal abnormalities caused by various etiologies including hyperglycemia. This study was conducted to determine whether nonspecific maternal immune stimulation could reduce diabetes-induced palate defects and orofacial clefts. Female ICR mice were immune stimulated before induction of hyperglycemia with Freund's complete adjuvant (FCA), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interferon-gamma (IFNgamma). Streptozocin was used to induce hyperglycemia (26-35 mmol blood glucose) in females before breeding. Fetuses from 12 to 18 litters per treatment group were collected on Day 17 of gestation. Palate width and length were measured, and the incidence of orofacial clefts was determined. Palate length and width were both decreased by maternal hyperglycemia. Maternal immune stimulation with GM-CSF or FCA limited the degree of palate shortening from the hyperglycemia. Each of the three immune stimulants attenuated significant narrowing of the palate. Rates of orofacial clefts were not significantly different between treatment groups. Palatogenesis is a complex process driven by cellular signals, which regulate cell growth and apoptosis. Dysregulation of cellular signals by maternal hyperglycemia can result in fetal malformations. Maternal immune stimulation may prevent dysregulation of these signaling pathways thus reducing fetal malformations and normalizing palate growth.

Neuromuscular development in the absence of programmed cell death: phenotypic alteration of motoneurons and muscle.

The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal death in vivo to ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from alpha- to gamma-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic gene Bax. However, there was an increase in intrafusal muscle fibers (spindles) in Bax knock-out versus both wild-type and MyoGDNF mice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.

Reduction in diabetes-induced craniofacial defects by maternal immune stimulation.

BACKGROUND: Maternal diabetes can induce a number of developmental abnormalities in laboratory animals and humans, including facial deformities and defects in neural tube closure. The incidence of birth defects in newborns of diabetic women is approximately 3-5 times higher than among non-diabetics. In mice, non-specific activation of the maternal immune system can reduce fetal abnormalities caused by diverse etiologies, including diabetes induced neural tube defects. This study was conducted to determine whether non-specific maternal immune stimulation could reduce diabetes-induced craniofacial defects as well. METHODS: Maternal immune function was stimulated before streptozocin (STZ) treatment by maternal footpad injection with Freund's complete adjuvant (FCA), maternal intraperitoneal (i.p.) injection with granulocyte-macrophage colony-stimulating factor (GM-CSF), or maternal i.p. injection with interferon-gamma (IFNgamma). Streptozocin (200 mg/kg i.p.) was used to induce hyperglycemia (26-35 mmol blood glucose) in female ICR mice before breeding. Fetuses from 12-18 litters per treatment group, were collected at Day 17 of gestation. RESULTS: Craniofacial defects were observed in fetuses from all hyperglycemic groups. The incidence of defects was significantly decreased in fetuses from dams immune stimulated with IFNgamma or GM-CSF. The most common defects were reduced maxillary and mandibular lengths. Both were prevented by maternal stimulation with GM-CSF. CONCLUSION: Maternal immune stimulation reduced the incidence of diabetic craniofacial embryopathy. The mechanisms for these protective effects are unknown but may involve maternal or fetal production of cytokines or growth factors that protect the fetus from the dysregulatory effects of hyperglycemia.