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.

Retinal microglia - A key player in healthy and diseased retina.

Microglia, the resident immune cells of the brain and retina, are constantly engaged in the surveillance of their surrounding neural tissue. During embryonic development they infiltrate the retinal tissues and participate in the phagocytosis of redundant neurons. The contribution of microglia in maintaining the purposeful and functional histo-architecture of the adult retina is indispensable. Within the retinal microenvironment, robust microglial activation is elicited by subtle changes caused by extrinsic and intrinsic factors. When there is a disturbance in the cell-cell communication between microglia and other retinal cells, for example in retinal injury, the activated microglia can manifest actions that can be detrimental. This is evidenced by activated microglia secreting inflammatory mediators that can further aggravate the retinal injury. Microglial activation as a harbinger of a variety of retinal diseases is well documented by many studies. In addition, a change in the microglial phenotype which may be associated with aging, may predispose the retina to age-related diseases. In light of the above, the focus of this review is to highlight the role played by microglia in the healthy and diseased retina, based on findings of our own work and from that of others.

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.

Biology of Microglia in the Developing Brain.

Microglia exist in different morphological forms in the developing brain. They show a small cell body with scanty cytoplasm with many branching processes in the grey matter of the developing brain. However, in the white matter such as the corpus callosum where the unmyelinated axons are loosely organized, they appear in an amoeboid form having a round cell body endowed with copious cytoplasm rich in organelles. The amoeboid cells eventually transform into ramified microglia in the second postnatal week when the tissue becomes more compact with the onset of myelination. Microglia serve as immunocompetent macrophages that act as neuropathology sensors to detect and respond swiftly to subtle changes in the brain tissues in pathological conditions. Microglial functions are broadly considered as protective in the normal brain development as they phagocytose dead cells and sculpt neuronal connections by pruning excess axons and synapses. They also secrete a number of trophic factors such as insulin-like growth factor-1 and transforming growth factor-ß among many others that are involved in neuronal and oligodendrocyte survival. On the other hand, microglial cells when activated produce a plethora of molecules such as proinflammatory cytokines, chemokines, reactive oxygen species, and nitric oxide that are implicated in the pathogenesis of many pathological conditions such as epilepsy, cerebral palsy, autism, and perinatal hypoxic-ischemic brain injury. Although many studies have investigated the origin and functions of the microglia in the developing brain, in-depth in vivo studies along with analysis of their transcriptome and epigenetic changes need to be undertaken to elucidate their full potential be it protective or neurotoxic. This would lead to a better understanding of their roles in the healthy and diseased developing brain and advancement of therapeutic strategies to target microglia-mediated neurotoxicity.

Glutamate Inhibits the Pro-Survival Effects of Insulin-Like Growth Factor-1 on Retinal Ganglion Cells in Hypoxic Neonatal Rat Retina.

Glutamate that accumulates in injured brain tissue has been shown to hinder the neuroprotection rendered by insulin-like growth factor-1 (IGF-1). However, its role in attenuating the neuroprotective effect of IGF-1 in the hypoxic retina is unknown and the current study was aimed at elucidating this. One-day-old Wistar rats were exposed to hypoxia for 2 h and the retinas were studied at 3 h to 14 days after exposure. Following hypoxia, the concentrations of glutamate and IGF-1 were significantly increased over control values in the immature retina and in cultured retinal ganglion cells (RGCs). In addition to IGF-1, the relative expression of insulin receptor substrate-1 (IRS1) phosphorylated at the tyrosine residue (p-IRS1tyr), phosphorylated protein kinase B (p-AKT) and phosphorylated protein kinase A (p-PKA), which are involved in IGF-1 signalling, was also studied in hypoxic retinas and in cultured RGCs. Glutamate-mediated inhibition of the IGF-1 pathway in hypoxic RGCs was evident with a reduced expression of p-IRS1tyr and p-AKT and an increased expression of p-PKA. However, the addition of exogenous IGF-1 reversed this. Glutamate enables the phosphorylation of IRS1 at the serine residue (p-IRS1ser) through a PKA-dependent pathway. The increased expression of p-IRS1ser and its increased association with IGF-1 receptors in hypoxic RGCs suggested a possible interference by glutamate with the IGF-1 pathway. Moreover, there was increased caspase-3/7 activity in hypoxic RGCs. These results suggest that glutamate, by blocking IGF-1-mediated neuroprotection, could cause the apoptosis of RGCs in the hypoxic neonatal retina.

The Choroid Plexus in Healthy and Diseased Brain.

The choroid plexus is composed of epithelial cells resting on a basal lamina. These cells produce the cerebrospinal fluid (CSF), which has many functions including rendering mechanical support, providing a route for some nutrients, removing by-products of metabolism and synaptic activity, and playing a role in hormonal signaling. The choroid plexus synthesizes many growth factors, including insulin-like, fibroblast, and platelet-derived growth factors. The tight junctions located between the apical parts of the choroid plexus epithelial cells form the blood-CSF barrier (BCSFB), which is crucial for the homeostatic regulation of the brain microenvironment along with the blood-brain barrier (BBB). Morphological changes such as atrophy of the epithelial cells and thickening of the basement membrane suggest altered CSF production occurs in aging and in Alzheimer disease. In brain injuries and infections, leukocytes accumulate in the CSF by passing through the choroid plexus. In inflammatory CNS diseases (eg, multiple sclerosis), pathogenic autoreactive T lymphocytes may migrate through the BBB and BCSFB into the CNS. The development of therapeutic strategies to mitigate disruption of the BCSFB may be helpful to curtail the entry of inflammatory cells into the CSF and hence reduce inflammation, thereby overcoming choroid plexus dysfunction in senescence and in various diseases of the CNS.

Hypoxia-Induced Iron Accumulation in Oligodendrocytes Mediates Apoptosis by Eliciting Endoplasmic Reticulum Stress.

This study was aimed at evaluating the role of increased iron accumulation in oligodendrocytes and its role in their apoptosis in the periventricular white matter damage (PWMD) following a hypoxic injury to the neonatal brain. In response to hypoxia, in the PWM, there was increased expression of proteins involved in iron acquisition, such as iron regulatory proteins (IRP1, IRP2) and transferrin receptor in oligodendrocytes. Consistent with this, following a hypoxic exposure, there was increased accumulation of iron in primary cultured oligodendrocytes. The increased concentration of iron within hypoxic oligodendrocytes was found to elicit ryanodine receptor (RyR) expression, and the expression of endoplasmic reticulum (ER) stress markers such as binding-immunoglobulin protein (BiP) and inositol-requiring enzyme (IRE)-1α. Associated with ER stress, there was reduced adenosine triphosphate (ATP) levels within hypoxic oligodendrocytes. However, treatment with deferoxamine reduced the increased expression of RyR, BiP, and IRE-1α and increased ATP levels in hypoxic oligodendrocytes. Parallel to ER stress there was enhanced reactive oxygen species production within mitochondria of hypoxic oligodendrocytes, which was attenuated when these cells were treated with deferoxamine. At the ultrastructural level, hypoxic oligodendrocytes frequently showed dilated ER and disrupted mitochondria, which became less evident in those treated with deferoxamine. Associated with these subcellular changes, the apoptosis of hypoxic oligodendrocytes was evident with an increase in p53 and caspase-3 expression, which was attenuated when these cells were treated with deferoxamine. Thus, the present study emphasizes that the excess iron accumulated within oligodendrocytes in hypoxic PWM could result in their death by eliciting ER stress and mitochondrial disruption.

Vascular changes in the developing rat retina in response to hypoxia.

This study was carried out to investigate the roles of tight junction (TJ) proteins and other factors in the increased permeability of the blood retinal barrier (BRB) affecting the immature neonatal retina following a hypoxic insult. The expression of endothelial TJ proteins such as claudin-5, occludin and zonula occludens-1 (ZO-1) and endothelial cell specific molecule-1 (ESM-1), and associated structural changes in the blood vessels were analyzed in the retinas of 1-day-old Wistar rats subjected to hypoxia for 2 h and subsequently sacrificed at different time points ranging from 3 h to 14 d. The mRNA and protein expression of claudin-5, occludin & ZO-1 was found to be reduced in the hypoxic retina, although, at the ultrastructural level, the TJ between the endothelial cells and retinal pigment epithelial cells appeared to be intact. Following the hypoxic insult vascular endothelial cells frequently showed presence of cytoplasmic vacuoles, vacuolated mitochondria and multivesicular aggregations projecting into the lumen of the capillaries. The expression of ESM-1 in the immature retinas was found to be increased following hypoxic exposure. The structural and molecular changes in the hypoxic neonatal retinas were consistent with a hypoxia induced impairment of the BRB. Hypoxia reduced the expression of TJ proteins in the neonatal retina, but the role of increased ESM-1 expression in this process warrants further investigation.

NF-κB-mediated nitric oxide production and activation of caspase-3 cause retinal ganglion cell death in the hypoxic neonatal retina.

PURPOSE: Hypoxic insult to the developing retina results in apoptosis of retinal ganglion cells (RGCs) through production of inflammatory mediators, nitric oxide (NO), and free radicals. The present study was aimed at elucidating the pathway through which hypoxia results in overproduction of NO in the immature retina, and its role in causing apoptosis of RGCs. METHODS: Wistar rats (1 day old) were exposed to hypoxia and their retinas were studied at 3 hours to 14 days after exposure. The protein expression of nuclear factor-κB (NF-κB) and neuronal nitric oxide synthase (nNOS) in the retina and primary cultures of RGCs was analyzed using Western blotting and double-immunofluorescence, whereas the concentration of NO was determined calorimetrically. In cultured RGCs, hypoxia-induced apoptosis was evaluated by caspase-3 immunolabeling. RESULTS: Following hypoxic exposure, NF-κB-mediated expression of nNOS, which was localized to the RGCs, and subsequent NO production was significantly increased in the developing retina. In primary cultures of RGCs subjected to hypoxia, the upregulation of nNOS and NO was significantly suppressed when treated with 7-nitroindazole (7-NINA), an nNOS inhibitor or BAY, an NF-κB inhibitor. Hypoxia-induced apoptosis of RGCs, which was evident with caspase-3 labeling, also was suppressed when these cells were treated with 7-NINA or BAY. CONCLUSIONS: Our results suggest that in RGCs, hypoxic induction of nNOS is mediated by NF-κB and the resulting increased release of NO by RGCs causes their apoptosis through caspase-3 activation. It is speculated that targeting nNOS could be a potential neuroprotective strategy against hypoxia-induced RGCs death in the developing retina.

Therapeutic implications of melatonin in cerebral edema.

Cerebral edema/brain edema refers to the accumulation of fluid in the brain and is one of the fatal conditions that require immediate medical attention. Cerebral edema develops as a consequence of cerebral trauma, cerebral infarction, hemorrhages, abscess, tumor, hypoxia, and other toxic or metabolic factors. Based on the causative factors cerebral edema is differentiated into cytotoxic cerebral edema, vasogenic cerebral edema, osmotic and interstitial cerebral edema. Treatment of cerebral edema depends on timely diagnosis and medical assistance. Pragmatic treatment strategies such as antihypertensive medications, nonsteroidal anti-inflammatory drugs, barbiturates, steroids, glutamate and N-methyl-D-aspartate receptor antagonists and trometamol are used in clinical practice. Although the above mentioned treatment approaches are being used, owing to the complexity of the mechanisms involved in cerebral edema, a single therapeutic strategy which could ameliorate cerebral edema is yet to be identified. However, recent experimental studies have suggested that melatonin, a neurohormone produced by the pineal gland, could be an effective alternative for treating cerebral edema. In animal models of stroke, melatonin was not only shown to reduce cerebral edema but also preserved the blood brain barrier. Melatonin's beneficial effects were attributed to its properties, such as being a potent anti-oxidant, and its ability to cross the blood brain barrier within minutes after its administration. This review summarizes the beneficial effects of melatonin when used for treating cerebral edema.

Hypoxia inducible factor-1α mediates iron uptake which induces inflammatory response in amoeboid microglial cells in developing periventricular white matter through MAP kinase pathway.

Iron accumulation occurs in tissues such as periventricular white matter (PWM) in response to hypoxic injuries, and microglial cells sequester excess iron following hypoxic exposure. As hypoxia has a role in altering the expression of proteins involved in iron regulation, this study was aimed at examining the interaction between hypoxia inducible factor (HIF)-1α and proteins involved in iron transport in microglial cells, and evaluating the mechanistic action of deferoxamine and KC7F2 (an inhibitor of HIF-1α) in iron mediated hypoxic injury. Treating the microglial cultures with KC7F2, led to decreased expression of transferrin receptor and divalent metal transporter-1. Administration of deferoxamine or KC7F2 to hypoxic microglial cells enhanced extracellular signal-regulated kinase (ERK) phosphorylation (p-ERK), but decreased the phosphorylation of p38 (p-p38). The increased p-ERK further phosphorylated the cAMP response element-binding protein (p-CREB) which in turn may have resulted in the increased mitogen activated protein kinase (MAPK) phosphatase 1 (MKP1), known to dephosphorylate MAPKs. Consistent with the decrease in p-p38, the production of pro-inflammatory cytokines TNF-α and IL-1ß was reduced in hypoxic microglia treated with deferoxamine and SB 202190, an inhibitor for p38. This suggests that the anti-inflammatory effect exhibited by deferoxamine is by inhibition of p-p38 induced inflammation through the pERK-pCREB-MKP1 pathway, whereas that of KC7F2 requires further investigation. The present results suggest that HIF-1α may mediate iron accumulation in hypoxic microglia and KC7F2, similar to deferoxamine, might provide limited protection against iron induced PWMD.

Consequences of iron accumulation in microglia and its implications in neuropathological conditions.

Iron is a vital element required by almost all cells for their normal functioning. The well-established role of iron in oxidative metabolism, myelination and synthesis of neurotransmitter makes it an indispensable nutrient required by the brain. Both iron deficiency and excess have been associated with numerous patho-physiologies of the brain, suggesting a need for iron homeostasis. Various studies have reported that the immune effector cells of the brain, the microglial cells, are involved in iron homeostasis in the brain. Microglial cells, which accumulate iron during the developmental period, have a role in myelination process. Along with the increased iron accumulation documented in neurodegenerative diseases, the striking finding is the presence of iron positive microglial cells at the foci of lesion. Though excess iron within activated microglia is demonstrated to enhance the release of pro-inflammatory cytokines and free radicals, a complete understanding of the role of iron in microglia is lacking. The present knowledge on iron mediated changes, in the functions of microglia is summarized in this review.

Roles of activated microglia in hypoxia induced neuroinflammation in the developing brain and the retina.

Amoeboid microglial cells (AMCs) in the developing brain display surface receptors and antigens shared by the monocyte-derived tissue macrophages. Activation of AMCs in the perinatal brain has been associated with periventricular white matter damage in hypoxic-ischemic conditions. The periventricular white matter, where the AMCs preponderate, is selectively vulnerable to hypoxia as manifested by death of premyelinating oligodendrocytes and degeneration of axons leading to neonatal mortality and long-term neurodevelopmental deficits. AMCs respond vigorously to hypoxia by producing excess amounts of inflammatory cytokines e.g. the tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) along with glutamate, nitric oxide (NO) and reactive oxygen species which collectively cause oligodendrocyte death, axonal degeneration as well as disruption of the immature blood brain barrier. A similar phenomenon is observed in the hypoxic developing cerebellum in which activated AMCs induced Purkinje neuronal death through production of TNF-α and IL-1ß via their respective receptors. Hypoxia is also implicated in retinopathy of prematurity in which activation of AMCs has been shown to cause retinal ganglion cell death through production of TNF-α and IL-1ß and NO. Because AMCs play a pivotal role in hypoxic injuries in the developing brain affecting both neurons and oligodendrocytes, a fuller understanding of the underlying molecular mechanisms of microglial activation under such conditions would be desirable for designing of a novel therapeutic strategy for management of hypoxic damage.

Iron and iron regulatory proteins in amoeboid microglial cells are linked to oligodendrocyte death in hypoxic neonatal rat periventricular white matter through production of proinflammatory cytokines and reactive oxygen/nitrogen species.

This study was aimed to examine the role of iron in causing periventricular white matter (PWM) damage following a hypoxic injury in the developing brain. Along with iron, the expression of iron regulatory proteins (IRPs) and transferrin receptor (TfR), which are involved in iron acquisition, was also examined in the PWM by subjecting 1-d-old Wistar rats to hypoxia. Apart from an increase in iron levels in PWM, Perls' iron staining showed an increase of intracellular iron in the preponderant amoeboid microglial cells (AMCs) in the tissue. In response to hypoxia, the protein levels of IRP1, IRP2, and TfR in PWM and AMCs were significantly increased. In primary microglial cultures, administration of iron chelator deferoxamine reduced the generation of iron-induced reactive oxygen and nitrogen species and proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß. Primary oligodendrocytes treated with conditioned medium from hypoxic microglia exhibited reduced glutathione levels, increased lipid peroxidation, upregulated caspase-3 expression, and reduced proliferation. This was reversed to control levels on treatment with conditioned medium from deferoxamine treated hypoxic microglia; also, there was reduction in apoptosis of oligodendrocytes. The present results suggest that excess iron derived primarily from AMCs might be a mediator of oligodendrocyte cell death in PWM following hypoxia in the neonatal brain.