■ Abstract Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process. The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among the death proteases. Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage via upstream proteases in an intracellular cascade. Regulation of caspase activation and activity occurs at several different levels: ( a) Zymogen gene transcription is regulated; ( b) antiapoptotic members of the Bcl-2 family and other cellular polypeptides block proximity-induced activation of certain procaspases; and ( c) certain cellular inhibitor of apoptosis proteins (cIAPs) can bind to and inhibit active caspases. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases. Collectively, these scissions disrupt survival pathways and disassemble important architectural components of the cell, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death.

Mammalian caspases: structure ,a ctivation ,s ubstrates, and functions during apoptosis

Autophagy is an important intracellular degradative process that delivers cytoplasmic proteins to lysosome for degradation. Dysfunction of autophagy is implicated in several human diseases, such as neurodegenerative diseases, infectious diseases, and cancers. Autophagy-related proteins are constitutively expressed in the eye. Increasing studies have revealed that abnormal autophagy is an important pathological feature of several ocular diseases. Pharmacological manipulation of autophagy may provide an alternative therapeutic target for some ocular diseases. In this manuscript, we reviewed the relevant progress about the role of autophagy in the pathogenesis of ocular diseases.

/pdf/repertoires-of-autophagy-in-the-pathogenesis-of-ocular-2lklvj9lx2.pdf

Repertoires of Autophagy in the Pathogenesis of Ocular Diseases

Located between vessels of the choriocapillaris and light-sensitive outer segments of the photoreceptors, the retinal pigment epithelium (RPE) closely interacts with photoreceptors in the maintenance of visual function. Increasing knowledge of the multiple functions performed by the RPE improved the understanding of many diseases leading to blindness. This review summarizes the current knowledge of RPE functions and describes how failure of these functions causes loss of visual function. Mutations in genes that are expressed in the RPE can lead to photoreceptor degeneration. On the other hand, mutations in genes expressed in photoreceptors can lead to degenerations of the RPE. Thus both tissues can be regarded as a functional unit where both interacting partners depend on each other.

The Retinal Pigment Epithelium in Visual Function

When a molecule absorbs a photon of appropriate energy, a chain of photophysical events ensues, such as internal conversion or vibrational relaxation (loss of energy in the absence of light emission), fluorescence, intersystem crossing (from singlet state to a triplet state) and phosphorescence, as shown in the Jablonski diagram for organic molecules (Fig. 1). Each of the processes occurs with a certain probability, characterized by decay rate constants (k). It can be shown that the average length of time τ for the set of molecules to decay from one state to another is reciprocally proportional to the rate of decay: τ = 1/k. This average length of time is called the mean lifetime, or simply lifetime. It can also be shown that the lifetime of a photophysical process is the time required by a population of N electronically excited molecules to be reduced by a factor of e. Correspondingly, the fluorescence lifetime is the time required by a population of excited fluorophores to decrease exponentially to N/e via the loss of energy through fluorescence and other non-radiative processes. The lifetime of photophycal processes vary significantly from tens of femotoseconds for internal conversion1,2 to nanoseconds for fluorescence and microseconds or seconds for phosphorescence.1






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Figure 1


Jablonski diagram and a timescale of photophysical processes for organic molecules.

/pdf/fluorescence-lifetime-measurements-and-biological-imaging-xfalzh4w7e.pdf

Fluorescence Lifetime Measurements and Biological Imaging

The culmination of apoptosis in vivo is phagocytosis of cellular corpses. During apoptosis, the asymmetry of plasma membrane phospholipids is lost, which exposes phosphatidylserine externally1,2,3,4. The phagocytosis of apoptotic cells can be inhibited stereospecifically by phosphatidylserine and its structural analogues, but not by other anionic phospholipids, suggesting that phosphatidylserine is specifically recognized1,5,6,7,8,9,10. Using phage display, we have cloned a gene that appears to recognize phosphatidylserine on apoptotic cells. Here we show that this gene, when transfected into B and T lymphocytes, enables them to recognize and engulf apoptotic cells in a phosphatidylserine-specific manner. Flow cytometric analysis using a monoclonal antibody suggested that the protein is expressed on the surface of macrophages, fibroblasts and epithelial cells; this antibody, like phosphatidylserine liposomes, inhibited the phagocytosis of apoptotic cells and, in macrophages, induced an anti-inflammatory state. This candidate phosphatidylserine receptor is highly homologous to genes of unknown function in Caenorhabditis elegans and Drosophila melanogaster, suggesting that phosphatidylserine recognition on apoptotic cells during their removal by phagocytes is highly conserved throughout phylogeny.

A receptor for phosphatidylserine-specific clearance of apoptotic cells

Purpose To determine whether the lipofuscin fluorophore A2E participates in blue light-induced damage to retinal pigmented epithelial (RPE) cells. Methods Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 microM concentrations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as assayed by quantitative high-performance liquid chromatography (HPLC). Restricted zones (0.5-mm diameter spots) of confluent cultures were subsequently exposed to 480 +/- 20-nm (blue) or 545 +/- 1-nm (green) light for 15 to 60 seconds. Phototoxicity was quantified at various periods after exposure by fluorescence staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure. Results Nonviable cells were located in blue light- exposed zones of A2E-containing RPE cells, whereas cells situated outside the illuminated areas remained viable. As shown by fluorescence labeling of the nuclei of membrane-damaged cells and by the presence of TUNEL-positive cells, the numbers of nonviable cells increased with exposure duration and as a function of the concentration of A2E used to load the cells before illumination. The numbers of blue light-induced TUNEL-positive cells also increased in advance of the increase in labeling of membrane-compromised cells, a finding that, together with Annexin V labeling, indicates an apoptotic form of cell death. Conversely, blue light- exposed RPE cells that did not contain A2E remained viable. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells. Conclusions These studies implicate A2E as an initiator of blue light-induced apoptosis of RPE cells.

The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.

RPE lipofuscin and its role in retinal pathobiology.

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.

https://www.pnas.org/content/pnas/95/25/14609.full.pdf

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

Recent studies have implicated local inflammation and activation of complement amongst the processes involved in the pathogenesis of age-related macular degeneration (AMD). Several lines of investigation also indicate that bis-retinoid pigments, such as A2E, that accumulate as lipofuscin in retinal pigment epithelial (RPE) cells, contribute to the disease process. In an investigation of a potential trigger for complement activation in AMD, we explored the notion that the complex mixture of products resulting from photooxidation of A2E might include a range of fragments that could be recognized by the complement system as “foreign” and that could serve to activate the complement system, leading to low-grade inflammation. To this end, we established an in vitro assay by using human serum as a source of complement, and we measured products of C3 activation by enzyme immunoassay. Accordingly, we found that the C3 split products inactivated C3b (iC3b) and C3a were elevated in serum, overlying ARPE-19 cells that had accumulated A2E and were irradiated to induce A2E photooxidation. Precoating of microtiter plates with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum, also activated complement. We suggest that products of the photooxidation of bis-retinoid lipofuscin pigments in RPE cells could serve as a trigger for the complement system, a trigger than would predispose the macula to disease and that, over time, could contribute to chronic inflammation. These findings link four factors that have been posited as being associated with AMD: inflammation, oxidative damage, drusen, and RPE lipofuscin.

https://www.pnas.org/content/pnas/103/44/16182.full.pdf

Janet R. Sparrow

Papers

The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.

RPE lipofuscin and its role in retinal pathobiology.

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

Complement activation by photooxidation products of A2E, a lipofuscin constituent of the retinal pigment epithelium

The bisretinoids of retinal pigment epithelium.