Lipofuscin Granule Bisretinoid Oxidation in the Human Retinal Pigment Epithelium forms Cytotoxic Carbonyls

Age-related macular degeneration (AMD) is the primary cause of central blindness among the elderly. AMD is associated with progressive accumulation of lipofuscin granules in retinal pigment epithelium (RPE) cells. Lipofuscin contains bisretinoid fluorophores, which are photosensitizers and are phototoxic to RPE and neuroretinal cells. In the presence of oxygen, bisretinoids are also oxidized, forming various products, consisting primarily of aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that in AMD, bisretinoid oxidation products are increased in RPE lipofuscin granules. The purpose of the present study was to determine if these products were toxic to cellular structures. The physicochemical characteristics of bisretinoid oxidation products in lipofuscin, which were obtained from healthy donor eyes, were studied. Raman spectroscopy and time-of-flight secondary ion mass spectrometry (ToF–SIMS) analysis identified the presence of free-state aldehydes and ketones within the lipofuscin granules. Together, fluorescence spectroscopy, high-performance liquid chromatography, and mass spectrometry revealed that bisretinoid oxidation products have both hydrophilic and amphiphilic properties, allowing their diffusion through lipofuscin granule membrane into the RPE cell cytoplasm. These products contain cytotoxic carbonyls, which can modify cellular proteins and lipids. Therefore, bisretinoid oxidation products are a likely aggravating factor in the pathogenesis of AMD.

A2E Distribution in RPE Granules in Human Eyes

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

Examining Cardiomyocyte Dysfunction Using Acute Chemical Induction of an Ageing Phenotype

Much effort is focussed on understanding the structural and functional changes in the heart that underlie age-dependent deterioration of cardiac performance. Longitudinal studies, using aged animals, have pinpointed changes occurring to the contractile myocytes within the heart. However, whilst longitudinal studies are important, other experimental approaches are being advanced that can recapitulate the phenotypic changes seen during ageing. This study investigated the induction of an ageing cardiomyocyte phenotypic change by incubation of cells with hydroxyurea for several days ex vivo. Hydroxyurea incubation has been demonstrated to phenocopy age- and senescence-induced changes in neurons, but its utility for ageing studies with cardiac cells has not been examined. Incubation of neonatal rat ventricular myocytes with hydroxyurea for up to 7 days replicated specific aspects of cardiac ageing including reduced systolic calcium responses, increased alternans and a lesser ability of the cells to follow electrical pacing. Additional functional and structural changes were observed within the myocytes that pointed to ageing-like remodelling, including lipofuscin granule accumulation, reduced mitochondrial membrane potential, increased production of reactive oxygen species, and altered ultrastructure, such as mitochondria with disrupted cristae and disorganised myofibres. These data highlight the utility of alternative approaches for exploring cellular ageing whilst avoiding the costs and co-morbid factors that can affect longitudinal studies.

Age-Related Changes in the Chorioretinal Junction: An Immunohistochemical Study

The chorioretinal junction comprises the retinal pigment epithelium, Bruch’s membrane (BM), and adjacent choroidal capillaries. Its significance lies in its ability to support the retina mechanically and metabolically. The aim of this cross-sectional study was to record the senescent changes affecting all the constituents of the chorioretinal junction in 40 histological specimens across the whole spectrum of the adult age range. This study included light microscopy, with hematoxylin and eosin and PAS stains, and fluorescent microscopy. Immunohistochemistry was done using antibodies against neurofilament, synaptophysin, S-100, and collagen IV. The descriptive microanatomy was corroborated by morphometry. The amount of melanin and lipofuscin granule and drusens were noted. The ratio of thickness of BM to capillary diameter reduced from 1:6 or less in the 2nd decade to 1:3 in the 10th decade. Complete hyalinization of intercapillary pillars was seen in the 10th decade. The accumulation of lipofuscin with age was documented with the diminution in the size of epithelial cells. The subepithelial accumulation of drusen was first noted in the specimen from the late 60s. We have described all senescent changes in the chorioretinal junction chronologically. Similar changes are found in a more pronounced form in age-related macular degeneration. These data might serve as a reference baseline for clinicians and pathologists.

Lipofuscin Quantification as a Potential Tool for Age Estimation in Snow Crabs,Chionoecetes opilio (O. Fabricius, 1788) (Decapoda, Oregoniidae)

Lipofuscin levels were investigated in snow crabs (Chionoecetes opilio) to assess the applicability of lipofuscin quantification as an age determining tool for this species. Localization and quantification of lipofuscin granules in the olfactory lobe cell mass (OLCM) of the brain were conducted by confocal microscopy and image analysis. The majority of immature crabs had no detectable lipofuscin. Crabs with a dirty carapace condition (an indication of greater age) had more lipofuscin (in terms of granule density, area fraction, and mean granule size) than crabs with a clean carapace condition; however, the amount of lipofuscin was much lower than that in more temperate species. Lipofuscin content was positively correlated with carapace size among crabs with a clean carapace but not among crabs with a dirty carapace. No correlation was observed between average lipofuscin granule fluorescent intensity and carapace width. OLCM neuron characteristics were also investigated. An inverse relationship was observed between neuron density and carapace width although it is unknown if this density reduction is due to a loss of neurons, an increase in size of the structures, or both. Furthermore, a positive relationship was observed between neuron size and carapace width. The range of neuron size also increased in larger crabs, suggesting the presence of mitotically active cells in the OLCM. Although lipofuscin levels were higher in dirty carapace condition (presumably older) crabs than in clean carapace crabs and increased with size (in mature, clean carapace snow crabs), the lack of detectable lipofuscin in most immature crabs, generally low levels of lipofuscin in mature crabs, and lack of increase with size in older (dirty carapace) crabs limits the applicability of this age estimation tool for this species.

Preliminary identification and quantification of the age-pigment lipofuscin in the brain of Farfantepenaeus paulensis (Crustacea: Decapoda)

A preliminary study was done on the age-pigment lipofuscin content in the brains of captive Farfantepenaeus paulensis juveniles (5 months old) and wild adults (estimated age of 12-15 months). Random samples of 6 individuals were obtained from each group (juvenile and adult) for histological analysis. Serial sections (6 mum) of the brains were mounted without staining and observed in an epifluorescent microscope. The fluorescent images of the five most central sections of the olfactory lobe cell mass (OLCM) of each individual were digitized for image analysis. The lipofuscin granule mean diameter was similar in both groups (p > 0.05), however the lipofuscin area fraction (percentage of the OLCM occupied by pigment granules) was significantly higher (p < 0.05) in the adult shrimp. The detection of lipofuscin granules in 5 month old F. paulensis indicates that lipofuscin deposition probably takes place even earlier in the juvenile phase. Our results suggested that the amount of granules in the F. paulensis OLCM is related to age, but further studies are necessary to evaluate the relationship between lipofuscin content and the age of captive F. paulensis.

The intragranular location of carboxyl groups in neuromelanin and lipofuscin in human brain and in meningeal melanosomes in mouse brain.

The intragranular location of carboxyl groups was tinctorially determined in human substantia nigra neuromelanin granules, human inferior olive lipofuscin granules, and mouse meningeal melanosomes. Soluble and insoluble lipid was stained with beta naphthol Sudans in unoxidized and oxidized frozen and paraffin sections containing neuromelanin or lipofuscin. Nile blue demonstrated carboxyls in unoxidized neuromelanin, lipofuscin, and melanin, and in oxidized neuromelanin and lipofuscin. Carbodiimide demonstrated carboxyls in unoxidized and oxidized lipofuscin and oxidized neuromelanin. In all instances, staining for carboxyls was inhibited by prior mild methylation, and proof of their presence was obtained by a pre-staining, stepwise, alternating, and repetitive mild demethylation, mild methylation sequence. Structurally, carboxyls were demonstrated in the neuromelanin granule's soluble lipid-free lipofuscin component, in the meningeal melanosome's melanin component, and virtually throughout the lipofuscin granule. The following structural and chemical basis was proposed for the different resistance of Nile blue staining of melanosomes and of neuromelanin and lipofuscin to acetone extraction. Nile blue forms an insoluble complex with melanosomal dopa-melanin's quinonoid, diphenolic, and undissociated carboxyl units. Such complex formation does not occur in neuromelanin's carboxyl-free dopamine-melanin component, however. Instead, Nile blue ionogenicly bonds with dissociated carboxyls belonging to the neuromelanin granule's lipofuscin component.