Detecting senescence: a new method for an old pigment.

Cellular senescence is a state of irreversible cell cycle arrest induced by different types of cellular stresses. The field of senescence has made significant advances in the understanding of many of the mechanisms governing this phenomenon; however, a universal biomarker that unambiguously distinguishes senescent from proliferating cells has not been found. In this issue of Aging Cell, Evangelou and colleagues developed a sensitive method for identification of senescent cells in different types of biological material based on the detection of lipofuscin using an analogue of Sudan Black B (SBB) histochemical dye coupled with biotin, which they named GL13. The authors propose that this method is more sensitive and versatile than using SBB alone. Lipofuscin, a nondegradable oxidation product of lipids, proteins and metals, is found in senescent cells. Detection of lipofuscin using GL13 staining may be a more feasible method than others currently used for identification of senescent cells both in cell culture and tissues.

Mitochondrial contribution to lipofuscin formation.

Mitochondria have been in the focus of oxidative stress and aging research for decades due to their permanent production of ROS during the oxidative phosphorylation. The hypothesis exists that mitochondria are involved in the formation of lipofuscin, an autofluorescent protein aggregate that accumulates progressively over time in lysosomes of post-mitotic and senescent cells. To investigate the influence and involvement of mitochondria in lipofuscinogenesis, we analyzed lipofuscin amounts as well as the mitochondrial function in young and senescent cells. In addition we used an aging model and Lon protease deficient HeLa cells to investigate the influence of mitochondrial degradation processes on lipofuscin formation. We were able to show that mitophagy is impaired in senescent cells resulting in an increased mitochondrial mass and superoxide formation. In addition, the inhibition of mitochondrial fission leads to increased lipofuscin formation. Moreover, we observed that Lon protease downregulation is linked to a higher lipofuscinogenesis whereas the application of the mitochondrial-targeted antioxidant mitoTEMPO is able to prevent the accumulation of this protein aggregate.

EGCG Inhibited Lipofuscin Formation Based on Intercepting Amyloidogenic ß-Sheet-Rich Structure Conversion.

BACKGROUND: Lipofuscin (LF) is formed during lipid peroxidation and sugar glycosylation by carbonyl-amino crosslinks with biomacrolecules, and accumulates slowly within postmitotic cells. The environmental pollution, modern dietary culture and lifestyle changes have been found to be the major sources of reactive carbonyl compounds in vivo. Irreversible carbonyl-amino crosslinks induced by carbonyl stress are essentially toxiferous for aging-related functional losses in modern society. Results show that (-)-epigallocatechin gallate (EGCG), the main polyphenol in green tea, can neutralize the carbonyl-amino cross-linking reaction and inhibit LF formation, but the underlying mechanism is unknown. METHODS AND RESULTS: We explored the mechanism of the neutralization process from protein, cell, and animal levels using spectrofluorometry, infrared spectroscopy, conformation antibodies, and electron microscopy. LF demonstrated an amyloidogenic ß-sheet-rich with antiparallel structure, which accelerated the carbonyl-amino crosslinks formation and disrupted proteolysis in both PC12 cells and D-galactose (D-gal)-induced brain aging mice models. Additionally, EGCG effectively inhibited the formation of the amyloidogenic ß-sheet-rich structure of LF, and prevented its conversion into toxic and on-pathway aggregation intermediates, thereby cutting off the carbonyl-amino crosslinks. CONCLUSIONS: Our study indicated that the amyloidogenic ß-sheet structure of LF may be the core driving force for carbonyl-amino crosslinks further formation, which mediates the formation of amyloid fibrils from native state of biomacrolecules. That EGCG exhibits anti-amyloidogenic ß-sheet-rich structure properties to prevent the LF formation represents a novel strategy to impede the development of degenerative processes caused by ageing or stress-induced premature senescence in modern environments.

Histopathology of melanosis coli and determination of its associated genes by comparative analysis of expression microarrays.

Melanosis coli (MC) refers to the condition characterized by abnormal brown or black pigmentation deposits on the colonic mucosa. However, the histopathological findings and genes associated with the pathogenesis of melanosis coli remain to be fully elucidated. The present study aimed to examine the histopathological features and differentially expressed genes of MC. This involved performing hematoxylin and eosin staining, specific staining and immunohistochemistry on tissues sections, which were isolated from patients diagnosed with MC. DNA expression microarray analysis, western blotting and immunofluorescence assays were performed to analyze the differentially expressed genes of melanosis coli. The results demonstrated that the pigment deposits in MC consisted of lipofuscin. A TUNEL assay revealed that a substantial number of apoptotic cells were present within the macrophages and superficial lamina propria of the colonic epithelium. Expression microarray analysis revealed that the significantly downregulated genes were CYP3A4, CYP3A7, UGT2B11 and UGT2B15 in melanosis coli. Western blotting and immunofluorescence assays indicated that the expression of CYP3A4 in the normal tissue was higher than in the MC tissue. The results of the present study provided a comprehensive description of the histopathological characteristics and pathogenesis of MC and for the first time, to the best of our knowledge, demonstrated that the cytochrome P450­associated genes were significantly downregulated in melanosis coli. This novel information can be used to assist in further investigations of melanosis coli.

Pigmented hepatocellular adenomas have a high risk of atypia and malignancy.

Pigment deposition is occasionally seen in hepatocellular adenomas. Several reports suggest that pigmented hepatocellular adenomas have increased risk of malignancy, but these tumors remain incompletely understood. To determine the frequency of pigment deposition, we evaluated and classified 109 well-differentiated hepatocellular neoplasms that were originally diagnosed or submitted in consultation as hepatocellular adenomas and found 27 (25%) pigmented tumors. All were negative on iron stain and in three cases electron microscopy confirmed the pigment was lipofuscin. The lipofuscin intensely stained with glypican-3 in most cases (89%). Of the 27 pigmented tumors, 11 cases (41%) were classified as hepatocellular adenomas, 7 cases (27%) were classified as atypical hepatocellular adenomas/hepatocellular neoplasms of uncertain malignant potential, and 9 cases (33%) were reclassified as well-differentiated hepatocellular carcinomas. Four (of 9) hepatocellular carcinomas arose in pigmented hepatocellular adenomas, giving a rate of malignant transformation in pigmented hepatocellular adenomas of 27%. Of the total 27 pigmented tumors, 78% were in women and 22% in men, but interestingly only men had tumors classified as hepatocellular carcinoma or hepatocellular neoplasm of uncertain malignant potential. Also of note, a total of 10 individuals (37%) had multiple hepatocellular neoplasms but in 9 of these cases the other adenomas were non-pigmented. Importantly, in cases with multiple hepatocellular neoplasms, only the pigmented hepatocellular neoplasms had atypia or malignancy. Genotype-phenotype classification of the pigmented tumors showed different subtypes: HNF1α inactivated (48%), ß-catenin activated (26%), inflammatory (15%), concurrently ß-catenin activated and inflammatory in 1 hepatocellular adenoma, concurrently HNF-1α inactivated and ß-catenin activated in 1 hepatocellular adenoma, and unclassified in 1 hepatocellular carcinoma. In conclusion, hepatocellular adenomas with lipofuscin pigment are a heterogeneous group of adenomas, with HNF-1α inactivation being the commonest genotype. They have an increased risk of atypia and malignancy, especially in males.

Rescue of the Stargardt phenotype in Abca4 knockout mice through inhibition of vitamin A dimerization.

Stargardt disease, an ATP-binding cassette, subfamily A, member 4 (ABCA4)-related retinopathy, is a genetic condition characterized by the accelerated accumulation of lipofuscin in the retinal pigment epithelium, degeneration of the neuroretina, and loss of vision. No approved treatment exists. Here, using a murine model of Stargardt disease, we show that the propensity of vitamin A to dimerize is responsible for triggering the formation of the majority of lipofuscin and transcriptional dysregulation of genes associated with inflammation. Data further demonstrate that replacing vitamin A with vitamin A deuterated at the carbon 20 position (C20-D3-vitamin A) impedes the dimerization rate of vitamin A--by approximately fivefold for the vitamin A dimer A2E--and subsequent lipofuscinogenesis and normalizes the aberrant transcription of complement genes without impairing retinal function. Phenotypic rescue by C20-D3-vitamin A was also observed noninvasively by quantitative autofluorescence, an imaging technique used clinically, in as little as 3 months after the initiation of treatment, whereas upon interruption of treatment, the age-related increase in autofluorescence resumed. Data suggest that C20-D3-vitamin A is a clinically amiable tool to inhibit vitamin A dimerization, which can be used to determine whether slowing the dimerization of vitamin A can prevent vision loss caused by Stargardt disease and other retinopathies associated with the accumulation of lipofuscin in the retina.

Structures and biogenetic analysis of lipofuscin bis-retinoids.

Age-related macular degeneration (AMD) is still an incurable blinding eye disease because of complex pathogenic mechanisms and unusual diseased regions. With the use of chemical biology tools, great progress has been achieved in improving the understanding of AMD pathogenesis. The severity of AMD is, at least in part, linked to the non-degradable lipofuscin bis-retinoids in retinal pigment epithelial (RPE). This material is thought to result from the lifelong accumulation of lysosomal residual bodies containing the end products derived from the daily phagocytosis of rod outer segments by RPE cells. Here, we present previously recognized bis-retinoids with focus on structures and biosynthetic pathways. In addition to a brief discussion on the mutual conversion relationships of bis-retinoids, future perspectives and the medical relevance of such studies on these lipofuscin constituents are also highlighted.

Lipofuscin: formation, effects and role of macroautophagy.

Oxidative stress plays a crucial role in the development of the aging process and age dependent diseases. Both are closely connected to disturbances of proteostasis by protein oxidation and an impairment of the proteasomal system. The final consequence is the accumulation of highly cross-linked undegradable aggregates such as lipofuscin. These aggregates of damaged proteins are detrimental to normal cell functions. Here we provide an overview about effect of these aggregates on the proteasomal system, followed by transcription factor activation and loss of cell viability. Furthermore, findings on the mechanism of radical genesis, proteasomal inhibition and the required components of lipofuscin formation were resumed.

Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium.

PURPOSE: The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE. METHODS: Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on flat-mounted RPE tissue sections and retinal cross-sections. RESULTS: Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased toward the central region. High-resolution MALDI-IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels. CONCLUSIONS: This report to our knowledge is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging.

Natto (fermented soybean) extract extends the adult lifespan of Caenorhabditis elegans.

We investigated the effects of a water extract of natto on the aging of the nematode Caenorhabditis elegans. The water extract significantly prolonged the adult lifespan of the wild-type worms and rendered them resistant to oxidative and thermal stress. In addition, treatment with natto extract significantly delayed the accumulation of lipofuscin, a characteristic of aging cells. Our findings suggest that components of natto have a beneficial anti-aging effect in vivo.

Lipofuscin accumulation and gene expression in different tissues of mnd mice.

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage diseases characterized by neurological impairment and blindness. NCLs are almost always due to single mutations in different genes (CLN1-CLN8). Ubiquitous accumulation of undigested material and of a hydrophobic inner mitochondrial membrane protein, the subunit c of mitochondrial ATP synthase, has been described. Although protein mutation(s) in the endoplasmic reticulum-lysosomes axis can modify the trafficking and the recycling of different molecules, one of the upstream targets in these diseases may be represented by the balance of gene expression. To understand if and how neurons modify the levels of important genes during the first phases of the disease, it is important to characterize the mechanisms of neurodegeneration. Due to the impossibility of performing this analysis in humans, alternative models of investigation are required. In this study, a mouse model of human NCL8, the mnd mouse has been employed. The mnd mice recapitulate many clinical and histopathological features described in NCL8 patients. In this study, we found an altered expression of different genes in both central and peripheral organs associated with lipopigment accumulation. This is a preliminary approach, which could also be of interest in providing new diagnostic tools for NCLs.

Analysis of aging in Caenorhabditis elegans.

This chapter is dedicated to the study of aging in Caenorhabditis elegans (C. elegans). The assays are divided into two sections. In the first section, we describe detailed protocols for performing life span analysis in solid and liquid medium. In the second section, we describe various assays for measuring age-related changes. Our laboratory has been involved in several fruitful collaborations with non-C. elegans researchers keen on testing a role for their favorite gene in modulating aging (Carrano et al., 2009; Dong et al., 2007; Raices et al., 2008; Wolff et al., 2006). But even with the guidance of trained worm biologists, this undertaking can be daunting. We hope that this chapter will serve as a worthy compendium for those researchers who may or may not have immediate access to laboratories studying C. elegans.

The bisretinoids of retinal pigment epithelium.

The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus autofluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-up of many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoid fluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscin found in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation is attributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occurs upon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even in healthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells. Nevertheless, the formation of this material is accelerated in some retinal disorders including recessive Stargardt disease and ELOVL4-related retinal degeneration. As such, these bisretinoid side-products are implicated in the disease processes that threaten vision. In this article, we review our current understanding of the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their related spectroscopic features and the biosynthetic pathways by which they form. We will revisit factors known to influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoid formation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it is not surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discuss recent findings that implicate the photodegradation of bisretinoid in the etiology of age-related macular degeneration.

Calcium overload is associated with lipofuscin formation in human retinal pigment epithelial cells fed with photoreceptor outer segments.

PURPOSE: To investigate the role of Ca²(+) in lipofuscin formation in human retinal pigment epithelial (RPE) cells that phagocytize bovine photoreceptor outer segments (POSs). METHODS: Cultured human RPE cells fed with 2 × 107per l bovine POS were treated with flunarizine, an antagonist of Ca²(+) channel, or/and centrophenoxine, a lipofuscin scavenger. The Ca²(+) changes and lipofuscin formation were measured with fluoresence dye Fluo-3/AM ester, laser scanning confocal microscopy (LSCM) and flow cytometry (FCM). The activity of RPE cells was measured by methyl thiazolyl tetrazolium (MTT) assay and argyrophilic nucleolar organizer regions (AgNORs) assay. RESULTS: The Ca²(+) fluorescence intensity (CFI) of RPE cells fed with POS was significantly increased compared with the controls (165.36 ± 29.92 U). It reached a peak with 777.33 ± 63.86 U (P<0.01) at 12 h, and then decreased but still maintained a high level of 316.90 ± 36.07 U (P<0.01) for 4 days. Flunarizine and centrophenoxine significantly decreased the Ca²(+) overload to 227.18 ± 14.00 U at 12 h and 211.06 ± 20.45 U at 4 days. FCM confirmed these changes. The drugs also showed an inhibitory effect on the lipofuscin formation. The proliferation rate of the cells fed with POS increased significantly. Both drugs had inhibitory effects on the activity of the cultured cells. This tendency was confirmed by AgNORs assay. CONCLUSIONS: The Ca²(+) inflow initiated lipofuscin accumulation in RPE cells fed with POS. Flunarizine and centrophenoxine can decrease Ca²(+) overload and lipofuscin formation in RPE cells, accompanied by maintaining cellular vitality.

C20-D3-vitamin A slows lipofuscin accumulation and electrophysiological retinal degeneration in a mouse model of Stargardt disease.

Stargardt disease, also known as juvenile macular degeneration, occurs in approximately one in 10,000 people and results from genetic defects in the ABCA4 gene. The disease is characterized by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE) of the eye and by vision loss. No cure or treatment is available. Although lipofuscin is considered a hallmark of Stargardt disease, its mechanism of formation and its role in disease pathogenesis are poorly understood. In this work we investigated the effects of long-term administration of deuterium-enriched vitamin A, C20-D(3)-vitamin A, on RPE lipofuscin deposition and eye function in a mouse model of Stargardt's disease. Results support the notion that lipofuscin forms partly as a result of the aberrant reactivity of vitamin A through the formation of vitamin A dimers, provide evidence that preventing vitamin A dimerization may slow disease related, retinal physiological changes and perhaps vision loss and suggest that administration of C20-D(3)-vitamin A may be a potential clinical strategy to ameliorate clinical symptoms resulting from ABCA4 genetic defects.

Deuterium enrichment of vitamin A at the C20 position slows the formation of detrimental vitamin A dimers in wild-type rodents.

Degenerative eye diseases are the most common causes of untreatable blindness. Accumulation of lipofuscin (granular deposits) in the retinal pigment epithelium (RPE) is a hallmark of major degenerative eye diseases such as Stargardt disease, Best disease, and age-related macular degeneration. The intrinsic reactivity of vitamin A leads to its dimerization and to the formation of pigments such as A2E, and is believed to play a key role in the formation of ocular lipofuscin. We sought a clinically pragmatic method to slow vitamin A dimerization as a means to elucidate the pathogenesis of macular degenerations and to develop a therapeutic intervention. We prepared vitamin A enriched with the stable isotope deuterium at carbon twenty (C20-D(3)-vitamin A). Results showed that dimerization of deuterium-enriched vitamin A was considerably slower than that of vitamin A at natural abundance as measured in vitro. Administration of C20-D(3)-vitamin A to wild-type rodents with no obvious genetic defects in vitamin A processing, slowed A2E biosynthesis. This study elucidates the mechanism of A2E biosynthesis and suggests that administration of C20-D(3)-vitamin A may be a viable, long-term approach to retard vitamin A dimerization and by extension, may slow lipofuscin deposition and the progression of common degenerative eye diseases.

Transmission of light to the young primate retina: possible implications for the formation of lipofuscin.

The purpose of this study was to determine the transmission properties of the anterior segment of young primate eyes and potentially relate those changes to photochemical processes in the retina that lead to the early, rapid formation of lipofuscin. A simple method has been developed to determine the optical properties of the anterior segment of the intact eye. Using this technique, the transmission/absorption properties of primate cadaver eyes were determined. A young primate anterior segment has a maximum absorption at 365nm due to the presence of the O-ß-glucoside of 3-hydroxykynurenine in the lens. This is synthesized in the last trimester of gestation. Although this compound filters out most of the UV light from reaching the retina, there is a small window of transmission centered on an absorption minimum at 320nm. This closes by the second decade of life. The window of transmission of UV light to the primate retina may explain the initial accelerated formation of lipofuscin in the young human retina by a photochemical process. This would be exacerbated by any decrease in the ozone layer with concomitant increase in UV-B reaching the earth's surface.

Neuroprotective effect of hydroalcoholic extract of dried fruits of Trapa bispinosa Roxb on lipofuscinogenesis and fluorescence product in brain of D-galactose induced ageing accelerated mice.

Effect of hydroalcoholic extract T. bispinosa (TB) was studied on fluorescence product and biochemical parameter like lipid peroxidation, catalase activity and glutathione peroxidase activity in the brain of female albino mice. Ageing was accelerated by the treatment of 0.5 ml 5% D-galactose for 15 days. This resulted in increased fluorescence product, increase lipid peroxidation and decrease antioxidant enzyme like glutathione peroxides and catalase in cerebral cortex. After cotreatment with hydroalcoholic extract of TB (500 mg/kg, po) there was decrease in fluorescence product in cerebral cortex. Moreover, TB inhibited increase lipid peroxidation and restores glutathione peroxidase and catalase activity in cerebral cortex as compare to ageing accelerated control group. To conclude TB found to be effective antioxidative agent which could to some extent reverse D-galactose induced ageing changes resulted due to oxidative damage.

Experimental approaches to the study of A2E, a bisretinoid lipofuscin chromophore of retinal pigment epithelium.

Bisretinoid lipofuscin compounds that accumulate in retinal pigment epithelial (RPE) cells are implicated in the pathogenesis of some forms of macular degeneration. In the development of approaches to the amelioration of retinal disorders characterized by enhanced RPE lipofuscin formation, attention is being given to therapies that reduce the production of these damaging pigments. An understanding of the biosynthetic pathways by which these molecules form is essential to the development of these therapies. Thus methods for studying the biosynthesis of these compounds are presented. A tissue culture model is also described whereby a human RPE cell line that is otherwise devoid of bisretinoid lipofuscin compounds is employed and synthesized A2E is delivered to the cells. This approach allows for a population of RPE cells that have accumulated the lipofuscin fluorophore A2E in addition to A2E-free cells.