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.

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.

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.

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.

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.

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.

Fat redistribution and adipocyte transformation in uninephrectomized rats.

Dyslipidemia complicates renal function leading to disturbances of major homeostatic organs in the body. Here we examined the effect of chronic renal dysfunction induced by uninephrectomy on fat redistribution and lipid peroxidation in rats treated with an angiotensin-converting enzyme (ACE) inhibitor (lisinopril) for up to 10 months. Uninephrectomized rats developed fat redistribution and hypercholesterolemia typical of chronic renal failure when compared with sham-operated rats or lisinopril-treated uninephrectomized rats. The weight of the peri-renal fat was significantly less in the untreated compared to the lisinopril-treated uninephrectomized rats or those rats with a sham operation. We also found that there was a shift of heat-protecting unilocular adipocytes to heat-producing multilocular fat cells in the untreated uninephrectomized rats. Similarly in these rats we found a shift of subcutaneous and visceral fat to ectopic fat with excessive lipid accumulation and lipofuscin pigmentation. Lisinopril treatment prevented fat redistribution or transformation and lipid peroxidation. This study shows that ACE inhibition may prevent the fat anomalies associated with chronic renal dysfunction.

Hypercapnia protects erythrocytes against free radical damage induced by hypoxia in exposed rats.

Several studies report that hypoxic exposure induces free radical oxidative damage in various tissues. The mechanism of this damage includes membrane lipid peroxidation which can be easily detected by measuring fluorescent end-products of the process, i.e. lipofuscin-like pigments. Four day exposure of rats to hypoxia (10% O(2)) increased the level of lipofuscin-like pigments in erythrocytes up to 9 fold. This increase was completely prevented when the animals were exposed to hypercapnia (4.3% CO(2)) in addition to hypoxia. We studied the possible mechanism of the hypercapnic protection on isolated erythrocyte membranes in vitro. Lipid peroxidation was initiated by incubation of the membranes with iron ions and ascorbate. Production of malonaldehyde, the precursor of lipofuscin-like pigments, was strongly inhibited in bicarbonate buffer. Similarly the production of lipofuscin-like products was damped. These experiments suggest that the protective effect of hypercapnia might consist in direct interaction of CO(2) with free radical processes.

A comparative study of artificial ceroid/lipofuscin from different tissue materials of rats.

The artificial ceroid/lipofuscin pigments originated from different organ tissues, including liver, brain, heart, and kidney of rats, and biomaterials were studied with improved fluorometric techniques. With all tissue materials exposed under ultraviolet (UV) light, a series of similar fluorescent colors were observed under microfluorometer. Analogous fluorescence spectra were also demonstrated with a three-dimensional (3-D) front-surface fluorometric technique despite of the tissue differences. Measured with 3-D fluorometry, relatively simple lipofuscin-like fluorophores were observed from the reactions of malondialdehyde (MDA) with critical biological macromolecules, such as bovine serum albumin (BSA) and DNA. Our results demonstrated that the biomaterials from different tissues have a similar fate under accelerated oxidative/carbonyl stresses but may be differentiated by a fluorescence intensity ratio.

Involvement of the tyrosinase gene in the deposition of cardiac lipofuscin in mice. Association with aortic fatty streak development.

Lipofuscin pigment, a terminal oxidation product, accumulates within cells during the normal aging process and under certain pathological conditions. We have analyzed a genetic cross between two inbred mouse strains, BALB/cJ and a subline of C57BL/6J, which differ in lipofuscin deposition. A comparison of the segregation pattern of cardiac lipofuscin with the albino locus (c) on mouse chromosome 7 revealed complete concordance. Analysis of spontaneous mutants of the tyrosinase gene, encoded by the albino locus, confirmed that the tyrosinase gene itself controls lipofuscin formation. Genetic analysis of other strains indicated that one or more additional genes cab contribute to the inheritance of lipofuscin. We also present evidence for an association between cardiac lipofuscin deposition and aortic fatty streak development in the mouse.

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.

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.

Lipofuscin-like fluorophores originated from malondialdehyde.

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of post-mitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. In the present mini-review, I provide a comprehensive summary of fluorescent adducts originated from malondialdehyde (MDA).

[Effect of peroxisome proliferator-activated receptor-alpha agonist on adipokines expression in rats fed with high-fat diet].

OBJECTIVE: To explore the effect of peroxisome proliferator-activated receptor-alpha ( PPAR-alpha) agonist fenofibrate on adipokines expression in high-fat diet fed SD rats and its relationship to insulin resistance (IR). METHODS: Rats were randomized into three groups (n = 10) : HD group, fed with high-fat diet; HDF group, fed with high fat diet and treated with fenofibrate; and control group, fed with normal diet. Animals were sacrificed after 4-week follow-up. Plasma lipids, fasting plasma insulin, free fatty acids (FFA), and insulin sensitivity were detected. Reverse transcription-polymerase chain reaction was used to semi-quantitatively determine the mRNA expression of adipokines including tumor necrosis factor-alpha (TNF-alpha) , interleukin-6 (IL-6), angiotensinogen (AGT), angiotensin 11 type 1 receptor (AT1R), and adiponectin in brown fat. RESULTS: The plasma level of FFA, TG, and homeostatic model approach-IR index were (2. 37+/-0. 60) vs (1. 59+/-0. 30) vs (1. 33+/-0. 34 ) mmol/L, (0. 48+/-0. 11) vs (0. 30+/-0. 04) vs (0. 36+/-0. 07) mmol/L, and 12. 30+/-3. 97 vs 5. 03 +/-1. 88 vs 4. 17+/-1. 27 in the HD group, HDF group, and control group after 4 weeks of treatment with fenofibrate, respectively. The mRNA expressions of TNF-alpha and adiponectin were 1. 726+/-1. 408 vs 0. 713+/-0. 711 vs 0. 593+/-0. 382 and 0. 660+/-0. 192 vs 0. 949+/-0. 35 vs 0. 936+/-0. 130 in these three groups, which showed significant difference between HD group and HDF group (P < 0. 05 ) , while no significant difference between HDF group and control group (P > 0. 05). The mRNA expressions of AGT, AT1 R, and IL-6 had no significant difference among these three groups (P > 0. 05 ). CONCLUSION: PPAR-alpha agonist fenofibrate may reverse high-fat diet induced lipid abnormalities, improve insulin sensitivity, and regulate the mRNA expressions of TNF-alpha and adiponectin in adipose tissues.

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.

Lysosomal dysfunction in muscle with special reference to glycogen storage disease type II.

The importance of proper lysosomal activity in cell and tissue homeostasis is underlined by "experiments of nature", i.e. genetic defects in one of the at least 40 lysosomal enzymes/proteins present in the human cell. The complete lack of 1-4 alpha-glucosidase (glycogen storage disease type II (GSD II) or Pompe disease) is life-threatening. Patients suffering from GSD II commonly die before the age of 2 years because of cardiorespiratory insufficiency. Striated muscle cells appear to be particularly vulnerable in GSD II. The high cytoplasmic glycogen content in muscle cells most likely gives rise to a high rate of glycogen engulfment by the lysosomes. The polysaccharides become subsequently trapped in these organelles when 1-4 alpha-glucosidase activity is absent. During the course of the disease, muscle wasting occurs. It is hypothesised that the gradual loss of muscle mass is caused by a combination of disuse atrophy and lipofuscine-mediated apoptosis of myocytes. Moreover, we hypothesise that in the remaining skeletal muscle cells, longitudinal transmission of force is hampered by swollen lysosomes, clustering of non-contractile material and focal regions with degraded contractile proteins, which results in muscle weakness.

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.

Lipofuscin.

Over time, postmitotic cells accumulate a non-degradable intralysosomal substance, lipofuscin, which forms due to iron-catalyzed oxidation/polymerization of protein and lipid residues. Lipofuscin is often considered a hallmark of aging, showing an accumulation rate that inversely correlates with longevity. There is an emerging impression that lipofuscin, although still typically considered a harmless wear-and-tear product, may have multiple negative effects. By interfering with the important autophagic process, by which most worn out cellular components are degraded, it may prevent cellular renewal and advance the accumulation of damaged cellular constituents. Due to binding of transition metals, such as iron and copper, lipofuscin also seems to sensitize lysosomes and cells to oxidative stress. Of importance for the pathogenesis of age-related macular degeneration, lipofuscin deposition interferes with the phagocytic activity of retinal pigment epithelial cells and also sensitizes their lysosomes to blue light.

The autofluorescent products of lipid peroxidation may not be lipofuscin-like.

The fluorescent molecules of cellular age pigment granules (lipofuscin) are commonly thought to be end products of membrane lipid autoxidation. Lipofuscin fluorophores of the retinal pigment epithelium (RPE) appear to be derived from photoreceptor outer segment membranes. Experiments were therefore conducted to determine whether the in vitro oxidation of retinal homogenates would generate fluorophores similar to the naturally occurring lipofuscin fluorophores of the RPE. Neural retina and RPE-choroid homogenates from young (2-3 month old) albino rats were subjected to an iron-ascorbate-air pro-oxidant reaction medium, and compared to unoxidized control samples from young age-matched animals as well as senescent (24 month old) rats. In addition, neural retina and RPE-choroid homogenates from 3 month old albino rats were subjected to a 100% oxygen atmosphere to test whether the fluorescent products of autoxidation differ substantially from those generated in the pro-oxidant medium. The chloroform-soluble fluorophores of chloroform-methanol sample extracts were analyzed by corrected fluorescence spectroscopy and thin-layer chromatography (TLC). In vitro pro-oxidation of both the neural retina and the RPE from young rats produced blue-emitting fluorophores which differed from the orange- and yellow-emitting fluorophores extracted from the RPE of senescent rats. Corrected fluorescence spectroscopy of aged tissue extracts revealed vitamin A-related fluorescence (330 nm excitation maximum; 515 nm emission maximum) and a spectrally resolvable age-related fluorescence (420 nm excitation maximum; 600 nm emission maximum). Only the vitamin A-related fluorescence could be measured in the control of young samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants.

The accumulation of lipofuscin by retinal pigment epithelium may be an important feature in the pathogenesis of age-related macular degeneration, suggesting the possibility that this common cause of blindness might be prevented or delayed by antioxidants. In support of this idea, we now report significantly reduced formation of lipofuscin when the antioxidant substances lutein, zeaxanthin, lycopene (carotenoids), or alpha-tocopherol were added to rabbit and bovine (calf) retinal pigment epithelial (RPE) cells exposed to normobaric hyperoxia (40%) and photoreceptor outer segments. Rabbit and calf RPE cells were grown for 2 weeks with addition of one of the test substances every 48 h. The cellular uptake of carotenoids and alpha-tocopherol was assayed by HPLC after 2 weeks. The lipofuscin-content was measured by static fluorometry (rabbit cells) or by image analysis (calf cells). Both rabbit and calf RPE showed similar results with significantly lower amounts of lipofuscin in antioxidant-treated cells. The effect of carotenoids is especially interesting, since the result is not dependent on their protective effect against photo-oxidative reactions. The chain-breaking abilities of these antioxidants in peroxidative reactions of lipid membranes and quenching of free radicals seem to be of importance for inhibition of lipofuscin formation.