Near-Infrared Autofluorescence in Atherosclerosis Associates With Ceroid and Is Generated by Oxidized Lipid-Induced Oxidative Stress.

[Figure: see text].

Occurrence and characterization of lipofuscin and ceroid in human atherosclerotic plaque.

Atherosclerotic plaque formation starts early in life, develops silently over decades, and often displays clear evidence of accelerated biological aging. Lipofuscin has been classically defined as "the most consistent and phylogenetically conserved cellular morphologic change of aging," however, despite this traditional view different lines of evidence have recently demonstrated that, besides aging, various noxious influences can engeder its accumulation in cells and also that specific experimental conditions can revert this effect. Lipofuscin has been also proven to interact with disease-related factors to enhance cell loss. Along with lipofuscin, ceroid, another autofluorescent lipopigment usually produced under various pathological conditions unrelated to aging, has been suggested to jeopardize cell performance and viability by inducing membrane fragility, mitochondrial dysfunction, DNA damage, and oxidative stress-induced apoptosis. With regard to atherosclerosis, very few investigations have been conducted to assess whether a link could exist between lipofuscin/ceroid accumulation and the progression of the disease and no information still exist regarding the anatomy and the ultrastructural diversification of lipofuscin and ceroid in the lesional vascular tissue. At the same time, data concerning their potential toxicity at the cellular level are fragmentary, dated, and scarce. The present study investigates the occurrence and distribution of lipofuscin and ceroid in human atherosclerotic plaque and adjacent healthy tissues and analyzes the ultrastructural changes associated with their accumulation within the cell.

CNS-expressed cathepsin D prevents lymphopenia in a murine model of congenital neuronal ceroid lipofuscinosis.

Deficiency in Cathepsin D (CtsD), the major cellular lysosomal aspartic proteinase, causes the congenital form of neuronal ceroid lipofuscinoses (NCLs). CtsD-deficient mice show severe visceral lesions like lymphopenia in addition to their central nervous system (CNS) phenotype of ceroid accumulation, microglia activation, and seizures. Here we demonstrate that re-expression of CtsD within the CNS but not re-expression of CtsD in visceral organs prevented both central and visceral pathologies of CtsD(-/-) mice. Our results suggest that CtsD was substantially secreted from CNS neurons and drained from CNS to periphery via lymphatic routes. Through this drainage, CNS-expressed CtsD acts as an important modulator of immune system maintenance and peripheral tissue homeostasis. These effects depended on enzymatic activity and not on proposed functions of CtsD as an extracellular ligand. Our results furthermore demonstrate that the prominent accumulation of ceroid/lipofuscin and activation of microglia in brains of CtsD(-/-) are not lethal factors but can be tolerated by the rodent CNS.

Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood.

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl approximately CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.

Identification of proteins in the ceroid-like autofluorescent aggregates from liver lysosomes of Beige, a mouse model for human Chediak-Higashi syndrome.

Chediak-Higashi syndrome is characterized by oculocutaneous albinism, a bleeding tendency and severe recurrent infections. Age-dependent formations of autofluorescent ceroid-like substances have been noted in a variety of tissues. In this study, we isolated an autofluorescent ceroid-like aggregate from purified Beige mouse liver lysosomes and analyzed the composition of the aggregate by ion trap mass-spectrometry. In addition to lysosomal proteins, this aggregate contains proteins normally localized in the ER, mitochondria, peroxisomes, and the cytosol. Bip, a luminal ER protein was abundant in lysosomal ceroid. The ER, mitochondria, and cytosol proteins could arise in lysosomes through stimulation of autophagy, but we found no differences between normal and CHS fibroblasts in the degree of lysosomal acidity and in the level of conversion of soluble microtubular-associated protein 1 light chain 3 type I to membrane-associated type II, an accepted probe for hyper-autophagy suggesting that ceroid formation is unlikely to arise via this mechanism.

Low oxygen: A (tough) way of life for Okavango fishes.

Botswana's Okavango Delta is a World Heritage Site and biodiverse wilderness. In 2016-2018, following arrival of the annual flood of rainwater from Angola's highlands, and using continuous oxygen logging, we documented profound aquatic hypoxia that persisted for 3.5 to 5 months in the river channel. Within these periods, dissolved oxygen rarely exceeded 3 mg/L and dropped below 0.5 mg/L for up to two weeks at a time. Although these dissolved oxygen levels are low enough to qualify parts of the Delta as a dead zone, the region is a biodiversity hotspot, raising the question of how fish survive. In association with the hypoxia, histological samples, collected from native Oreochromis andersonii (threespot tilapia), Coptodon rendalli (redbreast tilapia), and Oreochromis macrochir (greenhead tilapia), exhibited widespread hepatic and splenic inflammation with marked granulocyte infiltration, melanomacrophage aggregates, and ceroid and hemosiderin accumulations. It is likely that direct tissue hypoxia and polycythemia-related iron deposition caused this pathology. We propose that Okavango cichlids respond to extended natural hypoxia by increasing erythrocyte production, but with significant health costs. Our findings highlight seasonal hypoxia as an important recurring stressor, which may limit fishery resilience in the Okavango as concurrent human impacts rise. Moreover, they illustrate how fish might respond to hypoxia elsewhere in the world, where dead zones are becoming more common.

Neuronal pigmented autophagic vacuoles: lipofuscin, neuromelanin, and ceroid as macroautophagic responses during aging and disease.

The most striking morphologic change in neurons during normal aging is the accumulation of autophagic vacuoles filled with lipofuscin or neuromelanin pigments. These organelles are similar to those containing the ceroid pigments associated with neurologic disorders, particularly in diseases caused by lysosomal dysfunction. The pigments arise from incompletely degraded proteins and lipids principally derived from the breakdown of mitochondria or products of oxidized catecholamines. Pigmented autophagic vacuoles may eventually occupy a major portion of the neuronal cell body volume because of resistance of the pigments to lysosomal degradation and/or inadequate fusion of the vacuoles with lysosomes. Although the formation of autophagic vacuoles via macroautophagy protects the neuron from cellular stress, accumulation of pigmented autophagic vacuoles may eventually interfere with normal degradative pathways and endocytic/secretory tasks such as appropriate response to growth factors.

Retinal pathology in a canine model of late infantile neuronal ceroid lipofuscinosis.

PURPOSE: Late infantile neuronal ceroid lipofuscinosis (NCL) is an inherited disorder characterized by progressive vision loss. The disease results from mutations in the TPP1 (CLN2) gene. Studies were undertaken to characterize the effects of a TPP1 frameshift mutation on the retina in Dachshunds. METHODS: A litter of four puppies consisting of one homozygous affected dog, two heterozygotes, and one homozygous normal dog were monitored for neurologic and retinal changes through 10 months of age. The affected and homozygous normal dogs, as well as one of the heterozygotes, were then euthanatized, and the retinas were examined morphologically. RESULTS: The affected dog exhibited normal visual behavior and retinal function at 3 months of age, but vision was clearly impaired by 7 months, with markedly reduced ERG b-wave amplitudes. Beyond 7 months of age, the affected dog was functionally blind, and pupillary light reflexes and ERG response amplitudes continued to decline through 10 months of age. Both rod and cone system functions were severely impaired. The retina exhibited accumulation of autofluorescent storage bodies with distinctive curvilinear contents. Substantial cell loss occurred in the inner nuclear layer, with a smaller reduction in photoreceptor cell density. CONCLUSIONS: The canine TPP1 mutation results in progressive vision loss and retinal degeneration similar to that which occurs in human late infantile NCL. With the canine model, the natural history of disease progression in the retina provides a better understanding of the pathologic course of the disease and provides objective markers that can be used to assess the efficacy of therapeutic interventions.

Processing, lysis, and elimination of brain lipopigments in rejuvenation therapies.

Cerebral lipopigments (LPs)--lipofuscin and ceroid--represent a significant marker in postmitotic normal and pathologic aging, connected with causal and associated neuropathologic damage. Therefore, LP processing, lysis, and elimination may be the main targets in anti-aging and rejuvenation therapies. The regenerative neuroactive factors improve neuron supply with specific nutrients from plasma. They enhance the antioxidative defense, have anti-LP-poietic actions, stimulate brain anabolism, support energetic metabolism, and elevate the reduced lysosomal enzymes. In the second stage, by cytoplasm rehydration, they initiate the breaking up of the neuronal aggregated LP conglomerates, by consecutive disintegration. Then, possibly by the localized exo-endocytosis process between neurons and adjacent glia (especially microglia), intercellular LP transfer can be realized. So, therapeutically activated glia turn into brain garbage collectors and transporters. Therapeutic processing of glial LPs increases in the capillary neighborhood. Highly processed LPs, by glio-endothelial transfer, reach capillary walls before being eliminated. Consequently, neuroactive therapies having these synergistic rejuvenative actions represent new prospects in deceleration of normal and pathological cerebral aging.

Diagnosis of neuronal ceroid lipofuscinosis (Batten disease) by electron microscopy in peripheral blood specimens.

Neuronal ceroid lipopofuscinosis (Batten disease, NCL) represents a group of common childhood neurodegenerative diseases with a shared feature of deposition of abnormal metabolic products in neurons and other tissues, including peripheral blood lymphocytes. In most forms of NCL no specific enzyme defect is known and the diagnosis relies primarily on ultrastructural identification of characteristic membrane-bound inclusions containing the abnormal metabolic product. All buffy-coat specimens examined during a 7-year period (1997-2004) for the exclusion or confirmation of the diagnosis NCL were reviewed. From a total of 265 samples, 9 were inadequate and NCL was diagnosed in 56. Five showed granular osmophilic deposits of infantile Batten disease (NCL1), 10 showed curvilinear profiles of classical late infantile Batten disease (NCL2), and 17 showed vacuolated lymphocytes with fingerprint profiles, indicating classical juvenile Batten disease (NCL3). 24 samples (43%) demonstrated compact electron-dense deposits with fingerprint profiles in the absence of vacuolated lymphocytes, indicative of variant forms NCL. Ultrastructual examination of peripheral blood allows reliable and specific diagnosis of subtypes of Batten disease, including variants, and is a useful, minimally invasive test for the diagnosis of NCL in childhood.

Inducible nitric oxide synthase colocalizes with signs of lipid oxidation/peroxidation in human atherosclerotic plaques.

OBJECTIVE: Advanced human atherosclerotic plaques are characterized by the abundant presence of the autofluorescent non-soluble lipid pigment ceroid, consisting of oxidized lipoproteins. The aim of the present study was to examine the topographical and cellular distribution of inducible nitric oxide synthase (iNOS or NOS II) within different stages of atherosclerosis and its colocalization with ceroid deposits and nitrotyrosine. METHODS AND RESULTS: Different stages of atherosclerosis were studied by immunohistochemistry on whole-mount longitudinal sections of carotid endarterectomy specimens. In the adaptive intimal thickening the predominant cell type were smooth muscle cells. The fatty streaks contained both smooth muscle cells and macrophages with an extremely low NOS II immunoreactivity. The advanced atherosclerotic plaques however, showed a very dense infiltration by macrophages, of which a subpopulation expressed NOS II as a vesicular immunoreactivity in their cytoplasm. These were mainly present around the necrotic core, in association with ceroid accumulation and nitrotyrosine. Fluorescence quenching microscopy showed the presence of NOS II on autofluorescent ceroid vesicles in the macrophages. Large extracellular ceroid granules were not NOS II immunoreactive. NOS II mRNA was detected by RT-PCR and the protein by Western blot in the plaque tissue but not in mammary arteries used as controls. CONCLUSION: Ceroid, nitrotyrosine and NOS II colocalized in late stages of atherosclerosis and were found around the necrotic core in the plaque. This could suggest that NOS II expression in macrophages is involved in oxidation and peroxidation of lipids, leading to ceroid formation.

Thioredoxin reductase activity in Hermansky-Pudlak syndrome: a method for identification of putative heterozygotes.

Recent studies indicate that membrane-associated thioredoxin reductase (TR) is a possible regulator of melanin biosynthesis via the inhibition of tyrosinase by reduced thioredoxin. In normal individuals, the levels of TR activity in skin correlate linearly to the Fitzpatrick classification of skin type, being lowest in type I skin and highest in skin type VI. In this study, TR was measured in 3-mm skin biopsies in Hermansky-Pudlak syndrome (HPS) patients and their relatives. Forty-five individuals from seven Puerto Rican kindreds were tested, including 12 homozygotes, nine obligate heterozygotes, and 24 unclassified individuals. In addition, seven separate nonkindred HPS patients were tested. With one exception, TR activity was markedly decreased in 18 homozygotes. TR activity was decreased in eight obligate heterozygotes and in 12 unclassified kindred members, whereas 10 subjects had normal TR activity when compared to the expected activity of their skin type. Four individuals were excluded from the analysis because of inadequate controls for their age group or immunosuppressive treatment for kidney transplant. The results indicate that decreased TR activity assayed in 3-mm skin punch biopsies is a useful method for detecting carriers of the HPS gene.

Decreased proteolysis caused by protein aggregates, inclusion bodies, plaques, lipofuscin, ceroid, and 'aggresomes' during oxidative stress, aging, and disease.

Protein aggregation seems to be a common feature of several neurodegenerative diseases and to some extent of physiological aging. It is not always clear why protein aggregation takes place, but a disturbance in the homeostasis between protein synthesis and protein degradation seems to be important. The result is the accumulation of modified proteins, which tend to form high molecular weight aggregates. Such aggregates are also called inclusion bodies, plaques, lipofuscin, ceroid, or 'aggresomes' depending on their location and composition. Such aggregates are not inert metabolic end products, but actively influence the metabolism of cells, in particular proteasomal activity and protein turnover. In this review we focus on the influence of oxidative stress on protein turnover, protein aggregate formation and the various interactions of protein aggregates with the proteasome. Furthermore, the formation and effects of protein aggregates during aging and neurodegeneration will be highlighted.

The morphology of lipopigment in rat Purkinje neurons after chronic acetyl-L-carnitine administration: a reduction in aging-related changes.

The aging-related accumulation of neuronal lipopigment is considered to be cellular debris from processes of renewal of cellular constituents, but it can also reflect cell damage and certain diseases. Acetyl-L-carnitine (AC) has been reported to reduce some morphological and behavioral associations of brain aging and the present study investigated the effects of 37 weeks of AC administration on lipopigment in rat Purkinje neurons. Lipopigment was identified by fluorescence microscopy and the area enclosed by an outline of each discrete region of lipopigment was measured. Acetyl-L-carnitine was associated with a significant (p = 0.05) reduction in the number of discrete lipopigment regions and there was a significant (p = 0.001) association of AC administration with numbers of lipopigment regions in various size categories. As AC administration was associated with a reduction in some of the aging-related morphological changes in lipopigment, this compound is a candidate for evaluation as a long-term prophylactic agent for the adverse effects of cerebral aging.

Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores.

Serious studies of the formation mechanisms of age-related pigments and their possible cellular influence have been hampered for a long time by discrepancies and controversies over the definition, fluorescence emission, origin, and composition of these pigments. This review discusses several critical controversies in this field and lay special emphasis on the cellular and biochemical reactions related to the formation mechanisms of lipofuscin, ceroid, advanced glycation end-products (AGEs), and age pigment like fluorophores (APFs). Various amino compounds and their reaction with secondary aldehydic products of oxygen free radical-induced oxidation, particularly lipid peroxidation, are important sources of the fluorophores of ceroid/lipofuscin, which progressively accumulate as a result of phagocytosis and autophagocytosis of modified biomaterials within secondary lysosomes of postmitotic and other cells. Lipofuscin is the classical age pigment of postmitotic cells, while ceroid accumulates due to pathologic and experimental processes. There are good reasons to consider both ceroid and lipofuscin as materials of the same principal origin. The age-related intracellular fluorophores of retinal pigment epithelium (RPE) seems to represent a special class of lipofuscin, which partly contains derivatives of retinoids and carotenoids. Saccharide-originated fluorophores, principally AGEs formed during glycation/Maillard reactions, may be mainly responsible for the extracellular fluorescence of long-lived proteins, such as collagen, elastin, and lens crystalline. Although lipofuscin, ceroid, AGEs, and APFs can be produced from different types of biological materials due to different side reactions of essential biology, the crosslinking of carbonyl-amino compounds is recognized as a common process during their formation.

Ascorbic acid oxidation: a potential cause of the elevated severity of atherosclerosis in diabetes mellitus?

The exposure of mouse peritoneal macrophages to cholesterol linoleate-containing artificial lipoproteins can lead to intracellular ceroid accumulation. This can be used as a model to study the role of oxidation in macrophage uptake of lipoproteins containing unsaturated fatty acids, considered by many as a primary event in atherosclerotic plaque formation. Our studies show that ascorbic acid can both inhibit and promote the formation of ceroid in such a model system. The transition metal copper (Cu(II)) further elevates ceroid accumulation and EDTA, a metal chelator, inhibits it. When trace levels of transition metals are present, low concentrations of ascorbic acid can elevate ceroid formation. This pro- and antioxidant characteristic of ascorbic acid was confirmed by monitoring the generation of oxidants by various concentrations of ascorbic acid, assessed by benzoic acid hydroxylation or the fragmentation of BSA. We discuss these observations in the context of an apparent increase in ascorbic acid oxidation and elevated severity of atherosclerosis in diabetes mellitus.

The cellular pathology of neuronal ceroid-lipofuscinosis. A golgi-electronmicroscopic study.

A cerebral biopsy specimen from a 4-year-old girl with a moderately advanced stage of the late infantile form of neuronal ceroid-lipofuscinosis was observed in routine cell and fiber stains and in Golgi and electronmicroscopic preparations. There was no evidence of neuronal degeneration or loss. Golgi impregnations identified a fusiform enlargement of proximal axon segments of most pyramidal neurons and some polymorphic neurons but not of other cortical neuronal classes. Typical curvilinear inclusions were found in all cells and appeared to be impacted within the dilated proximal axon segments of pyramidal neurons. The numbers of type II synapses on the axon hillock and dilated proximal axon segments of pyramidal neurons were much reduced, whereas type I synapses remained abundant in the neuropil.

On the degradability and exocytosis of ceroid/lipofuscin in cultured rat cardiac myocytes.

The accumulation of lipofuscin (LF)--a polymeric, electron-dense, autofluorescent substance--within postmitotic cells is a characteristic manifestation of aging. It is generally believed that LF is undegradable and formed due to peroxidative alterations of various macromolecules under intralysosomal autophagic degradation. We report here that a short-term exposure of cultured neonatal rat cardiac myocytes to the thiol protease-inhibitor leupeptin, causes an accumulation of numerous electron-dense autophagic lysosomes within the cells. Although very similar to LF by ultrastructure, these inclusions do not display LF-specific, yellow-orange autofluorescence when excited with blue light. Moreover, they rapidly disappear from the cells upon re-establishment of normal culture conditions. In contrast, prolonged leupeptin treatment results in an accumulation of dense lysosomes that also show LF-typical autofluorescence. This autofluorescent material remains in the cells after the end of leupeptin action. The results suggest that: (i) a certain amount of time is needed for autophagocytosed material to become peroxidized, autofluorescent and undegradable, i.e. to acquire properties typical of LF; (ii) protease-inhibition by itself does not lead to LF-formation but rather allows the prolonged time needed for oxidative modification of autophagocytosed material; (iii) mature LF is probably not subjected to either degradation or exocytosis.

Neuronal ceroid-lipofuscinosis: ultrastructural study of lymphocytic dense bodies.

We studied the ultrastructural alterations of the dense bodies of peripheral blood lymphocytes in one case of neuronal ceroid-lipofuscinosis. These cytoplasmic vesicles were characterized by vacuolar transformation and "fingerprint" content. When we tilted the specimens, the "fingerprint" arrangement was shown to be the same as the arrangement of the crystalloid inclusions found in the same cells. Both vacuoles and periodic cytoplasmic inclusions could be due to a disorder of secondary lysosomes--the lymphocytic dense bodies.

Sea-blue histiocyte and posterior column dysfunction: a familial disorder.

This is a report of a new familial neurologic disorder characterized by ceroid-lipofuscin storage, sea-blue histiocytes, and associated neurologic findings. Neurologic manifestations in the family members examined were varied, but posterior column involvement was the most common finding. The presence of sea-blue histiocytes was genetically determined by an autosomal dominant trait. Vitamin E blood concentrations were decreased in the absence of absorption difficulties in two sisters who were most seriously affected. The relationship of ceroid-lipofuscin, lipid peroxidation, and vitamin E is discussed.