High levels of brain dolichols in neuronal ceroid-lipofuscinosis and senescence

Dolichols as unesterified alcohols were identified as significant components of lipid extracts from storage cytosomes isolated post-mortem from the brains of patients with the infantile, late infantile, and juvenile types of neuronal ceroid-lipofuscinosis (NCL). Very small amounts of dolichols were present in the corresponding subcellular fractions of non-NCL brains. The nuclear fraction from NCL cerebral cortex contained the highest dolichol content expressed per milligram protein or lipid, whereas the crude mitochondrial fraction was the richest in normal brain. Highly significant elevations of dolichol levels were found in human cerebral cortex of patients with NCL and Alzheimer's disease compared with age-matched controls, but the levels were normal in Pick's disease. In human non-NCL cerebral cortex, dolichols increased from 16 micrograms/g at age 5 to over 200 at age 81. Rat cerebral cortex showed a similar progressive increase in dolichol content with age. The high dolichol values in NCL, Alzheimer's disease, and senescence appears to be related to the increase of lipofuscin in brain. This is the first time a uniform biochemical abnormality has been found in all childhood forms of NCL, but the enzyme defect is still unidentified. It may lie on pathways where dolichols and retinyl compounds are recycled in Golgi membranes and derived organelles during the biosynthesis of glycoproteins.     

Dolichyl-pyrophosphoryloligosaccharide protein oligosaccharide transferase in neuronal ceroid-lipofuscinosis.

The kinetics of the oligosaccharide transfer from oligosaccharyl pyrophosphoryldolichol to endogenous protein acceptors in human fibroblasts were studied. No alterations in the transferase activity and enzyme characteristics could be observed in fibroblasts from neuronal ceroid-lipofuscinosis (NCL) patients. Analysis of urinary dolichol of two NCL patients also did not reveal substantial differences with respect to controls.     

Tay-Sachs disease with atypical chronic course and limited brain storage: Alpha-locus hexosaminidase genetic compound

A 19-year-old Irish-Jewish male had a slow neurologic regression starting at age 4 1/2 years with stuttering. The chronic course resembled that of Spielmeyer-Vogt (juvenile ceroid-lipofuscinosis) disease. The brain was atrophic with neuronal losses and huge compound inclusions in the remaining neurons. Lipid NANA was within normal limits in gray and white matter and G_M2 gangliosides were moderately elevated at 11.5% lipid NANA. Beta-hexosaminidase A activity was lipid composition showed nonspecific abnormalities. Exhaustive tissue extraction ruled out the possibility of tightly bound gangliosides to account for the relatively low G_M2 ganglioside concentration. The extract contained unidentified chromogenic substances interfering with the resorcinol reaction. The similarly affected patient's sister lived to age 26 years and her brain was even more atrophic. No biochemical abnormality to account for progressive neuronal losses and relative lack of G_M2 ganglioside storage was found.Deceased.Special issue dedicated to Dr. Leon S. Wolfe.     

Sea-blue histiocytes in canine ceroid-lipofuscinosis (CCL)

Cells corresponding to sea-blue histiocytes are described in bone marrow aspirates from dogs suffering hereditary ceroid-lipofuscinosis. Sea-blue histiocytes also occur in humans with so-called neuronal ceroid-lipofuscinosis and both exhibit strong autofluorescence. By quantitative fluorometric techniques, each species have similar spectral characteristics and, thus, the English setter with CCL can serve as a useful animal model.     

Ultrastructural study in the diagnosis of various neurolipidoses

We have studied the sphingolipidosis, principally, ceroid-lipofuscinosis and other complex lipid storage diseases. We examined biopsy tissue of fourteen patients with diseases of the various types given above. The biopsy tissue was usually skin, muscle or peripheral nerve, and occassionally brain or various visceral tissue.     

Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging

The activities of Cu,Zn superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase in neuronal and glial cell-enriched fractions obtained from the cerebral cortex of rat brain during aging (15, 30, 90, 350, 750 days of age) were assayed. Our results showed that glutathione peroxidase, catalase and glutathione reductase activities varied little during the examined periods. Only the Cu,Zn superoxide dismutase activity decreased notably from 15th to 750th day of age in both neuronal and glial cells, moreover the activities of all enzymes studied were always detected at lower levels in neuronal cells with respect to glial cells. In agreement with diminished SOD activity, the lipid peroxidation showed an elevated increase with aging; this fact is more evident in neuronal than in glial cells. In conclusion our data show that Cu,Zn superoxide dismutase is the most affected antioxidant enzymatic system of brain aging and it could be responsible for the increased lipid peroxidation in both cell types examined.A preliminary report of these results was presented at the 19th Meeting F.E.B.S. Rome July 2–7, 1989.     

Scavenging of reactive oxygen species and prevention of oxidative neuronal cell damage by a novel gallotannin,pistafolia A

Pistafolia A is a novel gallotannin isolated from the leaf extract of Pistacia weinmannifolia. In the present investigation, the ability of Pistafolia A to scavenge reactive oxygen species including hydroxyl radicals and superoxide anion was measured by ESR spin trapping technique. The inhibition effect on iron-induced lipid peroxidaiton in liposomes was studied. The protective effects of Pistafolia A against oxidative neuronal cell damage and apoptosis induced by peroxynitrite were also assessed. The results showed that Pistafolia A could scavenge both hydroxyl radicals and superoxide anion dose-dependently and inhibit lipid peroxidation effectively. In cerebellar granule cells pretreated with Pistafolia A, peroxynitrite-induced oxidative neuronal damage and apoptosis were prevented markedly. The antioxidant capacity of Pistafolia A was much more potent then that of the water-soluble analog of vitamin E, Trolox. The results suggested that Pistafolia A might be used as an effective natural antioxidant for the prevention and cure of neuronal diseases associated with the production of peroxynitrite and related reactive oxygen species.     

Neuronal ceroid-lipofuscin storage in a cynomolgus monkey (Macaca fascicularis)

The light and electron microscopical characteristics of non-age-related neuronal ceroid-lipofuscinosis in a young cynomolgus monkey (Macaca fascicularis) are described. The pigment in the neuronal cytoplasm was PAS-positive, sudanophilic, weakly acid-fast, and positive with Schmorl's and aldehydefuchsin stains for lipofuscin. Ultra-structurally, it appeared as membrane-bound, electron-dense aggregates within the cytoplasm of neurones of both brain and spinal cord.     

LEUKOCYTE PPD-PEROXIDASE ACTIVITY WITH POLYUNSATURATED FATTY ACID HYDROPEROXIDES: NORMAL VALUES IN BATTEN''S DISEASE

Abstract— The activity of leukocyte p -phenylenediamine (PPD)-dependent peroxidase with respect to 3 peroxidic substrates was investigated in three patients with Batten's disease (ceroid-lipofuscinosis) as compared with normal controls: 1. The activity of PPD-peroxidase, using H₂O₂ as the peroxidic substrate, was found to be normal in our patients with Batten's disease. 2. PPD-peroxidase was shown to be active towards arachidonic acid hydroperoxide (AAHPO) and linoleic acid hydroperoxide (LAHPO) as peroxidic substrates. No difference could be detected between patients and normals. 3. Determination of Michaelis constants with respect to H₂O₂, AAHPO and LAHPO in normal leukocytes revealed that PPD-peroxidase was more active towards AAHPO (lower K _m) than towards LAHPO. The same kinetic properties were found for PPD-peroxidase in patients with ceroid-lipofuscinosis.     

Infantile neuronal ceroid-lipofuscinosis is not an allelic form of Batten disease: exclusion of chromosome 16 region with linkage analyses

Infantile neuronal ceroid-lipofuscinosis (CLN1) is the form of neuronal ceroid-lipofuscinoses (NCL) with the earliest onset of symptoms. The locus of the most common form of these disorders, juvenile NCL (CLN3), has been mapped to chromosome 16. We report here linkage data of the same region in Finnish CLN1 families. Our results indicate that CLN1 is not allelic with CLN3 but represents a different locus, which is not located within about 70 cM in chromosome 16.     

Antioxidant enzymes and lipoperoxide in blood in uremic children and adolescents

To determine whether oxidant-antioxidant balance is altered in chronic renal failure, antioxidant enzymes and lipid peroxide in peripheral blood cells and lipid peroxide in plasma were measured. Nine children and adolescents maintained on hemodialysis (HD), 9 on continuous ambulatory peritoneal dialysis (CAPD), and 14 controls were studied. Lipid peroxide was assayed fluorimetrically as thiobarbituric acid-reactive substances, superoxide dismutases by radioimmunoassays. Both manganese and copper-zinc superoxide dismutases in lymphocytes and monocytes in the HD and CAPD patients, and manganese superoxide dismutase in polymorphs in the HD patients were higher than in the controls. Copper-zinc superoxide dismutase, glutathione peroxidase, and catalase in erythrocytes were unaltered. The lipid peroxide level in plasma in the dialyzed patients was increased, whereas those in polymorphs and lymphocytes were unaltered. Triglyceride and total cholesterol in plasma in the dialyzed patients were also increased. The plasma lipid peroxide in the patients correlated with the triglyceride and total cholesterol level. This is the first study in which manganese superoxide dismutase is measured in nucleated cells of the patients with chronic renal failure. The present results suggest that increased superoxide dismutases protect against oxidative stress induced by chronic renal failure in nucleated cells but in neither erythrocytes nor plasma.     

Juvenile neuronal ceroid-lipofuscinosis (Batten disease): a brief review and update

Juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease, Spielmeyer-Vogt-Sjogren disease, CLN3) is the most common inherited, autosomal recessive, neurodegenerative disorder in man. Like the other neuronal ceroid-lipofuscinoses, it is characterized by progressive loss of vision, seizures, and loss of cognitive and motor functions, leading to premature demise. JNCL is caused by mutations of CLN3, a gene that encodes a hydrophobic transmembrane protein, which localizes to membrane lipid rafts in lysosomes, endosomes, synaptosomes, and cell membrane. While the primary function of the CLN3 protein (CLN3P) may be debated, its absence affects numerous cellular functions including pH regulation, arginine transport, membrane trafficking, and apoptosis. We have recently suggested that the unifying primary function of CLN3P may be in a novel palmitoyl-protein Delta-9 desaturase (PPD) activity that in our opinion could explain all of the various functional abnormalities seen in the JNCL cells. Another group of researchers has recently shown a correlation between the CLN3P expression and the synthesis of bis(monoacylglycerol)phosphate (BMP) and suggested that CLN3P may play a role in the biosynthesis of BMP. In this review, following an introduction to the neuronal ceroid-lipofuscinoses, we provide a brief overview and an update of the most recent research in JNCL, specifically that related to the function of CLN3P.     

Stimulation and clustering of cytochrome b5 reductase in caveolin-rich lipid microdomains is an early event in oxidative stress-mediated apoptosis of cerebellar granule neurons

The apoptosis of cerebellar granule neurons (CGN) induced by low potassium in the extracellular medium is a model of neuronal apoptosis where an overshot of reactive oxygen species (ROS) triggers the neuronal death. In this work, using dihydroethidium and L-012 as specific dyes for superoxide anion detection we show that this ROS overshot can be accounted by an increased release of superoxide anion to the extracellular medium. The amplitude and time course of the increase of superoxide anion observed early during apoptosis correlated with the increase of the content of soluble cytochrome b(5), a substrate of the NADH-dependent oxidase activity of the cytochrome b(5) reductase associated with lipid rafts in CGN. Western blotting and immunofluorescence microscopy approaches, including fluorescence energy transfer, pointed out an enhanced clustering of cytochrome b(5) reductase within caveolins-rich lipid rafts microdomains. Protein/protein docking analysis suggests that cytochrome b(5) reductase can form complexes with caveolins 1α, 1β and 2, playing electrostatic interactions a major role in this association. In conclusion, our results indicate that overstimulation of cytochrome b(5) reductase associated with lipid rafts can account for the overshot of plasma membrane-focalized superoxide anion production that triggers the entry of CGN in the irreversible phase of apoptosis. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Oxidative stress in the hippocampus after pilocarpine-induced status epilepticus in Wistar rats

The role of oxidative stress in pilocarpine-induced status epilepticus was investigated by measuring lipid peroxidation level, nitrite content, GSH concentration, and superoxide dismutase and catalase activities in the hippocampus of Wistar rats. The control group was subcutaneously injected with 0.9% saline. The experimental group received pilocarpine (400 mg.kg(-1), subcutaneous). Both groups were killed 24 h after treatment. After the induction of status epilepticus, there were significant increases (77% and 51%, respectively) in lipid peroxidation and nitrite concentration, but a 55% decrease in GSH content. Catalase activity was augmented 88%, but superoxide dismutase activity remained unaltered. These results show evidence of neuronal damage in the hippocampus due to a decrease in GSH concentration and an increase in lipid peroxidation and nitrite content. GSH and catalase activity are involved in mechanisms responsible for eliminating oxygen free radicals during the establishment of status epilepticus in the hippocampus. In contrast, no correlations between superoxide dismutase and catalase activities were observed. Our results suggest that GSH and catalase activity play an antioxidant role in the hippocampus during status epilepticus.     

Lipid peroxidation-mediated oxidative stress and dopamine neuronal apoptosis in the substantia nigra during development.

The cellular pathways underlying naturally occurring neuronal apoptosis in the rat substantia nigra (SN) during the perinatal period remain largely unknown. Determining the mediators of this process in development may shed light on causes of premature neuronal death in adult neurodegenerative disorders, including the loss of dopamine neurons in Parkinson's disease. In the present study, we investigated whether lipid peroxidation-mediated oxidative stress mediates developmental death of nigral neurons by (1) establishing the profile of lipid peroxidation and other oxidative stress markers throughout the postnatal period both in the SN and striatum, and (2) examining whether the inhibitor of lipid peroxidation, alpha-tocopherol, protects these neurons from death. In addition to monitoring, the level of lipid peroxidation throughout development, we also measured the activities of three antioxidant enzymes, namely superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). We have shown that lipid peroxidation and SOD activity progressively increased from postnatal day (PND) 3 to PND 42 in both SN and striatum. During this period, GPx activity remained stable, while catalase activity transiently increased at PND 8 only in the SN. Furthermore, alpha-tocopherol treatment from embryonic day 18 to PND 2 did not reduce the number of apoptotic neurons at PND 3. These results do not support the hypothesis that lipid peroxidation-mediated oxidative stress is the major mediator of nigral dopamine neuronal apoptosis during the perinatal period.     

NADPH-dependent drug redox cycling and lipid peroxidation in microsomes from human term placenta

1. NADPH-dependent iron and drug redox cycling, as well as lipid peroxidation process were investigated in microsomes isolated from human term placenta. 2. Paraquat and menadione were found to undergo redox cycling, catalyzed by NADPH:cytochrome P-450 reductase in placental microsomes. 3. The drug redox cycling was able to initiate microsomal lipid peroxidation in the presence of micromolar concentrations of iron and ethylenediaminetetraacetate (EDTA). 4. Superoxide was essential for the microsomal lipid peroxidation in the presence of iron and EDTA. 5. Drastic peroxidative conditions involving superoxide and prolonged incubation in the presence of iron were found to destroy flavin nucleotides, inhibit NADPH:cytochrome P-450 reductase and inhibit propagation step of lipid peroxidation. 6. Reactive oxo-complex formed between iron and superoxide is proposed as an ultimate species for the initiation of lipid peroxidation in microsomes from human term placenta as well as for the destruction of flavin nucleotides and inhibition of NADPH:cytochrome P-450 reductase as well as for impairment of promotion of lipid peroxidation under drastic peroxidative conditions.     

Cytochrome P-450 involvement in the NADPH-dependent lipid peroxidation in human placental mitochondria

The NADPH-dependent lipid peroxidation in human placental mitochondria has been found to be inhibited strongly by amphenone B, aminoglutethimide and carbon monoxide, inhibitors of cytochrome P-450-mediated reactions, but was hardly affected by respiratory chain inhibitors. Cytochrome c, an exogenous electron acceptor which is known to compete with cytochrome P-450 for the reducing equivalents, showed an inhibitory effect on NADPH-dependent lipid peroxidation. The observed NADPH-dependent superoxide generation was also strongly inhibited by amphenone B and aminoglutethimide. Moreover, the lipid peroxidation in placental mitochondria was demonstrated to be stimulated by xanthine/xanthine oxidase added as superoxide generating system. This peroxidation was not affected by amphenone B and aminoglutethimide. On the other hand, the superoxide dismutase was found to inhibit both the xanthine oxidase- and NADPH-dependent lipid peroxidation. These data provide evidence that cytochrome P-450 is involved in NADPH-dependent mitochondrial lipid peroxidation. It is suggested that superoxide liberated from cytochrome P-450, in combination with iron, may be responsible for initiation of NADPH-dependent lipid peroxidation in human placental mitochondria.     

Lipid peroxidation and antioxidant status in blood of patients with uterine myoma, endometrial polypus, hyperplastic and malignant endometrium

Oxidative stress is considered to be involved in pathogenesis of many disorders of the female genital tract. In this study, we explored the lipid peroxidation levels and antioxidant enzyme activities in women diagnosed with different forms of uterine diseases in order to evaluate the extent of oxidative stress in blood of such patients. Blood samples of healthy subjects and gynecological patients were collected and subjected to assays for superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and lipid hydroperoxides. The results show that alterations of measured parameters vary with the enzyme type and diagnosis. However, both reduction in antioxidants and elevation of lipid peroxidation were observed in general. Lipid hydroperoxides level was negatively correlated to superoxide dismutase and glutathione peroxidase activities, as well as positively correlated to catalase activity. In addition, the lipid hydroperoxides/ glutathione peroxidase ratio was found to be increased, according to the type of uterine disease. The obtained results show that perturbation of antioxidant status is more pronounced in blood of patients with premalignant (hyperplastic) and malignant (adenocarcinoma) lesions, compared to those with benign uterine changes such as polypus and myoma.     

Methylated lysine in storage body protein of sheep with hereditary ceroid-lipofuscinosis.

Juvenile ceroid-lipofuscinosis (Batten disease) is a hereditary storage disease with an autosomal-recessive mode of transmission. This disorder has been identified in humans, dogs and sheep. It is characterized by massive accumulations of autofluorescent storage bodies in many tissues. This storage body accumulation is accompanied by functional decline and degeneration of the affected tissues, and ultimately results in premature death. The primary defect responsible for juvenile ceroid-lipofuscinosis has not been identified. Previous studies have indicated that the storage material is primarily protein. Why this protein accumulates in storage bodies remains to he determined. In affected humans, the storage body protein appears to be abnormally rich in a methylated derivative of lysine (epsilon-N-trimethyllysine). Studies were undertaken to determine whether the storage bodies from sheep with hereditary ceroid-lipofuscinosis were also characterized by the presence of this modified amino acid. Chromatographic and mass spectral analyses of hydrolysates of the storage body protein indicated a significant fraction of the lysine residues in this protein were present as the epsilon-N-trimethyl derivative. This modified amino acid was not detected in hydrolysates of protein from normal sheep tissues or from tissues of sheep with GM1 gangliosidosis, nor did it appear to be present in the storage body protein from a human subject with the late infantile form of ceroid-lipofuscinosis. Thus, it is apparently specific to the storage body protein that accumulates in the juvenile type of this disease. The abnormal presence of epsilon-N-trimethyllysine in proteins could interfere with their sorting or degradation within cells and thus cause them to accumulate in the storage bodies characteristic of the human juvenile and ovine ceroid-lipofuscinoses.