Background-Asthma is an increasingly common disorder responsible for considerable morbidity and mortality. Although obesity is a risk factor for asthma and weight loss can improve symptoms, many patients do not adhere to low calorie diets and the impact of dietary restriction on the disease process is unknown.

The plasma membrane (PM) contains redox enzymes that provide electrons for energy metabolism and recycling of antioxidants such as coenzyme Q and ␣-tocopherol. Brain aging and neurodegenerative disorders involve impaired energy metabolism and oxidative damage, but the involvement of the PM redox system (PMRS) in these processes is unknown. Caloric restriction (CR), a manipulation that protects the brain against aging and disease, increased activities of PMRS enzymes (NADH-ascorbate free radical reductase, NADH-quinone oxidoreductase 1, NADH-ferrocyanide reductase, NADH-coenzyme Q10 reductase, and NADH-cytochrome c reductase) and antioxidant levels (␣-tocopherol and coenzyme Q10) in brain PM during aging. Age-related increases in PM lipid peroxidation, protein carbonyls, and nitrotyrosine were attenuated by CR, levels of PMRS enzyme activities were higher, and markers of oxidative stress were lower in cultured neuronal cells treated with CR serum compared with those treated with ad libitum serum. These findings suggest important roles for the PMRS in protecting brain cells against age-related increases in oxidative and metabolic stress.Alzheimer's disease ͉ reactive oxygen species ͉ coenzyme Q10 ͉ oxidoreductase

Mutations in a-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of a-synuclein overexpression on the response of cells to various insults. Wild-type a-synuclein and a-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type a-synuclein delayed cell death induced by serum withdrawal or H 2 O 2 , but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP 1 ). By contrast, wild-type a-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-transnonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type a-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant a-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant a-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal a-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.

The ubiquitin/proteasome pathway plays an essential role in protein turnover in vivo, and contributes to removal of oxidatively damaged proteins. We examined the effects of proteasome inhibition on viability, oxidative damage and antioxidant defences in NT-2 and SK-N-MC cell lines. The selective proteasome inhibitor, lactacystin (1 microM) caused little loss of viability, but led to significant increases in levels of oxidative protein damage (measured as protein carbonyls), ubiquitinated proteins, lipid peroxidation and 3-nitrotyrosine, a biomarker of the attack of reactive nitrogen species (such as peroxynitrite, ONOO(-)) upon proteins. Higher levels (25 microM) of lactacystin did not further increase the levels of carbonyls, lipid peroxidation, 3-nitrotyrosine, or ubiquitinated proteins, but produced increases in the levels of 8-hydroxyguanine (a biomarker of oxidative DNA damage) and falls in levels of GSH. Lactacystin (25 microM) caused loss of viability, apparently by apoptosis, and also increased production of nitric oxide (NO.) (measured as levels of NO2- plus NO3-) by the cells; this was inhibited by N-nitro-L-arginine methyl ester (L-NAME), which also decreased cell death induced by 25 microM lactacystin and decreased levels of 3-nitrotyrosine. The NO. production appeared to involve nNOS; iNOS or eNOS were not detectable in either cell type. Another proteasome inhibitor, epoxomicin, had similar effects.

Mutations in Parkin (a ubiquitin protein ligase) are involved in autosomal recessive juvenile parkinsonism, but it is not known how they cause nigral cell death. We examined the effect of Parkin overexpression on cellular levels of oxidative damage, antioxidant defenses, nitric oxide production, and proteasomal enzyme activity. Increasing expression of Parkin by gene transfection in NT-2 and SK-N-MC cells led to increased proteasomal activity, decreased levels of protein carbonyls, 3-nitrotyrosine-containing proteins, and a trend to a reduction in ubiquitinated protein levels. Transfection of these cells with DNA encoding three mutant Parkins associated with autosomal recessive juvenile parkinsonism (Del 3-5, T240R, and Q311X) gave smaller increases in proteasomal activity and led to elevated levels of protein carbonyls and lipid peroxidation. Turnover of the mutant proteins was slower than that of the wild-type protein, and both could be blocked by the proteasome inhibitor, lactacystin. A rise in levels of nitrated proteins and increased levels of NO 2 ؊ /NO 3 ؊ was also observed in cells transfected with mutant Parkins, apparently because of increased levels of neuronal nitricoxide synthase. The presence of mutant Parkin in substantia nigra in juvenile parkinsonism may increase oxidative stress and nitric oxide production, sensitizing cells to death induced by other insults. Parkinson's disease (PD)1 results from degeneration of dopaminergic neurones in the substantia nigra (1). Although most cases appear sporadic and of unknown cause, oxidative stress and apoptosis are associated with disease progression (1). Consistent with this view, increased levels of oxidative damage to DNA, proteins, and lipids and decreased levels of GSH are found in substantia nigra in PD (1-5).Autosomal recessive juvenile parkinsonism (AR-JP), an early onset form of PD, is characterized by loss of tyrosine hydroxylase-immunoreactive neurones in substantia nigra pars compacta and locus ceruleus, usually without Lewy body formation (6). Various mutations (including deletion or point mutations) in the Parkin gene located on chromosome 6 (6q25.2-q27) have been found in AR-JP patients, but no clear correlations exist between types of Parkin mutations and clinical or pathologic features (7-14).Parkin has been identified as a ubiquitin-protein ligase containing 465 amino acids, which consists of a ubiquitin-like (UBL) domain in the N terminus, two ring-finger motifs (termed RING1 and RING2) flanking a Cys-rich domain, named as the in-between RING (IBR) (9, 14). An additional segment is a linker region that connects two regions of UBL and RING1-IBR-RING2 (named as the RING box). Deletional analysis of Parkin revealed that UBL and the linker region are not necessary for association with a specific E2 enzyme. In contrast, the full region of the RING box is necessary for noncovalent association with E2. Therefore, missense mutations in the RING box of Parkin in AR-JP patients have almost completely lost the specific E2-binding activity.2 Thus, ...

No abstract

Summary The cause of elevated level of amyloid β-peptide (Aβ42) in common late-onset sporadic (AD) has not been established. Here we show that the membrane lipid peroxidation product 4-hydroxynonenal (HNE) is associated with amyloid and neurodegenerative pathologies in AD, and that it enhances γ-secretase activity and Aβ42 production in neurons. The γ-secretase substrate receptor, nicastrin was found to be modified by HNE in cultured neurons and in brain specimens from AD patients, in which HNE-nicastrin levels were found to be correlated with increased γ-secretase activity and Aβ plaque burden. Furthermore, HNE modification of nicastrin enhanced its binding to the γ-secretase substrate, amyloid precursor protein (APP) C99. In addition, the stimulation of γ-secretase activity and Aβ42 production by HNE were blocked by an HNE-scavenging histidine analog in a 3xTgAD mouse model of AD. These findings suggest a specific molecular mechanism by which oxidative stress increases Aβ42 production in AD, and identify HNE as a novel therapeutic target upstream of the γ-secretase cleavage of APP.

Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 lM) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated-or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated a-tubulin, SOD-1, a-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311¢X). The nitric oxide synthase inhibitor, L-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.