Generation of reactive oxygen species by mitochondrial complex I: implications in neurodegeneration.

Mitochondrial Complex I [NADH Coenzyme Q (CoQ) oxidoreductase] is the least understood of respiratory complexes. In this review we emphasize some novel findings on this enzyme that are of relevance to the pathogenesis of neurodegenerative diseases. Besides CoQ, also oxygen may be an electron acceptor from the enzyme, with generation of superoxide radical in the mitochondrial matrix. The site of superoxide generation is debated: we present evidence based on the rational use of several inhibitors that the one-electron donor to oxygen is an iron-sulphur cluster, presumably N2. On this assumption we present a novel mechanism of electron transfer to the acceptor, CoQ. Complex I is deeply involved in pathological changes, including neurodegeneration. Complex I changes are involved in common neurological diseases of the adult and old ages. Mitochondrial cytopathies due to mutations of either nuclear or mitochondrial DNA may represent a useful model of neurodegeneration. In this review we discuss Parkinson's disease, where the pathogenic involvement of Complex I is better understood; the accumulated evidence on the mode of action of Complex I inhibitors and their effect on oxygen radical generation is discussed in terms of the aetiology and pathogenesis of the disease.

Characterization of highly toxic type 2 ribosome-inactivating proteins from Adenia lanceolata and Adenia stenodactyla (Passifloraceae).

From the caudices of the Passifloraceae Adenia lanceolata and A. stenodactyla, two lectins called lanceolin and stenodactylin, respectively, were purified by affinity chromatography on CL Sepharose 6B. The lectins are glycoproteins with M(r) 61,243 (lanceolin) and 63,131 (stenodactylin), consisting of an enzymatic A chain linked to a larger B chain with lectin properties, with N-terminal amino acid sequences similar to that of volkensin, the toxic lectin from A. volkensii. The lectins agglutinate red blood cells, inhibit protein synthesis both by a cell-free system and by whole cells, and depurinate ribosomes and DNA, but not tRNA or poly(A). They are highly toxic to cells, in which they induce apoptosis, and to mice, with LD(50)s 8.16 microg/kg (lanceolin) and 2.76 microg/kg (stenodactylin) at 48 h. Thus, lanceolin and stenodactylin have all the properties of the toxic type 2 ribosome-inactivating proteins and are amongst the most potent toxins of plant origin.

Lesions caused by ricin applied to rabbit eyes.

PURPOSE: Ricin, a highly potent toxin from castor beans, is a potential biological weapon that could be dispersed in the air as dust or aerosol. In these forms, ricin, besides being inhaled, could reach unprotected eyes. The present research was performed to ascertain the lesions that the toxin causes when applied to rabbit eyes. METHODS: Ricin was applied to rabbit eyes in solution, in quantities ranging from 1 to 100 mug. Animals were observed until death, when eyes and internal organs were removed and fixed. Sections were stained and examined microscopically. RESULTS: Ricin caused inflammation of the eyes and adnexa, visible both macroscopically and histologically. The damage was greatly reduced by rinsing the eyes with 10% lactose, provided the rinsing was done almost immediately after application of the toxin. Rinsing with phosphate-buffered saline (PBS) had no effect. With the highest dosage, congestion of internal organs was also apparent. CONCLUSIONS: Application of ricin to eyes causes local damage, mainly of the inflammatory type. The ineffectiveness of rapid rinsing with PBS and the partial efficacy of rapid rinsing with lactose indicate that the toxin quickly binds to and is taken up by cells. The lesions of internal organs show that ricin applied to the eyes can be absorbed, pass into the circulation, and, at least at some dosages, damage internal organs.

Neuronal apoptosis is accompanied by amyloid beta-protein accumulation in the endoplasmic reticulum.

A series of evidences suggests that enhanced susceptibility to programmed cell death (PCD) is a major pathogenetic factor in Alzheimer's disease (AD). We investigated this issue, analyzing amyloid beta-protein (A beta) production in a model of neuronal PCD, induced by staurosporine in a murine neuroblastoma cell line. When PCD was induced, a 280-290% secreted A beta occurred, in spite of a 20% metabolism and an unchanged A betaPP expression. The increased intracellular A beta reactivity largely colocalized with a marker of ER. Inhibition of caspases blocked the cleavage at the C-terminus of beta PP, but only partially rescued A beta overproduction caused by staurosporine treatment. Our findings suggest that PCD fosters the physiological pathways of A beta production characteristic of neuronal cells, and they confirm the theory that unbalance of PCD is a central event in AD pathogenesis. Moreover, our data indicate that still unidentified cellular mechanisms, other than caspases activation, are responsible of the specific alteration of A betaPP processing during PCD.

Protein levels of glycogen synthase 3 kinase are normal in progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by pure neurofibrillary tau pathology involving mainly basal ganglia and brain stem nuclei. One of the kinases involved in tau phosphorylation is glycogen synthase 3 kinase (GSK3). In mammals GSK3 is present in two isoforms, alpha and beta regulated by phosphorylation: phosphorylation of Ser9 in GSK3beta or Ser21 in GSK3alpha leads to inactivation while phosphorylation of Tyr216 in GSK3beta or Tyr279 in GSK3alpha leads to activation. We analyzed the protein levels of GSK3alpha/beta and of the phosphorylated forms GSK3beta S(9), GSK3beta Y(216), GSK3alpha Y(279) in brain tissues of subjects with PSP. The analysis failed to show significant differences of all GSK3 isoforms in PSP in comparison to age-matched control cases. This negative result argues against the role of GSK3 in the pathogenesis of PSP.

Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay.

Ribosome-inactivating proteins (RIPs) are plant proteins with enzymatic activity, classified as type 1 (single chain) or type 2 (two chains). They are identified as rRNA N-glycosidases (EC 3.2.2.22) and cause an irreversible inhibition of protein synthesis. Among type 2 RIPs, there are potent toxins (ricin is the best known) that are considered as potential biological weapons. The development of a fast and sensitive method for the detection of biological agents is an important tool to prevent or deal with the consequences of intoxication. In this article, we describe a very sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of RIPs-a type 1 RIP (dianthin) and a type 2 RIP (ricin)-that combines the specificity of immunological analysis with the exponential amplification of PCR. The limit of detection (LOD) of the technique was compared with the LODs of the conventional immunological methods enzyme-linked immunosorbent assay (ELISA) and fluorescent immunosorbent assay (FIA). The LOD of IPCR was more than 1 million times lower than that of ELISA, allowing the detection of 10 fg/ml of dianthin and ricin. The possibility to detect ricin in human serum was also investigated, and a similar sensitivity was observed (10 fg/ml). IPCR appears to be the most sensitive method for the detection of ricin and other RIPs.

Clusterin up-regulation following sub-lethal oxidative stress and lipid peroxidation in human neuroblastoma cells.

Clusterin/apolipoprotein J is a multifunctional protein up-regulated during various pathophysiological states. Since oxidative stress plays an important role in brain aging, and in many neurodegenerative disorders, to further understand the mechanistic underpinnings of clusterin expression, in this study, we examined clusterin expression in human neuroblastoma cells under conditions of increased production of reactive oxygen species and lipid peroxidation. Specifically, we analyzed clusterin mRNA and protein levels in human neuroblastoma IMR-32 and SH-SY5Y cells following exposure to sub-lethal amounts of iron-ascorbate to induce an increase in reactive oxygen species generation and lipid peroxidation. Under such conditions, we observed a time-dependent up-regulation of clusterin protein and mRNA levels, detected by immunoblot analysis and RT-PCR, respectively. Given the known roles of clusterin, the results of the present study support the notion that an increase in clusterin expression may be a physiological defence mounted to reduce cell damage and maintain cell viability during periods of increased oxidative stress.

Up-regulation of cDK5/p35 by oxidative stress in human neuroblastoma IMR-32 cells.

Cdk5, a member of the cyclin-dependent kinase (cdk) family, is predominantly active in neurons, where its activity is tightly regulated by the binding of its neuronal activators p35 and p39. Cdk5 is implicated in regulating the proper neuronal function; a deregulation of cdk5 has been found associated with Alzheimer's disease and amyotrophic lateral sclerosis. As oxidative stress products have been seen co-localized with pathological hallmarks of neurodegenerative diseases, we studied the effect of oxidative stress on the cdk5 enzyme in human neuroblastoma IMR-32 cells. We evaluated the effects of 4-hydroxynonenal and Ascorbate plus FeSO(4) on cdk5 activity and on the expression of cdk5 and p35 proteins. We report here that oxidative stress stimulates cdk5 activity and induces an upregulation of its regulatory and catalytic subunit expression in IMR-32 vital cells, showing that the cdk5 enzyme is involved in the signaling pathway activated by oxidative stress.

Regulation of glycogen synthase kinase-3beta by products of lipid peroxidation in human neuroblastoma cells.

The potential role of 4-hydroxynonenal (HNE), a major product of membrane lipid peroxidation, in regulating glycogen synthase kinase-3beta (GSK3beta) activity was examined in human neuroblastoma IMR-32 cells. The inhibition of GSK3beta activity by HNE was observed by in vitro kinase assays with two substrates, the synthetic glycogen synthase peptide-2 and the human recombinant tau. GSK3beta activity is regulated by Ser9 (inhibitory) and Tyr216 (stimulatory) phosphorylation. By using specific activity-dependent phospho-antibodies, immunoblot analysis revealed that HNE induces an increase in phosphorylation of GSK3beta in Ser9, enhancing basal phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 2 (ERK2) signalling pathways. Ser9-GSK3beta phosphorylation induced by HNE was abolished by treatment with LY294002 or U0126, two inhibitors of PI3K/AKT and ERK pathways, respectively. These experiments provide evidence for a crucial role of the PI3K/AKT and ERK2 pathways as intracellular targets of HNE that mediate the inhibition of GSK3beta activity in regulating cellular response to HNE in viable cells under conditions in which membrane lipid peroxidation occurs. These data support a key role for GSK3beta as a mediator of the signalling pathways activated by oxidative stress, and therefore it may be included among the redox-sensitive enzymes.