N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.     

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

Neuronal nicotinic receptor deficits in Alzheimer patients with the Swedish amyloid precursor protein 670/671 mutation

The influence of beta-amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (-73 to -87%) as well as in sporadic Alzheimer's disease (AD) cases (-37 to -57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both alpha3 and alpha4 and to alpha4 nAChR subtypes, respectively. Saturation binding studies with [3H]epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in Bmax (-82%) for the high-affinity site was observed in APP 670/671 subjects with no change in K(D) compared with controls (0.018 nM APP 670/671; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.     

The phosphatidylinositol 3-kinase inhibitor wortmannin alters the metabolism of the Alzheimer's amyloid precursor protein

One of the hallmarks of Alzheimer's disease is the accumulation of senile plaques in brain, extracellular lesions comprised mostly of aggregates of the amyloid beta-peptide (Abeta). Abeta is proteolytically derived from the Alzheimer's amyloid precursor protein (APP). The generation of Abeta and nonamyloidogenic derivatives of APP involves utilization of alternative processing pathways and multiple subcellular compartments. To improve our understanding of the regulation of APP processing, we investigated the effects of wortmannin, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, on APP processing. PI3-kinases form a multifaceted family of enzymes that represent converging points for multiple signal transduction pathways and also act as key regulators of vesicular trafficking. In N2a neuroblastoma cells expressing either wild-type APP or the "Swedish" familial Alzheimer's disease-associated mutant variant of APP, wortmannin treatment resulted in decreased release of both Abeta and soluble APPalpha. In parallel, full-length APP and both processed derivatives accumulated inside the cells. These effects were not present at nanomolar concentrations of wortmannin, but only at micromolar concentrations, implying the possible involvement of a recently described trans-Golgi network (TGN)-associated PI3-kinase that is resistant to nanomolar concentrations of the inhibitor, but sensitive to micromolar concentrations. All effects were reversible when the drug was removed from the cell culture medium. Given the suspected site of action of this novel PI3-kinase activity at the TGN, it is tempting to speculate that the unexpected increase in the levels of both intracellular soluble APPalpha and intracellular Abeta might be due to wortmannin-induced covesiculation of APP together with its respective secretase enzymes within the TGN, leading to the execution of alpha-, beta-, and gamma-secretase reactions.     

Alteration of Phosphoinositide Degradation by Cytosolic and Membrane-Bound Phospholipases after Forebrain Ischemia - Reperfussion in Gerbil: Effects of Amyloid Beta Peptide

The reperfusion of previously ischemic brain is associated with exacerbation of cellular injury. Reperfusion occasionally potentates release of intracellular enzymes, influx of Ca²⁺, breakdown of membrane phospholipids, accumulation of amyloid precursor protein or amyloid -(like) proteins, and apolipoprotein E. In this study, the effect of reperfusion injury on the activity of cerebral cortex enzymes acting on phosphatidyl [³H] inositol (PI) and [^l4C-arachidonoyl] PI was investigated. Moreover the effect of amyloid 25–35 on PI degradation by phospholipase(s) of normoxic brain and subjected to ichemia-reperfussion injury was determined. Brain ischemia in gerbils (Meriones unguiculatus) was induced by ligation of both common carotid arteries for 5 min and then brains were perfused for 15 min, 2 h and 7 days. Statistically significant activation of enzyme(s) involved in phosphatidylinositol degradation in gerbils subjected to ischemia-reperfusion injury was observed. Nearly all gerbils showed a higher activity of cytosolic PI phos-pholipase C (PLC) at 15 min after ischemia. Concomitantly, the significant enhancement of the level of DAG and AA radioactivity at this short reperfusion time confirmed the active PI degradation by phospholipase(s) in cerebral cortex and hippocampus. After a prolonged reperfusion time of 7 days after ischemia, both cytosolic and membrane-bound forms of PI-PLC were activated. The question arises if alteration of membranes by the degradation of phospholipids occurring after an ischemic episode potentates the effect of A on membrane-bound enzymes. A neuro-toxic fragment of amyloid, A 25–35, incubated in the presence of endogenous Ca²⁺, increased significantly the PI-PLC activity of normoxic brain. In its non-aggregated form, A 25–35 activates PI-PLC but in the aggregated form the enzymatic activity decreased. Thus, A 25–35 exerts a similar effect on the membrane-bound PI-PLC from normoxic brain or subjected to ischemia reperfussion injury. We conclude that the degradation of phosphatidylinositol by cytosolic phosphoinositide-phospholipase C may contribute to the pathophysiology of delayed neuronal death following cerebral ischemia. Thus, a specific inhibitor of this enzyme(s) may offer therapeutic strategies to protect the brain from damage triggered by ischemia. Ischemia-reperfusion injury had no effect on A-evoked alterations of synaptic plasma membrane-bound PI-PLC.     

Trace metal contamination initiates the apparent auto-aggregation,amyloidosis, and oligomerization of Alzheimer’s Aβ peptides

Nucleation-dependent protein aggregation (seeding) and amyloid fibril-free formation of soluble SDS-resistant oligomers (oligomerization) by hydrophobic interaction is an in vitro model thought to propagate -amyloid (A) deposition, accumulation, and incur neurotoxicity and synaptotoxicity in Alzheimers disease (AD), and other amyloid-associated neurodegenerative diseases. However, A is a high-affinity metalloprotein that aggregates in the presence of biometals (zinc, copper, and iron), and neocortical A deposition is abolished by genetic ablation of synaptic zinc in transgenic mice. We now present in vitro evidence that trace (0.8 µM) levels of zinc, copper, and iron, present as common contaminants of laboratory buffers and culture media, are the actual initiators of the classic A1–42-mediated seeding process and A oligomerization. Replicating the experimental conditions of earlier workers, we found that the in vitro precipitation and amyloidosis of A1–40 (20 µM) initiated by A1–42 (2 µM) were abolished by chelation of trace metal contaminants. Further, metal chelation attenuated formation of soluble A oligomers from a cell-free culture medium. These data suggest that protein self-assembly and oligomerization are not spontaneous in this system as previously thought, and that there may be an obligatory role for metal ions in initiating A amyloidosis and oligomerization.     

Acidic-rich region of amyloid precursor protein induces glial cell apoptosis

Amyloid precursor protein (APP) has several caspase cleavage sites in its C-terminal cytoplasmic domain and N-terminal extracellular domain. Caspase cleavages of APP at its cytosolic tail may result in releasing the domain and inducing cell death. During apoptosis, the N-terminal domain may also be processed at amino acids 197 and 219 by caspases leading to unmasking of an acidic-rich region (AR). In this study, AR-exposing APP was shown to inhibit cell growth after transfection into RBA-1 astrocytes and BV-2 microglial cells. The recombinant AR from residue 220 to 288 of APP (APP220-288) was produced and its biological activities were analyzed. APP220-288 induced morphological changes, cell death, and DNA fragmentation in BV-2 and RBA-1 cells. However, AR was determined to have no apparent effects in suspension cells, erythroleukemia K562 cells, and Jurkat T cells. The cytotoxicity was depending on negative charge cluster and the apoptotic activity of AR was attributed to the inhibition of cell adhesion. In BV-2 microglial cells, AR significantly stimulated Fas expression, although expressions of the pro-inflammatory cytokine genes were not detected. APP220-288 also induced nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. These findings indicate that the acidic-rich domain of APP may have apoptotic activity due to inhibition of cell adhesion and induction of iNOS and Fas expressions. Moreover, unmasking the apoptosis-induced AR may activate and exacerbate glial cells which in turn lead to further progression of the death program.     

Insulin amyloid fibrillation at above 100 degrees C: new insights into protein folding under extreme temperatures

To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140 degrees C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100 degrees C. Amyloid formation was gradually replaced by random coil generation after approximately 80 degrees C until no amyloid was detected at 140 degrees C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140 degrees C and incubated at 100 degrees C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation.     

Observation of sequence specificity in the seeding of protein amyloid fibrils

It is well established that the rate of formation of fibrils by amyloidogenic proteins is enhanced by the addition of preformed fibrils, a phenomenon known as seeding. We show that the efficiency of seeding fibril formation from solutions of hen lysozyme by a series of other proteins depends strongly on the similarity of their sequences. This observation is consistent with the importance of long-range interactions in stabilizing the core structure of amyloid fibrils and may be associated with the existence of a species barrier observed in the transmissible spongiform encephalopathies. In addition, it is consistent with the observation of a single dominant type of protein in the deposits associated with each form of amyloid disease.     

Recombinant human serum amyloid P component from Pichia pastoris: production and characterization

Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.