Replicating neuroblastoma cells in different cell cycle phases display different vulnerability to amyloid toxicity.

A key role of mitotic activation in neuronal cell death in early stages of Alzheimer's disease (AD) has been suggested. Apparently, terminally differentiated neurons are precluded from mitotic division, yet some phenotypic markers of cell cycling are present in AD-vulnerable brain areas. In this paper, we investigated whether dividing human neuroblastoma cells are preferentially vulnerable to amyloid aggregate toxicity in some specific cell cycle stage(s). Our data indicate that Abeta1-40/42 aggregates added to the cell culture media bind to the plasma membrane and are internalized faster in the S than in the G2/M and G1 cells possibly as a result of a lower content in membrane cholesterol in the former. Earlier and sharper increases in reactive oxygen species production triggered a membrane oxidative injury and a significant impairment of antioxidant capacity, eventually culminating with apoptotic activation in S and, to a lesser extent, in G2/M exposed cells. G1 cells appeared more resistant to the amyloid-induced oxidative attack possibly because of their higher antioxidant capacity. The high vulnerability of S cells to aggregate toxicity extends previous data suggesting that neuronal loss in AD could result from mitotic reactivation of terminally differentiated neurons with arrest in the S phase.

Differentiation increases the resistance of neuronal cells to amyloid toxicity.

A substantial lack of information is recognized on the features underlying the variable susceptibility to amyloid aggregate toxicity of cells with different phenotypes. Recently, we showed that different cell types are variously affected by early aggregates of a prokaryotic hydrogenase domain (HypF-N). In the present study we investigated whether differentiation affects cell susceptibility to amyloid injury using a human neurotypic SH-SY5Y cell differentiation model. We found that retinoic acid-differentiated cells were significantly more resistant against Abeta1-40, Abeta1-42 and HypF-N prefibrillar aggregate toxicity respect to undifferentiated cells treated similarly. Earlier and sharper increases in cytosolic Ca(2+) and ROS with marked lipid peroxidation and mitochondrial dysfunction were also detected in exposed undifferentiated cells resulting in apoptosis activation. The reduced vulnerability of differentiated cells matched a more efficient Ca(2+)-ATPase equipment and a higher total antioxidant capacity. Finally, increasing the content of membrane cholesterol resulted in a remarkable reduction of vulnerability and ability to bind the aggregates in either undifferentiated and differentiated cells.

Prefibrillar amyloid aggregates could be generic toxins in higher organisms.

More than 40 human diseases are associated with fibrillar deposits of specific peptides or proteins in tissue. Amyloid fibrils, or their precursors, can be highly toxic to cells, suggesting their key role in disease pathogenesis. Proteins not associated with any disease are able to form oligomers and amyloid assemblies in vitro displaying structures and cytotoxicity comparable with those of aggregates of disease-related polypeptides. In isolated cells, such toxicity has been shown to result from increased membrane permeability with disruption of ion homeostasis and oxidative stress. Here we microinjected into the nucleus basalis magnocellularis of rat brains aggregates of an Src homology 3 domain and the N-terminal domain of the prokaryotic HypF, neither of which is associated with amyloid disease. Prefibrillar aggregates of both proteins, but not their mature fibrils or soluble monomers, impaired cholinergic neuron viability in a dose-dependent manner similar to that seen in cell cultures. Contrary to the situation with cultured cells, however, under our experimental conditions, cell stress in tissue is not followed by a comparable level of cell death, a result that is very likely to reflect the presence of protective mechanisms reducing aggregate toxicity. These findings support the hypothesis that neurodegenerative disorders result primarily from a generic cell dysfunction caused by early misfolded species in the aggregation process.

Differing molecular mechanisms appear to underlie early toxicity of prefibrillar HypF-N aggregates to different cell types.

Considerable attention has been paid to the high cytotoxic potential of small, prefibrillar aggregates of proteins/peptides, either associated or not associated with amyloid diseases. Recently, we reported that different cell types are variously affected by early aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N), a protein not involved in any disease. In this study, we provide detailed information on a chain of events triggered in Hend murine endothelial cells and IMR90 fibroblasts, which have previously been shown to be highly vulnerable or very resistant, respectively, to HypF-N aggregates. Initially, both cell lines displayed impaired viability upon exposure to HypF-N toxic aggregates; however, at longer exposure times, IMR90 cells recovered completely, whereas Hend cells did not. In particular, significant initial mitochondrial permeability transition (MPT) pore opening was found in IMR90 cells followed by a sudden repair of membrane integrity with rapid and efficient inhibition of cytochrome c and AIF release, and upregulation of Bcl-2. The greater resistance of IMR90 fibroblasts may also be due to a higher cholesterol content in the plasma membrane, which disfavours interaction with the aggregates. In contrast, Hend cells, which have less membrane cholesterol, showed delayed MPT opening with prolonged translocation of cytochrome c into the cytosol. Finally, the caspase 9 active fragment was increased significantly in both Hend and IMR90 cells; however, only Hend cells showed caspase 8 and caspase 3 activation with DNA fragmentation. From our data, the different responses of the two cell types to the same aggregates appear to be associated with two key events: (a) aggregate interaction with the plasma membrane, disfavoured by a high level of membrane cholesterol; and (b) alterations in mitochondrial functionality, leading to the release of pro-apoptotic stimuli, which are counteracted by upregulation of Bcl-2.

Environmental monitoring of occupational exposure to N,N-dimethylformamide: comparison between active and diffusive sampling.

OBJECTIVES: The objective of this study is to optimize the evaluation of the exposure to N,N-dimethylformamide (DMF) in synthetic leather factories by diffusive samplers. The DMF exposure was monitored in synthetic leather factories by two sampler types: active and diffusive. METHODS: Air measurements were carried out using two different personal air samplers, a diffusive and an active one. The diffusive sampling method, TK200 with charcoal filters, was examined in comparison with pumping through NIOSH silica gel tubes workplace air as with the currently available "gold standard". The evaluation was carried out, in two different years but in the same season, for all the duration of the shift, i.e. 8 h on workers employed in five different factories in the district of Florence and Prato (Italy). RESULTS: The statistical and graphical analysis of data show a good correlation between active and passive samplers (r = 0.96, P < 0.001, n = 91), a good linear regression (DMF(diffusive )= 0.95 DMF(active) + 0.15, R (2) = 0.92), a not statistically significant difference between data (tested by paired t test and non-parametric Wilcoxon test). Moreover, all these results are confirmed for data lower and higher than TLV-TWA, in particular we found a significant Pearson correlation (r = 0.92, P < 0.001, n = 83; r = 0.92, P < 0.05, n = 8, respectively) and a significant linear regression (DMF(diffusive )= 0.88 DMF(active) + 0.73, R (2 )= 0.86; DMF(diffusive )= 0.90 DMF(active) + 3.76, R (2 )= 0.85). Besides, the analysis of graphical representations confirmed the previous evidences. Finally, we can not find a significant difference between different types of job. CONCLUSIONS: Due to the good agreement between the two groups of data, the TK200 samplers can be considered as a simpler approach than the pump for screening worker exposures to DMF.

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts.

Much experimental evidence suggests that an imbalance in cellular redox status is a major factor in the pathogenesis of Alzheimer's disease (AD). Our previous data showed a marked increase in membrane lipoperoxidation in primary fibroblasts from familial AD (FAD) patients. In the present study, we demonstrate that when oligomeric structures of Abeta 1-40 and Abeta 1-42 are added to the culture media, they accumulate quicker near the plasma membrane, and are internalized faster and mostly in APPV717I fibroblasts than in age-matched healthy cells; this results in an earlier and sharper increase in the production of reactive oxygen species (ROS). Higher ROS production leads in turn to an increase in membrane oxidative-injury and significant impairment of cellular antioxidant capacity, giving rise to apoptotic cascade activation and finally to a necrotic outcome. In contrast, healthy fibroblasts appear more resistant to amyloid oxidative-attack, possibly as a result of their plasma membrane integrity and powerful antioxidant capacity. Our data are consistent with increasing evidence that prefibrillar aggregates, compared to mature fibrils, are likely the more toxic species of the peptides. These findings provide compelling evidence that cells bearing increased membrane lipoperoxidation are more susceptible to aggregate toxicity as a result of their reduced ability to counteract amyloid oligomeric attack.

Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates.

It has been reported that different tissue or cultured cell types are variously affected by the exposure to toxic protein aggregates, however a substantial lack of information exists about the biochemical basis of cell resistance or susceptibility to the aggregates. We investigated the extent of the cytotoxic effects elicited by supplementing the media of a panel of cultured cell lines with aggregates of HypF-N, a prokaryotic domain not associated with any amyloid disease. The cell types exposed to early, pre-fibrillar aggregates (not mature fibrils) displayed variable susceptibility to damage and to apoptotic death with a significant inverse relation to membrane content in cholesterol. Susceptibility to damage by the aggregates was also found to be significantly related to the ability of cells to counteract early modifications of the intracellular free Ca2+ and redox status. Accordingly, cell resistance appeared related to the efficiency of the biochemical equipment leading any cell line to sustain the activity of Ca2+ pumps while maintaining under control the oxidative stress associated with the increased metabolic rate. Our data depict membrane destabilization and the subsequent early derangement of ion balance and intracellular redox status as key events in targeting exposed cells to apoptotic death.