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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.
The amyloidoses are a group of protein-folding diseases in which specific peptides or proteins, which are either incorrectly folded or unfolded, aggregate intra- or extracellularly into polymeric assemblies rich in β sheet, and are eventually deposited in tissue as amyloid fibrils [1,2]. Amyloid diseases include a number of sporadic, familial or transmissible degenerative pathologies affecting either the central nervous system (Alzheimer's, Parkinson's and Creutzfeldt–Jakob diseases) or a variety of peripheral tissues and organs (systemic amyloidoses and type II diabetes) . Since 1998, a growing number of peptides and proteins not associated with known protein deposition diseases have been shown to aggregate in vitro, under suitable experimental conditions, into fibrils that are indistinguishable from those associated with pathological conditions [3,4]. This has led to the proposal that the ability to form amyloid assemblies can be considered a generic property inherent in any polypeptide chain .
Currently, considerable attention is focused on the cytotoxic potential of small prefibrillar protein aggregates arising initially in the protein fibrillization pathway. This cytotoxic potential appears to be higher than that of mature fibrils [2,3]. These early assemblies share basic structural features that, in most cases at least, seem to underlie the common biochemical mechanisms of cytotoxicity [5,6]. Cells exposed to toxic prefibrillar aggregates apparently die as a consequence of apoptosis [7–9] or, less frequently, by secondary necrosis [10–13]. Recent studies have shown that cells experiencing prefibrillar aggregates undergo similar early biochemical modifications; these include interaction between the aggregates and cell membranes and, possibly, interaction with membrane receptors [14–16], followed by an imbalance in the intracellular redox status [13,15] and ion levels [1,17], and mitochondria impairment [9,18], together with other modifications such as lipid homeostasis. Prefibrillar aggregates of a number of peptides associated with amyloid diseases can also induce mitochondrial permeability transition (MPT) pore opening in exposed cells, allowing molecules smaller than 1500 Da to diffuse freely between the matrix and the cytosol [18–23]. These modifications can result in the collapse of the transmembrane electrochemical gradient with loss of solutes from the matrix, mitochondrial swelling, release of proapoptotic factors such as cytochrome c and AIF, and activation of procaspase 2, 3 and 9. Cytochrome c, in complex with the cytosolic factor Apaf-1 activates the caspase-dependent apoptotic pathway, whereas AIF translocates to the nucleus inducing chromatin condensation and large-scale fragmentation of DNA [23,24].
Similar modifications have also been found in cells exposed to prefibrillar amyloid aggregates of proteins that are not associated with disease, including the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) [5,25]. In particular, when added to the cell culture media, early HypF-N aggregates can be internalized by the cells , where they induce modifications in intracellular free Ca2+ and reactive oxygen species (ROS) levels [10–13,26], reducing the potential across the inner mitochondrial membrane. In turn, ROS trigger the intrinsic or extrinsic apoptotic pathways , or in some cases lead to cell death by necrosis [13,26]. Data on the toxicity of HypF-N prefibrillar aggregates suggest a mechanism of cell death that is possibly shared with the prefibrillar aggregates of most peptides and proteins .
Much research is currently being carried out into molecules that are able to avoid the appearance of misfolded proteins and their initial aggregates in tissue. Notwithstanding the validity of such an approach, better knowledge of the biochemical basis of cell vulnerability to protein aggregates may also provide clues to possible interventions aimed at increasing the resistance of cells to these toxic assemblies. We sought to provide information on the chain of events that leads to death in cells experiencing toxic aggregates by investigating features of the apoptotic pathways triggered in two different cell lines upon exposure to toxic HypF-N prefibrillar aggregates. Although different cell types often show similar biochemical alterations, they are variously affected by exposure to the same toxic protein aggregates, such that only specific cell populations are stressed [14,15,28,29]. Such differences in vulnerability reflect the inherent ability of any cell to disfavour aggregate interaction with the plasma membrane, and possibly other membranes, and the subsequent early modifications by using its specific biochemical equipment. This equipment includes the specific membrane lipid composition, the total antioxidant defences (TAC), the efficiency of Ca2+ extrusion membrane pumps and the energy load (ATP availability).
A recent study showed large variations in the toxic effects of HypF-N prefibrillar aggregates on a panel of cultured cell lines , leading us to rank the cell lines according to their vulnerability. This study was carried out using murine endothelial Hend cells and human IMR90 fibroblasts; these were chosen as examples of cells that are very vulnerable or very resistant to toxic HypF-N aggregates, respectively. The different vulnerability of the cell lines was associated with different plasma membrane cholesterol content, which has been shown to disfavour membrane interaction with aggregates . We found that both cell lines showed early activation of a programmed cell death following exposure to the aggregates; however, IMR90 cells were able to counteract the toxic insult and recover despite initial impairment. Details of the apparent differences in the specific apoptotic pathways in the two cell lines are also discussed.
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It is known that only specific cell types are impaired in tissues facing amyloid deposits [29,36] and that cell stress eventually leads to cell death by apoptosis or, in some cases, to secondary necrosis [12,37]. We previously reported that the vulnerability of different cell lines to toxic HypF-N prefibrillar aggregates appears to be related to intrinsic biochemical features of the cells . We also provided data suggesting that the choice between an apoptotic and a necrotic outcome depends on the timing and severity of mitochondria impairment . In this study, we investigated the apoptotic pathways activated in two different cell lines, Hend and IMR90, chosen as examples of cells that are highly vulnerable or highly resistant to insult by toxic prefibrillar aggregates, respectively. The differing susceptibility to the damage by the aggregates was not an artefact due to a different dose–response in each cell line, as shown by the substantial resistance of IMR90 cells to much higher amounts of aggregates than those impairing Hend cells. Both cell lines appeared significantly stressed after 3 h exposure to the aggregates. At this time, cell damage appeared substantially reversible even for the most heavily affected Hend cells; however, at longer exposure times cell recovery was increasingly less complete, indicating a progressive deterioration in cell viability. At longer exposure times, IMR90 cells recovered completely despite early activation of the apoptotic programme, whereas a significant fraction of Hend cells underwent apoptotic death at 24 h exposure. Therefore, the differing vulnerability seen in the two cell lines following 24 h exposure to the aggregates appears to be associated with the greater ability of IMR90 cells to counteract the early biochemical modifications underlying activation of the apoptotic pathway, rather than an effect of a lower sensitivity to similar amounts of aggregates.
Severe alterations in many biochemical parameters, including intracellular redox status, energy load and free Ca2+ homeostasis , as well as membrane lipid composition [14,38], appear to be key factors in favouring cell impairment or resistance to the toxic aggregates of peptides and proteins either associated [1,39] or not associated with amyloid diseases [13,14]. It is also well known that protein prefibrillar aggregates can interact with the plasma membrane of exposed cells inducing modifications in the lipid or proteolipid structure, or self-assembling into pores thus inducing alterations in membrane selective permeability . In this scenario, it is conceivable that cells endowed with higher basal antioxidant defences and efficient Ca2+ pumps are better suited to resist any increase in free Ca2+ (or other ion) and the consequent biochemical modifications .
We found that the highly vulnerable Hend cells exposed to HypF-N toxic aggregates displayed earlier and greater increases in both intracellular ROS and free Ca2+ when compared with the more resistant IMR90 cells. The early Ca2+ increase may induce ROS overproduction by speeding up oxidative metabolism to supply energy for the increased activity of the membrane Ca2+ pumps . The resulting oxidative stress may subsequently favour entry of Ca2+ into the cell with endoplasmic reticulum stress and mitochondrial impairment eventually targeting the cell for apoptotic death [40,41]. Resistance of IMR90 to aggregate damage was previously found to be significantly related to the high efficiency of these cells in counteracting early modifications of the intracellular free Ca2+ and redox status . Under our experimental conditions, both exposed cell lines displayed ATP depletion supporting mitochondria involvement; however, Hend cells, endowed with a lower basal energy load, showed much more serious and prolonged loss of ATP than IMR90 cells, indicating that the former were less suited to counteracting ion balance derangement, which may explain their higher vulnerability to apoptotic death.
The higher resistance of IMR90 fibroblasts to toxic insult by the aggregates may also result from a significant upregulation of Bcl-2. Such an antiapoptotic factor acts as an endogenous inhibitor of MPT pore opening and mitochondrial apoptotic channel (MAC) formation by Bax and Bak [42,43], resulting in the release of proapoptotic factors such as AIF and cytochrome c[1,23] and inhibition of the proteolytic processing of AIF . Interestingly, nuclear AIF was unchanged in Hend cells, suggesting that it is not involved in the apoptotic cascade. The partial release of cytochrome c not associated with AIF release found in Hend cells agrees with previous data on infrared-irradiated human fibroblasts . AIF was significantly increased in the nuclei of IMR90 cells after 3 h exposure, where it matched, although in a delayed manner, cytochrome c release. However, the release of AIF and cytochrome c was not sustained at longer exposure times, where upregulation of Bcl-2 occurred. The latter could disassemble MAC, the proposed channel allowing cytochrome c to translocate to the cytosol , thus explaining the complete recovery in mitochondrial function, which is also supported by the recovery in ATP levels, and hence cell viability.
As pointed out above, both exposed cell lines displayed early translocation of cytochrome c from the mitochondria to the cytosol. However, cytochrome c release was much higher and decreased rapidly in IMR90 cells, whereas in Hend cells it increased progressively up to 24 h exposure. Once released from the mitochondria, cytochrome c, in association with Apaf-1, is involved in the activation of caspase 9. Indeed, both Hend and IMR90 cells showed a significant increase in the caspase 9 active fragment, however, the latter occurred earlier and was higher in IMR90 cells than in Hend cells, where a sharp increase in caspase 9 activation was observed after 24 h exposure. Moreover, the extrinsic apoptotic pathway triggered by caspase 8 cleavage was activated only in Hend cells after just 20 min exposure to aggregates. It is known that activation of the effector caspase 3 occurs downstream of caspase 8 and caspase 9 cleavages in response to differing apoptotic stimuli; once activated, caspase 3 can activate caspase 9 directly in a feedback loop, and caspase 8 indirectly . Indeed, in Hend cells exposed to HypF-N aggregates the increase in caspase 3 active fragment was earlier and sharper than in IMR90 cells and was probably responsible for the late activation of caspase 8 in Hend cells. It seems likely, therefore, that the different response of either cell line to the same toxic insult can be traced to, among others, a differing interaction between the aggregates and the plasma membrane, resulting in differing activation of the apoptotic extrinsic pathway.
We have previously shown that HypF-N prefibrillar aggregates interact with the plasma membrane of Hend cells more extensively than with the membrane of more resistant cell lines, apparently due to a different lipid composition, including cholesterol . In this study, we confirmed the dependence of cell resistance on membrane cholesterol content, which is considerably higher in IMR90 cells than in Hend cells. Therefore, according to our previous results, we expect a reduced interaction between the aggregates and the plasma membrane in IMR90 cells compared with Hend cells. In IMR90 cells, reduced membrane fluidity with increased resistance to the destabilizing effects of the aggregates can also be hypothesized. These considerations may explain the lack of activation of the apoptotic extrinsic pathway and cell recovery after the initial insult; they also agree with recent findings indicating that cholesterol can modulate membrane-associated Aβ fibrillogenesis and neurotoxicity, and that decreasing the fluidity of brain lipid bilayers reduces the interaction of Aβ40 with the bilayer surface and insertion of the latter inside the bilayer itself .
Caspase 3 activation targeted exposed Hend cells to apoptotic death. In these cells, the amount of histone-associated oligonucleosomes released into the cytoplasm confirmed a significant increase in DNA fragmentation, in agreement with the severe impairment of cell viability and the increases in ROS and active caspase 3 fragment. Caspase 3 activation resulted in PARP cleavage and inactivation. In contrast, IMR90 cells appeared more resistant to DNA oxidative attack, possibly because of their higher basal antioxidant capacity  and/or reduced damage and permeabilization of the cell membrane by the aggregates.
Overall, the data support differing scenarios for the responses of Hend and IMR90 cells to the toxic aggregates. These differences can, at least in part, be traced to differing involvement of the plasma membranes with the aggregates (Fig. 9). In Hend cells, reduced membrane cholesterol content favours interaction of the aggregates with the plasma membrane  leading to membrane destabilization and permeabilization. The structural and biochemical modifications of the plasma membrane result in early and transient increases in cytosolic free Ca2+ that appears sufficient to trigger the extrinsic apoptotic pathway. In this case, the metabolic efficiency of the mitochondria appears to be impaired at late exposure times, possibly following the oxidative stress accompanying the initial high energy demand to counteract the altered membrane permeability. However, mitochondria do not release large amounts of proapoptotic factors, nor is the antiapoptotic Bcl-2 upregulated, and activation of the effector caspase 3 appears to result mainly from the early activation of caspase 8.
Figure 9. Representative flow-chart of the molecular events underlying cell impairment upon exposure to HypF-N prefibrillar aggregates. Membrane cholesterol content, increases in intracellular free Ca2+ and ROS, mitochondrial status, cytochrome c and AIF release, Bcl-2 expression, and caspase 8 and caspase 9 activation support different scenarios in the response of Hend and IMR90 cells to the same toxic aggregates.
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In IMR90 cells, whose plasma membranes are richer in cholesterol, the extrinsic pathway does not appear to be activated. Nevertheless, early involvement of the mitochondria is apparent in this cell line, with a significant but transient release of cytochrome c and, slightly later, of AIF, suggesting that a signal arising from the plasma membrane may trigger transient MAC organization in the mitochondria outer membrane. At the same time, a significant but transient activation of caspase 9 was seen with subsequent recovery to basal values (Fig. 9). The ability of these cells to recover after an initial insult can be tentatively traced to the higher resistance of a plasma membrane rich in cholesterol to the destabilizing effects of the aggregates, and to the early upregulation of Bcl-2, possibly counteracting the initial formation of MAC in the outer mitochondrial membrane.