The results observed in this work demonstrated the fundamental role of PrPC under different conditions of oxidative stress in astrocyte cells. Astrocytes provide essential metabolic and functional support for neurons (55, 102, 103). However, after CNS injury or illness, overproduction of ROS or defects in detoxification may activate a coordinated astrocytic response: a series of biochemical and morphological changes collectively referred to as reactive astrogliosis. (104, 105). It was found here that the astrocyte cell line with PrPC (wild-type) is more resistant to oxidative stress. This fact showed that PrPC is essential for activating the cell death mechanism via apoptosis, and consequently, neuroprotection. The results obtained for cell viability demonstrated a slight cell vulnerability regarding the cell viability of PrPC-null compared to wild-type astrocytes under oxidative stress conditions. The results indicated cumulative dysregulation in proliferation and probably self-regeneration caused by the absence of PrPC, affecting the cell signaling cascade to maintain survival and cell homeostasis. Evidence in the literature showed the antioxidant capacity of the cellular prion protein, and in knockout cells for PrPC, their sensitivity to oxidative stress becomes greater (79, 80). PrPC is believed to mediate signaling for the expression of antioxidant enzymes such as SOD1 and thus plays a protective role against oxidative stress generated by ROS (13, 81, 82). In addition, astrocytes are described as copper accumulators, as they have a high affinity with the metal carriers in question, making them highly resistant to toxicity caused by excess copper (57). The prion protein is also cited as a contributor to regulating the entry and distribution of copper in astrocytes (57, 83, 84). Here, we can observe in the copper measurements that the PrPC-null astrocytes had less intracellular copper than the wild-type cell, corroborating this copper-transport role of PrPC. The iron imbalance exhibited by the PrPC-null cells under oxidative conditions indicated that the absence of the PrPC caused the spread of damage to astrocyte cells can also be due to iron misregulation.
Studies showed that PrPC is one of the proteins involved in the regulation of iron by astrocytes involved in the redox process of Fe3+ to Fe2+ (89). PrPC-null cells had fewer calcium levels than wild-type cells, compromising the signaling for cellular defense mechanisms to induce apoptosis, thus showing neuroprotective and antioxidant action of PrPC. The dysregulation in calcium homeostasis is also directly linked to A.D. The calcium dysfunction can influence the accumulation of Aβ and hyperphosphorylation of the tau protein (66). Fragments containing toxic Aβ peptides increase intracellular calcium. Disruption of calcium homeostasis may be one of the main mechanisms by which Aβ manifests its neurotoxicity (92–94). We showed that in the presence of PrPC under oxidative stress, the calcium levels were decreased, indicating the possible paralysis of cellular replication processes, leading to the programmed cell death process (apoptosis)(95).
The observation of higher p-Tau levels compared to wild-type astrocytes confirmed the hypothesis of the neuroprotective function of PrPC. It is known that an increase in iron levels due to oxidative stress is a factor that can trigger neurodegeneration. Astrocytes could internalize and degrade toxic β-amyloid peptides, as a crucial defense role in developing and advancing A.D. (41, 96, 97). However, it is not known how this degradation happens. Apolipoprotein E (APOE) is involved in the process (97–99). Almost nothing is known if the PrPC participates in the degradation of Aβo in astrocytes (72, 100).
The results obtained in this work showed that the absence of cellular prion protein in astrocytes leads to large amounts of protein aggregates. This fact was observed by the large band of aggregates formed in the PrPC-null cells under the induction of neurodegeneration in the absence of the reducing agent β-mercaptoethanol when compared to the wild-type cell. These results agree with those observed by Nieznanski et al., indicating that the PrPC and its N-terminal fragment inhibits Aβo 1–42 amyloidogenesis and cytotoxicity (101).
In cell viability results, cell death was observed only with STS condition (used as a control experiment for damage and cell death) and H2O2 for both cell lines. However, the results obtained for the expression of Caspase 9 protein (Fig. 4 - A), showed increased expression for wild-type cells in the incubation with STS and H2O2 compared to its negative control. This result agrees with the results obtained for significant changes in calcium levels in the wild-type, correlating them as a response caused by the induction of cell death via apoptosis, where through the calcium channels (cell signaling through intracellular release) the cascade that leads to the activation of caspases can be initiated (56). In this work, the cascade of caspases was activated in the wild-type cells, in contrast to the PrPC-null cells. This fact can be due to the balanced signaling between Bcl2-Bak mediated by the participation of PrPC, allowing the mitochondrial release of cytochrome c and the release of intracellular calcium to the extracellular medium (in agreement with results obtained in the determination of metals). All these events recruited the activation of procaspase and led to apoptosis.
It was observed that the SOD1 expression was not affected by the absence of PrPC. Thus, the Cu reduction in CCS may contribute to the decrease in SOD1 activity already observed in null mice for PrPC (75, 107). The link between PrPC expression and SOD1 points to common pathways in regulating its expression and indicates the role between PrPC and cell protection from oxidative stress (27, 28, 108). The cellular prion protein has been shown to have similar activity to SOD1 and has been demonstrated by unequivocal evidence that PrPC can regulate the copper redox cycle. Thus, changes in PrPC expression result in unbalanced proteins associated with copper metabolism to compensate for the loss in PrPC expression and consequently changes in intracellular copper levels (107, 108).
A recent discovery indicates a new role for SOD1 as a nuclear transcription factor to control the overall response to oxidative stress (109). Under oxidative stress induction with H2O2, it was found that SOD1 expression was not altered. Still, SOD1 translocation to the cell nucleus occurred, regulating the expression of a large set of oxidative response genes known to provide resistance to oxidative stress and repair DNA damage. The results obtained in this work showed that the cellular prion protein is responsible for bringing SOD1 to the cell nucleus under the induction of oxidative stress in astrocyte cells. Both wild-type and PrPC-null cells exhibit similar baseline levels of SOD1 expression. The presence of PrPC promoted antioxidant activity in the wild-type cell. In the PrPC-null cells, we observed lower Aβo internalization. The prion protein led to less intracellular ROS generation and less required antioxidant activity, contrary to the PrPC-null cells, which required the antioxidant defense performed by SODs. The translocation of SOD1 to the nucleus occurred only in the presence of PrPC in the wild-type cell, indicating the crucial link to explain the SOD1 translocation to the nucleus in oxidative stress (109).
Like PrPC, RAGEs bind to β-amyloid peptides and transport them by BBB. More evidence suggested that they can act on their accumulation in brain tissue (113), stimulating the activation of pro-inflammatory cytokines (such as NFĸB) and ROS release, which leads to damage to neurons and causes dysfunction in the BBB. RAGE is then described as a central mediator of Aβ cytotoxicity (114–116). From the results obtained here, RAGEs activation was observed in the presence of the PrPC in the nuclear region of the astrocyte. However, such an isolated result does not infer what culminates such interaction. The p53 activation leads to modifications such as phosphorylation and acetylation, resulting in isoforms that initiate cell signaling for cell senescence and apoptosis cascade by the positive regulation of genes from pro-apoptotic proteins like Bax and Bak and inhibits the gene for Bcl2 (anti-apoptotic protein)(117, 118). However, it was reported that a different cellular response to oxidative stress was observed independent of p53 through the overexpression of SODs, precisely by inducing oxidative stress and consequent DNA damage. Another apoptosis signaling pathway was observed in which the dependence drastically decreases by activating the caspases displayed by p53 (119). The NFĸB protein has been reported to modulate p53 activity. However, NFĸB expression can be downregulated by overexpression of SOD1 (120). As PrPC exhibits antioxidant activity similar to SOD1, the response observed here for p53 and NFĸB in the wild-type cells indicated that the apoptosis activation mechanism is not dependent on p53.
Consequently, the apoptotic mechanism is followed by the cascade of anti/pro-apoptotic proteins released by cytochrome c. Still, another mechanism that possibly the PrPC acts as the protagonist is not yet unraveled. The levels of p53 observed in the absence of PrPC in the null cells can be justified as an alternative protective mechanism for the cell to defend itself against DNA damage. But it fails because it cannot eliminate the damage by apoptosis.
Through the expression and localization of the analyzed proteins, it was possible to notice that the internalization of Aβo occurs mainly in the PrPC-null cells, consequently leading to a higher probability of protein aggregation. The internalization of the oligomers may also have appeared for the native cells. However, if such an event had occurred, the PrPC would have been responsible for their degradation. Furthermore, it was found that under Aβo incubation, the ATP7B activation occurred in the presence of PrPC. This result possibly relates to signaling displayed by PrPC to control and balance intracellular copper, preventing the neurodegenerative process, which the accumulation of copper may accentuate. The activation of RAGEs (described as the main mediating factor for Aβ cytotoxicity) has been observed in the wild-type cells in Aβo incubation and colocalization in the nuclear region. Still, it is not yet clear what effect of such an interaction is kept. The results obtained for NFĸB protein expression suggested the hypothesis that the presence of PrPC during Aβo incubation controls its entry intracellularly. In the PrPC-null cells, the internalization and non-degradation of Aβo promote the generation of ROS as a neurotoxicity performer. The NFĸB cell signaling pathway is then activated in response to the inflammation generated by ROS. The analysis performed for the SOD1 protein connected with several results obtained led us to elucidate part of the neuroprotection mechanism exhibited by PrPC. It has been shown that the cellular prion protein is responsible for bringing SOD1 to the cell nucleus under the induction of oxidative stress in astrocyte cells. The translocation of SOD1 to the nucleus occurred only in the presence of PrPC, indicating the crucial link, since resistance to oxidative stress and DNA damage repair, in this case, seems to be mediated by the SOD1-PrPC interaction in the astrocyte cells, and together with the other results of this work, proved that cellular prion protein is fundamental in the neuroprotective mechanism in oxidative conditions. Regarding the participation of PrPC against neurodegenerative processes caused by stimulated protein aggregation, the protein aggregation assay detected broad bands of protein aggregation in the absence of PrPC under Aβo incubation, proving that the interaction of PrPC with Aβo contributes as a neuroprotective event in astrocyte cells, and consequently protecting neurons in the progression of neurodegenerative diseases. Finally, the results clarified that the cellular prion protein is a fundamental agent for neural protection from oxidative damage that can lead to neurodegeneration.
As a result, using several variables, this work provides solid confirmatory support for PrPc being protective to astrocytes. Various interesting findings include PrPC-null cells, despite being more vulnerable to some doses or timepoints after H202, respond robustly to stressors in protective pathways e.g. to SOD, and less to apoptotic pathways. Interesting effects of α,β-amyloid oligomer (αβo) are seen as well without a paradigm demonstrating the clear-cut vulnerability of PrPC-null cells.