Cellular prion protein offers neuroprotection in astrocytes under oxidative stress

doi:10.21203/rs.3.rs-1533314/v1

Download PDF

Research Article

Cellular prion protein offers neuroprotection in astrocytes under oxidative stress

https://doi.org/10.21203/rs.3.rs-1533314/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 28 Dec, 2022

Read the published version in Molecular and Cellular Biochemistry →

You are reading this latest preprint version

The cellular prion protein (PrP^C), in its native conformation, performs numerous cellular and cognitive functions in brain tissue. However, despite the cellular prion research in recent years, there are still doubts about its activities and how its molecular interface occurs in oxidative and neurodegenerative processes. Also, its metal-protein interaction is not apparent. This study aims to elucidate the participation of PrP^C in the neuroprotection cascade in the presence of oxidative stressors. For that, astrocytes from wild-type mice and knockout to PrP^C were subjected to the induction of oxidative stress with hydrogen peroxide (H₂O₂) and with the toxic oligomer of the beta-amyloid protein (Aβo). We observed that the presence of PrP^C showed resistance in the cell viability of astrocytes. It was also possible to monitor changes in basic levels of metals and associate them with an induced damage condition, indicating the precise role of PrP^C in metal homeostasis, where the absence of PrP^C leads to metallic unbalance culminating in cellular vulnerability to oxidative stress. Activation of specific proteins at different levels was also observed, such as caspase 3, Tau, p-Tau, p53, and Bcl2, which may establish a relationship between a PrP^C and an induced damage condition. Complementarily, it has been shown that PrP^C prevents the internalization of AβO and/or promotes its degradation under oxidative stress induction and thus prevents protein aggregation in astrocytes. It was also observed that the PrP^C is responsible for translocating SOD1 to cell nuclei under conditions of oxidative stress, probably controlling DNA damage. The results obtained in this study proved that PrP^C acts against oxidative stress, activating the cellular response and defense by displaying neuroprotection to neurons and ensuring the functionality of astrocytes.

prion

astrocytes

oxidative stress

neurodegeneration

neuroprotection

The Cellular Prion Protein (PrP^C) is a 30–35 kDa cell surface glycoprotein (1, 2), with 208 amino acid residues and a GPI (glycophosphatidylinositol) anchor (3, 4). It is mainly expressed in the Central Nervous System (CNS) through the transcription of the PRNP gene, but it can also be found in other cells and tissues at lower levels (1). PrP^C is expressed in neurons and astrocytes, glial and microglial cells (5, 6). Astrocytes are the primary neural cell responsible for maintaining cerebral homeostasis. Astrocytes dynamically modulate neuronal activity by expressing a wide variety of receptors, transporters, and ion channels (7). Astrocytes act with neurons in the trafficking and recycling neurotransmitters, ion homeostasis, viability, and defense against oxidative stress (8, 9). However, pathogenic stimuli can disturb astrocytic function, compromising neuronal functionality and viability (6, 7). Its distribution in brain tissue suggests that PrP^C can play a regulatory role in forming and maintaining synapses (10, 11). PrP^C participates in transmembrane signaling processes in different phenomena, including neuronal survival, neurite proliferation, and response to neurotoxicity (2, 6, 12), also the protection against oxidative stress-induced by Reactive Oxygen Species (ROS), DNA damage, neuroprotective activity, and anti-apoptotic activity (13–18).

It is also known that PrP^C has an affinity for divalent metals through its octapeptide domain, such as copper, zinc, iron, and stands out as a possible candidate for metal homeostasis (19–23). Still, copper interacts with the prion protein binding domains and has great importance for its structure and function. Some studies indicated that PrP^C modulates antioxidant enzymes that convert ROS into less toxic products (4, 24–26). Brown et al. reported the ability of PrP^C to act as an antioxidant in an activity similar to Cu,Zn-superoxide dismutase, the SOD1 (27, 28). However, these studies showing PrPC as a SOD1 activity have been barely reproducible and later abandoned.

Conversely, the expression of PrPC has been related to the exhibition of neurotoxicity by the affinity of interaction with β-amyloid oligomers (Aβo). Aβo is considered in the literature to be the primary toxic species associated with the development of Alzheimer's Disease (A.D.), and they accumulate as a consequence of the unregulated proteolytic cleavage of Amyloid Precursor Protein (APP)(29). The cellular prion protein is located on β-amyloid plaques and acts as a receptor for oligomers, enhancing the aggregation process (30–36). PrP^C was identified as an Aβo ligand, but not to monomers or fibrils (31), with high affinity with its structural interaction at the N-terminal (37). These findings attracted significant attention because they represent a potential link of PrP^C to A.D. However, although independent groups have confirmed the interaction between the Aβo and PrP^C, the role of PrP^C in the pathophysiology of A.D. remains controversial. The same polybasic region in the N-terminal of the PrP^C identified itself as a crucial region of Aβo binding. It was previously considered critical for the interaction of the PrP^C with β-secretase. This interaction resulted in β-secretase activity inhibition and cleavage of APP, giving rise to toxic or non-amyloid peptides (38, 39). The observation that the same PrP^C region mediates the potential protective effect of the protein on A.D. (by inhibiting β-secretase cleavage) and its toxic role (blocking the potentiation of oligomer production) is highly intriguing, suggesting that can exist a delicate balance between these effects, controlled by additional cellular cofactors (37). A group of potential cofactors is glycosaminoglycans, which compete for the same local polybasic bond at the N-terminal of PrP^C (40) and appear to be necessary for the inhibitory action exerted by PrP^C on the cleavage of APP by β-secretase (38). The interaction between PrP^C and Aβo is shown to be independent of the toxic consequences exhibited by protein aggregation (41–43). Evidence suggested that PrP^C can recognize and bind to Aβo, preventing fibrillation (44). Also, pointing to the participation of PrP^C in neurodegenerative processes by protein aggregation, it is still not possible to conclude its role due to the conflicting nature of the evidence obtained so far (45–48). However, the facts observed over the decades attribute the PrP^C to primordial functions such as recruiting binding molecules involved in specific cell signaling and mediating signaling pathways (47).

This study aimed to put light on the role of PrP^C during cellular oxidative stress process and submitted to two different stressors, hydrogen peroxide (H₂O₂) and α,β-amyloid oligomer (αβo), including the investigation of cell signaling of proteins, cell cycle, metal internalization, and protein aggregation, using an in vitro cell model from immortalized astrocytes of mice of the native Wild Type (W.T.) and Knockout mice, PrP^C-null (K.O.) for the expression of PrP^C.

2.1 PrP^C in the control of cellular maintenance

Astrocytes are essential to the CNS. The main objective of this study was to assess the participation of the PrP^C in cell signaling processes in astrocytes and its adjacent mechanisms under conditions of oxidative stress. The in vitro models chosen for such evaluation were cultured wild-type and PrP^C-null mice astrocytes, previously described in other studies (13, 18, 49–52). These cells are confident in terms of stability, characterization, and morphology, differing exclusively in the presence of PrP^C, thus making it a reliable study in vitro model. Cell viability was carried out in different concentrations and times of exposure to H₂O₂. According to Fig. 1-A, higher cell viability was noted after 48 hours of incubation with H₂O₂, indicating possible recovery of cell viability. Thus, the 24 hours H₂O₂ incubation time was chosen best to represent the effect of oxidative stress in astrocytes. The result obtained was 800 µM of H₂O₂ for the wild-type astrocytes and 640 µM of H₂O₂ for PrP^C-null astrocytes. In Fig. 1-B, the oxidative stress brought with α,β-amyloid oligomer (αβo) demonstrated that it was impossible to reach a 50% reduction in cell viability. So we used the microscopic visual analysis of the cells with αβo incubation. The concentration of 0.5 µM αβo had a damaging effect on the morphology of the cells, making it possible to identify swelling and loss of its characteristic morphology compared to the control, a fact previously observed in the literature (53).

To evaluate the cell cycle, samples of astrocytes from both cell lines were analyzed, and results were reported in Fig. 1C-K. Data showed the characteristic cell cycle profile, with a cell population distributed between the cycle phases without significant differences between cell lines (Fig. 1C-D) after 24 hours. As expected, the cells incubated with H₂O₂ presented a high population in Sub-G1, indicating cell death (Fig. 1E-F). Cells incubated only with αβo (Fig. 1G-H) did not show significant changes in their cell cycle compared to their respective negative controls. A different result was observed for cells incubated concomitantly with αβo and H₂O₂ (Fig. 1 – I-J). The literature reported that β-amyloid toxicity could be mediated by oxidative stress conditions (54). The population was totaled in the SubG1 phase for the wild-type cells, indicating DNA fragmentation. The presence of PrP^C can act to control the damage leading to programmed cell death.

In contrast, for PrP^C-null cells it was observed a different profile including the decrease in the G1 phase, a slight reduction in the S phase, and an increase in the G2/M phase, indicating many cells in the final stage of cell division when compared to the values presented on Fig. 1-K. Such data showed that the response to the damage induced by αβo + H₂O₂ followed by cell death was not effective for the PrP^C-null cell line. When cells receive some harmful stimulus, it can lead to death, entering the first phase of apoptosis, the genetic control point of cell death. Therefore, in this case, the absence of PrP^C evidences its participation in response processes to cell damage, highlighting its neuroprotective function in astrocyte cells (55, 56).

2.2 The control in metal regulation exhibited by the PrP^C

PrP^C has an affinity for divalent metals in its octapeptide region (20). Based on this interaction, Cu, Fe, Zn, and Ca were analyzed in the wild type and PrP^C-null astrocytes cells under different stress conditions (H₂O₂ and αβo) using the ICP-MS (inductive coupled plasma – mass spectrometry) technique. Graphs demonstrated the metal ng/mg of protein results in Fig. 2A-D. It was possible to observe in Fig. 2A significant changes in Cu levels only in PrP^C-null cells compared to the wild-type cells, indicating that the intracellular copper efflux was compromised under αβo incubation. It is known that astrocytes regulate Cu levels in the brain due to the copper-binding affinity with the repeating units of the octapeptide present in the structure of the PrP^C (57). Our results can be related to the change in intracellular copper homeostasis caused by αβo. In the PrP^C-null cells, Cu levels were similar to control (without oxidative conditions).

Still complementing the results obtained for Cu, Fig. 2-E exposes the results obtained for the copper-transporter ATPase ATP7B, an essential protein for copper homeostasis regulation in mammals and thus monitored to assess copper efflux in cells. In the cell incubation with αβo, the activation of ATP7B occurred only in the wild-type cells (arrow: a), indicating the probable participation of PrP^C to balance the intracellular copper efflux. For the incubation with H₂O₂, activation of ATP7B was noted in both cell lines, but to a lesser extent in the PrP^C-null cells. In this case, it was not possible to show that PrP^C worked on the activation of ATP7B. PrP^C is supposed to assist the input and output copper control in cells (58) due to its copper-binding affinity. However, as seen in the image results mainly under αβo incubation, the wild-type astrocyte showed greater activation of ATP7B. This fact was possibly related to some signaling displayed by PrP^C requesting the release of intracellular copper excess, preventing the neurodegenerative process.

The accumulation of intracellular copper is a crucial factor in the progression of neurodegenerative diseases (59). Astrocytes also participate in cerebral iron homeostasis, an essential metal in numerous physiological functions, inducing oxidative damage in unstable conditions. Astrocytes control iron absorption in the brain through the Blood-Brain Barrier (BBB) and distribute iron to neuronal cells (60). However, as schematically illustrated in Fig. 2F, excess Fe²⁺ may participate in the Fenton reaction (Fe²⁺ + H₂O₂ → Fe³⁺ + • O.H. + O.H.⁻), generating highly reactive hydroxyl radicals. The hydroxyl radical is highly reactive, and when produced in vivo, it reacts close to its formation site (61). Figure 2B showed few variations in iron levels between induced oxidative conditions in the wild-type astrocytes.

In contrast, in the PrP^C-null astrocytes, iron increased under incubation with H₂O₂. These results corroborated with what is reported in the literature regarding the participation of PrP^C in the process of regulation of iron levels, the metallic imbalance, and the generation of ROS (62–64). Zinc levels with significant changes were observed in Fig. 2-C, for both cell lines, mainly in PrP^C-null astrocytes. The Zn decrease occurred with the incubation of H₂O₂ in the wild-type astrocytes. In the PrP^C-null cells, the reduction in the Zn levels was observed in both oxidative conditions. It was also noted that the PrP^C-null cells have higher Zn baseline levels than the negative control of wild-type. Astrocytes can also quickly accumulate zinc (which is closely involved in cognition) (65).

However, excess Zn can influence the accumulation of Zn²⁺ ions within amyloid plaques. These processes lead to cellular damage by excitotoxicity or oxidative stress, directly affecting the synapse, where the presence or absence of Zn can be crucial (63, 64). Because the PrP^C-null cells presented high baseline levels of Zn, they may be more vulnerable to such a condition. It possibly indicates that astrocytes regulate Zn homeostasis. PrP^C may be acting in these cases since the decrease in Zn levels was significant in the wild-type cells, mainly under oxidative stress. In Fig. 2 – D, there was a substantial decrease in calcium levels under oxidative stress in wild-type cells. Calcium levels observed in the PrP^C-null cells had lower basal levels regarding wild-type cells. The implication of this data can impact many cellular processes delimited by calcium signaling, which is a vital mediator of physiological events, such as synaptic transmission and plasticity, pathological events, such as excitotoxicity (66, 67). It was reported that PrP^C acts directly on calcium levels in the endoplasmic reticulum. The release of intracellular reserves can lead to transmitters such as glutamate to the extracellular medium and work on cell death signaling (56, 68).

2.3 PrP^C prevents protein aggregation in astrocytes

To better understand the effects caused in astrocytes exposed to oxidative conditions regarding mechanisms and cellular signaling, Fig. 3A - E illustrates relevant data obtained. Tau protein is commonly reported in the literature due to its phosphorylated form (p-Tau), which becomes prone to the process of protein aggregation in neurodegenerative diseases. This evaluation becomes relevant because of the interaction between PrP^C and Tau (46, 69). We observed the expression levels of the Tau protein for wild-type cells by Western Blotting (Fig. 3 - A). Tau levels were higher when compared to the PrP^C-null cells and upon exposure to oxidative agents. A comparative condition for the induction of cell death was generated by incubation with Staurosporine (STS), a pro-apoptotic agent, widely used for this purpose (70–73). This control experiment allowed us to evaluate known mechanistic responses concerning cell death by caspases activation. Also, it was possible to verify the effectiveness of each method used and thus compare them with other conditions induced by other agents in this work to obtain answers about the participation of PrP^C in these conditions. The concentration and exposure time for the cells used in this study were previously tested and set at 4 µM for 3 hours incubation.

In comparison, for PrP^C-null cells, the Tau levels are lower. The phosphorylated Tau (p-Tau) protein levels (Fig. 3 - B) also differ between the wild-type and PrP^C-null. Higher p-Tau levels were observed in the PrP^C-null than the wild-type cells in oxidative conditions, mainly indicating that the cells incubated with αβo and STS showed even greater expression. For the wild-type cells, much lower p-Tau levels were observed in all conditions. Interestingly, the p-Tau/Tau ratio differs between the cells at baseline levels. The results presented for the p-Tau and Tau proteins and their relationship (Fig. 3 - E) showed that the PrP^C-null cells were more susceptible to oxidative processes by stimulated aggregation, corroborating with data reported in the literature (46, 70, 71). Specifically, the protein aggregation caused by the induction of oxidative stress with αβo is demonstrated through immunocytochemistry illustrated in Fig. 3 – D. The results reflect a more acute internalization of β-amyloid protein αβo in the PrP^C-null cells (arrow: b, showing that the peptides had been internalized not degraded).

So, it was observed that the presence of PrP^C in astrocytes was essential in the degradation of β-amyloid oligomers 1–42. Consequently, possible inhibition of protein aggregation that leads to the progression of AD Foley and collaborators observed that the internalization of β-amyloid oligomers mediated by the prion protein did not generate protein aggregation-dependent (72). In addition, protein aggregation was evaluated using a commercial gel STAIN-FREE BioRad Mini-PROTEAN TGX Stain-free. The results obtained (Fig. 3 - E) allowed us to observe, through the presence and absence of the reducing agent β-mercaptoethanol, the formation of protein aggregates in both cell lines under the induction of oxidative stress conditions. It was noted that the protein aggregates in the PrP^C-null cells occurred after inducing stress with αβo, observed by the formation of a large band of high molecular weight, as indicated in Fig. 3-E. Such observation confirmed the hypothesis that the absence of PrP^C causes the accumulation of β-amyloid oligomers and the formation of toxic aggregates in astrocytes.

2.4 PrP^C has a neuroprotective function against oxidative stress

It is well established in the literature that caspase proteins are involved in cell death by apoptosis (73). However, the relationship between the expression of these proteins and the correlation with PrP^C is still not entirely clear. Caspase 9 and Caspase 3 (Fig. 4 - A and B), the apoptosis initiator and effector proteins, were more expressed in wild-type cells. At the same time, for the PrP^C-null cells, no significant changes were observed in caspases expression levels, demonstrating that the PrP^C absence resulted in the lack of response by activating the apoptosis cascade during oxidative stress damage stimuli. This fact agreed with reported in the literature regarding the participation of PrP^C in apoptosis mechanisms (56). The increase in Bcl2 protein expression was higher under a typical condition in the induction of cell death (STS incubation) and oxidative stress in the wild-type cells (Fig. 4 - C). However, in the PrP^C-null cells, the Bcl2 expression levels remained constant. No changes of Bak protein expression were observed through the conditions induction in wild-type cells (Fig. 4 - D). However, the Bak expression levels rose in the PrP^C-null cell with αβo incubation. These results indicate the activation of Bak in response to oxidative stress caused by αβo. The answer obtained emphasizes that there is probably a regulation mechanism in the proportion of Bak and Bcl2 mediated by PrP^C, as previously reported in the literature (74). This involves a link mechanism between PrP^C-Bcl2 that mediates the cascade of apoptotic activation in situations of damage and injury. In the results obtained of SOD1 expression (Fig. 4 - E), it was observed that SOD1 basal levels were similar for the wild-type and PrP^C-null cell.

An increase in SOD1 expression upon exposure to oxidative conditions was observed in PrP^C-null cells, caused mainly for STS and H₂O₂. The literature reported that PrP^C exhibits antioxidant activity similar to SOD1 (13, 26, 28, 75). Thus, the activation of SOD1 may be linked to the role of PrP^C in response to oxidative damage. Here it was shown that the absence of PrP^C in astrocytes leads to higher levels of SOD1 expression in response cell survival by an antioxidant compensation mechanism, independent of PrP^C. Relevant results of SOD1 were observed in immunocytochemistry for both cell lines (Fig. 4 - F). Negative controls showed similar baseline levels between cell lines, exhibiting marked fluorescence. For the induction of oxidative stress in the wild-type cells, a similar response was noted compared to its respective negative control, without SOD1 activation. For the PrP^C-null cells in the same oxidative stress condition, a slight increase in fluorescence was observed, indicating the activation of SOD1. Under oxidative stress induction, the wild-type astrocyte showed not only the SOD1 activation but its focus in the nuclear region (arrow: c).

2.5 The involvement of PrP^C in signaling mechanisms during stress in astrocytes

Cell signaling mechanisms were reported on Fig. 5A-C. Figure 5 - A showed the results obtained for the expression of Nuclear Factor-kappa B (NFĸB). Both cell lines showed similar NFĸB levels in the negative control, highlighting a little more for PrP^C-null cells. With Aβo incubation, there was also intense fluorescence for the PrP^C-null cells, indicating greater activation of NFĸB (arrow: c). NFĸB levels of fluorescence were also observed for the wild-type cells but slightly higher concerning its negative control. The wild-type cells also showed activation of NFĸB, however to a lesser extent. This fact can be explained because the presence of PrP^C controls the αβo intracellularly, preventing its toxic effects, demonstrating neuroprotective function. Once the αβo were internalized in the PrP^C-null cells, the generation of ROS occurs as a neurotoxicity agent.

Consequently, the NFĸB cell signaling pathway could be activated in response to the inflammation generated by ROS. This result agrees with the hypothesis that the cellular prion protein exhibits neuroprotective function against αβo in astrocytes through inhibiting its intracellular accumulation, generation of ROS, neurotoxicity, and consequently protein aggregation initially caused by this agent. For oxidative conditions induced by H₂O₂, NFĸB activation occurred proportionally for both cell lines, indicating that the H₂O₂ could cause oxidative stress and activate pro-inflammatory signaling via the NFĸB pathway. In this case, the presence of PrP^C did not influence the activation mechanism of this cell signaling pathway.

Figure 5-B shows the results obtained to RAGEs (final receptors for advanced glycation products). They can interact with αβo and increase neurotoxicity, acting as AGEs (advanced glycation end-products). AGEs can exert cellular effects simply by targeting RAGEs in a probable repetitive cycle on the cell surface. The binding of αβo-RAGE induces oxidative stress that positively regulates RAGE, further increasing the αβo-RAGE interaction (76). The evaluation of the expression and location of RAGEs was performed to infer the interaction of PrP^C-RAGE under stress caused by αβo and H₂O₂. The results showed that under αβo incubation, in the wild-type cell, the activation and localization of RAGEs occurred in the nuclear region of astrocytes (arrow: d). This result indicated a probable interaction between PrP^C and RAGE, mediated by the presence of αβo. As for the oxidative condition caused by H₂O₂, with RAGE located in the nucleus for both wild-type and PrP^C-null cells.

The p53 protein is a central regulator of cellular stress responses and senescence (77). The p53 isoforms can regulate neuroprotective and neurotoxic functions of human astrocytes. The p53 also regulates the progression of the cell cycle, the action of DNA repair mechanisms, and the removal of damaged cells through apoptosis. When DNA damage occurs, there is an increase in the expression of this protein (78). In Fig. 5 - C, it can be observed that the PrP^C-null astrocytes presented higher baseline levels of p53 expression than wild-type cells. The baseline expression levels of p53 were lower for the wild-type cells, and under the induction of neurodegenerative or oxidative stress, their levels decrease or remain equivalent, respectively. This behavior showed the hypothesis of the neuroprotective function of PrP^C, through excitotoxicity, in a mechanism independent of p53 expression, leading to the induction of apoptosis in the wild-type cells. In contrast, in the PrP^C-null astrocytes, the absence of PrP^C under stress conditions and neurodegeneration induction implied an increase in p53 expression levels. Due to some failure in cell signaling, it allows the cell to enter a neurodegenerative process instead of cell death as a defense mechanism, a fact confirmed by the values obtained from the relationship between p-Tau/total Tau in the PrP^C-null astrocytes.

The results observed in this work demonstrated the fundamental role of PrP^C 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 PrP^C (wild-type) is more resistant to oxidative stress. This fact showed that PrP^C 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 PrP^C-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 PrP^C, 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 PrP^C, their sensitivity to oxidative stress becomes greater (79, 80). PrP^C 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 PrP^C-null astrocytes had less intracellular copper than the wild-type cell, corroborating this copper-transport role of PrP^C. The iron imbalance exhibited by the PrP^C-null cells under oxidative conditions indicated that the absence of the PrP^C caused the spread of damage to astrocyte cells can also be due to iron misregulation.

Studies showed that PrP^C is one of the proteins involved in the regulation of iron by astrocytes involved in the redox process of Fe³⁺ to Fe²⁺ (89). PrP^C-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 PrP^C. 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 PrP^C 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 PrP^C. 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 PrP^C 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 PrP^C-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 PrP^C 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 PrP^C-null cells. This fact can be due to the balanced signaling between Bcl2-Bak mediated by the participation of PrP^C, 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 PrP^C. Thus, the Cu reduction in CCS may contribute to the decrease in SOD1 activity already observed in null mice for PrP^C (75, 107). The link between PrP^C expression and SOD1 points to common pathways in regulating its expression and indicates the role between PrP^C 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 PrP^C can regulate the copper redox cycle. Thus, changes in PrP^C expression result in unbalanced proteins associated with copper metabolism to compensate for the loss in PrP^C 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 PrP^C-null cells exhibit similar baseline levels of SOD1 expression. The presence of PrP^C promoted antioxidant activity in the wild-type cell. In the PrP^C-null cells, we observed lower Aβo internalization. The prion protein led to less intracellular ROS generation and less required antioxidant activity, contrary to the PrP^C-null cells, which required the antioxidant defense performed by SODs. The translocation of SOD1 to the nucleus occurred only in the presence of PrP^C in the wild-type cell, indicating the crucial link to explain the SOD1 translocation to the nucleus in oxidative stress (109).

Like PrP^C, 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 PrP^C 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 PrP^C 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 PrP^C 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.

CONCLUSION

Through the expression and localization of the analyzed proteins, it was possible to notice that the internalization of Aβo occurs mainly in the PrP^C-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 PrP^C would have been responsible for their degradation. Furthermore, it was found that under Aβo incubation, the ATP7B activation occurred in the presence of PrP^C. This result possibly relates to signaling displayed by PrP^C 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 PrP^C during Aβo incubation controls its entry intracellularly. In the PrP^C-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 PrP^C. 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 PrP^C, indicating the crucial link, since resistance to oxidative stress and DNA damage repair, in this case, seems to be mediated by the SOD1-PrP^C 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 PrP^C against neurodegenerative processes caused by stimulated protein aggregation, the protein aggregation assay detected broad bands of protein aggregation in the absence of PrP^C under Aβo incubation, proving that the interaction of PrP^C 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 PrP^C-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 PrP^C-null cells.

4.1 Chemicals

Chemicals used were of analytical grade, obtained from standard commercial suppliers. Ultrapure water (18.2 MΩ resistivity) obtained by a Milli-Q purification system (Millipore, Bedford, MA, USA), hydrogen peroxide (H2O2 – Merck – 1.07210.1000); beta-amyloid protein oligomeric fragment 1–42 (Sigma Aldrich - A9810); Hexafluoroisopropanol (HFIP - Sigma Aldrich − 105228); Dimethylsulfoxide (DMSO - Merck − 6768-5); Staurosporine (Sigma Aldrich – S4400); Dulbecco's Modified Eagle's Medium High Glucose Culture Medium (DMEM - Sigma Aldrich − 5648); Fetal Bovine Serum (SFB -Gibco − 12657029), trypsin-EDTA (Sigma Aldrich - T4049); penicillin (Gibco − 15140); Sodium Phosphate Monobasic (Usb − 20229); Dibasic sodium phosphate (Sigma Aldrich – S5136); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol) bromide (MTT – Sigma Aldrich – M2128); Trypan Blue (Sigma Aldrich – M2128); Dithiothreitol (DTT – Fisher – BP1725); Sodium orthovanadate (Sigma Aldrich – 56508); Phenylmethanesulfonyl fluoride (PMSF – Sigma Aldrich – 56508); Hydroxymethyl aminomethane (TRIS - Sigma Aldrich – 252859); Formaldehyde (Sigma Aldrich – S220702); Sodium dodecyl sulfate (SDS – Fisher – BP166-500); Protease inhibitor (Sigma Aldrich – P8340); Folin&Ciocalteu's Phenol (Sigma Aldrich – F9252); Bis-acrylamide (Acros − 164855000); Albumin Serum Bovine (BSA – Fluka − 05480); β-mercaptoethanol (β-ME - Sigma Aldrich – M3148); Propidium iodide (Sigma Aldrich – P4864); RNAse (Calbiochem − 55674). The other reagents were used with a purity standard of ≥ 98% from Sigma-Aldrich, Vetec, Thermo Fisher, or similar. We used antibodies for western blot assay, α-tubulin (Sigma-Aldrich T6074—1:1000), β-actin (Sigma-Aldrich A2228—1:1000), p53 (Abcam Ab90363—1:500), SOD1 (Ab13498—1:500), Caspase 9 (Sigma-Aldrich C4356— 1:1000), Caspase 3 (Sigma-Aldrich C84871:500), Bcl2 (Millipore-UPSTATE 05729-MN—1:500), Tau (Sigma-Aldrich T9450—1:1000), phosphorylated Tau (p-Tau) (Sigma-Aldrich T6819—1:1000), anti-PrP^C (antibody produced directly from the wild type animal, which provided the cell line in this study—1:5000), anti-mouse IgG Peroxidase (Sigma-Aldrich A9044—1:10,000) or Anti-rabbit IgG Peroxidase (Sigma-Aldrich A0545—1:10,000). For immunocytochemistry, primary polyclonal antibody ATP7B produced in rabbit (Sigma-Aldrich HPA013187–1:50); or primary polyclonal SOD1 antibody produced in goat (Sigma-Aldrich SAB2500976–1:50); or primary polyclonal RAGE antibody produced in rabbit (Abcam Ab3611–1: 200); or primary polyclonal beta-amyloid 1–42 antibody produced in rabbit (Abcam Ab10148–1: 100), Alexa Fluor 488 Donkey anti-Rabbit IgG or Alexa Fluor 546 Donkey anti-Mouse IgG (Invitrogen, 1: 500).

4.2 Cell culture and conditions

The immortalized astrocyte cell lines from mice used in this work were PrP^C-null (Prnp^0/0; ZrchI) and wild-type control (Prnp^+/+; ZrchI). It was obtained from primary astrocyte cultures from the cerebral hemispheres of embryonic day 17 (E17) wild type, and PrP^C-null (knockout) mice were prepared as previously described (13, 50). The verification of the expression/absence of the prion protein in the astrocyte cells was carried out by the Western Blotting assay, data shown in the Supporting information. Both cell lines (wild type for PrP^C: wild-type, and knockout for PrP^C: PrP^C-null cells) were cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM; Gibco/Life Technologies, Grand Island, NY, USA) and supplemented with 10% fetal bovine serum (FBS; Gibco/Life Technologies), 100 U/mL penicillin and 10.0 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cell cultures were maintained at 37°C in a controlled atmosphere of 5% CO₂ (cell culture greenhouse-Forma Series II, Hepa Class 200, Thermo Scientific). Culture flasks with a confluence of 80% cell growth were plated according to the density required for each assay.

The oxidative stress condition was induced by incubation with an aqueous hydrogen peroxide solution. The exposure time established was 24h, at a concentration of 800 µM for wild-type cell line and at a concentration of 640 µM for PrP^C-null cells (concentrations obtained through the determination of IC₅₀). To perform the preparation of the aqueous solution, we proceeded with the molar quantification of the commercial solution by UV-Vis spectrophotometry at 240 nm, a method adapted from Wolff and Ou (121). The neurodegenerative condition was induced with β-amyloid protein oligomers of the toxic peptide segment 1–42 (Aβo) at a concentration of 0.5µM (in DMEM culture medium) for both cells lines, with an exposure time of 24 hours. The preparation of the oligomers was carried out according to the protocol described by Stine and collaborators (122). The method consists of the solubilization of the commercial aliquot of oligomeric fragments 1–42 of the β-amyloid protein in Hexafluoroisopropanol (HFIP), in sequence, resting time of 30 minutes and after drying the aliquots in the desired concentration with argon gas. After the procedure, the aliquots were stored at -80ºC and thawed just before the cell incubation.

4.3 Cellular viability

For the cell viability tests, the MTT test was used and alternatively also the Trypan Blue test. Both tests were performed according to the protocol described by Matias et al. (127) and Magrini et al. (128).

For the Trypan Blue assay, the cell lines were plated at a density of 2×10⁴ cells/cm² in 24-well plates and exposed to the proposed incubation with Aβo. After 24 and 48 hours, the cells were stained with trypan blue and counted under an optical microscope using a Neubauer chamber (129). The cell count data were represented graphically as the total number of live cells.

For MTT assay, cells from wild type and PrP^C-null were cultured and plated in 96-well plates at densities of 2×10⁴ cells/cm² and exposed to the proposed incubations with H₂O₂ and Aβo for 24 hours. After the induced conditions, the MTT salt (5 µg/mL) was added directly to the medium and incubated for 1 hour and 30 minutes in the dark. After the time, the culture medium was removed entirely, and 150 µL of DMSO was added for incubation with shaking and under the cover for 15 minutes. Then, the samples were read in a microplate reader (CELER Polaris) at a wavelength of 570 nm (130). In addition to assessing cell viability from this assay, IC₅₀ values for induced conditions were also determined when applicable. The calculations were performed using the program A linear Interpolation method for sublethal toxicity: The inhibition concentration (ICP) approach V.2.0 June 1993 (U.S. Environmental Protection Agency, Environmental Research Laboratory, Duluth, MN 55804) (Linear interpolation).

4.4 Cell cycle assay

Flow cytometry (FACS) was used to analyze the cell cycle. For this, the cells of both lines were plated at a density of 4×10⁴ cells/cm² in 6-well plates. The cells were incubated with H₂O₂ and Aβo, and after 24 h, they were trypsinized and washed with PBS three times at 1300 rpm for 5 min. The cell pellets were resuspended with propidium iodide (P.I., 5 µg/ml), Triton X-100 (0.1%) and RNAse (0.2 mg/ml) and incubated for 1 hour, and then continued for analysis flow cytometer (BD.FACS Canto II - B.D. Biosciences), according to the protocol described by Bertuchi and collaborators (13). The data were analyzed using FlowJo software v.X.0.7.

4.5 Determination of metal levels

To determine intracellular metal levels, the inductively coupled plasma mass spectrometry (ICP-MS) was used, according to the protocol described by Lago et al. (131). The cell lines were plated at a density of 2×10⁴ cells/cm² in 75 cm² flasks, and after 24 h the cells were trypsinized, washed with PBS (an aliquot was separated for protein quantification) and freeze-dried (Liobras L101, Brazil). Then, the samples (in triplicate per cell line) were pre-digested with 50 µL double-distilled concentrated (65% w/v) HNO₃ for 24 h. After that, the samples were heated in a water bath at 90°C for 2 h. After, 950 µL of ultrapure water (Milli-q, Millipore) was added, homogenized, and heated in a water-bath at 90°C for 2 h. All analytical standard calibration curves and blanks were made by serial dilution of standard stock solutions in 5% v/v HNO₃. Finally, the samples were analyzed by an inductively coupled plasma mass spectrometer (DRC-ICP-MS Elan DRCII; Perkin-Elmer, Boston, MA, USA). The results were converted using the protein concentration of each sample and the dilution factor, yielding the result in ng/metal per mg of sample protein. Samples were analyzed by ICP-MS (Agilent 7900, Hachioji, Japan). The results were converted using the protein concentration of each sample and the dilution factor, yielding the result in ng/metal per mg of sample protein.

4.6 Western blot analysis

To evaluate the levels of expression of proteins of interest, the western blotting technique was used, according to the protocol described in the previous work (132). Cellular proteins (50ug) were subjected to SDS–PAGE and blotted onto nitrocellulose (G.E. Healthcare Life Sciences), with α-tubulin or β-actin loading control. The primary and secondary antibodies were employed, and the complexes formed after incubation with the secondary antibodies were detected using SuperSignal West Pico (Thermo Fisher Scientific) or SuperSignal Femto substrate (Thermo Fisher Scientific) in a Biorad photographer BioRad ChemiDoc XR + and quantified by Software ImageLabTM 6.0.1.

4.7 Immunocytochemistry

Cell lines were grown on chamber slides at a density of 0.5×10⁴ cells/cm² and then induced conditions described above. After 24 h incubation with inductor agents, the cells were pre-fixed with 2% paraformaldehyde diluted in DMEM (v/v) followed by a 1% paraformaldehyde post-fixation. Briefly, the slides were washed twice with ice-cold PBS for 3 minutes and then permeabilized with PBS containing 0.2% Triton X-100 for 30 minutes. The permeabilized cells were incubated with a blocking solution (4% bovine serum albumin (BSA), in PBS and 0.2% Triton X-100) and then incubated with PBS containing 1% BSA and the specific primary antibody at 4°C overnight. After washing with PBS, the cells were incubated in PBS containing Alexa Fluor 488 Donkey anti-Rabbit IgG ou Alexa Fluor 546 Donkey anti-Mouse IgG (Invitrogen, 1:500), and 600nM DAPI solution (Life Technologies) at 37°C for 3 hours. Then, the cells were washed with PBS. The coverslips were mounted in a glycerol/ PBS solution (1:1 v/v), and the cells were examined using a fluorescence microscope (Leica AF6000). All images were analyzed by Leica Application Suite – Advanced Fluorescence Lite 2.6.3). Immunocytochemical images were done at least in triplicate independent experiments.

4.8 Protein aggregation

To detect protein aggregates and relate them to neurodegenerative processes, the protocol described by Dantas and collaborators was followed (133). A commercial STAIN-FREE BioRad Mini-PROTEAN TGX Stain-free, 12% gel, was used. Similar to the procedure performed in the Western blotting assay, cells were plated at a density of 2×10⁴ cells/cm² in 25 cm² bottles. They were incubated with Aβo only for 24 hours. Cell aliquots of samples containing 50 µg were incubated with sample buffer (62 mM Tris-HCl, pH 6.8 containing 10% glycerol, 2% SDS, 0.01% bromophenol blue) in the absence and presence of the agent β-mercaptoethanol reducer (200 mM) for 5 minutes at 95°C. The samples were standing and then applied to the gel. The run was carried out for about 1 h (fixed voltage of 150V), in running buffer (25mM TRIS base, 250mM glycine, 0.1% SDS, pH 8.3). The development of the gel was obtained through the photo-documenter BioRad ChemiDoc XR + through the incidence of U.V. The images were incubated by the ImageLabTM 6.0.1 Software.

4.9 Statistical analyses

All experiments were repeated at least three times in independent replicates, and the results are expressed as the mean values ± standard deviations. Westerns Blottings are done each one with its respective mass control anti-α-tubulin or anti-β-actin for quantification purposes, always in the same gel and blotting for comparison. The analysis of variance (ANOVA) with Bonferroni's test was used to evaluate the differences between the cell lines groups for all experiments with the level of significance set at P ≤ 0.05.

Supporting information

This article contains supporting information.

Acknowledgments

The authors are grateful to the multiuser central facilities (UFABC) for the experimental support. We thank Dr. Vilma R. Martins and Dr. Tiago Góss dos Santos, from AC Camargo (São Paulo, Brazil), for donating anti-PrP^C produced in Wild type mice.

Author contributions

CMSM, RNG, T.P., BLB, and G.C. designed the experiments, performed research, and analyzed the data. G.C. conceived the study. CMSM and G.C. wrote the paper. All authors read and approved the final manuscript.

Funding and additional information

The authors thank Sao Paulo State Foundation (FAPESP grant 2018/14152-0 and 2020/14175-0), and CNPq. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Linden, R., Martins, V. R., Prado, M. A. M., Cammarota, M., Izquierdo, I., and Brentani, R. R. (2008) Physiology of the prion protein. Physiol. Rev. 88, 673–728
Poggiolini, I., Saverioni, D., and Parchi, P. (2013) Prion Protein Misfolding, Strains, and Neurotoxicity : An Update from Studies on Mammalian Prions. Int. J. Cell Biol. 10.1155/2013/910314
Roland, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., and Wüthrich, K. (1996) NMR structure of the mouse prion protein domain PrP(121-231). Nature. 382, 180–182
Sakudo, A., Xue, G., Kawashita, N., Ano, Y., Takagi, T., Shintani, H., Tanaka, Y., Onodera, T., and Ikuta, K. (2010) Structure of the Prion Protein and Its Gene : An Analysis Using Bioinformatics and Computer Simulation. Curr. Protein Pept. Sci. 11, 166–179
Adle-biassette, H., Verney, C., Peoc, K., Gressens, P., Budka, H., and Henin, D. (2006) Immunohistochemical Expression of Prion Protein ( PrPC ) in the Human Forebrain During Development. J Neuropathol Exp Neurol. 65, 698–706
Hartmann, C. A., Martins, V. R., and Lima, F. R. S. (2013) High levels of Cellular Prion Protein improve astrocyte development. FEBS Lett. 587, 238–244
Guillamón-Vivancos, T., Gómez-Pinedo, U., and Matías-Guiu, J. (2015) Astrocytes in neurodegenerative diseases (I): function and molecular description. Neurol. (English Ed. 30, 119–129
Bélanger, M., and Magistretti, P. J. (2009) The role of astroglia in neuroprotection. Dialogues Clin. Neurosci. 11, 281–296
Bylicky, M. A., Mueller, G. P., and Day, R. M. (2018) Mechanisms of Endogenous Neuroprotective Effects of Astrocytes in Brain Injury. Oxid. Med. Cell. Longev. 2018, 6501031
Collinge, J., Whittington, M. A., Sidle, Katie, C. L., Smith, C. J., Palmer, M. S., Clarke, A. R., and Jefferys, J. G. R. (1994) Prion protein is necessary for normal synaptic function. Lett. to Nat. 370, 295–297
Llorens, F., and Del Río, J. A. (2012) Unraveling the neuroprotective mechanisms of PrPC in excitotoxicity. Prion. 6, 245–251
Amin, L., Nguyen, X. T. A., Rolle, I. G., D'Este, E., Giachin, G., Tran, T. H., Šerbec, V. Č., Cojoc, D., and Legname, G. (2016) Characterization of prion protein function by focal neurite stimulation. J. Cell Sci. 129, 3878–3891
Bertuchi, F. R., Bourgeon, D. M. G., Landemberger, M. C., Martins, V. R., and Cerchiaro, G. (2012) PrPC displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrP C knockout mice. Biochem. Biophys. Res. Commun. 418, 27–32
Fantini, D., Bravard, A., Daynac, M., Xu, Z., Bernardino-sgherri, J., Sisso, L., Etienne, O., Dehen, C., Comoy, E., and Boussin, D. (2015) The prion protein is critical for DNA repair and cell survival after genotoxic stress. 43, 904–916
Gourdain, P., Ballerini, C., Nicot, A. B., and Carnaud, C. (2012) Exacerbation of experimental autoimmune encephalomyelitis in prion protein (PrPc)-null mice: Evidence for a critical role of the central nervous system. J. Neuroinflammation. 10.1186/1742-2094-9-25
Jarosz-Griffiths, H. H., Corbett, N. J., Rowland, H. A., Fisher, K., Jones, A. C., Baron, J., Howell, G. J., Cowley, S. A., Chintawar, S., Cader, M. Z., Kellett, K. A. B., and Hooper, N. M. (2019) Proteolytic shedding of the prion protein via activation of metallopeptidase ADAM10 reduces cellular binding and toxicity of amyloid-βoligomers. J. Biol. Chem. 294, 7085–7097
Parrie, L. E., Crowell, J. A. E., Moreno, J. A., Suinn, S. S., Telling, G. C., and Bessen, R. A. (2020) The cellular prion protein promotes neuronal regeneration after acute nasotoxic injury. Prion. 14, 31–41
Marques, C. M. S., Pedron, T., Batista, B. L., and Cerchiaro, G. (2021) Cellular prion protein activates Caspase 3 for apoptotic defense mechanism in astrocytes. Mol. Cell. Biochem. 10.1007/s11010-021-04078-5
Ashok, A., and Singh, N. (2018) Prion protein modulates glucose homeostasis by altering intracellular iron. Sci. Rep. 8, 1–15
Choi, C. J., Kanthasamy, A., Anantharam, V., and Kanthasamy, A. G. (2006) Interaction of metals with prion protein : Possible role of divalent cations in the pathogenesis of prion diseases. 27, 777–787
Krebs, B., Wiebelitz, A., Balitzki-korte, B., Vassallo, N., Paluch, S., Mitteregger, G., Onodera, T., Kretzschmar, H. A., and Herms, J. (2007) Cellular prion protein modulates the intracellular calcium response to hydrogen peroxide. J. Neurochem. 100, 358–367
Singh, A., Haldar, S., Horback, K., Tom, C., Zhou, L., and Singh, N. (2013) Prion Protein Regulates Iron Transport by Functioning as a Ferrireductase. J Alzheimers Dis. 35, 541–552
Wulf, M., Senatore, A., and Aguzzi, A. (2017) The biological function of the cellular prion protein : an update. BMC Biol. 15, 1–13
Rachidi, W., Vilette, D., Guiraud, P., Arlotto, M., Riondel, J., Laude, H., Lehmann, S., and Favier, A. (2003) Expression of Prion Protein Increases Cellular Copper Binding and Antioxidant Enzyme Activities but Not Copper Delivery *. 278, 9064–9072
Paterson, A. W. J., Curtis, J. C., and MacLeod, N. K. (2008) Complex I specific increase in superoxide formation and respiration rate by PrP-null mouse brain mitochondria. J. Neurochem. 105, 177–191
Brown, D. R., Schulz-schaeffer, W. J., Schmidt, B., and Kretzschmar, H. A. (1997) Prion Protein-Deficient Cells Show Altered Response to Oxidative Stress Due to Decreased SOD-1 Activity. 112, 104–112
Brown, D. R., Clive, C., and Haswell, S. J. (2001) Antioxidant activity related to copper binding of native prion protein. J. Neurochem. 76, 69–76
Brown, D. R., Wong, B.-S., Hafiz, F., Clive, C., Haswell, S. J., and Jones, I. M. (1999) Normal prion protein has an activity like that of superoxide dismutase. Biochem. J. 344, 1–5
Walsh, D. M., and Selkoe, D. J. (2007) Aβoligomers - A decade of discovery. J. Neurochem. 101, 1172–1184
Schwarze-Eicker, K., Keyvani, K., Görtz, N., Westaway, D., Sachser, N., and Paulus, W. (2005) Prion protein (PrPc) promotesβ-amyloid plaque formation. Neurobiol. Aging. 26, 1177–1182
Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Cellular prion protein mediates impairment of synaptic plasticity by amyloid-Βoligomers. Nature. 457, 1128–1132
Voigtländer, T., Klöppel, S., Birner, P., Jarius, C., Flicker, H., Verghese-Nikolakaki, S., Sklaviadis, T., Guentchev, M., and budka, H. (2001) Marked increase of neuronal prion protein immunoreactivity in Alzheimer's disease and human prion diseases. Acta Neuropathol. 101, 417–423
Kellett, K. A. B., and Hooper, N. M. (2009) Prion protein and Alzheimer disease. Prion. 3, 190–194
Salazar, S. V., and Strittmatter, S. M. (2017) Cellular Prion Protein as a Receptor for Amyloid-βOligomers in Alzheimer's Disease. Biochem. Biophys. Res. Commun. 483, 1143--1147
Brody, A. H., and Strittmatter, S. M. (2018) Synaptotoxic Signaling by Amyloid Beta Oligomers in Alzheimer's Disease Through Prion Protein and mGluR5 A.H. Adv Pharmacol. 182, 293–323
Purro, S. A., Nicoll, A. J., and Collinge, J. (2018) Prion Protein as a Toxic Acceptor of Amyloid-βOligomers. Biol. Psychiatry. 83, 358–368
Chen, S., Yadav, S. P., and Surewicz, W. K. (2010) Interaction between human prion protein and amyloid-β(Aβ) oligomers: Role of N-terminal residues. J. Biol. Chem. 285, 26377–26383
Parkin, E. T., Watt, N. T., Hussain, I., Eckman, E. A., Eckman, C. B., Manson, J. C., Baybutt, H. N., Turner, A. J., and Hooper, N. M. (2007) Cellular prion protein regulatesβ-secretase cleavage of the Alzheimer's amyloid precursor protein. Proc. Natl. Acad. Sci. U. S. A. 104, 11062–11067
Griffiths, H. H., Whitehouse, I. J., and Hooper, N. M. (2012) Regulation of amyloid-βproduction by the prion protein. Prion. 6, 2885–2895
Pan, T., Wong, B. S., Liu, T., Li, R., Petersen, R. B., and Sy, M. S. (2002) Cell-surface prion protein interacts with glycosaminoglycans. Biochem. J. 368, 81–90
Forloni, G., and Balducci, C. (2011)β-amyloid oligomers and prion protein: Fatal attraction? Prion. 5, 10–15
Balducci, C., Beeg, M., Stravalaci, M., Bastone, A., Sclip, A., Biasini, E., Tapella, L., Colombo, L., Manzoni, C., Borsello, T., Chiesa, R., Gobbi, M., Salmona, M., and Forloni, G. (2010) Synthetic amyloid-βoligomers impair long-term memory independently of cellular prion protein. Proc. Natl. Acad. Sci. U. S. A. 107, 2295–2300
Calella, A. M., Farinelli, M., Nuvolone, M., Mirante, O., Moos, R., Falsig, J., Mansuy, I. M., and Aguzzi, A. (2010) Prion protein and Ab-related synaptic toxicity impairment. EMBO Mol. Med. 2, 306–314
Nieznanski, K., Surewicz, K., Chen, S., Nieznanska, H., and Surewicz, W. K. (2014) Interaction between prion protein and Aβamyloid fibrils revisited. ACS Chem. Neurosci. 5, 340–345
Castle, A. R., Gill, A. C., and Jackson, W. S. (2017) Physiological Functions of the Cellular Prion Protein. Front. Mol. Biosci. 4, 1–25
Vergara, C., Ordóñez-Gutiérrez, L., Wandosell, F., Ferrer, I., del Río, J. A., and Gavín, R. (2015) Role of PrPC Expression in Tau Protein Levels and Phosphorylation in Alzheimer’s Disease Evolution. Mol. Neurobiol. 51, 1206–1220
Linden, R. (2017) The biological function of the prion protein: A cell surface scaffold of signaling modules. Front. Mol. Neurosci. 10, 1–19
Onodera, T. (2017) Dual role of cellular prion protein in normal host and alzheimer's disease. Proc. Japan Acad. Ser. B Phys. Biol. Sci. 93, 155–173
Arantes, C., Nomizo, R., Lopes, M. H., Hajj, G. N. M., Lima, F. R. S., and Martins, V. R. (2009) Prion Protein and Its Ligand Stress Inducible Protein 1 Regulate Astrocyte Development. Glia. 1449, 1439–1449
Büeler, H., Fischer, M., Lang, Y., Bluethmann, H., Lipp, H., DeArmond, S., Prusiner, S., Aguet, M., and Weissmann, C. (1992) Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature. 359, 710–713
Lima, F. R. S., Arantes, C. P., Muras, A. G., Nomizo, R., Brentani, R. R., and Martins, V. R. (2007) Cellular prion protein expression in astrocytes modulates neuronal survival and differentiation. J. Neurochem. 103, 2164–2176
Martins, V. R., Linden, R., Prado, M. A. M., Walz, R., Sakamoto, A. C., Izquierdo, I., and Brentani, R. R. (2002) Cellular prion protein: On the road for functions. FEBS Lett. 512, 25–28
Jalonen, T. O., Charniga, C. J., and Wielt, D. B. (1997)β-Amyloid peptide-induced morphological changes coincide with increased K+ and Cl- channel activity in rat cortical astrocytes. Brain Res. 746, 85–97
Behl, C., Davis, J. B., Lesley, R., and Schubert, D. (1994) Hydrogen peroxide mediates amyloidβprotein toxicity. Cell. 77, 817–827
Zeng, L., Zou, W., and Wang, G. (2015) Cellular prion protein (PrPC) and its role in stress responses. Int. J. Clin. Exp. Med. 8, 8042–8050
Liang, J., Fl, G., Yy, L., Wang, J., Hh, Z., Lp, Y., Tt, L., Ji, Q., Xy, G., Zx, L., and Fan, D. (2006) Review article : Role of PrPc Related to Apoptosis. EXCLI J. 5, 11–24
Dringen, R., Scheiber, I., and Bulcke, F. (2015) Copper metabolism of astrocytes. Springerplus. 4, 1–32
Salzano, G., Giachin, G., and Legname, G. (2019) Structural Consequences of Copper Binding to the Prion Protein. Cells. 8, 770
Angelova, M., Asenova, S., and Nedkova, V. (2011) Copper in the human organism. Mol. Biol. Int. 9, 88–98
Qian, Z. M., and Shen, X. (2001) Brain iron transport and neurodegeneration. Trends Mol. Med. 7, 103–108
Valko, M., Rhodes, C. J., Moncol, J., Izakovic, M., and Mazur, M. (2006) Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160, 1–40
Singh, N. (2014) The Role of Iron in Prion Disease and Other Neurodegenerative Diseases. PLOS Pathog. 10, 9–11
Adlard, P. A., and Bush, A. I. (2018) Metals and Alzheimer's Disease: How Far Have We Come in the Clinic? J. Alzheimer's Dis. 62, 1369–1379
Wong, B. X., and Duce, J. A. (2014) The iron regulatory capability of the major protein participants in prevalent neurodegenerative disorders. Front. Pharmacol. 5 APR, 1–10
Nolte, C., Gore, A., Sekler, I., Kresse, W., Hershfinkel, M., Hoffmann, A., Kettenmann, H., and Moran, A. (2004) ZnT-1 expression in astroglial cells protects against zinc toxicity and slows the accumulation of intracellular zinc. Glia. 48, 145–155
LaFerla, F. M. (2002) Calcium dyshomeostasis and intracellular signalling in alzheimer's disease. Nat. Rev. Neurosci. 3, 862–872
Morris, G. P., Clark, I. A., Zinn, R., and Vissel, B. (2013) Microglia: A new frontier for synaptic plasticity, learning and memory, and neurodegenerative disease research. Neurobiol. Learn. Mem. 105, 40–53
Westergard, L., Christensen, H. M., and Harris, D. A. (2007) The cellular prion protein (PrP(C)): its physiological function and role in disease. Biochim. Biophys. Acta. 1772, 629–44
Corbett, G. T., Wang, Z., Hong, W., Colom-Cadena, M., Rose, J., Liao, M., Asfaw, A., Hall, T. C., Ding, L., DeSousa, A., Frosch, M. P., Collinge, J., Harris, D. A., Perkinton, M. S., Spires-Jones, T. L., Young-Pearse, T. L., Billinton, A., and Walsh, D. M. (2020) PrP is a central player in toxicity mediated by soluble aggregates of neurodegeneration-causing proteins. Acta Neuropathol. 139, 503–526
Rubenstein, R., Chang, B., Yue, J. K., Chiu, A., Winkler, E. A., Puccio, A. M., Diaz-Arrastia, R., Yuh, E. L., Mukherjee, P., Valadka, A. B., Gordon, W. A., Okonkwo, D. O., Davies, P., Agarwal, S., Lin, F., Sarkis, G., Yadikar, H., Yang, Z., Manley, G. T., Wang, K. K. W., Cooper, S. R., Dams-O'Connor, K., Borrasso, A. J., Inoue, T., Maas, A. I. R., Menon, D. K., Schnyer, D. M., and Vassar, M. J. (2017) Comparing plasma phospho tau, total Tau, and phospho tau–total tau ratio as acute and chronic traumatic brain injury biomarkers. JAMA Neurol. 74, 1063–1072
Meeter, L. H. H., Vijverberg, E. G., Del Campo, M., Rozemuller, A. J. M., Donker Kaat, L., de Jong, F. J., van der Flier, W. M., Teunissen, C. E., van Swieten, J. C., and Pijnenburg, Y. A. L. (2018) Clinical value of neurofilament and phospho-tau/tau ratio in the frontotemporal dementia spectrum. Neurology. 0, 10.1212/WNL.0000000000005261
Foley, A. R., Roseman, G. P., Chan, K., Smart, A., Finn, T. S., Yang, K., Scott Lokey, R., Millhauser, G. L., and Raskatov, J. A. (2020) Evidence for aggregation-independent, PrPC-mediated Aβcellular internalization. Proc. Natl. Acad. Sci. U. S. A. 117, 28625–28631
Mcilwain, D. R., Berger, T., and Mak, T. W. (2015) Caspase Functions in Cell Death and Disease. 10.1101/cshperspect.a008656
Roucou, X., Giannopoulos, P. N., Zhang, Y., Jodoin, J., Goodyer, C. G., and LeBlanc, A. (2005) Cellular prion protein inhibits pro-apoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells. Cell Death Differ. 12, 783–795
Brown, D. R., and Besinger, A. (1998) Prion protein expression and superoxide dismutase activity. Biochem. J. 334 ( Pt 2, 423–9
Yan, S. D., Chen, X., Fu, J., Chen, M., Zhu, H., Roher, a, Slattery, T., Zhao, L., Nagashima, M., Morser, J., Migheli, a, Nawroth, P., Stern, D., and Schmidt, a M. (1996) RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature. 382, 685–91
Mijit, M., Caracciolo, V., Melillo, A., Amicarelli, F., and Giordano, A. (2020) Role of p53 in the regulation of cellular senescence. Biomolecules. 10, 1–16
Turnquist, C., Horikawa, I., Foran, E., Major, E. O., Vojtesek, B., Lane, D. P., Lu, X., Harris, B. T., and Harris, C. C. (2016) P53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration. Cell Death Differ. 23, 1515–1528
Anantharam, V., Kanthasamy, A., Choi, C. J., Martin, D. P., Latchoumycandane, C., Richt, J. A., and Kanthasamy, A. G. (2008) Opposing roles of prion protein in oxidative stress- and E.R. stress-induced apoptotic signaling. Free Radic. Biol. Med. 45, 1530–1541
Roucou, X., Gains, M., and LeBlanc, A. C. (2004) Neuroprotective Functions of Prion Protein. J. Neurosci. Res. 75, 153–161
Klamt, F., Dal-Pizzol, F., Conte Da Frota, M. L., Walz, R., Andrades, M. E., Da Silva, E. G., Brentani, R. R., Izquierdo, I., and Fonseca Moreira, J. C. (2001) Imbalance of antioxidant defense in mice lacking cellular prion protein. Free Radic. Biol. Med. 30, 1137–1144
Senator, A., Rachidi, W., Lehmann, S., Favier, A., and Benboubetra, M. (2004) Prion protein protects against DNA damage induced by paraquat in cultured cells. Free Radic. Biol. Med. 37, 1224–1230
Brown, D. R. (2004) Role of the prion protein in copper turnover in astrocytes. Neurobiol. Dis. 15, 534–543
Hare, D. J., Grubman, A., Ryan, T. M., Lothian, A., Liddell, J. R., Grimm, R., Matsuda, T., Doble, P. A., Cherny, R. A., Bush, A. I., White, A. R., Masters, C. L., and Roberts, B. R. (2013) Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry. Metallomics. 5, 1656–1662
Kozlowski, H., Janicka-Klos, A., Brasun, J., Gaggelli, E., Valensin, D., and Valensin, G. (2009) Copper, iron, and zinc ions homeostasis and their role in neurodegenerative disorders (metal uptake, transport, distribution and regulation). Coord. Chem. Rev. 253, 2665–2685
Jomova, K., Vondrakova, D., Lawson, M., and Valko, M. (2010) Metals, oxidative stress and neurodegenerative disorders. Mol. Cell. Biochem. 345, 91–104
Bagheri, S., Squitti, R., Haertlé, T., Siotto, M., and Saboury, A. A. (2018) Role of copper in the onset of Alzheimer's disease compared to other metals. Front. Aging Neurosci. 9, 1–15
Crisponi, G., Nurchi, V. M., Fanni, D., Gerosa, C., Nemolato, S., and Faa, G. (2010) Copper-related diseases: From chemistry to molecular pathology. Coord. Chem. Rev. 254, 876–889
Singh, N., Haldar, S., Tripathi, A. K., Horback, K., Wong, J., Sharma, D., Beserra, A., Suda, S., Anbalagan, C., Dev, S., Mukhopadhyay, C. K., and Singh, A. (2014) Brain Iron Homeostasis: From Molecular Mechanisms To Clinical Significance and Therapeutic Opportunities. Antioxid. Redox Signal. 20, 1324–1363
Ide-Ektessabi, A., and Rabionet, M. (2005) The role of trace metallic elements in neurodegenerative disorders: quantitative analysis using XRF and XANES spectroscopy. Anal. Sci. Int. J. Japan Soc. Anal. Chem. 21, 885–892
Bousejra-Elgarah, F., Bijani, C., Coppel, Y., Faller, P., and Hureau, C. (2011) Iron(II) binding to amyloid-β, the Alzheimer's peptide. Inorg. Chem. 50, 9024–9030
Kuchibhotla, K. V., Lattarulo, C. R., Hyman, B. T., and Bacskai, B. J. (2009) Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science (80-. ). 323, 1211–1215
Lazzari, C., Kipanyula, M. J., Agostini, M., Pozzan, T., and Fasolato, C. (2015) Aβ42 oligomers selectively disrupt neuronal calcium release. Neurobiol. Aging. 36, 877–885
Berridge, M. J. (2010) Calcium hypothesis of Alzheimer disease. Signal. CELL Physiol. 459, 441–449
Jacobson, J., and Duchen, M. R. (2002) Mitochondrial oxidative stress and cell death in astrocytes--requirement for stored Ca2+ and sustained opening of the permeability transition pore. J. Cell Sci. 115, 1175–1188
Guénette, S. Y. (2003) Astrocytes: A cellular player in Aβclearance and degradation. Trends Mol. Med. 9, 279–280
Ries, M., and Sastre, M. (2016) Mechanisms of Aβclearance and degradation by glial cells. Front. Aging Neurosci. 8, 1–9
Koger, D., Malkani, S., Higgs, R., Hanson, J., Paul, S. M., Bales, K. R., Koistinaho, M., Wu, X., Liu, F., Esterman, M., and Lin, S. (2004) Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-βpeptides. Nat. Med. 10, 719–726
Nielsen, H. M., Mulder, S. D., Beliën, J. A. M., Musters, R. J. P., Eikelenboom, P., and Veerhuis, R. (2010) Astrocytic Aβ1-42 uptake is determined by Aβ-aggregation state and the presence of amyloid-associated proteins. Glia. 58, 1235–1246
Cline, E. N., Bicca, M. A., Viola, K. L., and Klein, W. L. (2018) The Amyloid-βOligomer Hypothesis: Beginning of the Third Decade. J. Alzheimer's Dis. 64, S567–S610
Nieznanski, K., Choi, J. K., Chen, S., Surewicz, K., and Surewicz, W. K. (2012) Soluble prion protein inhibits amyloid-β(Aβ) fibrillization and toxicity. J. Biol. Chem. 287, 33104–33108
Maragakis, N. J., and Rothstein, J. D. (2006) Mechanisms of Disease: Astrocytes in neurodegenerative disease. Nat. Clin. Pract. Neurol. 2, 679–689
Grad, L. I., and Cashman, N. R. (2014) Prion-like activity of Cu/Zn superoxide dismutase Implications for amyotrophic lateral sclerosis. Prion. 10.4161/pri.27602
McBean, G. J. (2017) Cysteine, glutathione, and thiol redox balance in astrocytes. Antioxidants. 6, 1–13
Rizor, A., Pajarillo, E., Johnson, J., Aschner, M., and Lee, E. (2019) Astrocytic oxidative/nitrosative stress contributes to parkinson's disease pathogenesis: The dual role of reactive astrocytes. Antioxidants. 10.3390/antiox8080265
Gasperini, L., Meneghetti, E., Legname, G., and Benetti, F. (2016) In absence of the cellular prion protein, alterations in copper metabolism and copper-dependent oxidase activity affect iron distribution. Front. Neurosci. 10, 1–14
Kralovicova, S., Fontaine, S. N., Alderton, A., Alderman, J., Ragnarsdottir, K. V., Collins, S. J., and Brown, D. R. (2009) The effects of prion protein expression on metal metabolism. Mol. Cell. Neurosci. 41, 135–147
Millhauser, G. L. (2007) Copper and the Prion Protein: Methods, Structures, Function, and Disease. Annu Rev Phys Chem. 58, 299–320
Tsang, C. K., Liu, Y., Thomas, J., Zhang, Y., and Zheng, X. F. S. (2014) Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance. Nat. Commun. 5, 3446
Banks, C. J., and Andersen, J. L. (2019) Mechanisms of SOD1 regulation by post-translational modifications. Redox Biol. 26, 101270
Li, X., Qiu, S., Shi, J., Wang, S., Wang, M., Xu, Y., Nie, Z., Liu, C., and Liu, C. (2019) A new function of copper zinc superoxide dismutase: As a regulatory DNA-binding protein in gene expression in response to intracellular hydrogen peroxide. Nucleic Acids Res. 47, 5074–5085
Tsang, C. K., Liu, Y., Thomas, J., Zhang, Y., and Zheng, S. (2015) Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance. Nat Commun. 344, 1173–1178
Deane, R., Du Yan, S., Submamaryan, R. K., LaRue, B., Jovanovic, S., Hogg, E., Welch, D., Manness, L., Lin, C., Yu, J., Zhu, H., Ghiso, J., Frangione, B., Stern, A., Schmidt, A. M., Armstrong, D. L., Arnold, B., Liliensiek, B., Nawroth, P., Hofman, F., Kindy, M., Stern, D., and Zlokovic, B. (2003) RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat. Med. 9, 907–913
Wan, W., Chen, H., and Li, Y. (2013) The potential mechanisms of Aβ-receptor for advanced glycation end-products interaction disrupting tight junctions of the blood-brain barrier in Alzheimer's disease. Int. J. Neurosci. 7454, 1–7
Takuma, K., Fang, F., Zhang, W., Yan, S., Fukuzaki, E., Du, H., Sosunov, A., McKhann, G., Funatsu, Y., Nakamichi, N., Nagai, T., Mizoguchi, H., Ibi, D., Hori, O., Ogawa, S., Stern, D. M., Yamada, K., and Yan, S. S. Du (2009) RAGE-mediated signaling contributes to intraneuronal transport of amyloid-βand neuronal dysfunction. Proc. Natl. Acad. Sci. U. S. A. 106, 20021–20026
Yan, S. Du, Bierhaus, A., Nawroth, P. P., and Stern, D. M. (2009) RAGE and Alzheimer's disease: A progression factor for amyloid-β- induced cellular perturbation? J. Alzheimer's Dis. 16, 833–843
Wyllie, A. H., Rich, T., and Allen, R. L. (2000) Defying death after DNA damage. Nature. 407, 777–783
Schuler, M., Bossy-Wetzel, E., Goldstein, J. C., Fitzgerald, P., and Green, D. R. (2000) P53 Induces Apoptosis By Caspase Activation Through Mitochondrial Cytochrome C Release. J. Biol. Chem. 275, 7337–7342
Drane, P., Bravard, A., Bouvard, V., and May, E. (2001) Reciprocal down-regulation of p53 and SOD2 gene expression-implication in p53 mediated apoptosis. Oncogene. 20, 430–439
Huang, C. Y., Fujimura, M., Noshita, N., Chang, Y. Y., and Chan, P. H. (2001) SOD1 down-regulates NF-κB and c-Myc expression in mice after transient focal cerebral ischemia. J. Cereb. Blood Flow Metab. 21, 163–173
Ou, P., and Wolff, S. P. (1996) A discontinuous method for catalase determination at "near physiological" concentrations of H2O2 and its application to the study of H2O2 fluxes within cells. J. Biochem. Biophys. Methods. 31, 59–67
Stine, W. B., Dahlgren, K. N., Krafft, G. a, and LaDu, M. J. (2003) In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J. Biol. Chem. 278, 11612–11622
Lee, J. K. H., Pearson, J. D., Maser, B. E., and Ingham, R. J. (2013) Cleavage of the JunB transcription factor by caspases generates a carboxyl-terminal fragment that inhibits activator protein-1 transcriptional activity. J. Biol. Chem. 288, 21482–21495
Ohno, Y., Koizumi, M., Nakayama, H., Watanabe, T., Hirooka, M., Tokumoto, Y., Kuroda, T., Abe, M., Fukuda, S., Higashiyama, S., Kumagi, T., and Hiasa, Y. (2017) Downregulation of ANP32B exerts anti-apoptotic effects in hepatocellular carcinoma. PLoS One. 12, 1–17
Belmokhtar, C. A., Hillion, J., and Segal-Bendirdjian, E. (2001) Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms. Oncogene. 107, 3354–3362
Simenc, J., and Lipnik-Stangelj, M. (2012) Staurosporine induces different cell death forms in cultured rat astrocytes. Radiol. Oncol. 46, 312–320
Matias, A. C., Manieri, T. M., Cipriano, S. S., Carioni, V. M. O., Nomura, C. S., Machado, C. M. L., and Cerchiaro, G. (2012) Diethyldithiocarbamate induces apoptosis in neuroblastoma cells by raising the intracellular copper level, triggering cytochrome c release and caspase activation. Toxicol. Vitr. 27, 349–357
Magrini, T. D., dos Santos, N. V., Milazzotto, M. P., Cerchiaro, G., and da Silva Martinho, H. (2012) Low-level laser therapy on MCF-7 cells: a micro-Fourier transform infrared spectroscopy study. J. Biomed. Opt. 17, 1015161
Strober, W. (2001) Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. Appendix 3, Appendix 3B
Liu, Y., Peterson, D. a, Kimura, H., and Schubert, D. (1997) Mechanism of Cellular 3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction. J. Neurochem. 69, 581–593
Lago, L., Nunes, E. A., Vigato, A. A., Souza, V. C. O., Barbosa, F., Sato, J. R., Batista, B. L., and Cerchiaro, G. (2017) Flow of essential elements in subcellular fractions during oxidative stress. Biometals. 10.1007/s10534-016-9988-3
Marques, C. M. S., Nunes, E. A., Lago, L., Pedron, C. N., Manieri, T. M., Sato, R. H., Oliveira, V. X., and Cerchiaro, G. (2017) Generation of Advanced Glycation End-Products (AGEs) by glycoxidation mediated by copper and ROS in a human serum albumin (HSA) model peptide: reaction mechanism and damage in motor neuron cells. Mutat. Res. - Genet. Toxicol. Environ. Mutagen. 824, 42–51
Dantas, L. S., Chaves-Filho, A. B., Coelho, F. R., Genaro-Mattos, T. C., Tallman, K. A., Porter, N. A., Augusto, O., and Miyamoto, S. (2018) Cholesterol secosterol aldehyde adduction and aggregation of Cu,Zn-superoxide dismutase: Potential implications in ALS.Redox Biol. 19, 105–115

No competing interests reported.

Download PDF

Journal Publication

published 28 Dec, 2022

Read the published version in Molecular and Cellular Biochemistry →

Editorial decision: Major revision
10 Sep, 2022
Reviews received at journal
22 Apr, 2022
Reviewers agreed at journal
12 Apr, 2022
Reviewers agreed at journal
11 Apr, 2022
Reviewers invited by journal
08 Apr, 2022
Editor assigned by journal
08 Apr, 2022
Submission checks completed at journal
08 Apr, 2022
First submitted to journal
07 Apr, 2022

You are reading this latest preprint version

Cellular prion protein offers neuroprotection in astrocytes under oxidative stress

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1 PrP^C in the control of cellular maintenance

2.2 The control in metal regulation exhibited by the PrP^C

2.3 PrP^C prevents protein aggregation in astrocytes

2.4 PrP^C has a neuroprotective function against oxidative stress

2.5 The involvement of PrP^C in signaling mechanisms during stress in astrocytes

3. Discussion

4. Experimental Procedures

4.1 Chemicals

4.2 Cell culture and conditions

4.3 Cellular viability

4.4 Cell cycle assay

4.5 Determination of metal levels

4.6 Western blot analysis

4.7 Immunocytochemistry

4.8 Protein aggregation

4.9 Statistical analyses

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Cellular prion protein offers neuroprotection in astrocytes under oxidative stress

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1 PrPC in the control of cellular maintenance

2.2 The control in metal regulation exhibited by the PrPC

2.3 PrPC prevents protein aggregation in astrocytes

2.4 PrPC has a neuroprotective function against oxidative stress

2.5 The involvement of PrPC in signaling mechanisms during stress in astrocytes

3. Discussion

4. Experimental Procedures

4.1 Chemicals

4.2 Cell culture and conditions

4.3 Cellular viability

4.4 Cell cycle assay

4.5 Determination of metal levels

4.6 Western blot analysis

4.7 Immunocytochemistry

4.8 Protein aggregation

4.9 Statistical analyses

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

2.1 PrP^C in the control of cellular maintenance

2.2 The control in metal regulation exhibited by the PrP^C

2.3 PrP^C prevents protein aggregation in astrocytes

2.4 PrP^C has a neuroprotective function against oxidative stress

2.5 The involvement of PrP^C in signaling mechanisms during stress in astrocytes