Activation of the Nrf2 Pathway Prevents Mitochondrial Dysfunction Induced by Caspase-3 Cleaved Tau: Implications for Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by memory and cognitive impairment, accompanied by the accumulation of extracellular deposits of amyloid β-peptide (Aβ) and the presence of neurofibrillary tangles (NFTs) composed of pathological forms of tau protein. Mitochondrial dysfunction and oxidative stress are also critical elements for AD development. We previously showed that the presence of caspase-3 cleaved tau, a relevant pathological form of tau in AD, induced mitochondrial dysfunction and oxidative damage in different neuronal models. Recent studies demonstrated that the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) plays a significant role in the antioxidant response promoting neuroprotection. Here, we studied the effects of Nrf2 activation using sulforaphane (SFN) against mitochondrial injury induced by caspase-3 cleaved tau. We used immortalized cortical neurons to evaluate mitochondrial bioenergetics and ROS levels in control and SFN-treated cells. Expression of caspase-3 cleaved tau induced mitochondrial fragmentation, depolarization, ATP loss, and increased ROS levels. Treatment with SFN for 24 h significantly prevented these mitochondrial abnormalities, and reduced ROS levels. Analysis of Western blots and rt-PCR studies showed that SFN treatment increased the expression of several Nrf2-related antioxidants genes in caspase-3 cleaved tau cells. These results indicate a potential role of the Nrf2 pathway in preventing mitochondrial dysfunction induced by pathological forms of tau in AD.


Introduction
AD is the most prevalent neurodegenerative disease worldwide. AD is a progressive disorder that causes cognitive defects and behavioral changes, where the earliest symptoms are memory impairment that progresses gradually into severe dementia [1]. AD is characterized by the accumulation of Aβ and NFTs composed of pathological forms of tau, including hyperphosphorylated and truncated types [2,3]. Tau is a microtubule-associated protein that contributes to axonal transport, synaptic function, and microtubule stability. The cleavage of tau at D421 by caspase-3 (truncation) is an important contributor to NFTs formation and neurodegenerative changes observed in AD [4,5]. In particular, tau truncation by caspase-3 (D421) is present in AD brains [6], and increasing levels of this tau form have been detected in aged mice contributing to the deficits in synaptic plasticity and cognitive impairment [7].
Thus, nowadays, AD is considered a multifactorial disease in which mitochondrial dysfunction actively contributes to AD onset and progression. These mitochondrial abnormalities include bioenergetics defects, mitochondrial respiratory impairment, oxidative damage, and defects in mitochondrial dynamics that contribute to synaptic dysfunction observed in AD [8][9][10]. Importantly, we have demonstrated that the expression of caspase-3 incubated at 33 • C and 5% CO 2 . In addition, these cells were transiently transfected with plasmids containing GFP, GFP-full-length tau (GFP-T4), and GFP-caspase-3 cleaved tau (GFP-T4C3), and after 48 h, transfected cells were treated with 10 µm of SFN (ChemCruz, Santa Cruz Biotechnology, Dallas, TX, USA) for 24 h.

Determination of Mitochondrial Length and Membrane Potential in Live Cells
The mitochondrial length was evaluated in CN 1.4 cells double transfected by GFP and GFP-tau (s) forms and Mito-mCherry construct [9,11]. The mitochondrial length was obtained by measuring the lengths of individual mitochondria present in fluorescence images, which were obtained using high-resolution fluorescence microscopy (Leica, Wetzlar, Germany). We analyzed the mitochondrial population of 40-60 cells, measuring 25 images for each experiment using ImageJ software for analysis [9,[11][12][13].
The mitochondrial membrane potential (MMP) levels were evaluated using the mitochondrial Mitotracker Red 2XRosH 2 (MitoRed) dye in CN 1.4 cells [12,13]. Transfected cells were loaded with MitoRed in Krebs-Ringer-HEPES (KRH) buffer (136 mM NaCl, 20 mM HEPES, 4.7 mM KCl, 1.5 mM MgSO4, 1.25 mM CaCl2, 5 mM glucose; pH = 7.4) for 35 min, and then fluorescence images were taken. Fluorescence intensity was captured using a fluorescence microscope LX6000 (Leica, Wetzlar, Germany) under a 63×oil objective. We analyzed 30-40 cells on average for the quantification process, measuring at least 25 images for each experiment. Mitochondrial potential levels are presented as the pseudo ratio (∆F/F0), where F is the average of the fluorescence signal (F) per area in every image, and (F0) represents the intensity of background fluorescence [11][12][13].

Determination of ROS Levels
ROS levels were evaluated using CellRox TM dye (Molecular Probes, Thermo Fisher Scientific) [24]. Transfected cultured cells were incubated with 0.5 µM CellRox in KRH-glucose buffer at 37 • C for 30 min [13]. Fluorescence images were acquired using the same exposure time and gain to minimize dye photo-bleaching [35,36]. CellRox fluorescence (F) was determined in each cell, and each image's background (F0) was subtracted before normalization per cell area. Normalization was performed to ameliorate differences in the dye distribution inside the cells that present differences in cellular morphology. Fluorescence images were acquired using a fluorescence microscope (Leica LX6000, Wetzlar, Germany).

Real-Time Polymerase Chain Reaction
The expression of Nrf2 (NM_010902.5), Keap1 (NM_001110307.1, HO-1 (NM_010442.2), NQO-1 (NM_008706.5), GR1 (NM_010344.4), TRXr1 (NM_001042523.1), and GCS (NM_010295.2) mRNA levels were analyzed by real-time polymerase chain reaction (RT-PCR) ( Table 1) [37]. Total RNA was isolated from 100 mg of CN 1.4 lysates transfected with GFP, GFP-T4, and GFP-T4C3 using the Trizol reagent (Life Technologies, Thermo Fisher Scientific), according to the manufacturer's protocol. RNA yield and purity were measured in a microplate reader (TECAN, Infinite 200 PRO series. One µg of total RNA was subjected to reverse transcription using the ImProm-II Reverse Transcription System (Promega), following the manufacturer's protocol. The cDNA was stored at −20 • C for further use. Each cDNA sample was diluted 10 times with nuclease-free water for qPCR analysis. The real-time PCR reaction was performed in triplicate in the Aria Max (Agilent technologies) in a final volume of 10 µL [13]. Amplification conditions consisted of an initial denaturation at 95 • C for 10 min, followed by amplification of 40 cycles (95 • C for 15 s, 60 • C for the 20 s, and 72 • C for 20 s). A melting curve analysis was performed immediately after amplification from 55 to 95 • C. Values were normalized to 18S expression levels using the ∆CT method. The following table presents the primers sequence of genes evaluated: Table 1. Primers sequences of Nrf2/ARE representative genes evaluated.

Gen
Forward Primer Reverse Primer

Measurement of ATP Concentration
ATP concentration was measured in cortical neurons transfected with GFP, GFP-T4, and GFP-T4C3 in the total lysates using a luciferin/luciferase bioluminescence assay kit (ATP determination kit #A22066, Molecular Probes, Invitrogen) [38]. The amount of ATP in each sample was calculated from standard curves and normalized to the total protein concentration.

Statistical Analysis
The data are expressed as the mean ± standard error (SEM), with at least three experiments indicated in the corresponding figures. All samples included in these studies were analyzed to normality distribution using the Kolmogorov-Smirnov test [37]. Later, the obtained data were analyzed using Student's t-test with Dunnett's post hoc test or, if analyzing more than two groups, ANOVA followed by Bonferroni's post hoc test. p < 0.05 and p < 0.001 were considered statistically significant. All statistical analyses were performed using Prism software (GraphPad Software Inc., San Diego, CA, USA).

Activation of the Nrf2 Pathway Prevent Mitochondrial Dysfunction Induced by Caspase-3 Cleaved Tau
Immortalized cortical neurons were co-transfected with Mito-mCherry to examine mitochondrial morphology in situ, as well as mitochondrial membrane potential levels ( Figure 1A-D). We previously showed that truncated tau expression reduced mitochondrial length and mitochondrial membrane potential levels (MMP) [10,11]. Here, we observed that GFP-T4C3 presented a reduced mitochondrial length than GFP and GFP-T4 cells ( Figure 1A,B). GFP and GFP-T4 present a uniform mitochondrial morphology distributed throughout the whole cell body ( Figure 1B). However, GFP-T4C3 cells showed more rounded and fragmented mitochondria ( Figure 1B). Quantification analysis shows that GFP-T4C3 cells present a decrease of two-fold in mitochondrial length, compared with GFP or GFP-T4 cells ( Figure 1C). Interestingly, when GFP-T4C3 cells were treated for 24 h with 10 µM of SFN, the mitochondrial fragmentation was prevented entirely, reaching similar values of mitochondrial length of GFP and GFP-T4 cells ( Figure 1B,D). For mitochondrial membrane potential determinations, GFP and GFP-tau (s) transfected cells were loaded with MitoRed, and treated with 0.5 µM thapsigargin (25 min) to induce a transient cytosolic calcium increase to test mitochondrial function [11,39]. Therefore, MitoRed fluorescence intensity levels were obtained from live-cell images taken in a fluorescence microscope (Leica, Germany). Mitochondrial potential levels were severely affected in GFP-T4C3 cells, reaching a more than four-fold decrease compared with GFP or GFP-T4 transfected cells. More importantly, GFP-T4C3 treated with SFN showed prevention of mitochondrial depolarization reaching similar mitochondrial potential levels to GFP and GFP-T4 cells ( Figure 1D). These studies suggest that SFN prevents mitochondrial impairment induced by caspase-3 cleaved tau.

Activation of the Nrf2 Pathway Improves Mitochondrial Health by Rescuing ATP Production Loss Induced by Caspase-3 Cleaved Tau
Due to their high activity and signaling, neuronal cells require a constant energy supply, which makes them particularly vulnerable to mitochondrial dysfunction [40]. Decreased ATP production is a common feature in neurodegenerative diseases, such as AD, and can be caused by various mechanisms, including the impaired activity of mitochondrial respiratory complexes, alterations in glucose uptake, glycolysis, and the TCA cycle or uncoupling [41]. To further understand how the presence of caspase-3 cleaved tau affected mitochondrial functions, we measured ATP production in immortalized cortical neurons that expressed GFP, GFP-T4, and GFP-T4C3 ( Figure 2). The expression of GFP-T4C3 presented a significant decrease in basal ATP levels compared to GFP and GFP-T4 expressing cells ( Figure 2A). Furthermore, we treated transfected cells with 0.5 µM of thapsigargin, and we observed that ATP production was markedly decreased, reaching similar values in all these three conditions (Figure 2A). Significantly, when cells were treated for 24 h with 10 µM SFN, the loss in ATP production was restored in GFP-T4C3 cells treated with thapsigargin ( Figure 2A).  prevented ATP loss induced by caspase-3 cleaved tau. (B) ATP levels determination in transfected cells exposed to SFN (10 µM, 24 h) and control conditions. Graphs bars represent Mean ± SE, n = 4. The statistical differences were calculated by a one-way ANOVA test. * p < 0.5, ** p < 0.01, *** p < 0.0007, **** p < 0.0001.

Sulforaphane Prevented ROS Increase Induced by Caspase-3 Cleaved Tau Expression
Complementary to mitochondrial function determinations, we analyzed ROS production levels in cells transfected with GFP, GFP-T4, and GFP-T4C3 using Cell Rox dye ( Figure 3). We observed that the expression of caspase-3 cleaved tau significantly increased basal ROS levels compared with GFP and GFP-T4 cells ( Figure 3A). Moreover, transfected cells treated with thapsigargin (0.5 µM, 30 min) showed an increase in ROS levels; however, under this condition, GFP-T4C3 cells showed a more significant increase in Cell Rox signal compared to GFP and GFP-T4 cells ( Figure 3B). Interestingly, ROS increase in GFP-T4C3 cells showed a fluorescence pattern similar to mitochondrial localization, indicating an essential role of this organelle in this effect ( Figure 3A). Significantly, SFN completely reduced ROS-increased levels in cells expressing caspase-3 cleaved tau (GFP-T4C3), compared with ROS levels shown in control or full-length tau cells ( Figure 3B). These studies suggest that incubation with SFN prevented the excess ROS production present in GFP-T4C3 cells, facilitating the protection of mitochondrial function, as observed by the normalization of ROS levels. (B) Cells expressing caspase-3 cleaved tau showed excess in ROS production compared to full-length tau. SFN treatment prevented this ROS increase from reaching similar values to those observed in cells expressing full-length tau. Data are mean ± SE, n = 4. p < 0.05 indicates differences between groups calculated by the one-way ANOVA test. *** p < 0.0005, **** p < 0.0001. Bar = 20 µm.

Treatment with SFN Induces Activation of the Nrf2 Pathway in Immortalized Cortical Neurons
To verify the activation of the Nrf2 pathway by SFN, we performed rt-PCR and analyzed the mRNA expression of central antioxidant response genes activated by Nrf2 in GFP, GFP-T4, and GFP-T4C3 cells (Figure 4). Under basal conditions, the mRNA levels of Nrf2 in the GFP condition and the presence of caspase-3 cleaved tau remained relatively similar, except for GFP-T4 cells, where lower levels of mRNA expression were observed ( Figure 4). As a part of this Nrf2 activation mechanism, SFN modifies Keap1 cysteine residues, functioning as sensors for numerous oxidants and electrophiles, leading to Nrf2 activation [18]. Notably, when the cells were treated with SFN (10 µM, 24 h), Nrf2 expression was significantly increased in all three conditions ( Figure 4A). To further analyze the Nrf2 pathway, we evaluated the mRNA levels of the KEAP1, the negative regulator of the Nrf2 pathway ( Figure 4B). When cells were exposed to SFN, a mild increase in the KEAP1 expression in GFP and GFP-T4 cells was shown. Moreover, caspase-3 cleaved tau cells exposed to SFN presented KEAP1 mRNA levels similar to the control condition.
Furthermore, we analyze the expression of primary Nrf2-ARE dependent genes, including NQO1, HO-1, Glutamate-cysteine ligase (GCS), glutathione-disulfide reductase (GR1), and thioredoxin reductase 1 (TRxR 1), [26] (Figure 4C-F). The analysis of NQO1, a cytosolic flavoprotein that catalyzes quinine detoxification, showed that its basal expression was diminished in GFP, GFP-T4, and GFP-T4C3 expressing cells ( Figure 4C). On the other hand, the treatment with SFN induced a marked increase in this gene expression, reaching an increase of more than three-fold in the GFP and GFP-T4C3 expressing cells ( Figure 4C). Although, this increase was lower in GFP-T4 cells than in control, maintaining a similar tendency in the expression of Nrf2. In addition, we evaluated HO-1 levels ( Figure 4D), showing that SFN treatment significantly increased its expression in the three cell conditions ( Figure 4D). Further analysis of GCS and GR1 mRNA levels showed that SFN treatment increased GCS and GR1 levels in GFP, GFP-T4, and GFP-T4C3 cells ( Figure 4E,F). Finally, we evaluated the levels of the mammalian selenoprotein, thioredoxin reductase 1 (TRxR 1) ( Figure 4G). Intriguingly, GFP-T4C3 cells showed a higher basal expression in TRxR 1 levels than GFP and GFP-T4 cells. Moreover, SFN treatment increased TRxR1 in all conditions indicated. However, this increase was higher in cells that expressed GFP-T4C3 ( Figure 4G).
These results indicate that SFN induces the expression of antioxidant genes dependent on Nrf2 activity in immortalized cortical neurons. These events could be related to the prevention of ROS increase and mitochondrial impairment induced by the treatment with SFN in GFP-T4C3 cells.

Nrf2 Pathway Activation Increase the Antioxidant Protein Expression
Complementarily, we checked protein expression levels of several antioxidant proteins regulated by the Nrf2 pathway [42,43]. As we previously observed for mRNA levels, under basal conditions, Nrf2 protein levels remained similar in the GFP and GFP-T4 cells, while a lower decrease in Nrf2 expression was observed in GFP-T4C3 cells. After the treatment with SFN (10 µm, 24 h), Nrf2 expression was significantly increased in all cell conditions indicated (control, full-length, and caspase-3 cleaved tau). NQO-1 expression in cortical neurons showed similar results, with lower levels in the control conditions and higher expression when they were treated with SFN. Moreover, we measured catalase levels, whose expression is also related to Nrf2 activity [42]. Catalase is one of the crucial antioxidant enzymes that mitigates oxidative stress to a considerable extent by converting hydrogen peroxide to produce water and oxygen [42,43]. Under control conditions, GFP, full-length, and caspase-3 cleaved tau expressing cells showed relatively similar protein expression, and SFN treatment significantly increased catalase levels in all three conditions. Differences were calculated by one-way ANOVA tests. * p < 0.5, ** p < 0.01, *** p < 0.001.

Discussion
This study aimed to elucidate the possible role of the Nrf2 pathway on mitochondrial impairment induced by caspase-3 cleaved in neuronal cells. Nrf2 plays a central role in the cytoprotective response to oxidative stress and is critical for maintaining mitochondrial redox homeostasis [44]. Here, we reported that, in cells expressing truncated tau, mitochondria presented a fragmented morphology and higher levels of ROS and mitochondrial depolarization [11]. These observations indicate that truncated tau impairs mitochondrial function, which likely contributes to the neuronal dysfunction observed in AD. Interestingly, SFN treatment, a natural activator of Nrf2, prevented mitochondrial fragmentation in caspase-3 cleaved tau expressing cells (Figure 1). In addition, mitochondrial depolarization presented in GFP-T4C3 cells was prevented by the treatment with SFN ( Figure 1). Moreover, the treatment with SFN for 24 h prevented ROS production in truncated tau-expressing cells, suggesting that Nrf2 activation improves mitochondrial health.
Related to our findings, Kovac and colleagues showed that mitochondrial-ROS production is increased in brain tissue of Nrf2-KO mice [45]. They also demonstrated that Nrf2 impacts cellular bioenergetics by controlling substrate availability and the efficiency of mitochondrial fatty acid oxidation, thus affecting oxidative phosphorylation and increasing mitochondrial-ROS production [45]. Here, we observed that caspase-3 cleaved tau induces a decrease in ATP production, which correlates with mitochondrial depolarization shown in truncated tau expressing cells (Figure 2). In the same context, Holmström et al. observed an impaired activity of complex I of the respiratory chain under conditions of Nrf2 deficiency in neuronal cells of Nrf2-knockout mice [46]. Furthermore, Nrf2 deficiency leads to mitochondrial depolarization, reduced ATP levels, and impaired respiration [46]. On the other hand, other studies showed that basal MMP was increased when Nrf2 was genetically activated by knockout or knockdown of Keap1 protein, indicating that Nrf2 could regulate mitochondrial activity. Furthermore, the Nrf2 pathway contributes to mitochondrial function by modulating NADH and FADH2, both of which are important substrates for mitochondrial respiration [47,48].
Interesting studies have suggested an essential role of the Nrf2 pathway in promoting mitochondrial integrity under oxidative conditions [49]. For example, the isolated brain of rats that were subjected to a single dose of the Nrf2 activator SFN showed resistance to opening the mitochondrial permeability transition pore (mPTP) caused by the oxidant tertbutyl hydroperoxide [49]. The formation and opening of the mPTP is a fundamental factor in mitochondrial dysfunction, and this action can induce mitochondrial depolarization, decrease ATP production, release mitochondrial contents, and lead to cell death [50][51][52][53][54][55][56]. Interestingly, some studies suggest an important action of the Nrf2 pathway modulating mPTP opening induced by oxidative stress [57,58]. For example, Greco and coworkers observed a slight inhibition of peroxide-induced mPTP opening in mitochondria isolated from the brains of normal rats injected with SFN [49].
Furthermore, additional studies from Greco et al. demonstrated that SFN administration to rats dramatically inhibits redox-regulated mPTP opening by liver mitochondria, and increases immunoreactive levels of mitochondrial antioxidant-related proteins [49]. More importantly, we previously described that the use of mPTP inhibitor cyclosporin A (CsA) was influential in preventing mitochondrial fragmentation, decrease in MMP, and mitochondrial calcium handling defects showed in neuronal cells that expressed caspase-3 cleaved tau [11]. However, further studies are needed to elucidate if the prevention of mitochondrial dysfunction induced by Nrf2 is produced by mPTP regulation in caspase-3 cleaved tau expressing cells.
Emerging evidence demonstrates that the accumulation of phosphorylated and oligomerized tau is a toxic event that affects neuronal function [59,60]. In this context, evidence obtained by Jo et al. showed that activating the Nrf2 pathway with SFN reduced the abnormal accumulation of hyperphosphorylated tau by inducing the expression of autophagy adapter protein NDP52 in neurons [36]. The NDP52 protein presents three ARE in its promoter region, and its expression is strongly induced by Nrf2 activation, facilitating the elimination of hyperphosphorylated tau to the autophagic degradative pathway [36,61]. Although we did not observe these effects in caspase-3 cleaved tau cells treated with SFN ( Figure 5), we cannot rule out the possibility that this pathway could be contributing to preventing mitochondrial dysfunction induced by truncated tau. Finally, our results showed that treating cortical neurons with SFN increased the Nrf2-ARE-dependent genes expression in all conditions indicated ( Figure 4). Interestingly, we detected a significant increase in mRNA TRX1 expression in caspase-3 cleaved tau cells, compared with GFP-T4 cells (Figure 4). TRX1 is a critical enzyme that regulates several processes involved in decreasing oxidative stress-reducing intracellular disulfides and upholding the redox homeostasis within the cell [62]. Previous findings have shown that TRX1 levels were elevated in the cerebrospinal fluid of AD patients [63]. Moreover, the immunohistochemical studies of TRX1 revealed that only cytosolic localization was observed in the hippocampus CA1 of AD patients, whereas TRX1 in the control patients was observed in the nucleus, with no difference in the expressing levels between control and AD patients [63]. These observations suggest that the translocation from the nucleus to the cytosol may diminish this reduction and, ultimately, the function of critical signaling molecules and transcription factors regulated by TRX1 [64]. Notably, under the effect of SFN, NQO-1, GCS, and GR1 levels were elevated in full-length and caspase-3 cleaved tau expressing cells, indicating activation of the Nrf2-ARE pathway.
Furthermore, previous findings reported a significant increase in HO-1 expression in the post-mortem brain of AD temporal cortex and hippocampus compared to agedmatched control [65]. Moreover, an increase in NQO1 activity and expression was found in astrocytes and neurons of the AD brain [66]. Additionally, studies in aged APP/PS1 AD mouse models showed a reduction in Nrf2, NQO1, GCL catalytic subunit (GCLC), and GCL modifier subunit (GCLM) mRNA levels [67]. These studies indicate that Nrf2-related genes contribute to neurodegeneration in AD, and they may be responsible for mitochondrial impairment observed during this disease.
Our findings presented here indicate the importance of the Nrf2 pathway in reducing mitochondrial and oxidative damage induced by pathological forms of tau. Furthermore, it is essential to understand the mechanism underlying the protection induced by Nrf2dependent genes, and to elucidate other protective effects on mitochondrial function and the consequences of this protection on neuronal dysfunction in AD.