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

Abstract

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.

Keywords: Alzheimer’s disease; Nrf2; antioxidant; mitochondrial dysfunction; neuroprotection; sulforaphane; tau.

Figure 1

Activation of the Nrf2 pathway prevents the mitochondrial failure induced by truncated tau. (A) CN1.4 cells were co-transfected with Mito-mCherry and GFP, GFP-T4, and GFP-T4C3 tau forms to evaluate mitochondrial length. Alternatively, GFP, GFP-T4, and GFP-T4C3 were treated with Sulforaphane (SFN, 10 μM 24 h) to evaluate mitochondrial length and mitochondrial membrane potential levels. (B) Magnification of mitochondria morphology from Mito-mCherry presented in A. Representative fluorescent images show that treatment with SFN prevented mitochondrial fragmentation induced by caspase-3 cleaved tau in immortalized cortical neurons. (C) Quantification of mitochondrial length obtained from fluorescent images obtained from double transfected CN 1.4 cells. (D) Determination of mitochondrial membrane potential levels. SFN treatment prevented mitochondrial depolarization induced by truncated tau. Data are presented as Mean ± SE, n = 5. Statistics differences were calculated by the t-Student test. * p < 0.001, ** p < 0.05. A, Bar = 20 μm; B Bar = 5 μm.

Figure 2

Treatment with sulforaphane prevents ATP loss induced by truncated tau. (A) CN 1.4 cells were transfected with GFP and GFP-tau(s) forms (full-length and truncated) and were treated with thapsigargin (0.5 μM, 1 h) to determine ATP levels. Treatment with Sulforaphane (SFN, 10 μM 24 h) 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.

Figure 3

Activation of the Nrf2 pathway decreased ROS production in cortical neurons that express caspase-3 cleaved tau. (A) Fluorescence representative images of cells transfected with GFP, GFP-T4, and GFP-T4C3 tau forms were treated with thapsigargin (0.5 μM, 1 h) and Sulforaphane (SFN, 10 μM 24 h). ROS production was measured using Cell Rox dye (see Materials and Methods section). (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.

Figure 4

Sulforaphane induces the activation of the Nrf2 pathway in immortalized cortical neurons. Relative mRNA expression of (A) Nrf2, (B) Keap1, (C) NQO1, (D) HO-1, (E) GCS, (F) GR1, and (G) TRxR in CN1.4 cells transfected with GFP, GFP-T4, and GFP-T4C3 tau forms. Treatment with SFN (10 μM, 24 h) increased antioxidant gene expression. Data are presented as the Mean ± SE, n = 4. Differences were calculated by one-way ANOVA tests. * p < 0.5, ** p < 0.01, *** p < 0.001.

Figure 5

Treatment with sulforaphane increases the antioxidants protein expression in immortalized cortical neurons. (A,C,E) are representative western blot images of immortalized cortical neurons transfected with GFP, GFP-T4, and GFP-T4C3, showing the protein expression of Nrf2, NQO-1, and Catalase. Treatment with SFN (10 μM 24 h) increased the expression of these proteins in all conditions indicated. (B,D,F) showed densitometry analyses for Nrf2, NQO-1, and Catalase expression. Data are presented as the Mean ± SE, n = 4. Statistical analysis were performed using one-way ANOVA test. * p < 0.01, ** p < 0.0061, *** p < 0.0003, **** p < 0.0001.