Spermidine Rescues Bioenergetic and Mitophagy Deficits Induced by Disease-Associated Tau Protein

Abstract

Abnormal tau build-up is a hallmark of Alzheimer's disease (AD) and more than 20 other serious neurodegenerative diseases. Mitochondria are paramount organelles playing a predominant role in cellular bioenergetics, namely by providing the main source of cellular energy via adenosine triphosphate generation. Abnormal tau impairs almost every aspect of mitochondrial function, from mitochondrial respiration to mitophagy. The aim of our study was to investigate the effects of spermidine, a polyamine which exerts neuroprotective effects, on mitochondrial function in a cellular model of tauopathy. Recent evidence identified autophagy as the main mechanism of action of spermidine on life-span prolongation and neuroprotection, but the effects of spermidine on abnormal tau-induced mitochondrial dysfunction have not yet been investigated. We used SH-SY5Y cells stably expressing a mutant form of human tau protein (P301L tau mutation) or cells expressing the empty vector (control cells). We showed that spermidine improved mitochondrial respiration, mitochondrial membrane potential as well as adenosine triphosphate (ATP) production in both control and P301L tau-expressing cells. We also showed that spermidine decreased the level of free radicals, increased autophagy and restored P301L tau-induced impairments in mitophagy. Overall, our findings suggest that spermidine supplementation might represent an attractive therapeutic approach to prevent/counteract tau-related mitochondrial impairments.

Keywords: autophagy; bioenergetics; mitochondria; mitophagy; spermidine; tau.

Figure 1

Effects of spermidine on cell metabolic activity and ATP production. (A) No effect of spermidine on cell metabolic activity or (B) ATP production was observed after 24 h of treatment. (C) Spermidine at 0.1 and 1 μM significantly increased the metabolic activity in SH-SY5Y cells after 48 h of treatment. (D) Spermidine at 0.1 and 1 μM significantly increased ATP production in SH-SY5Y cells after 24 h of treatment. Values represent the mean and SD of four independent experiments (N = 20–25 replicates per condition). * p < 0.05; ** p < 0.01; one-way ANOVA and post hoc Dunnett’s multiple comparison test versus control condition (vehicle-treated cells). MTT: MTT assay.

Figure 2

Improved mitochondrial bioenergetics and decreased reactive oxygen species levels by spermidine in control and P301L cells. Cells were treated with 0.1 μM spermidine for 48 h. (A) Cell metabolic activity in vector and P301L cells. (B) ATP concentration in vector and P301L cells. (C) Mitochondrial membrane potential in vector and P301L cells. (D) Total superoxide anion level in vector and P301L cells. (E) Mitochondrial superoxide anion level in vector and P301L cells. Data are presented as mean ± SD (N = 25–30 replicates/conditions). * p < 0.05; ** p < 0.01; *** p < 0.001; one-way ANOVA and post hoc Tukey’s multiple comparisons test. MTT: MTT assay, SPD: spermidine, TMRM: tetramethylrhodamine, methyl ester and perchlorate, DHE: dihydroethdium.

Figure 3

Effects of spermidine on the bioenergetic phenotype of P301L tau-expressing cells and control cells. (A) Oxygen consumption rate (OCR) of vector and P301L tau cells was measured using a Seahorse XF HS Mini Analyzer (Agilent). The sequential injection of mitochondrial inhibitors, namely oligomycin (O), FCCP (F) and rotenone/antimycin A (RA), is indicated (see details in the “Materials and Methods” section). Values represent the mean ± SEM. (B) Values corresponding to the different respiratory parameters of vector cells and P301L cells are represented as mean ± SD (n = 8–10 replicates/condition). * p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA + post hoc Tukey’s multiple comparisons test. (C) Bioenergetic phenotype (OCR versus ECAR) of vector cells and P301L cells revealed an increase in metabolic activity after a 48 h treatment with 0.1 μM spermidine. Values represent the mean of each group (mean of the ECAR in abscissa/mean of the OCR in ordinate). (D) Extracellular acidification rate (ECAR) corresponding to the basal glycolysis with or without spermidine (SPD) treatment are represented as mean ± SD (n = 8–10 replicates). ** p < 0.01, *** p < 0.001, one-way ANOVA + post hoc Tukey’s multiple comparisons test.

Figure 4

Effects of spermidine on autophagy in P301L tau-expressing cells and vector cells. (A) Autophagic flux was assessed in vector and P301L cells by stimulating them with the autophagosome–lysosome fusion inhibitor Bafilomycin A1 (BAFA1, 100 nM) or DMSO alone. LC3 puncta (green), indicators of autophagosome formation, were then quantified. (B) P301L tau-expressing cells exhibit increased LC3 puncta levels when compared with vector cells at baseline. Spermidine (SPD) attenuated increased LC3 levels in P301L cells in basal conditions and increased the autophagic flux following treatment with Bafilomycin A1 in both cell lines. Data are represented as mean ± SD (n = 30–50 cells/conditions). * p < 0.05, ** p < 0.01; *** p < 0.001; three-way ANOVA + post hoc Tukey’s multiple comparisons test, ### p < 0.001, three-way ANOVA baseline vs. BAFA1, LC3: microtubule-associated protein 1A/1B-light chain 3.

Figure 5

Effects of spermidine on mitophagy in P301L tau-expressing cells and control cells. (A) To assess the levels of basal and carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP)-induced mitophagy, P301L and vector cells were transfected with LC3-RFP (green) and then stained with mitotracker (red) to measure the colocalization of LC3 puncta with mitochondria. (B) In basal conditions, no significant effect of P301L tau on cellular mitophagy was observed. However, following stimulation with the protonophore FCCP, which induces mitophagy by collapsing the mitochondrial membrane potential, P301L cells exhibited significantly reduced mitophagy compared with controls. Spermidine (SPD) significantly increased mitophagy in P301L cells following FCCP treatment. (C–F) Impact of mutant tau and SPD on the mRNA level of proteins involved in mitophagy, namely PINK1 (C), PARK2 (D), LC3 (E) and P62 (F). * p < 0.05, *** p < 0.001; three-way ANOVA + post hoc Tukey’s multiple comparisons test, ### p < 0.001, three-way ANOVA baseline vs. FCCP. PINK1: PTEN-induced putative kinase 1; PARK2: Parkin; LC3: microtubule-associated protein 1A/1B-light chain 3; P62: Sequestosome-1.

Figure 6

Schematic representation of the effects of abnormal tau protein and spermidine on mitochondrial function and autophagy. P301L mutant tau protein impairs mitochondrial bioenergetics, leading to decreased mitochondrial respiration, mitochondrial membrane potential (MMP) and ATP production, as well as an increase in reactive oxygen species (ROS) level. Spermidine seems to counteract the effects of abnormal tau on these parameters. P301L mutant tau protein also impairs autophagy (possibly by inhibiting the fusion between lysosomes and autophagosomes) as well as mitophagy. These effects may be, at least in part, due to the abnormal tau-induced dysregulation of genes involved in mitophagy/autophagy. Spermidine improves these processes in mutant tau-expressing cells and increases the expression of genes involved in mitophagy/autophagy. PINK1: PTEN-induced putative kinase 1; PARK2: Parkin; LC3: microtubule-associated protein 1A/1B-light chain 3; P62: Sequestosome-1. Created with BioRender.com.