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Human Mitochondrial Transcriptional Factor A Breaks the Mitochondria-Mediated Vicious Cycle in Alzheimer's Disease

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Human Mitochondrial Transcriptional Factor A Breaks the Mitochondria-Mediated Vicious Cycle in Alzheimer's Disease

Sugako Oka et al. Sci Rep.

Abstract

In the mitochondria-mediated vicious cycle of Alzheimer's disease (AD), intracellular amyloid β (Aβ) induces mitochondrial dysfunction and reactive oxygen species, which further accelerate Aβ accumulation. This vicious cycle is thought to play a pivotal role in the development of AD, although the molecular mechanism remains unclear. Here, we examined the effects of human mitochondrial transcriptional factor A (hTFAM) on the pathology of a mouse model of AD (3xTg-AD), because TFAM is known to protect mitochondria from oxidative stress through maintenance of mitochondrial DNA (mtDNA). Expression of hTFAM significantly improved cognitive function, reducing accumulation of both 8-oxoguanine, an oxidized form of guanine, in mtDNA and intracellular Aβ in 3xTg-AD mice and increasing expression of transthyretin, known to inhibit Aβ aggregation. Next, we found that AD model neurons derived from human induced pluripotent stem cells carrying a mutant PSEN1(P117L) gene, exhibited mitochondrial dysfunction, accumulation of 8-oxoguanine and single-strand breaks in mtDNA, and impaired neuritogenesis with a decreased expression of transthyretin, which is known to be downregulated by oxidative stress. Extracellular treatment with recombinant hTFAM effectively suppressed these deleterious outcomes. Moreover, the treatment increased expression of transthyretin, accompanied by reduction of intracellular Aβ. These results provide new insights into potential novel therapeutic targets.

Figures

Figure 1
Figure 1. Expression of hTFAM improved mitochondrial function in the ADh/WT cerebral cortex and cognitive impairment in ADh/WT mice.
(a) Expression of hTFAM improved mitochondrial function in the ADh/WT cerebral cortex. Mitochondrial function was determined by enzymatic staining for cytochrome c oxidase (COX). Frozen sections from the cortex of 13–14-month-old Non-Tg, ADh/WT, and ADh/hTFAMh mice were stained for COX (reddish brown). Scale bar, 20 μm. (b) COX enzymatic staining in each mitochondrion in the cortex was visualized by electron microscopy. Arrowheads show degenerating mitochondria characterized by diminished COX staining and disrupted membranes and cristae in the ADh/WT cortex. Scale bars, 1 μm (upper panels); 500 nm (lower panels). (c) Latency to cross over the previously hidden platform 24 h after the training trial in the d2 protocol of the Morris water maze using 11–12-month-old Non-Tg, ADh/WT, and ADh/hTFAMh mice. One-way ANOVA, p = 0.0076. Tukey’s HSD test (vs. ADh/WT), *p < 0.05, **p < 0.01. Data represent means ± SEM, n = 5–8. (d) The number of crossings over the previously hidden platform 24 h after the training trial in the d2 protocol of the Morris water maze using 11–12-month-old Non-Tg, ADh/WT, and ADh/hTFAMh mice. Student’s t-test (vs. ADh/WT), #p = 0.0463. Data represent means ± SEM, n = 5–8. (e) Latency to reach the platform in the d5 protocol of the Morris water maze using 11–12-month-old ADh/WT and ADh/hTFAMh mice. Multivariate analysis of variance (MANOVA), genotype, p = 0.0011. Data represent means ± SEM, n = 5–8.
Figure 2
Figure 2. hTFAM suppresses Aβ accumulation in ADh/WT mice.
(a) Immunofluorescence microscopy of Aβ in cortex and hippocampus of 13–14-month-old ADh/WT and ADh/hTFAMh mice using anti-human Aβ (N). Nuclei are stained with DAPI. Scale bar, 20 μm. (b) Intracellular localization of Aβ in the ADh/WT cortex. Cell morphology was confirmed by differential interference contrast microscopy (DIC) with Aβ immunofluorescence. Scale bar, 5 μm. (c) Quantitative measurement of Aβ immunofluorescence. Aβ immunofluorescence was measured as described in Supplementary Fig. S3, and the relative intensity (Aβ index) is shown in whisker-box plot. Kruskal–Wallis rank sum test: cortex, #p = 0.0433, hippocampus, ##p = 0.0209, n = 3–4.
Figure 3
Figure 3. hTFAM markedly decreases accumulation of mitochondrial 8-oxoG in ADh/WT mice.
(a) Immunofluorescence microscopy of 8-oxoG in cortex and hippocampus of 13–14-month-old ADh/WT and ADh/hTFAMh mice. hTFAM decreases mitochondrial 8-oxoG in the cortex and hippocampus. Scale bar, 20 μm. (b) Quantitative measurement of 8-oxoG immunofluorescence. 8-OxoG immunofluorescence was measured as described in Supplementary Fig. S3, and the relative intensity of 8-oxoG immunoreactivity (8-oxoG index) is shown in bar graphs. Cortex, one-way ANOVA, p = 0.0028. Tukey-Kramer’s HSD test: *p = 0.0193, **p = 0.0025; Hippocampus, one-way ANOVA, p = 0.0198. Tukey-Kramer’s HSD test: *p = 0.0394, **p = 0.0277. Data represent means ± SEM, n = 4.
Figure 4
Figure 4. hTFAM markedly increases expression of a set of genes in ADh/WT mice.
(a) Hierarchical portioning and clustering of 166 transcript clusters demonstrate 1.6-fold or greater difference between hippocampi from ADh/WT and ADh/hTFAMh mice. Student’s t-test p < 0.05, n = 3. (b) Top significant network. Solid and dashed lines indicate direct and indirect interactions, respectively. Molecular interactions involving only binding were excluded. Downregulated molecules are shown in green and upregulated molecules are shown in red. (c) hTFAM increases the level of transthyretin in ADh/WT mice. Scale bar, 20 μm. (d) Western blot analysis of extracts prepared from cerebral cortices using anti-transthyretin. Ponceau S staining was used as the loading control (lower panel). (e) Double-immunostaining with anti-human TFAM and anti-transthyretin in ADh/hTFAMh cortex. Scale bar, 20 μm.
Figure 5
Figure 5. Recombinant human TFAM inhibits mitochondrial dysfunction and 8-oxoG accumulation in a human neuron model of AD.
(a) Recombinant human TFAM [rhTFAM ( + )], but not mutant rhTFAM (ΔMTS-rhTFAM) lacking MTS, ameliorates mitochondrial dysfunction in PS1P117L cells. Mitochondrial membrane potential was detected using JC-1. Intensities of green fluorescence in ΔMTS-rhTFAM-treated PS1P117L neurons were as high as those seen in control PS1P117L neurons (rhTFAM[−]), whose levels were higher than those observed in wild-type neurons with or without rhTFAM or ΔMTS-rhTFAM. Upper panels: JC-1 aggregates with intense red fluorescence, indicating energized mitochondria with high membrane potentials. Lower panels: JC-1 monomers with green fluorescence, indicating damaged mitochondria with low membrane potentials. Scale bar, 100 μm. (b) rhTFAM decreases mitochondrial 8-oxoG in PS1P117L cells. COX IV was used as a mitochondrial marker. Fluorescence intensities of 8-oxoG in 50 cells were measured, and the 8-oxoG index is shown in the bar graph. One-way ANOVA, p < 0.0001. Tukey-Kramer HSD test, *p < 0.0026, **p < 0.0001. Data represent means ± SEM. (c) rhTFAM inhibits single-strand breaks in mitochondrial DNA in PS1P117L cells. VDAC was used as a mitochondrial marker. (d) rhTFAM decreases mitochondrial superoxide production in PS1P117L cells. Wild-type and PS1P117L cells were treated with or without rhTFAM for 24 h, and then incubated with MitoTracker Green, a mitochondrial marker (upper panels, green) and MitoSOX, a mitochondrial superoxide indicator (lower panels, red). Nuclei were counterstained with Hoechst 33342 (upper panels, blue). Fluorescence intensities of MitoSOX in 50 cells were measured, and the MitoSOX index is shown as a bar graph. Two-way ANOVA, p < 0.0001. Tukey’s HSD test, *p < 0.0001. Data represent means ± SEM. (e) rhTFAM protein suppresses mitochondrial membrane lipid peroxidation in PS1P117L cells. MitoPeDPP, a fluorescent probe specific for lipophilic peroxide in the mitochondrial inner membrane was applied to PS1P117L cells with or without rhTFAM treatment. Nuclei were counterstained with Hoechst 33342. Fluorescence intensities of MitoPeDPP in 50 cells were measured, and the MitoPeDPP index is shown as a bar graph. Student’s t-test: ##p < 0.0001. Data represent means ± SEM. Scale bars, 40 μm (be).
Figure 6
Figure 6. Recombinant human TFAM inhibits Aβ accumulation in a human neuron model of AD with increased expression of transthyretin.
(a) rhTFAM decreases the accumulation of Aβ in PS1P117L cells. Wild-type and PS1P117L cells were treated with (rhTFAM[+]) or without rhTFAM (rhTFAM[-]) for 24 h. (b) rhTFAM has little effect on Aβ secretion in PS1P117L cells. Secretion of Aβ(1–40) and Aβ(1–42) was measured by ELISA using the supernatant 24 h after the treatment of 100 nM ΔMTS_rhTFAM (open bars) or rhTFAM (closed bars) protein. Means ± SEM, n = 4. Two-way ANOVA, p < 0.0001. Tukey’s HSD test, **p < 0.0001 vs. wild-type cells (wild) with or without treatment. *p < 0.05. (c) rhTFAM increases transthyretin expression in PS1P117L but not in wild-type neurons. (d) Interaction of transthyretin and rhTFAM in mitochondria of PS1P117L cells treated with rhTFAM. PS1P117L cells were treated with 100 nM rhTFAM for 24 h and then fractionated into mitochondrial (Mt) and nuclear (Nu) fractions (left panels). Proliferating nuclear antigen (PCNA, 35.5 kDa) or heat shock protein 60 (HSP60, 60 kDa) was detected as a nuclear or mitochondrial marker, respectively. Co-immunoprecipitation of transthyretin was performed using anti-hTFAM or normal IgG as a negative control (IgG) and the mitochondrial fraction prepared from PS1P117L cells treated with rhTFAM (right panels). (e) Phenylalanine significantly decreased expression of transthyretin in rhTFAM-treated PS1P117L cells. Cells were treated with 100 nM rhTFAM in the presence (Phe[+]) or absence (Phe[–]) of phenylalanine. Transthyretin index is shown in the bar graph. Student’s t-test, ##p < 0.0001. (f) Phenylalanine increased ROS production in rhTFAM-treated PS1P117L cells. Student’s t-test, ##p < 0.0001. (g) Phenylalanine increased Aβ immunofluorescence in rhTFAM-treated PS1P117L cells. Aβ index is shown in the bar graph. Student’s t-test, ##p < 0.0001. Data represent means ± SEM from 50 cells. (h) Phenylalanine did not alter Aβ secretion in rhTFAM-treated PS1P117L cells. No significant alteration was observed for the Aβ (1–42)/Aβ (1–40) ratio or the level of each Aβ peptide. Data represent means ± SD, n = 3. All scale bars (a,c,e,f,g), 40 μm.
Figure 7
Figure 7. Recombinant human TFAM improves neuritogenesis with increased expression of neuritogenesis-related genes in a neuron model of AD.
(a) Hierarchical partitioning and clustering of 138 clusters exhibited 8-fold or more increased expression (average raw expression level [log2] >6.6 in PS1P117L + rhTFAM), in PS1P117L cells with rhTFAM treatment. Student’s t-test: p < 0.05, Means ± SD, n = 3. (b) Top significant network consisted of 25-upregulated genes among 132 functional/pathway eligible genes in the IPA. (c) Effect of rhTFAM on the 16 neuritogenesis-related gene expression in PS1P117L cells. Student’s t-test: p < 0.05. (d) rhTFAM improves neuritogenesis. Immunofluorescence microscopy of iPSCs-derived neurons treated with or without rhTFAM, using anti-MAP2. Nuclei are stained with DAPI. Scale bar, 20 μm. (e) MAP2-positive cells treated with or without rhTFAM or ΔMTS-rhTFAM were classified into three stages: stage 1, lacking neurites; stage 2, one or more minor neurites; stage 3, one neurite at least twice as long as any other. Fifty MAP2-positive cells in each culture condition were examined, and their distribution is shown in bar graphs. Fisher’s Exact test: *p = 0.0281, **p = 0.0062, ***p < 0.0001 versus PS1P117L rhTFAM(–). (f) Phenylalanine inhibits neuritogenesis in rhTFAM-treated PS1P117L cells. Fifty MAP2-positive cells in each culture condition were examined, and their distribution is shown in bar graphs. Fisher’s exact test, *p = 0.0017 vs. rhTFAM(+)/Phe(−).
Figure 8
Figure 8. TFAM breaks the mitochondria-mediated vicious cycle of Alzheimer’s disease.
hTFAM binds mtDNA and forms a nucleoid-like structure, thereby protecting mtDNA from ROS generated under conditions of Aβ toxicity. Improved maintenance of mitochondrial homeostasis reduced ROS generation from mitochondria, and results in suppression of oxidative modification of transthyretin and/or increase in its expression. Transthyretin reduces intracellular Aβ accumulation, thus further contributes to the break of mitochondria- mediated vicious cycle. hTFAM also improves neuronal function such as neuritogenesis through suppression of ATP depletion caused by mitochondrial dysfunction and increased expression of neuritogenesis-related genes. hTFAM effectively suppresses the vicious cycle of the neuronal mitochondrial dysfunction, thereby ameliorating the AD pathophysiology.

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