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. 2024 Jun 29;14(7):369.
doi: 10.3390/metabo14070369.

Impaired Mitochondrial Energy Metabolism Regulated by p70S6K: A Putative Pathological Feature in Alzheimer's Disease

Wenyu Gu  1   2   3 , Xinli Cong  1   2   3 , Yechun Pei  1   2   3 , Nuela Manka'a Che Ajuyo  1   2 , Yi Min  1   2   3 , Dayong Wang  1   2
Affiliations

Impaired Mitochondrial Energy Metabolism Regulated by p70S6K: A Putative Pathological Feature in Alzheimer's Disease

Wenyu Gu et al. Metabolites. .

Abstract

Alzheimer's disease (AD) is a neurodegenerative disease. Mitochondrial energy metabolism and p70 ribosomal protein S6 kinase (p70S6K) play significant roles in AD pathology. However, the potential relationship between them is unclear. In this study, bioinformatics methods were initially applied to analyze the transcriptomic data in the CA1 and the primary visual cortex of patients with AD and Aβ42-treated SH-SY5Y cells. By applying secreted Aβ42 and p70S6K gene silencing in cells, we explored disorders in mitochondrial function and the regulatory roles of p70S6K by flow cytometry, laser scanning confocal microscopy, high-performance liquid chromatography, Western blotting, and quantitative reverse transcription PCR. The study reveals that impaired mitochondrial energy metabolism is a potential pathological feature of AD and that p70S6K gene silencing reversed most of the changes induced by Aβ42, such as the activities of the electron transport chain complexes I and III, as well as ATP synthase, ATP production, generation of reactive oxygen species, mitochondrial membrane potential, and phosphorylation of AMPK, PINK1, and Parkin, all of which are required for mitochondria to function properly in the cell.

Keywords: Alzheimer’s disease; RNA sequencing; mitochondrial energy metabolism; oxidative phosphorylation; p70S6K; secreted Aβ42.

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Conflict of interest statement

There are no conflicts of interest to declare. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
DEG enrichment in the Aβ42–treated group versus the control group. (a) Volcano map of DEGs in the Aβ42–treated group. The red dots represent upregulated gene expressions; blue dots indicate downregulated gene expressions; and gray dots represent nonsignificant gene expressions. (b) Top DEG clusters in the BP, CC, and MF aspects of the Gene Ontology. (c) Top DEG clusters in the EIP, OS, HD, and CP aspects of the KEGG pathway. (d) Top 20 DEG clusters in the Disease Ontology (DO). The dots on the folded line represent the numbers of genes in the respective DO terms.
Figure 2
Figure 2
GO and KEGG pathway analyses of common DEGs in the hippocampal CA1 area in patients with Alzheimer’s disease and Aβ42–treated SH–SY5Y cells: (a) Venn diagram showing the DEGs in the hippocampal CA1 area of patients with Alzheimer’s disease and Aβ42–treated SH–SY5Y cells; (b) top clusters of common DEGs in the EIP, OS, HD, and M sections of the KEGG pathway; (c) chord plots showing the top clustered GO terms for the common DEGs, and the genes.
Figure 3
Figure 3
GO and KEGG pathway analyses of the common DEGs in the hippocampal CA1 area and primary visual cortex of patients with Alzheimer’s disease and Aβ42–treated SH–SY5Y cells: (a) Venn diagram showing the DEGs in the hippocampal CA1 area and primary visual cortex of patients with Alzheimer’s disease and Aβ42–treated SH–SY5Y cells; (b) top clusters of common DEGs in the KEGG pathway, where the pink color signifies upregulated gene expressions; blue represents downregulated gene expressions; and black dots signify the numbers of genes enriched in respective terms; (c) transcriptome sequencing and qRT–PCR analyses of the transcription levels of AK4, mt–ND4, mt–ND4L, mt–ND5, SMAD6, and METRNL genes. # p < 0.05 and ## p < 0. 01. compared with the respective control group by one–way ANOVA, followed by the Tukey–Kramer post hoc test for multiple comparisons. The results from three independent experiments are expressed as the means ± SD.
Figure 4
Figure 4
Differential gene expressions induced by p70S6K gene knockdown in Aβ42–treated cells: (a) volcano map of the DEGs in the p70S6K siRNA and the Aβ42–treated group, and the Aβ42–treated control group, where red dots represent upregulated gene expressions; blue dots indicate downregulated gene expressions; and gray dots show nonsignificant gene expressions; (b) KEGG pathway analysis of the DEGs; (c) top 5 enriched terms of the GSEA of DEGs.
Figure 5
Figure 5
Effects of the p70S6K gene knockdown on the levels of NAD+, NADH, ATP, and ROS in Aβ42–treated SH–SY5Y cells: (a) HPLC assay of the ATP levels in p70S6K siRNA and the Aβ42–treated SH–SY5Y cells; (b) ROS levels detected by flow cytometry in the cells; (c) levels of NADH in the cells; (d) levels of NAD+ in the cells; (e) ratio of NAD+/NADH in the cells. The SH–SY5Y cells were treated with Aβ42 for 24 h with or without knockdown of the p70S6K gene expression. * p < 0.05 and ** p < 0.01, compared with the Aβ42–treated group; # p < 0.05 and ## p < 0.01 compared with the control group, by one–way ANOVA, followed by the Tukey–Kramer post hoc test for multiple comparisons. The results from three independent experiments are expressed as the means ± SD.
Figure 6
Figure 6
Effects of the p70S6K gene knockdown on the activities of the ETC complexes in Aβ42–treated SH–SY5Y cells: (a) NADH dehydrogenase activities in the p70S6K siRNA and the Aβ42–treated SH–SY5Y cells; (b) succinate dehydrogenase activity in the cells; (c) cytochrome c reductase activities in the cells; (d) cytochrome C oxidase activities in the cells; (e) ATP synthase activities in the cells. The SH–SY5Y cells were treated with Aβ42 for 24 h with or without knockdown of the p70S6K gene expression. * p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the Aβ42–treated group; # p < 0.05, ## p < 0.01 and ### p < 0.01, compared with the control group, by one–way ANOVA, followed by the Tukey–Kramer post hoc test for multiple comparisons. The results from three independent experiments are expressed as the means ± SD.
Figure 7
Figure 7
Laser confocal microscopic detection of the effects of the p70S6K gene knockdown on the mitochondrial membrane potential (MMP) in Aβ42–treated SH–SY5Y cells: (a) representative figures of the MMP detected by the JC–1 fluorescent staining; (b) statistical analysis of the JC–1 fluorescence intensity. The SH–SY5Y cells were treated with Aβ42 for 24 h with or without knockdown of the p70S6K gene expression. ** p < 0.01, compared with the Aβ42–treated group; ## p < 0.01, compared with the control, by one–way ANOVA, followed by the Tukey–Kramer post hoc test for multiple comparisons. The results from three independent experiments are expressed as the means ± SD, n = 3.
Figure 8
Figure 8
Effects of the p70S6K gene knockdown on the autophagy protein expression levels in the Aβ42–treated SH–SY5Y cells: (a) protein expression levels of p70S6K in p70S6K siRNA and the Aβ42–treated SH–SY5Y cells; (b) p70S6K Thr389 phosphorylation levels in the cells; (c) AMPK α1 Thr183/α2 Thr172 phosphorylation levels in the cells; (d) protein expression levels of PINK 1 in the cells; (e) protein expression levels of Parkin in the cells; (f) protein expression levels of p62 in the cells; (g) protein expression levels of LC3 I in the cells; (h) protein expression levels of LC3 II in the cells. The SH–SY5Y cells were treated with Aβ42 for 24 h with or without knockdown of the p70S6K expression. * p < 0.05 and ** p < 0.01 compared with the Aβ42–treated group; # p < 0.05 ## p < 0.01 and ### p < 0.001 compared with the control, by (a) Student’s t–test or (b–h) one–way ANOVA, followed by the Tukey–Kramer post hoc test for multiple comparisons. The results from three independent experiments are shown as the means ± SD.
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