Abnormal Regulation of Mitochondrial Sphingolipids during Aging and Alzheimer's Disease

Abstract

During pathogenesis of Alzheimer's disease (AD), mitochondria suffer alterations that lead to low energy production and reactive oxygen species formation. However, the mechanism of impaired mitochondria homeostasis in AD is not fully understood. We hypothesized that abnormal sphingolipid metabolism in mitochondria could be one of the contributing factors to mitochondrial dysfunction. Synaptic and non-synaptic mitochondria were isolated from 5xFAD and wild type (WT) mice at 3 and 7 months using Ficoll gradient ultracentrifugation, and their function was analyzed using Seahorse assay. Additionally, mitochondria were analyzed using mass spectrometry for proteomics and sphingolipidomics analyses. Sphingolipid levels were also determined in synaptic and non-synaptic mitochondria isolated from AD patients and healthy controls. We found that synaptic mitochondria isolated from 3-months old 5xFAD mice manifest diminished oxygen consumption as compared to WT. Consistently, proteomics analysis showed that proteins related to respiratory electron transport and oxidative phosphorylation were altered in 5xFAD mice. When quantifying the main sphingolipids in mitochondria, we found that Cer 18:0, Cer 22:0, and Cer 24:1 were increased already at 3 months in 5xFAD mice. No increase in ceramides was detected in mitochondria isolated from AD patients. However, increased levels of sphingosine were found in both 5xFAD mice and AD patients when compared to respective controls. We report that the regulation of sphingolipids in mitochondria is abnormal at 3 months of age in 5xFAD mice, as indicated by the accumulation of long-chain ceramides, which increases with age. Sphingosine levels are increased in both the mitochondria of 5xFAD mice and AD patients. Our data suggest that the sphingolipid composition is dysregulated in mitochondria early during AD pathogenesis.

Keywords: 5xFAD; Aging; Alzheimer’s disease; ceramide; mitochondria; sphingosine.

Figure 1.

Very long chain and long-chain ceramides are upregulated in synaptic and non-synaptic mitochondria isolated from 5xFAD mice. Quantification of (Sph), sphinganine, sphingosine-1-phosphate (S1P), sphinganine-1-phosphate, Cer (N-acyl chain lengths = C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0) by mass spectrometry of brain synaptic and non-synaptic mitochondria isolated from WT and 5xFAD at 3 and 7 months. Bar graphs represent average ± SD with each N = >4/group. Two-way ANOVA followed by LSD (*p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.0001).

Figure 2.

Synaptic mitochondria of 3 months old 5xFAD manifest impaired oxygen consumption. The OCR measured by seahorse on synaptic and non-synaptic mitochondria isolated from WT (n = 5) and 5xFAD (n = 5) isolated at 3 months of age Unpaired t-test (*p < 0.05).

Figure 3.

Proteomic analysis of non-synaptic mitochondria shows dysregulation of protein related to respiratory electron transport and ATP synthesis. (A) Volcano plots of proteomic data showing log2- fold change and log10 of the p values of WT vs 5xFAD at 3 and 7 months, and 3- vs 7-months old WT. n = 3/group. In red are shown the dysregulated proteins used for gene enrichment analysis. (B) Gene enrichment analysis was run on list of genes dysregulated at 7 months comparing WT and 5xFAD. (C) Gene enrichment analysis was run on list of genes dysregulated comparing 3- and 7-months WT.

Figure 4.

Proteomic analysis of synaptic mitochondria shows dysregulation of proteins related to pyruvate kinase complex and the mitochondrial double-membrane system. (A) Volcano plots of proteomic data showing log2- fold change and log10 of the p values of WT vs 5xFAD at 3 and 7 months, and 3- vs 7-months old WT. N = 3/group. In red is shown the dysregulated proteins used for gene enrichment analysis. (B) Gene enrichment analysis was run on list of genes dysregulated at 7 months comparing WT and 5xFAD. (C) Gene enrichment analysis was run on list of genes dysregulated comparing 3- and 7-months WT. (D) Venn Diagram illustrating the intersection of genes between the Synaptic and Non-synaptic mitochondrial fractions. (E) Pathway enrichment analyses of Synaptic and Non-synaptic intersect genes demonstrating significant correlations with multiple gene ontologies (GO) (Molecular Function (MF), biological process (BP) and cellular component (CC)) related to mitochondria functions and components.