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Sphingolipid accumulation causes mitochondrial dysregulation and cell death.

ABSTRACT: Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2? mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2? cells depends on nutritional status. Abnormal Ras activation in csg2? cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2? cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways.

SUBMITTER: Knupp J

PROVIDER: S-EPMC5686345 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Sphingolipid accumulation causes mitochondrial dysregulation and cell death.

Knupp Jeffrey J Martinez-Montañés Fernando F Van Den Bergh Francoise F Cottier Stephanie S Schneiter Roger R Beard Daniel D Chang Amy A

Cell death and differentiation 20170811 12

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In thes ...[more]

PMID: 28800132

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Project description:Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations.

Project description:Lipid intermediates derived from sphingolipid metabolism are crucial regulators of mitochondrial function from yeast to humans. Among these intermediates, trans-2-hexadecenal (t-2hex) within the sphingolipid degradation pathway exhibits remarkable efficiency in inducing mitochondria-mediated cell death. In yeast cell cultures, the addition of t-2-hex triggers complete disintegration of the mitochondrial network, leading to subsequent cell death. This effect is particularly pronounced in yeast cells lacking the activity of the t-2-hex degrading enzyme, Hfd1. However, the molecular mechanisms of t-2-hex induction of mitochondrial dysfunction are completely unknown. In this project, we want to exploit the unprecedented power of yeast genetics to unveil novel genetic determinants involved in t-2-hex's pro-apoptotic function. To accomplish this, we employed the SATAY method, which combines saturated transposon mutagenesis with high-throughput sequencing to functionally explore the yeast genome. In our screening approach, hfd1 mutant cells harboring a plasmid-encoded inducible MiniDs transposon were induced by galactose, resulting in extensive integration of the transposon throughout the yeast genome. Cells with the plasmid excised and the transposon genomically integrated were pooled together, creating a high-density transposon library comprising approximately 2.3E+06 independent insertion mutants. Subsequently, the pooled mutant library was subjected to treatment with the mitochondria-mediated death inducer, t-2-hexadecenal. As a control, cells were also incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Following the treatments, cells were collected for genomic DNA extraction and digestion, using restriction enzymes with frequent four-base pair recognition sites. The resulting library fragments were circularized using T4 DNA ligase, and the transposon-genome junctions were selectively amplified through PCR with outward-facing primers specific to the transposon. Finally, the pooled and purified amplicons were subjected to massive sequencing on an Illumina MiSeq platform. The obtained sequences were then aligned to the reference genome of Saccharomyces cerevisiae, allowing for the mapping of transposon insertions and the calculation of transposon counts per gene. This project enabled the identification of genes required for the resistance and toxicity to t-2hex.

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Sphingolipid accumulation causes mitochondrial dysregulation and cell death.

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Sphingolipid accumulation causes mitochondrial dysregulation and cell death.

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