J Lipid Res. 2017 Jan;58(1):42-59. doi: 10.1194/jlr.M068676.

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction.

Alecu I1,2, Tedeschi A3, Behler N4, Wunderling K4, Lamberz C5, Lauterbach MA6, Gaebler A4, Ernst D1, Van Veldhoven PP7, Al-Amoudi A5, Latz E6, Othman A8, Kuerschner L4, Hornemann T1,2, Bradke F3, Thiele C4, Penno A9.

1 Institute for Clinical Chemistry, University of Zurich, Zurich, Switzerland.
2 Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
3 Axonal Growth and Regeneration, German Center for Neurodegenerative Diseases, Bonn, Germany.
4 LIMES Life and Medical Sciences Institute, University of Bonn, Bonn, Germany.
5 Cyro-Electron Microscopy and Tomography, German Center for Neurodegenerative Diseases, Bonn, Germany.
6 Institute of Innate Immunity, University Hospital Bonn, Bonn, Germany.
7 Laboratory for Lipid Biochemistry and Protein Interactions, Campus Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium.
8 Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany.
9 LIMES Life and Medical Sciences Institute, University of Bonn, Bonn, Germany anke.penno@uni-bonn.de.

 

Abstract

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

KEYWORDS:

ES-285; chemical synthesis; diabetes; inborn errors of metabolism; lipids/chemistry; metabolic syndrome; mitotoxicity; neurons; peripheral neuropathy; sphingolipids

PMID: 27881717

 

Supplements:

1-Deoxysphingolipids (deoxySLs) are a new class of lipids that have only recently been described in humans. Although it remains unclear why human cells produce deoxySLs, a pathological elevation of these lipids in plasma has been linked with HSAN1-, diabetes- and paclitaxel-induced neurotoxicity. The neurotoxicity of 1-deoxySLs has been validated in vitro in several studies and injection of 1-deoxy-sphinganine (doxSA) as a potential anti-cancer treatment in clinical trials led to diverse cytotoxic events in the patients, including reversible neurotoxicity.

Due to the implication of deoxySLs in human health, combined with the great lack of knowledge regarding their physiological metabolism and function, we aimed to study the metabolism and transport of these lipids. However, the tracking and visualization of lipids inside cells is an especially challenging task. As antibodies rarely exist for lipids, the lipid in question very often needs to be directly fluorescently labeled. Currently, NBD and BODIPY are the most commonly used fluorophores for tagging lipids. However, both of these are very bulky and relatively polar molecules, and thus they strongly alter the biophysical properties of the lipid. They may therefore change uptake, transport and metabolism of the molecule greatly (Figure 1). Furthermore, first attempts to study the metabolism and localization of deoxySLs by using doxSA tagged with Nile red, a more lipophilic fluorescent tag, failed greatly in our hands. Although the probe was taken up by cells, it showed an unexpected and exclusive nuclear staining. Also, even after 48 hours, it was not at all metabolized to any downstream deoxySL products. We therefore chose to synthetize an alkyne-probe of doxSA and showed that the uptake and metabolism of this newly synthesized probe is similar to that of the natural lipid. The new probe enabled us to study deoxySL biology via the “click”-reaction.

 

Figure 1: Lipid tracer structures. Structure of the alkyne-lipid probe in comparison with the natural lipid and directly fluorescently labeled doxSA.

 

The results of this paper and of the one that is published in collaboration [1] shed some important light into the metabolism of deoxySLs. Firstly, no direct conversion pathway of deoxySLs to canonical sphingolipids or fatty acids was observed. Rather, deoxySLs were oxidized by a cytochrome P450-dependent pathway. This pathway is likely part of Phase I of the detoxification process that takes place in the liver, followed by the addition of more hydrophilic compounds in Phase 2 to increase the water solubility of the metabolites and make elimination possible. This pathway is entirely independent of the degradation pathway of canonical sphingolipids which is sphingosine-1-phosphate lyase dependent. The observed metabolic pathways for deoxySLs are summarized in Figure 2.

Concerning the localization of deoxySLs, we observed that exogenously added doxSA was taken up by the cells and accumulated predominately in their mitochondria. The up-take and localization of doxSA to mitochondria was accompanied by great changes in mitochondrial morphology. In detail, we first observed the mitochondria to undergo hyperfusion, leaving a dense and highly connected meshwork of mitochondria after 1 hour incubation with a cytotoxic doxSA concentration (Figure 3A). At later time points (6-24 hours), the hyperfusion state was lost and mitochondria fragmented into spherical balloon-like structures. The induction of mitochondrial fragmentation in general led to detachment and cell death. Interestingly, cells could persist in the hyper-fragmented mitochondrial state, especially when being in close contact to neighboring cells (Figure 3B). Indeed, we consistently observed that in a dish in which the fibroblasts were not evenly spread, cells that were seeded in contact with each other would survive the doxSA-insult, while cells that grew in less populated areas of the dish would undergo cell death. This notion, that doxSA-induced cell death greatly depends on cellular density, has been observed before but has so far not been throughfully investigated.

DeoxySL-induced changes in mitochondrial dynamics and function could be part of the pathomechanism puzzle leading to the afore-mentioned neurotoxicity. Therefore, the further elucidation of deoxySL metabolism and localization, and the distinctions that exist compared to canonical sphingolipids, could prove important in the attempts to diminish the toxicity of these lipids in patients with elevated deoxySL levels.

 

 

Figure 2: Summarized deoxySL metabolism. 1-Deoxysphingolipids are formed when the enzyme serine palmitoyltransferase uses L-alanine instead of L-serine in the condensation with palmitoyl-CoA. These atypical sphingolipids lack the C1-hydroxyl group, precluding them from degradation via sphingosine-1-phosphate, which occurs at the cytosolic surface of the ER. They are instead further metabolized to more hydrophilic compounds by the cytochrome P450 4F enzyme subfamily. Toxic 1-deoxysphingolipid metabolites accumulate in mitochondria, leading to mitochondrial dysfunction and neurotoxicity.

 

 

 

Figure 3: Changes in mitochondrial morphology upon doxSA treatment in mouse fibroblasts. A. Quickly upon doxSA treatment, mitochondrial morphology in mouse fibroblasts changed from a lose network-like structure to a highly interconnected dense mesh (hyperfusion). Further exposure to doxSA led to loss of mitochondrial hyperfusion with greatly increased fragmentation towards spherical balloon-like mitochondria (hyper-fragmentation). B. DoxSA-induced toxicity depends on cell-to-cell contacts as cell survival greatly depended on cell density at the time of exposure to the lipid insult. Cells could persist in the hyperfragmented mitochondrial state without undergoing cell death.

 

 

References: 

[1] Alecu I, Othman A, Penno A, Saied EM, Arenz C, von Eckardstein A, Hornemann T. Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway. J Lipid Res. 2017 Jan;58(1):60-71. doi: 10.1194/jlr.M072421.