We developed and applied a novel lipid-labeling approach for 3D-electron microscopy aiming to understand the enigmatic sub-cellular action of non-canonical 1-deoxy-sphingolipids (deoxySLs) − A lipid class relevant for diseases such as type 2 diabetes mellitus (T2DM), hereditary sensory and autonomic neuropathy type 1 (HSN-1) and cancer. [1,2,3,4]
Earlier studies indicated that intracellular deoxySL accumulation is related to mitochondrial dysfunction and defects in sub-cellular membrane structure and trafficking [2,6].
In the present study, we investigated altered organelle dynamics by applying 1 µM DOXSA and organelle live dyes to MEF cells. Live-cell imaging showed impaired cell motion, mitochondrial dysfunction and tube formation of ER and auto-lysosomal compartments.
Based on these findings, cultured mammalian cells (MEF cells) were treated with alkyne -1- deoxysphinganine (alkyne-DOXSA) − A traceable analogue of 1-deoxysphinganine (DOXSA).
Alkyne-lipids are lipid tracer which act essentially native in biological systems [5].
Subsequently we investigated deoxySL related lipid membrane defects on ultrastructural level using TEM tomography. For visualization of alkyne-lipids, we employed a novel labeling method. We call that approach the "Golden-Click-Method" (GCM). GCM is based on functionalized gold-nanoparticles containing azide groups as electron-dense reporters. These are covalently conjugated to alkyne-deoxySLs during a copper-catalyzed “click-reaction”. This labeling approach for EM was further complemented by STED microscopy of fluorescently tagged alkyne-deoxySLs enabling co-localization studies of alkyne-lipids and specific organelle markers at the sub-organelle level. In conclusion, our examinations revealed auto-lysosomal activity in MEF cells after exogeneous DOXSA-application. Emerging intra-cellular membrane distortion and toxic acting lipid aggregates may be a plausible explanation for deoxySL-mediated organelle dysfunctions leading ultimately to neurodegeneration. − Here, we emphasize on sub-cellular processes that connect deoxySL mediated membrane damage with cell biological mechanisms, such as ER-Stress and mitochondrial dysfunction. Moreover, our findings in adherent MEF cells corroborate that deoxySL mediated autophagy and intra-lysosomal enrichment of deoxySL derivates may display relevant factors for neurodegenerative diseases.
[1] Penno et al., Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids (2010)
[2] Alecu et al., Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction (2017)
[3] Othman et al., Plasma 1-deoxysphingolipids are predictive biomarkers for type 2 diabetes mellitus. (2015)
[4] Massard et al. Phase I dose-escalating study of ES-285 given as a three-hour intravenous infusion every three weeks in patients with advanced malignant solid tumors. (2012)
[5] Thiele et al., Tracing Fatty Acid Metabolism by Click-Chemistry (2012)
[6] Jimenez-Rojo et al., Biophysical Properties of Novel 1-Deoxy-(Dihydro)ceramides Occurring in Mammalian Cells.” (2014) Cells.” (2014).
