ABSTRACT

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system.

Abbreviations: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N₃635P: azido-STAR635P; N₃Cy3: azido-cyanine 3; N₃picCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.

 抽象的

1-脱氧鞘脂 (deoxySL) 是具有临床意义的非典型鞘脂，因为它们在患有遗传性感觉和自主神经病 (HSAN1) 或 2 型糖尿病的患者血浆中升高。它们的神经毒性得到了最好的描述，但它们通过不确定的病理机制对各种细胞类型造成损害。使用小鼠胚胎成纤维细胞和 1-脱氧二氢鞘氨醇 (doxSA) 的炔类似物（所有脱氧SL 的代谢前体），我们在此研究脱氧SL 对巨自噬/自噬（功能失调或消耗性细胞成分的调节降解）的影响。我们发现 deoxySLs 诱导自噬体和溶酶体积累，表明自噬通量增加。自噬体机制以积累了 N-酰化 doxSA 代谢物（可能是脱氧神经酰胺和脱氧二氢神经酰胺）的受损线粒体为目标，并表现出异常肿胀和小管形成。自噬体和溶酶体还与细胞脂质聚集体和晶体相互作用，这种相互作用是在细胞摄取单体 doxSA 和 N-酰化作用时发生的。由于已知进入吞噬细胞溶管体装置的晶体会触发 NLRP3 炎症小体，因此我们还用 doxSA 处理巨噬细胞。我们证明了 doxSL 激活 NLRP3 炎症小体，促使原代巨噬细胞释放 IL1B。综上所述，我们的数据确定了 doxSL 对自噬的影响，并将 doxSL 病理生理学与炎症和先天免疫系统联系起来。

缩写： alkyne-doxSA：(2S,3R)-2-aminooctadec-17yn-3-ol；炔-SA：(2S,3R)-2-氨基十八-17yn-1,3-二醇； aSA：炔-二氢鞘氨醇； ASTM-BODIPY：叠氮基-磺基-四甲基-BODIPY； CerS：神经酰胺合酶； CMR：克隆巨噬细胞报告基因； deoxySLs：1-脱氧鞘脂； dox(DH)Cer：1-脱氧二氢神经酰胺； doxCer：1-脱氧神经酰胺； doxSA: 1-脱氧二氢鞘氨醇； FB1：伏马菌素B1； HSAN1：遗传性感觉和自主神经病1型； LC3：MAP1LC3A 和 MAP1LC3B； LPS：脂多糖； MEF：小鼠胚胎成纤维细胞； MS：质谱； N ₃ 635P：叠氮基-STAR635P； N ₃ Cy3：叠氮基花青3； N ₃ picCy3：叠氮基-吡啶甲基氰基3； NLRP3：含有蛋白 3 的 NOD 样受体热蛋白结构域； P4HB：脯氨酰4-羟化酶亚基β； PINK1：PTEN 诱导的推定激酶 1； PYCARD/ASC：包含PYD和CARD域； SPTLC1：丝氨酸棕榈酰转移酶长链碱基亚基1； SQSTM1: 隔离体 1; TLC：薄层色谱法。