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Abstract

Sphingolipids have important physiological functions and a tightly regulated and spatially distributed anabolic and catabolic enzymatic network. Dysfunction within this enzymatic network is associated with many diseases. The exact mechanisms how these metabolic dysregulations cause pathological alterations are often unknown and an in-depth investigation of these mechanisms may provide novel therapeutic targets for treatments. Therefore, I investigated biochemical consequences of dysfunction for two enzymes of the sphingolipid metabolic network – 3-ketodihydrosphingosine reductase (KDSR) and Gb3 synthase (Gb3S). Dysfunction of both is associated with a specific organ pathology in mammals.

(I) Loss of globo-series glycosphingolipids by knockout of Gb3S was associated with albuminuria but was also shown to convey renoprotective effects in models of glycerol-induced and gentamicin-induced acute kidney injury in mice. How globo-series glycosphingolipids mediate these effects remains unexplained. I used the immortalized proximal tubular epithelial cell line HK-2 as an in vitro model to identify – after introducing genetic modifications with the CRISPR/Cas9 system - protein interaction partners of globo-series glycosphingolipids using a bifunctional, metabolic sphingolipid probe (pacSph). I found indications of an interaction of Gb3Cer with the Kunitz-type serine-protease inhibitor SPINT1, an inhibitor of HGF activator (HGFAC) and therefore modulator of MET signalling. Interestingly, activation of MET signalling was shown before to mediate renoprotective effects in models of acute kidney injury. Although the presented data is not comprehensive, it may stimulate further investigations of the role of globo-series glycosphingolipids in signalling in this context. Establishing a link between Gb3Cer and the regulation of MET signalling may help to identify strategies to reduce acute kidney damage in CRUSH syndrome, during pharmacological interventions, or with diabetes, but could also provide novel targets for cancer treatment. (II) Furthermore, I examined the role of the sphingolipid de novo synthesis enzyme KDSR in skin of two patients with biallelic mutations in KDSR and symptomatic palmoplantar keratoderma. Following analysis of stratum corneum by LC-MS/MS and chemical synthesis of keto-type ceramides, I revealed the biochemical consequences of KDSR dysfunction and found that significant amounts of a novel, yet undescribed, class of sphingolipids - 3-ketodihydroceramide - are formed in the skin of both patients. Analysis of skin biopsies and blood samples further revealed that 3-ketodihydroceramides are exclusively formed in the epidermis and cannot be detected in the dermis or blood indicating a close link to the observed skin pathologies. Further analysis revealed a significant reduction in the length of epidermal sphingoid bases and ceramides, not only in KDSR patients, but also in atopic dermatitis and psoriasis vulgaris indicating dysfunctional keratinocyte differentiation. A mechanistic link between the presence of pathological keto-type compounds and pathogenesis in KDSR patients remains to be established. It may be speculated, that reactive 3-ketosphingolipids have a negative impact on reactive oxygen species (ROS) scavenging systems in the epidermis, but this remains to be further addressed. If a mechanistic link can be established, a careful reduction of serine-palmitoyl-CoA transferase (SPT) activity to match the reduced activity of KDSR or pharmacological targeting of the oxidative stress response may be a feasible strategy to improve the burden of these patients. Reduction of SPT activity may be achieved with inhibitors like myriocin.

Concluding, these findings demonstrate the relevance of sphingolipid metabolism for physiology and pathology and provide a foundation for further research.