Specific sphingolipid content decrease in Cerkl knockdown mouse retinas

Abstract

Sphingolipids (SPLs) are finely tuned structural compounds and bioactive molecules involved in membrane fluidity and cellular homeostasis. The core sphingolipid, ceramide (CER), and its derivatives, regulate several crucial processes in neuronal cells, among them cell differentiation, cell-cell interactions, membrane conductance, synaptic transmission, and apoptosis. Mutations in Ceramide Kinase-Like (CERKL) cause autosomal recessive Retinitis Pigmentosa and Cone Rod Dystrophy. The presence of a conserved lipid kinase domain and the overall similarity with CERK suggested that CERKL might play a role in the SPL metabolism as a CER kinase. Unfortunately, CERKL function and substrate(s), as well as its contribution to the retinal etiopathology, remain as yet unknown. In this work we aimed to characterize the mouse retinal sphingolipidome by UPLC-TOF to first, thoroughly investigate the SPL composition of the murine retina, compare it to our Cerkl -/- model, and finally assess new possible CERKL substrates by phosphorus quantification and protein-lipid overlay. Our results showed a consistent and notable decrease of the retinal SPL content (mainly ranging from 30% to 60%) in the Cerkl -/- compared to WT retinas, which was particularly evident in the glucosyl/galactosyl ceramide species (Glc/GalCer) whereas the phospholipids and neutral lipids remained unaltered. Moreover, evidence in favor of CERKL binding to GlcCer, GalCer and sphingomyelin has been gathered. Altogether, these results highlight the involvement of CERKL in the SPL metabolism, question its role as a kinase, and open new scenarios concerning its function.

Fig. 1

Diagram of the sphingolipid metabolism. The de novo synthesis pathway starts at the endoplasmic reticulum by the condensation of palmitate and serine to produce 3-ketosphinganine, which is then reduced to sphinganine, and converted to DhCER by the addition of a fatty acid. CER is the core molecule of SPL metabolism and is formed by the desaturation of DhCER. CER is the precursor of SPH, C1P, and a collection of complex SPLs: SM, GlcCer, GalCer and its derivatives. The SPL groups analyzed and quantified in this work are depicted in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Sphingolipid quantification in WT and KO retinas. The SPL content was resolved and quantified from P60 retinas of 6 WT and Cerkl −/− retinas. Note that the y axis (pmol/mg prot) is discontinuous, divided in three segments that overall encompass 4 orders of magnitude. Decreased levels of several SPLs were observed in the KO retinas (**p < 0.01, Mann–Whitney test). Note that LacCer and GM3 values were quantified using MHCer standards, whereas CER standards were used for sulfatides.

Fig. 3

Retinal ceramide, sphingomyelin, dihydroceramide, and dihydrosphingomyelin species in WT and Cerkl −/− mice. (A) Ceramide species quantification. (B) Sphingomyelin species quantification. (C) Dihydroceramide (the CER precursor) species quantification. (D) Dihydrosphingomyelin (produced from DhCER by the addition of phosphocholine group) species quantification. Note that the y axis (pmol/mg prot) is discontinuous in (A) and (B), divided in three segments that overall encompass 3 or 4 orders of magnitude. Significant differences were observed (*p < 0.05, **p < 0.01, Mann–Whitney test).

Fig. 4

Glycolipid species determination and quantification in WT and Cerkl −/− retinas. (A) Monohexosyl ceramide (glucosyl- and galactosylceramide) species. GlcCer and GalCer are directly synthesized from CER. (B) Lactosyl ceramide (derived from GlcCer) species determination and quantification. (C) GM3 species analysis and quantification. GM3 is produced from LacCer by the addition of a sialic acid. (D) Sulfatide (produced from GalCer) species determination and quantification. Significant differences were observed in Glc/GalCer, LacCer species, and GM3 (**p < 0.01, Mann–Whitney test). Note that LacCer and GM3 values were quantified using MHCer standards, whereas CER standards were used for sulfatides.

Fig. 5

Retinal phospholipid and neutral lipid content in WT and Cerkl −/− retinas. DAG, TAG and CE levels were quantified using PC standards whereas PI was referred to (C17:0/C17:0-PE). Note that the y axis (nmol/mg prot) is discontinuous, divided in three segments that overall encompass 3 orders of magnitude. No significant changes in any of these lipid groups were observed in WT and KO retinas.

Fig. 6

Semiquantitative expression analysis of SPL genes. Transcription levels of the genes involved in the synthesis of CER, SM, GlcCer and GalCer, as well as SPL kinases and transporters were analyzed on WT and KO retinas. No differences were observed. Gapdh and Cerkl were used for sample normalization and control, respectively.

Fig. 7

CERKL kinase activity assessment. (A) Validation of the sensitivity of the phosphorus incorporation assay using ceramide-containing micelles mixed with protein lysates of cells overexpressing either CERKL or CERK (positive control). CERKL did not phosphorylate CERs under standard conditions. (B) Custom-made synthetic micelles containing several commercial lipids and SPLs combined according to their structure and similarity. Group 0 corresponded to the negative control with empty micelles. (C) Phosphorus incorporation assays using the micelles listed in (B) together with, either no protein lysate (light gray), empty vector protein lysate (medium gray) or CERKL-transfected cell lysate (dark gray). No significant differences were observed at incorporated phosphorus levels (Mann–Whitney test).

Fig. 8

Protein–lipid overlay analysis. One representative replicate (of at least three) is shown in this figure. (A) Membranes spotted with bovine retinal lipids (RL) (positive control), SM, GlcCer, cis-retinol (cis-R) and retinoic acid (RA) were incubated with lysates of HEK293T cells transfected with either the empty vector (pcDNA), CERKLa (considered the reference CERKL transcript isoform) or 4 CERKL isoforms co-transfection (CERKLa, b, c and d). B indicates Blank (negative control). Immunodetection showed CERKL binding with RL, SM and GlcCer. (B) Free fatty acids (FFA), very long chain fatty acids (VLCFA), phosphatidylinositol mixture (PI) and other SPLs were also tested. Positive signal was only detected in RL (positive control), GlcCer and GalCer. The graphics on the right show the lipid distribution on the membranes.