Alterations of the Ceramide Metabolism in the Peri-Infarct Cortex Are Independent of the Sphingomyelinase Pathway and Not Influenced by the Acid Sphingomyelinase Inhibitor Fluoxetine

Abstract

Ceramides induce important intracellular signaling pathways, modulating proliferation, migration, apoptosis, and inflammation. However, the relevance of the ceramide metabolism in the reconvalescence phase after stroke is unclear. Besides its well-known property as a selective serotonin reuptake inhibitor, fluoxetine has been reported to inhibit the acid sphingomyelinase (ASM), a key regulator of ceramide levels which derives ceramide from sphingomyelin. Furthermore, fluoxetine has shown therapeutic potential in a randomized controlled rehabilitation trial in stroke patients. Our aim was to investigate and modulate ceramide concentrations in the peri-infarct cortex, whose morphological and functional properties correlate with long-term functional outcome in stroke. We show that certain ceramide species are modulated after experimental stroke and that these changes do not result from alterations of ASM activity, but rather from nontranscriptional induction of the ceramide de novo pathway. Unexpectedly, although reducing lesion size, fluoxetine did not improve functional outcome in our model and had no significant influence on ASM activity or the concentration of ceramides. The ceramide metabolism could emerge as a potential therapeutic target in the reconvalescence phase after stroke, as its accumulation in the peri-infarct cortex potentially influences membrane functions as well as signaling events in the tissue essential for neurological recovery.

Figure 1

Ceramide, its precursors, and its derivate levels are altered in the peri-infarct cortex after photothrombotic stroke. (a) Total ceramide levels, (b) dihydroceramide levels, (c) sphinganine levels, (d) sphingosine levels, (e) glucosylceramide levels, and (f) lactosylceramide levels. Sphingolipids were measured at the indicated time points by tandem mass spectrometry. Differences between sham and PT group were analyzed using Student's unpaired two-tailed t-test. Data are presented as means ± SD; sham values are indicated by dotted line (for individual sham-SD's, see Section 3); values are not significantly different compared to sham if not marked otherwise; ^∗ p ≤ 0.05; ^∗∗ p ≤ 0.01; n.s., nonsignificant; n = 8–10/group.

Figure 2

Ceramide subspecies are differentially regulated in the peri-infarct cortex. (a) Total ceramide, ceramide 16:0, 18:0, and 24:0 in the time course after photothrombotic stroke compared to sham. Asterisks for p values as well as ceramide 18:1 and 24:1 are not shown (see Section 3). (b) Ratios for ceramide 16:0/ceramide 24:0 + 24:1. Data are presented as means ± SD. Differences between sham and PT group were analyzed using Student's unpaired two-tailed t-test.^∗∗ p ≤ 0.01; ^∗∗∗ p ≤ 0.001; n.s., nonsignificant; n = 8–10.

Figure 3

Sphingomyelinase activity is not altered in the peri-infarct cortex. (a) ASM activity and (b) NSM activity. Sphingomyelinase activity was measured at different time points by an enzyme activity assay with radioactive labeled sphingomyelin. Differences between sham and PT group were analyzed using Student's unpaired two-tailed t-test. Data are presented as means ± SD; n.s., nonsignificant; n = 5–10/group.

Figure 4

Expression of ceramide-metabolizing enzymes is differentially regulated in the peri-infarct cortex. (a) ASM mRNA, (b) neutral sphingomyelinase- (NSM-) 2 mRNA, (c) NSM-1 mRNA, (d) Acid ceramidase (ACER) mRNA, (e) neutral ceramidase (NCER) mRNA, (f) glucocerebrosidase- (GBA-) 1 mRNA, (g) GBA-2 mRNA, and (h)–(l) CerS1–6 mRNA, mRNA-levels were measured at the indicated time points by Taqman-PCR. Differences between sham and PT group were analyzed using Student's unpaired two-tailed t-test. Data are presented as means ± SD; sham values are indicated by dotted line (for individual sham-SD's, see Section 3); values are not significantly different compared to sham if not marked otherwise; ^∗ p ≤ 0.05; ^∗∗ p ≤ 0.01; n.s., nonsignificant; n = 8–10/group.

Figure 5

Fluoxetine treatment from day 3 to day 28 reduces infarct size, but has no impact on functional outcome, ASM activity or ceramide levels in the peri-infarct cortex. (a) Stroke size at day 28, (b) functional outcome, measured by the cylinder test, (c) functional outcome, measured by the forelimb grid-walking test, (d) total ceramide levels, and (e) ASM activity. Stroke size was determined by measuring the infarcted cortical area, ceramide level, ASM activity was measured at different time points as indicated above. Differences between saline and fluoxetine were analyzed using Student's unpaired two-tailed t-test. Data are presented as means ± SD; ^∗∗∗ p ≤ 0.001; n.s., nonsignificant; (a), (d), and (e): n = 5–10/group; (b) and (c): n = 10/group.