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. 2011 Mar;89(3):352-64.
doi: 10.1002/jnr.22564. Epub 2010 Dec 29.

Persistence of psychosine in brain lipid rafts is a limiting factor in the therapeutic recovery of a mouse model for Krabbe disease

A B White  1 , F Galbiati, M I Givogri, A Lopez Rosas, X Qiu, R van Breemen, E R Bongarzone
Affiliations

Persistence of psychosine in brain lipid rafts is a limiting factor in the therapeutic recovery of a mouse model for Krabbe disease

A B White et al. J Neurosci Res. 2011 Mar.

Abstract

Sphingolipids are intrinsic components of membrane lipid rafts. The abnormal accumulation of these molecules may introduce architectural and functional changes in these domains, leading to cellular dysfunction. Galactosylsphingosine (psychosine) is a pathogenic lipid raft-associated molecule whose accumulation leads to brain deterioration and irreversible neurological handicap in the incurable leukodystrophy Krabbe disease (KD). The relevance of clearing excessive levels of pathogenic psychosine from lipid rafts in therapy for KD has not been investigated. The work presented here demonstrates that psychosine inhibits raft-mediated endocytosis in neural cells. In addition, although in vitro enzyme reconstitution is sufficient for the reversal of related endocytic defects in affected neural cells, traditional in vivo enzyme therapies in the mouse model of KD appear to be insufficient for complete removal of pathogenic levels of raft-associated psychosine. This work describes a mechanism that may contribute to limiting the in vivo efficacy of traditional therapies for KD.

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Figures

Figure 1
Figure 1
Lipid raft-mediated/caveolar endocytosis is inhibited in GALC deficient cells and in cells treated with psychosine. Endocytosis was examined by measuring the uptake of fluorescent cholera toxin B (CTB) and lactosyl ceramide (LacCer) in differentiated twitcher neural stem cells (NSC) and in NSC34 cells treated with psychosine. A–C. Uptake of CTB measured in wild type and twitcher NSCs after 30 minute exposures. D–F. LacCer uptake in Wild type and twitcher NSCs after 15 minutes of exposure to the fluorescent probe. G–J. CTB uptake is measured in NSC34 cells exposed to 0.5 and 1μM psychosine for 1 hour. K–N. Uptake of LacCer in NSC34 cells exposed 0.5 and 1 μM psychosine for 1 hour. In I and M, sphingosine is used as a non-toxic sphingolipid control for the specificity of the activity of psychosine in these cells. Representative cell images are shown for relevant experimental conditions. All data is expressed as the mean fluorescence intensity per cell +/− SE with an N of at least 25 for all conditions. *P=<0.0001.
Figure 2
Figure 2
Non raft-mediated endocytosis is not altered by GALC deficiency or psychosine exposure. Mannose-6-phosphate receptor/clathrin mediated endocytosis was measured by quantification of the uptake of either HA-tagged GALC or fluorescent transferrin in differentiated twitcher NSCs and in NSC34 cells exposed to psychosine. A–C. Analysis of the uptake of GALC-HA by twitcher and wild type NSCs after 1 hour of exposure. D–F. Analysis of transferrin uptake in twitcher and wild type NSCs after 1 hour exposure. G–J. Uptake of GALC-HA is measured in NSC34 cells treated with 0.5 or 1 μM psychosine for 1 hour. K–N. Fluorescent transferrin uptake in NSC34 cells treated with 0.5 or 1 μM psychosine for 1 hour. In I and M, sphingosine is used as a non-toxic sphingolipid control for the specificity of the activity of psychosine in these cells. Representative cell images are shown for relevant experimental conditions. All data is expressed as the mean fluorescence intensity per cell +/− SE with an N of at least 25 for all conditions.
Figure 3
Figure 3
Psychosine is removed from lipid rafts and endocytic defects are reversed in NSCs incubated with GALC conditioned medium. Enzymatic correction was tested in vitro by incubating twitcher NSCs with conditioned medium from HeLa cells overexpressing GALC. A. Analysis of GALC activity in NSCs after 8, 30 and 72 hours of exposure to GALC conditioned medium. Recovery of enzymatic activity is represented as a percent recovery of GALC activity with respect to WT levels. B. Measurement of psychosine levels in twitcher NSCs after GALC exposure. Data is expressed as the percentage of psychosine retained in treated cells with respect to untreated twitcher NSCs at time zero. C. Total levels of psychosine are shown for twitcher cells in non-conditioned medium (NCM), GALC-conditioned medium (GCM) and wild type cells in NCM. Data is shown as nmol/mg protein and was collected after 72 hours of exposure to conditioned medium. D. Levels of psychosine in lipid rafts are shown after exposure to either non-conditioned medium (NCM) or GALC conditioned medium (GCM) for 30 and 72 hours. Data is expressed as a fold change with respect to normal wild type levels. Data for A–D is represented as the mean +/− SE.
Figure 4
Figure 4
Lipid raft-mediated/caveolar endocytosis and lipid raft architecture are recovered in vitro with GALC enzyme replacement. The uptake of markers of raft-mediated endocytosis was assayed after incubation of twitcher neural cells with GALC conditioned medium (GCM). A–C Uptake of cholera toxin B (CTB) is measured in wild type NSCs and in twitcher NSCs incubated in GCM for 48 hours. D–F NBD-Lactosyl Ceramide (NBD-LacCer) uptake assays in wild type NSCs and in twitcher NSCs after 48 hour incubation in GCM. Data in A and D are expressed as the mean fluorescence intensity per cell +/− SE with an N of at least 25 for all conditions. B–C and E–F are representative cell images for each experimental condition. The recovery of raft architecture was assayed by western blot analysis of the levels and distribution of the lipid raft marker flotillin 2. G. Blots were performed using antibodies against Flotillin 2 (raft marker) and P115 (non-raft control). Samples were collected by lipid raft preparation of wild type, twitcher, and treated twitcher NSCs.
Figure 5
Figure 5
Inhibition of Lipid raft-mediated/caveolar endocytosis and its recovery by enzyme replacement are independent of neural lineage specificity in GALC deficient cells. Wild type and twitcher cells were cultured in differentiating conditions for 7–9 days. A subset of twitcher cells was exposed to GALC conditioned medium for the final 48 hours. Endocytosis was measured by uptake of cholera toxin B-488 (CTB-488) after 30 minutes of exposure. Cell types were identified by immunocytochemical staining using specific markers for each lineage. A–D. Measurement of CTB-488 uptake in glial fibrillary acidic protein (GFAP) positive astrocytes. EH. CTB-488 uptake assayed in oligodendrocytes that were positive for the lineage marker O4. I–L. Analysis of CTB-488 uptake in neurons that were identified by labeling with NeuN. Representative cell images are shown for all experimental conditions. All data is expressed as the mean fluorescence intensity per cell +/− SE. *P=<0.001.
Figure 6
Figure 6
Lipid raft disruption persists after enzyme replacement therapy in the twitcher mouse. A. Analysis of psychosine concentrations in lipid rafts prepared from twitcher and wild type mice. Enzyme replacement in the twitcher mice was achieved by bone marrow transplant (BMT), injection of lentiviral GALC (LV) or a combination of the two (BMT+LV). Results indicate that significant amounts of psychosine can be detected in twitcher lipid rafts in all conditions with respect to normal wild type levels. Data is shown for raft and non-raft membranes and represents the average concentration of fractions 4–5 and 8–12, respectively. Psychosine concentrations in rafts of P20 twitchers are not significantly different from P45 twitcher+BMT+LV or twitcher+BMT. However, P45 twitcher, twitcher+BMT+LV at max survival and twitcher+LV all show psychosine concentrations that are significantly above these P20 levels. B. Signal intensity is shown for representative mass spectrometric peaks resulting from the detection of psychosine in each treatment group. C. Western blots of total levels of raft markers are shown with respect to actin for each of the twitcher treatment groups as well as for the wild type animals. D–E. Western blots are shown for each treatment group and represent loading of fractions 4–12 from lipid raft preparations. Fractions 4–5 are labeled as rafts and 6–12 are notated as non-raft fractions. The levels of both markers are quantified in fraction 4 and 5 for each of the treatment groups and are labeled as follows: twitcher (TWI), twitcher+BMT+LV (TWI+BMT+LV), twitcher+LV (TWI+LV), twitcher+BMT (TWI+BMT), wild type (WT).
Figure 7
Figure 7
Psychosine in lipid rafts is concomitant with a loss of raft-associated protein kinase C activity in treated twitcher animals. Lipid raft fractions from wild type, twitcher and treated twitcher animals were analyzed for total protein kinase C (tPKC) and phosphorylated protein kinase C (pPKC). A. Western blots show the distribution of pPKC and tPKC in lipid raft fractions from wild type and twitcher animals including twitchers treated with a combination of lentiviral GALC and bone marrow transplant (+LV+BMT). B. Quantification of the ratio of pPKC/tPKC in fractions 4–5 from wild type mice or twitcher mice that are either untreated or treated with lentiviral GALC (LV), bone marrow transplant (BMT) or a combination of the two (BMT+LV). C. Quantification of PKC activity in whole brain extracts as indicated by the ratio of pPKC/tPKC. This was done for each treatment group and shows no detectable changes in PKC activity indicating that loss of localization of active PKC in lipid rafts may be the mechanism of its inhibition by psychosine.
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