Anomalous Surface Distribution of Glycosyl Phosphatidyl Inositol–anchored Proteins in Neurons Lacking Acid Sphingomyelinase

Abstract

Acid sphingomyelinase (ASM) converts sphingomyelin (SM) into ceramide. Mutations in the ASM gene cause the mental retardation syndrome Niemann Pick type A (NPA), characterized as a lysosomal disorder because of the SM accumulation in these organelles. We here report that neurons from mice lacking ASM (ASMKO) present increased plasma membrane SM levels evident in detergent-resistant membranes. Paralleling this lipidic alteration, GPI-anchored proteins show an aberrant distribution in both axons and dendrites instead of the axonal enrichment observed in neurons from wild-type mice. Trafficking analysis suggests that this is due to defective internalization from dendrites. Increasing the SM content in wild-type neurons mimics these defects, whereas SM reduction in ASMKO neurons prevents their occurrence. Moreover, expression of active RhoA, which membrane attachment is affected by SM accumulation, rescues internalization rates in ASMKO neurons. These data unveil an unexpected role for ASM in neuronal plasma membrane organization and trafficking providing insight on the molecular mechanisms involved. They also suggest that deficiencies in such processes could be key pathological events in NPA disease.

High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A

“…To this aim, SM was added to cultured primary neurons from wt mice. Lysenin staining 25 indicated 0.85-fold increase of SM levels in the treated neurons, similar to that detected in ASMko neurons, 10 without affecting cell viability (Figure 3a, see also Galvan et al 10 ). SM rise resulted in 0.52-fold higher LC3-II levels ( Figure 3b) and led to 0.6-fold increase in ubiquitinated proteins (Figure 3c) in wt neurons.…”

“…[20][21][22][23][24] On the other hand, the lipid that most significantly accumulates in neurons from ASMko mice is SM. 10 Hence, we directly tested whether SM accumulation was responsible for the autophagosome-lysosome alterations observed in the ASMko mice brain. To this aim, SM was added to cultured primary neurons from wt mice.…”

“…and resulted in a 0.6-fold decrease in LC3-II levels ( Figure 5a). As SM accumulates in the lysosomes of ASM-deficient cells as much as at the plasma membrane, 10 Impaired autophagy in Niemann Pick disease type A E Gabandé-Rodrı´guez et al the formation of autophagosomes, 27 we sought to determine whether reduction of SM levels at this cellular compartment could also lessen autophago-lysosomal buildup. We treated NPA fibroblasts with dexamethasone, which specifically reduces SM levels at the plasma membrane by activating the neutral sphingomyelinase.…”

“…The following compounds (all obtained from Sigma-Aldrich) were added to the cell media for the different treatments: SM at 40 mM for 24 h; 10 BafA1 at 0.1 mM for 16 h; protease inhibitors diluted 1 : 200 for 8 h; FB1 at 25 mM for 5 days; dexamethasone at 0.1 mM for 5 days; methyl b-cyclodextrin at 300 mM for 24 h. SM labeling. Lysenin staining was carried out as in Galvan et al 10 and analyzed in an Axiovert200 microscope (Carl Zeiss AG) for neurons or in a confocal LSM510 META microscope (Carl Zeiss AG) for fibroblasts. Quantification in neurons was done by determining pixel intensity with ImageJ software in areas chosen randomly along neurites.…”

“…8 In mice lacking ASM (acid sphingomyelinase knockout mice, ASMko), which are a model for NPA, 9 gradual accumulation of SM occurs in brain lysosomes and at the plasma and synaptic membranes of neurons. 10,11 Here we have analyzed the autophagy-lysosomal systems in the brain of ASMko mouse and in fibroblasts from NPA patients. Our work demonstrates the existence of autophagy alterations in this disease, defines SM excess as a key determinant in these alterations and proposes strategies to revert them.…”