To test if this is the case when ASM activity is lacking, synaptic transmission and short-term synaptic plasticity of Schaffer collateral (Sc)-CA1 synapses were next investigated in hippocampal slices from 7-month-old wt and ASMko mice. These results evidence that ASM deficiency alters the synaptic membrane lipid composition by drastically increasing SM levels.ĪSM deficiency leads to the alteration of short-term synaptic plasticity eventsĪlterations in the lipid composition of the synapses could affect synaptic transmission. In contrast, the amounts of other lipids such as cholesterol (362☖6 and 270±53 nmol/mg protein), ceramide (3.2☐.1 and 2.7☑ nmol/mg protein), phosphatidylcholine (306☑07 and 266☗9 nmol/mg protein), phosphatidylserine (64☒8 and 38☖ nmol/mg protein) and phosphatidylethanolamine (247☗4 and 200☒9 nmol/mg protein), were not significantly altered. This study revealed a 3-fold increase in SM levels in ASMko synaptosomes compared to wt (176±40 and 59☑7 nmol/mg protein, respectively, mean±SD, n = 3). The synaptosome isolation procedure was refined so that no traces of myelin and lysosomes, which are loaded with SM in ASMko conditions, could interfere with our measurements (see methods and Supplementary Figure S1). To determine whether lack of ASM activity affects synaptic membrane lipid composition, mass analysis of lipids was performed on synaptosomes of age-matched (7months) wild type (wt) and ASMko mice. The absence of ASM causes a drastic increase of SM in synaptic membranes This moved us to consider these mice a suitable model to investigate the involvement of sphingolipids in synaptic function. Furthermore, accumulation of SM and its derivatives also occurs at the plasma membrane of hippocampal neurons. These mice recapitulate the human disease symptoms showing axonal dystrophy and neurodegeneration, particularly dramatic in cerebellar Purkinje cells. SM-loaded lysosomes also characterize the cells of ASMko mice.
Its absence causes the accumulation of SM in these organelles, which is a hallmark in NPA patients. ASM is the enzyme responsible for the conversion of SM into ceramide in the lysosomes. Among them, NPA results from loss of function mutations in the acid sphingomyelinase (ASM) gene leading to severe mental retardation. This knowledge is essential to understand not only the molecular mechanisms of cognition but also the defects underlying the cognitive impairment that accompany most lipidosis. However, much still remains to be learned about the mechanisms by which lipids influence synaptic function and about the enzymatic activities regulating their action. Moreover, arachidonic and phosphatidic acids stimulate vesicle fusion by interacting with the SNARE exocytic complex –. Phosphoinositides and cholesterol regulate the synaptic vesicle cycle. Thus, glial-derived cholesterol was identified as an essential factor promoting synapse formation whereas pharmacological reduction of cholesterol and sphingolipids levels leads to synapse loss. Increasing evidence suggests a key role for lipids in the establishment and functionality of synapses. These data provide with a novel mechanism for synaptic vesicle control by sphingolipids and could explain cognitive deficits of NPA patients. Moreover, Se reduces vesicle docking in primary neurons and increases paired-pulse facilitation when added to wt hippocampal slices. In vitro reconstitution assays demonstrated that Se changes syntaxin1 conformation enhancing its interaction with Munc18.
Biochemical analysis of ASMko synaptic membranes unveiled higher amounts of SM and sphingosine (Se) and enhanced interaction of the docking molecules Munc18 and syntaxin1. Consistently, electron microscopy revealed reduced number of docked vesicles. Electrophysiological recordings showed that ASMko hippocampi have increased paired-pulse facilitation and post-tetanic potentiation. Acid sphingomyelinase knockout mice (ASMko) are a suitable model to address the role of sphingolipids in synaptic regulation as they recapitulate a mental retardation syndrome, Niemann Pick disease type A (NPA), and their neurons have altered levels of sphingomyelin (SM) and its derivatives. Consensus exists that lipids must play key functions in synaptic activity but precise mechanistic information is limited.
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