Role of sphingolipids in the biogenesis and biological activity of extracellular vesicles

Abstract

Extracellular vesicles (EVs) are membrane vesicles released by both eukaryotic and prokaryotic cells; they not only serve physiological functions, such as disposal of cellular components, but also play pathophysiologic roles in inflammatory and degenerative diseases. Common molecular mechanisms for EV biogenesis are evident in different cell biological contexts across eukaryotic phyla, and inhibition of this biogenesis may provide an avenue for therapeutic research. The involvement of sphingolipids (SLs) and their enzymes on EV biogenesis and release has not received much attention in current research. Here, we review how SLs participate in EV biogenesis by shaping membrane curvature and how they contribute to EV action in target cells. First, we describe how acid and neutral SMases, by generating the constitutive SL, ceramide, facilitate biogenesis of EVs at the plasma membrane and inside the endocytic compartment. We then discuss the involvement of other SLs, such as sphingosine-1-phosphate and galactosyl-sphingosine, in EV formation and cargo sorting. Last, we look ahead at some biological effects of EVs mediated by changes in SL levels in recipient cells.

Keywords: ceramide; ectosomes; endocannabinoids; exosomes; microvesicles; psychosine; sphingomyelinase; sphingosine-1-phosphate.

Fig. 1.

MV and exosome biogenesis: the role of SMase and its product Cer. A: On the left, SM (cylinder shaped) stabilizes membrane structure; on the right, when a-SMase translocates from the lysosomal compartment to the outer leaflet of the plasma membrane, SM hydrolysis and Cer accumulation into the inner leaflet of the plasma membrane favor blebbing and MV evagination, thanks to the inverted cone shape of Cer. B: n-SMase resides on the cytosolic leaflet of endosomes/MVBs. As in A, before n-SMase action SM stabilizes membrane structure, while Cer production by n-SMase induces spontaneous negative curvature to the membrane of endosomes, thus promoting the formation of internal vesicles inside MVBs.

Fig. 2.

MV-dependent activation of SL metabolism in neurons. A: The SL cascade on the plasma membrane and the targets of genetic/pharmacological inhibitors. B: The cartoons elucidate the mode of action of MV released by microglial cells on excitatory glutamatergic synapses and the effects of genetic/pharmacological inhibitors. Microglial MVs interact with the plasma membrane of neurons through surface contact facilitated by receptor-ligand interaction between PS residues (typically exposed on the MV membrane) and PS neuronal receptor (in blue). Subsequently, unidentified lipid(s) on the surface of MVs activate a-SMase (translocated to the outer leaflet of plasma membrane from lysosomes), thus stimulating SL metabolism in neurons. Sph and S1P have been identified as the SLs responsible for the MV effects on the excitatory presynaptic terminal (see also the “a-SMase KO animals,” “CDase inhibitor,” and “SK inhibitor/S1P₃ inhibitor/S1P₃ siRNA” panels). Sph acts on the synaptic vesicle-associated SNARE protein, synaptobrevin, activating it and thereby promoting the assembly of the SNARE complex (which is necessary for synaptic vesicle fusion at the presynaptic terminal and, therefore, neurotransmitter release). This event increases the probability of neurotransmitter release, empowering excitatory synaptic transmission in neurons targeted by MVs. On the other hand, S1P, a SL able to move outside the membranes, travels extracellularly to engage its receptor on the presynaptic terminal (likely the S1P₃ receptor). S1P₃ receptor activation translates into downstream signaling in the target neuron, inducing ERK activation and synapsin I phosphorylation. Synapsin I is a presynaptic protein responsible for anchoring synaptic vesicles of the reserve pool to the actin cytoskeleton, therefore preventing their exocytosis. Dephosphorylated synapsin I binds synaptic vesicles and the actin cytoskeleton, tying the two of them together. When phosphorylated, synapsin I breaks the link between vesicles and actin, leaving synaptic vesicles free to migrate to the presynaptic terminal, ready for exocytosis upon stimulation. This increases the number of synaptic vesicles that undergo exocytosis when neurotransmitter release is triggered. The actions of genetic/pharmacological inhibitors are described in the a-SMase KO animals, CDase inhibitor, and SK inhibitor/S1P₃ inhibitor/S1P₃ siRNA panels.

Fig. 3.

Effects on inhibitory/excitatory synapses of MV-associated bioactive lipids. Center: Microglial cells release EVs (MVs and exosomes). MV interaction with the neuronal surface is mediated by PS residues, typically exposed on the MV membrane, which bind their specific neuronal receptor (in blue). Left: On inhibitory synapses, vesicular eCB(s) (likely AEA exposed on the surface of MVs) interact with presynaptic CB₁ receptors. CB₁ receptor activation induced by vesicular eCBs causes an increase in phosphorylated ERK levels and a downregulation of inhibitory synaptic transmission. Right: As already described in Fig. 2, on excitatory synapses, microglial MVs activate SL metabolism. Sph and S1P mediate MV action, inducing an increase in excitatory transmission.