Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function and degeneration. Their precise role in these processes, however, remains obscure. Serine Palmitoyl-transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although neuromuscular junction (NMJ) length is normal in lace mutants, the number of boutons per NMJ is reduced to ~50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig-domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. In support of this, Bsg was found to be enriched in lipid rafts. The data points to a role for sphingolipids in the regulation and fine-tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg (West, 2018).

In 1967 a prominent enrichment was identified of glycosphingolipids within synaptic structures in the mammalian brain. To date understanding of the role of these enigmatic lipids in synapse structure and function has yet to be fully elucidated. Sphingolipids are major lipid components of the plasma and endomembrane system and have been implicated in many forms of neuropathy and neurodegeneration (for review see (Sabourdy, 2015). Sphingolipids are proposed to generate structure in membranes due to their rigidity and association with cholesterol. They are also known to be potent signalling molecules regulating processes such as apoptosis, proliferation, migration and responses to oxidative stress. Numerous neurological and neurodegenerative conditions are directly attributable to the inability to synthesise or catabolise sphingolipids. The failure to synthesise all or particular sphingolipids gives rise to a number of neurological conditions such as infant Fonset symptomatic epilepsy (loss of GM3 ganglioside synthesis); bovine spinal muscular atrophy (loss of 3-ketohydrosphingosine reductase) and hereditary sensory and autonomic neuropathy type 1 (HSAN1); recessive and dominant mutations in serine palmitoyl transferase subunit 1 (SPTLC1). Conversely, failure to catabolise sphingolipids in the lysosome generates a subset of lysosomal storage diseases/disorders (LSD's) known as sphingolipidoses, of which there are approximately 14 identified separate genetic conditions. Sphingolipids are now suggested to have a prominent role in the onset and progression of Alzheimer's disease while the production after bacterial infection of autoimmune antibodies to gangliosides present at the neuromuscular synapse is likely to cause the dramatic and often lethal paralysis seen in Guillain Barré and Miller Fisher syndromes. The presence of sphingolipids at the synapse is further attested by the ability of tetanus and botulinal toxins to effect their entry to synapses via co attachment to synaptic glycosphingolipids. While the presence of sphingolipids (in particular, glycosphingolipids) at the synapse is well established, little is known about their functional or structural role in the operational life of the synapse. Some in vitro studies have addressed the role of sphingolipids at synapses in the context of sphingolipid/cholesterol microdomains and indicate roles in the function and localisation of neurotransmitter receptors, and synaptic exocytosis. The prominence of sphingolipids in neurological disease suggests that absence or accumulation of sphingolipids can exert an influence in synaptic function and indicates an inappropriately large gap in knowledge regarding the actions of these lipids at the synapse. In the above outlined context, roles for sphingolipids in synapse structure and function remain to be determined. To this end, this study ha undertaken an analysis of sphingolipid function at a model synapse, the third instar neuromuscular junction of Drosophila. Mutations were examined in SPT2/SPTLC2 (Serine Palmitoyltransferase, Long Chain Base Subunit 2), which encodes an essential subunit of the Serine Palmitoyltransferase (SPT) heterodimer necessary for the initial step in sphingolipid synthesis, for defects in neuromuscular synapse structure. Evidence is presented to suggest that sphingolipids are essential for synaptic structure and function, and structural regulation may be mediated partially through function of the Ig domain adhesion protein Basigin/CD147 (Bsg) (West, 2018).

On examination of sphingolipid deficient synapses, a disruption to the normal synaptic structure was observed. Synaptic boutons were found to be enlarged and the overall numbers of boutons reduced by ~50% while the length of the neuromuscular synapse remained indistinguishable from wildtype. This phenotype is highly reminiscent of the reduction of synapse number, but increase in synapse size observed in hippocampal neurons in culture depleted for lipid rafts (Hering, 2003). Nevertheless, it was surprising that beyond the structural deficit of the synapse, the ultrastructure of the synapse was remarkably intact, suggesting a role in fine-tuning of synaptic properties (West, 2018).

Synapses depleted for sphingolipids were capable of greater growth when combined with the synaptic overgrowth mutation highwire (hiw). The data suggests the mutations in lace and sphingolipid depletion decouples bouton structure from normal synaptic length. Large boutons are observed in mutants of mothers against dpp (mad), thick veins (tkv), saxophone (sax) medea (med), and glass-bottom-boat (gbb), components of the TGF-β pathway that is known to regulate synaptic growth. However these mutations reduce synaptic length by ~50% and ultrastructural synaptic defects such as non-plasma membrane attached active zones (T-bars), large endosomal vesicles and ripples in pre-synaptic peri-active membranes are observed. One obvious ultrastructural defect that is present in sphingolipid depleted synapses is enlarged mitochondria. Enlarged mitochondria are observed in a number of sensory neuropathies and it is of interest that dominant mutations in SPTLC1 that generate aberrant sphingolipids give rise to Hereditary and Sensory Neuropathy Type 1 (HSAN1) where enlarged mitochondria are often observed. This may be attributable to a recognised role for sphingolipids in mitochondrial fission (West, 2018).

To dissect the spatial requirement for sphingolipid regulation of synapse structure the lace mutant was rescued with a rescue transgene, expressed globally, pre- or post-synaptically. Synaptic bouton size and number were rescued with a global expression of the rescue transgene, but no aspects of the phenotype could be rescued with a pre-synaptic expression. Perturbed NMJ morphology could also be rescued by glial or post-synaptic expression of lace, however post-synaptic muscle expression generated a partial rescue, with an excess of 'satellite' boutons, a phenotype normally associated with integrin dysfunction or endocytic defects. Previous data feeding lace mutant larvae with sphingosine, the product of the SPT enzyme, partially rescued lace mutant associated phenotypes (Adachi-Yamada, 1999). Taken together with the current analysis, there is a strong suggestion that sphingolipid precursors such as sphingosine may be able to act non-cell autonomously, and traffic between cells to support synapse structure and function, but not when supplied from the nervous system. Sphingolipids are required for regulation of synaptic output (West, 2018).

Analysis of EJP and miniEJP characteristics at the 3rd instar larval NMJ reveals mutations in lace produce, at the Ca2+/Mg2+ concentrations that were used used, a small but significant increase in synaptic strength, accompanied by a change in short-term plasticity, with synaptic depression predominating over synaptic facilitation. NMJs with high-quantal content EJPs normally show synaptic depression during paired or short-train repetitive stimulation, while those with a low basal quantal content show synaptic facilitation. Further analysis is required, for instance using a range of Ca2+ concentrations, to establish whether this apparent change in synaptic plasticity is commensurate with a greater basal synaptic strength in the lace mutant larvae, or whether it represents a specific effect of the mutation, disrupting the normal link between mechanisms that couple basal quantal content to short-term synaptic plasticity (West, 2018).

In vitro and in vivo analysis has suggested a role for sphingolipids in synaptic vesicle endocytosis in addition to a role in neurotransmitter distribution. No evident defects were observed in neurotransmitter receptor distribution. Interestingly, ablation of major subsets of gangliosides and subsequent analysis of synaptic function at the NMJ in a mouse model reveals a more pronounced run-down of neurotransmitter release upon sustained stimulation, consistent with the data presented in this study. The apparent deficit in synaptic facilitation observed in this study in lace mutants cannot be attributed to exo- or endocytosis, at this point (West, 2018).

A strong phenotypic similarity was noticed at the larval neuromuscular synapse between the lace mutants and mutations in the small Ig domain adhesion protein Basigin/CD147. Bsg is a glycoprotein localised in the plasma membrane that is known to genetically interact with integrins during development of the Drosophila eye. In Bsg mutants, synaptic boutons at the larval neuromuscular junction are enlarged in size and reduced in number with a modest reduction in synaptic span. Bsg has previously been localised to sphingolipid enriched lipid rafts in invading epithelial breast cells and this study observed that Bsg is abundant in the lipid raft associated membrane fraction, co-sedimenting with syntaxin, a known component of lipid rafts. At this juncture it cannot be said if Bsg function is directly regulated by sphingolipids. Indeed, recruitment of Bsg to lipid rafts can be critical for the recruitment of other protein factors such as claudin-5 in retinal vascular epithelial cellsg. However, given the genetic interaction between Bsg and lace, with bsg;lace transheterozygous double mutants phenocopying both lace and bsg mutants, the data suggests Bsg and sphingolipids genetically interact to regulate synaptic structure. This interaction is intrepeted as indirect; the loss of sphingolipid generated in the lace mutant affecting Bsg function to regulate synapse structure and function (West, 2018).

Synaptic sphingolipids have previously been implicated in synaptic vesicle release, endocytosis, neurotransmitter receptor localisation and maintenance of synaptic activity. However other roles at the synapse for these enigmatic lipids remain elusive. Two potential functions for sphingolipid at the synapse are suggested by the current study. Mitochondrial uptake of Ca2+ shapes Ca2+ dependent responses. The enlarged mitochondria observed in lace mutants may impinge on Ca2+ uptake to affect synaptic facilitation. A further deficit in Ca2+ handling at the synapse is suggested by the recent finding that Bsg is an obligatory subunit of plasma membrane Ca2+-ATPases (PMCAs). PMCAs extrude Ca2+ to the extracellular space, and knock-out of Bsg considerably affects Ca2+ handling by PMCAs. Sphingolipid deficient synapses in the lace mutant have deficits in Bsg function which may in turn have an effect on Ca2+ dynamics via PMCA function (West, 2018).

Ablation of sphingolipid synthesis at a Drosophila model synapse supports a role for sphingolipids in maintenance of synaptic activity and regulation of synaptic structure. This analysis also points to sphingolipid dependent regulation of synaptic structure via function of the small Ig-domain protein Bsg. The precise regulation of synapse structure and function is a potent mechanism underlying synaptic plasticity and it is suggested that the presence of sphingolipids at synapse may partially reflect this function (West, 2018).

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Using glial-specific RNAi knockdown in Drosophila, this study silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among the screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, this RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia (Ghosh, 2013).

Serine palmitoyltransferase catalyzes the condensation of serine and palmitoyl-CoA to generate 3-ketosphinganine, the rate-limiting step in de novo sphingolipid synthesis. Mutations in the two human subunits of the serine palmitoyltransferase are associated with hereditary sensory and autonomic neuropathy. A common feature of the mutations is the loss of canonical enzyme activity and the generation of toxic lipid intermediates. Glial inhibition of lace function (repo>mCD8-GFP/lace RNAi) resulted in glial bulging and in an alteration of the axonal packing in all eight pairs of abdominal nerves in all larval PNS examined. The bulging of glia was localized to focal regions, but appeared randomly along the entire peripheral nerves with diameters ranging from 10 μm to 30 μm. Nerves of repo-GAL4/+ control flies were straight and packed in bundles with a uniform diameter of 5-8 μm. In contrast, knockdown of lace in neurons did not result in any visible alterations of axonal morphology . The average cross-section area of the nerve were similar in the knockdown (elav-GAL4/lace RNAi) and in the elav-GAL4/+ control flies (Ghosh, 2013).

Notably, the ensheathment defect was not due to a compromised blood-nerve-barrier as has for example been observed in null fray mutants. In addition, the number of glial cells in the peripheral nerves was comparable to control. It is important to note glial cell death affects neuronal survival and results in embryonic lethality. The absence of embryonic lethality and the comparable glial cell number suggested that glial cell death did not occur at the larval stage after knockdown of lace. The expression of lace in glia was confirmed by double immunolabeling of lace⁵(LacZ enhancer trap line) with anti-β-galactosidase and anti-repo in L3 larval peripheral nerves. In addition, by RT-PCR analysis of the fly brain and PNS lace transcript was identified in the nervous system of both male and female flies (Ghosh, 2013).

Two independent RNAi lines (Transformant ID 21803 and 110181, VDRC) against lace showed identical swelling and wrapping defects. In addition, hypomorphic lace mutant (lace²/lace5) resulted in axonal defasciculation, ruling out off-target effects of the RNAi lines. As in the lace-RNAi knockdown, the average cross-section area of the nerves was increased in the hypomorphic lace mutant animals. The mutant phenotypes appeared to be subtle, which is not surprising as complete loss of lace during the development is lethal, while this hypomorphic combination are viable even into adulthood. However, 100% penetrance of the phenotype was observed both for the RNAi knockdownand the hypomorphic mutants. Importantly, the lace mutant phenotype was rescued by expressing UAS-lace specifically in the glial cells (repo-GAL4), pointing to an essential function of lace in glia (Ghosh, 2013).

Next, an analysis was performed to see in which of the different glial subtype lace was required. Glia subtype specific GAL4 drivers were used to silence lace function. A phenotype was only observed when lace was depleted in wrapping glia (Nrv2-GAL4). Quantification of GFP signal intensity revealed that membrane area was significantly reduced as compared to control (Nrv2>mCD8GFP/lace RNAi versus Nrv2>mCD8GFP). Similar results were observed when the Nrv2>mCD8-mcherry driver line was used to knockdown lace in the wrapping glia. In contrast, knockdown of lace in the two other glial subtypes, the subperineurial (gliotactin-GAL4) and the perineurial (NP6293-GAL4), did not lead to any visible changes (glial swellings or decrease in the GFP signal intensity) in glia or in axons (-G), suggesting a predominant role of lace in the encapsulation of peripheral nerves (Ghosh, 2013).

To examine the ultrastructure in more detail electron microscopy was performed. Electron micrographs clearly showed that knockdown of lace in glia (repo/lace RNAi) severely impaired axonal enwrapping compared to control (repo/+). Notably, also in the non-swollen regions (A2-A3 segment) of the nerve much less glial processes covered the axons. Quantification demonstrated a significant increase in the number of completely unwrapped axons in this region. A similar phenotype was observed when lace was knocked down in wrapping glia (Nrv2/lace RNAi). Again, a clear increase in the completely unwrapped axons was detected as compared to the controls. Importantly, TUNEL assay could not detect any apoptotic glial nuclei (Nrv2>laceRNAi) suggesting that loss of axonal ensheathment is not because of dying wrapping glial cells. Together, these results indicate that sphingolipids or intermediates of the sphingolipid pathway are necessary for membrane expansion of wrapping glia (Ghosh, 2013).

In order to search for the specific sphingolipid (SL) species required by the wrapping glia to mediate axonal ensheathment, a genetic dissection study was performed by expressing RNAi against all known SL metabolic enzymes selectively in glia. Out of 12 genes, knockdown of Spt-I, schlank, Des1 and Pect in glia (repo>mCD8-GFP/RNAi) with two different RNAi lines (except Des1 due to unavailability) phenocopied the glial swelling and axonal defasciculation as observed upon loss of lace function. Interestingly, all four genes that show 100% penetrance are known to be involved in the biosynthesis of ceramide-phosphoethanolamine (CerPE). The specificity of the effect was demonstrated by the absence of any visible phenotype after neuronal specific knockdown of Spt-I, schlank, Des1 and Pect. Additionally, glial specific knockdown of different ceramide derivative synthesizing enzymes (GlcT1, CGT, CerK) and PE synthesizing enzyme (bbc) did not show any visible defects of axon or glial morphology. Moreover, when Spt-I, schlank, Des1 or Pect were knocked down specifically in wrapping glia, wrapping defects similar to the lace phenotype were observed . The quantification revealed that the GFP signal intensity was significantly reduced in all four experiments as observed after lace knockdown. Ultrastructural analysis by transmission electron microscopy also showed that wrapping glia failed to extend their membrane around the axons; and consequently there was an increase of the completely unwrapped axons. Hence, the data strongly suggests an essential function of glial CerPE in axonal ensheathment by wrapping glia (Ghosh, 2013).

In order to analyze whether knockdown of lace, schlank, Des1 and Pect resulted in depletion of CerPE levels, a detailed lipidomics analysis of the nervous system was performed. This is particularly important in RNAi studies targeting enzymes, because residual enzyme activity due to inefficient RNAi-silencing is often sufficient for their function. Since the nervous system of Drosophila only contains 10% of glia, the RNAi was expressed both in neurons and glia using repo-GAL4 and elav-GAL4 drivers to deplete the enzymes in the entire nervous system. L3 larval brain and peripheral nerves were dissected and lipidomics analysis was performed with high-resolution shotgun mass spectrometry. Importantly, lipidomics analysis confirmed that knockdown of lace, schlank, Des1 and Pect reduced CerPE levels significantly, whereas triacylglycerol (TAG) and diacylglycerol (DAG) and sterol levels were unaltered. Ceramide levels were reduced upon downregulation of lace and Des1, whereas knockdown of Pect lead to increased ceramide levels consistent with its function as a phosphoethanolamine cytidylyltransferase. Phosphatidylcholines (PC) and Phosphatidylethanolamine (PE) levels were slightly changed possibly due to compensatory mechanisms (Ghosh, 2013).

Next, tests were performed to see whether it was possible to rescue the morphological phenotype induced by knockdown of lace in glia by supplementing sphingosine (re-converted to ceramide by condensation with a fatty-acylCoA catalyzed by the various ceramide synthases) into the diet of the flies. Indeed, the phenotype of glia-specific knockdown of lace was efficiently rescued by the exogenous addition of sphingosine (300 μM) to the food. Double Immunolabelling of glia and neuronal membrane reveals that the glial bulging and axonal unpacking was rescued upon addition of sphingosine to the diet. Orthogonal projections and the quantification demonstrated the rescue of the neuropathy like phenotype in flies. It was furthermore observed with the ultrastructural analysis that the glial enwrapment defect was recovered upon sphingosine addition to the diet. Quantitative analysis of the peripheral nerves using the confocal and electron microscopy showed that the oral administration of sphingosine can restore the enwrapping defect and the neuropathy-like phenotype (Ghosh, 2013).

Sphingolipids have both structural and signalling functions in cells. CerPE is a relatively low abundant lipid constituting only around 1% of the total fly lipidome. Interestingly, CerPE appears to be enriched in the fly brain membrane lipidome (4%). In mammals, CerPE is only found in trace amounts, since sphingolipids are in general built on ceramide phosphatidylcholine in higher organisms. There are different possibilities of how CerPE could exert its function in glia. CerPE might be required for signal transduction pathways that control membrane synthesis in wrapping glia. Recently, a mutation in egghead, an enzyme that extends the glycosphingolipids (GSLs) in flies, causes the proliferation and overgrowth of subperineurial glia mediated by aberrant activation of phosphatidylinositol 3-kinase-Akt pathway. CerPE may also increase the packing density of the lipids in the membrane, thereby helping to build up an efficient barrier for the electrical insulation of the axons. In vertebrates, a related sphingolipid, galactocylceramide, is critical for the formation of an insulating myelin sheath in oligodendrocytes. Galactosylceramide and/or its sulphated form are required for the tight sealing of the glial paranodal membrane to the axon. Interestingly, mice lacking ceramide synthase 2, a vertebrate homolog of schlank, have myelination defects (Ghosh, 2013).

Alterations of enzyme function or enzyme deficiencies do not only result in a reduction in the amount of an essential product, but can also lead to the accumulation of a toxic intermediate, or the production of a toxic side-product. For example, mutations in human serine palmitoyltransferase result in a loss of normal enzyme function causing a shift in the substrate specificity, which increase the accumulation of atypical, toxic lipid products. Thus, gain-of-toxic-function is another possibility of how knockdown of lace may cause the axonal ensheathment defects (Ghosh, 2013).

Interestingly, supplementing sphingosine to the diet restored the ability of wrapping glia to extend their membrane around axons. How diets affect the distribution of lipids in cells and thereby modulate biological processes will be an important question for future investigations. Drosophila is an ideal system to pursue such studies because of the short life span and the powerful genetics, which enable rapid and detailed analysis. In summary, the current study illustrates that a large-scale screen in Drosophila, in combination with concomitant morphological and electrophysiological analysis has the potential to dissect the basic mechanisms of neuron-glia communication. Detailed knowledge of neuron-glia interactions is a pre-requirement for the rational design of treatment strategies for neuropathies or other diseases in the future (Ghosh, 2013).