A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons

Abstract

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. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our 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, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia.

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

“…CPE is concentrated in neural tissue of certain insects, comprising about 4% of the total lipid content of brain tissue (49) in Drosophila and about 6 -7% of total lipid content of the nervous system in Musca domestica (50). In Drosophila, CPE is required by glial cells to properly insulate axons, and lack of CPE leads to neuropathy (48). Another future research question is whether the neurotoxic effects of isolated SicTox proteins on different prey will depend on a match between enzyme specificity and the content of SM and/or CPE in neurological tissue.…”

“…In addition to SM, the panel included the phosphosphingolipid ceramide phosphoethanolamine (CPE), which differs from SM only in replacement of the choline headgroup with ethanolamine (see Scheme 1). The ethanolamine-containing substrates LPE and CPE were particularly interesting to us as possible alternative substrates to LPC and SM, because ethanolamine is a common headgroup in insect lipids (47)(48)(49)(50). We included LPG and LPS as examples of lipids with neutral/zwitterionic headgroups.…”

Differentiation of Drosophila glial cells

“…Recently, a large set of genes (6930) has been tested for effects on glial differentiation in an RNAi‐based screen . A few of the genes identified in this screen have been further analyzed.…”

Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates

“…In particular, the highest levels of CPE have been detected during the onset of the larval stage, indicating its important role during early development. Specific depletion of CPE levels in wrapping glia impairs axonal ensheathment of the peripheral nerves, giving rise to a neuropathy‐like phenotype (14).…”