Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

  1. Hilary Scott
  2. Boris Novikov
  3. Berrak Ugur
  4. Brooke Allen
  5. Ilya Mertsalov
  6. Pedro Monagas-Valentin
  7. Melissa Koff
  8. Sarah Baas Robinson
  9. Kazuhiro Aoki
  10. Raisa Veizaj
  11. Dirk J Lefeber
  12. Michael Tiemeyer
  13. Hugo Bellen
  14. Vladislav Panin

  • Curated by eLife

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    Scott et al use Drosophila as a model to study the sialylation pathway and its role in nervous system function. Surprisingly, they find that the critical substrate for sialylation, CMP-Neu5Ac, is 'outsourced' to glia. This significant study presents a new twist in mechanisms underlying protein glycosylation and uncovers a new layer in the complex interplay of neurons and glia.

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Abstract

Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila , CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.

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  1. Version published to 10.7554/elife.78280 on eLife
  2. eLife

    eLife assessment

    Scott et al use Drosophila as a model to study the sialylation pathway and its role in nervous system function. Surprisingly, they find that the critical substrate for sialylation, CMP-Neu5Ac, is 'outsourced' to glia. This significant study presents a new twist in mechanisms underlying protein glycosylation and uncovers a new layer in the complex interplay of neurons and glia.

  3. eLife

    Reviewer #1 (Public Review):

    Here, the authors generated a CSAS-LexA driver line to investigate the expression pattern of CSAS and showed that CSAS expression is confined to glia and does not overlap with DSiaT expression. DSiaT expression is presumed to be in neurons, but this was not evaluated with specific markers in this study.

    The authors showed that restoring CSAS expression specifically in glia but not neurons could rescue the mutant phenotype of temperature sensitive paralysis and confirmed that glial (and not neuronal) CSAS expression could rescue excitatory junction potentials at neuromuscular junctions in CSAS mutants. In addition to rescue experiments, the authors also performed RNAi knockdowns in glia vs. neurons to show that CSAS function is required in glia and DSiaT in neurons for the same paralysis phenotype.

    Next, the authors performed mass spec to analyse sialylated proteins in larval brains and found that sialylated proteins could not be detected in DSiaT and CSAS mutants. However, sialylated proteins were only barely detectable in wildtypes.

    Of note, the authors show that CSAS functions normally in glia and cannot function in neurons due to low endogenous NANS activity (sialic acid synthase).

    Finally, the authors explore the hypothesis that the temperature-sensitive paralysis CSAS phenotype is due to oxidative stress with a paraquat exposure paradigm. This could be strengthened by examining ROS levels in vivo in CSAS or DSiaT mutants. The specific genetic background of these experiments seemed to be a major factor in the results obtained and more stringent controls or backcrossing to isogenize the genetic background would be required to be fully confident in the conclusions drawn from these experiments.

    The authors also demonstrate a link between sialylation and Para (protein) expression. Although intriguing, there is very little data provided on this aspect of the story, though it does not detract from the broader message of the manuscript.

  4. eLife

    Reviewer #2 (Public Review):

    The function of many proteins depends on posttranslational modifications. Protein glycosylation is widespread and glycosylated proteins are mostly found on the outer surface of cells, where it is frequently implicated in cell-to-cell adhesion. It involves the addition of often complex and branched sugar chains to a protein backbone. Sialic acid is a particular relevant sugar as it is negatively charged and occupies terminal positions at the glycan chain. The enzymatic cascade leading to sialylated proteins is known. Unlike mammals, flies have only one sialyltransferase (SiaT), thus, Drosophila is a particularly well-suited model to study protein sialylation. The penultimate enzymatic steps in sialylation are mediated by N-acetlyneuraminic acid synthetase (NANS) and sialic acid synthetase (CSAS).

    Scott et al., start with careful and state-of-the-art dissection of the expression patterns of the relevant genes. They first generated transgenic flies harboring a BAC covering the CSAS gene - which was able to rescue the mutant phenotype. They then replaced the CSAS coding sequence with LexA and demonstrated that LexA expression was sufficient to drive LexAop-CSAS to a full rescue of the CSAS mutant. CSAS-LexA was found to be active only in Repo expressing glial cells. The authors performed further experiments employing another BAC harboring an HA-tagged SiaT gene and found complementary expression in neurons (here I missed a comment on why the endogenously tagged SiaT gene (Repnikova 2010) was not used).

    To study cell-type specific requirements UAS-based rescue experiments were conducted. The CSAS mutant phenotype could be rescued not only by panglial expression of CSAS but also by expression exclusively in subperineurial or ensheathing glial cells. Whether astrocytes or cortex glial cells are similarly able to rescue the mutant phenotype has not been addressed. No rescue was observed when CSAS was expressed in neurons, but co-expression of CSAS and NANS led to a partial rescue, further validating the split of the biosynthetic pathway leading to sialylated proteins to glial and neuronal cells.
    In addition to the rescue experiment, the authors also performed RNAi-based knockdown experiments for both, CSAS and SiaT which together support the conclusion that sialylation requires a split of the biosynthesis pathway.

    In a subsequent mass spec approach, the authors analyzed sialylated proteins in larval brains. Whereas in wild type brains sialylated proteins were barely detected, they could not be seen in SiaT or CSAS mutant brains. However, according to Flybase, the highest expression of both genes is in adult flies. Why not look at these stages? It would also be good to use the cell type-specific knockdown flies for such experiments to fully support the notion that sialylation requires a glia-neuron transfer of intermediates. Possibly, low (and thus undetected) levels of SiaT in glia could be sufficient for function. In this respect it is interesting that the presence of a UAS-SiaT element is sufficient to rescue the SiaT mutant phenotype, suggesting that only very low levels of SiaT are needed for function.

    Subsequently, Scott et al., demonstrate that the paralysis phenotype of CSAS mutants is sensitive to gene dose and that CSAS activity protects flies from oxidative stress. Quite interesting, they also demonstrate that sialylation is required - directly or indirectly - to maintain protein expression of the voltage gate sodium ion channel Para.

  5. eLife

    Reviewer #3 (Public Review):

    In this work, the authors find that similar to mammals, sialylation is critical in neurons within flies, yet in flies the critical substrate for sialylation, CMP-Neu5Ac, is 'outsourced' to glial cells. These findings are shown through an extensive array of knockout, knockdown, and transgenic flies where CMP-Neu5Ac biosynthesis and sialyltransferase expression is modulated in either glial cells or neurons. The importance of sialylation in neurons is demonstrated by showing that sialylation impacts the expression levels of a critical voltage-gated ion channel.

    This elegant work dissecting sialylation in the fly brain convincingly demonstrates the requirement for glial cells in the process of sialylation of neurons and deserves to be published. The major unaddressed question remaining is precisely how the CMP-Neu5Ac is delivered from the glial cells to neurons with several possibilities that merit further discussion including (but not limited to): extracellular vesicles, receptor-mediated uptake (unlikely but can't be ruled out), or exocytosis. The authors could make the point stronger that CMP-Neu5Ac should not be able to cross the neuronal membrane (or the Golgi membrane for that matter), requiring specific transport mechanisms.

  6. Version published to 10.1101/2022.03.29.486211v1 on bioRxiv