Tissue-specific O-GlcNAcylation profiling identifies substrates in translational machinery in Drosophila mushroom body contributing to olfactory learning
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eLife assessment
This work describes a valuable new technique involving proximity labelling to identify Drosophila proteins modified by GlcNAcylation in subsets of cells in vivo. A solid set of experiments shows that several ribosomal proteins are modified in the fly mushroom body. Consistent with a role for GlcNAcylation of ribosomal proteins in control of memory related translational control, the authors show that perturbation of GlcNAc modification in KCs prevents efficient consolidation of long-term memory.
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Abstract
O- GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an O- GlcNAc binding activity derived from Clostridium perfringens OGA ( Cp OGA) with TurboID proximity labeling in Drosophila , we developed an O- GlcNAcylation profiling tool that translates O- GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the O- GlcNAc interactome in major brain regions of Drosophila and found that components of the translational machinery, particularly ribosomal subunits, were abundantly O- GlcNAcylated in the mushroom body of Drosophila brain. Hypo- O- GlcNAcylation induced by ectopic expression of active Cp OGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of O- GlcNAcylation in Drosophila , and suggests a possibility that O- GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.
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Author Response
Reviewer #1 (Public Review):
The author's goal was to determine the role of O-GlcNAc modification in associate learning in Drosophila using an odor discriminatory task. In particular, they sought to determine the population of O-GlcNAc modified proteins in a region of the brain critical for memory, the mushroom body. They provide compelling evidence that there are brain-region-specific populations of O-GlcNAc modified proteins and that in the mushroom body, proteins involved in translation represent a sizable, and larger fraction than elsewhere in the central nervous system. Using expression of a bacterial protein that cleaves O-GlcNAc in the mushroom body, they show both reductions in the levels of this modification and effects on associative learning. Further exploration of new protein synthesis in situ supports the …
Author Response
Reviewer #1 (Public Review):
The author's goal was to determine the role of O-GlcNAc modification in associate learning in Drosophila using an odor discriminatory task. In particular, they sought to determine the population of O-GlcNAc modified proteins in a region of the brain critical for memory, the mushroom body. They provide compelling evidence that there are brain-region-specific populations of O-GlcNAc modified proteins and that in the mushroom body, proteins involved in translation represent a sizable, and larger fraction than elsewhere in the central nervous system. Using expression of a bacterial protein that cleaves O-GlcNAc in the mushroom body, they show both reductions in the levels of this modification and effects on associative learning. Further exploration of new protein synthesis in situ supports the hypothesis that O-GlcNAc modification affects the activity of the translational machinery and could provide the basis for learning deficits when O-GlcNAc levels are compromised. Rescue of deficits resulting from reductions in O-GlcNAc was achieved by over-expression of dMyc, a known regulator of ribosome biogenesis and translation. While the critical role of protein synthesis in learning is long established, and that O-GlcNAc modification regulates protein synthesis, this work connects O-GlcNAc modification in a specialized region of the brain to translation regulation and associative learning. The authors also provide a method for identification of O-GlcNAc modified proteins using a tissue-specific and inducible proximity-labelling method. This will provide a useful tool for further functional studies of O-GlcNAc modification.
Thank you for summarizing our main findings and recognizing the usefulness of the tool reported here.
Reviewer #2 (Public Review):
In this report Yu et al. try to demonstrate how O-GlcNAcylation of ribosomal proteins in the mushroom body (MB) is required for protein synthesis and olfactory learning. The authors develop a new method combining the O-GlcNAc binding activity of an OGlcNAcase (OGN) and TurboID for efficient isolation. This novel method is a useful tool for the identification of O-GlcNAc modified proteins and closely interacting partners. Transgenic expression of this binder allows the authors to perform a profiling that can be time and tissue/region/cell specific. This novel tool is thoroughly tested to show it works in cultured cells, whole Drosophila and in a tissue specific manner expressing it pan-neuronally or specific regions of the brain.
The authors had previously shown that reduced O-GlcNAcylation through transgenic expression of a highly active OGN affected olfactory learning. In this work the same approach is used to reduce O-GlcNAcylation in different brain regions to show that specific reduction in the adult MB reduced olfactory learning performance. As control OGN expression in the ellipsoid body has no effect on olfactory learning. Optic and antennal lobes could not be tested as OGN expression affected olfactory acuity. The most critical part of this finding is time specific expression of OGN in the adult in a tissue specific manner given the developmental defects it induces with earlier expression. The MB has a widely reported role in associative learning, therefore this finding while not unexpected it is satisfying.
Thank you for recognizing the significance of our work.
Yu et al. use their TurboID-OGA to identify O-GlcNAcylated proteomes in different brain regions. The authors focus on the MB given its role in associative learning and the effect of reduced O-GlcNAcylation in this region. Among other substrates several ribosomal proteins are found to be specifically O-GlcNAcylated to a greater extent in the MB compared to other brain regions.
To demonstrate the role of MB O-GlcNAcylated ribosomes in protein synthesis an ex vivo OPP fluorescent assay is used in brains of flies expressing OGN or a mutant form lacking its catalytic and binding activities. The experiment shows reduced protein synthesis in the MB. In addition, the authors can increase protein synthesis inducing ribosomal biogenesis through the expression of dMyc. Flies expressing of dMyc and OGN together do not present the learning deficits of flies carrying only OGN. Protein synthesis in MB has been previously reported to be required for associative learning (for example Wu et al.2017 or Lin et al. 2022) and the present results bring further support. A link between ribosomal O-GlcNAcylation and protein synthesis could be a really interesting finding but, unfortunately the experiments presented in this work are still too preliminary.
The experiments presented just focus on ribosomal proteins while these are just some of the O-GlcNAcylation substrates in the MB. While a correlation between ribosomal modification and protein synthesis is shown, a demonstration is not provided. Many other mechanisms and O-GlcNAcylation of other substrates could account for the same observations. For example, O-GlcNAcylation has been reported to have a role in protein synthesis affecting different translation initiation factors (Li et al 2018, Shu et al 2022). In vitro experiments where specific O-GlcNAcylation ribosomal components could be targeted are required. In addition, O-GlcNAcylation is also known to modify ribosomal-associated mRNAs. Experiments where specific mutations preventing O-GlcNAcylation in ribosomes could demonstrate a direct link of such ribosomal modifications in olfactory learning.
We appreciate that you bring up a crucial point that our data fall short for a causal connection between O-GlcNAcylation of ribosomes and translational activity. We have made significant changes to the text throughout the manuscript to make our description more accurate.
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eLife assessment
This work describes a valuable new technique involving proximity labelling to identify Drosophila proteins modified by GlcNAcylation in subsets of cells in vivo. A solid set of experiments shows that several ribosomal proteins are modified in the fly mushroom body. Consistent with a role for GlcNAcylation of ribosomal proteins in control of memory related translational control, the authors show that perturbation of GlcNAc modification in KCs prevents efficient consolidation of long-term memory.
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Reviewer #1 (Public Review):
The author's goal was to determine the role of O-GlcNAc modification in associate learning in Drosophila using an odor discriminatory task. In particular, they sought to determine the population of O-GlcNAc modified proteins in a region of the brain critical for memory, the mushroom body. They provide compelling evidence that there are brain-region-specific populations of O-GlcNAc modified proteins and that in the mushroom body, proteins involved in translation represent a sizable, and larger fraction than elsewhere in the central nervous system. Using expression of a bacterial protein that cleaves O-GlcNAc in the mushroom body, they show both reductions in the levels of this modification and effects on associative learning. Further exploration of new protein synthesis in situ supports the hypothesis that …
Reviewer #1 (Public Review):
The author's goal was to determine the role of O-GlcNAc modification in associate learning in Drosophila using an odor discriminatory task. In particular, they sought to determine the population of O-GlcNAc modified proteins in a region of the brain critical for memory, the mushroom body. They provide compelling evidence that there are brain-region-specific populations of O-GlcNAc modified proteins and that in the mushroom body, proteins involved in translation represent a sizable, and larger fraction than elsewhere in the central nervous system. Using expression of a bacterial protein that cleaves O-GlcNAc in the mushroom body, they show both reductions in the levels of this modification and effects on associative learning. Further exploration of new protein synthesis in situ supports the hypothesis that O-GlcNAc modification affects the activity of the translational machinery and could provide the basis for learning deficits when O-GlcNAc levels are compromised. Rescue of deficits resulting from reductions in O-GlcNAc was achieved by over-expression of dMyc, a known regulator of ribosome biogenesis and translation. While the critical role of protein synthesis in learning is long established, and that O-GlcNAc modification regulates protein synthesis, this work connects O-GlcNAc modification in a specialized region of the brain to translation regulation and associative learning. The authors also provide a method for identification of O-GlcNAc modified proteins using a tissue-specific and inducible proximity-labelling method. This will provide a useful tool for further functional studies of O-GlcNAc modification.
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Reviewer #2 (Public Review):
In this report Yu et al. try to demonstrate how O-GlcNAcylation of ribosomal proteins in the mushroom body (MB) is required for protein synthesis and olfactory learning. The authors develop a new method combining the O-GlcNAc binding activity of an OGlcNAcase (OGN) and TurboID for efficient isolation. This novel method is a useful tool for the identification of O-GlcNAc modified proteins and closely interacting partners. Transgenic expression of this binder allows the authors to perform a profiling that can be time and tissue/region/cell specific. This novel tool is thoroughly tested to show it works in cultured cells, whole Drosophila and in a tissue specific manner expressing it pan-neuronally or specific regions of the brain.
The authors had previously shown that reduced O-GlcNAcylation through transgenic …
Reviewer #2 (Public Review):
In this report Yu et al. try to demonstrate how O-GlcNAcylation of ribosomal proteins in the mushroom body (MB) is required for protein synthesis and olfactory learning. The authors develop a new method combining the O-GlcNAc binding activity of an OGlcNAcase (OGN) and TurboID for efficient isolation. This novel method is a useful tool for the identification of O-GlcNAc modified proteins and closely interacting partners. Transgenic expression of this binder allows the authors to perform a profiling that can be time and tissue/region/cell specific. This novel tool is thoroughly tested to show it works in cultured cells, whole Drosophila and in a tissue specific manner expressing it pan-neuronally or specific regions of the brain.
The authors had previously shown that reduced O-GlcNAcylation through transgenic expression of a highly active OGN affected olfactory learning. In this work the same approach is used to reduce O-GlcNAcylation in different brain regions to show that specific reduction in the adult MB reduced olfactory learning performance. As control OGN expression in the ellipsoid body has no effect on olfactory learning. Optic and antennal lobes could not be tested as OGN expression affected olfactory acuity. The most critical part of this finding is time specific expression of OGN in the adult in a tissue specific manner given the developmental defects it induces with earlier expression. The MB has a widely reported role in associative learning, therefore this finding while not unexpected it is satisfying.
Yu et al. use their TurboID-OGA to identify O-GlcNAcylated proteomes in different brain regions. The authors focus on the MB given its role in associative learning and the effect of reduced O-GlcNAcylation in this region. Among other substrates several ribosomal proteins are found to be specifically O-GlcNAcylated to a greater extent in the MB compared to other brain regions.
To demonstrate the role of MB O-GlcNAcylated ribosomes in protein synthesis an ex vivo OPP fluorescent assay is used in brains of flies expressing OGN or a mutant form lacking its catalytic and binding activities. The experiment shows reduced protein synthesis in the MB. In addition, the authors can increase protein synthesis inducing ribosomal biogenesis through the expression of dMyc. Flies expressing of dMyc and OGN together do not present the learning deficits of flies carrying only OGN. Protein synthesis in MB has been previously reported to be required for associative learning (for example Wu et al.2017 or Lin et al. 2022) and the present results bring further support. A link between ribosomal O-GlcNAcylation and protein synthesis could be a really interesting finding but, unfortunately the experiments presented in this work are still too preliminary.
The experiments presented just focus on ribosomal proteins while these are just some of the O-GlcNAcylation substrates in the MB. While a correlation between ribosomal modification and protein synthesis is shown, a demonstration is not provided. Many other mechanisms and O-GlcNAcylation of other substrates could account for the same observations. For example, O-GlcNAcylation has been reported to have a role in protein synthesis affecting different translation initiation factors (Li et al 2018, Shu et al 2022). In vitro experiments where specific O-GlcNAcylation ribosomal components could be targeted are required. In addition, O-GlcNAcylation is also known to modify ribosomal-associated mRNAs. Experiments where specific mutations preventing O-GlcNAcylation in ribosomes could demonstrate a direct link of such ribosomal modifications in olfactory learning.
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