Glial Cell-Surface Proteomics of the Intact Brains of Young and Old Flies.

A: Transgenic expression of HRP fused to the transmembrane protein CD2 targets CD2-HRP to the extracellular side of the glial plasma membrane. In the presence of H2O2, CD2-HRP converts BxxP substrate into phenoxyl radicals that biotinylate nearby endogenous proteins. Biotinylated proteins are then enriched from brain lysates using streptavidin beads, followed by on-bead trypsin digestion, TMT labeling, and liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS). B: To confirm HRP-dependent biotinylation of glial cells, Repo-GAL4 were crossed with either UAS-HRP-CD2 or wildtype (w1118) flies and adult brains were stained with neutravidin. Proteins were biotinylated in the experimental group (UAS-HRP-CD2) but not in the control group (w1118). C: Biochemical characterization by streptavidin blot showed that the glial cell-surface-targeted HRP biotinylated a wide range of proteins compared to the negative control lacking HRP-CD2 expression. D: For each time point (5d and 50d), two biological replicates and two negative controls (one lacking HRP-CD2 expression and one lacking H2O2 in the reaction) were profiled. In total, we detected 2,007 proteins with two or more unique peptides. Potential contaminants were removed by comparing differential enrichment between the experimental and negative control groups (i.e., the TMT ratio for each protein identified; see Methods). E: Strong correlations between biological replicates were observed, further validating the quality of the proteome profiles. F: The receiver operating characteristic (ROC) curve depicts the true-positive rate against the false-positive rate of detected proteins, for each biological replicate (only 126:127C pictured). G: Gene Ontology (GO) analysis was performed based on the 872 proteins identified following ratiometric and cutoff (see Methods). Top 5 GO terms on cellular compartments with the lowest false discovery rates are shown.

Quantitative comparison of young and old proteomes and selection of candidate genes.

A: Genes encoding proteins associated with the largest old/young fold changes were identified and 48 candidate genes were selected for functional screening. Down-regulated candidate genes selected for functional screening are labeled in blue whereas up-regulated candidate genes are labeled in red. B: A Gene Ontology (GO) analysis investigating proteome biological processes revealed that genes encoding the most up-regulated proteins were primarily associated with cell localization, transport, and maintaining homeostasis, while most down-regulated proteins were primarily associated with neural development, synapse organization, and neuroplasticity. C: Genes encoding the most up- and down-regulated proteins were clustered by their reported protein-protein interactions (PPIs) and corresponding confidence scores (using a Markov clustering algorithm, inflation value set to 2.5). Each protein in the PPI plot was then assessed individually to determine if it had reported function in synapse organization, synapse regulation, cell adhesion, development and cell migration, or localization and transport.

Functional screening of candidate genes predicted to be involved in normal brain aging.

A: Confocal images showing that the glial-GS line only induced transgene expression in the presence of the inducible drug Ru486. B: 48 candidate genes were selected for functional screening. For each of the 19 up-regulated genes, two RNAi lines were used to control for possible off target effects. Of the 29 down-regulated genes selected, we were able to acquire and screen lines for 19 genes. C: No obvious lifespan effect was observed in control lines: a w1118 wildtype control, a UAS-luciferase RNAi control, a UAS-lacZ control, and a flySAM control. D: Pie charts summarizing the results of our broadscale lifespan screen for male and female flies. E: Of the 19 up-regulated candidate genes screened, RNAi knockdown of 4 genes led to consistent lifespan decreases for males. Alternatively, RNAi knockdown of 3 genes led to consistent lifespan increases for females. F: Of the 19 down-regulated candidate genes screened, overexpression of 2 genes led to lifespan increases for males, whereas overexpression of 3 genes led to lifespan decreases. In parallel, overexpression of 10 genes led to lifespan increases for females, whereas overexpression of 1 gene led to decreased lifespan.

Validating that pan-glial overexpression of DIP-β increases lifespan and serves a protective function during aging.

A-C: Glial overexpression of DIP-β (flySam 2.0 line) significantly extends lifespan for both male and female flies, compared to the negative controls (only the w1118 and UAS-lacZ controls are shown here). D: Glial overexpression of DIP-β (UAS-cDNA line) significantly extends lifespan for both male and female flies E: To further validate that glial overexpression of DIP-β is serving a protective function during brain aging, climbing (i.e., negative geotaxis) assays were conducted. Glial overexpression of DIP-β was also associated with improved climbing and motor control ability in aged flies.

Single-nucleus RNA-sequencing - investigating the downstream transcriptomic effects of glial overexpression of DIP-β during aging.

A: Two whole-head samples (20 heads each) were prepared for snRNA-seq: one where DIP-β had been overexpressed in glia from day 5-50 and one control. Single-nucleus RNA-seq libraries were prepared using the 10X Genomics Chromium Single Cell 3’ HT v3.1 protocol. B: In total, approximately 49,000 high-quality cells were obtained after quality control and filtering from our fly head samples, which consist of 8 broad cell types. C: Broad cell-type cluster compositions were largely consistent between the glial DIP-β overexpression treatment (Ru486+) and the control (Ru486-) groups. D: A broad analysis of differentially expressed genes (DEGs) revealed that the glial cell-type cluster showed the second highest number of DEGs between the glial DIP-β overexpression treatment (Ru486+) and the negative control (Ru486-) groups. E: A more detailed analysis investigating DEGs across 90 different cell subtypes revealed that of the glial cell subtypes, cortex glia (i.e., “adult brain cell body glia”) and ensheathing glia showed the highest number of DEGs between the DIP-β overexpression treatment (Ru486+) and the control (Ru486-) groups. F: Of the 252 DEGs identified for glial cells, 186 were unique to cortex glia, 27 were unique to ensheathing glia, and 40 were found to be differentially expressed in both cortex and ensheathing glia. G: As fat cells exhibited the highest number of DEGs between the DIP-β overexpression and control groups, we conducted a KEGG analysis to evaluate the biological functions of these genes. Most fat cell DEGs were up-regulated, and the top altered metabolic and signaling pathways for these genes were found to be related to homeostasis, signal integration, stress resistance, and longevity. H: Cell-cell communication (CCC) analysis was performed using FlyPhoneDB2. DIP-β overexpression was generally associated with an increase in cell-cell communication, particularly glia-neuron and glia-fat cell communication. The top altered glial signaling pathways are shown. I: Our proposed model: Glial overexpression of DIP-β leads to improved glia-neuron and glia-fat cell communication as flies age, which may contribute to the observed lifespan extensions.

Glial cell-surface proteomics

A: We searched the 872 proteins included in our proteomic profiles against three databases: FlyBase, UniProt, and the Gene Ontology Data Archive (DOI 10.5281/zenodo.1205166; Version 2023-05-10; Ashburner et al., 2000) to determine which proteins had previous plasma membrane or extracellular matrix annotations. We found the 682 out of the 872 proteins could be validated by at least one database, further validating the quality of our cell-surface proteomic labeling. B: We plotted the receiver operating characteristic (ROC) curve for each biological replicate answer observed high specificity. C: We identified the top 10 “hub” genes in our PPI network and found that of the top 10 hub genes identified, 7 had reported function in cell adhesion (klg, kst) and synaptic wiring (DIP-β, DIP-δ, DIP-γ, DIP-κ, DIP-ɑ), while the remaining three had reported function in neuron projection (CG33543), axon guidance (CG34353), and dendrite guidance (Toll-6).

Functional screening of candidate genes.

A-C: In our initial functional lifespan screen, glial overexpression of DIP-β was found to significantly extend lifespan for both male and female flies, compared to the negative controls (only the luciferase RNAi and flySAM controls are pictured here).

Functional screening of up-regulated candidate genes.

A-E: We selected 19 up-regulated candidate genes for functional screening, which we selectively knocked down in glia to examine their influence on lifespan. For each up-regulated gene, we screened two RNAi lines (to control for possible off target effects). Of the 19 up-regulated candidates screened, we found that RNAi knockdown of 4 genes led to consistent lifespan decreases for males. Alternatively, for female flies, RNAi knockdown of 3 genes led to consistent lifespan increases.

Functional screening of up-regulated candidate genes.

A-E: We selected 19 up-regulated candidate genes for functional screening, which we selectively knocked down in glia to examine their influence on lifespan. For each up-regulated gene, we screened two RNAi lines (to control for possible off target effects). Of the 19 up-regulated candidates screened, we found that RNAi knockdown of 4 genes led to consistent lifespan decreases for males. Alternatively, for female flies, RNAi knockdown of 3 genes led to consistent lifespan increases.

Functional screening of up-regulated candidate genes.

A-E: We selected 19 up-regulated candidate genes for functional screening, which we selectively knocked down in glia to examine their influence on lifespan. For each up-regulated gene, we screened two RNAi lines (to control for possible off target effects). Of the 19 up-regulated candidates screened, we found that RNAi knockdown of 4 genes led to consistent lifespan decreases for males. Alternatively, for female flies, RNAi knockdown of 3 genes led to consistent lifespan increases.

Functional screening of up-regulated candidate genes.

A-E: We selected 19 up-regulated candidate genes for functional screening, which we selectively knocked down in glia to examine their influence on lifespan. For each up-regulated gene, we screened two RNAi lines (to control for possible off target effects). Of the 19 up-regulated candidates screened, we found that RNAi knockdown of 4 genes led to consistent lifespan decreases for males. Alternatively, for female flies, RNAi knockdown of 3 genes led to consistent lifespan increases.

Functional screening of down-regulated candidate genes.

A-J: We selected 29 down-regulated candidate genes for functional screening, which we selectively overexpressed in glia to examine their influence on lifespan. For each of the 29 down-regulated genes, we screened one overexpression line (using either UAS-cDNA lines or flySAM2.0 lines), when available. In total, we were able to acquire and screen lines for 19 genes. Of these candidates, overexpression of 2 genes led to lifespan increases for males, whereas overexpression of 3 genes led to lifespan decreases. Alternatively, overexpression of 10 genes led to lifespan increases for females, whereas overexpression of 1 gene led to decreased lifespan. We found that constitutive overexpression of DIP-β in glia significantly increased lifespan in both males and females compared to controls.

Functional screening of down-regulated candidate genes.

A-J: We selected 29 down-regulated candidate genes for functional screening, which we selectively overexpressed in glia to examine their influence on lifespan. For each of the 29 down-regulated genes, we screened one overexpression line (using either UAS-cDNA lines or flySAM2.0 lines), when available. In total, we were able to acquire and screen lines for 19 genes. Of these candidates, overexpression of 2 genes led to lifespan increases for males, whereas overexpression of 3 genes led to lifespan decreases. Alternatively, overexpression of 10 genes led to lifespan increases for females, whereas overexpression of 1 gene led to decreased lifespan. We found that constitutive overexpression of DIP-β in glia significantly increased lifespan in both males and females compared to controls.

Single-nucleus RNA-sequencing - investigating the downstream transcriptomic effects of pan-glial DIP-β overexpression.

A: In total, we sequenced approximately 49,000 cells from our fly head samples, with approximately 23,000 from the glial DIP-β overexpression, (i.e., Ru486+) sample and approximately 26,000 from the negative control (i.e., Ru486-) sample. B: Detailed cell subtype cluster composition annotations. C: A detailed analysis investigating differentially expressed genes (DEGs) across cell subtypes.