Insulin-like Signaling Promotes Glial Phagocytic Clearance of Degenerating Axons through Regulation of Draper

Abstract

Neuronal injury triggers robust responses from glial cells, including altered gene expression and enhanced phagocytic activity to ensure prompt removal of damaged neurons. The molecular underpinnings of glial responses to trauma remain unclear. Here, we find that the evolutionarily conserved insulin-like signaling (ILS) pathway promotes glial phagocytic clearance of degenerating axons in adult Drosophila. We find that the insulin-like receptor (InR) and downstream effector Akt1 are acutely activated in local ensheathing glia after axotomy and are required for proper clearance of axonal debris. InR/Akt1 activity, it is also essential for injury-induced activation of STAT92E and its transcriptional target draper, which encodes a conserved receptor essential for glial engulfment of degenerating axons. Increasing Draper levels in adult glia partially rescues delayed clearance of severed axons in glial InR-inhibited flies. We propose that ILS functions as a key post-injury communication relay to activate glial responses, including phagocytic activity.

Figure 1. InR/Akt1 is required in adult glia for proper glial clearance of degenerating axons

(A) Schematic of experimental method to temporally control UAS-driven gene expression. (B) Maximum intensity projections of the antennal lobe region show axons of OR85e ORNs expressing membrane-tethered GFP (green). (C) Quantification of GFP+ axonal material shown in B. N ≥ 18 for each genotype and condition. (D) Confocal projections of OR85e axons in control, InR^RNAi or dominant negative InR (dnInR)-expressing flies. White arrowheads point to persistent axonal debris post-injury. (E) Quantification of GFP shown in B. N ≥ 22 for each genotype and condition. ****p < 0.0001. Pooled data plotted as mean ± SEM. All image scale bars represent 20 μm. Genotypes: Control (OR85e-mCD8::GFP, tub-Gal80^ts/+; repo-Gal4/+), dnInR (OR85e-mCD8::GFP, tub-Gal80^ts/+; repo-Gal4, InR^ex15/UAS-Dominant Negative InR), InR^RNAi (OR85e-mCD8::GFP, tub-Gal80^ts/+; repo-Gal4, InR^ex15/UAS-InR^RNAi), Akt^RNAi (OR85e-mCD8::GFP, tub-Gal80^ts/+; repo-Gal4, InR^ex15/UAS-Akt^RNAi). See Figure S1.

Figure 2. InR and Akt1 are acutely activated in local glia responding to axon injury

(A) Single confocal slice images of the antennal lobe region in uninjured, antennal nerve axotomized and maxillary nerve axotomized animals. Phospho-InR signal (magenta) overlaps with GFP-labeled glial membranes (green) expanding after antennal nerve axotomy (white arrowheads) or accumulating on severed maxillary glomeruli post-maxillary nerve axotomy (white arrowheads). Insets: magnified view of outlined area (yellow rectangle). (B) Phospho-InR signal in the dorsal half of antennal lobes was quantified after computationally segmenting to GFP (antennal injury in A). N ≥ 20 for each condition. (C) Quantification of phospho-InR signal in OR85e-innervated glomeruli in A (maxillary injury). N ≥ 16 for each condition. (D) Single confocal slice images of antennal lobes. Phospho-Akt (magenta) intensity is increased in ensheathing glia (green) responding to antennal (white arrowheads, middle panels) or maxillary nerve injury (lower panels). Insets: magnification of yellow boxed areas. (E) Quantification of phospho-Akt signal in dorsal antennal lobes after computationally segmenting to GFP. N ≥ 18 for each condition. (F) Quantification of phospho-Akt signal in OR85e-innervated glomeruli in D (bottom panels). N ≥ 20 for each condition. **p<0.01, ***p<0.001, ****p < 0.0001. Pooled data plotted as mean ± SEM. All image scale bars represent 20 μm. Genotypes: UAS-mCD8::GFP, repo-Gal4/+. See Figure S2.

Figure 3. Glial InR signaling positively regulates Draper expression and Draper recruitment to severed axons

(A) Western blot of fly brain lysates. (B) Quantification of normalized Draper in A. (C) Representative images of adult antennal lobes. Brains stained with Draper (magenta) and GFP to visualize OR85e axons (green). Robust Draper accumulation typically observed post-injury (white arrowheads) is attenuated following glial InR^RNAi expression. (D) Volumetric quantification of Draper fluorescence intensity on injured OR85e axons (arrowheads) in C. (E) Q-PCR analysis of draper-I transcript in central brains. 2^−ΔΔCt ± SEM values are plotted. N= 6 independent samples of pooled animals for each time point and genotype. (F) Images of single antennal lobes (dotted line) show activation of of 10XSTAT92E-dGFP (destabilized GFP) reporter 18 hours after antennal nerve axotomy in control animals and glial Akt^RNAi or InR^RNAi -expressing flies. (G) Antennal lobe region of uninjured brains (single confocal slice) and 1 day post axotomy (maximum intensity projection) immunostained for Draper. (H) Volumetric quantification of basal Draper levels in the cortex region of uninjured flies (ROI shown with white dotted circle in panel G). (I) Volumetric quantification of Draper accumulation on injured maxillary palp glomeruli (see arrowheads in G). (J) Glial overexpression (OE) of Draper-I reverses glial clearance defects in InR-depleted animals. Representative Z-stack projections of OR85e-innervated glomeruli show GFP+ OR85e axons. (K) Quantification of GFP in J. (L) Images of each experimental genotype shown in J immunostained for Draper. (M) OR85e axons (green) before and after axotomy. (N) Quantification of GFP⁺ OR85e axonal debris in M. Genotypes in A–E: control (OR85e-mCD8::GFP, tub-Gal80^ts/+; repo-Gal4/+), dnInR (OR85e-mCD8::GFP, tub-Gal80^ts/+; repo-Gal4, InR^ex15/UAS-Dominant Negative InR), InR^RNAi (OR85e-mCD8::GFP, tub-Gal80^ts/+; repo-Gal4, InR^ex15/UAS-InR^RNAi). F: control (10XSTAT92E-dGFP/+; repo-Gal4/+), Akt^RNAi (10XSTAT92E-dGFP/+; repo-Gal4/UAS-Akt^RNAi), InR^RNAi (10XSTAT92E-dGFP/+; repo-Gal4/UAS-InR^RNAi). G–I: control (+/tub-Gal80^ts/+; repo-Gal4), caInR (UAS-caInR/ tub-Gal80^ts/+; repo-Gal4). J–L: control, InR^RNAi (OR85e-mCD8::GFP, tub-Gal80^ts/UAS-LacZ; repo-Gal4, InR^ex15/UAS- InR^RNAi), InR^RNAi + draper-I OE (OR85e-mCD8::GFP, UAS-Draper-I/tub-Gal80^ts; repo-Gal4, InR^ex15/UAS- InR^RNAi). M, N: control (OR85e-mCD8::GFP/+; repo-Gal4/+). caInR (OR85e-mCD8::GFP/UAS-LacZ::NLS, UAS-caInR; repo-Gal4/+). caInR+draperRNAi (OR85e-mCD8::GFP/UAS-draperRNAi, UAS-caInR; repo-Gal4/+). *p < 0.05; ***p < 0.001. ****p< 0.0001. All pooled data are represented as mean ± SEM. All scale bars represent 20 μm. N ≥ 18 for each condition. See Figure S3 and S4.

Figure 4. InR is required in ensheathing glia, not astrocytes, for proper clearance of degenerating axonal debris

(A) Representative confocal images of single antennal lobes. Brains immunostained with anti-phospho-InR (magenta) reveal little overlap between increased InR activity (white arrowheads) and astrocyte membranes labeled with mCD8::GFP (green). (B) Zoomed image of region outlined in A (white box). (C) Representative confocal images of single antennal lobes. Brains immunostained with anti-phospho-InR (magenta) reveal overlap between increased InR activity (white arrowheads) and ensheathing glial membranes labeled with mCD8::GFP (green). (D) Zoomed image of region outlined in C (white box). (E) Maximum intensity projections of OR85e axons. (F) Quantification of GFP⁺ OR85e axonal debris in E. **p < 0.01. N ≥ 16 for each condition. Scale bar =20 μm. Pooled data plotted as mean ± SEM. Genotypes: A, B: (UAS-mCD8::GFP/+; alrm-Gal4/+). C, D: (UAS-mCD8::GFP/+; TIFR-Gal4/+), InR^RNAi. E, F: control (OR85e-mCD8::GFP, tub-Gal80^ts/+; InR^ex15/+), ensheathing dnInR (OR85e-mCD8::GFP, tub-Gal80^ts/+; TIFR-Gal4, InR^ex15/UAS-Dominant Negative InR), astrocyte dnInR (OR85e-mCD8::GFP, tub-Gal80^ts/+; alrm-Gal4, InR^ex15/UAS-Dominant Negative InR).

Figure 5. Knockdown of PC2 or Cadps in olfactory neurons attenuates phospho-InR activation and Draper upregulation in ensheathing glia

(A) GFP-tagged atrial natriuretic factor (ANF::GFP) neuropeptide distribution along uninjured OR22a axons (white arrows). Grayscale converted confocal images (red represents maximum intensity) are shown. Fewer ANF::GFP+ puncta (65%) are observed 30 minutes after antennal nerve axotomy. Synaptic rich OR22a glomeruli are highlighted with brackets. Genotype: y, w, UAS-ANF::GFP/+; OR22a-Gal4/+; N>15 brains for each condition. Scale bar =20 microns. (B) PCR analysis of ilp1–7 ligands in w¹¹¹⁸ adult third antennal segments. +/− correspond to + and − RT samples. L= molecular ladder. (C, E) Quantification of phosphor-InR in dorsal antennal lobe ensheathing glia 18 hrs after antennal nerve axotomy. N ≥ 18 for each condition. (D, F) Quantification of Draper levels in dorsal antennal lobe ensheathing glia 18 hrs after antennal nerve axotomy. N ≥ 21 for each condition. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Pooled data plotted as mean ± SEM. Genotypes in B–E: control (UAS-dicer2/+; orco-Gal4/+), PC2 RNAi #1 (UAS-dicer2/UAS-PC2^RNAi VDRC 110788; orco-Gal4/+); PC2 RNAi #2 (UAS-dicer2/+; orco-Gal4/UAS-PC2^RNAi Bl28583); Cadps^RNAi (UAS-dicer2/UAS-Cadps^RNAi VDRC 110055; orco-Gal4/+).