Progress of endocytic CHRN to autophagic degradation is regulated by RAB5-GTPase and T145 phosphorylation of SH3GLB1 at mouse neuromuscular junctions in vivo

Abstract

Endocytosed nicotinic acetylcholine receptors (CHRN) are degraded via macroautophagy/autophagy during atrophic conditions and are accompanied by the autophagic regulator protein SH3GLB1. The present study addressed the functional role of SH3GLB1 on CHRN trafficking and its implementation. We found an augmented ratio of total SH3GLB1 to threonine-145 phosphorylated SH3GLB1 (SH3GLB1:p-SH3GLB1) under conditions of increased CHRN vesicle numbers. Overexpression of T145 phosphomimetic (T145E) and phosphodeficient (T145A) mutants of SH3GLB1, was found to either slow down or augment the processing of endocytic CHRN vesicles, respectively. Co-expression of the early endosomal orchestrator RAB5 largely rescued the slow processing of endocytic CHRN vesicles induced by T145E. SH3GLB1 phosphomutants did not modulate the expression or colocalization of RAB5 with CHRN vesicles, but instead altered the expression of RAB5 activity regulators. In summary, these findings suggest that SH3GLB1 controls CHRN endocytic trafficking in a phosphorylation- and RAB5-dependent manner at steps upstream of autophagosome formation.

Keywords: AChR; Bif-1; NMJ; RAB5; SH3GLB1; acetylcholine receptor; endophilin B1; neuromuscular junction; skeletal muscle; synapse.

Figure 1.

Denervation induces accumulation of endocytic CHRN vesicles that is further augmented in the presence of chloroquine. TA muscles from innervated (inn) and 4-d denervated (den) legs were injected with BGT-AF647 to stain surface-exposed CHRN. One d later, muscles were imaged in situ using confocal microscopy to observe surface-exposed (on NMJs, large pretzel-like structures) and endocytosed (on vesicles, small puncta) CHRNs. Animals were either treated with saline or chloroquine for 5 d prior to imaging. (A-D) Representative maximum-z projections displaying individual NMJs and their corresponding internalized CHRN vesicles. Scale bar: 20 µm. (E) Quantification of CHRN puncta per NMJ. Given is mean ± SEM (n = 3 muscles for each condition; * P< 0.05; ** P < 0.01; a total of 8874 puncta was analyzed). Statistical significance was probed using ANOVA.

Figure 2.

Denervation or chloroquine augment the ratio of total SH3GLB1 to T145-phosphorylated SH3GLB1 as well as levels of RAB5. Mice were unilaterally denervated and daily injected with saline or chloroquine for 5 d. TA muscles from innervated (inn) and denervated (den) legs were then homogenized and equal amounts of protein subjected to western blot. (A) Displayed is a representative blot showing the relative amounts of total SH3GLB1, SH3GLB1 phosphorylated at position T145 (p-SH3GLB1), RAB5, and MAP1LC3B. GAPDH served as the loading control. (B-D) Quantitative analysis of the relative band intensities for SH3GLB1:p-SH3GLB1 ratio (B), RAB5 (C), and MAP1LC3B-II (D). All values were normalized to the internal GAPDH loading controls. Shown are mean ± SEM of values obtained from 3 independent experiments. Statistical significance was probed using ANOVA, * P< 0.05; ** P < 0.01.

Figure 3.

The number of internalized CHRN puncta is differently affected by phosphomutants of SH3GLB1 but not in the presence of chloroquine. TA muscles were transfected as indicated with SH3GLB1^T145A or SH3GLB1^T145E mutants fused with mCherry. After 9 d, CHRN were labeled using BGT-AF647. Another day later, muscles were imaged with confocal in vivo microscopy. During the 5 d before microscopy, mice either received saline (A) or chloroquine (B). (A-B) Three left panel columns show maximum-z projections of representative fluorescence signals of BGT-AF647 (CHRN), SH3GLB1 phosphomutants fused with mCherry (T145A or T145E), and overlays of both (overlay). The right panel column depicts zoom images of single optical layers of the boxed regions in the overlay images. CHRN panels are shown without contrast enhancement. Overlay zoom panels were contrast enhanced to better visualize CHRN-positive vesicles. In overlay and overlay zoom panels BGT-AF647 and mCherry signals are displayed in white/gray and red, respectively. Scale bars: 20 µm. (C) Quantitative analysis of CHRN puncta per NMJ. Shown are mean ± SEM (n = 3 muscles for each condition without chloroquine, n = 4 muscles for T145A + chloroquine, n = 6 muscles for T145E + chloroquine; Statistical significance was probed using ANOVA; * P< 0.05, ** P < 0.01; a total of 33,443 puncta was analyzed).

Figure 4.

CHRN interacts with RAB5, and CHRN endocytosis is a RAB5-dependent process. (A) TA muscles were transfected with RAB5-GFP. After 10 d, CHRN were labeled using BGT-AF647. Confocal in vivo microscopy commenced 1 h later. Upper panels depict maximum-z projections of representative fluorescence signals of BGT-AF647 (CHRN), RAB5-GFP (RAB5), and overlays of both (overlay). Lower panels show a detail of a single optical slice of the boxed region in the upper panels focusing on puncta positive for internalized CHRN and RAB5-GFP. In overlay panels, BGT-AF647 and RAB5-GFP signals are displayed in white/gray and green, respectively. Green and red arrowheads indicate CHRN puncta colocalizing or not colocalizing with RAB5-GFP, respectively. To better visualize CHRN-positive vesicles, zoom panels were contrast enhanced. Scale bar: 20 µm. (B) Untransfected gastrocnemius muscles were injected with BGT-biotin. Five h later, muscles were harvested, homogenized and CHRN affinity precipitated with Neutravidin beads. Western blot signals upon exposure to primary antibodies against ADRB2 (negative control), RAB5, and CHRNA1 are shown. (C-E) TA muscles were transfected with RAB5 mutants S34N (C) or Q79L (D), both fused to GFP. Nine days later, CHRN were labeled using BGT-AF647. Twenty-four h later, confocal in vivo microscopy was performed. Upper panels depict maximum-z projections of representative fluorescence signals of BGT-AF647 (CHRN), RAB5 mutants fused to GFP (RAB5^S34N and RAB5^Q79L), and overlays of both (overlay). Lower panels show details of single optical slices of the boxed regions in the upper panels. In overlay panels, BGT-AF647 and GFP signals are displayed in white/gray and green, respectively. Green and red arrowheads indicate CHRN puncta colocalizing or not colocalizing with RAB5-GFP, respectively. To better visualize CHRN-positive vesicles, zoom and overlay panels were contrast enhanced. Scale bar: 20 µm. (E-F) Quantitative analysis of the number (E) and colocalization with GFP signals (F) of CHRN-positive puncta. Depicted are mean ± SEM (n = 5 muscles for RAB5^S34N, n = 3 muscles for RAB5^Q79L; statistical analysis employed ANOVA; ** P < 0.01).

Figure 5.

Overexpression of RAB5-GFP reduces the number of internalized CHRN puncta in the presence of T145E in a chloroquine-dependent manner. TA muscles were cotransfected with RAB5-GFP and SH3GLB1^T145A (A and C) or SH3GLB1^T145E (B and D) mutants coupled to mCherry. Nine d after transfection, CHRN were labeled using BGT-AF647. Another day later, muscles were imaged with confocal in vivo microscopy. During the 5 d before microscopy, mice either received saline (A, B) or chloroquine (C, D). (A-D) Upper panels show representative NMJs and their CHRN puncta (CHRN), mCherry (T145A or T145E), and GFP (RAB5) signals, as indicated. Lower panels depict details of single optical layers from the boxed regions of upper panels. In the overlay pictures, mCherry, RAB5-GFP, and CHRN signals are shown in red, green, and gray/white, respectively. CHRN, mCherry, and RAB5-GFP triple-positive puncta are indicated with green arrowheads. CHRN and mCherry puncta not positive for RAB5-GFP are indicated with red arrowheads. Scale bars: 20 µm. (E) Quantitative analysis of CHRN puncta per NMJ. Mean ± SEM (n = 11 muscles for T145A and n = 8 muscles for T145E in the absence of chloroquine; n = 4 muscles each for T145A and T145E in the presence of chloroquine; statistical analysis employed ANOVA; ** P < 0.01; a total of 38,808 puncta was analyzed). (F) Colocalization analysis of RAB5-GFP-positive CHRN puncta. Mean ± SEM (n-values as in [E]; statistical analysis employed ANOVA; ** P < 0.01).