Research ArticleAlzheimer’s Disease

Gga3 deletion and a GGA3 rare variant associated with late onset Alzheimer’s disease trigger BACE1 accumulation in axonal swellings

See allHide authors and affiliations

Science Translational Medicine  18 Nov 2020:
Vol. 12, Issue 570, eaba1871
DOI: 10.1126/scitranslmed.aba1871
  • Fig. 1 GGA3 is sorted to both dendrites and axon.

    (A) Representative neuron expressing GFP-GGA3 (green, grayscale inverted left) immunostained for MAP2B (red). Arrowheads point to the axon. Stitched confocal z-stacks. Magnification, ×63 oil. Scale bar, 100 μm. (B) Representative neuron expressing AP1(μ1A)-GFP (grayscale inverted). Vector-mCherry (red) and MAP2B (blue). Arrowheads point to the axon. Stitched confocal z-stacks. Magnification, ×40 oil. Scale bar, 50 μm. (C) PI analysis: GFP-GGA3 (n = 18) and AP1(μ1A)-GFP (n = 31). D/A, Dendrites/Axon. (D) Top left: DIV10 neurons grown in a microfluidics device stained for MAP2B (red), NFH (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Stitched confocal z-stacks. Magnification, ×20 oil. Scale bar, 50 μm. Bottom left and right: Western blot of cell and axonal proteins probed for MAP2B, TubβIII, and GGA3.

  • Fig. 2 BACE1 and GGA3 are cotransported in axons.

    (A) BACE1-mCherry (red) and GFP-GGA3 (green) colocalization analysis in wild-type neurons. Z-stack confocal images. Magnification, ×63 oil. Scale bar, 100 μm. Manders’ coefficient, n = 8. Single frames from movie S1 (A and B) [top; total internal reflection fluorescence (TIRF) microscope, ×60 oil, 2 fps] and corresponding kymographs (bottom) of (B) GFP-GGA3- (C) and BACE1-mCherry–positive vesicles (nv = 397 and 897) moving for 3 min along 100-μm axons in wild-type neurons (n = 14 and 15). In kymographs, lines with negative/positive slopes represent particles moving anterograde/retrograde. Vertical lines represent stationary vesicles. Percentage of anterograde (Antero), retrograde (Retro), and stationary (Stat) particles were calculated, along with the time spent by the vesicles moving/pausing and their velocity/run length. For percentage of frequency and time (%), values are means ± SEM of the number of neurons analyzed (n). Statistical analysis: one-way analysis of variance (ANOVA), Tukey’s post hoc test. For velocity/run length, only moving vesicles were analyzed. Black line, median of the number of vesicles analyzed (nv). Statistical analysis: two-tailed Mann-Whitney test. (D) Single frame from movie S1C (top; TIRF microscope, ×60 oil, 2 fps) and kymograph (bottom) of a neuron coexpressing GFP-GGA3 and BACE1-mCherry. (E) Analysis of the frequency distribution of particle velocities, n = 13, nv = 195. Only moving vesicles were analyzed. Statistical analysis: chi-square test. *P < 0.05, **P ≤ 0.01, and ****P ≤ 0.0001.

  • Fig. 3 Gga3 deletion induces BACE1 axonal accumulation.

    (A) Representative images of BACE1-mCherry in Gga3+/+ and Gga3−/− neurons. Arrowheads indicate axons. PI analysis, n = 19. Stitched confocal z-stacks. Magnification, ×63 oil. Scale bar, 50 μm. (B) Gga3+/+ and Gga3−/− neurons expressing synaptophysin (Syn)–GFP (green, grayscale inverted left). MAP2B in blue. Arrowheads indicate axons. Stitched confocal z-stacks. Magnification, ×40 oil. Scale bar, 50 μm. PI summary table. Representative images are in fig. S2. (C) BACE1 (green, D10E5) and DAPI staining (blue). Mean gray intensity fluorescence analysis: Gga3+/+, n = 5 mice; Gga3−/−, n = 6 mice. At least two coronal sections per animal (four hippocampi) were analyzed. Stitched epifluorescent z-stacks. Magnification, ×20. Scale bar, 250 μm. Statistical analysis: two-tailed unpaired t test. ***P ≤ 0.001 and ****P ≤ 0.0001.

  • Fig. 4 Lack of BACE1 axonal motility in Gga3−/− neurons.

    (A) Single frames from movie S3A (top; TIRF microscope, ×60 oil, 2 fps) and corresponding kymographs (bottom) of BACE1-mCherry axonal particles in Gga3+/+ (n = 15) and Gga3−/− (n = 17) neurons. Syn-GFP motility (B) (TIRF microscope, ×60 oil, 2 fps): Gga3+/+ and Gga3−/− (n = 13) neurons (movie S3B). (C) Coexpression of BACE1-mCherry (red) and Syn-GFP (green; TIRF microscope, ×60 oil, 2 fps) in Gga3−/− (n = 13) and Gga3+/+ (n = 12) neurons (movie S3C). Statistical analysis: two-way ANOVA, Bonferroni’s multiple comparison test. ***P ≤ 0.001 and ****P ≤ 0.0001.

  • Fig. 5 BACE1 accumulates in axonal swellings in Gga3−/− neurons.

    (A) Single frame images from movie S4A showing Gga3−/− BACE1-positive axonal swellings (arrowheads). Neurons: Gga3−/−, n = 30; Gga3+/+, n = 35. Statistical analysis: two-tailed unpaired t test; two-way ANOVA, Bonferroni’s multiple comparison test. (B) Syn-GFP expression in Gga3+/+ (n = 25) and Gga3−/− (n = 21) axons (movie S4B). BACE1 and synaptophysin coexpression in Gga3−/− (n = 20) and Gga3+/+ (n = 21) neurons. Arrowheads indicate spheroids. Statistical analysis: one-way ANOVA, Tukey’s post hoc test; two-way ANOVA, Bonferroni’s multiple comparison test. (A and B) Pseudo-colored, TIRF microscope, ×60 oil. Scale bars, 10 μm. Insets are consecutive frames from corresponding movie. Axonal swelling linear density and frequency of neurons with/without swellings were calculated. (C) Representative images of cerebella from Gga3+/+ and Gga3−/− mice stained for calbindin. PcL, Purkinje cell; ML, molecular layer; IGL, internal granular layer. Purkinje cells axonal spheroids in Gga3−/− neurons (insets, arrowheads). Confocal z-stacks. Magnification, ×40 oil. Scale bars, 100 μm. Gga3−/−, n = 6 mice; Gga3+/+, n = 7 mice. Three sections per mouse were analyzed. (D) Image adapted from Allen Mouse Brain Connectivity Atlas: GFP-positive afferents from the medial entorhinal cortex to CA1 stratum lacunosum-moleculare (Slm). Axonal spheroids (arrowheads) in Gga3−/− mice (Bielschowsky’s stain). Bright-field images. Magnification, ×20. Scale bar, 100 μm. n = 6. At least two sections per mouse were analyzed (four hippocampi). (C and D) Statistical analysis: two-tailed unpaired t test. ***P ≤ 0.001 and ****P ≤ 0.0001.

  • Fig. 6 β-Secretase and γ-secretase inhibition prevents axonal dystrophies in Gga3−/− neurons.

    (A) Representative axonal pseudo-colored images from live-cell imaging movies. Analyzed conditions: Gga3+/+ (n = 29), Gga3−/− (n = 24), Gga3−/−_GFP-GGA3 (n = 27), Gga3−/−_C3 (n = 26), and Gga3−/−_DAPT (n = 32). TIRF microscope, ×60 oil. Scale bar, 10 μm. Statistical analysis: one-way ANOVA, Tukey’s post hoc test; two-way ANOVA, Bonferroni’s multiple comparison test. (B) Single frames from movie S5 (top; TIRF microscope, ×60 oil, 2 fps) and corresponding kymographs (bottom) of BACE1-mCherry particles. Analyzed conditions: Gga3+/+ (n = 19), Gga3−/− (n = 19), Gga3−/−_GFP-GGA3 (n = 11), Gga3−/−_C3 (n = 20), and Gga3−/−_DAPT (n = 23). Table reports statistical analysis/significance: two-way ANOVA, Bonferroni’s multiple comparison test. Analysis of dystrophic phenotype after pharmacological inhibition of β-secretase in the cerebellum (C) and hippocampal CA1 (D). Number of mice analyzed is indicated in the graphs. Representative images in fig. S6. Statistical analysis: one-way ANOVA, Tukey’s post hoc test. n.s. indicates P > 0.05, *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

  • Fig. 7 An AD-linked GGA3 rare insertion results in loss of function.

    (A) Schematic of human GGA3 domains. Hinge domain sequence alignment of human (NM_138619-3) wild-type and mutated (Ins545T) GGA3. (B) Neuron expressing GFP-Ins545T (green, grayscale inverted left) and stained for MAP2B (red). Arrowheads point to the axon. Confocal z-stacks. Magnification, ×40 oil. Scale bar, 100 μm. (C) Representative axonal pseudo-colored images from live-cell imaging movies. BACE1 axonal swelling phenotype was analyzed under different conditions: Gga3−/− (n = 24), Gga3−/−_GFP-GGA3 (n = 28), Gga3−/−_GFP-Ins545T (n = 31), and Gga3−/−_GFP-Ins545T_C3 (n = 28). TIRF microscope, ×60 oil. Statistical analysis: one-way ANOVA, Tukey’s post hoc test; two-way ANOVA, Bonferroni’s multiple comparison test. (D) Single frames from movie S6 (top; TIRF microscope, ×60 oil, 2 fps) and corresponding kymographs (bottom) of BACE1-mCherry particles. Analyzed conditions: Gga3−/− (n = 19), Gga3−/−_GFP-GGA3 (n = 11), Gga3−/−_GFP-Ins545T (n = 19), and Gga3−/−_GFP-Ins545T_C3 (n = 13). Table reports statistical analysis/significance: two-way ANOVA, Bonferroni’s multiple comparison test. n.s. indicates P > 0.05, *P < 0.05, **P ≤ 0.01, and ****P ≤ 0.0001.

  • Fig. 8 Gga3 deletion exacerbates axonal pathology in 5XFAD brains and induces endogenous BACE1 accumulation in axonal swellings.

    (A) Representative images of the somatosensory cortex from 5X/Gga3+/+ and 5X/Gga3−/− mice (Bielschowsky’s stain). White boxes highlight axonal swellings. Bright-field images. Magnification, ×40. Scale bar, 10 μm. 5X/Gga3+/+, n = 7 mice; 5X/Gga3−/−, n = 9 mice. At least two sections per mouse were analyzed. Statistical analysis: two-tailed unpaired t test. (B) IF staining for NFH (purple) and BACE1 3D5 (green) in 5X/Gga3−/− mice. DAPI in blue. Confocal z-stacks. Magnification, ×63 oil. Scale bar, 10 μm. ****P ≤ 0.0001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/570/eaba1871/DC1

    Materials and Methods

    Fig. S1. Representative kymographs for BACE1-mCherry and GFP-GGA3 cotransport experiments in Fig. 2D.

    Fig. S2. Representative images for PI analysis in Fig. 3B.

    Fig. S3. Colocalization analysis for BACE1-mCherry with EEA1 and LAMP2.

    Fig. S4. Representative image for BACE1-mCherry and LAMP2 in Gga3−/− axonal swelling.

    Fig. S5. NFH staining of hippocampal CA1 axonal bundles and BACE1 accumulation in the cerebellum and hippocampus of Gga3−/− mice.

    Fig. S6. Images for BACE1-mcherry and GFP-GGA3 channels corresponding to axon in Fig. 6A.

    Fig. S7. Supplementary data and analysis related to BACE inhibition experiments.

    Fig. S8. Representative images of cerebella and hippocampi from MBi-3–treated mice.

    Fig. S9. Images for BACE1-mcherry and GFP-GGA3 channels corresponding to axons in Fig. 7C.

    Table S1. [MBi-3] micromolars—Pharmacokinetics (PK) analysis summary.

    Table S2. Genetics dataset description.

    Table S3. Selected single variants, showing nominal association with AD in one of the datasets.

    Table S4. Summary of the antibodies used throughout this study.

    Movie S1. BACE1-mCherry and GFP-GGA3 axonal trafficking in wild-type neurons.

    Movie S2. BACE1-mCherry and synaptophysin-GFP axonal trafficking in Gga3+/+ and Gga3−/− neurons.

    Movie S3. Three-dimensional reconstruction of BACE1-mCherry and LAMP2 in Gga3−/− axonal swelling.

    Movie S4. BACE1-mCherry and synaptophysin-GFP accumulation in axonal swellings in Gga3−/− neurons.

    Movie S5. Representative movies for BACE1-mCherry trafficking experiments in Fig. 6.

    Movie S6. Representative movies for BACE1-mCherry trafficking experiments in Fig. 7.

    Data file S1. Individual-level data file n < 20 (provided as separate Excel file).

    References (6064)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Representative kymographs for BACE1-mCherry and GFP-GGA3 cotransport experiments in Fig. 2D.
    • Fig. S2. Representative images for PI analysis in Fig. 3B.
    • Fig. S3. Colocalization analysis for BACE1-mCherry with EEA1 and LAMP2.
    • Fig. S4. Representative image for BACE1-mCherry and LAMP2 in Gga3−/− axonal swelling.
    • Fig. S5. NFH staining of hippocampal CA1 axonal bundles and BACE1 accumulation in the cerebellum and hippocampus of Gga3−/− mice.
    • Fig. S6. Images for BACE1-mcherry and GFP-GGA3 channels corresponding to axon in Fig. 6A.
    • Fig. S7. Supplementary data and analysis related to BACE inhibition experiments.
    • Fig. S8. Representative images of cerebella and hippocampi from MBi-3–treated mice.
    • Fig. S9. Images for BACE1-mcherry and GFP-GGA3 channels corresponding to axons in Fig. 7C.
    • Table S1. [MBi-3] micromolars—Pharmacokinetics (PK) analysis summary.
    • Table S2. Genetics dataset description.
    • Table S3. Selected single variants, showing nominal association with AD in one of the datasets.
    • Table S4. Summary of the antibodies used throughout this study.
    • Legend for data file S1
    • References (6064)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). BACE1-mCherry and GFP-GGA3 axonal trafficking in wild-type neurons.
    • Movie S2 (.mp4 format). BACE1-mCherry and synaptophysin-GFP axonal trafficking in Gga3+/+ and Gga3−/− neurons.
    • Movie S3 (.mov format). Three-dimensional reconstruction of BACE1-mCherry and LAMP2 in Gga3−/− axonal swelling.
    • Movie S4 (.mp4 format). BACE1-mCherry and synaptophysin-GFP accumulation in axonal swellings in Gga3−/− neurons.
    • Movie S5 (.mp4 format). Representative movies for BACE1-mCherry trafficking experiments in Fig. 6.
    • Movie S6 (.mp4 format). Representative movies for BACE1-mCherry trafficking experiments in Fig. 7.
    • Data file S1. Individual-level data file n < 20 (provided as separate Excel file).

Stay Connected to Science Translational Medicine

Navigate This Article