Inhibition of Lysosome Membrane Recycling Causes Accumulation of Gangliosides that Contribute to Neurodegeneration

Maxime Boutry, Julien Branchu, Céline Lustremant, Claire Pujol, Julie Pernelle, Raphaël Matusiak, Alexandre Seyer, Marion Poirel, Emeline Chu-Van, Alexandre Pierga, Kostantin Dobrenis, Jean-Philippe Puech, Catherine Caillaud, Alexandra Durr, Alexis Brice, Benoit Colsch, Fanny Mochel, Khalid Hamid El Hachimi, Giovanni Stevanin, Frédéric Darios, Maxime Boutry, Julien Branchu, Céline Lustremant, Claire Pujol, Julie Pernelle, Raphaël Matusiak, Alexandre Seyer, Marion Poirel, Emeline Chu-Van, Alexandre Pierga, Kostantin Dobrenis, Jean-Philippe Puech, Catherine Caillaud, Alexandra Durr, Alexis Brice, Benoit Colsch, Fanny Mochel, Khalid Hamid El Hachimi, Giovanni Stevanin, Frédéric Darios

Abstract

Lysosome membrane recycling occurs at the end of the autophagic pathway and requires proteins that are mostly encoded by genes mutated in neurodegenerative diseases. However, its implication in neuronal death is still unclear. Here, we show that spatacsin, which is required for lysosome recycling and whose loss of function leads to hereditary spastic paraplegia 11 (SPG11), promotes clearance of gangliosides from lysosomes in mouse and human SPG11 models. We demonstrate that spatacsin acts downstream of clathrin and recruits dynamin to allow lysosome membrane recycling and clearance of gangliosides from lysosomes. Gangliosides contributed to the accumulation of autophagy markers in lysosomes and to neuronal death. In contrast, decreasing ganglioside synthesis prevented neurodegeneration and improved motor phenotype in a SPG11 zebrafish model. Our work reveals how inhibition of lysosome membrane recycling leads to the deleterious accumulation of gangliosides, linking lysosome recycling to neurodegeneration.

Keywords: autophagic lysosome recovery; autophagy; glycosphingolipids; induced pluripotent stem cells; knockout; lipid metabolism; lysosomes; membrane trafficking; neurodegenerative disease; organoids.

Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Spatacsin Loss Promotes Lysosomal Accumulation of Gangliosides in Neurons of the Cortex (A and B) GM2 (green) and Lamp1 (magenta) immunostaining of Spg11+/+ and Spg11−/− neurons of the layer V of the motor cortex from 6-week-old and 8-month-old mice observed by confocal microscopy with a 60× (A) or 20× (B) objective. Images showing the accumulation of GM2-positive staining in lysosomes surrounded by Lamp1 staining in 6-week-old and 8-month-old Spg11−/− animals. Insets show the view along the z axis (A). Scale bars: 10 μm (A) and 50 μm (B). (C–F) Quantification of the mean of the GM2 (C), GM3 (D), GD2 (E), and GD3 (F) immunostaining intensity per neuron. Quantification was performed in neurons of the layer V of motor cortex that were detected by their large soma. The graphs show the mean ± SEM values. N = 10–15 neurons quantified per cortex slices in five cortex slices of five independent mice. Differences between Spg11+/+ and Spg11−/− were analyzed by a Kruskal-Wallis test at each time point; ∗ p ≤ 0.05, ∗∗∗p ≤ 0.0001. See also Table S1 and Figures S1 and S2.
Figure 2
Figure 2
Spatacsin Loss Promotes Lysosomal Accumulation of Gangliosides in Neurons Derived from SPG11 Patients (A) Immunostaining of brain organoids differentiated for 90 days in vitro with antibodies against the progenitor marker Pax6 and the neuron-specific marker βIII-tubulin. Note that neuronal cells are concentrated at the periphery of the organoids. Scale bar: 50 μm. (B) GM2 (green) and Lamp1 (magenta) immunostaining in the neuronal layer of organoids derived from healthy subjects or SPG11 patients. βIII-Tubulin (yellow) shows the neuronal identity of the cells that were analyzed. Confocal microscopy images showing the accumulation of GM2-positive staining in lysosomes labeled by Lamp1 staining of organoids derived from SPG11 patients (arrowheads). Scale bar: 10 μm. (C–F) Quantification of the mean of the GM2 (C), GM3 (D), GD2 (E), and GD3 (F) immunostaining intensity per neuron. The graph shows mean ± SEM values. N = 10–15 neurons quantified per slices in five slices obtained from three independent organoids. One-way ANOVA; ∗∗∗p ≤ 0.001. See also Figure S3.
Figure 3
Figure 3
Spatacsin Loss Induces Lysosomal Accumulation of Gangliosides in Primary Cultures of Mouse Cortical Neurons (A) GM2 (green) and Lamp1 (magenta) immunostaining of Spg11+/+ and Spg11−/− neurons cultured for 6 days in vitro. Confocal microscopy images showing the accumulation of GM2-positive staining of lysosomes labeled by Lamp1 staining. Scale bar: 10 μm. (B–E) Quantification of the proportion of GM2 (B), GM3 (C), GD2 (D), and GD3 (E) staining that is localized in lysosomes. The graphs show the mean ± SEM values. N = 58–71 neurons quantified in three independent neuron preparations. t test; ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001. (F) Immunostaining of GM2 ganglioside (green), Lamp1 (cyan), and Spatacsin-V5 (magenta) in wild-type neurons co-transfected with vectors allowing expression of Spatacsin-V5 and either control miRNA or miRNA downregulating Neu1. Spatacsin is enriched around GM2-positive vesicles (arrowheads). Scale bar: 10 μm. (G) Quantification of the number of lysosomes detected with Lamp1 immunostaining and with a diameter larger than 1 μm in Spg11+/+ and Spg11−/− neurons cultured for 6 days in vitro and treated or not with miglustat (100 μM). The graphs show the mean ± SEM values. N = 62–64 neurons quantified in four independent neuron preparations (control); N = 24–34 neurons quantified in three independent neuron preparations (miglustat). One-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p ≤ 0.001. See also Figure S4.
Figure 4
Figure 4
Spatacsin Promotes Clearance of Gangliosides from Lysosomes (A) Quantification of the number of lysosomes with a diameter larger than 1 μm in Spg11+/+ and Spg11−/− neurons expressing control miRNA or miRNA downregulating clathrin heavy chain (CHC). The graphs show the mean ± SEM values. N = 35–50 neurons quantified in two independent neuron preparations. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗p = 0.03 and ∗∗∗p ≤ 0.001. (B) Quantification of the proportion of GM2 staining that is localized in lysosomes. The graphs show the mean ± SEM values. N = 36–49 neurons quantified in two independent neuron preparations. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗p = 0.0017. (C) Immunostaining of Spg11−/− neurons with Lamp1 (cyan), GM2 (yellow), and clathrin (magenta) antibodies. Arrowheads point to lysosomes positive for the GM2 marker that also show recruitment of clathrin. Scale bar: 5 μm. (D) Quantification of the proportion of clathrin colocalized with GM2-positive vesicles. The graphs show the mean ± SEM values. N = 88 (Spg11+/+) and 102 (Spg11−/−) neurons quantified in three independent neuron preparations. t test; ∗∗p = 0.0023. (E) Confocal microscopy images of autofluorescence (green), Lamp1 (magenta), and clathrin (cyan) immunostaining of Spg11+/+ and Spg11−/− cortical motor neurons from 8-month-old animals showing the accumulation of clathrin surrounding autofluorescent lysosomes. Scale bars: 10 μm. (F) Western blot showing the interaction of GFP-spatacsin (domain 1943–2443) with dynamin in HeLa cells expressing GFP or GFP-spatacsin 1943–2443. Western blot signals were detected by chemiluminescence. (G) Left: western blots showing the relative amount of Lamp1, p62, clathrin heavy chain (CHC), and dynamin in whole-brain lysates and lysosome-enriched fractions obtained from Spg11+/+ and Spg11−/− mouse brains. Western blot signals were detected using an infrared imaging system. Right: relative quantification of the band intensities. The graphs show the mean ± SEM. Preparations from N = 3 independent animals. One-tailed Mann-Whitney U test; ∗p = 0.05. (H) Quantification of the proportion of GM2 staining that is localized in lysosomes in neurons treated with 40 μM dynasore. The graphs show the mean ± SEM values. N = 73–124 neurons quantified in three independent neuron preparations. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗p = 0.02 and ∗∗p = 0.01. (I) Quantification of the number of lysosomes with a diameter larger than 1 μm in neurons treated with 40 μM dynasore. The graphs show the mean ± SEM values. N = 49–54 neurons quantified in two independent neuron preparations. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗p = 0.001 and ∗∗∗p ≤ 0.001. (J) Immunostaining of GM2 ganglioside (yellow) and Spatacsin-V5 (magenta) in neurons transfected with vector allowing expression of Spatacsin-V5 and treated with either vehicle or dynasore (40 μM) for 2 hr. Spatacsin is enriched around GM2-positive vesicles (arrowheads). Scale bar: 10 μm. See also Figure S5.
Figure 5
Figure 5
GM2 Accumulation in Lysosomes Promotes the Accumulation of Autophagy Marker p62 in Lysosomes (A) Proportion of lysosomes stained with Lamp1 that were also positive for p62 in Spg11+/+ and Spg11−/− neurons after 3 and 6 days in vitro. The graph shows the mean ± SEM values. Two-way ANOVA, followed by Holm-Sidak post hoc test; ∗p = 0.04 and ∗∗∗p < 0.001. (B) Immunostaining of Spg11−/− neurons with GM2 antibody (cyan), lysosomal marker Lamp1 (magenta), and the autophagy marker p62 (yellow). Arrowheads indicate lysosomes, which were positive for p62 and GM2 staining. Scale bar: 10 μm. (C) Proportion of lysosomes stained with Lamp1 that were also positive for both p62 and GM2 in Spg11+/+ and Spg11−/− neurons after 3 and 6 days in vitro. The graph shows the mean ± SEM values. Two-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001. (D) Effect of miglustat treatment (100 μM) on the proportion of lysosomes stained with Lamp1 that were also positive for p62 in Spg11+/+ and Spg11−/− neurons after 6 days in vitro. The graph shows the mean ± SEM values. Two-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001. (E) Effect of the downregulation of GM3 synthase with two independent miRNAs (GM3S-1 and GM3S-2) on the proportion of lysosomes stained with Lamp1 that were also positive for p62 in control neurons after 6 days in vitro. The graph shows the mean ± SEM values. Two-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001. (F) Effect of Neu1 downregulation with two independent miRNAs (Neu1-1 and Neu1-2) on the proportion of lysosomes stained with Lamp1 that were also positive for p62 in control neurons after 6 days in vitro. The graph shows the mean ± SEM values. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001. In (A–D), day in vitro 3 (DIV3), N = 47–50 neurons quantified in two independent neuron preparations; DIV6, N = 173–193 neurons quantified in four independent neuron preparations. In (E), N = 7–29 neurons quantified in two independent neuron preparations. In (F), N = 57–68 neurons quantified in three independent neuron preparations.
Figure 6
Figure 6
Decreasing Ganglioside Synthesis Prevents Neuronal Death and Improves Motor Dysfunction in Zebrafish Deficient for Spatacsin and Modeling SPG11 (A) Quantification of neuronal death 30 hr after incubation of neurons with glutamate (200 μM) in primary cultures of Spg11+/+ or Spg11−/− cortical neurons treated with miglustat. The graph shows the mean ± SEM values. N = 5–14 independent experiments. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗p = 0.002 and ∗∗∗p < 0.001. (B) Quantification of neuronal death 30 hr after incubation of neurons with glutamate (200 μM) in primary cultures of Spg11+/+ and Spg11−/− cortical neurons transfected with vectors expressing control miRNA or two different miRNAs against GM3 synthase. The graph shows the mean ± SEM. N = 3–8 independent experiments. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗p < 0.02 and ∗∗∗p < 0.001. (C) Quantification of neuronal death 30 hr after incubation of neurons with glutamate (200 μM) in primary cultures of Spg11+/+ and Spg11−/− cortical neurons transfected with vectors that downregulate Neu1 with two independent miRNAs (Neu1-1 and Neu1-2). The graph shows the mean ± SEM values. N = 6 independent experiments. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001. (D and E) Immunostaining (D) and quantification (E) of GM2 immunostaining in the telencephalon of morphants injected with 1.2-pmol zspg11spl antisense or 1.2-pmol mismatch (mm) morpholino (MO). Injection of zspg11spl morpholino increased the mean and variance of GM2 immunostaining intensity. This phenotype was corrected when morphants were treated with miglustat. N = 6–12 morphants analyzed in each condition. One-way ANOVA; ∗∗∗p < 0.001. (F) Phenotype of morphants that were non-injected or injected with 1.2-pmol zspg11spl or 1.2-pmol mismatch (mm) morpholino. 48 hr post-fertilization, morphants were classified as normal phenotype, slowly swimming, paralyzed, or curly morphants. Injection of zspg11spl morpholino leads a large proportion of paralyzed or slowly swimming phenotypes. Treatment with miglustat decreased the proportion of paralyzed morphants. N = 102–366 morphants analyzed in each groups. Chi square test; ∗∗∗p < 0.0001. (G and H) Tracking (G) and quantification of distance traveled (H) by larvae following a touch-evoked escape response. Injection of zspg11spl morpholino impaired the swimming of morphants, which was corrected when treated by miglustat. The graph shows the mean ± SEM. N = 12 morphants analyzed in each condition. One-way ANOVA; ∗∗∗p < 0.001. See also Figure S6.

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