Spastic paraplegia proteins spastizin and spatacsin mediate autophagic lysosome reformation

Abstract

Autophagy allows cells to adapt to changes in their environment by coordinating the degradation and recycling of cellular components and organelles to maintain homeostasis. Lysosomes are organelles critical for terminating autophagy via their fusion with mature autophagosomes to generate autolysosomes that degrade autophagic materials; therefore, maintenance of the lysosomal population is essential for autophagy-dependent cellular clearance. Here, we have demonstrated that the two most common autosomal recessive hereditary spastic paraplegia gene products, the SPG15 protein spastizin and the SPG11 protein spatacsin, are pivotal for autophagic lysosome reformation (ALR), a pathway that generates new lysosomes. Lysosomal targeting of spastizin required an intact FYVE domain, which binds phosphatidylinositol 3-phosphate. Loss of spastizin or spatacsin resulted in depletion of free lysosomes, which are competent to fuse with autophagosomes, and an accumulation of autolysosomes, reflecting a failure in ALR. Moreover, spastizin and spatacsin were essential components for the initiation of lysosomal tubulation. Together, these results link dysfunction of the autophagy/lysosomal biogenesis machinery to neurodegeneration.

Figures

Figure 11. Model of effects of spastizin and spatacsin loss in ALR.

(A) In control cells, autophagosomes containing autophagic materials fuse with the lysosome to form the autolysosome. After degrading materials, the lysosomal tubule emanates from the autolysosome and becomes the protolysosome, which is destined to become a lysosome after maturation. This machinery for maintaining the population of cellular lysosomes is called ALR. In cells from patients with SPG15 or SPG11, fusion of the lysosome and the autophagosome occurs normally, but ALR is blocked by impaired initiation of lysosomal tubulation from the autolysosome, which eventually results in accumulation of autolysosomes and exhaustion of free lysosomes. (B) Schematic model of lysosome reformation in starvation and feeding conditions. In feeding conditions, basal autophagy occurs to maintain cell homeostasis. At autolysosomes, PI4KB plays a critical role by converting phosphatidylinositol (PI) to PI(4)P, which suppresses uncontrolled lysosomal tubulation from autolysosomes and facilitates lysosome budding/vesiculation managed by clathrin (or other coat proteins) and Dyn2, reminiscent of endocytosis. The spastizin-spatacsin complex localizes to the lysosome/autolysosome by interaction of the spastizin FYVE domain and PI(3)P, and this complex is also an essential component for lysosome vesiculation. In prolonged starvation conditions, to rapidly generate energy sources cells choose an enhanced pathway, ALR, which reforms lysosomes more efficiently. In this case, PI(4,5)P2 produced from PI(4)P by PIP5K1B is an essential component for tubule initiation. The spastizin-spatacsin complex may function downstream of PI(4,5)P2 or work independently during ALR. The AP-5 protein complex that coprecipitates with spastizin and spatacsin is not shown.

Figure 10. Spastizin and spatacsin interact with PI4KB.

(A) HeLa cells were transfected with siCTL or siPI4KB siRNAs and then coimmunostained for spastizin (green) and LAMP1 (red). Merged images are to the right. (B) HeLa cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs and then lysates were immunoblotted as shown. (C) Cells from B were coimmunostained for PI4KB (green) and GM130 (red). (D) Myc-PI4KB was coexpressed with HA vector, HA-spastizin, or HA-spatacsin in HEK293T cells, and lysates were immunoprecipitated and immunoblotted with the indicated antibodies (right). (E) Myc-PI4KB was coexpressed with indicated HA-spastizin wild-type or mutant constructs in HEK293T cells, and lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (F) Myc-PI4KB was coexpressed with HA vector or indicated HA-spastizin truncation mutants in HEK293T cells, and lysates were immunoprecipitated (anti-HA) and immunoblotted for HA- and Myc-epitopes. Scale bar: 10 μm. Arrows identify specific proteins, while asterisks denote the IgG heavy chain. Molecular weight standards (in kDa) are shown to the left in B and D–F.

Figure 9. Spastizin and spatacsin are essential for ALR initiation.

(A) HeLa cells stably expressing LAMP1-GFP were starved in EBSS (0 or 8 hours) and imaged. (B) HeLa cells stably expressing LAMP1-GFP were transfected with RFP-SPNS1, starved in EBSS for 8 hours, and imaged. (C) HeLa cells stably expressing LAMP1-GFP were transfected with siCTL, siSPG15, or siSPG11 siRNAs; starved with EBSS (0 or 8 hours); and imaged. (D) Cells with >5 lysosomal tubules from C were quantified (n = 3; >100 cells per experiment). (E and F) HeLa cells stably expressing LAMP1-GFP were transfected with siCTL or siSPG15 siRNAs and then treated with DMSO, EBSS, or EBSS with Dynasore (40 μM) for 2 hours before imaging. (E) Representative images of Dynasore-treated control or spastizin-depleted (SPG15-depleted) cells. (F) Cells with >5 lysosomal tubules longer than 2 μm were quantified (n = 3; >100 cells per experiment). (G) HeLa cells were transfected with siCTL, siSPG15, PI4KB (siPI4KB), or both spastizin and PI4KB (Double KD) siRNAs, and cell lysates were immunoblotted. Molecular weight standards (in kDa) are shown to the left. (H) HeLa cells stably expressing LAMP1-GFP were transfected with siCTL, siSPG15, siPI4KB, or both spastizin and PI4KB siRNAs and then imaged. (I) Cells with >5 lysosomal tubules from H were quantified (n = 3; >100 cells per experiment). Scale bar: 10 μm. Mean ± SD are shown. One-way ANOVA followed by Tukey’s multiple comparison test, ***P < 0.001.

Figure 8. Spastizin- and spatacsin-depleted cells exhibit impaired autophagosome clearance.

(A) HeLa cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs and then BODIPY-FL-pepstatin A (1 mM) was added for 30 minutes to stain lysosomes containing active cathepsins. To neutralize the acidic lysosomes, bafilomycin A1 (40 μM) was added in serum-free media to siCTL-transfected cells for 16 hours before imaging. (B) HeLa cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs and coimmunostained for CI-MPR (green) and LAMP1 (red). Bafilomycin A1–treated, siCTL-transfected cells were included for comparison. (C) Fibroblasts derived from a control subject (II-1) or patients with SPG15 (II-2 and II-3) were coimmunostained for CI-MPR (green) and LAMP1 (red). (D) HeLa cells transfected with siCTL, siSPG15, or siSPG11 siRNAs were treated with DMSO, bafilomycin A1 (0.4 μM), or leupeptin (50 μg/mL) for 6 hours before harvesting. Lysates were immunoblotted as shown. Molecular weight standards (in kDa) are shown to the left. (E) LC3-II flux from D was measured (n = 3). Scale bar: 10 μm. Mean ± SD are shown. One-way ANOVA followed by Tukey’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7. Autolysosomes in spastizin- or spatacsin-depleted cells are functional.

(A) HeLa cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs. LysoSensor Green DND-189 (1 mM) was added for 1 hour to stain acidic lysosomes. To neutralize acidic lysosomes, bafilomycin A1 (Baf A1, 40 μM) was added in serum-free media for 16 hours before imaging. (B) HeLa cells stably expressing mRFP-GFP-LC3 were transfected with siCTL, siSPG15, or siSPG11 siRNAs. To elevate the lysosomal pH, chloroquine (CQ, 50 μM) was added to siCTL-transfected, serum-deprived cells for 4 hours. Cells were visualized by confocal microscopy, and images from red and green channels were merged. (C) Cells with >20 yellow puncta from B were quantified (n = 3; >200 cells per experiment). (D) Lysates were prepared from control (II-1), SPG15 (II-3), or SPG11 (SPG11-1 and SPG11-2) fibroblasts. HeLa cell lysates were obtained from cells transfected with siCTL, siSPG15, siSPG11, or AP5B1 (siAP5B1-1) siRNAs. Lysates were immunoblotted as shown. Molecular weight standards (in kDa) are shown to the left. Scale bar: 10 μm. Mean ± SD are shown. One-way ANOVA followed by Tukey’s multiple comparison test, ***P < 0.001.

Figure 6. Enlarged LPOs induced by depletion of spastizin or spatacsin in nutrient conditions are autolysosomes.

(A and B) HeLa cells maintained in serum-containing media were transfected with siCTL, siSPG15, or siSPG11 siRNAs and then coimmunostained either for (A) LC3 (green) and LAMP1 (red) or (B) CD63 (green) and LC3 (red). Insets in the images are enlarged (original magnification, ×2.8) to the right in A. (C) LC3 puncta overlapping with CD63 from B were quantified (10 cells; >30 particles per cell). Scale bar: 10 μm. Mean ± SD are shown.

Figure 5. A failure of clearance of autophagosomes in spastizin- or spatacsin-depleted cells occurs downstream of mTOR activation.

(A and B) HeLa cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs; starved by serum deprivation; and immunostained for LC3. (A) Representative images of starved control cells for the indicated time periods. (B) Starved cells with >20 autophagosomes were quantified (n = 3; >200 cells per experiment). (C) Lysates from A were immunoblotted with the indicated antibodies. Molecular weight standards (in kDa) are shown to the left. Molecular weight of S6K is approximately 59 kDa. (D) Phospho-S6K levels from C were normalized to total S6K (n = 3). (E) HeLa cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs and then coimmunostained for mTOR (green) and LAMP1 (red). Scale bar: 10 μm. Mean ± SD are shown.

Figure 4. Depletion of spastizin or spatacsin induces accumulation of autophagosomes under nutrient conditions.

(A) HeLa cells cultured in serum-containing media were transfected with siCTL, siSPG15, or siSPG11 siRNAs and then coimmunostained for p62 (green) and LC3 (red). Insets in the merged images are enlarged (original magnification, ×3) to the right. Scale bar: 10 μm. (B) Cells with >20 autophagosomes from A were quantified (n = 3; >200 cells per experiment). One-way ANOVA followed by Tukey’s multiple comparison test, ***P < 0.001. (C) HeLa cells maintained in serum-containing media were transfected with siCTL, siSPG15, or siSPG11 siRNAs, and cell lysates were immunoblotted. Molecular weight standards (in kDa) are shown to the left. Arrows identify specific proteins, while the asterisk denotes a cross-reacting band.

Figure 3. Prolonged starvation does not restore normal LPO size in cells lacking spastizin or spatacsin.

(A) Representative images of control HeLa cells immunostained for LAMP1 for the indicated times of starvation. (B) Cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs; starved by serum deprivation; and immunostained for LAMP1. Cells with >20 enlarged LPOs were quantified (n = 3; >200 cells per experiment). (C and D) Fibroblasts derived from a control subject (II-1), a patient with SPG15 (II-3), and 2 patients with SPG11 (SPG11-1 and SPG11-3) were starved by serum deprivation and then coimmunostained for LAMP1 (red) and spastizin (green). (C) Representative images of 0 hour–starved cells. (D) Cells with >20 enlarged LPOs were quantified (n = 3; >200 cells per experiment). Boxes within images are enlarged (original magnification, ×4) in the bottom row. Scale bar: 10 μm. Mean ± SD are shown.

Figure 2. Depletion of spastizin or spatacsin causes prominent LPO enlargement.

(A) HeLa (left) or hTERT RPE-1 (right) cells transfected with control (siCTL), spastizin (siSPG15), or spatacsin (siSPG11) siRNAs were immunostained for LAMP1. Nuclei were stained with Hoechst 33342 (blue). Boxes within images in the left columns are enlarged (original magnification, ×4) in the right columns. (B and C) Numbers of (B) HeLa or (C) hTERT RPE-1 cells from A with >20 enlarged LPOs (LPOs with > 0.5 μm diameter) were quantified (n = 3; >200 cells per experiment). (D) HeLa cells were transfected with siCTL, siSPG15, or siSPG11 siRNAs, and cell lysates were immunoblotted. Molecular weight standards (in kDa) are at the left. (E) LAMP1 protein levels from D were normalized to β-tubulin (n = 3). (F) HeLa cells were transfected with siCTL or siSPG15 siRNAs and subsequently with the indicated HA-spastizin constructs or an empty vector and then immunostained for LAMP1 (green) and HA-epitope (red). Merged images are shown in the bottom row. (G) Cells with >20 enlarged LPOs from F were quantified (n = 3; >100 cells per experiment). Scale bar: 10 μm. Mean ± SD are shown. One-way ANOVA followed by Tukey’s multiple comparison test, ***P < 0.001.

Figure 1. Spastizin localizes to lysosomes through its FYVE domain.

(A and B) HeLa cells transiently expressing HA-spastizin were coimmunostained for HA-epitope (green) and either endogenous (A) LAMP1 or (B) CD63 (red). (C and D) HeLa cells were coimmunostained for endogenous spastizin (green) and either (C) LAMP1 or (D) CD63 (red). (E) HeLa cells were coimmunostained for either EEA1 or CI-MPR (red) with endogenous spastizin (green). (F) Skin fibroblasts derived from a control subject and a patient with SPG15 were coimmunostained for LAMP1 (red) and endogenous spastizin (green). (G) HeLa cells were transfected with wild-type or mutant HA-spastizin constructs as shown and coimmunostained for HA-epitope (green) and LAMP1 (red). Merged images are to the right in A–D and G. Boxed areas in the images in the top rows are enlarged in the bottom rows in A–D (original magnification, ×2.5), and boxed areas in the images in the left columns are enlarged in the right columns in E and F (original magnification, ×4). Scale bar: 10 μm.