Phosphoinositides (PtdIns) control fundamental cell processes, and inherited defects of PtdIns kinases or phosphatases cause severe human diseases, including Lowe syndrome due to mutations in OCRL, which encodes a PtdIns(4,5)P2 5-phosphatase. Here we unveil a lysosomal response to the arrival of autophagosomal cargo in which OCRL plays a key part. We identify mitochondrial DNA and TLR9 as the cargo and the receptor that triggers and mediates, respectively, this response. This lysosome-cargo response is required to sustain the autophagic flux and involves a local increase in PtdIns(4,5)P2 that is confined in space and time by OCRL. Depleting or inhibiting OCRL leads to an accumulation of lysosomal PtdIns(4,5)P2, an inhibitor of the calcium channel mucolipin-1 that controls autophagosome–lysosome fusion. Hence, autophagosomes accumulate in OCRL-depleted cells and in the kidneys of Lowe syndrome patients. Importantly, boosting the activity of mucolipin-1 with selective agonists restores the autophagic flux in cells from Lowe syndrome patients.
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We thank S. Aydin (UCL Brussels), N. Demoulin (UCL Brussels), M.-C. Gubler (INSERM Paris), G. Montini (Univ. Bologna), U. Querfeld (Charité Berlin), B. Rudolph (Charité Berlin) and F. Schaeffer (Univ. Heidelberg) for providing renal biopsies from patients with Lowe syndrome and appropriate controls, C. Wilson, F. Emma, C. Settembre, N. B. Pierri and R. Venditti for insightful discussions, G. Diez-Roux for critical reading of the manuscript, the TIGEM Bioinformatics Core and M. Failli for the analysis of the gene expression profiles, the Advanced Microscopy and Imaging Core of TIGEM for the EM analysis, and the Central Proteomics Facility, Sir William Dunn Pathology School, Oxford University. M.A.D.M. acknowledges the support of Telethon (grant TGM11CB1), the Italian Association for Cancer Research (AIRC, grant IG2013_14761), European Research Council Advanced Investigator grant no. 670881 (SYSMET), Associazione Italiana Sindrome di Lowe (AISLO), Lowe Syndrome Association, USA (LSA) and Programma Operativo Nazionale (PON) 01_00862. M.G.D.L. received a Fellowship from AIRC. E. Levtchenko was supported by the fund for Scientific Research, Flanders (F.W.O. Vlaanderen) grant 1801110N. F.O. was supported by a grant from the UK Lowe Syndrome Trust (NoMU/ML/1010) awarded to M.L. T.S. was supported by the Wellcome Trust (088785/Z/09/Z). O.D. was supported by the Fonds National de la Recherche Scientifique and the Fonds de la Recherche Scientifique Médicale (Brussels, Belgium); the European Community’s Seventh Framework Programme under grant agreement no. 305608 (EURenOmics); the Cystinosis Research Foundation (Irvine, California, USA); the Rare Disease Initiative Zürich (RADIZ), and the Swiss National Science Foundation project grant 310030_146490 (O.D. and A.L.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) HK-2 cells were treated with non-targeting siRNA (CTRL) or with Sigma Aldrich (S) or Ambion (A) OCRL siRNA pools for 96 h. Cell lysates (50 μg/sample) were analysed by SDS-PAGE and immunoblotted using an anti-OCRL antibody or an anti-β-actin antibody as a loading control. A representative blot is shown. An unprocessed scan of the blot is shown in Supplementary Fig. 9. The graph reports the results of the quantitative analysis of OCRL levels from n = 10 independent experiments for CTRL (mock) and OCRLKD (S) and n = 5 independent experiments for CTRL (non-targeting) and OCRLKD (A). The data are expressed as the level of OCRL normalized for β-actin as a% of the CTRL. Mean values ± s.d. (b) Representative images of CTRL (non-targeting siRNA) and OCRL-KD HK-2 cells stained for OCRL, TGN46 (a Trans Golgi Network marker) and EEA1 (an Early Endosome marker). Insets represent OCRL-TGN46 and OCRL-EEA1 merged enlargements of the boxed areas. Scale bars, 10 μm. (c) HK-2 cells were treated with the indicated siRNAs (Listed in Supplementary Table 1). Total RNA (1 μg/sample) was analysed by qRT–PCR using specific primers (Listed in Supplementary Table 4). Expression levels in siRNA-treated cells are reported relative to the expression of the corresponding gene in mock-treated cells (CTRL) normalized to β-actin. All qPCR reactions were carried out in triplicate and the data represent mean values ± s.d. from n = 5 independent experiments. Statistical significance calculated by Student’s t-test.
Supplementary Figure 2 OCRL depletion induces upregulation of lysosomal genes and TFEB nuclear translocation.
(a) Fold change expression of the indicated lysosomal genes (selected amongst those most upregulated in the microarray) in OCRL-KD (Ambion siRNAs pool) cells relative to non-targeting siRNA-treated cells (CTRL) as assessed by quantitative RT-PCR carried out in triplicate and the data represent mean values ± s.d. from n = 6 independent experiments. p values calculated using Student’s t test (unpaired). (b) CTRL, OCRL-KD (treated either with Sigma (S) or Ambion (A) pools) and YU142670-treated HK-2 cells were stained with an anti-TFEB antibody (green). Mean values ± s.d. n = 150 cells pooled from 3 independent experiments. Numbers on the images report the percentage of cells with nuclear TFEB. P < 1.6 × 10−7 for CTRL versus OCRLKD (S), OCRLKD (A) and YU142670-treated cells calculated by One-way ANOVA with Tukey’s post hoc test. Scale bars, 10 μm. Statistic source data for Supplementary Fig. 2b can be found in Supplementary Table 2. (c) The position of the TFEB binding site (CLEAR motif, blue boxes) relative to the transcriptional start site (0) in the promoters of the indicated upregulated lysosomal genes in OCRL-KD cells, assessed as described in Methods. The colour intensity is proportional to the match score (the darker the higher). (d) Residual upregulation of the indicated lysosomal genes in HK-2 cells knocked down for both OCRL and TFEB relative to OCRL-KD cells as assessed by quantitative RT-PCR carried out in triplicate. The data represent mean values ± s.d. from n = 6 independent experiments. p values were calculated by Student’s t-test. NS, not significant. Notice the inverse correlation between the presence of a CLEAR motif and the extent of residual upregulation on TFEB depletion.
Supplementary Figure 3 OCRL, AP2 and clathrin recruitment to lysosomes during autophagy requires the synthesis of PI(4,5)P2 and is independent of mTOR activation or inactivation.
(a) Representative image of the colocalization of PI4KIIIβ with LAMP1 in HBSS. Scale bar, 10 μm. The graph reports quantification of PI4KIIIβ with LAMP1 colocalization in growth medium and HBSS. Mean values ± s.d. n = 200 cells pooled from 3 independent experiments. (b) Quantification of PI(4,5)P2, AP2, clathrin and OCRL colocalization with LAMP1 during starvation (HBSS for 3 h) after impairing PI(4,5)P2 synthesis (PI4KIIIβ-KD, PIP5K1α-KD and PIP5K1β-KD). Mean values ± s.d. n = 200 cells per condition pooled from 3 independent experiments. (c) Cell lysates (50 μg) from HK-2 cells treated with HBSS and then re-fed by addition of complete growth medium for the indicated time points were analysed by SDS-PAGE and immunoblotted using an anti-p70, an anti-phospho-p70 (p-p70), or an anti-GAPDH antibody as a loading control. A representative blot is shown and an unprocessed scan of the blot is shown in Supplementary Fig. 9. (d) Starvation-independent autophagy stimuli induce an increase in PI(4,5)P2 and recruitment of AP2, clathrin and OCRL to lysosomes. HK-2 cells were treated with the mTOR inhibitor Torin-1, with Tat-beclin 1 peptide (see Methods), or left untreated (Growth medium) and then stained for OCRL and LAMP1. The insets are enlargements of the boxed areas. Scale bars, 10 μm. (e) Quantification of PI(4,5)P2, AP2, clathrin and OCRL colocalization with LAMP1 and of the average number of autophagosomes (LC3 structures) per cell in untreated cells and in cells treated with Torin-1 and Tat-beclin 1 peptide. Mean values ± s.d. n = 100 cells per condition pooled from 3 independent experiments. p values calculated by one-way ANOVA with Tukey’s post hoc test. Statistic source data for Supplementary Fig. 3 can be found in Supplementary Table 2.
Supplementary Figure 4 Inhibiting TLR9 or depleting mtDNA inhibits the lysosomal recruitment of OCRL and impairs the recycling of the autophagosomal SNARE STX17.
(a) IEM of fed or starved TLR9-YFP expressing HK-2 cells and stained with an anti-GFP antibody (scale bar, 200 nm). Arrows, TLR9 in the ER (white), in Multivesicular bodies/Lysosomes (black), or in autolysosomes (red). (b) Control, TFAM-KD and EtBr treated51,58 HK-2 cells stained with PicoGreen58 to visualize mitochondrial DNA. Scale bars, 10 μm. (c) Expression of the indicated nuclear (α-SMA) and mitochondrial (ND-6, D-loop, CoxI and CoxII) genes evaluated by qPCR in EtBr-treated cells versus control cells. All qPCR reactions were carried out in triplicate and the data represent mean values ± s.d. from n = 3 independent experiments. (d) OCRL (red) association with lysosomes on starvation in CTRL, TFAM-KD, and HK-2 cells treated with EtBr. Scale bars, 10 μm. Insets, enlargements of the boxed areas. (e) Fed or starved HK-2 cells expressing TIRAP-GFP stained for LAMP1. Insets, enlargements of the boxed areas. Scale bars, 10 μm. Graph, colocalization of TIRAP with LAMP1. Mean ± s.d. n = 100 cells per condition pooled from 3 independent experiments. (f) Fed or starved HK-2 cells KD for adaptors (MyD88 and TIRAP) or transducers (IRAK4) of TLR9 signalling and for UNC93B1. LAMP1-OCRL colocalization. Mean values ± s.d. n = 300 cells per condition pooled from 3 independent experiments. (g) Representative images of control or TLR9-KD HK-2 cells expressing the STX17-GFP incubated in HBSS for 3 h. Insets, enlargements of the boxed areas. Scale bars, 10 μm. Right graph, quantification of LAMP1-STX17 colocalization. n = 75 cells per condition pooled from 3 independent experiments. The data show that STX17 is enriched in LAMP1-positive compartments in TLR9-depleted cells as compared to control cells. The LAMP1 structures containing STX17 are, by definition, autolysosomes: since the total number of autolysosomes is lower in TLR9-KD cells (Fig. 5b, e), then the fraction of autolysosomes still containing STX17 is markedly increased on TLR9 deletion, suggesting that recycling of STX17 from autolysosomes is under control of TLR9. The p values are indicated, calculated using Student’s t-test in c, e, and by one-way ANOVA with Tukey’s post hoc test in f, g. Statistic source data in Supplementary Table 2.
Supplementary Figure 5 HK-2 cells depleted of OCRL accumulate mature autophagosomes following impairment of autophagosome/lysosome fusion.
(a) Representative images of CTRL and OCRL-KD cells incubated in growth medium or in HBSS for 3 h, stained for ATG16L1 (an early and transient autophagosomal marker). Scale bars, 10 μm. The graph reports the quantification of the ATG16L1-positive structures, means ± s.d. n = 150 cells pooled from 3 independent experiments. (b) Immunoblot analysis of cell lysates (50 μg/sample) from CTRL, OCRL-KD (Sigma (S) or Ambion (A) siRNA pools) and YU142670-treated cells with or without 100 nM Bafilomycin A1 (Baf A1) for 2 h with the indicated antibodies. β-actin was used as a loading control. A representative blot is shown and an unprocessed scan of the blot is shown in Supplementary Fig. 9. Mean values ± s.d. n = 3 lysates per condition pooled from 3 independent experiments. (c) HK2 cells were depleted of OCRL using Ambion siRNA pool and treated as described in Fig. 6d. Mean values ± s.d. n = 120 cells per condition pooled from 3 independent experiments. (d) HK2 cells were depleted of OCRL using Ambion siRNA pool and treated as described in Fig. 6e. The arrow indicates an autolysosome. Mean values ± s.d. n = 150 cells per condition pooled from 3 independent experiments. (e) CTRL and OCRL-KD cells were incubated in HBSS for 3 h and labelled with an anti-LAMP1 (green) antibody together with an anti-GST antibody to detect recombinant GST-PHPLCδ (a probe for PI(4,5)P2)5. White arrows indicate structures positive for both markers. Scale bars, 10 μm. (f) Representative images and quantification of PI(4,5)P2-LAMP1 colocalization in OCRL-KD cells expressing an siRNA-resistant wild-type (GFP-OCRL wt) or catalytically inactive (GFP-OCRL V527D) OCRL and stained with antibodies against PI(4,5)P2 (red) and LAMP1 (green). Transfected cells are outlined. Scale bars, 10 μm. Mean values ± s.d. n = 300 cells pooled from 3 independent experiments. p values are indicated, calculated by One-way ANOVA with Tukey’s post hoc test in a, c, d, f, and by Student’s t-test in b; NS (not significant). Statistic source data for Supplementary Fig. 5 can be found in Supplementary Table 2.
Supplementary Figure 6 Accumulation of autophagosomes and expansion of lysosomal compartments in kidneys of Lowe syndrome patients.
(a) Representative confocal micrographs of proximal tubules from two patients with Lowe syndrome and one control immunostained with an anti-LC3 (red) and anti-AQP1 (green, a proximal tubule marker) antibody. Quantification of the LC3-positive structures is given in f. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (b) Representative confocal micrographs of proximal tubules from two patients with Lowe syndrome and one control immunostained with an anti-LAMP1 (red) and anti-AQP1 (green) antibody. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. The graph reports the quantification of LAMP1-positive structures in AQP1-positive proximal tubules in human kidney biopsies from three Lowe syndrome patients and four controls. Data are means ± s.e.m., n = 50 proximal tubules pooled from 10 fields per biopsy. ∗P < 0.0025 calculated using one-way ANOVA/post-hoc tests.
Supplementary Figure 7 The MCOLN1 agonist SF-51 specifically reverts autophagosome accumulation in OCRL-depleted cells and in PTCs from Lowe syndrome patients.
(a) OCRL depletion does not affect MCOLN1 localization at lysosomes. Mock-treated (CTRL) and OCRL-KD HK-2 cells transiently expressing MCOLN1-myc were immunostained with anti-myc (green) and anti-LAMP1 (red) antibodies. Numbers indicate the percentage of LAMP1-MCOLN1 colocalization. Means ± s.d., n = 150 transfected cells per condition pooled from 3 independent experiments. Scale bar, 10 μm. (b) SF-5147 does not rescue the autophagosome accumulation caused by depleting components of the autophagosome–lysosome tethering/fusion machinery. Mock-treated (CTRL), OCRL-KD, MCOLN1-KD, RAB7-KD and VPS16-KD HK-2 cells incubated in growth medium with or without 200 μM of the mucolipin-1 agonist SF-51 for 2 h were immunostained for LC3 (red) and LAMP1 (green). The graph reports the quantification of the number of LC3-positive structures per cell. Means ± s.d. n = 300 cells per condition pooled from 3 independent experiments. p values are indicated calculated by One-way ANOVA with Tukey’s post hoc test. Scale bar, 10 μm.
At steady state OCRL associates with clathrin-coated pits (CCP), clathrin-coated vesicles (CCV), early endosomes and the Trans Golgi Network (TGN) (and only marginally with lysosomes) and the phosphoinositides that are mainly present in lysosomes are PI3P and PI(3,5)P2. When autophagosomes (AV) fuse with lysosomes (LY) during starvation, the mtDNA released into lysosomes activates TLR9, which translocates from the endoplasmic reticulum (ER) to autolysosomes (AL). TIRAP, a known component of the TLR9 signalling pathway, also translocates to AL. The activation of TLR9 in AL induces the recruitment of PI(4,5)P2-producing enzymes (PIP5Kα and β) that leads to an increase in PI(4,5)P2. This increase is restricted by the simultaneous recruitment of OCRL, a PI(4,5)P2 5-phosphatase. The lysosomal translocation of OCRL is driven by the clathrin adaptor AP2 and clathrin, which associate with lysosomes in a PI(4,5)P2-dependent manner. The fine spatial control of PI(4,5)P2 in lysosomes is required to preserve the activity of MCOLN1 (required for autophagosome–lysosome fusion) which is inhibited by PI(4,5)P2. OCRL, which interacts with MCOLN1, ensures PI(4,5)P2-free microdomains around MCOLN1.
Supplementary Figure 9 Unprocessed scans of original blots shown in figures and supplementary figures.
The uncropped and unprocessed scans of original blots shown in this study are reported.
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De Leo, M., Staiano, L., Vicinanza, M. et al. Autophagosome–lysosome fusion triggers a lysosomal response mediated by TLR9 and controlled by OCRL. Nat Cell Biol 18, 839–850 (2016). https://doi.org/10.1038/ncb3386
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