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CLN3 is required for the clearance of glycerophosphodiesters from lysosomes

Nature volume 609, pages 1005–1011 (2022)Cite this article

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Abstract

Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2,3,4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)—the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.

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Fig. 1: LysoTag mouse for proteomic and metabolite profiling of tissue lysosomes.
Fig. 2: Loss of CLN3 leads to significant alterations in metabolite levels in brain lysosomes.
Fig. 3: GPDs accumulate in lysosomes after CLN3 loss.
Fig. 4: CLN3 is required for the efflux of GPDs out of lysosomes.

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Data availability

The MS proteomics data were deposited to the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifier PXD018624. Known lysosomal proteins in our proteomics analysis were defined based on Gene Ontology Cellular Component or UniProt subcellular localization annotation (https://www.uniprot.org/). The conditional LysoTag mouse was deposited at the Jackson Laboratory (strain 035401). Other unique biological materials in the form of plasmids or cell lines are available from the corresponding author on request. Detailed lipidomics and metabolomics data are provided in Supplementary Tables 3 and 4, respectively. Other data generated are available from the corresponding author on request. Source data are provided with this paper.

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Acknowledgements

We thank the members of the Abu-Remaileh and Sabatini laboratories for insights; J. M. Kirkpatrick and the FLI Core Proteomics; and the participants and families in this study for their invaluable contributions. This work was supported by grants from the NIH (DP2-CA271386), Stanford Alzheimer’s Disease Research Center (ADRC), Charles King fellowship, BeatBatten (Netherlands) and NCL-Stiftung (Germany) foundations to M.A.-R. and from the NIH (R01 CA103866 and R01 CA129105) to D.M.S. N.N.L. and W.D. were also supported by the BeatBatten (Netherlands) and NCL-Stiftung (Germany) foundations; U.N.M. by the Stanford ChEM-H Chemistry/Biology Interface Program, O’Leary-Thiry Graduate Fellowship and NIH T32 training grant (T32GM120007); A.L.C. by the F31 predoctoral NRSA (5F31DK113665); and C.G. and R.T. were partially supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Projektnummer 23928380—TRR 152 and the NCL-Stiftung (Germany); A.O. by DFG through the Research Training Group ProMoAge (GRK 2155), the Else Kröner Fresenius Stiftung (no. 2019_A79), the Fritz-Thyssen foundation (no. 10.20.1.022MN) and the Chan Zuckerberg Initiative Neurodegeneration Challenge Network (nos 2020-221617 and 2021-230967). The FLI is a member of the Leibniz Association and is financially supported by the Federal Government of Germany and the State of Thuringia. The CLN3 natural history study is funded by an NIH Clinical Center Bench to Bedside award and the NICHD intramural research programme (ZIA HD008989). M.A.-R. is a Terman Faculty Fellow at Stanford University.

Author information

Author notes

  1. Sze Ham Chan

    Present address: Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA, USA

  2. These authors contributed equally: David M. Sabatini, Monther Abu-Remaileh

  3. Unaffiliated: David M. Sabatini

Authors and Affiliations

  1. Department of Chemical Engineering, Stanford University, Stanford, CA, USA

    Nouf N. Laqtom, Wentao Dong, Uche N. Medoh & Monther Abu-Remaileh

  2. Department of Genetics, Stanford University, Stanford, CA, USA

    Nouf N. Laqtom, Wentao Dong, Uche N. Medoh & Monther Abu-Remaileh

  3. The Institute for Chemistry, Engineering & Medicine for Human Health (ChEM-H), Stanford University, Stanford, CA, USA

    Nouf N. Laqtom, Wentao Dong, Uche N. Medoh & Monther Abu-Remaileh

  4. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA

    Uche N. Medoh

  5. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Andrew L. Cangelosi

  6. Koch Institute for Integrative Cancer Research, Cambridge, MA, USA

    Andrew L. Cangelosi

  7. Whitehead Institute for Biomedical Research, Cambridge, MA, USA

    Andrew L. Cangelosi, Vimisha Dharamdasani, Sze Ham Chan, Tenzin Kunchok & Caroline A. Lewis

  8. Leibniz Institute on Aging—Fritz Lipmann Institute, Jena, Germany

    Ivonne Heinze & Alessandro Ori

  9. Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, Ludwig-Maximilians-Universität, Munich, Germany

    Rachel Tang & Christian Grimm

  10. Division of Translational Medicine, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA

    An N. Dang Do & Forbes D. Porter

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  1. Nouf N. Laqtom
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Contributions

M.A.-R. and D.M.S. initiated the project and, with help from N.N.L., designed the research plan. N.N.L. and W.D. performed most of the experiments and analysed the data with help from A.L.C., V.D. and S.H.C.; S.H.C., T.K. and C.A.L. had a critical role in LC–MS analysis. W.D. also helped with analysing LC–MS data and U.N.M. generated GPD chemical standards. I.H. and A.O. designed the proteomic experiments and analysed the lysosomal proteomic data. R.T. and C.G. were consulted on CLN3 function and edited the manuscript. A.N.D.D. and F.D.P. supervised the CLN3 natural history study and provided the CSF samples. M.A.-R. and N.N.L. wrote the manuscript and D.M.S. edited it.

Corresponding author

Correspondence to Monther Abu-Remaileh.

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Competing interests

M.A.-R. is a scientific advisory board member of Lycia Therapeutics. A.N.D.D. and F.D.P. have a collaborative research agreement with Amicus Therapeutics. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 The effects of LysoTag expression on lysosomes in vivo.

a, In the constitutive LysoTag mice, TMEM192–3×HA is expressed across all tissues examined as determined by immunoblotting using antibodies to the HA epitope. No difference in the expression of lysosomal markers LAMP1, Cathepsin B, C, and D is observed upon the expression of TMEM192–3×HA. Vinculin and AKT were used as loading controls. Numbers indicate molecular weights in kDa according to protein standards run on the same gel. Immunoblot is representative of two independent experiments. b, Normalized body weights of mice with the indicated genotypes at 5-9 weeks of age. Mice were weighed individually. The body mass of each mouse was normalized to the mean mass of sex-matched wild-type littermates before pooling by genotype for statistical analyses using a two-tailed unpaired t-test. (n = 25, 19, and 10 for Wild type, LysoTag+, and LysoTagLSL mice, respectively.). c,d, Measurement of lysosomal hydrolase activity in brain and liver tissues of the constitutive LysoTag mice shows no effect of TMEM192–3×HA expression on lysosomal function. For measuring Cathepsin B activity, its fluorogenic substrate was incubated with homogenates of the corresponding tissues (see methods). For determining β-hexosaminidase activity, fluorogenic 4-methylumbelliferyl (MUF)- N-acetyl-β-Glucosaminide was used as a substrate (see methods). Fluorescence was measured at 37 °C and data were shown following the subtraction of background fluorescence observed in the absence of homogenate (mean ± s.e.m.; n=4). e,f, Representative transmission electron micrographs showing lysosomes from brain and liver tissues of LysoTag- (control) and LysoTag+ mice. Lysosomes were identified by the presence of a single membrane and granular, electron-dense appearance. White and Red arrowheads indicate lysosomes in cells from LysoTag- and LysoTag+ mice, respectively. n = 2 mice per group and scale bar, 500 nm. The graph showing the mean diameter of lysosomes in multiple quantified cells. The diameter of lysosome in each group was generated by measuring lysosomes using ImageJ v1.52. (For brain, n = 42 and n = 55 measured lysosomes from LysoTag- and LysoTag+ mice, respectively. For liver, n = 51 and n = 56 measured lysosomes LysoTag- and LysoTag+ mice, respectively. Data presented as mean ± s.e.m. n.s: non-significant; Two-tailed unpaired t-test (e,f). g, Heatmap presentation of the enrichment of early- and late-endosomal markers as well as those for lysosome in the LysoIP performed from mouse liver tissue. Enrichment values are derived from Supplementary Table 1. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 2 Generation and validation of LysoTag mouse model for Batten disease studies.

a, Schematic depicting the breeding strategy to generate LysoTag models for Batten disease studies. Cln3−/− mice were crossed with heterozygous constitutive LysoTag mice and their progeny were then crossed back to Cln3−/− animals to generate the indicated experimental mouse genotypes. Mouse drawing was created with BioRender. b, Expression and localization of the TMEM192–3×HA fusion protein to lysosomes in brain neurons of Cln3+/+, Cln3+/− and Cln3−/− LysoTag mice. TMEM192–3×HA, lysosomes and neurons were detected in an immunofluorescence assay using antibodies to the HA epitope the lysosomal marker, LAMP1 and the neuronal marker Neu-N, respectively. Scale bar = 5 µm, and magnified insets are labelled with white boxes in the main image. Percentage of colocalization between TMEM192–3×HA and LAMP1 shown in the right panel was measured in n = 7 cells per genotype. Data are shown as mean ± s.e.m. Micrographs are representative of at least three independent experiments. c , Volcano plot comparing the lipidomic data from whole brain homogenates of Cln3+/− and Cln3−/− mice shows that only few lipids change significantly and they belong to phospholipids (yellow) and lysophosphatidylglycerol (LPG) in red. Loss of CLN3 also led to a decrease in the brain of bis(monoacylglycero)phosphate (BMP), a class of lysosomal lipids (blue). BMP/PG (cyan) annotation was used when MS/MS fragmentation was not acquired (see Supplementary Table 3 and methods for details). Horizontal line indicates a p-value of 0.05, and vertical dotted lines a fold change of 2. Each dot represents a lipid species. Data were acquired in negative ion mode and normalized to internal lipid standards for the best-matched lipid class (n = 5 and 4 for Cln3+/− and Cln3−/− mice, respectively). Female mice with an average age of 7 months were used. Data are in Supplementary Table 3. Two-tailed unpaired t-test was used.

Source data

Extended Data Fig. 3 Validation of glycerophosphodiesters (GPDs) as the metabolites that accumulate in lysosomes upon CLN3 loss.

a, Identification and annotation of metabolites using untargeted metabolomics analyses. Overview of our untargeted metabolomics experimental workflow. MS1 data were collected for every sample using full scan mode on a high-resolution accurate-mass instrument. MS/MS data were collected from pooled samples only to aid with compound identification. Data were then analysed using Thermo Compound Discoverer v3.1 to generate a list of unique features which were deconvoluted into a list of unique compounds. Differential and statistical analyses were performed. Compounds which were statistically significant in the differential analyses were validated and identified using authentic standards, when available. If no authentic standard was available, compounds were annotated by comparing MS/MS data to a database, or in silico fragmentation prediction (competitive fragmentation modelling-ID, CFM-ID). Data were then reported according to the Metabolomics Standards Initiative (MSI) guidelines. b,c, Mirror plots for GPC and GPI in negative ion mode, respectively. Fragments common to the glycerol-phosphate group are indicated with an asterisk*. Fragments common to those found in the MS/MS spectra reported by Kopp et al.32 are indicated with †. d, EICs for GPE and GPG across a range of samples were analysed alongside in-house generated standards (Std) and showed a matching retention time (RT). e,f, Mirror plots for GPE and GPG, respectively. Fragments common to the glycerol-phosphate group are indicated with an asterisk*. Fragments common to those found in the MS/MS spectra reported by Kopp et al.32 are indicated with †. g, Mirror plots showing MS/MS spectra for glycerophosphoserine from experimental samples compared to MS/MS spectra generated in silico using CFM-ID. Fragments common to the glycerol-phosphate group are indicated with an asterisk*. Fragments common to those found in the MS/MS spectra reported in Kopp et al.32 are indicated with †. h, Amplicon sequencing was used to validate CLN3 knock out cells generated using CRISPR-Cas9. Three mutant alleles were identified and all were found to generate frameshift deletions in the CLN3 coding sequence as compared to the wild-type sequence. i, Localization of Flag-CLN3 protein to lysosomes. Flag-CLN3 and lysosomes were detected in an immunofluorescence assay using antibodies to the Flag epitope and the lysosomal marker LAMP2, respectively. Scale bar = 5 μm. Micrographs are representative images of three experiments. j, Changes in GPD levels in CLN3-deficient cells expressing TMEM192–3×HA tag or LAMP1–3×HA tag. Data presented as a comparison between the increase in the lysosomal abundance of GPDs upon CLN3 loss in LAMP1–3×HA tagged lysosomes relative to that in TMEM192–3×HA tagged lysosomes (mean ± s.e.m.; n = 4 biologically independent samples). n.s: non-significant; Two-tailed unpaired t-test.

Source data

Extended Data Fig. 4 Testing the effects of CLN3 loss on lysosomal pH and cellular lipid metabolism.

a to d, CLN3 loss does not increase lysosomal pH. a, A standard calibration curve of the ratiometric dye LysoSensor Yellow/Blue DND-160. See methods for experimental details. b, Lysosomal pH in wild-type and CLN3 KO HEK293T cells as calculated using the standard curve in a. Data are presented as mean ± s.d., n = 12 biologically independent samples. c, Targeted analyses of the fold changes in whole-cell and lysosomal levels of GPDs upon treatment with 500 nM Bafilomycin A1 for 6 h in CLN3 expressing HEK293T cells. Data are presented as mean ± s.d., n = 3 biologically independent samples, (Two-tailed unpaired t-test). d, The levels of several amino acids whose egress from lysosomes is sensitive to the proton gradient across the lysosomal membrane are not affected by CLN3 loss. The levels of proline, alanine, and glutamate in whole cells and lysosomes were compared between CLN3 KO cells with and without CLN3 cDNA addback and upon treatment with 500 nM Bafilomycin A1 (BafA1) for 6 h. Data are presented as mean ± s.d., n = 3 biologically independent samples, (Two-tailed unpaired t-test). e, The recombinant CLN3 protein does not have glycerophosphodiesterase activity. Deuterated GPC (D9-GPC) was incubated with recombinant 3×Flag–CLN3 or the positive control glycerophosphodiesterase1 (3×Flag–GDE-1) for the indicated time points. The extent of D9-GPC hydrolysis was determined by measuring the decline in its level and the increase in the levels of the product D9-choline. Data presented as mean ± s.e.m. of n = 3 biologically independent samples. f, CLN3 loss does not decrease tracer uptake. Fold change in tracer levels (D9-16:0-16:0 PC) normalized to total protein for samples measured in Fig. 4f and g. Data presented as mean ± s.e.m. of n = 4 biologically independent samples. g, CLN3 loss does not affect the biosynthesis or turnover of PC and sphingomyelin (SM) in cells. Free D9-choline was used as a tracer. Data are presented as fold changes in the whole-cell molar percent enrichment (MPE) of D9-choline-containing lipids in cortical neuron cultures prepared from Cln3−/− mice relative to those from Cln3+/− animals (mean ± s.e.m., n = 3 biologically independent samples, (Two-tailed unpaired t-test)).

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Laqtom, N.N., Dong, W., Medoh, U.N. et al. CLN3 is required for the clearance of glycerophosphodiesters from lysosomes. Nature 609, 1005–1011 (2022). https://doi.org/10.1038/s41586-022-05221-y

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