CLN7 protein functions at the interface between endolysosomes and stress granules to promote cell survival

CLN7 protein functions at the interface between endolysosomes and stress granules to promote cell survival | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CLN7 protein functions at the interface between endolysosomes and stress granules to promote cell survival Tristan Tristan McKay, Aseel Sharaireh, Marta Guevara-Ferrer, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6520859/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Cell Death & Disease → Version 1 posted 8 You are reading this latest preprint version Abstract Inherited biallelic mutations in the CLN7 gene result in the variant late infantile onset neuronal ceroid lipofuscinosis, a subtype of Batten disease (BD), a severe and fatal childhood neurodegenerative disease. Intriguingly, CLN7 genetic variants have also been associated with retinopathies, amyotrophic lateral sclerosis, and frontotemporal dementia. CLN7 encodes a transmembrane protein localizing to endolysosomal membranes with outward-facing chloride channel activity. Loss of CLN7 function results in cortical neurons accumulating swollen lipofuscin-containing lysosomes, leading to neuroinflammation and neurodegeneration. The molecular mechanisms underlying CLN7 BD neuropathology are not understood. We have generated iPSC lines from two CLN7 BD patients and age-matched unaffected controls to interrogate intracellular molecular phenotypes in iPSC-derived neural progenitor cells (iNPC). Taking a multi-omics approach we have identified disease-modified activities in endolysosomal transport in iNPCBD that lead to a dysfunctional lysosome and decreased mitophagy resulting in the accumulation of metabolically defective mitochondria. We further observe a breakdown in nuclear functions that centres on RNA processing and nuclear export, which links to CLN7 protein interactions at the stress granule. We have identified dual and distinct functions for CLN7 promoting cell survival during the cellular stress response. CLN7 loss of function in BD results in neuronal apoptosis. Biological sciences/Neuroscience/Cellular neuroscience Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations Supplementary Table 7 is not available with this version. (Not answered) Supplementary Files SuppFig.1.jpg Supplementary Figure 1 SuppFig2A.jpg Supplementary Figure 2A SuppFig2B.jpg Supplementary Figure 2B SuppFig3.jpg Supplementary Figure 3 SuppFig4.jpg Supplementary Figure 4 SuppFig5.jpg Supplementary Figure 5 SuppTable13.jpg Supplementary Table 1-3 SuppTable4.jpg Supplementary Table 4 SuppTable56.jpg Supplementary Table 5-6 SuppTable8.jpg Supplementary Table 8 SuppTable8Cont.jpg Supplementary Table 8 (Cont) Cite Share Download PDF Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 23 Jun, 2025 Review # 1 received at journal 06 Jun, 2025 Reviewer # 1 agreed at journal 25 May, 2025 Reviewers invited by journal 14 May, 2025 Submission checks completed at journal 28 Apr, 2025 First submitted to journal 25 Apr, 2025 Unknown event 25 Apr, 2025 Editor assigned by journal 24 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6520859","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":456370377,"identity":"2eb919e8-2b8b-4476-b7eb-c072f7eb5e38","order_by":0,"name":"Tristan Tristan 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2","display":"","copyAsset":false,"role":"figure","size":1264902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6520859/v1/ec2863ae0b36c6b64cb1bed2.png"},{"id":82900078,"identity":"36bbe759-9bcf-4a8f-91a4-51f90d5a9f3f","added_by":"auto","created_at":"2025-05-16 13:23:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":533354,"visible":true,"origin":"","legend":"\u003cp\u003eUnbiased transcriptomic comparison of iNPCWT and iNPCBD . (A) Schematic workflow of long-read RNA sequencing. (Bi) Pathways including upregulated transcripts in iNPCBD compared to iNPCWT are ranked by z-score and -log(p-value). (Bii) p62 and the mTORC1 \u0026nbsp;downstream target p70S6K are upregulated at the protein level on western blot. (C) Pathways \u0026nbsp;including downregulated transcripts in iNPCBD compared to iNPCWT ranked by z-score and -log(p-value). (D) Volcano plot of transcript fold change (Log2) and significance (-Log10) for iNPCBD versus iNPCWT . (Ei \u0026amp; ii) The expression of top decile upregulated and downregulated transcripts \u0026nbsp;was evaluated in an independent experimental comparison of iNPCBD versus iNPCWT with 3 \u0026nbsp;biological replicates. All data presented as mean ± SEM, * p≤0.05, **≤0.01, ***≤0.001 and \u0026nbsp;****≤0.0001, (p-values in Table 8).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6520859/v1/2bb11ff97405d603c1dc70cf.png"},{"id":82901418,"identity":"75b7e953-ef9d-48fd-98c8-5997424bdacf","added_by":"auto","created_at":"2025-05-16 13:31:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":752594,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of autophagy in iNPCWT and iNPCBD . (A) Comparison of iNPCWT1 and \u0026nbsp;iNPCDBD1\u0026amp;2 with established markers of autophagy by dual immunocytochemistry. Images are \u0026nbsp;representative of 3 biological repeats. (B) Quantitation of autophagic marker staining was \u0026nbsp;conducted from a minimum of 6 images containing multiple cells using ImageJ software. Data is expressed as corrected total cell fluorescence (CTCF). Error bars indicate S.E.M., pairwise \u0026nbsp;comparisons were made using a one-way ANOVA with post-hoc Tukey’s test. All size bars=30µm.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6520859/v1/6f53ecd645bef15f56361cd6.jpg"},{"id":82900119,"identity":"4c543056-7dab-4fbd-aa6a-8de11297f680","added_by":"auto","created_at":"2025-05-16 13:23:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":628337,"visible":true,"origin":"","legend":"\u003cp\u003eAltered mitochondrial bioenergetics leading to apoptosis in iNPCBD . (A) Immunocytochemistry comparison of iNPCWT with iNPCBD1 and iNPCBD2 for the mitochondrial \u0026nbsp;marker ATP5A. Images are representative of 3 independent experiments. Size-bar=30µm. \u0026nbsp;Mitochondrial membrane potential was measured by TMRM (B, n=6) and mitochondrial super \u0026nbsp;oxide, quantified using MitoSOX (C, n=6), are elevated in iNPCBD1 compared to iNPCWT . \u0026nbsp;Mitochondrial respiration was assayed following 6h of Baf A (10 nM) or DMSO vehicle using the \u0026nbsp;Seahorse MitoStress test to quantify (D) ATP production and (E) maximal respiration in (i) iNPCBD1 \u0026nbsp;compared to iNPCWT1 (n=6) and (ii) Cln7∆ex2 compared to wild-type cortical neurons (n=4) using \u0026nbsp;the Seahorse MitoStress test. (F) Quantitation of NADH/NAD+ ratio by MTT assay in the presence \u0026nbsp;and absence of Baf A in iNPCBD1 compared to iNPCWT1 (n=6). (G) Apoptosis assay using Apotracker \u0026nbsp;staining of gated viable cells that exclude eFluor-780 dye in the presence or absence of BafA in \u0026nbsp;iNPCBD1 and iNPCBD2 compared to iNPCWT1 (n=3). All data presented as mean ± SEM with pairwise \u0026nbsp;comparisons using a one-tailed t-test. * p≤0.05, **≤0.01, ***≤0.001 and ****≤0.0001, (p-values \u0026nbsp;in Table 8).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6520859/v1/85b649691e6b7cc3e7bb500f.png"},{"id":82900115,"identity":"474bf2bd-9f42-4183-a65a-a5b3f8f12285","added_by":"auto","created_at":"2025-05-16 13:23:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":656066,"visible":true,"origin":"","legend":"\u003cp\u003eUnbiased global proteomics comparison of iNPCBD to iNPCWT with and without BafA \u0026nbsp;treatment. (A) Schematic workflow of iTRAQ mass spectrometry, proteomic quantification and downstream bioinformatic analyses. Proteins with differential regulation of \u0026gt;2-fold with a p-value\u0026lt;0.05 in iNPCBD versus iNPCWT were subjected to hierarchical Bayesian modelling then \u0026nbsp;pathway changes identified using Cytoscape gProfiler through Reactome Pathway. (Bi) \u0026nbsp;Upregulated protein pathways ranked by p-value and (Bii) separated by cellular localization. (Biii) \u0026nbsp;Upregulated protein pathways ranked by p-value following Baf A (10nM for 12h) treatment. (C) A \u0026nbsp;Minimal Essential Network identified in differentially upregulated proteins focussed around \u0026nbsp;vesicular transport. (Di) Downregulated protein pathways ranked by p-value and (Dii) separated \u0026nbsp;by cellular localization. (Diii) Downregulated protein pathways ranked by p-value following Baf A \u0026nbsp;(10nM for 12h) treatment. (E) A Minimal Essential Network identified from differentially \u0026nbsp;downregulated proteins focussed on RNA interactions.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6520859/v1/243165f0608f08e42faa2d3b.png"},{"id":82901414,"identity":"dab524e2-90c8-40f4-b4ad-22cc0394a912","added_by":"auto","created_at":"2025-05-16 13:31:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1074775,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of putative CLN7 associating proteins using co-immunoprecipitation \u0026nbsp;mass spectrometry in SH-SY5Y neuroblastoma cells. (A) Schematic workflow of CLN7 co-immunoprecipitation, mass spectrometry and downstream bioinformatics. (B) CLN7 co-immunoprecipitation from SH-SY5Y cell lysates is specific in identifying CLN7 (57kDa) and its N-glycosylated higher molecular weight forms on western blot alongside IgG light (~25kDa) and \u0026nbsp;heavy chain (50kDa) bands. A lower molecular weight band in cell lysates likely represents a \u0026nbsp;protease cleaved isoform. (C) Biocarta, Biological Process (BP), Cellular Component (CC) and \u0026nbsp;Molecular Function (MF) displays from DAVID analysis. (D) STRING analysis of putative CLN7 \u0026nbsp;interactors reveals associations with RNA processing and translation initiation. (E) co-localization of mScarlet-CLN7 fusion protein with the stress granule associating G3BP1-GFP \u0026nbsp;fusion protein. Images are of selected dual transfected cells, but representative of multiple \u0026nbsp;experiments. Size bar= 10µm. Pathway terms that are shared between the interactome and the \u0026nbsp;transcriptome, and proteome for upregulated (Fi) and downregulated (Fii) differential expression.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6520859/v1/6fa982eb9e3e09b868b5841a.png"},{"id":94904493,"identity":"9bcb8b76-f72b-4b02-b84b-3fa9dca8201c","added_by":"auto","created_at":"2025-11-01 07:09:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2356282,"visible":true,"origin":"","legend":"Article File","description":"","filename":"SharairehAetal.25.04.2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6520859/v1_covered_00c2a5e6-dd43-469f-955f-669fc11d3f75.pdf"},{"id":82900085,"identity":"64939557-2314-46bf-a30f-39b23ec28b93","added_by":"auto","created_at":"2025-05-16 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13:23:13","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":176694,"visible":true,"origin":"","legend":"Supplementary Table 8 (Cont)","description":"","filename":"SuppTable8Cont.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6520859/v1/b3991dad4916a1be841f3940.jpg"}],"financialInterests":"\u003cp\u003eSupplementary Table 7 is not available with this version.\u003c/p\u003e\n\u003cp\u003e(Not answered)\u003c/p\u003e","formattedTitle":"CLN7 protein functions at the interface between endolysosomes and stress granules to promote cell survival","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6520859/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6520859/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Inherited biallelic mutations in the CLN7 gene result in the variant late infantile onset neuronal ceroid lipofuscinosis, a subtype of Batten disease (BD), a severe and fatal childhood neurodegenerative disease. Intriguingly, CLN7 genetic variants have also been associated with retinopathies, amyotrophic lateral sclerosis, and frontotemporal dementia. CLN7 encodes a transmembrane protein localizing to endolysosomal membranes with outward-facing chloride channel activity. Loss of CLN7 function results in cortical neurons accumulating swollen lipofuscin-containing lysosomes, leading to neuroinflammation and neurodegeneration. The molecular mechanisms underlying CLN7 BD neuropathology are not understood. We have generated iPSC lines from two CLN7 BD patients and age-matched unaffected controls to interrogate intracellular molecular phenotypes in iPSC-derived neural progenitor cells (iNPC). Taking a multi-omics approach we have identified disease-modified activities in endolysosomal transport in iNPCBD that lead to a dysfunctional lysosome and decreased mitophagy resulting in the accumulation of metabolically defective mitochondria. We further observe a breakdown in nuclear functions that centres on RNA processing and nuclear export, which links to CLN7 protein interactions at the stress granule. We have identified dual and distinct functions for CLN7 promoting cell survival during the cellular stress response. 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