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Cell pellets were reconstituted in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 % NP40, 0.5 % sodium deoxycholate, 0.1 % SDS, 10 mM EGTA, 10 mM MgCl2,) containing 1% phosphatase inhibitor cocktails 1 and 3 (Sigma), 2% Complete EDTA-free (Roche) and a nuclease cocktail composed by Benzonase (≥ 250 units/ µL, Millipore), Micrococcal nuclease (229 units/ µL Thermo Scientific) and RNase (10 mg/ml, Sigma-Aldrich) added at 1 µL per 10 mL RIPA buffer. Protein concentration of supernatants was determined by using the BioRad protein assay (BioRad laboratories). Automated protein purification and trypsin digestion were performed using a KingFisher Flex system (Thermo Scientifics) using 96 deep-well plates. For each sample, 25 μg protein were mixed with SDC buffer (1% Sodium deoxycholate, 10 mM TCEP (tris(2-carboxyethyl)phosphine)), 40 mM Chloroacetamide, 100 mM Tris-HCl pH8.5), for simultaneous protein reduction and alkylation for 30 min. Protein binding to MagReSyn HILIC microspheres (20 μg, resyn biosciences), which were pre-equilibrated in 15 % acetonitrile (ACN) 100 mM ammonium acetate (NH4Ac) pH 4.5 buffer, was performed in binding buffer (0.2 % SDS, 30 % ACN, 200 mM NH4Ac). After washes in 95 % ACN, on bead trypsin digestion was performed in 15 mM NH4HCO2 pH 8.2 containing 5 ng/μL trypsin protease MS-grade (Thermo Scientific) for 6 hours at 37oC. Samples were dried out in a speed vac, reconstituted in 45 μL of 0.1 % FA, centrifuged for 30 minutes at 16000 g and 4 oC, and supernatants were transferred to HPLC vials."],"repository":["Pride"],"modification":[""],"quantification_method":["Not available"],"data_protocol":["LC-MS/MS analysis was performed on a timsTOF Pro 2 (Bruker Daltonics) connected to a nanoElute (Bruker Daltonics) HPLC. Peptide separation was performed using a Bruker25 (150um*25cm) column with running mobile phases A (0.1% formic acid) and B (0.1% formic acid in acetonitrile). The nano-flow LC gradient was delivered at 250 nL/min and consisted of a 120 minute gradient starting with mobile phase B increasing from 2% to 17% B in 60 min, followed by increases to 25% B in 30 min, 37% in 10 min, and to 95% B in 10 min, where it was hold for 10 min. HPLC was coupled to MS by a Captiva Ion Source with a ZDV Sprayer with ID 20 um (Bruker Daltonics). The timsTOF instrument was operated in the DDA PASEF mode with 10 PASEF scans per acquisition cycle and accumulation and ramp times of 100 ms each. MS and MS/MS data were acquired over an m/z range of 100–1,700. The ‘target value’ was set to 20,000 and dynamic exclusion was activated and set to 0.4 min. The quadrupole isolation width was set to 2 Th for m/z < 700 and 3 Th for m/z > 800 (77, 78). Database search was performed with MaxQuant. Open workflow (79) was used to inspect the raw data to determine optimal search parameters for MaxQuant v 2.0.3.0 (80). Along with defaults, deamidation of asparagine/glutamine as dynamic post-translational modification was appended. Search performed against the Human proteome including isoforms downloaded from Uniprot (https://www.uniprot.org/proteomes/UP000005640) in June 2022 and MaxQuant’s internal contaminants database using Andromeda built into MaxQuant. Match-between-runs (MBR) functionality is utilized to reduce missing values in raw data. MBR verifies the presence of a peptide signal in one sample that wasn’t identified in another. This is achieved by utilizing high mass precision and a restricted retention time period of 1-minute with 20-minutes overall sliding window. If this particular signal is detected (MS1 intensity threshold 30), it is labeled as ‘By matching’ instead of ‘By MS/MS’ in the output. Peptide group identifications false discovery rate was kept at 1% and only unique peptides with high confidence were used for downstream protein group identification. The peptide intensity data was cleaned, log-transformed, normalized by robust quantiles, following to be aggregated into protein level (by “robustSummary” method in the function of “aggregateFeatures”) using a R package Qfeatures (v 1.12.0), according to Sticker, A. et al. (81). The DE proteins were identified by a linear regression model in limma package (v 3.58.1).  The significant DE protein was defined by the log2FoldChange > 1 and adjusted p_value < 0.05."],"omics_type":["Proteomics"],"labhead":["Mirta Mittelstedt Leal de Sousa"],"instrument_platform":[""],"labhead_affiliation":["Senior Forsker Institutt for klinisk og molekylær medisin Laboratoriesenteret, 231.05.032, Øya, Erling Skjalgssons g 1 Department of Cancer Research and Molecular Medicine, NTNU, Norway."],"species":["Homo Sapiens (human)"],"submission_type":["PARTIAL"],"publication":["42129767 Yang M, Wang W, Cámara-Quílez M, Farsund BH, Andersen NN, Garten K, Sharma A, Lin X, Åmellem I, Ravlo E, Ye J, Bjørås M, de Sousa MML. Modeling CLN3 Batten disease in astrocytes reveals alterations in mitochondria homeostasis, fatty acid metabolism and oxidative stress response. J Biomed Sci. 2026 33(1):50 10.1186/s12929-026-01253-y"],"submitter_mail":["sharma.animesh@gmail.com"],"submitter_affiliation":["Engineer at NTNU, Norway"],"submitter_country":["India"],"pubmed_abstract":["<h4>Background</h4>CLN3 Batten disease is a severe pediatric neurodegenerative disorder caused by mutations in the CLN3 gene, most commonly a 1 kb deletion encompassing exons 7 and 8. CLN3 deficiency is associated with lysosomal dysfunction, impaired cellular clearance and disrupted metabolism. While neurons are particularly vulnerable in CLN3 Batten disease and have been the primary focus of research, glial cells are increasingly recognized as active contributors to disease pathology. Among them, astrocytes-the most abundant glial cell type in the brain-play critical roles in maintaining neuronal health and homeostasis. However, astrocytes remain understudied in CLN3 patient-derived models.<h4>Methods</h4>We present the first iPSC-derived astrocyte model from a skin biopsy of a CLN3 patient carrying the common 1 kb deletion. Cellular and molecular features of iPSC and astrocytes derived from both healthy controls and the CLN3 patient were characterized via qPCR, immunocytochemistry and targeted mass spectrometry. In addition, comprehensive omics-based profiling, through transcriptomic and label-free quantitative proteomics, was performed to uncover novel molecular mechanisms and generate hypotheses that can guide future mechanistic and functional studies.<h4>Results</h4>Transcriptomic and proteomic analyses during astrocyte differentiation revealed an upregulation of mitochondrial respiratory chain complexes I and IV-contrasting with the downregulation typically observed in CLN3-deficient neurons. We also identified a metabolic shift favoring the elongation of very-long-chain saturated fatty acids, accompanied by reduced lipid synthesis and enhanced fatty acid oxidation. These metabolic alterations were paralleled by an upregulation of proteins involved in oxidative stress responses, likely reflecting a compensatory adaptation to mitochondrial and lipid metabolic dysregulation. Furthermore, we observed significant changes in chromatin organization during astrocyte differentiation in CLN3 cells, suggesting epigenetic remodeling as a contributing factor to disease pathology.<h4>Conclusion</h4>Our findings prompt the hypothesis that mitochondrial dysfunction may precede lysosomal defects in CLN3-deficient astrocytes. Restoring mitochondrial health could improve brain metabolism, inflammation control, neurotransmitter regulation, and neuronal survival, highlighting mitochondria as promising therapeutic targets in CLN3 Batten disease."],"pubmed_title":["Modeling CLN3 Batten disease in astrocytes reveals alterations in mitochondria homeostasis, fatty acid metabolism and oxidative stress response."],"pubmed_authors":["Yang Mingyi M, Wang Wei W, Cámara-Quílez María M, Farsund Borghild Hvesser BH, Andersen Niklas Nonboe NN, Garten Karin K, Sharma Animesh A, Lin Xiaolin X, Åmellem Ingrid I, Ravlo Erlend E, Ye Jing J, Bjørås Magnar M, de Sousa Mirta Mittelstedt Leal MML"],"additional_accession":[]},"is_claimable":false,"name":"Modeling CLN3 Batten disease in astrocytes reveals dysfunctions in mitochondria, fatty acid metabolism and chromatin dynamics","description":"CLN3 Batten disease is a severe pediatric neurodegenerative disorder caused by mutations in the CLN3 gene, most commonly a 1 kb deletion affecting exons 7 and 8. While research has focused on neuronal dysfunction, glial cells are increasingly recognized as key contributors to disease pathology. Among them, astrocytes, the most abundant glial cell type in the brain, are essential for neuron health and may play critical roles in disease progression. Here, we present the first iPSC-derived astrocyte model from a patient with the 1 kb deletion. Transcriptomic and proteomic analyses during astrocyte differentiation revealed significant disruptions in mitochondrial function, fatty acid metabolism, and oxidative stress, with minimal impact on lysosomal homeostasis. These findings prompt the hypothesis that mitochondrial dysfunction may precede lysosomal defects in CLN3-deficient astrocytes. Restoring mitochondrial health could improve brain metabolism, inflammation control, neurotransmitter regulation, and neuronal survival, highlighting mitochondria as promising therapeutic targets in CLN3 Batten disease.","dates":{"publication":"2026-06-08","submission":"2025-05-22"},"accession":"PXD064202","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2042546","NEWT:148942","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:44270","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:661410","NEWT:488003","NEWT:224326","NEWT:376619","NEWT:3765","NEWT:2433","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:44250","NEWT:317447","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:1177","NEWT:11308","NEWT:12637","NEWT:2164133","NEWT:1180","NEWT:2767846","NEWT:1343074","NEWT:314344","NEWT:108061","NEWT:224308","NEWT:49928","NEWT:160621","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:1410384","NEWT:1310173","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:135666","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:65121","NEWT:3716","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:118221","NEWT:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:1423687","NEWT:657322","NEWT:10886","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:1301262","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:28196","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:272831","NEWT:987059","NEWT:491","NEWT:422203","NEWT:460523","NEWT:2423","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:291146","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:476","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:63363","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:2003758","NEWT:29486","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:1325292","NEWT:219798","NEWT:548","NEWT:150475","NEWT:546","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:8673","NEWT:550","NEWT:10804","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:40686","NEWT:1225786","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:536","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:51315","NEWT:52641","NEWT:353152","NEWT:116597","NEWT:353153","NEWT:116598","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:13037","NEWT:519","NEWT:941638","NEWT:511","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:13064","NEWT:529120","NEWT:300641","NEWT:39655","NEWT:38323","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639","NEWT:52638","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:1196866","NEWT:6039","NEWT:1045775","NEWT:1123869","NCBITaxon:1335626","NEWT:7370","NEWT:280406","NEWT:83906","NEWT:99488","NEWT:99487","NEWT:1134506","NEWT:255470","NEWT:762903","NEWT:27697","NEWT:86185","NEWT:9935","NEWT:266940","NEWT:287889","NEWT:9938","NEWT:1028307","NEWT:52603","NEWT:92867","NEWT:92866","NEWT:1344414","NEWT:202950","NEWT:741158","NEWT:1293091","NEWT:202956","NEWT:483179","NEWT:315750","NEWT:99490","NEWT:632957","NEWT:265619","NEWT:11294","NEWT:754466","NEWT:9925","NCBITaxon:9606","NEWT:1436183","NEWT:1479235","NEWT:4232","NEWT:9922","NEWT:416348","NEWT:105812","NEWT:11298","NEWT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