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Lee"],"software":[""],"tissue":["Brain"],"sample_protocol":["Approximately 5-10 mg of weighted frozen mouse brain tissue was resuspended in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NEM, 1% NP40 containing complete protease inhibitor cocktail (4693159001, Sigma-Aldrich) and phosSTOP (4906837001, Sigma-Aldrich), and homogenised for 30 seconds using an Omni TH220 tissue homogenizer. The tissue homogenate was probe-sonicated (10 s, setting 3, Branson Sonifier 450) and centrifuged at 14,000g for 30 mins at 4oC. The clarified supernatants were transferred to new tubes and protein quantification was carried out using BCA. Approximately 20 μg protein were denatured by boiling at 95°C for 5 minutes in Laemmli SDS Running buffer containing 40 mM dithiothreitol (DTT). Denatured proteins were separated by gel electrophoresis using 4–15% Tris-glycine gels (4565083, BioRad, CA, United States) according to manufacturer instructions for approximately 3-5 mins (1-3cm into the gel) and stained with Coomassie Brilliant Blue. Stained proteins were excised into 3-5 fractions for each sample and prepared for in-gel trypsin digestion as described previously. In-gel trypsin digestion  Each gel fraction was destained with 50 mM ammonium bicarbonate, 50% acetonitrile, followed by dehydration with 100% acetonitrile. The gel pieces were dried under vacuum centrifugation. Proteins were reduced with 10 mM DTT for 30 mins at 55oC, alkylated with 55 mM IAA for 60 mins at room temperature in the dark. Trypsin digestion (1:50 enzyme:protein) was added to the gel pieces and incubated overnight at 37 °C (V5111, Promega). Extracted peptides were lyophilized and then resuspended in 0.1% formic acid (FA) for desalting using C18 OMIX tips (Agilent). Samples were lyophilized again and resuspended in 0.1% FA, bath-sonicated for 20 min, and then centrifuged at 14,000× g for 15 min to remove insoluble debris, and analysed by LC-MS/MS. Reverse phase C18 liquid chromatography mass spectrometry (RP-LC-MS/MS) and Data Processing The Ultimate 3000 nanoLC (Thermo Fisher Scientific) fitted with the Acclaim PepMap RSLC column particle size of 2 μm, diameter of 0.075 mm, and length of 150 mm (Thermo Fisher Scientific) was used, employing a 60 min gradient (2–80% v/v ACN, 0.1% v/v FA) running at a flow rate of 300 nL/min to separate peptides. Subsequently, eluted peptides were ionized into the Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) that had an electrospray source fitted with an emitter tip 10 μm in diameter (New Objective) and maintained at 1.6 kV electrospray voltage. The capillary temperature was set to 250 °C. A data-dependent “Top 10” method operating in FT acquisition mode with HCD fragmentation was used for MS/MS fragmentation to select precursor ions. On the Q Exactive Plus, FT-MS analysis was carried out at 70,000-times resolution with an AGC target of 1 × 106 ions in full MS and a maximum injection time of 30 milliseconds. Additionally, MS/MS scans were carried out at 17,500-times resolution with an AGC target of 2 × 104 ions with the maximum injection time set to 50 milliseconds. The ion selection threshold was set to 25,000 counts to trigger MS/MS fragmentation. HCD fragmentation was performed using an isolation width of 2.0 Da with a normalized collision energy of 27."],"repository":["Pride"],"labhead":["Albert Lee"],"instrument_platform":[""],"submission_type":["COMPLETE"],"labhead_affiliation":["Motor Neuron Disease Research Centre, Macquarie University, NSW, 2113, Australia"],"publication":["10.1186/S12974-026-03827-X"],"submitter_affiliation":["Macquarie University"],"labhead_mail":["albert.lee@mq.edu.au"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Gel-based experiment","Bottom-up proteomics"],"disease":["Amyotrophic Lateral Sclerosis"],"submitter_keywords":["Brain","Mnd","Mouse","Neurodegeneration","Proteomics","Ccnf","Als","Cyclin f"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD064648"],"quantification_method":[""],"modification":[""],"data_protocol":["Raw files were searched with Proteome Discoverer 2.4 (Thermo Fisher Scientific) against Uniprot Mus musculus FASTA database (Release 2018_03, 28th March 2018, 16,929 sequences and Release 2024_07, 24th July 2024, 17196 sequences) incorporating the Sequest search algorithm. Search parameters accounted for 20 ppm precursor ion tolerance and 0.1 Da MS/MS fragment ion tolerance. The search allowed for static modifications of cysteine carbamidomethylation and variable modifications of methionine oxidation, asparginine, and glutamine deamidation on acetylated N-terminal residues. Two missed cleavages were allowed for. The data were processed through Percolator for estimation of false discovery rates. Protein identifications were validated employing a q-value of 0.05. Protein identification required at least one unique peptide per protein. Statistically significant differences in protein, peptide sequence, and peptide groups identified were calculated using Proteome Discoverer 2.4. High-confidence proteins with abundance ratios having p < 0.05 were selected and included for pathway analysis. An absolute expression fold-change cutoff of 2 was used to identify analysis-ready molecules"],"omics_type":["Proteomics"],"species":["Mus Musculus (mouse)"],"submitter_mail":["albert.lee@mq.edu.au"],"submitter_country":["Australia"],"doi":["10.6019/PXD064648"],"additional_accession":[]},"is_claimable":false,"name":"ALS-FTD-linked CCNFS621G Drives Increased Astrocyte Ramification, Alterations in the Proteome Related to Translation, Mitochondrial Function and Neuroinflammation, and Reduced Motor Neuron Excitability","description":"Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with interlinked pathophysiology. Mutations in CCNF, encoding the E3 ubiquitin ligase, Cyclin F, can cause either ALS or FTD or both, within the same family. The majority of previous published research on CCNFS621G in ALS/FTD has used overexpression of exogenous Cyclin F, and as a result may have encountered artefacts of pathology caused by dysregulation of native or wild-type Cyclin F. In this study, we generated the first reported mouse model of endogenous CcnfS621G using CRISPR/Cas9. Neither heterozygous nor homozygous CcnfS621G mice exhibited motor decline or neuronal loss within 18 months. However, through this period mice presented behavioural alterations congruent with early FTD phenotypes. Immunohistochemistry identified alterations in hippocampal astrocyte morphology, including increased ramification, and proteomic analyses identified significant alterations in pathways linked to translation, mitochondrial function, cytoskeletal remodeling and neuroinflammatory signaling. Consistent with this, increased astrocyte ramification was also identified in human sporadic ALS and ALS-FTD post mortem tissue, as well as in human CCNFS621G induced pluripotent stem cell (iPSC)-derived astrocytes. A common phenotype observed in familial and sporadic ALS is alterations in neuronal excitability and loss of repetitive action potential firing. ALS patients and cell and animal models display an early increase in neuronal excitability (hyperexcitability), followed by a decrease in excitability (hypoexcitability) as neurodegeneration progresses. Thus, we reasoned that alterations in CCNFS621G iPSC-derived astrocytes may impact neuronal excitability in co-cultures. CCNFS621G iPSC-derived motor neurons cultured without astrocytes exhibited increased action potential firing, compared to isogenic control neurons, consistent with neuronal hyperexcitability. However, the addition of CCNFS621G astrocytes, but not isogenic control astrocytes, abolished repetitive action potential firing in both CCNFS621G and isogenic control neurons. CCNFS621G astrocytes also increased the population of neurons that failed to fire action potentials and reduced voltage-gated sodium currents in either CCNFS621G or isogenic control neurons. Together, these results suggest that astrocyte activation is an early feature of neurodegenerative disease, occurring in the absence of neuronal loss in CcnfS621G mice and is present with region-specific severity in sporadic ALS and ALS-FTD post mortem tissue. Furthermore, astrocyte-mediated loss of repetitive firing could play a critical role in pre-symptomatic disease stages of CCNF-mediated ALS and FTD pathology.","dates":{"publication":"2026-05-07","submission":"2025-06-05"},"accession":"PXD064648","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:108018","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:1126","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:247480","NEWT:1211601","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:1367493","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:46429","NEWT:475174","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:2736291","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:294671","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:2086595","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:44250","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:3357","NEWT:7719","NEWT:29073","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:2767846","NEWT:108061","NEWT:60711","NEWT:2020713","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:1220585","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1916073","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:468822","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:342808","NEWT:2579791","NEWT:614666","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:99802","NEWT:1211620","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:709964","NEWT:242605","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:156974","NEWT:3714","NCBITaxon:37162","NEWT:3715","NEWT:2065263","NEWT:199225","NEWT:1549675","NEWT:177437","NEWT:717966","NEWT:9014","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:29022","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:96795","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:1883426","NEWT:8165","NEWT:96794","NEWT:10401","NEWT:10886","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:1955249","NEWT:215451","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:2714896","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:1608994","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:329032","NEWT:56636","NEWT:2499525","NEWT:55307","NEWT:1122981","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:2830","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:31345","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:141469","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:1578","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:291146","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:1116234","NEWT:415978","NEWT:235455","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:1092586","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:911022","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:717989","NEWT:33952","NEWT:1255232","NEWT:11723","NEWT:445","NEWT:153009","NEWT:1004879","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:1871049","NEWT:215402","NEWT:1547","NEWT:1290454","NE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