{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T01:51:53Z","timestamp":1781661113999,"version":"3.54.5"},"reference-count":51,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2025,10,17]],"date-time":"2025-10-17T00:00:00Z","timestamp":1760659200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JPMJPF2213"],"award-info":[{"award-number":["JPMJPF2213"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]},{"name":"JSPS KAKENHI","award":["JP24K08627"],"award-info":[{"award-number":["JP24K08627"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>This study focused on the abnormal phosphorylation of tau and its aggregation process, characteristic of Alzheimer\u2019s disease, and aimed to compare the morphology and formation process of phosphorylated tau aggregates produced by four kinases: Cdk5\/p25, GSK3\u03b2, MARK4, and p38\u03b1. Using quantum dots for 2D and 3D structural analysis, tau aggregates were confirmed in non-phosphorylated tau (non p-tau), as well as tau phosphorylated by GSK3\u03b2 and MARK4. Aggregation initiation times were observed around 72 h for non-p-tau, and around 96 h for GSK3\u03b2 and MARK4 phosphorylated tau. The thickness of non-p-tau aggregates was approximately 11 \u03bcm, while GSK3\u03b2 aggregates were significantly thicker (13 \u03bcm) and exhibited increased density. TEM analysis suggested that tau forming wavy filaments was less prone to forming large aggregates. ThT assays and CD spectra showed an increased \u03b2-sheet structure for all kinases. Non-p-tau and GSK3\u03b2 exhibited an increased right-twisted \u03b2-sheet structure, while Cdk5\/p25, MARK4, and p38\u03b1 showed an increased left-twisted \u03b2-sheet structure. The direct correlation between kinase activity and tau aggregate morphology revealed in this study provides a potential mechanistic basis for understanding disease heterogeneity and establishing novel therapeutic targets for AD specifically or for other neurodegenerative diseases as well.<\/jats:p>","DOI":"10.3390\/ijms262010122","type":"journal-article","created":{"date-parts":[[2025,10,17]],"date-time":"2025-10-17T13:07:22Z","timestamp":1760706442000},"page":"10122","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Aggregation Characteristics of Tau Phosphorylated by Various Kinases as Observed by Quantum Dot Fluorescence Imaging"],"prefix":"10.3390","volume":"26","author":[{"given":"Eisuke","family":"Ishibashi","sequence":"first","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koki","family":"Araya","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kota","family":"Nakamura","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keiya","family":"Shimamori","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1385-9001","authenticated-orcid":false,"given":"Koji","family":"Uwai","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4958-1286","authenticated-orcid":false,"given":"Masahiro","family":"Kuragano","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7747-4808","authenticated-orcid":false,"given":"Kiyotaka","family":"Tokuraku","sequence":"additional","affiliation":[{"name":"Graduate School of Engineering, Muroran Institute of Technology, Muroran 050-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1016\/S0140-6736(15)01124-1","article-title":"Alzheimer\u2019s Disease","volume":"388","author":"Scheltens","year":"2016","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Alzheimer\u2019s Association (2016). 2016 Alzheimer\u2019s Disease Facts and Figures. Alzheimers Dement., 12, 459\u2013509.","DOI":"10.1016\/j.jalz.2016.03.001"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1017\/S135561771700100X","article-title":"Alzheimer\u2019s Disease: Past, Present, and Future","volume":"23","author":"Bondi","year":"2017","journal-title":"J. Int. Neuropsychol. Soc."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1186\/s13024-021-00460-5","article-title":"\u201cDon\u2019t Phos Over Tau\u201d: Recent Developments in Clinical Biomarkers and Therapies Targeting Tau Phosphorylation in Alzheimer\u2019s Disease and Other Tauopathies","volume":"16","author":"Xia","year":"2021","journal-title":"Mol. Neurodegener."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5721","DOI":"10.1242\/jcs.01558","article-title":"Tau Phosphorylation in Neuronal Cell Function and Dysfunction","volume":"117","author":"Johnson","year":"2004","journal-title":"J. Cell Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1699","DOI":"10.1016\/j.cell.2020.10.029","article-title":"Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer\u2019s Disease","volume":"183","author":"Wesseling","year":"2020","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"S123","DOI":"10.3233\/JAD-2012-129031","article-title":"Abnormal Hyperphosphorylation of Tau: Sites, Regulation, and Molecular Mechanism of Neurofibrillary Degeneration","volume":"33","author":"Wang","year":"2013","journal-title":"J. Alzheimers Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.bj.2018.01.003","article-title":"Tau and Neuroinflammation: What Impact for Alzheimer\u2019s Disease and Tauopathies?","volume":"41","author":"Laurent","year":"2018","journal-title":"Biomed. J."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1126\/science.1072994","article-title":"The Amyloid Hypothesis of Alzheimer\u2019s Disease: Progress and Problems on the Road to Therapeutics","volume":"297","author":"Hardy","year":"2002","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/S1474-4422(12)70291-0","article-title":"Tracking Pathophysiological Processes in Alzheimer\u2019s Disease: An Updated Hypothetical Model of Dynamic Biomarkers","volume":"12","author":"Jack","year":"2013","journal-title":"Lancet Neurol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Fan, X., Xia, L., Zhou, Z., Qiu, Y., Zhao, C., Yin, X., and Qian, W. (2022). Tau Acts in Concert with Kinase\/Phosphatase Underlying Synaptic Dysfunction. Front. Aging Neurosci., 14.","DOI":"10.3389\/fnagi.2022.908881"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"463","DOI":"10.1002\/1097-4547(20001101)62:3<463::AID-JNR16>3.0.CO;2-7","article-title":"Phosphorylation of Human Tau Protein by Microtubule-Associated Kinases: GSK3\u03b2 and Cdk5 Are Key Participants","volume":"62","author":"Flaherty","year":"2000","journal-title":"J. Neurosci. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2197","DOI":"10.1038\/s41380-023-02113-z","article-title":"Does Modulation of Tau Hyperphosphorylation Represent a Reasonable Therapeutic Strategy for Alzheimer\u2019s Disease? From Preclinical Studies to the Clinical Trials","volume":"28","author":"Basheer","year":"2023","journal-title":"Mol. Psychiatry"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2181","DOI":"10.7150\/ijbs.57078","article-title":"Interaction between A\u03b2 and Tau in the Pathogenesis of Alzheimer\u2019s Disease","volume":"17","author":"Zhang","year":"2021","journal-title":"Int. J. Biol. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2321","DOI":"10.2174\/092986708785909111","article-title":"Hyperphosphorylation of Microtubule-Associated Protein Tau: A Promising Therapeutic Target for Alzheimer Disease","volume":"15","author":"Gong","year":"2008","journal-title":"Curr. Med. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e2217864120","DOI":"10.1073\/pnas.2217864120","article-title":"A Cdk5-Derived Peptide Inhibits Cdk5\/P25 Activity and Improves Neurodegenerative Phenotypes","volume":"120","author":"Pao","year":"2023","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Pao, P.-C., and Tsai, L.-H. (2021). Three Decades of Cdk5. J. Biomed. Sci., 28.","DOI":"10.1186\/s12929-021-00774-y"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Kimura, T., Ishiguro, K., and Hisanaga, S.-I. (2014). Physiological and Pathological Phosphorylation of Tau by Cdk5. Front. Mol. Neurosci., 7.","DOI":"10.3389\/fnmol.2014.00065"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4013","DOI":"10.1091\/mbc.02-03-0046","article-title":"Protein Kinase MARK\/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity","volume":"13","author":"Biernat","year":"2002","journal-title":"Mol. Biol. Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"966","DOI":"10.1093\/jnen\/59.11.966","article-title":"Microtubule-Affinity Regulating Kinase (MARK) Is Tightly Associated with Neurofibrillary Tangles in Alzheimer Brain: A Fluorescence Resonance Energy Transfer Study","volume":"59","author":"Chin","year":"2000","journal-title":"J. Neuropathol. Exp. Neurol."},{"key":"ref_21","first-page":"100259","article-title":"Gain-of-Function MARK4 Variant Associates with Pediatric Neurodevelopmental Disorder and Dysmorphism","volume":"5","author":"Samra","year":"2024","journal-title":"HGG Adv."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"fcae136","DOI":"10.1093\/braincomms\/fcae136","article-title":"Mark4 Ablation Attenuates Pathological Phenotypes in a Mouse Model of Tauopathy","volume":"6","author":"Sultanakhmetov","year":"2024","journal-title":"Brain Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2431","DOI":"10.1002\/j.1460-2075.1990.tb07419.x","article-title":"Molecular Cloning and Expression of Glycogen Synthase Kinase-3\/Factor A","volume":"9","author":"Woodgett","year":"1990","journal-title":"EMBO J."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Sayas, C.L., and \u00c1vila, J. (2021). GSK-3 and Tau: A Key Duet in Alzheimer\u2019s Disease. Cells, 10.","DOI":"10.3390\/cells10040721"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Germann, U.A., and Alam, J.J. (2020). P38\u03b1 MAPK Signaling-A Robust Therapeutic Target for Rab5-Mediated Neurodegenerative Disease. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21155485"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Detka, J., P\u0142achtij, N., Strzelec, M., Manik, A., and Sa\u0142at, K. (2024). P38\u03b1 Mitogen-Activated Protein Kinase-An Emerging Drug Target for the Treatment of Alzheimer\u2019s Disease. Molecules, 29.","DOI":"10.3390\/molecules29184354"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"eabl8809","DOI":"10.1126\/sciadv.abl8809","article-title":"Alzheimer\u2019s Disease: Ablating Single Master Site Abolishes Tau Hyperphosphorylation","volume":"8","author":"Stefanoska","year":"2022","journal-title":"Sci. Adv."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4231","DOI":"10.1038\/s41467-021-24362-8","article-title":"Co-Factor-Free Aggregation of Tau into Seeding-Competent RNA-Sequestering Amyloid Fibrils","volume":"12","author":"Chakraborty","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e76494","DOI":"10.7554\/eLife.76494","article-title":"Assembly of Recombinant Tau into Filaments Identical to Those of Alzheimer\u2019s Disease and Chronic Traumatic Encephalopathy","volume":"11","author":"Koh","year":"2022","journal-title":"eLife"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Sasaki, R., Tainaka, R., Ando, Y., Hashi, Y., Deepak, H.V., Suga, Y., Murai, Y., Anetai, M., Monde, K., and Ohta, K. (2019). An Automated Microliter-Scale High-Throughput Screening System (MSHTS) for Real-Time Monitoring of Protein Aggregation Using Quantum-Dot Nanoprobes. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-38958-0"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Tokuraku, K., Marquardt, M., and Ikezu, T. (2009). Real-Time Imaging and Quantification of Amyloid-Beta Peptide Aggregates by Novel Quantum-Dot Nanoprobes. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0008492"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"29609","DOI":"10.1074\/jbc.M302186200","article-title":"The Number of Repeat Sequences in Microtubule-Associated Protein 4 Affects the Microtubule Surface Properties","volume":"278","author":"Tokuraku","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1093\/jb\/mvm063","article-title":"An Isoform of Microtubule-Associated Protein 4 Inhibits Kinesin-Driven Microtubule Gliding","volume":"141","author":"Tokuraku","year":"2007","journal-title":"J. Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1093\/jb\/mvaa046","article-title":"Microtubule Elongation along Actin Filaments Induced by Microtubule-Associated Protein 4 Contributes to the Formation of Cellular Protrusions","volume":"168","author":"Doki","year":"2020","journal-title":"J. Biochem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1513","DOI":"10.1038\/nprot.2009.154","article-title":"Separation and Detection of Large Phosphoproteins Using Phos-Tag SDS-PAGE","volume":"4","author":"Kinoshita","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Ishigaki, Y., Tanaka, H., Akama, H., Ogara, T., Uwai, K., and Tokuraku, K. (2013). A Microliter-Scale High-Throughput Screening System with Quantum-Dot Nanoprobes for Amyloid-\u03b2 Aggregation Inhibitors. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0072992"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Lin, X., Galaqin, N., Tainaka, R., Shimamori, K., Kuragano, M., Noguchi, T.Q.P., and Tokuraku, K. (2020). Real-Time 3D Imaging and Inhibition Analysis of Various Amyloid Aggregations Using Quantum Dots. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21061978"},{"key":"ref_38","unstructured":"Uversky, V.N., and Lyubchenko, Y.L. (2014). Bio-Nanoimaging Protein Misfolding and Aggregation, Elsevier."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1186\/s12964-021-00704-3","article-title":"Baicalein Inhibits Heparin-Induced Tau Aggregation by Initializing Non-Toxic Tau Oligomer Formation","volume":"19","author":"Sonawane","year":"2021","journal-title":"Cell Commun. Signal."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1016\/j.neulet.2018.11.011","article-title":"Phosphorylation of Serine 305 in Tau Inhibits Aggregation","volume":"692","author":"Strang","year":"2019","journal-title":"Neurosci. Lett."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"5915","DOI":"10.1074\/jbc.M304528200","article-title":"MARK4 Is a Novel Microtubule-Associated Proteins\/Microtubule Affinity-Regulating Kinase That Binds to the Cellular Microtubule Network and to Centrosomes","volume":"279","author":"Trinczek","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e2414176121","DOI":"10.1073\/pnas.2414176121","article-title":"GSK3\u03b2 Phosphorylation Catalyzes the Aggregation of Tau into Alzheimer\u2019s Disease-like Filaments","volume":"121","author":"Chakraborty","year":"2024","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1038\/s41586-021-03911-7","article-title":"Structure-Based Classification of Tauopathies","volume":"598","author":"Shi","year":"2021","journal-title":"Nature"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.sbi.2020.05.011","article-title":"Cryo-EM Structures of Tau Filaments","volume":"64","author":"Scheres","year":"2020","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Jin, N., Yin, X., Yu, D., Cao, M., Gong, C.-X., Iqbal, K., Ding, F., Gu, X., and Liu, F. (2015). Truncation and Activation of GSK-3\u03b2 by Calpain I: A Molecular Mechanism Links to Tau Hyperphosphorylation in Alzheimer\u2019s Disease. Sci. Rep., 5.","DOI":"10.1038\/srep08187"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1016\/S0014-5793(02)03487-7","article-title":"Involvement of Aberrant Glycosylation in Phosphorylation of Tau by cdk5 and GSK-3\u03b2","volume":"530","author":"Liu","year":"2002","journal-title":"FEBS Lett."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1093\/emboj\/20.1.27","article-title":"Decreased Nuclear \u03b2-Catenin, Tau Hyperphosphorylation and Neurodegeneration in GSK-3\u03b2 Conditional Transgenic Mice","volume":"20","author":"Lucas","year":"2001","journal-title":"EMBO J."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/0896-6273(93)90279-Z","article-title":"Phosphorylation of Ser262 Strongly Reduces Binding of Tau to Microtubules: Distinction between PHF-like Immunoreactivity and Microtubule Binding","volume":"11","author":"Biernat","year":"1993","journal-title":"Neuron"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"17138","DOI":"10.1074\/jbc.RA120.014420","article-title":"Microtubule Affinity-Regulating Kinase 4 with an Alzheimer\u2019s Disease-Related Mutation Promotes Tau Accumulation and Exacerbates Neurodegeneration","volume":"295","author":"Oba","year":"2020","journal-title":"J. Biol. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0021-9258(19)52451-6","article-title":"Protein Measurement with the Folin Phenol Reagent","volume":"193","author":"Lowry","year":"1951","journal-title":"J. Biol. Chem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/20\/10122\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,21]],"date-time":"2025-10-21T04:26:52Z","timestamp":1761020812000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/20\/10122"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,17]]},"references-count":51,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["ijms262010122"],"URL":"https:\/\/doi.org\/10.3390\/ijms262010122","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,17]]}}}