{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:12:16Z","timestamp":1785413536779,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,11]]},"group-title":"Neuroscience","reference-count":63,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T00:00:00Z","timestamp":1781136000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"id":[{"id":"https:\/\/ror.org\/0185drb55","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32AG000222-27"],"award-info":[{"award-number":["T32AG000222-27"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["5R56AG080525"],"award-info":[{"award-number":["5R56AG080525"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1R01AG086914"],"award-info":[{"award-number":["1R01AG086914"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P30AG062421"],"award-info":[{"award-number":["P30AG062421"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/05nzwyq50","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Alzhimer's san diego"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Apolipoprotein E (ApoE) is the primary genetic risk modifier of late-onset Alzheimer\u2019s disease, with the \u03b54 allele increasing risk up to 15-fold relative to \u03b53. The structural differences between isoforms are thought to underlie their distinct effects on lipid transport, receptor binding, and disease risk. ApoE4 exhibits reduced thermodynamic stability compared to ApoE3, but prior characterisation has relied on purified recombinant protein, leaving open whether these differences are preserved in native cellular environments and how they relate to rare disease-associated variants. Here, we employed the cellular thermal shift assay (CETSA) and a bioluminescence-based thermal stability assay (BiTSA) to systematically characterise ApoE thermal stability across isoforms and variants. Using CETSA on brain tissue from humanised APOE knock-in mice and post-mortem human brain, we confirm that ApoE4 exhibits significantly reduced thermal stability compared to ApoE3 in native tissue, with this difference conserved across species despite variation in absolute melting temperatures. We developed BiTSA, which leverages a split-luciferase HiBiT tag to quantify soluble ApoE across a thermal gradient in living cells, providing a higher-throughput platform that faithfully recapitulates isoform stability differences. Applying BiTSA to rare AD-associated variants, we found that L28P exerts divergent, isoform-dependent effects, destabilising ApoE3 while paradoxically stabilising ApoE4\u2014a finding supported by AlphaFold modelling revealing isoform-specific differences in helix 1 architecture. These results establish BiTSA as a robust cellular tool for ApoE variant characterisation and demonstrate that isoform background critically modulates the structural consequences of rare mutations.<\/jats:p>","DOI":"10.64898\/2026.06.09.730809","type":"posted-content","created":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T22:10:16Z","timestamp":1781215816000},"source":"Crossref","is-referenced-by-count":0,"title":["Utilizing a cell culture based novel cellular thermal shift assay to understand the isoform-dependent thermal stability of ApoE variants"],"prefix":"10.64898","author":[{"given":"Rosemary J.","family":"Jackson","sequence":"first","affiliation":[{"name":"Department of Neuroscience, Faculty of Medicine, University of Dundee, Dundee UK"},{"name":"Alzheimer Research Unit, The Massachusetts General Hospital Institute for Neurodegenerative Disease, Charlestown, MA, USA"},{"name":"Department of Neurology, The Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven","family":"Dierksmeier","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, The Massachusetts General Hospital Institute for Neurodegenerative Disease, Charlestown, MA, USA"},{"name":"Department of Neurology, The Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maryam","family":"Nishtar","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, The Massachusetts General Hospital Institute for Neurodegenerative Disease, Charlestown, MA, USA"},{"name":"Department of Neurology, The Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonah","family":"Meltzer","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, The Massachusetts General Hospital Institute for Neurodegenerative Disease, Charlestown, MA, USA"},{"name":"Department of Neurology, The Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frieda","family":"Balduin","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, The Massachusetts General Hospital Institute for Neurodegenerative Disease, Charlestown, MA, USA"},{"name":"Department of Neurology, The Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bobby","family":"Beaumont","sequence":"additional","affiliation":[{"name":"Department of Neuroscience, Faculty of Medicine, University of Dundee, Dundee UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhanyun","family":"Fan","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, The Massachusetts General Hospital Institute for Neurodegenerative Disease, Charlestown, MA, USA"},{"name":"Department of Neurology, The Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eduard","family":"Sergienko","sequence":"additional","affiliation":[{"name":"Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery Institute,La Jolla, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steve","family":"Olson","sequence":"additional","affiliation":[{"name":"Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery Institute,La Jolla, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael R.","family":"Jackson","sequence":"additional","affiliation":[{"name":"Conrad Prebys Center for Chemical Genomics, Sanford Burnham Prebys Medical Discovery Institute,La Jolla, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7959-9401","authenticated-orcid":false,"given":"Bradley T.","family":"Hyman","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, The Massachusetts General Hospital Institute for Neurodegenerative Disease, Charlestown, MA, USA"},{"name":"Department of Neurology, The Massachusetts General Hospital and NeuroDiscovery Center, Harvard Medical School, Boston, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026061514001285000_2026.06.09.730809v1.1","doi-asserted-by":"publisher","DOI":"10.1126\/science.3283935"},{"key":"2026061514001285000_2026.06.09.730809v1.2","doi-asserted-by":"crossref","unstructured":"Liu, C. C. , Kanekiyo, T. , Xu, H. & Bu, G . Apolipoprotein E and Alzheimer disease: risk, mechanisms, and therapy. Nature reviews. Neurology 9, 106 (2013).","DOI":"10.1038\/nrneurol.2012.263"},{"key":"2026061514001285000_2026.06.09.730809v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)52510-8"},{"key":"2026061514001285000_2026.06.09.730809v1.4","doi-asserted-by":"publisher","DOI":"10.1126\/science.8346443"},{"key":"2026061514001285000_2026.06.09.730809v1.5","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.90.5.1977"},{"key":"2026061514001285000_2026.06.09.730809v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/0896-6273(93)90070-8"},{"key":"2026061514001285000_2026.06.09.730809v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/ng0694-180"},{"key":"2026061514001285000_2026.06.09.730809v1.8","unstructured":"Reiman, E. M. et al. Exceptionally low likelihood of Alzheimer\u2019s dementia in APOE2 homozygotes from a 5,000-person neuropathological study. 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