BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

• Keywords
• 3-sulfopropanoic acid, Aggregation, Alzheimer’s disease, Apolipoprotein E, Cerebral organoids, HDX-MS, Lipidomics, Molecular dynamics, Neurodegeneration, Protein crystallography, Proteomics, Tramiprosate,
• MeSH
• Alzheimer Disease * drug therapy   genetics   metabolism   MeSH
• Apolipoprotein E3 genetics   MeSH
• Apolipoprotein E4 * genetics   metabolism   MeSH
• Apolipoproteins E genetics   MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Apolipoprotein E3 MeSH
• Apolipoprotein E4 * MeSH
• Apolipoproteins E MeSH
• tramiprosate MeSH Browser

Cyanobacterial blooms are known sources of environmentally-occurring retinoid compounds, including all-trans and 9-cis retinoic acids (RAs). The developmental hazard for aquatic organisms has been described, while the implications for human health hazard assessment are not yet sufficiently characterized. Here, we employ a human neural stem cell model that can differentiate in vitro into a mixed culture of neurons and glia. Cells were exposed to non-cytotoxic 8-1000 nM all-trans or 9-cis RA for 9-18 days (DIV13 and DIV22, respectively). Impact on biomarkers was analyzed on gene expression (RT-qPCR) and protein level (western blot and proteomics) at both time points; network patterning (immunofluorescence) on DIV22. RA exposure significantly concentration-dependently increased gene expression of retinoic acid receptors and the metabolizing enzyme CYP26A1, confirming the chemical-specific response of the model. Expression of thyroid hormone signaling-related genes remained mostly unchanged. Markers of neural progenitors/stem cells (PAX6, SOX1, SOX2, NESTIN) were decreased with increasing RA concentrations, though a basal population remained. Neural markers (DCX, TUJ1, MAP2, NeuN, SYP) remained unchanged or were decreased at high concentrations (200-1000 nM). Conversely, (astro-)glial marker S100β was increased concentration-dependently on DIV22. Together, the biomarker analysis indicates an RA-dependent promotion of glial cell fates over neural differentiation, despite the increased abundance of neural protein biomarkers during differentiation. Interestingly, RA exposure induced substantial changes to the cell culture morphology: while low concentrations resulted in a network-like differentiation pattern, high concentrations (200-1000 nM RA) almost completely prevented such network patterning. After functional confirmation for implications in network function, such morphological features could present a proxy for network formation assessment, an apical key event in (neuro-)developmental Adverse Outcome Pathways. The described application of a human in vitro model for (developmental) neurotoxicity to emerging environmentally-relevant retinoids contributes to the evidence-base for the use of differentiating human in vitro models for human health hazard and risk assessment.

• Keywords
• Developmental neurotoxicity, Retinoid signaling, Thyroid hormone signaling,
• MeSH
• Alitretinoin * toxicity   MeSH
• Cell Differentiation MeSH
• Humans MeSH
• Neural Stem Cells * drug effects   MeSH
• Receptors, Retinoic Acid genetics   metabolism   MeSH
• Retinoids pharmacology   MeSH
• Tretinoin * toxicity   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alitretinoin * MeSH
• Receptors, Retinoic Acid MeSH
• Retinoids MeSH
• Tretinoin * MeSH