The main results of the project can be summarized as follows:
a) We showed that apoE4 and its pathogenic variants (apoE4[L28P], apoE4-165) exert differential, cell-type-specific effects on AD pathologic processes, underscoring the intricate interplay between apoE4, Aβ, tau and inflammatory responses. Furthermore, we found that specific diet-originating fatty acids can ameliorate AD-related pathogenic effects of apoE4 in brain cells and may have a role against AD development or progression. Specifically, we showed that short-chain fatty acids, which are the main metabolites generated by gut fermentation of dietary fibers and display important AD-related biological effects, effectively reduced all the studied AD-related pathogenic functions of lipid-free apoE4 variants.
b) We examined whether there is a structural basis behind the properties of the protective apoE variants (R136S, V236E and R251G), by analyzing their structural and thermodynamic integrity in APOE3 and APOE4 allelic background and in comparison to their wild-type counterparts. CD spectroscopy showed that only the V236E variation significantly alters the secondary structure of apoE3 and apoE4 in lipid-free form. This variant was also less prone to oligomerization. Chemical denaturation analysis indicated changes in the unfolding profile of V236E and R251G apoE variants in lipid-free form. Thermal unfolding analysis revealed small thermodynamic alterations in each variant compared to their wild-type apoE counterparts in lipid-free form, but a thermodynamic stabilization in lipoprotein-associated form. Additionally, following lipidation, all protective apoE variants were found to enhance the viability of SK-N-SH neuroblastoma cells and reduce the production of TNFα from BV2 microglia cells. Overall, these findings suggest that the specific amino acid substitutions found in AD-protective apoE variants can induce changes in the molecule’s stability and conformation that may underlie common functional consequences, which are independent of the apoE background.
c) We performed a longitudinal study using humanized apoE-targeted replacement mouse lines (apoE3TR and apoE4TR) to assess how ApoE variants influence Alzheimer’s disease (AD)-related outcomes. Biochemical analyses of brain tissue showed that hippocampal TNFα was significantly elevated in apoE4TR females, suggesting a sex-specific increase in inflammatory signaling associated with ApoE4. Overall, the findings suggest that ApoE4 alone does not produce measurable cognitive impairment under baseline conditions, but it may contribute to sex-dependent inflammatory and oxidative stress changes in the brain.
d) We showed that under a high fat diet ApoE4TR female mice emerged as the most vulnerable cohort, exhibiting robust obesity susceptibility and a persistent “metabolic memory” of elevated lipids even after returning to a regular diet. Cognitively, the diet impaired spatial working memory in both sexes, but selectively disrupted non-spatial recognition and recency memory in females, particularly those with the ApoE4 genotype. At the cellular level, these cognitive failures correlated with increased microglial and astrocytic reactivity, significant synaptic depletion, and localized pTau accumulation within the prefrontal cortex and hippocampus. Ultimately, these findings suggest that the ApoE4 genotype “primes” the brain for an aggressive neuroinflammatory response to dietary stress, leading to a structural weakening of the prefrontal cortex-hippocampal axis and accelerated neurocognitive decline.
e) We showed that overexpression of human ApoC-III in mice induces severe hypertriglyceridemia and that introducing this dyslipidemic state into ApoE3- or ApoE4-targeted replacement mice produces genotype-dependent effects on cognition and brain inflammation relevant to Alzheimer’s disease. ApoC-III improved novel object recognition in ApoE3TR mice while significantly impairing temporal order recognition in ApoE4TR mice, suggesting ApoE4-specific vulnerability of certain memory circuits. Preliminary brain data showed ApoC-III expression in multiple regions (especially hippocampus and cerebellum) along with microglial activation markers consistent with a pro-inflammatory phenotype, although hippocampal TNF-α levels were unchanged. Overall, the findings support ApoC-III–driven dyslipidemia as a potential modifier of ApoE-related Alzheimer’s risk through metabolic and immune mechanisms.
f) We performed chromatin immunoprecipitation experiments in brain tissues from the ApoE3TR and ApoE4TR mice and identified chromatin regions that interact with ApoE3 or ApoE4 in the brains of ApoETR mice.
g) We identified intracellular proteins that interact with ApoE3 or ApoE4 in the cells by performing immunoprecipitation followed by proteomics analysis. LC-MS/MS analysis identified 46.928 peptides that correspond to 3.720 individual proteins, which were immunoprecipitated with the ApoE ab relative to the control IgG. Using bioinformatics, we identified the apoE-interacting proteins with the highest statistical significance and fold-difference in each group of comparison and classified these proteins in different pathways, processes and diseases.
In conclusion, apoE4 contributes to Alzheimer’s disease through complex, cell-type-specific effects on Aβ, tau phosphorylation, inflammation, and brain lipid homeostasis, and these effects can be significantly shaped by metabolic comorbidities such as dyslipidemia and high-fat diet. The findings support a synergistic relationship between apoE4 genetic background and metabolic stress, with apoE4-targeted replacement mice showing diet-related cognitive impairments and inflammatory changes. Importantly, this work also reveals non-canonical nuclear roles of apoE in microglia, including regulation of TNFα transcription and direct chromatin interactions, adding a new layer of mechanistic complexity. Finally, structural and functional analyses of rare apoE variants suggest that specific single amino acid changes can stabilize apoE and produce protective effects, highlighting apoE variants and diet-related metabolites (especially short-chain fatty acids) as promising tools and targets for future AD therapies