X33 - ApoE4 Mutation alters the Inflammatory and Metabolic Properties of Extracellular Vesicles

Introduction: : Alzheimer's Disease is a leading cause of dementia, leading to 60-80% of all cases in the United States. Apolipoprotein E (ApoE) is a lipid transporter whose mutant form, ApoE4, is the strongest predictor of Alzheimer’s disease development. ApoE’s role as a lipid transporter is disrupted in the E4 mutant due to differences in its binding affinity, altering ApoE’s preferred substrate to vLDLs from the HDLs that ApoE3 prefers. This alters the cholesterol homeostasis of neural cells such as astrocytes, leading to fatty acid buildup and lipid droplet formation, which contribute to neuroinflammation. Amyloid β (Aβ), a cytotoxic protein that is believed to be one of the main mediators of neurotoxicity in Alzheimer’s Disease, serving as a seeding spot to promote Aβ fibril formation and promoting neurodegeneration.
ApoE mutation also affects endosome formation and trafficking, leading to dysregulation of lysosomal activity. This also affects the formation of extracellular vesicles through the disruption of ESCRT activity. In addition, ApoE has been identified as a cargo protein carried within exosomes, suggesting exosomes also play a role in regulating neural lipid metabolism. However, the extent and further results of ApoE disruption of exosome excretion and cargo are not well- established. The extent by which multivesicular body formation and packing of proteins and miRNAs are not understood, though disruptions in communication can have profound impacts. This study seeks to identify differences in exosome secretion and bioactivity between ApoE3 and ApoE4 genotypes in various neurological cell types and the role they play in Alzheimer’s Disease pathogenesis.

Materials and

Methods: : Isogenic iPSCs (ApoE3 vs. ApoE4) differentiated into astrocytes (iAS) and mesenchymal stem cells (iMSC). Cells underwent flow cytometry to confirm they were of the correct identity. Conditioned media from mature cultures was collected and underwent a PEG precipitation protocol to isolate exosomes. The final exosome pellet was resuspended and underwent nanoparticle tracking analysis to identify exosome size profile and concentration. An in vitro model of neuroinflammation was generated by treating THP-1 monocytes containing a NF- luciferase reporter with LPS for 6 hours. After LPS activation, exosomes from astrocytes or MSCs with different ApoE genetic backgrounds were added to THP-1 culture overnight before adding a luciferase substrate to measure relative activation of NF-Κβ mediated inflammation using a microplate reader. Additionally, exosomes were added to THP-1 cells that were not activated with LPS to assess any differences in innate inflammatory properties that may arise from differences in ApoE genotype and measures as before.
Because of the importance of ApoE in mediating fatty acid metabolism, oleic acid was added to astrocyte cultures from both genotypes. DMSO was also added to astrocyte cultures from each genotype as a vehicle control. After 3 days of treatment, the conditioned media from each culture was collected and used for exosome isolation and characterization as before. Exosomes from each culture were added to THP-1 cells left untreated with LPS to determine how differences in fatty acid metabolism affect the inflammatory properties of exosomes in ApoE4 mutant astrocytes. Data was then analyzed and plotted using GraphPad Prism.

Results, Conclusions, and Discussions:: Initial measurements of EV secretion found that in astrocytes, EV secretion is significantly higher in E3 homozygous cells compared to E4 mutants. No difference can be observed between genotypes in MSCs (Figure A,B). When treating THP-1 cells, ApoE4 derived EVs hold an innate inflammatory capability across cell types, increasing the levels of NF-Kß activation significantly compared to untreated controls (Figure C,D). Additionally, EVs from ApoE3 astrocytes suppressed NF- Kß activation when THP-1 cells were pretreated with LPS, a feature not observed in ApoE4 derived EVs (Figure E). The addition of oleic acid to ApoE3 and ApoE4 parent cells significantly increased secretion in both genotypes, though ApoE3 retained higher exosome secretion than ApoE4 in both conditions (Figure F). Additionally, the addition of oleic acid increased the degree by which ApoE4 astrocyte exosomes induce NF-Kß activation, while exosomes from ApoE3 astrocytes did not significantly increase NF-Kß activation when pretreated with oleic acid (Figure G).
Our current results suggest that ApoE4 mutation influences the levels of secretion from astrocytes, which is hypothesized to be a result of disrupted endosomal trafficking via interruptions in ESCRT pathways. It is also possible that altered cell membrane composition resulting from disrupted lipid metabolism in ApoE4 mutants could affect ESCRT- independent exosome formation. In addition, ApoE4- mutant derived exosomes have innate inflammatory properties that are exacerbated in the presence of excess fatty acid. This suggests that exosomes are at least partially responsible for the inflammation observed in Alzheimer’s disease. It is unsure whether this increase in inflammation is due to the cargo contained within exosomes or if the lipid profile of the exosomes’ bilayers increases the lipid burden in receiving cells. Moving forward, we are seeking to establish how ApoE4 mutants disrupt exosome formation by PCR and Western blotting for ESCRT and ESCRT- independent exosome biogenesis proteins. It is also planned to investigate other aspects of Alzheimer’s progression, including the role of ApoE in the formation of amyloid plaques and lipid droplets in glial cells. This study paves the way for understanding the role of exosomes in mediating inflammation and lipid metabolism in ApoE4 related Alzheimer’s Disease.

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