Apolipoprotein E4-driven effects on inflammatory and neurotrophic factors in peripheral extracellular vesicles from cognitively impaired, no dementia participants who converted to Alzheimer's disease

Abstract

Introduction: In brain, extracellular vesicles (EVs) play an essential role in the neuron-glia interface and ensure the crosstalk between the brain and the periphery. Some studies now link the pathway dysfunction of the EVs to apolipoprotein E gene variant (APOE ε4) and the risk of progression to Alzheimer's disease (AD). To better understand the role of APOE ε4 in pre-clinical AD, we have determined levels of pathogenic, neurotrophic and inflammatory proteins in peripheral EVs (pEVs) and in plasma from cognitively impaired, no dementia (CIND) participants stratified upon the absence (APOE ε4^-) or the presence (APOE ε4⁺ ) of the ε4 allele of APOE.

Methods: Levels of 15 neurodegenerative, neurotrophic and neuroinflammatory proteins were quantified in pEVs and compared to their plasma levels from cognitively normal and CIND participants.

Results: Levels of neurotrophic and inflammatory markers were reduced in pEVs from APOE ε4⁺. The pentraxin-2/α-synuclein ratio measured in pEVs was able to predict AD 5 years before the onset among APOE ε4⁺-CIND individuals.

Discussion: Our findings suggest an alteration of the endosomal pathway in APOE ε4⁺ and that pEVs pentraxin-2/α-synuclein ratio could serve as a useful early biomarker for AD susceptibility.

Keywords: Alzheimer's disease; DJ‐1; S100B; biomarkers; extracellular vesicles; lipocalin; pentraxin‐2; α‐synuclein.

FIGURE 1

Characterization and visualization of pEVs. (A) TEM images of pEVs; magnification 40.000×. (B) Analysis of the size distribution and concentration. (C) Western blotting for exosomal proteins TSG101 and CD63. (D) Confocal imaging of EVs labeled with PKH67. (E) TSG101 antibodies using Alexa Fluor 594. (F) Co‐localization of PKH67 and TSG101. (G) Anti‐rabbit antibodies with pEVs

FIGURE 2

Effects of APOE ε4 variant on pEVs and plasma characteristics. (A) Effect on pEVs number. (B) Effect on pEVs size distribution. (C) Effect on pEVs protein content. (D) Effect on plasma protein content. ^** P < .01, ^*** P < .001

FIGURE 3

Levels of neurotrophic factors in pEVs and in plasma. Levels of DJ‐1, progranulin, and α‐synuclein measured in pEVs (A, C, E) and in plasma (B, D, F). Graph points indicate individual values for each participant. (−), ε4 non‐carriers; (+), ε4 carriers; apoE, apolipoprotein E; CIND, cognitively impaired, no dementia; EVs, extracellular vesicles. Statistical analysis was performed using the Student t test for DJ‐1 (EVs and plasma) and plasma α‐synuclein. For progranulin (EVs and plasma) and EV α‐synuclein, the Mann‐Whitney test was used. Values are mean ± SEM with ^* P < .05 versus CIND‐ApoE4(−)

FIGURE 4

Levels of inflammatory factors in pEVs and in plasma. Levels of lipocalin, S100B, pentraxin‐2, and ANGPTL‐4 measured in pEVs (A, C, E, G) and in plasma (B, D, F, H). Graph points indicate individual values for every participant. (−), ε4 non‐carriers; (+), ε4 carriers; apoE, apolipoprotein E; CIND, cognitively impaired, no dementia; EVs, extracellular vesicles. Statistical analysis was performed using the Student t test for S100B and pentraxin‐2 (EVs and plasma) and plasma lipocalin and the Mann‐Whitney test for ANGPTL‐4 (EVs and plasma) and EVs lipocalin. Values are mean ± SEM with ^* P < .05 versus CIND‐ApoE4(−)

FIGURE 5

Levels and statistical correlation of pentraxin‐2 and α‐synuclein in pEVs with cognitive performance. (A) EVs levels of α‐Syn between controls and CIND‐AD patients. (B) Correlation between NPTX‐2 and α‐Syn levels in EVs. (C) EVs NPTX‐2/α‐Syn ratio between controls and CIND‐AD patients. (D) Correlation between EVs NPTX‐2/ α‐Syn ratio and MMSE score. Graph points indicate individual values for every participant. CIND‐AD, cognitively impaired, no dementia‐Alzheimer's disease; CTR, controls; EVs, extracellular vesicles; NPTX‐2, pentraxin‐2; p, significance (Student t test); r, Pearson correlation coefficient; α‐Syn, α‐synuclein. The correlation coefficient (Pearson r) and P values were determined using Pearson correlation. Statistical analysis was performed using the student t test for EVs α‐Syn levels and EVs NPTX‐2/α‐Syn ratio. Values are mean ± SEM