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Page 134 Eitan et al. Extracell Vesicles Circ Nucleic Acids 2023;4:133-150 https://dx.doi.org/10.20517/evcna.2023.13

neuronal origin was demonstrated by showing enrichment for neuronal markers (proteins, mRNA) and recovery of
spiked neuronal EVs. We performed NDEV isolation retrospectively from plasma samples from two cohorts of
early AD patients (N = 19 and N = 40) and controls (N = 20 and N = 19) and measured p181-Tau, amyloid-beta (A
β) 42, brain-derived neurotrophic factor (BDNF), precursor brain-derived neurotrophic factor (proBDNF),
glutamate receptor 2 (GluR2), postsynaptic density protein (PSD) 95, GAP43, and syntaxin-1.

Results: p181-Tau, Aβ42, and NRGN were elevated in AD samples, whereas proBDNF, GluR2, PSD95, GAP43, and
Syntaxin-1 were reduced. Differences for p181-Tau, proBDNF, and GluR2 survived multiple-comparison correction
and were correlated with cognitive scores. A model incorporating biomarkers correctly classified 94.7% of AD
participants and 61.5% of control participants. The observed differences in NDEVs-associated biomarkers are
consistent with previous findings.

Conclusion: NDEV isolation by GAP43 and NLGN3 immunocapture offers a robust novel platform for biomarker
development in AD, suitable for large-scale validation.

Keywords: Biomarkers, exosomes, neuron-derived exosomes, Alzheimer’s disease

INTRODUCTION
Developing effective treatments for Alzheimer’s disease (AD) and AD-related dementias (ADRD)
represents an unmet medical need of major socioeconomic importance. Approved symptomatic treatments
have no impact on disease progression, whereas therapeutic development remains exceptionally challenging
and costly . Only recently have two purportedly disease-modifying drugs, Lecanemab and Aducanumab,
[1]
[2]
received FDA approval for the treatment of AD ; however, the clinical significance of their effects is
questionable.

Complicating things further, postmortem analyses have shown that most dementia patients present with
mixed underlying pathologies . Therefore, as with cancer, a precision strategy informed by the patient’s
[3-5]
underlying biology is highly desirable but currently underdeveloped. Detection of AD-specific pathologies
in living patients currently relies on expensive and/or invasive biomarkers obtained through intensive
positron-emission tomography (PET) scans or lumber punctures and the analysis of biomarkers in
cerebrospinal fluid (CSF) . And in both cases, these approaches measure and reflect only a limited number
[6]
of pathologies focused on amyloid-beta (Aβ) and tau proteins. Blood biomarkers are inherently
advantageous because blood draws are minimally invasive and can be performed repeatedly for early-stage
diagnosis, monitoring of disease progression, and assessment of therapeutic responses . A key challenge in
[7]
developing blood biomarkers for neurodegenerative diseases is to achieve specificity for changes occurring
in neurons and other brain cells rather than non-neuronal sources. One way to address this challenge is the
enrichment of EVs isolated from blood plasma or serum for neuronal origin in order to analyze NDEVs.
Because EVs are nanosized particles surrounded by a lipid bilayer membrane that contain proteins and
RNA that are representative of their cells of origin [8-12] , they may provide a molecular snapshot of the brain .
[9]
The use of NDEVs as a biomarker platform relies on a few assumptions backed with experimental support:
the first is that the ability of NDEVs to cross the blood-brain barrier (BBB) has been demonstrated [13-15] ;
[16]
second, only NDEVs contain neuron-specific surface proteins ; and third, NDE cargo reflects the cell of
NDE origin; and lastly, modifications to NDEV cargo are minimal following the release of NDEVs. Previous
studies on biomarkers in EVs used antibodies against L1CAM (L1 cell adhesion molecule, CD171), a surface
marker predominantly expressed by neurons but also by cells in the kidney, dermis, and peripheral
lymphocytes (https://www.proteinatlas.org/ENSG00000198910-L1CAM/tissue), as a means to isolate