Synaptic proteins in neuron-derived extracellular vesicles as biomarkers for Alzheimer’s disease: novel methodology and clinical proof of concept

Abstract

Aims: Blood biomarkers can improve drug development for Alzheimer’s disease (AD) and its treatment. Neuron-derived extracellular vesicles (NDEVs) in plasma offer a minimally invasive platform for developing novel biomarkers that may be used to monitor the diverse pathogenic processes involved in AD. However, NDEVs comprise only a minor fraction of circulating extracellular vesicles (EVs). Most published studies have leveraged the L1 cell adhesion molecule (L1CAM) for NDEV immunocapture. We aimed to develop and optimize an alternative, highly specific immunoaffinity method to enrich blood NDEVs for biomarker development. Methods: After screening multiple neuronal antigens, we achieved NDEV capture with high affinity and specificity using antibodies against Growth-Associated Protein (GAP) 43 and Neuroligin 3 (NLGN3). The EV identity of the captured material was confirmed by electron microscopy, western blotting, and proteomics. The specificity for 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 (Aβ) 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.

Comparative assessment of Alzheimer’s disease-related biomarkers in plasma and neuron-derived extracellular vesicles: a nested case-control study

“…Unlike proBDNF, mature BDNF showed no differences between groups. These findings are in agreement with the study by Eitan et al (2023) , in which levels of NDEV-associated proBDNF were also found lower in AD compared to control individuals and were positively correlated with MMSE scores. Our results, also, indicate that levels of NDEV-associated proBDNF may reflect true relative concentrations in brain cells, since similar decreases in MCI and AD dementia compared to the cognitively normal state were found in an autopsy study using cerebral tissue samples ( Peng et al, 2005 ).…”

“…Plasma aliquots were received and processed blindly by investigators at the National Institute on Aging, Baltimore, MD. NDEVs were isolated following a two-step procedure based on previous studies by our group and others validating L1 Cell Adhesion Molecule (L1CAM), the axonal protein Growth-Associated Protein 43 (GAP43), and the neuron cell surface protein Neuroligin 3 (NLGN3) as targets for the immunocapture of NDEVs from plasma ( Kapogiannis et al, 2019 ; Eitan et al, 2023 ). Slight differences between the protocol employed here and that described by Eitan et al include the isolation of crude plasma EVs via size exclusion chromatography (SEC) instead of polymer-based sedimentation, and targeting the neuronal surface proteins GAP43 and NLGN3, in addition to L1CAM, for the immunocapture of NDEVs, expected to increase the purity and yield of recovered NDEVs, respectively.…”

Extracellular Vesicles for Alzheimer’s Disease and Dementia Diagnosis

“…Also, a subsequent study showed that anti-L1CAM antibody clone UJ127 does not elute with EVs isolated by size-exclusion chromatography [22], exists mostly in soluble forms and cross-reacts with antibodies employed for biomarker quantification. For these reasons, many groups have been exploring alternative neuronal markers, such as ATP1A3 [80], GAP43, and NLGN3 [131], GABRD and GPR162 [81] and NRXN3 [163]. Meanwhile, markers like GLAST1 are sometimes employed to enrich speculative astrocytic EVs.…”

Emergence of Extracellular Vesicles as “Liquid Biopsy” for Neurological Disorders: Boom or Bust

“…Although western blotting of typical EV markers has been extensively used to characterize EVs isolated from different biofluids and cell-cultured media, the low recovery of specific cell-derived EVs, including BDEs, limits its utility, especially when isolated from human biofluids. Still, the expression of neuron-specific markers, such as L1CAM, SNAP25, NeuN, and GluR2, as well as general EV markers, like CD9, Flot-1, CD9, and TSG101, has been confirmed by western blotting in many studies ( Dagur et al, 2019 ; Serpente et al, 2020 ; Saeedi et al, 2021 ; Eitan et al, 2023 ). Similarly, the expression of typical EV markers, like Alix, flotillin-1, Annexin-A2, and CD81, has been reported in ADEs isolated from cultured astrocytes media ( Pascua-Maestro et al, 2019 ; D’Arrigo et al, 2021).…”

“…BDEs have been extensively characterized for their concentration (particle number per ml), size distribution, expression of EV biomarkers, and cargos (protein, metabolites, and nucleic acids) to identify disease-specific biomarkers ( Goetzl et al, 2015b , 2016b ; Doyle and Wang, 2019 ; Kumar et al, 2021a , 2022 , 2023 ; Eitan et al, 2023 ) ( Fig. 3 ).…”