Synaptic Proteins in Neuron-derived Extracellular Vesicles As Biomarkers for Alzheimer's Disease: Novel Methodology and Clinical Proof of Concept

Overview

Journal Extracell Vesicles Circ Nucl Acids

Date 2023 Oct 16

PMID 37842184

Authors

Erez Eitan

Tricia Thornton-Wells

Katya Elgart

Eren Erden

Eve Gershun

Amir Levine

Olga Volpert

Mitra Azadeh

Daniel G Smith

Dimitrios Kapogiannis

Affiliations

Soon will be listed here.

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.

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References

Sodero A . 24S-hydroxycholesterol: Cellular effects and variations in brain diseases. J Neurochem. 2020; 157(4):899-918. DOI: 10.1111/jnc.15228. View

Korkut C, Li Y, Koles K, Brewer C, Ashley J, Yoshihara M . Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron. 2013; 77(6):1039-46. PMC: 3626103. DOI: 10.1016/j.neuron.2013.01.013. View

Banks W, Sharma P, Bullock K, Hansen K, Ludwig N, Whiteside T . Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation. Int J Mol Sci. 2020; 21(12). PMC: 7352415. DOI: 10.3390/ijms21124407. View

Binderup H, Madsen J, Heegaard N, Houlind K, Andersen R, Brasen C . Quantification of microRNA levels in plasma - Impact of preanalytical and analytical conditions. PLoS One. 2018; 13(7):e0201069. PMC: 6053236. DOI: 10.1371/journal.pone.0201069. View

Mustapic M, Eitan E, Werner Jr J, Berkowitz S, Lazaropoulos M, Tran J . Plasma Extracellular Vesicles Enriched for Neuronal Origin: A Potential Window into Brain Pathologic Processes. Front Neurosci. 2017; 11:278. PMC: 5439289. DOI: 10.3389/fnins.2017.00278. View

Palviainen M, Saraswat M, Varga Z, Kitka D, Neuvonen M, Puhka M . Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery. PLoS One. 2020; 15(8):e0236439. PMC: 7446890. DOI: 10.1371/journal.pone.0236439. View

Sodar B, Kittel A, Paloczi K, Vukman K, Osteikoetxea X, Szabo-Taylor K . Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection. Sci Rep. 2016; 6:24316. PMC: 4834552. DOI: 10.1038/srep24316. View

Jia L, Qiu Q, Zhang H, Chu L, Du Y, Zhang J . Concordance between the assessment of Aβ42, T-tau, and P-T181-tau in peripheral blood neuronal-derived exosomes and cerebrospinal fluid. Alzheimers Dement. 2019; 15(8):1071-1080. DOI: 10.1016/j.jalz.2019.05.002. View

Goetzl E, Abner E, Jicha G, Kapogiannis D, Schwartz J . Declining levels of functionally specialized synaptic proteins in plasma neuronal exosomes with progression of Alzheimer's disease. FASEB J. 2017; 32(2):888-893. PMC: 5888398. DOI: 10.1096/fj.201700731R. View

10.

Zhou R, Chen K, Zhang J, Xiao B, Huang Z, Ju C . The decade of exosomal long RNA species: an emerging cancer antagonist. Mol Cancer. 2018; 17(1):75. PMC: 5861621. DOI: 10.1186/s12943-018-0823-z. View

11.

Khodashenas S, Khalili S, Moghadam M . A cell ELISA based method for exosome detection in diagnostic and therapeutic applications. Biotechnol Lett. 2019; 41(4-5):523-531. DOI: 10.1007/s10529-019-02667-5. View

12.

Jack Jr C, Bennett D, Blennow K, Carrillo M, Dunn B, Budd Haeberlein S . NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease. Alzheimers Dement. 2018; 14(4):535-562. PMC: 5958625. DOI: 10.1016/j.jalz.2018.02.018. View

13.

Goetzl E, Kapogiannis D, Schwartz J, Lobach I, Goetzl L, Abner E . Decreased synaptic proteins in neuronal exosomes of frontotemporal dementia and Alzheimer's disease. FASEB J. 2016; 30(12):4141-4148. PMC: 5102122. DOI: 10.1096/fj.201600816R. View

14.

Ohrfelt A, Brinkmalm A, Dumurgier J, Brinkmalm G, Hansson O, Zetterberg H . The pre-synaptic vesicle protein synaptotagmin is a novel biomarker for Alzheimer's disease. Alzheimers Res Ther. 2016; 8(1):41. PMC: 5048479. DOI: 10.1186/s13195-016-0208-8. View

15.

Shaw L, Vanderstichele H, Knapik-Czajka M, Clark C, Aisen P, Petersen R . Cerebrospinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects. Ann Neurol. 2009; 65(4):403-13. PMC: 2696350. DOI: 10.1002/ana.21610. View

16.

Jamaly S, Ramberg C, Olsen R, Latysheva N, Webster P, Sovershaev T . Impact of preanalytical conditions on plasma concentration and size distribution of extracellular vesicles using Nanoparticle Tracking Analysis. Sci Rep. 2018; 8(1):17216. PMC: 6249294. DOI: 10.1038/s41598-018-35401-8. View

17.

Wen S, Lima L, Lobb R, Norris E, Hastie M, Krumeich S . Breast Cancer-Derived Exosomes Reflect the Cell-of-Origin Phenotype. Proteomics. 2019; 19(8):e1800180. DOI: 10.1002/pmic.201800180. View

18.

OBrien K, Breyne K, Ughetto S, Laurent L, Breakefield X . RNA delivery by extracellular vesicles in mammalian cells and its applications. Nat Rev Mol Cell Biol. 2020; 21(10):585-606. PMC: 7249041. DOI: 10.1038/s41580-020-0251-y. View

19.

Haynes W, Vallania F, Liu C, Bongen E, Tomczak A, Andres-Terre M . EMPOWERING MULTI-COHORT GENE EXPRESSION ANALYSIS TO INCREASE REPRODUCIBILITY. Pac Symp Biocomput. 2016; 22:144-153. PMC: 5167529. DOI: 10.1142/9789813207813_0015. View

20.

Hogan D, Fiest K, Roberts J, Maxwell C, Dykeman J, Pringsheim T . The Prevalence and Incidence of Dementia with Lewy Bodies: a Systematic Review. Can J Neurol Sci. 2016; 43 Suppl 1:S83-95. DOI: 10.1017/cjn.2016.2. View