Abstract
L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The data supporting the findings of this study are available within the paper and its Extended Data files. Source data are provided with this paper.
Code availability
The custom Python code used in this study is available as Supplementary Software.
References
Raposo, G. & Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200, 373–383 (2013).
Hlavin, M. L. & Lemmon, V. Molecular structure and functional testing of human L1CAM: an interspecies comparison. Genomics 11, 416–423 (1991).
Angiolini, F. et al. A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing. eLife 8, e44305 (2019).
Rissin, D. M. et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol. 28, 595–599 (2010).
Lobb, R. J. et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J. Extracell. Vesicles 4, 27031 (2015).
Mechtersheimer, S. et al. Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins. J. Cell Biol. 155, 661–673 (2001).
Zhou, L. et al. The neural cell adhesion molecules L1 and CHL1 are cleaved by BACE1 protease in vivo. J. Biol. Chem. 287, 25927–25940 (2012).
Carithers, L. J. et al. A novel approach to high-quality postmortem tissue procurement: the GTEx project. Biopreserv. Biobank. 13, 311–319 (2015).
Shi, M. et al. Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson’s disease. Acta Neuropathol. 128, 639–650 (2014).
Thery, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).
Thompson, A. G. et al. Extracellular vesicles in neurodegenerative disease—pathogenesis to biomarkers. Nat. Rev. Neurol. 12, 346–357 (2016).
Shi, M., Sheng, L., Stewart, T., Zabetian, C. P. & Zhang, J. New windows into the brain: central nervous system-derived extracellular vesicles in blood. Prog. Neurobiol. 175, 96–106 (2019).
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).
Peng, J. & Gygi, S. P. Proteomics: the move to mixtures. J. Mass Spectrom. 36, 1083–1091 (2001).
Eng, J. K., McCormack, A. L. & Yates, J. R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).
Busskamp, V. et al. Rapid neurogenesis through transcriptional activation in human stem cells. Mol. Syst. Biol. 10, 760 (2014).
Thorvaldsdottir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief. Bioinform. 14, 178–192 (2013).
Acknowledgements
We thank A. Ng for help with stem cell differentiation and J. Van Deun for help with DGC. We also thank the Taplin Biological Mass Spectrometry Facility at Harvard Medical School and the Harvard Center for Mass Spectrometry for help with proteomic experiments. This work was supported by funding from the Chan Zuckerberg Initiative Neurodegeneration Challenge Network (to D.R.W., G.M.C., A.S.C.-P.), Good Ventures (to D.R.W.), the NIH Center for Excellence in Genomic Science (to G.M.C., RM1HG008525), the Howard Hughes Medical Institute (to A.R.) and the Klarman Cell Observatory (to A.R.). These funding agencies had no role in conceptualization, design, data collection, analysis, decision to publish or preparation of the manuscript.
Author information
Authors and Affiliations
Contributions
M.N. and D.T.-O. conceived the study and designed experiments. D.T.-O., M.N., W.T., J.H.L., E.J.K.K. and R.L. performed experiments. D.T.-O., M.N. and D.R.W. analyzed data and wrote the manuscript with input from all authors. G.M.C., A.S.C.-P., A.R. and D.R.W. supervised the study and provided funding support.
Corresponding author
Ethics declarations
Competing interests
D.R.W. is a founder and equity holder of Quanterix. A.R. is an SAB member of Thermo Fisher Scientific, Neogene Therapeutics, Asimov and Syros Pharmaceuticals. A.R. is a cofounder of and equity holder in Celsius Therapeutics and an equity holder in Immunitas. From 1 August 2020, A.R. is an employee of Genentech. G.M.C. is a founder, consultant or advisory board member to companies listed here: http://arep.med.harvard.edu/gmc/tech.html. These companies had no influence over any aspect of this research. We have filed intellectual property on methods for EV analysis and isolation. M.N., D.T.-O. and D.R.W. filed a provisional patent for the measurement of EVs using single-molecule arrays as described in this study. Additionally, D.T.-O., E.J.K.K., A.R. and G.M.C. filed intellectual property relating to the identification and use of new candidate markers for NDEV isolation.
Additional information
Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Proteinase protection assays.
(a) Schematic overview of a Proteinase K protection assay analyzing the integrity of EVs after fractionation with SEC, using four different conditions: a No treatment, two-hour incubation, lyse with Triton X, one-hour incubation b Proteinase K application, two-hour incubation, lyse with Triton X, one-hour incubation. c No treatment, one-hour delay, add PMSF for one hour, lyse with Triton X, one-hour incubation. d Proteinase K application, one-hour incubation, add PMSF to inhibit Proteinase K for one hour, lyse with Triton X, one-hour incubation. (b) Alix and Albumin concentrations after the Proteinase K protection assay, as measured with Simoa. Data represent the average of two technical replicates of the Simoa assay measurements from a single experiment with one sample. This experiment was conducted 3 times with similar results.
Extended Data Fig. 2 Electron Microscopy of SEC fractions.
Transmission Electron Microscopy of a. Fraction 9 and b. Fraction 12 from plasma fractionated using SEC and negatively stained with uranyl formate. Representative images are shown at 6000x magnification (top) and 20,000x magnification (bottom). Arrows indicate ‘cup-shaped’ EVs. This experiment was conducted once.
Extended Data Fig. 3 Mass spectrometry of L1CAM immunocaptured from plasma.
Mass spectrometry analysis shows peptides mapping to different parts of the L1CAM protein. Full length sequence of L1CAM displayed with peptides detected by mass spectrometry shown in green. Blue box indicates L1CAM transmembrane domain and red box indicates amino acid sequence encoded by Exon 25. Top: full length recombinant L1CAM protein standard shows peptides matching an isoform which includes Exon 25. Bottom: Mass Spectrometry of L1CAM immunocaptured from human plasma shows peptides matching the cytosolic domain at the C terminus (emphasized with black arrow). This experiment was conducted once.
Extended Data Fig. 4 Analysis of RNA-seq data for L1CAM.
(a) L1CAM intro-exon gene structure including Exon 25, which contains the only transmembrane domain. Alternative splicing skipping Exon 25 (L1CAM isoform without a transmembrane domain) would lead to transcripts with an exon-exon junction across Exon 24 and Exon 26. (b) Reads from GTEx RNA-Seq data of human Tibial Artery loaded in Integrative Genome Browser (IGV) aligning to Exon 25 of L1CAM, which contain the transmembrane domain (highlighted in red). Aligned junction reads supporting the skipping of Exon 25 are indicated with black arrows.
Extended Data Fig. 5 Analysis of reads from GTEx RNA-Seq Data indicating Exon 25 skipping in alternative splicing of L1CAM.
Fraction of reads mapping to L1CAM isoform supporting skipping of L1CAM Exon 25 (junction reads spanning Exon 24 and Exon 26) vs. inclusion of Exon 25 from RNA-Seq GTEx data of various human organs.
Extended Data Fig. 6 Affinity of L1CAM for recombinant alpha-synuclein.
Concentration of Alpha Synuclein recombinant protein captured with control (mIgG) and L1CAM (UJ12) antibodies in a pull-down experiment. Data shown is the average of two technical replicates from a single experiment.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2, Table 1 and Note 1
Source data
Source Data Fig. 1
Statistical source data.
Source Data Fig. 2
Statistical source data and western blots.
Source Data Extended Data Fig. 1
Statistical source data.
Source Data Extended Data Fig. 6
Statistical source data.
Rights and permissions
About this article
Cite this article
Norman, M., Ter-Ovanesyan, D., Trieu, W. et al. L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma. Nat Methods 18, 631–634 (2021). https://doi.org/10.1038/s41592-021-01174-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41592-021-01174-8
This article is cited by
-
Blood-based CNS regionally and neuronally enriched extracellular vesicles carrying pTau217 for Alzheimer’s disease diagnosis and differential diagnosis
Acta Neuropathologica Communications (2024)
-
Blood extracellular vesicles carrying brain-specific mRNAs are potential biomarkers for detecting gene expression changes in the female brain
Molecular Psychiatry (2024)
-
Neuron enriched extracellular vesicles’ MicroRNA expression profiles as a marker of early life alcohol consumption
Translational Psychiatry (2024)
-
Analysis of biomarkers in speculative CNS-enriched extracellular vesicles for parkinsonian disorders: a comprehensive systematic review and diagnostic meta-analysis
Journal of Neurology (2024)
-
Neuron-derived extracellular vesicles in blood reveal effects of exercise in Alzheimer’s disease
Alzheimer's Research & Therapy (2023)