Proc Natl Acad Sci U S A 116(32): 16086-16094

PMC: PMC6689941

PMID: 31320591

Exosomes regulate neurogenesis and circuit assembly

Supplementary Material

^{Neuroscience Department, The Scripps Research Institute, La Jolla, CA, 92093;}

^{The Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA, 92093;}

^{Department of Pediatrics/Rady Children’s Hospital San Diego, University of California San Diego School of Medicine, La Jolla, CA, 92093;}

^{Department of Cellular & Molecular Medicine, University of California San Diego School of Medicine, La Jolla, CA, 92093;}

^{Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92093;}

^{Kavli Institute for Brain and Mind, University of California San Diego, La Jolla, CA, 92093;}

^{Center for Academic Research and Training in Anthropogeny, La Jolla, CA, 92093}

^{To whom correspondence may be addressed. Email:}ude.sppircs@enilc.

Edited by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 19, 2019 (received for review February 16, 2019)

Author contributions: P.S. and H.T.C. designed research; P.S., P.M., C.C., D.R.M., and L.S. performed research; P.S., J.R.Y., and A.R.M. contributed new reagents/analytic tools; P.S. and L.S. analyzed data; and P.S. and H.T.C. wrote the paper.

^{P.S. and P.M. contributed equally to this work.}

^{A.R.M. and H.T.C. contributed equally to this work.}

Edited by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 19, 2019 (received for review February 16, 2019)

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

Exosomes have been implicated in intercellular communication in cancer and neurodegenerative disorders. We explored their function in brain development. Proteomic analysis demonstrated that exosomes from isogenic control cultures contain neurodevelopmental signaling proteins, which are lacking in exosomes from MECP2 loss-of-function (MECP2LOF) cultures. Treating MECP2LOF neural cultures with control exosomes rescues neurodevelopmental deficits, increasing neurogenesis, synaptogenesis, and network activity. Exosomes function similarly in vivo: injecting purified exosomes into the lateral ventricles of P4 mouse brains increased hippocampal neurogenesis. These findings significantly advance the field by demonstrating that neural exosomes contain diverse protein cargo predicted to affect multiple outcome measures of neural development and that exosomes signal between cells in developing neural circuits to promote neural circuit development and function.

Keywords: exosomes, Rett syndrome, neuronal development, synaptogenesis, extracellular vesicle

Significance

Abstract

Exosomes are thought to be released by all cells in the body and to be involved in intercellular communication. We tested whether neural exosomes can regulate the development of neural circuits. We show that exosome treatment increases proliferation in developing neural cultures and in vivo in dentate gyrus of P4 mouse brain. We compared the protein cargo and signaling bioactivity of exosomes released by hiPSC-derived neural cultures lacking MECP2, a model of the neurodevelopmental disorder Rett syndrome, with exosomes released by isogenic rescue control neural cultures. Quantitative proteomic analysis indicates that control exosomes contain multiple functional signaling networks known to be important for neuronal circuit development. Treating MECP2-knockdown human primary neural cultures with control exosomes rescues deficits in neuronal proliferation, differentiation, synaptogenesis, and synchronized firing, whereas exosomes from MECP2-deficient hiPSC neural cultures lack this capability. These data indicate that exosomes carry signaling information required to regulate neural circuit development.

Abstract

Exosomes are small vesicles secreted by all cells in the brain, including neurons, and have been hypothesized to play a critical role in cell–cell communication (1, 2). Exosomes can signal over short range within brain tissue (3), and can signal widely throughout the brain through the cerebrospinal fluid (4, 5). Strong evidence indicates that exosomes impart biological activity to neurons (3, 6). For instance, in the Drosophila larval neuromuscular junction, exosome-mediated protein transport is required for coordinated development of pre- and postsynaptic components of the neuromuscular junction (7, 8). Exosomes secreted by oligodendrocytes affect firing rate, signaling pathways, and gene expression in cultured primary neurons (9, 10). Although there is evidence of biological roles of exosomes secreted by neurons and other cell types in the brain, their function in the development of human neural circuits is largely unexplored.

To investigate the role of exosomes in neural circuit development, we developed a reductionist experimental paradigm in which we added purified exosomes isolated from human induced pluripotent stem cell (hiPSC)-derived neurons onto human primary neural cultures to assay their capacity to influence neuronal and circuit development. We found that treatment with exosomes increased neurogenesis by promoting cell proliferation and neuronal differentiation, suggesting that exosomes carry signaling components that influence cell fate in developing neural circuits. We injected purified rodent exosomes into the lateral ventricle of P4 mice to test their role in hippocampal neurogenesis in an in vivo model. Consistent with in vitro observations, exosome treatment increases in proliferation in the granule cell layer (GCL) of dentate gyrus. Based on these observations that exosomes affect neural circuit development in vitro and in vivo, we hypothesized that conditions that disrupt neural circuit development may arise from defective exosome signaling. The loss of function of the X-linked Methyl-CpG-binding protein 2 (MECP2) gene results in aberrant neural circuit development, leading to Rett syndrome (11 –14). As disruption of a single protein, MECP2, leads to widespread pleiotropic deficits in neural circuit development, we further hypothesized that altered exosome-mediated intercellular communication may contribute to the impaired neural circuit development seen with MECP2 loss of function (MECP2 LOF). We had previously shown that neurons derived from MECP2 LOF hiPSC-derived neurons have fewer synapses, reduced spine density, smaller soma size, altered calcium signaling, and electrophysiological defects compared with control neurons (15), whereas CRISPR/Cas9 isogenic rescue of the MECP2 mutation (referred to as isogenic control) results in normal neural cultures (16, 17). Here, we compared exosomes released from MECP2 LOF donor neural cultures with exosomes from isogenic control donor neural cultures by using quantitative proteomic analysis. Isogenic control exosomes differed significantly from MECP2 LOF exosomes in their protein signaling content. Bioinformatic analyses revealed signaling complexes in control exosomes capable of eliciting neurodevelopmental outcomes, such as neural cell proliferation, neurogenesis, and synaptic development, that were lacking in MECP2 LOF exosomes. Treating recipient MECP2 LOF cultures with exosomes from isogenic control donor cultures increased cell proliferation, neurogenesis, synaptogenesis, and synchronized firing, whereas treating control recipient neural cultures with exosomes from MECP2 LOF donor cultures did not have adverse effects on neural phenotypes. These data show that exosomes play a significant role in neuronal circuit development and can be used to reverse deficits in neurological disease models.

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Acknowledgments

We thank Kristin Baldwin and Ardem Patapoutian from comments on the manuscript and members of the laboratory of H.T.C. for helpful discussions. This work was supported by grants from the National Institutes of Health (R01MH108528, R01MH094753, R01MH109885, R01MH100175, and U19MH107367), SFARI Grant 345469, and a NARSAD Independent Investigator Grant to A.R.M.; an International Rett syndrome Foundation (IRSF) fellowship to P.M.; NIH grants (R01MH103134 and R01EY011261) and an endowment from the Hahn Family Foundation to H.T.C.; a fellowship from the California Institute of Regenerative Medicine (CIRM; TG2-01165) and a Fellowship from the Helen Dorris Foundation to P.S.; and NIH Grants 5R01MH067880 and 5R01MH100175 to J.R.Y.

Acknowledgments

Footnotes

Conflict of interest statement: The authors declare a conflict of interest. A.R.M. is a cofounder and has equity interest in TISMOO, a company dedicated to genetic analysis focusing on therapeutic applications customized for autism spectrum disorder and other neurological disorders with genetic origins. The terms of this arrangement have been reviewed and approved by the University of California San Diego, in accordance with its conflict of interest policies. The remaining authors declare that they have no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1902513116/-/DCSupplemental.

Footnotes

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