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“EVs in Neurological Diseases,” with Focus on Rett Syndome, Is Second Plenary Address on Opening Day of International Society for Extracellular Vesicles (ISEV) 2020 Virtual Annual Meeting (July 20-22) for 1,600 Virtual Attendees from 52 Countries

(Written for BioQuick News by Michael A. Goldman, PhD, Professor & Former Chair, Biology, San Francisco State University, The International Society for Extracellular Vesicles (ISEV) 2020 Virtual Annual Meeting (July 20-22) (, with a record 1,600 virtual attendees from 52 countries around the world, and offering ~600 presentations of various types (Plenary Addresses, “Hot-Topic” Panel Sessions, Featured Abstracts, Oral Abstract Talks, Poster Chats, & Education Sessions), both live-streamed and on-demand, showcased its second Plenary Address on Monday, July 20. This address was titled “EVs in Neurological Diseases,” and was delivered by Hollis Cline (photo), PhD, the Hahn Professor of Neuroscience and Co-Chair of the Department of Neuroscience at Scripps Research in La Jolla, California, USA. Dr. Cline ( is also a Counselor for the National Institute of Neurological Disorders and Stroke (NINDS) and a Past President of the Society for Neuroscience. She received her BA from Bryn Mawr College and her PhD from the University of California at Berkeley, followed by postdoctoral training at Yale University and Stanford University. Dr. Cline has served on the faculty of the University of Iowa and the Cold Spring Harbor Laboratory, where she served as the Director of Research from 2002-2006. Dr. Cline’s research has demonstrated the roles of a variety of activity-dependent mechanisms in controlling structural plasticity of neuronal dendrites and axons, synaptic maturation, and topographic map formation. This body of work has helped to generate a comprehensive understanding of the role of experience in shaping brain development. Two key points to emerge from her research are that circuit formation in vivo is a dynamic process throughout development that is continuously guided by experience, and that the basic mechanisms governing brain development, plasticity, information processing, and organizational principles of brain circuits are highly conserved across vertebrates. Dr. Cline was introduced by outgoing ISEV President Andy Hill, PhD, Professor & Director, La Trobe Institute for Molecular Science, LaTrobe University, Melbourne, Australia. Early in his career, Dr. Hill held post-doctoral positions in the Medical Research Council (MRC) Prion Unit (London) and in the Department of Pathology at the University of Melbourne as a Wellcome Trust Prize Traveling Research Fellow.

Dr. Cline's Plenary Presentation focused on EVs and Neurological Diseases. She outlined an elegant study showing the potential role of naturally-occurring exosomes as a therapeutic delivery system for neurological signaling molecules to correct Rett syndrome, a single-gene, X-linked neurodegenerative disease. The approach may have much wider applicability.

Exosomes can carry signals or other molecular cargoes from one cell to another within the healthy brain. Some of these signals may play crucial roles in the development of neural circuitry. In neurodegenerative degenerative diseases like Alzheimer's, exosomes may be involved in the transport of toxic proteins, like prions, alpha-synuclein, tau, and A-beta. Exosomes are able to cross the blood-brain barrier, and it is therefore possible that alterations in exosome-mediated signals could, in principle, be implicated in a wide variety of brain disorders. The Cline laboratory is especially interested in the normal functioning of exosome signaling in the brain, as well as the breakdown of these processes in diseases.

Are exosome cargoes cell-type and physiological-state specific? Do these cargoes differ during the process of development and aging? And are exosome characteristics altered in a genotype-specific manner? The characterization of exosomes within the brain represents a new and exciting approach to the understanding, and potentially the treatment, of neurodegenerative disease.

Professor Cline described her laboratory's work with Rett syndrome [RTT, OMIM #312750], an X-linked, dominant genetic disease resulting from a loss of function (LOF) mutation in the methyl-CpG-binding protein-2 (MECP2) locus [OMIM Gene #300005] at Xq28. First described in 1966, Rett syndrome entails a variety of characteristic neurological features beginning at about 6 to 18 months. In contrast to X-linked recessive diseases, such as hemophilia A and color blindness, the Rett phenotype is seen overwhelmingly in females, with few exceptions.

In females, one of the two X chromosomes is silenced, through a process called X-chromosome inactivation (XCI), in each cell early in embryonic development. The fetus, and the adult, are effectively mosaics of cells having either the maternal X silent and the paternal X expressed, or vice versa. In an individual carrying an MECP2 mutation on one X chromosome, about half the cells, on average, will have the functional copy of the gene, while the other half will suffer from the loss of function. In males, with only one X chromosome, loss of function of MECP2 is lethal.

The MECP2 protein is implicated in widespread transcriptional silencing of human genes, and it is therefore expected that the expression of many genes would be abnormal in a LOF mutation. It appears to be particularly crucial in the development of the nervous system, and abnormalities in the gene may also affect autism susceptibility and some forms of mental retardation. Rett syndrome has been refractory to treatment.

In order to study the cells of a Rett patient in vitro, Professor Cline and her colleagues employed induced pluripotent stem cells (iPSCs) produced by de-differentiating the patient's fibroblast cells. These iPSCs simulate an embryonic-like cell that could subsequently be induced to enter neural development.

In the process of de-differentiation, the X chromosome is reactivated so that this iPSC line has two active X chromosomes. Female embryos begin life with both X chromosomes active, and random inactivation of either the paternal X or the maternal X occurs during the process of differentiation.

The iPSCs were induced to differentiate into neurons, in which each nucleus has only one active X chromosome. These neurons, derived from Rett syndrome iPSCs, exhibited defects such as a deficiency in synapse formation, small size, and other electrophysiological issues. These cells, and the process of differentiation from iPSCs, serve as a model for the study of Rett syndrome neural development.

It is ideal to compare these Rett syndrome cells to a cell line that is identical, except for having two normal copies of the Rett syndrome gene (MeCP2). This was achieved using a CRISPR technique to correct the mutant MeCP2 allele in the Rett-patient-derived iPSC's. These cells were then considered an isogenic (genetically-identical) "control" cell line. When differentiated, these cells again undergo X-chromosome inactivation, but become essentially normal neurons, exhibiting none of the effects typical of the Rett-derived cells.

Professor Cline and colleagues isolated exosomes from the Rett and control neurons, and characterized those exosomes with respect to the proteins they contained. There were approximately 2,570 proteins in neural exosomes, 750 of which had annotated neural functions. Comparing the two types of cells, about 240 proteins exhibited at least 1.5-fold difference in quantitative studies, suggesting that the nature of the exosomes differs between Rett and control neurons.

Remarkably, the exosomes isolated from control cultures could "rescue" the phenotypic defects in the Rett neurons, including the development of neural circuitry, suggesting that the exosome proteins play a crucial role in neurobiology. In contrast, if exosomes isolated from Rett neurons were used to treat control neurons, there were no deleterious phenotypic effects. It therefore appears that Rett-derived exosomes do not harbor a toxic substance involved in neural function.

The work by Professor Cline and colleagues helps to explain some of the genetics of Rett syndrome. Affected individuals are heterozygous for a normal copy of the gene and a loss-of-function mutation. Due to X-chromosome inactivation, each cell has only one of these two alleles transcriptionally "active." If the normal allele is silenced, then the cell is without MeCP2 activity, and produces abnormal exosomes. If the normal allele is active, then the cell is essentially normal and produces normal exosomes. With these exosomes involved in signaling between neurons in the brain, inadequate signaling occurs because some of the exosomes are not functioning properly. Because the X-inactivation process is random, sometimes, by chance, most cells happen to have the normal allele active, producing a nearly-normal population of exosomes and leading to a relatively normal phenotype. In other cases, most of the normal alleles might be silenced, producing an extreme of the Rett syndrome phenotype. This is sometimes called "unfortunate Lyonization," in recognition of Mary F. Lyon, who originally described the X-inactivation process.

While some experiments targeting exosomes to the brain have been done in mice, experiments in humans have been confined to cells in culture. It is quite exciting that exosomes from normal neurons had a therapeutic effect on Rett syndrome neurons, although it is not yet clear how early in the disease development the therapy must, or can, be applied. It is also possible that pharmaceuticals could be delivered to neurons using modified exosomes.

We are just beginning to understand the broad-ranging roles of exosomes and their potential as therapeutic agents, and this work provides evidence that the work might help with some of the diseases that have been the most devastating and difficult to treat.

Suggested reference: Sharma, P., et al. (2019). "Exosomes Regulate Neurogenesis and Circuit Assembly," PNAS 116(32): 16086-16094 (

Dr. Cline’s presentation was followed by a Q&A session moderated Alissa Weaver, MD, PhD, ISEV Co-Chair, International Organizing Committee (IOC); Professor & Chair, Cell & Developmental Biology; Professor, Pathology, Microbiology, & Immunology, Vanderbilt University School of Medicine, USA.