Harnessing Exosomes as a Therapeutic for Rett Syndrome

I am a scientist at Scripps Research Institute in La Jolla, California working in the lab of Professor Hollis Cline. A thirst for knowledge is what originally attracted me to science. The potential to contribute, even in a small way, to alleviating suffering drives that thirst and passion even more.

Human biology has always fascinated me. Imagine for a moment how the human body is created. It starts with a single cell that multiplies to create a complex organism of trillions of cells. The human brain alone is estimated to contain more than 150 billion cells, 86 billion neurons and about an equal number of non-neuronal cells, all of a wide variety of specializations. It is mind boggling to imagine that a few founder cells contain the programming information that, through a series of cell fate decisions, produces a complex organ like the brain. What kind of communication and logistics are required to orchestrate the development and function of this behemoth?

One requirement is a package delivery mechanism; think of it as the body’s UPS, which allows information and material transfer between cells. Over the past decade, researchers have discovered that our bodies employ amazing inter-cellular couriers called exosomes or extracellular vesicles to transport fundamental biomolecules like proteins, nucleic acids and lipids. Exosomes can also perform additional duties, such as scouting and laying the path for a growing axon or migrating cells. For example, cancer cells use exosomes to lay the foundation of their migration out of a tumor, leading to metastasis.

Our work has uncovered a fundamental role of exosome communication in brain development. We show that exosomes secreted by neurons contain signals to direct the development and function of neural circuits.  Importantly we have discovered that exosomes have the potential to  become therapeutics for neurodevelopmental disorders, including Rett Syndrome.

Our brain works like a musical ensemble. The neurons fire to produce a pattern of activity very much like an ensemble of musicians playing together to produce a melody. Historically, a vast majority of studies directed towards understanding brain function focused on the skills of the individual neurons or their training together in producing a melody. We found that when these musicians in our brain called neurons, hang together and socialize, they use exosomes to communicate between themselves. These exosomes contained messages that provided them great collective motivation and were extremely helpful in their training and performance. Extending this analogy to the case of Rett Syndrome, Rett neurons practice very hard but are unable to play together and produce a melody. Rett neurons not only lacked some music skills, they had problems coordinating their music with each other. We found that the Rett exosome no longer contained motivating messages to help the neurons with their music skills and coordination.

We thought that maybe if we take exosomes from healthy neurons and give them to Rett neurons, it will provide them the message they are lacking and help motivate them to play a melody. Remarkably, the exosome message from healthy neurons let Rett Syndrome neurons overcome their shortcomings and fire together in a synchronous way to produce a melody.

For the scientifically inclined readers I’ll provide a more scientific description.  All cells in the brain secrete exosomes. However, it was not very clear what function the exosomes perform in the brain. We purified exosomes from functional neural cultures and asked, could these exosomes contain a bioactivity to perform any function in a developing neural circuit? We observed that exosome treatment led to an increase in neuronal number. This led to a further question – if exosomes have a role in developing neural circuits, what happens when the neural development is deficient?  A good way to find that out is to compare exosomes from healthy neurons to exosomes from neurons with a neurodevelopmental disorder.

We decided to explore this question by experimenting with induced pluripotent stem cells (iPSC) from a Rett Syndrome patient. This audience knows very well that Rett is caused by disruption of a single gene, MECP2. We restored the function of the MECP2 gene in the iPSCs using CRISPR gene editing. We therefore had two human iPSC neural cultures that are identical to each other genetically except in the function of just one protein, MECP2. This was an ideal setup to study the fundamental role of exosomes in normal neural circuit development and compare it to a condition where neural circuit development is deficient.

The Rett patient iPSC derived neural cultures displayed cellular and circuit manifestations of Rett Syndrome, whereas CRISPR corrected controls were normal. We then purified exosomes secreted by each culture yielding normal control exosomes and Rett exosomes and compared them. Our results were so remarkable that it took us a while to appreciate them.

First, exosomes were full of proteins that are important in development of neurons and formation and maintenance of synapses. Synapses are conduits of electrochemical information flow between neurons, and are critical to proper brain function.

Second, the Rett exosomes displayed specific alterations in the signaling capacities, like proliferation, neural development, and synaptic function. In short, we found that normal exosomes could potentially guide the proliferation, neuron development and synapse function, and Rett exosomes are somewhat deficient in that function.

Taking cues from these results, we compared the bioactivity and found that normal exosomes boosted proliferation of neural stem cells and Rett exosomes did not. In addition, normal exosome treatment led to a big increase in neural progeny and modest increase in astrocyte progeny; astrocytes are another cell type in the brain that have a range of ancillary functions. In comparison, Rett exosome treatment, while it lacked the capability to increase neural progeny, still directed the modest increase of astrocyte progeny. This result shows that Rett exosomes retain some functions, but their neural specific functions are lacking.

However, the most important question was still nagging us. Could treatment with normal control exosomes rescue deficits in Rett Syndrome neural cultures? After an onerous journey of problem solving and establishment of assays, we successfully demonstrated that treatment of Rett neural cultures with normal control exosomes could increase neuron number, boost the number of synapses and make neurons fire in a more synchronized way. Importantly, exosome treatment showed improvements at the cellular, synaptic, and functional level.

While a very exciting result, we wanted to take this a step further into live animals. So we took healthy exosomes and injected them into the brain of developing mice and monitored neuronal proliferation in hippocampus, a brain area important for learning and memory. Exosome injections led to a remarkable boost in neuronal proliferation in hippocampus, just like human in-vitro disease models. This showed that if delivered to the brain in live animals, the exosomes can deliver the promised bioactivity.

I belive exosomes have immense therapeutic potential as they have inherent advantages. Unlike stem cells, there is no possibility that they can go rogue and form tumors. Importantly, exosomes do not elicit an immunune response when injected into the patient. Exosomes can be sourced from cultured neurons made from the patient’s own cells, providing personalized medicine.

Neural exosomes are thought to contain signals that guide the exosome to the brain. They can be loaded with any therapeutic drugs or molecules developed for Rett Syndrome and delivered to the brain. Our future work will focus on optimizing exosomes for specific and efficient delivery to the brain; finding the least invasive way of delivering exosomes to the brain; and showing that exosomes can be used to rescue disease in a mouse model of Rett Syndrome.

Acknowledgements: This symphony would have been impossible without our musical ensemble of Hollis T. Cline, Alysson R. Muotri, John R. Yates III, Pinar Mesci, Cassiano Carromeu, Daniel B. McClatchy and Lucio Schiapparelli. I sincerely thank Monica Coenraads for help in providing better voice to my words.