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Further experiments suggested that the only source
of Syt4 is the neuron. These observations were consistent
with the model that Syt4 is actually transferred from neurons
to muscle cells. As a transmembrane protein, however,
Syt4 was thought to be unable to move from one cell
to another through traditional avenues.
How the Syt4 protein was moving from neuron
to muscle cell was unclear.
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Riding the exosome shuttle from neuron to muscle
A newly discovered intercellular transportation system has the potential to deliver RNAi and other gene-based therapies using these tiny vehicles, such as RNA interference (RNAi), to directly target disease-carrying cells.
Important new research from UMass Medical School demonstrates how exosomes shuttle proteins from neurons to muscle cells where they take part in critical signaling mechanisms, an exciting discovery that means these tiny vehicles could one day be loaded with therapeutic agents, such as RNA interference (RNAi), and directly target disease-carrying cells. The study, published this month in the journal Neuron, is the first evidence that exosomes can transfer membrane proteins that play an important role in cell-to-cell signaling in the nervous system.
"There has been a long-held belief that certain cellular
materials, such as integral membrane proteins, are unable
to pass from one cell to another, essentially trapping them
in the cell where they are made.
What we've shown in this study is that these cellular materials can actually move between different cell types by riding in the membrane of exosomes.
"What is so exciting about this discovery is that these
exosomes can deliver materials from one cell, over a
distance, to a very specific and different cell. Once inside the recipient cell, the materials contained in the exosome
can influence or perform processes in the new cell.
This raises the enticing possibility that exosomes can be
packed with gene therapies, such as RNAi, and delivered
to diseased cells where they could have a
therapeutic effect for people."
Vivian Budnik, PhD
Professor of Neurobiology
lead author
University of Massachusetts Medical School
Discovered in the mid-80s, exosomes have only recently attracted the attention of scientists at large, according to Budnik. Exosomes are small vesicles containing cellular materials such as microRNA, messenger RNAs (mRNAs) and proteins, packaged inside larger, membrane-bound bodies called multivesicular bodies (MVBs) inside cells. When MVBs containing exosomes fuse with the cell plasma membrane, they release these exosome vesicles into the extracellular space. Once outside the cell, exosomes can then travel to other cells, where they are taken up. The recipient cells can then use the materials contained within exosomes, influencing cellular function and allowing the recipient cell to carry out certain processes that it might not be able to complete otherwise.
Budnik and colleagues made this startling discovery while investigating how the synapses at the end of neurons and nearby muscle cells communicate in the developing Drosophila fruit fly to form the neuromuscular junction (NMJ). The NMJ is essential for transmitting electrical signals between neurons and muscles, allowing the organism to move and control important physiological processes. Alterations of the NMJ can lead to devastating diseases, such as muscular dystrophy and Amyotrophic lateral sclerosis (ALS). Understanding how the NMJ develops and is maintained is important for human health.
As organisms develop, the synapse and muscle cell need to grow in concert. If one or the other grows too quickly or not quickly enough, it could have dire consequences for the ability of the organism to move and survive. To coordinate development, signals are sent from the neuron to the muscle cell (anterograde signals) and from the muscle cell to the neuron (retrograde signals). However, the identity of these signals and how their release is coordinated is poorly understood.
Normally, the vesicle protein Synaptotagmin 4 (Syt4)
is found in both the synapse and the muscle cells.
Previous knockout experiments eliminating the Syt4 protein
from Drosophila have resulted in stunted NMJs
(or the neuromuscular junction).
Suspecting that Syt4 played an important role in retrograde
signaling at the developing NMJ, Budnik and colleagues used
knockdown experiments to decrease Syt4 protein levels
in either the neurons or the muscle cells.
Surprisingly, when RNAi was used to knockdown Syt4 in the
neurons alone, Syt4 protein was eliminated in both neurons
and muscles. The opposite was not the case. When Syt4 was
knocked down in muscle cells only, there was no change
in the levels of Syt4 in either muscles or neurons.
To confirm this, Budnik and colleagues inserted a Syt4 gene
into the neurons of a Drosophila mutant completely lacking
the normal protein. This restored Syt4 in both
neurons and muscle cells.
Further experiments suggested that the only source
of Syt4 is the neuron. These observations were consistent
with the model that Syt4 is actually transferred from neurons
to muscle cells. As a transmembrane protein, however,
Syt4 was thought to be unable to move from one cell
to another through traditional avenues.
How the Syt4 protein was moving from neuron
to muscle cell was unclear.
Knowing that exosomes had been observed to carry transmembrane proteins in other systems and from their own work on the Drosophila NMJ, Budnik and colleagues began testing to see if exosomes could be the vehicle responsible for carrying Syt4 form neurons to muscles. "We had previously observed that it was possible to transfer transmembrane proteins across the NMJ through exosomes, a process also observed in the immune system," said Budnik. "We suspect this was how Syt4 was making its way from the neuron to the muscle."
When exosomes were purified from cultured cells containing
Syt4, they found that exosomes indeed contained Syt4.
In addition, when these purified exosomes were applied
to cultured muscle cells from fly embryos, these cells were
able to take up the purified Syt4 exosomes.
Taken together, these findings indicate that Syt4 plays a
critical role in the signaling process between synapse
and muscle cell that allows for coordinated
development of the NMJ.
While Syt4 is required to release a retrograde signal from
muscle to neuron, a component of this retrograde signal
must be supplied from the neuron to the muscle.
This establishes a positive feedback loop that ensures
coordinated growth of the NMJ. Equally important is the
finding that this feedback mechanism is enabled by the use
of exosomes, which can shuttle transmembrane
proteins across cells.
"While this discovery greatly enhances our understanding of how the neural muscular junction develops and works, it also has tremendous promise as a potential vector for targeted genetic therapies," said Budnik. "More work needs to be done, but this study significantly supports the possibility that exosomes could be loaded with therapeutic agents and delivered to specific cells in patients."
About the University of Massachusetts Medical School
The University of Massachusetts Medical School, one of the fastest growing academic health centers in the country, has built a reputation as a world-class research institution, consistently producing noteworthy advances in clinical and basic research. The Medical School attracts more than $250 million in research funding annually, 80 percent of which comes from federal funding sources. The mission of the Medical School is to advance the health and well-being of the people of the commonwealth and the world through pioneering education, research, public service and health care delivery with its clinical partner, UMass Memorial Health Care. For more information, visit http://www.umassmed.edu.
Original article: http://www.eurekalert.org/pub_releases/2013-03/uomm-rte032713.php
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