Posts tagged exosomes

A specific MicroRNA, a short set of RNA (ribonuclease) sequences, naturally packaged into minute (50 nanometers) lipid containers called exosomes, are released by stem cells after a stroke and contribute to better neurological recovery according to a new animal study by Henry Ford Hospital researchers.

The important role of a specific microRNA transferred from stem cells to brain cells via the exosomes to enhance functional recovery after a stroke was shown in lab rats. This study provides fundamental new insight into how stem cells affect injured tissue and also offers hope for developing novel treatments for stroke and neurological diseases, the leading cause of long-term disability in adult humans.

The study was published in the journal Stem Cells.

Although most stroke victims recover some ability to voluntarily use their hands and other body parts, nearly half are left with weakness on one side of their body, while a substantial number are permanently disabled.

Currently no treatment exists for improving or restoring this lost motor function in stroke patients, mainly because of mysteries about how the brain and nerves repair themselves.

“This study may have solved one of those mysteries by showing how certain stem cells play a role in the brain’s ability to heal itself to differing degrees after stroke or other trauma,” says study author Michael Chopp, Ph.D., scientific director of the Henry Ford Neuroscience Institute and vice chairman of the department of Neurology at Henry Ford Hospital.

The researchers noted that Henry Ford’s Institutional Animal Care and Use Committee approved all the experimental procedures used in the new study.

The experiment began by isolating mesenchymal stem cells (MSCs) from the bone marrow of lab rats. These MSCs are then genetically altered to release exosomes that contain specific microRNA molecules. The MSCs then become “factories” producing exosomes containing specific microRNAs. These microRNAs act as master switches that regulate biological function.

The new study showed for the first time that a specific microRNA, miR-133b, carried by these exosomes contributes to functional recovery after a stroke.

The researchers genetically raised or lowered the amount of miR-133b in MSCs and, respectively, treated the rats. When these MSCs are injected into the bloodstream 24 hours after stroke, they enter the brain and release their exosomes. When the exosomes were enriched with the miR-133b, they amplified neurological recovery, and when the exosomes were deprived of the miR-133b, the neurological recovery was substantially reduced.

Stroke was induced under anesthesia by inserting a nylon thread up the carotid artery to occlude a major artery in the brain, the middle cerebral artery. MSCs were then injected 24 hours after the induction of stroke in these animals and neurological recovery was measured.

As a measure on neurological recovery, rats were given two types of behavioral tests to measure the normal function of their front legs and paws – a “foot-fault test,” to see how well they could walk on an unevenly spaced grid; and an “adhesive removal test” to measure how long it took them to remove a piece of tape stuck to their front paws.

Researchers then separated the disabled rats into several groups and injected each group with a specific dosage of saline, MSCs and MSCs with increased or decreased miR-133b, respectively. The two behavioral tests were again given to the rats three, seven and 14 days after treatment.

The data demonstrated that the enriched miR-133b exosome package greatly promoted neurological recovery and enhanced axonal plasticity, an aspect of brain rewiring, and the diminished miR-133b exosome package failed to enhance neurological recovery

While the research team was careful to note that this was an animal study, its findings offer hope for new ways to address the single biggest concern of stroke victims as well as those with neural injury such as traumatic brain injury and spinal cord damage – regaining neurological function for a better quality of life.

(Source: henryford.com)

Exosomes are small, virus-like particles that can transport genetic material and signal substances between cells. Researchers at Lund University, Sweden, have made new findings about exosomes released from aggressive brain tumours, gliomas. These exosomes are shown to have an important function in brain tumour development, and could be utilised as biomarkers to assess tumour aggressiveness through a blood test.

“Current wisdom says that cells are closed entities that communicate through the secretion of soluble signalling molecules. Recent findings indicate that cells can exchange more complex information – whole packages of genetic material and signalling proteins. This is an entirely new conception of how cells communicate”, says Dr Mattias Belting, Professor of Oncology at Lund University and senior consultant in oncology at Skåne University Hospital, Lund, Sweden.

Exosomes are small vesicles of only 30–90 nm. They are produced inside cells and act as “transport vehicles” of genetic material that can be transferred to surrounding cells. Since their first discovery, exosomes have been found in blood, saliva, urine, breast milk and other body fluids.

Mattias Belting’s research group has investigated exosomes released from tumour cells of patients with gliomas. The tiny exosome particles are delivered from the tumour to healthy cells of the brain and may prime normal tissue for efficient spreading of the tumour. The researchers in Lund have now shown that the aggressiveness of the tumour is reflected in the exosome molecular profile.

“We have succeeded in developing a method for the isolation of exosomes from brain tumour patients through a relatively simple blood test. Our analyses indicate that the content of exosomes mirrors the aggressiveness of the tumour in a unique manner”, says postdoctoral researcher Paulina Kucharzewska.

Exosomes could thus be utilised as biomarkers, i.e. to provide guidance on how the patient should be treated and to monitor treatment response. This possibility is particularly attractive with brain tumours that are not readily accessible for tissue biopsy. However, analysis of exosomes from the blood may also prove important with other tumour types. The value of conventional tumour biopsies is limited by the heterogeneity of tumour tissue, i.e. the tissue specimen may not be fully representative of the biological characteristics of a particular tumour. Exosomes, however, may offer more comprehensive information, according to the researchers.

The second international meeting on exosomes has just opened in Boston, and Mattias Belting and members of his team are there.

“It is very exciting to be part of the emergence of a novel research field. It can be anticipated that the most influential researchers in this area may one day be awarded the Nobel Prize”, says Dr Belting.

The results are published in Proceedings of the National Academy of Sciences (PNAS).

(Source: lunduniversity.lu.se)

Novel intercellular transportation system may have potential for delivering RNAi and other gene-based therapeutics

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,” said Vivian Budnik, PhD, professor of neurobiology and lead author of the study. “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,” said Dr. Budnik. “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.”

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. 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.”

(Source: umassmed.edu)