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Extracellular vesicles for clinical diagnostics of nervous system diseases
Atai, N.
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Atai, N. (2014). Extracellular vesicles for clinical diagnostics of nervous system diseases
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Download date: 18 Jun 2017
Chapter 3: Extracellular vesicles: shedding new insights on their role in the central
nervous system
Running title: Extracellular vesicles in the central nervous system
Nadia A. Atai MD candidate, PhD candidate1,2,3, Ganesh M. Shankar MD, PhD1, William
E. Butler MD1, Stefano Pluchino PhD4 , Pamela S. Jones MD1, Daniel P. Cahill MD,
PhD1, Bob S. Carter MD, PhD5,6, Fred H. Hochberg MD*1,2,7
1
Department of Neurosurgery, Massachusetts General Hospital, Boston, Massachusetts,
USA,
2
Department of Neurology and Program in Neuroscience, Massachusetts General
Hospital and Harvard Medical School, Boston, Massachusetts, USA,
3
Department of Cell Biology and Histology, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands,
4
Department of Clinical Neurosciences, John van Geest Cambridge Centre for Brain
Repair and Wellcome Trust-Medical Research Council Stem Cell Institute, University of
Cambridge, United Kingdom.
5
Department of Neurosurgery, University of California, San Diego, California, USA
6
Center for Theoretic and Applied Neuro-oncology, University of California, San Diego,
California,
7
Pappas Center, Massachusetts General Hospital, Boston, Massachusetts
Key
words:
Exosomes,
Extracellular
vesicles,
nervous
system,
glioma,
neurodegenerative, infection, biomarker.
*Corresponding author:
Fred H. Hochberg, MD,
Massachusetts General Hospital Cancer Center
Stephen E. & Catherine Pappas Center for Neuro-Oncology
55 Fruit Street, YAW 9E
Boston, Massachusetts 02114
E-mail: [email protected]
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!
47
Abstract
The biofluid of the central nervous system is composed of intercellular fluid released
from cells, intercellular fluid as ultrafiltrate of plasma regulated by the “blood-brain
barrier”, and “cerebrospinal fluid” (CSF) as ultrafiltrate of the vascularized choroid
plexus. The second and third fluid compartments have been evaluated, whereas little is
known of the first space nor the communication between it and the CSF. Understanding
of this relationship is fundamental for the development of biomarker analytics utilizing
these
biofluids
to
diagnose
and
provide
metrics
of
brain
trauma,
stroke,
neurodegenerative diseases, mental disorders, and infectious diseases of the brain and
coverings. Over the last decade, insights have been gained into the role of extracellular
vesicles (EVs) budding from the plasma membrane of cells that can influence
neighboring cells, to affect immune responses and to activate molecular and
neuroendocrine pathways. Knowledge of these vesicles may open the venue for the
development of diagnostic or prognostic neurologic biomarkers while offering
explanations for neural development, immune reactions and neurophysiology. The 2013
Frye-Halloran Symposia offered the basis for novel translational studies of EVs. This
symposium provided the first discussion of the potential role of EVs as diagnostic tool for
neuroscientists and clinicians.
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48
1. Introduction
The Frye-Halloran Symposium 2013
The annual Frye-Halloran Symposium provides a forum for international scientists to
translate basic insights to the care of patients with tumors of the central nervous system
(CNS). The symposia have been organized over a decade with topics including the use of
microdialysis to measure levels of chemotherapeutic drugs in brain tumors, novel
approaches in vascular imaging of brain tumors, stem cells in brain tumors, primary CNS
lymphoma, biomarkers of brain tumors as well as mutations in isocitrate dehydrogenase 1
and 2 (IDH1/2) in glioma. The 2013 Symposium at Massachusetts General
Hospital/Harvard Medical School was an educational forum at the meeting of the
International
Society
for
Extracellular
Vesicles
2013
(ISEV;
http://www.isevmeeting.org/). The meeting was attended by over 700 participants. A fifth
of the contributions were directly relevant for neuroscientists offering the prospect of
novel diagnostics and therapy of neurologic disorders. The presentations were
particularly relevant in the light of the recent NIH Common Fund initiative towards a
better understanding of EVs and their analysis for the diagnosis of brain tumors. We
review the highlights presented at this meeting focusing on EVs in healthy and diseased
brain.
2. Extracellular vesicles history
Douglas Mulhall Av-Gay Laboratory at the University of British Columbia as well as
the Delft University of Technology and the Erasmus University) reviewed the history of
EVs. His review focused on how to use historical successes to generate faster and more
practical results to avoid reinventing the wheel. He stressed that successful EV vaccines
already exist, such as those against bacterial meningitis, and are important practical
models to prevent and treat a range of human diseases. Another pathway to success is to
re-examine the large body of literature on membrane-bound viruses, which only recently
were identified to be EVs whose mechanisms have been hijacked by viruses. Those
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49
under-appreciated data, when combined with more recent data on RNA and other factors
suggest that EVs may be the new drivers of genetic therapies.
The relevance of EVs for neurologists and neurosurgeons may be that EV vaccines point
at a non-invasive low-risk potential for preventing neurological disorders. By studying
how EV are being used for vaccine and therapy development in, e.g., infection and cancer
outside the neurological field, it may be possible to accelerate the development of new
therapies for neurological disorders. An important tissue for future development is the
discovery that EVs distinct from synaptic vesicles can traffick between nerve cells,
opening new avenues to explore intercellular neuronal communication1. He finished his
lecture emphasizing the potential importance of the greatly under-explored role of EV in
ecology.
Shortly after the Symposium, the 2013 Nobel Prize for Medicine was awarded for
discoveries of vesicle cargo delivery mechanisms. The discovery by one of the laureates
Thomas Südhof how neurotransmitters are released from vesicles that fuse with the outer
membrane of nerve cells underlined the significance of vesicles for neuroscience.
However, the Nobel Prize did not yet recognize EVs as a mechanism for transporting
more than neurotransmitters; they allow precise packaging and intercellular trafficking of
complex aggregations. Mulhall described in his presentation how the latter EV discovery
was delayed for years due to an artefact of the acridine orange staining technique that
caused EVs to explode when prepared to electron microscopy (EM), which led to the
concept that vesicles were used only for exocytosis2. Surprisingly, acridine orange is used
until today in EM to study EVs/exocytosis and this stresses the importance of
interdisciplinary studies of EVs.
Alain Brisson, University of Bordeaux, characterized EVs from blood plasma by Cryo
EM and immuno-gold labeling to provide a comprehensive structural description of EVs
in normal plasma. He reported that plasma-derived EVs range in size from 50 nm to 8 !m
with 90% of them being smaller than 500 nm. He identified the main populations of EVs,
namely EVs that expose phosphatidylserine and bind annexin-5, and EVs of platelet or
erythrocyte origin. His data provided novel structural information on plasma-derived EVs
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50
and may open avenues for characterizing EVs from different cell types, including cancer
cells.
EVs were first discovered in 1946 by Chargaff and West in a study of the coagulation
system3. In 1967, they were described as “cell dust in human plasma”4. The vesicles are
produced by most eukaryotic (Fig. 1A) and prokaryotic cells and are considered to be
important in the biology5 of cells and in particular of platelets, lymphocytes and
endothelial cells6 (Fig. 3C). Currently, a broad terminology is used without consensus
referring to these EVs as exosomes, exosome-like vesicles, microvesicles, shedding
microvesicles, epididimosomes, argosomes, promininosomes, prostasomes, dexosomes,
texosomes, archaeosomes, and oncosomes7.
It is generally accepted that their size range from 30 to 1000 nm in diameter; however,
recent studies indicate that larger EVs exist (Brisson et al., unpublished data) and may be
precursors of smaller vesicles. It is believed that most EVs are formed by inward budding
of endosomal membranes giving rise to intracellular multivesicular bodies that later fuse
with the plasma membrane to be released to the exterior7 of cells. EVs can be isolated by
differential centrifugation of serum, plasma, saliva, urine, breast milk, (Fig. 2)8 (Atai et
al., unpublished data). So far, EVs have been isolated from human normal brain, brain
tumors, CSF, vitreal fluids and cochlear fluids (Fig. 1B). Thus EVs have been classified
by their origin (donor cell or fluid), density, size, morphology, and lipid composition but
classifications have not been performed yet using either EM (Fig.1), laser light,
NanoSight (Fig. 3C) analysis or structural chemistry. Virtually no studies have been
performed on biofluids of the central nervous system from normal individuals. EVs
appear to play a pivotal role in cellular homeostasis such as intercellular communication,
waste management, the presentation of antigens and the response to environmental
changes5. Teleologically, EV-mediated delivery of substances provides more stable
conformational conditions for the contents, a higher bioactivity of the biomolecules
inside, an efficient distribution in the microenvironment and more efficient interactions
with target cells, as compared to free biomolecules in biofluids9;10. EVs traffick between
cells and provide pre-packaged DNA, mRNA, microRNA (miRNA), other non-coding
RNAs (ncRNAs) and proteins (Fig. 3A). For example, EVs derived from normal cells
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51
such as B cells and dendritic cells have potent immune-stimulatory and antitumor effects
in vivo and have been used as antitumor vaccines11. Dendritic cell-derived EVs contain
co-stimulatory proteins necessary for T-cell activation whereas EVs released from tumor
cells may contain oncogenic components and can suppress the immune response and
accelerate tumor growth12-15. EVs derived from platelets may play a role in coagulation16.
As EVs convey donor cell-derived information, they serve as potential diagnostic
biomarkers for various disease states of which the most promising are tumors and those
of the nervous system (Hochberg et al., submitted). For instance, peripheral blood EVderived DNA, RNA and protein contents provide a unique fingerprint for monitoring
dynamic changes in tumors of the lung and melanoma (Fig. 3B). EVs obtained from
peripheral blood or CSF enable detection of EGFRvIII mutations and CSF-derived EVs
enable the detection of IDH1/IDH2 mutations in glioma or BRAF mutation in pediatric
nervous system tumors17. Recognition of the diagnostic importance of EVs in disease can
be followed by novel therapies which harness EVs for immunization and immune
regulation of neurologic diseases, inhibition of aberrant intercellular communication, and
targeted drug delivery.
3. Extracellular vesicles in healthy brain
The first function that was dedicated to EVs was waste management of cellular content,
such as microglia-mediated clearance of toxins or aggregates18 and abnormalities in this
mechanisms are associated with neurodegenerative diseases19. Fauré et al. demonstrated
that EVs promote transfer of the L1 cell adhesion molecule (L1CAM), a neuronal
transmembrane protein which guides synapse development and neuronal cell migration20.
Lachenal et al. showed in vitro that EVs are part of normal physiology of a synapse and
contribute to synaptic transmission and plasticity1. In this study, cortical neurons released
EVs containing glutamate receptor (GluR2) subunits and "-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor, key modulators of the synaptic glutamatergic
activity system. As consequence of their observations, Smalheiser et al. proposed that the
epigenetic content of EVs sustain cell-to-cell communication in the CNS and contribute
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52
to synaptic plasticity21;22. In another study, it was shown that oligodendrocytes secrete
myelin protein in their EV, which could be used as marker for oligodendrocyte-derived
EVs. Furthermore, these EVs contain proteins that may function to protect cell against
stress23-25. A very recent study suggests that oligodendroglial-derived EVs contribute to
neuronal integrity. Here, activity-dependent release of the neurotransmitter glutamate
triggers oligodendroglial EVs secretion mediated by the entry of calciumions via
oligodendroglial NMDA and AMPA receptors. Target neurons internalize the released
EVs in vivo, and by endocytosis in vitro, and show increased viability under conditions
of cell stress23. Koles and Budnikobserved show in neuromuscular junctions of
Drosophila that EVs transfer the Drosophila wnt1 homolog wingless (wg) across the
synapse, which binds to Drosophila frizzled 2 (DFz2) receptors leading to uptake by the
postsynaptic muscle recipient cells26-29.
4. Role of extracellular vesicles in glioma and brain tumor metastases
Johan Skog, Exosome Diagnostics, New York, who was the first to show that tumorderived mutations such as EGFRvIII can be detected in serum-derived EVs of glioma
patients, discussed the role of EVs in various biofluids as biomarker for different type of
tumors such as glioma and melanoma (http://www.exosomedx.com/).
Alain Charest, Tufts University School of Medicine, Boston, highlighted how animal
models can support the biofluid-derived EVs for detection of tumor-specific markers,
such as EGFRvIII. In addition, he also reviewed the use of gene-based technologies to
develop and test RNA interference-mediated therapeutics in mouse models of brain
cancer30.
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Hector Peinado, Weill Cornell Medical College, New York, presented his work on the
role of EVs generated by tumor cells and their effect on metastatic progression of
malignant melanoma. He identified that tumor-derived EVs instruct bone marrow
progenitor cells to form a pro-metastatic and pro-vasculogenic phenotype to sustain the
tumor progression in a process that they defined as “education of the tumor
microenvironment through tumor-derived exosomes”31. Importantly, his work was the
first to demonstrate that oncoproteins, i.e. c-Met, are increased in bone marrow-derived
progenitor cells via tumor exosome transfer or effects.
Bruce Chabner, Harvard Medical School and Massachusetts General Hospital, Boston,
who has extensive experience in the field of cancer drug discovery and development,
discussed the clinical implication of EVs in cancer treatment such as glioma.
He
concluded that current diagnostic and therapeutic interventions are restricted by the lack
of access to tissue during treatment and at the time of tumor progression. He suggested
that EVs could provide an easily accessible window into molecular changes induced by
experimental treatments.
Glioma affect over 20,000 Americans each year. The incidence of glioblastoma is
approx. 3.5 per 100,000 people per year with a mean overall survival of 1.5 year as a
consequence of recurrence (Hochberg et al., submitted). Glioma are divided into tumors
of astrocytic origin and subdivided by grade of severity into low-grade astrocytoma
(WHO grade II), anaplastic astrocytoma (WHO grade III), glioblastoma (GBM: WHO
grade IV). Glioma of oligodendrocytic origin are oligodendro. Glioma (WHO grade II)
and anaplastic oligodendroglioma (WHO grade III). GBM is histopathologically
characterized by nuclear atypia, high mitotic activity, proliferation of blood vessels and
necrosis32. About 90% of these tumors develop rapidly (de novo or primary GBM)
without evidence of a low-grade precursor33. Secondary GBM develops slowly,
representing transformation from low-grade astrocytomas. Standard treatment involves
resection followed by concurrent chemotherapy with temozolamide and conformal
radiation34. Glioma may possess genetic mutations that can affect prognosis and response
to therapy. These mutations including EGFRvIII and IDH1/2 can be found by analysis of
glioma tumor tissue removed from brain as well as in EVs obtained from plasma or
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serum or CSF from these patients35;36. A second class of intramedullary neoplasms is
metastasis, which represents an incidence of 100,000 people/year in the United States.
As an example, intracranial lesions are noted in nearly 50 percent of people with
metastatic melanoma which has a frequently-occurring mutation in the V600E of BRAF
gene and could be detected in EVs from plasma or serum37.
5. Neurodegenerative disease
Neurodegenerative diseases represent a wide array of pathological conditions such as
Alzheimer’s disease, Parkinson’s disease (PD), prion disease, Huntington’s disease,
dementia that are mainly caused by damaged neurons in the brain. These diseases are
incurable resulting in progressive degeneration and finally loss of neuronal circuits,
which cannot be regenerated leading to neurological dysfunction such as mental and
movement disorders. Currently, the role of EVs in neurodegenerative diseases has not
been elucidated; however, some studies show that EVs could be used as vehicle to
diagnose and treat these diseases38.
Parkinson’s disease
Parkinson’s disease (PD) is a neurodegenerative disorder considered to be exclusively
triggered by environmental factors up to 1997 when mutations in the SNCA gene were
identified. In addition, more recent genome-wide association studies (GWAS) have
shown genetic contributions in the development of sporadic disease, such as mutations in
the LRRK2 gene39. PD is characterized by the degeneration and death of dopaminergic
neurons in the substantia nigra pars compacta that project to the striatum, where
specialized neuronal circuits control body movement. Another pathological hallmark of
the disease is the presence of intraneuronal " -synuclein-containing aggregates called
Lewy bodies40. A recent study reported calcium-dependent secretion of "-synuclein in
cell culture, invoking a pathway for secretion of the protein by EVs41. It is speculated
that this intraneuronal transfer of "-synuclein aggregates could propagate dissemination
of the aggregates throughout the brain42. Microglial activation is commonly observed in
postmortem PD brain tissue, suggesting that inflammation may play a role in disease,
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although it is unclear whether this inflammation is causative or a marker of earlier
pathological events43.
Alzheimer disease
Alzheimer disease (AD) is a neurodegenerative disease that is clinically characterized by
progressive dementia, and neuropathologically, by loss of synapses and neurons, gliosis,
and the presence of both amyloid plaques and neurofibrillary tangles44. Plaques consist of
amyloid ß (A!), a peptide generated by proteolysis of the type I protein, amyloid !
precursor protein (A!PP), upon sequential cleavage by !- and "-secretases. "-secretase
has been characterized as a multiprotein complex, of which presenilin is a component,
mediating intramembrane proteolysis.
Familial AD (FAD) is caused by the
overexpression of or mutations in the A!PP gene, or by mutations in the presenilin genes.
Sporadic AD is the most frequent form of dementia in the elderly with an exponential
age-dependent increase in incidence of AD. Aß has been shown to be associated with
EVs, possibly arising from early endosomal processing of AßPP45. EVs also contain the
proteases involved in processing AßPP into Aß; however, the pathologic implication of
this finding is unclear46.
Prion disease
Prion disease is a neurodegenerative disorder resulting from an infectious agent. This
condition results from progressive transformation of the native prion protein (PrPc) into
the disease associated scrapie (PrPsc). These proteins have been found in EVs47-49 and are
proposed to play role in EV-mediated transfer via tunneling nanotubes, which may
contain and transfer these misfolded proteins to their microenvironment50;51. In animal
studies, intracerebral injection of these EVs copies the prion protein disease course49.
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Amyotrophic lateral sclerosis
Stefano Pluchino, University of Cambridge, Cambridge presented stem cell- and EVbased prospective therapies to promote CNS repair in neurodegenerative diseases. He
first reviewed the most recent evidence that some of the functions of grafted stem cells,
including immune regulation, require sophisticated mechanisms of intercellular
communication52. Then, he discussed that while some communication is delivered by
secreted cytokines and/or growth factors53, his groups most recent work suggests that a
key role may be attributed to a novel mechanism of intercellular communication through
the transfer of EVs from grafted stem cells to target host cells. He stressed that this
represents one of the most challenging areas in the field of regenerative medicine and that
EVs could be used as vehicle for intercellular communication to initiate tissue repair in a
number of potential target neurological diseases, such as multiple sclerosis.
Samir El Andaloussi, Karolinska Institutet, Stockholm and Oxford University, Oxford,
discussed EV-mediated delivery of siRNA in animal models of neurodegenerative and
neuromuscular disorders to halt progression of these diseases. He discussed the potential
of using engineered EVs displaying targeting peptides for macromolecular drug delivery
to the CNS and the challenges associated with drug loading into EVs (i.e. inefficient
loading of exogenous drugs). Alternative strategies of drug loading were discussed where
endogenous small RNAs or proteins (for example, surface receptors) are enriched in or
on EVs. Furthermore, he introduced the concept of combining the innate
immunosuppressive properties of specific EVs with further engineering of EVs for
treatment of inflammatory diseases. Exploiting the innate regenerative potential of EVs,
further loading them with drugs and target them to CNS can be an effective strategy to
target various neurological diseases.
The pathological signature of amyotrophic lateral sclerosis (ALS) is the presence of
misfolded copper-zinc superoxide dismutase 1 (SOD1) and TAR DNA-binding protein
43 (TDP43). There is increasing evidence that these proteins are misfolded and can be
transferred between different cells in CNS54. It has been shown in mouse neuron cultures
and motorneuron-like cell lines that these cells secrete SOD1 in EVs55. It has also been
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demonstrated that co-culturing neurons in medium containing EVs with SOD1 results in
uptake of SOD1 and subsequent neurotoxicity56-58.
6. Infectious diseases of the central nervous system
Guido Grandi, Chiron Vaccines/Novartis Vaccines, HollySprings, NC, discussed the use
of bacterial outer membrane vesicles for developing vaccines to prevent meningitis. He
described ongoing clinical trials in Europe.
Stephen Gould, John Hopkins University, Baltimore, MD, presented his work on
intercellular vesicle traffic. He explained that this pathway allows for transfer of lipids
and proteins, a process that has obvious implications on intercellular communication. He
discussed whether EV formation requires the class E VPS proteins (a group of 20
proteins that generate ‘reverse’ oriented vesicles at endosomal membranes) and factors
that have been implicated in the fusion of late endosomes with the plasma membrane
(Rab27 proteins, their effectors, such as Slp1 and myosin V). He recently introduced
‘Trojan exosome hypothesis’59, which proposes that retroviruses exploit EVs exchange
for (i) the synthesis of retroviral particles, and (ii) a low-efficiency pathway of horizontal
transfer that does not depend on retroviral proteins. This mechanism may explain the
ability of retroviruses to infect cells that lack their receptor and the horizontal
transmission of Env-deleted retroviruses such as HIV/AIDS.
As with any organ system, the CNS can be infected by bacterial, viral, fungal and
protozoal vectors that are diagnosed by imaging and by CSF/plasma analyses.
Approximately 2500-3500 cases of bacterial meningitis is caused annually by Neisseria
meningitidis, a commensal organism that colonizes the nasopharynx in approx. 20% of
the adults with a virulence mediated by the polysaccharide capsule. The molecular
composition of this capsule distinguishes the 12 variants of N. meningitidis, of which 6
have been shown to cause epidemics. The development of vaccines targeting these
capsule antigens have been challenging given the variability between strains. Outer
membrane vesicles released by N. meningitidis have been in focus in the vaccine
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development by Guido Grandi of Novartis.
7. Other neurologic diseases
The relevance of EVs and pathologies of the CNS that were not addressed in the FryeHalloran Symposium 2013 are briefly discussed here.
Cerebrovascular disorders
Cerebrovascular disease (CVD) are common conditions in the elderly including stroke
and transient ischemic attack that are further classified on the basis of etiology, location,
and duration of symptoms60. Lee et al observed that platelet-derived EVs are elevated in
patients with lacunar infarcts61. Others demonstrated that high amounts of these EVs are
associated with an elevated risk to develop stroke62-64. In addition, it seems that
endothelium-derived EVs can predict risk for cerebral ischemia and may have a potential
role as a stroke biomarker65;66. In another study, endothelium-derived EVs predicted risk
for cerebral vasospasm in patients with spontaneous subarachnoid hemorrhage67.
Epilepsy
Epilepsy is characterized by recurrent seizures and can be a result of brain trauma,
strokes, neoplasm, infection, and toxins. A recent study found elevated amounts of
prominin-1/CD133-positive EVs in CSF in patients with partial temporal epilepsy
secondary to neoplasms or hippocampal sclerosis68. Prominin-1 (rodent) /CD133 (human)
is a somatic stem cell marker and it is also often associated with malignant tumors such
as glioma.
Trauma
Every year in the United States, approx. 1.7 million traumatic brain injury (TBI) cases are
reported. EVs may serve as a tool to monitor recovery following TBI. Recent studies
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reported that phosphatidylserine (PS)-positive EVs are detectable in CSF and plasma, and
enhance pro-coagulant effects. It has been shown that these EV levels reach a peak in the
acute phase and decline at 10 days after ictus69. These EVs seem to be mainly derived
from platelets and endothelial cells. In addition, persistently high amounts of these EVs
are associated with poor clinical outcome, suggesting that these EVs are used as a
prognostic factor.
8. Extracellular vesicles and international collaboration
Dr. Bob Carter, University of Califoria San Diego, CA, emphasized the importance of
an EV-consortium. He is one of the leaders in the clinical brain tumor program at UCSD
bringing together the efforts of 50 faculty and staff members who are focused on brain
tumor clinical care, research, and education. He introduced the first brain tumor
consortium studying the EGFRvIII mutation, which is found in 30% of glioma patients.
This consortium aims to collect CSF and plasma to study the role of EV-derived
EGFRvIII as a biomarker for glioma. This consortium provides special kits and protocols
to process samples for the mutation. This method may be transferred to any other tumor
class and marker.
Dr. Susan Windham-Bannister, Massachusetts Life Sciences Center, Boston, MA,
highlighted the funding opportunities for the researches in field of EVs and how the
institute could be approached for personal and non-personal funding.
9. Discussion
As highlighted at the 2013 Frye Halloran Symposium, understanding of EVs is evolving
rapidly. Harnessing these insights for diagnostic and therapeutic purposes in CNS tumors,
degeneration and infection will be the phase of translational research. Drs. Carter and
Windham-Bannister emphasized that these developments hinge on international
collaboration and funding. These developments will also be facilitated by improved
informatics to categorize and analyze EVs that have been described thus far. The success
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60
of the recent large-scale genomic efforts through the Cancer Genome Atlas, was
dependent on algorithms to identify biologically-relevant mutations and copy number
variations. Similarly, a central repository for EVs and their biological characterization
will allow for identifying their roles in diseases described above.
10. The future implication of EVs in healthy and diseased brain
In conclusion, the field of EVs is rapidly expanding. In the next 10 years scientists will
focus on different aspects EVs and their role in normal cells and cancer cells. This will
fill the gap in our understanding of how EVs act in biological systems. In addition, there
are increasingly more investigations in standardized methods to obtain EVs, novel
analytic techniques such as digital PCR and DMR, the evaluation of large numbers of
normative controls and the development of disease-specific nervous system biofluids and
data repositories. Clinicians such as neurologists and neurosurgeons will apply these tools
in the clinic to treat illnesses as diverse as CNS trauma, Alzheimer’s dementia and it’s
surrogates, motor system disease and multiple sclerosis.
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Acknowledgements
The authors thank Ms. Emily Mills (Millstone Design; http://www.millstone-design.com)
for preparation of figures and Prof. Dr. C.J.F. Van Noorden for editing the manuscript.
This work was supported by a 2009 AMC Scholarship, University of Amsterdam, the
Netherlands (N.A.A.). This work was supported by Prof. dr. Xandra O. Breakefield
(NIH/NCI P01 CA069246).
Disclosure
Dr. Bob Carter serves on the Scientific Advisory Committee of Exosome Diagnostics,
Inc. NY. USA.
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Reference
1. Lachenal G, Pernet-Gallay K, Chivet M et al. Release of exosomes from
differentiated neurons and its regulation by synaptic glutamatergic activity. Mol
Cell Neurosci 2011;46:409-418.
2. Jaiswal JK, Fix M, Takano T et al. Resolving vesicle fusion from lysis to monitor
calcium-triggered lysosomal exocytosis in astrocytes. Proc Nat Acad Sci USA
2007;104:14151-14156.
3. Chargaff E, West R. The biological significance of the thromboplastic protein of
blood. J Biol Chem 1946;166:189-197.
4. Wolf P. The nature and significance of platelet products in human plasma. Br J
Haematol 1967;13:269-288.
5. van der Pol E, Boing AN, Harrison P et al. Classification, functions, and clinical
relevance of extracellular vesicles. Pharmacol Rev 2012;64:676-705.
6. van Doormaal FF, Kleinjan A, Di NM et al. Cell-derived microvesicles and
cancer. Neth J Med 2009;67:266-273.
7. Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and
function. Nat Rev Immunol 2002;2:569-579.
8. Keller S, Sanderson MP, Stoeck A et al. Exosomes: from biogenesis and secretion
to biological function. Immunol Lett 2006;107:102-108.
9. D'Asti E, Garnier D, Lee TH et al. Oncogenic extracellular vesicles in brain tumor
progression. Front Physiol 2012;3:294.
10. Muralidharan-Chari V, Clancy JW, Sedgwick A et al. Microvesicles: mediators of
extracellular
communication
during
cancer
progression.
J
Cell
Sci
2010;123:1603-1611.
!
!
63
11. Baj-Krzyworzeka M, Szatanek R, Weglarczyk K et al. Tumour-derived
microvesicles carry several surface determinants and mRNA of tumour cells and
transfer some of these determinants to monocytes. Cancer Immunol Immunother
2006;55:808-818.
12. Clayton A, Mitchell JP, Court J et al. Human tumor-derived exosomes selectively
impair lymphocyte responses to interleukin-2. Cancer Res 2007;67:7458-7466.
13. Wieckowski E, Whiteside TL. Human tumor-derived vs dendritic cell-derived
exosomes have distinct biologic roles and molecular profiles. Immunol Res
2006;36:247-254.
14. Chaput N, Taieb J, Andre F et al. The potential of exosomes in immunotherapy.
Expert Opin Biol Ther 2005;5:737-747.
15. Atai NA, Balaj L, Van Veen H et al. Heparin blocks transfer of extracellular
vesicles between donor and recipient cells. J Neurooncol 2013;115:343-351.
16. Bastida E, Ordinas A, Escolar G et al. Tissue factor in microvesicles shed from
U87MG human glioblastoma cells induces coagulation, platelet aggregation, and
thrombogenesis. Blood 1984;64:177-184.
17. Balaj LC, W; Liau, Lm; Soto, H; Garret, M; Zhu, Ld; Sivaraman, S; Wong, Et;
Carter, B; Hochberg, F; Breakefield, Xo; Skog, J. BEAMing qRT-PCR analysis
of IDH1 mutant in tumor microvesicles. Mol Ther Nucleic Acids 2013;2:e109.
18. Jantzen PT, Connor KE, DiCarlo G et al. Microglial activation and beta -amyloid
deposit reduction caused by a nitric oxide-releasing nonsteroidal antiinflammatory drug in amyloid precursor protein plus presenilin-1 transgenic mice.
J Neurosci 2002;22:2246-2254.
19. Grathwohl SA, Kalin RE, Bolmont T et al. Formation and maintenance of
Alzheimer's disease beta-amyloid plaques in the absence of microglia. Nat
Neurosci 2009;12:1361-1363.
!
!
64
20. Faure J, Lachenal G, Court M et al. Exosomes are released by cultured cortical
neurones. Mol Cell Neurosci 2006;31:642-648.
21. Smalheiser NR. Do neural cells communicate with endothelial cells via secretory
exosomes and microvesicles? Cardiovasc Psychiatry Neurol 2009;2009:383086.
22. Smalheiser NR. Exosomal transfer of proteins and RNAs at synapses in the
nervous system. Biol Direct 2007;2:35.
23. Fruhbeis C, Frohlich D, Kuo WP et al. Neurotransmitter-triggered transfer of
exosomes
mediates
oligodendrocyte-neuron
communication.
PLoS
Biol
2013;11:e1001604.
24. Fruhbeis C, Frohlich D, Kramer-Albers EM. Emerging roles of exosomes in
neuron-glia communication. Front Physiol 2012;3:119.
25. Kramer-Albers EM, Bretz N, Tenzer S et al. Oligodendrocytes secrete exosomes
containing major myelin and stress-protective proteins: Trophic support for
axons? Proteomics Clin Appl 2007;1:1446-1461.
26. Koles K, Budnik V. Exosomes go with the Wnt. Cell Logist 2012;2:169-173.
27. Koles K, Budnik V. Wnt signaling in neuromuscular junction development. Cold
Spring Harb Perspect Biol 2012;4:a008045.
28. Koles K, Nunnari J, Korkut C et al. Mechanism of evenness interrupted (Evi)exosome release at synaptic boutons. J Biol Chem 2012;287:16820-16834.
29. Korkut C, Li Y, Koles K et al. Regulation of postsynaptic retrograde signaling by
presynaptic exosome release. Neuron 2013;77:1039-1046.
30. Zhu H, Acquaviva J, Ramachandran P et al. Oncogenic EGFR signaling
cooperates with loss of tumor suppressor gene functions in gliomagenesis. Proc
Natl Acad Sci U S A 2009;106:2712-2716.
!
!
65
31. Peinado H, Aleckovic M, Lavotshkin S et al. Melanoma exosomes educate bone
marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat
Med 2012;18:883-891.
32. Bleeker FE, Atai NA, Lamba S et al. The prognostic IDH1( R132 ) mutation is
associated with reduced NADP+-dependent IDH activity in glioblastoma. Acta
Neuropathol 2010;119:487-494.
33. Atai NA, Bansal M, Lo C et al. Osteopontin is up-regulated and associated with
neutrophil
and
macrophage
infiltration
in
glioblastoma.
Immunology
2011;132:39-48.
34. Atai NA, Renkema-Mills NA, Bosman J et al. Differential activity of NADPHproducing dehydrogenases renders rodents unsuitable models to study IDH1R132
mutation effects in human glioblastoma. J Histochem Cytochem 2011;59:489-503.
35. Chen WW, Balaj L, Liau LM et al. BEAMing and Droplet Digital PCR Analysis
of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid
Extracellular Vesicles. Mol Ther Nucleic Acids 2013;2:e109.
36. Baldewpersad Tewarie NM, Burgers IA, Dawood Y et al. NADP+ -dependent
IDH1 R132 mutation and its relevance for glioma patient survival. Med
Hypotheses 2013;80:728-731.
37. Menzies AM, Long GV, Murali R. Dabrafenib and its potential for the treatment
of metastatic melanoma. Drug Des Devel Ther 2012;6:391-405.
38. Aguzzi A, Rajendran L. The transcellular spread of cytosolic amyloids, prions,
and prionoids. Neuron 2009;64:783-790.
39. Deng X, Choi HG, Buhrlage SJ et al. Leucine-rich repeat kinase 2 inhibitors: a
patent review (2006-2011). Expert Opin Ther Pat 2012;22:1415-1426.
40. Chinta SJ, Lieu CA, Demaria M et al. Environmental stress, ageing and glial cell
senescence: a novel mechanistic link to Parkinson's disease? J Intern Med
2013;273:429-436.
!
!
66
41. Emmanouilidou E, Melachroinou K, Roumeliotis T et al. Cell-produced alphasynuclein is secreted in a calcium-dependent manner by exosomes and impacts
neuronal survival. J Neurosci 2010;30:6838-6851.
42. Brundin P, Olsson R. Can alpha-synuclein be targeted in novel therapies for
Parkinson's disease? Expert Rev Neurother 2011;11:917-919.
43. Sanchez-Guajardo V, Febbraro F, Kirik D, Romero-Ramos M. Microglia acquire
distinct activation profiles depending on the degree of alpha-synuclein
neuropathology in a rAAV based model of Parkinson's disease. PLoS One
2010;5:e8784.
44. Carreiras MC, Mendes E, Perry MJ et al. The multifactorial nature of Alzheimer's
disease for developing potential therapeutics. Curr Top Med Chem 2013;13:17451770.
45. Rajendran L, Honsho M, Zahn TR et al. Alzheimer's disease beta-amyloid
peptides are released in association with exosomes. Proc Natl Acad Sci U S A
2006;103:11172-11177.
46. Sharples RA, Vella LJ, Nisbet RM et al. Inhibition of gamma-secretase causes
increased secretion of amyloid precursor protein C-terminal fragments in
association with exosomes. FASEB J 2008;22:1469-1478.
47. Vella LJ, Sharples RA, Nisbet RM et al. The role of exosomes in the processing
of proteins associated with neurodegenerative diseases. Eur Biophys J
2008;37:323-332.
48. Vella LJ, Sharples RA, Lawson VA et al. Packaging of prions into exosomes is
associated with a novel pathway of PrP processing. J Pathol 2007;211:582-590.
49. Fevrier B, Vilette D, Archer F et al. Cells release prions in association with
exosomes. Proc Natl Acad Sci U S A 2004;101:9683-9688.
50. Gousset K, Zurzolo C. Tunnelling nanotubes: a highway for prion spreading?
Prion 2009;3:94-98.
!
!
67
51. Gousset K, Schiff E, Langevin C et al. Prions hijack tunnelling nanotubes for
intercellular spread. Nat Cell Biol 2009;11:328-336.
52. Pluchino S, Cossetti C. How stem cells speak with host immune cells in
inflammatory brain diseases. Glia 2013;61:1379-1401.
53. Drago D, Cossetti C, Iraci N et al. The stem cells secretome and its role in brain
repair. Biochimie 2013;95:2271-2285.
54. Schneider A, Simons M. Exosomes: vesicular carriers for intercellular
communication in neurodegenerative disorders. Cell Tissue Res 2013;352:33-47.
55. Gomes C, Keller S, Altevogt P, Costa J. Evidence for secretion of Cu,Zn
superoxide dismutase via exosomes from a cell model of amyotrophic lateral
sclerosis. Neurosci Lett 2007;428:43-46.
56. Grad LI, Guest WC, Yanai A et al. Intermolecular transmission of superoxide
dismutase 1 misfolding in living cells. Proc Natl Acad Sci U S A 2011;108:1639816403.
57. Munch C, O'Brien J, Bertolotti A. Prion-like propagation of mutant superoxide
dismutase-1 misfolding in neuronal cells. Proc Natl Acad Sci U S A
2011;108:3548-3553.
58. Haidet-Phillips AM, Hester ME, Miranda CJ et al. Astrocytes from familial and
sporadic ALS patients are toxic to motor neurons. Nat Biotechnol 2011;29:824828.
59. Nguyen DG, Booth A, Gould SJ et al. Evidence that HIV budding in primary
macrophages occurs through the exosome release pathway. J Biol Chem
2003;278:52347-52354.
60. Latorre JG, Greer DM. Management of acute intracranial hypertension: a review.
Neurologist 2009;15:193-207.
!
!
68
61. Lee YJ, Jy W, Horstman LL et al. Elevated platelet microparticles in transient
ischemic attacks, lacunar infarcts, and multiinfarct dementias. Thromb Res
1993;72:295-304.
62. Cherian P, Hankey GJ, Eikelboom JW et al. Endothelial and platelet activation in
acute ischemic stroke and its etiological subtypes. Stroke 2003;34:2132-2137.
63. Pawelczyk M, Van LL, Fransen E et al. Analysis of gene polymorphisms
associated with K ion circulation in the inner ear of patients susceptible and
resistant to noise-induced hearing loss. Ann Hum Genet 2009;73:411-421.
64. Kuriyama N, Nagakane Y, Hosomi A et al. Evaluation of factors associated with
elevated levels of platelet-derived microparticles in the acute phase of cerebral
infarction. Clin Appl Thromb Hemost 2010;16:26-32.
65. Simak J, Gelderman MP. Cell membrane microparticles in blood and blood
products: potentially pathogenic agents and diagnostic markers. Transfus Med Rev
2006;20:1-26.
66. Simak J, Gelderman MP, Yu H et al. Circulating endothelial microparticles in
acute ischemic stroke: a link to severity, lesion volume and outcome. J Thromb
Haemost 2006;4:1296-1302.
67. Lackner P, Dietmann A, Beer R et al. Cellular microparticles as a marker for
cerebral
vasospasm
in
spontaneous
subarachnoid
hemorrhage.
Stroke
2010;41:2353-2357.
68. Huttner HB, Corbeil D, Thirmeyer C et al. Increased membrane shedding-indicated by an elevation of CD133-enriched membrane particles--into the CSF in
partial epilepsy. Epilepsy Res 2012;99:101-106.
69. Morel N, Morel O, Petit L et al. Generation of procoagulant microparticles in
cerebrospinal fluid and peripheral blood after traumatic brain injury. J Trauma
2008;64:698-704.
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Figure 1. TEM images of EVs detected in vitro and in vivo.
(A) TEM images of normal and a cancer cell shedding EVs in vitro (Skog et a., 2008 and
Van der Pol et al., 2012)
(B) TEM of EVs detected in different human biofluids (scale bar= 500 nm)
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Figure 2. EVpurification protocol in vitro and in vivo.
A. EV purification from cell lines and biofludis according to recent protocols.
B. Different method to detect EVs in biofluids.
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Figure 3. Cell to cell communication mediated by EVs.
A. Tumor cell to normal cell communication mediated by EVs
B. Circulating tumor and normal EVs
C. Different detection methods for validation of circulating EVs
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Figure 4. Role of EVs in different brain disorders.
Localization of different neurodegenerative diseases. Frontal lobe (red, F); Parietal lobe (orange,
P); Occipital (purple, O); Temporal (green; T); Cerebellum (blue; C). CSF (light blue)
A. Stroke in brain with ischemic area (grey)
B. Brain tumor localized in right hemisphere with diffused inflammation and micro tumors.
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Figure 5. EV-derived uptake of EGFRvIII mRNA in recipient cells.
Glioma-derived EVs enriched with oncogene EGFRvIII transfer their content into recipient cells.
This figure shows relative uptake of EGFRvIII mRNA after treatment with EGFRvIII positive
EVs (Atai et al., 2013).
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