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by blood contamination [56-59] . Some studies have reported that total α-synuclein levels were reduced in
the saliva of PD patients compared with control subjects, whereas oligomeric α-synuclein appeared to be
elevated in the saliva of PD patients [56,57] .

MEASUREMENT OF α-SYNUCLEIN FROM NEURONAL EXOSOMES AS A POTENTIAL
BIOMARKER
α-synuclein was considered to be localized mostly in the cytoplasm of neuronal cells, until several studies
demonstrated its presence in human CSF, human plasma and in the conditioned medium of various cell
lines [23,60] . Many studies have shown that α-synuclein is physiologically secreted in the extracellular space,
but the mechanism of α-synuclein release is still unclear. Evidence from recent studies has also suggested
that extracellular α-synuclein can confer to the progression of PD [1-3] and it has been proposed that
α-synuclein secretion, either in a monomeric or oligomeric state, induces α-synuclein propagation via cell-
[61]
to-cell transfer . Thus, elucidating the mechanism by which α-synuclein is secreted in the extracellular
space is of great importance in understanding cellular pathways that may cause PD.

Release of α-synuclein via extracellular vesicles termed exosomes has been demonstrated by our group and
others [62,63] . Exosomes are extracellular vesicles of ~50 to 200 nm diameter and can mediate proximal and
distal cellular communication through the transfer of biological molecules between cells. They originate
from the inward budding of multi-vesicular bodies (MVBs) and are released to the extracellular space upon
fusion of MVBs with the plasma membrane in an exocytic manner. Exosomes are released from numerous
[64]
cell types including neurons and glia and in several studies have been observed to be associated with
pathologic proteins including α-synuclein [63,65] . Based on the current knowledge, exosomes are functionally
active entities, with a highly versatile role, ranging from intercellular communication by delivering
specific protein, lipid or RNA cargo, and removal of obsolete or misfolded proteins, as a means of cell
[66]
detoxification, to deleterious shuttles that impair cell homeostasis .

Some well-characterized functions of exosomes are protein secretion and intracellular uptake, immune
[67]
response regulation and toxicity induction . Interestingly, Danzer et al. demonstrated that exosome
[65]
associated α-synuclein is more potent in transmitting aggregation pathology between neurons than
free-secreted α-synuclein. One study has shown that patients with PD have higher α-synuclein levels in
[68]
[69]
plasma exosomes compared to healthy controls , while Stuendl et al. found decreased neural exosome
α-synuclein levels in PD patients, consistent with the total α-synuclein levels in CSF. In addition, the
quantification of CSF exosomal α-synuclein exhibited distinct differences between patients with PD
and DLB. Moreover, exosomal α-synuclein levels correlated with the severity of cognitive impairment
[69]
in cross-sectional samples from patients with DLB. In the same study, Stuendl et al. showed that
exosomes from PD and DLB patients contain pathogenic α-synuclein species which serve as seeds to
[70]
induce the oligomerization of soluble α-synuclein in recipient cells. Shi et al. have shown that CNS-
derived exosomes can efflux into blood. Importantly, they found a substantially augmented α-synuclein
concentration in the plasma-isolated exosomes from PD patients compared to healthy control subjects,
despite the fact that no differences were detected in plasma total α-synuclein levels. Additionally, they
report a significant increase of plasma exosomal α-synuclein/total α-synuclein ratio in PD patients,
negatively correlated with the disease severity, further supporting the importance of the disease-related
exosomal cargos as PD biomarkers with high sensitivity and specificity. The authors concluded that plasma,
[70]
CNS-derived exosomal α-synuclein can serve as a PD biomarker with high sensitivity and specificity .
The same group has also shown that CNS-derived exosomal tau in plasma is significantly higher in PD
[71]
patients than in controls and is correlated with CSF total tau and phosphorylated tau . Furthermore,
[72]
distinct circulating exosome entities have been identified in the serum of patients with PD . A recent
study demonstrated that the levels of DJ-1 and α-synuclein in plasma CSF-derived exosomes and the