Summary:
α-Synuclein is an abundant cytosolic protein which exerts very important
functions in presynaptic nerve terminals. It has been directly linked to
idiopathic and familial forms of Parkinson’s disease through biochemical and
genetic studies. Parkinson’s disease is a progressive neurodegenerative
disorder which is characterized by the spread of proteinaceous inclusions, the
main constituent of which is α-synuclein. During the past years a plethora of
findings suggests that α-synuclein has toxic paracrine effects. This protein is
secreted via non-classical pathways which have not been fully identified yet.
Recently it was shown that one of them is the exosome secretion pathway.
Exosomes are cell-secreted vesicles of endocytic biogenesis and specific
biochemical and morphological characteristics. They have rightfully taken their
place in the center of scientific research as functional mediators of
intercellular communication. Proteins that are related to neurodegeneration,
such as the Aβ amyloid peptide and α-synuclein, have been found to be secreted
in association with exosomes. Assuch, the “Trojan Horse” hypothesis proposes
that exosomes carry and deliver toxic protein species to healthy recipient
cells.
Inthepresentstudy,thesecretionofα-synucleinunder physiological and
disease-mimicking conditions is quantitatively assessed for the first time.
Initially,the normal topology of α-synuclein secretion was investigated in
healthy primary cortical neurons. The culture of cells in compartmentalization
devices was optimized. α-Synuclein was assessed with an ultra-sensitive
in-house ELISA. Next, we investigated how the physiological secretion of the
protein is altered by conditions which mimic Parkinson’s disease. Two
cell-models of the disease were used, for the overexpression of the wild type
and A53T mutant form of α-synuclein, upon induction. The secretion of α-
synuclein in a free form and in association with exosomes was quantitatively
assessed and comparisons were made as to the effect of the levels and mutation
to either of the secreted forms. To this end, two sensitive quantitative
methods were optimized: micro-Bradford and the Ellman esterase assay.Using
these methods together,the comparison of the number of exosomes secreted
between different experimental conditions was achieved. Likewise, the quantity
of α-synuclein associated with the same number of exosomal particles was
calculated with precision. Last, a protocol for exosome labeling was optimized
in order to study the kinetics of exosome uptake.
α-Synuclein is normally secreted by both the soma and neuronal axons. The
extracellular protein is encountered in a free and an exosome-associated form.
The intracellular induction of α-synuclein expression led to a significant
increase of free α-synuclein and at comparable levels between the wild type and
mutant form. This suggests that the mutation does not alter the mechanism of α-
synuclein secretion. Under these conditions, the exosome number remains
constant yet, more α-synuclein load is packed into exosomes. Also in this case,
the mutation does not affect the mechanism of exosome-mediated secretion.
Another important finding is that exosomes are not the main non-classical
secretion mechanism by which the protein is secreted out of the cell under
these experimental conditions. This may signify that other mechanisms are
activated when the intracellular levels of the protein are increased.
Nevertheless, the question of whether exosomes are an important means by which
toxic species of the protein exit the cell remains to be answered. Finally, it
was demonstrated thatexosomes are uptaken by differentiated cells and primary
neurons and that their uptake is a time-dependent process.
The findings of the present study suggest that the maintenance of the
equilibrium ofα-synuclein in and outside of the cell is a potent thepareutic
target against the spread of Parkinson’s-related pathology. Exosomes could be
very promising carriers of targeted therapy, due to their unique ability to
cross the plasma membrane and to functionally deliver their cargo intact.
Keywords:
Parkinson's Disease, α-synuclein, characterization of secretion, exosomes, neuronal cells
