Isolation And Identification Of Serum Exosomes And The Relationship Between Exosomal Proteins And Parkinson’s Disease

Background:Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. Clinical features of the disease include bradykinesia, resting tremor, muscle tone rigidity, and possible cognitive involvement. However, the mechanism underlying disease onset and progression are still unclear. Evidences from genetic, environment and aging may associate with the pathogenesis of the disease. Pathologically, PD is characterized by selective degeneration of dopaminergic neurons in the substantia nigra pars compact (SNpc) and the presence of Lewy bodies (LBs), primarily composed of fibrillar a-synuclein (a-syn) in the surviving neurons.Exosomes are small membranous vesicles with a size ranging from30to100nm and appear with a characteristic round or cup-shaped morphology as observed by transmission or cryo-electron microscopy, with a buoyant density of1.13-1.19g/ml in sucrose sulution. These nanovesicles are generated via the inward budding of endosomes to form multi-vesicular bodies (MVBs) that fuse with the plasma membranes to release exosomes into the extracellular environment. Exosomes are secreted by multiple living cells which contain a variety of proteins, lipids and RNAs. Exosomes were first reported in association with sheep reticulocytes in the1980s, and in2005Caby provided the first evidence for the presence of exosomes in vivo, in the blood from healthy donors. Exosomes represent a novel form of intercellular communication among cells without cell-to-cell direct contact. Growing evidence indicates that the role of exosomes in a variety of physiological pathways as conveyors of biological materials from cell-to-cell. Recently, it is emerging that exosomes are involved in pathological processes as potential carriers in the progression of neurodegenerative pathologies associated with misfold protein. Increase our knowledge on the implication of exosomes in the neurodegeneration and propagation from cell-to-cell transmissions, and a novel strategy for diagnosis and therapeutic intervention in Parkinson and other neurodegenerative diseases.Here, MVBs can either fuse with the lysosome membrane to degrade ILVs and its content, or alternatively with the plasma membrane to release the ILVs as exosomes into the extracellular space. A number of genes linked to PD, either carrying causal mutations (LRRK2, ATP13A2and VPS35) or more common variants acting as risk factors (heterozygous mutation in GBA), have been suggested to play a role in lysosomal-endosomal pathways. LRRK2plays an important role in exosome secretion and fusion of MVBs with plasma membrane as it has been found to co-localize with MVBs. It has been known for several years that α-synuclein and can be detected in cerebrospinal fluid and plasma, as well as in the media of cultured cells. Emmanouilidou showed for the first time that cell-produced α-synuclein was secreted via an exosomal, calcium-dependent mechanism. Then Alvarez was found that α-synuclein is also secreted via exosomes in SH-SY5Y cells. These findings highlight that a-synuclein secretion via exosomes serves to amplify and propagate PD-related pathology.Although studied in several biofluids from healthy donors or tumor patients, exosomes have not been extensively studied in the serum from PD patients. Previous studies found that α-synuclein was detected in serum and was secreted via exosomes in SH-SY5Y cells, there are no studies focusing on wether exosomes in serum from PD patients contain α-synuclein. Objective:Determine whether serum from Parkinson’s disease patients contained exosomes or not, and clarify the significance of the levels of α-synuclein and LRRK2in exosomes extracted from Parkinson’s disease patients. Exosomes may provide a suitable target for diagnosis and therapeutic intervention of Parkinson’s disease. Methods: Serum was collected from20Parkinson’s disease patients and20age-matched controls and exosomes were extracted by ultracentrifugation and ExoQuick Exosome Precipitation Solution. The presence of exosomes was determined by western blot for marker proteins and transmission electron microscopy for its size and appearance. Total protein was purified from the exosomes, and expression levels of a-synuclein and LRRK2were analyzed by western blot.Results:1. Notably, both isolations contained30-100nm vesicles, and were positive for exosome markers (CD63, CD9) based on transmission electron microscopy and western blot. We found ExoQuick Exosome Precipitation Solution to be more effective method to isolate exosomes. Compared to ultracentrifugation, the levels of exosomes’ proteins were significantly higher(P<0.001) than those concentrated by ExoQuick kit. CD63andCD9, two exosomal marker proteins, were identified in the ExoQuick Exosome Precipitation Solution pellet using western blot. Transmission electron microscopy visualized structures consistent with exosomes in size and appearance.2. The expression levels of exosomal a-synuclein were significantly lower in patients with Parkinson’s disease than healthy donors (P<0.05). However, our study did not detect that LRRK2was present in serum exosomes from Parkinsin’s disease patients or healthy donors. Conclusion:1. Our data provieded first evidence that exosomes were present in serum from PD patients.2. This was the first report of a-synuclein in the exosomal fraction from PD patients’ serum. The expression levels of exosomal α-synuclein were significantly lower in patients with Parkinson’s disease than healthy donors.3. Of interst, we did not detect LRRK2in serum exosomes from Parkinson’s disease patients.