Analytica chimica acta · 2026
Extracellular vesicles released in vitro from a Parkinson's disease-like model: a combined biochemical and spectroscopic approach as proof of concept for the diagnosis of neurodegenerative diseases.
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Romanò Sabrina, Becerril Rodriguez David, Spahiu Amanda, Domenici Fabio, Mazzuca Claudia, Cricenti Antonio, Krasnowska Ewa Krystyna, Serafino Annalucia
Аннотация
BACKGROUND: Extracellular vesicles (EVs) are stable carriers of molecular signals and can cross the blood-brain barrier, making them promising non-invasive biomarkers for diseases of the central nervous system. While their diagnostic potential is already established and is present in many clinical trials for brain tumors, applications in neurodegenerative disorders such as Parkinson's disease (PD) are still emerging. Although researchers have shown that EVs are involved in the intercellular diffusion of aggregated α-synuclein, a hallmark of PD pathology, the clinical use of EVs as disease biomarkers is still limited by the lack of standardized and selective protocols for EV isolation and analysis.
RESULTS: Here, we proposed a dual biochemical and spectroscopic approach to selectively detect PD-associated EVs. As a cellular model of dopaminergic neurons, we employed the human neuroblastoma cell line SH-SY5Y differentiated with retinoic acid and then subjected to 6-hydroxydopamine (6-OHDA) insult to mimic PD-like neurodegeneration. Western blot analysis of SH-SY5Y cells exposed to the neurotoxin revealed high-molecular-weight α-synuclein species consistent with oligomeric forms, which are known to be highly toxic and were also detected in EVs. Moreover, a dot-blot assay with selective sensitivity for aggregated α-synuclein revealed its localization on the membrane surface of fresh, intact PD-like-derived EVs. In addition, Fourier transform infrared spectroscopy identified biochemical signatures that may correlate with pathological states, distinguishing EVs carrying aggregated α-synuclein.
SIGNIFICANCE: By combining these biochemical and spectroscopic methods, which required minimal sample volumes, we were able to selectively identify the class of vesicles carrying the aggregated, toxic-form of α-synuclein. As proof of concept, our findings highlight the diagnostic and non-invasive potential of these biomarkers for distinguishing pathology, supporting their possible use in liquid biopsy for neurodegenerative diseases.
RESULTS: Here, we proposed a dual biochemical and spectroscopic approach to selectively detect PD-associated EVs. As a cellular model of dopaminergic neurons, we employed the human neuroblastoma cell line SH-SY5Y differentiated with retinoic acid and then subjected to 6-hydroxydopamine (6-OHDA) insult to mimic PD-like neurodegeneration. Western blot analysis of SH-SY5Y cells exposed to the neurotoxin revealed high-molecular-weight α-synuclein species consistent with oligomeric forms, which are known to be highly toxic and were also detected in EVs. Moreover, a dot-blot assay with selective sensitivity for aggregated α-synuclein revealed its localization on the membrane surface of fresh, intact PD-like-derived EVs. In addition, Fourier transform infrared spectroscopy identified biochemical signatures that may correlate with pathological states, distinguishing EVs carrying aggregated α-synuclein.
SIGNIFICANCE: By combining these biochemical and spectroscopic methods, which required minimal sample volumes, we were able to selectively identify the class of vesicles carrying the aggregated, toxic-form of α-synuclein. As proof of concept, our findings highlight the diagnostic and non-invasive potential of these biomarkers for distinguishing pathology, supporting their possible use in liquid biopsy for neurodegenerative diseases.