Objective: To establish a method that allows for investigating early synaptic dysfunction in Parkinson’s disease (PD) by analyzing CNS-derived extracellular vesicles and assessing their effects on synaptic transmission and membrane properties in autaptic cultured neurons using electrophysiological recordings.

Background: PD is typically diagnosed after major dopaminergic neuron loss, although early pathology may begin at the synapse with presynaptic α-synuclein aggregation and altered synaptic function. Extracellular vesicles (EVs) from PD patients can induce α-syn pathology in neurons in vitro and in vivo (Stuendl et al. 2016; Guo et al. 2020; Herman et al. 2023), suggesting that patient-derived EVs may model early synaptic dysfunction in PD.

Method: To establish the method EVs were isolated from serum and CSF of healthy controls. EVs were fluorescently labeled with a membrane dye to enable visualization of cellular uptake. Primary excitatory neurons were cultured in an autaptic configuration and incubated with different constant doses of labeled EVs. To enhance neuronal uptake and reduce astrocytic interference, a modified autaptic culture system based on the Banker method was used, which spatially separates neurons from astrocytes while maintaining trophic support. Confocal microscopy was performed to confirm successful EV uptake by neurons.

Results: We demonstrated that total serum EVs were readily taken up in standard autaptic co-cultures containing neurons and astrocytes, with preferential uptake by astrocytes. In contrast, in the modified Banker culture system, total serum EVs were efficiently taken up by neurons. Eletrophysiological recordings in standard co-cultures revealed no detectable changes in synaptic properties following EV treatment. In the Banker culture, total serum EVs induced measurable effects in neurons, with possible indications of reduced numbers of functional synapses. Importantly, total CSF-derived EVs did not induce comparable changes in synaptic function under the same experimental conditions.

Conclusion: Using the modified Banker system, neuronal uptake of EVs was successfully achieved, whereas in standard co-cultures EV uptake was predominantly observed in astrocytes. Total serum EVs showed functional effects in neurons, while total CSF-derived EVs did not demonstrate comparable effects under the same conditions.

Standard Cultures Show No EV Effect

EV Effects in Banker Autaptic Neurons

Confocal Images of EV Uptake in Cultures

References: Guo, Min, Jian Wang, Yanxin Zhao, et al. 2020. “Microglial Exosomes Facilitate α-Synuclein Transmission in Parkinson’s Disease.” Brain: A Journal of Neurology 143 (5): 1476–97. https://doi.org/10.1093/brain/awaa090.
Herman, Shay, Ruth Djaldetti, Brit Mollenhauer, and Daniel Offen. 2023. “CSF-Derived Extracellular Vesicles from Patients with Parkinson’s Disease Induce Symptoms and Pathology.” Brain 146 (1): 209–24. https://doi.org/10.1093/brain/awac261.
Stuendl, Anne, Marcel Kunadt, Niels Kruse, et al. 2016. “Induction of α-Synuclein Aggregate Formation by CSF Exosomes from Patients with Parkinson’s Disease and Dementia with Lewy Bodies.” Brain 139 (2): 481–94. https://doi.org/10.1093/brain/awv346.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/extracellular-vesicle-based-characterization-of-synaptic-dysfunction-in-early-parkinsons-disease/