Stimulation of synaptoneurosome glutamate release by monomeric and fibrillated α‐synuclein

• Published in: Journal of neuroscience research Vol. 95; no. 9; pp. 1871 - 1887
• Main Authors: Sarafian, Theodore A., Littlejohn, Kaitlyn, Yuan, Sarah, Fernandez, Charlene, Cilluffo, Marianne, Koo, Bon‐Kyung, Whitelegge, Julian P., Watson, Joseph B.
• Format: Journal Article
• Language: English
• Published: United States Wiley 01-09-2017
• Subjects: BASIC BIOLOGICAL SCIENCES; electron microscopy; excitotoxicity; fibrillated; fluorescence; glutamate oxidase; neurosciences & neurology; synaptoneurosome; α-synuclein; glutamate oxidase; fluorescence; fibrillated; electron microscopy; excitotoxicity; synaptoneurosome; α-synuclein
• ISSN: 0360-4012; 1097-4547
• DOI: 10.1002/jnr.24024

The α‐synuclein protein exists in vivo in a variety of covalently modified and aggregated forms associated with Parkinson's disease (PD) pathology. However, the specific proteoform structures involved with neuropathological disease mechanisms are not clearly defined. Since α‐synuclein plays a role in presynaptic neurotransmitter release, an in vitro enzyme‐based assay was developed to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) enriched in synaptic endings. Glutamate measurements utilizing SNs from various mouse genotypes (WT, over‐expressers, knock‐outs) suggested a concentration dependence of α‐synuclein on calcium/depolarization‐dependent presynaptic glutamate release from forebrain terminals. In vitro reconstitution experiments with recombinant human α‐synuclein proteoforms including monomers and aggregated forms (fibrils, oligomers) produced further evidence of this functional impact. Notably, brief exogenous applications of fibrillated forms of α‐synuclein enhanced SN glutamate release but monomeric forms did not, suggesting preferential membrane penetration and toxicity by the aggregated forms. However, when applied to brain tissue sections just prior to homogenization, both monomeric and fibrillated forms stimulated glutamate release. Immuno‐gold and transmission electron microscopy (TEM) detected exogenous fibrillated α‐synuclein associated with numerous SN membranous structures including synaptic terminals. Western blots and immuno‐gold TEM were consistent with SN internalization of α‐synuclein. Additional studies revealed no evidence of gross disruption of SN membrane integrity or glutamate transporter function by exogenous α‐synuclein. Overall excitotoxicity, due to enhanced glutamate release in the face of either overexpressed monomeric α‐synuclein or extrasynaptic exposure to fibrillated α‐synuclein, should be considered as a potential neuropathological pathway during the progression of PD and other synucleinopathies. © 2017 Wiley Periodicals, Inc.
The proteoform structures of α‐synuclein that modulate presynaptic neurotransmitter release in Parkinson's disease (PD) pathology are not clearly defined. In this study, an in vitro enzyme‐based assay was used to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) utilizing various mouse genotypes and reconstituted human α‐synuclein proteoforms (monomers, oligomers, fibrils). Notably, exogenous applications (SN Treated) of fibrillated forms of α‐synuclein preferentially enhanced glutamate release, lending support to synaptic excitotoxicity as a neuropathological pathway during the progression of PD and other synucleinopathies.