title: Stimulation of synaptoneurosome glutamate release by monomeric and fibrillated α-synuclein.
abstract: 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.
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
authors.forename: Theodore A
authors.initials: TA
authors.lastname: Sarafian
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
authors.forename: Kaitlyn
authors.initials: K
authors.lastname: Littlejohn
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Yuan
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
authors.forename: Charlene
authors.initials: C
authors.lastname: Fernandez
authors.affiliation: Brain Research Institute, UCLA, Los Angeles, CA, USA.
authors.forename: Marianne
authors.initials: M
authors.lastname: Cilluffo
authors.affiliation: Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, USA.
authors.forename: Bon-Kyung
authors.initials: BK
authors.lastname: Koo
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.affiliation: Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Watson
mesh_terms: D000818:Animals; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010300:Parkinson Disease; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism*; D051844:alpha-Synuclein / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: C497604:SNCA protein, human; D051844:alpha-Synuclein; D018698:Glutamic Acid
keywords: electron microscopy; excitotoxicity; fibrillated; fluorescence; glutamate oxidase; synaptoneurosome; α-synuclein