Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis

Synucleinopathies are neurodegenerative diseases characterized by pathological inclusions called "Lewy pathology" (LP) that consist of aggregated alpha-synuclein predominantly phosphorylated at serine 129 (PSER129). Despite the importance for understanding disease, little is known about the endogenous function of PSER129 or why it accumulates in disease. Here we conducted several observational studies using a sensitive tyramide signal amplification (TSA) technique to determine PSER129 distribution and function in the non-diseased mammalian brain. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb (OB) and several brain regions across the neuroaxis (i.e., OB to brain stem). In contrast, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. We found evidence of PSER129 positive structures in OB mitral cells of non-diseased mice, rats, non-human primates, and healthy humans. Using TSA multiplex fluorescent labeling we show that PSER129 positive punctate structures occur within inactive (i.e., cfos negative) T-box transcription factor 21 (TBX21) positive mitral cells and PSER129 within these cells was spatially associated with PK-resistant alpha-synuclein. Ubiquitin was found in PSER129 mitral cells but was not closely associated with PSER129. Biotinylation by antigen recognition (BAR) identified 125 PSER129-interacting proteins in the OB of healthy mice, which were significantly enriched for presynaptic vesicle trafficking/recycling, SNARE, fatty acid oxidation, oxidative phosphorylation, and RNA binding. TSA multiplex labeling confirmed the physical association of BAR identified protein Ywhag with PSER129 in the OB and in other regions across the neuroaxis. We conclude that PSER129 accumulates in mitral cells of the healthy OB as part of alpha-synuclein normal cellular functions. Incidental LP has been reported in the OB, and therefore we speculate that for synucleinopathies either; the disease processes begin locally in OB mitral cells or a systemic disease process is most apparent in the OB because the natural tendency to accumulate PSER129. Significance StatementMultiple lines of evidence have suggested that the disease process in some synucleinopathies begins in the olfactory bulb. Here we demonstrated that disease-associated phosphorylated alpha-synuclein preferentially occurs in mitral cells of the healthy mammalian olfactory bulb. We identified the protein interactome of phosphorylated alpha-synuclein in the healthy mouse olfactory bulb and established phosphorylated alpha-synuclein associates with presynaptic glutamatergic vesicles, SNARE machinery, and RNA metabolism machinery. Our data implicates olfactory bulb mitral cells in synucleinopathy pathogenesis. These findings advance our understanding of synucleinopathy disease origins and set the stage for new experimental models to interrogate the pathogenesis of synucleinopathies.