Investigating α-Synuclein Changes in the Olfactory Circuitry and Inflammatory Responses in Mice Nasally Inoculated by a Neurotropic Virus

Abstract

The importance of the olfactory circuitry in Parkinson disease (PD) is supported by the presence of frequent, prodromal hyposmia and of Lewy inclusion-type pathology (LP) in the olfactory bulb (OB) at an early disease stage. Our team previously published that α-synuclein is highly expressed in the olfactory epithelium (OE) and several groups reported that the protein reduces the impact of bacterial and viral infections. We hypothesized that viral encounters could act as a seeding event that induces changes in α-synuclein metabolism, thereby promoting its aggregation and pathological alterations. In the present study, I sought to investigate the state and modifications of endogenous α-synuclein in C57BL/6 mice nasally inoculated with the Indiana serotype of vesicular stomatitis virus [that expresses green fluorescent protein (VSV-GFP)]. Skull sections of animals at different days post-inoculation (DPIs) were analyzed utilizing immunohistochemical and immunofluorescent microscopy to examine the spatiotemporal progression of VSV-GFP signals and α-synuclein modifications in anterior olfactory structures. Immunoreactivity was quantified to probe for morphological changes in glia. This, to further understand the degree of inflammation and juxtapose it to the viral burden in different regions of the brain. Further, microscopic co-localization studies were carried out to elucidate the nature of α-synuclein distribution during infection, including testing for its aggregation. Immunoblotting of brain homogenates was also employed to gauge any quantitative differences in the total amounts of α-synuclein between mock-treated and virus-infected animals. I found that mice showed variable, but progressively stronger reactivity for VSV-GFP along the olfactory circuitry with sequential involvement of more caudal brain structures, such as the cerebellum and midbrain. This occurred at different days post-inoculation, with viral titers peaking at day 2 post inoculation (DPI) in the anatomical regions analyzed, and with titer fluctuation seen at 6DPI. I found a significant increase in glial reactivity in the olfactory epithelium and bulb between days 2 and 10 post-inoculation, peaking at 6DPI for microglia and 10DPI for astrocytes; further, α-synuclein reactivity overlapped partially with viral proteins in some axons of olfactory sensory neurons and synapses of olfactory bulb glomeruli, i.e., at rostral points of entry into the mouse brain. I found pathological modifications of α-synuclein comprising Ser129 phosphorylation and co-labeling with the scaffolding protein p62; further, α- synuclein aggregates could be observed in extra- as well as intracranial neurons at 6DPI, albeit with considerable variability. Affected sites included olfactory sensory neurons in the olfactory epithelium, glomeruli, and juxtaglomerular cells, and in mitral cells of the olfactory bulb. Finally, the amount of endogenously expressed, total α-synuclein was increased in 6DPI mice compared to mock-treated animals. In summary, singular nasal inoculation with VSV-GFP of adult mice generated a progressive - but variable - infection in the rhinencephalon (and more caudal brain structures), which was accompanied by robust microglial and astrocytic responses. In parallel, this led to the rare formation of murine α-synuclein aggregates within olfactory sensory neurons, alongside positive reactivity for Ser129-phosphorylated-α-synuclein in glomeruli and mitral cells, and the detection of p62 reactivity at 6DPI. Future studies will determine whether these aggregates are transient, whether they have the capacity to grow in size over time within the cell (or are degraded or sloughed off during epithelial renewal), whether they promote neural death at the same site or whether they are propagated to other areas of the brain.