Figure 3. Conditional human α-synuclein expression induces α-synuclein seeding activity in gut organoids.

(A) The SNCAbow expression construct contains 4 tandem cassettes downstream of a chicken β-actin promoter (not shown). The first cassette (not shown) expresses a chemically inducible near-infrared fluorogen-activating peptide (FAP-Mars1). The next 3 cassettes encode a unique fluorescent protein (TagBFP: blue, mTFP1: cyan, or mKO: orange) and a corresponding human synuclein protein SNCA^WT, SNCA^A30P, and SNCA^A53T. When Tg mice are mated to the Villin-Cre (Vil-Cre) strain, Cre-mediated recombination by 3 pairs of orthogonal lox sites (LoxN, Lox2272, LoxP) results in the expression of a single fluorescent protein marker and the corresponding human α-synuclein in any given mucosal cell. (B) Photomicrograph of a small intestine organoid illustrates 3 fluorescent proteins in the mucosa of an SNCAbow mouse indicating the expression of SNCA^WT TagBFP (blue), SNCA^A30P mTFP1 (turquoise), and SNCA^A53T mKO (orange). Scale bar = 30 μm. (C and D) RT-QuIC endpoint ThT fluorescence analysis of nodose ganglia from Snca^–/–, SNCA^A53T, and SNCAbow mice at 6 months of age. (C) A representative ThT fluorescence profile for these genotypes is provided. (D) Endpoint values were collected after 100 hours of RT-QuIC relative to negative controls. Data were collected and combined from 3 mice for each strain in triplicate. All the group analyses are shown as mean ± SEM. All RT-QuIC curves shown are representative of the mean from all the groups analyzed. Significance was determined by 1-way ANOVA with a Dunnett’s post hoc analysis relative to Snca^–/–; *P < 0.05, **P < 0.01, n = 3.