Journal: bioRxiv

Article Title: Gut mucosal cells transfer α-synuclein to the vagus nerve

doi: 10.1101/2023.08.14.553305

Figure Lengend Snippet: (A,B) Immunostaining of duodenum harvested from SNCA A53T mice. Enteroendocrine cells (EECs) expressing green fluorescent protein (green) are scattered among other mucosal cells (DAPI labeled nuclei, blue) and are in proximity to α-synuclein (red) containing fibers stained with the pan-neuronal marker PGP9.5 (cyan) in the lamina propria of the villus. ELISA quantification of human α-synuclein in (C) duodenum (α-synuclein quantification in nanograms per mg of nodose tissue), (D) nodose ganglia (α-synuclein quantification in picograms per mg of nodose tissue), and (E) hindbrain (α-synuclein quantification in nanograms per mg of nodose tissue), from Snca −/− and SNCA A53T mice. RT-QuIC analysis of (F) duodenum, (G) nodose ganglia, and (H) hindbrain of Snca −/− and SNCA A53T mice. Scale bars are 30 µm in panel A, 10 µm and 1 µm (inset) in panel B. Significance was determined by unpaired t-test, where ***P<0.001.

Article Snippet: Primary antibodies used for immunostaining included: Rabbit CCK , rabbit α-synuclein (Abcam Cat# ab138501, RRID:AB_2537217, at 1:1000), guinea pig PGP9.5 (Abcam Cat# ab10410, RRID:AB_297150, at 1:100), chick beta-tubulin III, Tuj1 (Neuromics Cat# CH23005, RRID:AB_2210684, at 1:100), and chick GFP (Abcam Cat# ab13970, RRID:AB_300798, at 1:1000).

Techniques: Immunostaining, Expressing, Labeling, Staining, Marker, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Gut mucosal cells transfer α-synuclein to the vagus nerve

doi: 10.1101/2023.08.14.553305

Figure Lengend Snippet: (A) Intestinal organoids were prepared from a SNCA A53T mouse in which CCK-containing cells express enhanced green fluorescent protein (eGFP), and vagal nodose ganglia neurons were isolated from a Snca −/− mouse lacking endogenous α-synuclein. (B) Representative images of organoids and neurons grown in co-culture for five days, with eGFP-positive cells (green) in the organoid, and β-tubulin (cyan) highlighting neuronal processes. (C) Representative high-magnification α-synuclein (red) staining of an eGFP-positive EEC. Red arrow indicates localization to a PGP9.5 (cyan) positive process in an Snca −/− mouse neuron. (D,E) Representative images with neuron-specific class III tubulin (Tuj1, cyan). Surface and (adjacent) intracellular confocal slices are shown. Scale bars are 30 μm for panel B, 3 μm for panel C, and 5 μm for panels D and E.

Article Snippet: Primary antibodies used for immunostaining included: Rabbit CCK , rabbit α-synuclein (Abcam Cat# ab138501, RRID:AB_2537217, at 1:1000), guinea pig PGP9.5 (Abcam Cat# ab10410, RRID:AB_297150, at 1:100), chick beta-tubulin III, Tuj1 (Neuromics Cat# CH23005, RRID:AB_2210684, at 1:100), and chick GFP (Abcam Cat# ab13970, RRID:AB_300798, at 1:1000).

Techniques: Isolation, Co-Culture Assay, Staining

Journal: bioRxiv

Article Title: Gut mucosal cells transfer α-synuclein to the vagus nerve

doi: 10.1101/2023.08.14.553305

Figure Lengend Snippet: (A) SNCAbow mouse duodenum illustrating expression of fluorescent protein markers BFP (blue), TFP (turquoise), and mKO2 (orange) in mucosal cells (enterocytes and EECs). Alpha-synuclein (magenta) immunostaining is present in a single intestinal mucosal cell consistent with an EEC (Scale bar = 10 μm). (B) Duodenum from SNCAbow mouse illustrating PGP9.5 positive neuronal fibers (magenta) innervating intestinal crypts and villi (Scale bar = 50 μm). (C) ELISA quantification of human α-synuclein protein in nodose ganglia of non-transgenic, Snca −/− , and SNCAbow mice. (D) A representative thioflavin T fluorescence profile (RT-QuIC) and end-point analysis of nodose ganglia from non-transgenic (nTg), Snca −/− and SNCAbow mice at 1 month of age. (E) RT-QuIC analysis of nodose ganglia from 6 month old nTg, Snca −/− , and SNCAbow mice. Significance was determined by one-way ANOVA with a Dunnett’s post hoc analysis relative to SNCAbow, **P<0.01, ***P<0.001, n=3.

Article Snippet: Primary antibodies used for immunostaining included: Rabbit CCK , rabbit α-synuclein (Abcam Cat# ab138501, RRID:AB_2537217, at 1:1000), guinea pig PGP9.5 (Abcam Cat# ab10410, RRID:AB_297150, at 1:100), chick beta-tubulin III, Tuj1 (Neuromics Cat# CH23005, RRID:AB_2210684, at 1:100), and chick GFP (Abcam Cat# ab13970, RRID:AB_300798, at 1:1000).

Techniques: Expressing, Immunostaining, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Fluorescence