Additional file 1 of Upregulating β-hexosaminidase activity in rodents prevents α-synuclein lipid associations and protects dopaminergic neurons from α-synuclein-mediated neurotoxicity
Creators: name: Oeystein R. Brekk
name: Korecka, Joanna A.
name: Crapart, Cecile C.
name: Mylene Huebecker
name: MacBain, Zachary K.
name: Rosenthal, Sara Ann
name: Sena-Esteves, Miguel
name: Priestman, David A.
name: Platt, Frances M.
name: Isacson, Ole
name: Hallett, Penelope J.
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Medicine
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: FOS: Health sciences

description: Additional file 1: Supplementary Fig. 1. aSYN-ubiquitination in the SD-TG mouse model and in AAV-aSYN-injected rat SNpc. (A) Representative micrograph of aSYN (green) and ubiquitin (magenta) in age-matched WT- and SD-TG mouse SNpc. Filled arrowheads indicate ubiquitin+ aSYN+ inclusions. Open arrowheads indicate ubiquitin+ aSYN- inclusions. Boxed inserts are shown in higher magnification with orthogonal view on right. Scale bar = 15um. (B) Representative micrograph of aSYN (green) and ubiquitin (magenta) in non-injected and AAV-CTRL+aSYN and HEX+aSYN injected rat SNpc. Arrowheads indicate ubiquitin+ aSYN+ inclusions. Boxed inserts are shown in higher magnification with orthogonal view on right. Scale bar = 15um. Supplementary Fig. 2. AAV6-mediated expression of HEX subunits A and B alone does not elicit upregulation of enzymatic activity (A) HEX activity histochemical detection assay (red precipitate) in a rat injected with AAV6 expressing HEX subunit B. (SNr = substantia nigra pars reticulata, SNpc = substantia nigra pars compacta, VTA = ventral tegmental area). Boxed inserts are shown in higher magnification below. (B) High magnification inserts of tissue prepared as in (A), in SNpc injected (1) with AAV6-HEX A, and the non-injected control hemisphere (2). Supplementary Fig. 3. Overexpressed HEX localizes to lysosomes in transduced TH+ neurons of the rat SNpc. Representative immunofluorescent micrograph showing HEX subunit A (red) and LAMP-1 (greyscale) in TH+ dopaminergic neurons (green) in the SNpc of AAV6-HEX injected- and non-injected hemispheres. Dotted outline represents TH+ cell profile. Boxed insert is shown in orthogonal view (YZ). Supplementary Fig. 4. Representative immunofluorescent micrograph showing AAV6-overexpressed human WT aSYN (magenta) in dopaminergic neurons (TH+) (green) in the rat striatum and SNpc of an animal injected with AAV6-aSYN, HEXA and HEXB (STR = striatum, VTA = ventral tegmental area, SNpc = substantia nigra pars compacta, SNr = substantia nigra pars reticulata.) Supplementary Fig. 5. Phosphorylated aSYN is a feature of both AAV-mediated aSYN overexpression and HEX loss-of-function. (A) Immunoblot of S129-phosphorylated aSYN (p-aSYN) in CTRL+aSYN and HEX+aSYN AAV-injected and non-injected rat SNpc. GAPDH is shown as loading control. (B) Immunoblot of p-aSYN CTRL+aSYN-injected SNpc containing lipids (+) or after lipid extraction at 65C for 16 h(−). (C) Immunoblot of p-aSYN in age-matched WT- or Sandhoff transgenic (SD) mouse whole-brain. GAPDH is shown as loading control.