Additional file 11 of Neuronal excitatory-to-inhibitory balance is altered in cerebral organoid models of genetic neurological diseases
Creators: name: Foliaki, Simote T.
name: Schwarz, Benjamin
name: Groveman, Bradley R.
name: Walters, Ryan O.
name: Ferreira, Natalia C.
name: Orrù, Christina D.
name: Smith, Anna
name: Wood, Aleksandar
name: Schmit, Olivia M.
name: Freitag, Phoebe
name: Yuan, Jue
name: Zou, Wenquan
name: Bosio, Catharine M.
name: Carroll, James A.
name: Haigh, Cathryn L.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Sociology
subject: FOS: Sociology
subject: 111714 Mental Health
subject: FOS: Health sciences

description: Additional file 11: Assessing for disease-related pathology. Representative images showing immunofluorescence detection of total amyloid beta (a; antibody: 6E10), alpha-synuclein (b; antibody: anti-alpha-synuclein), PrP (c; antibody: EP1802Y), and THT (d) in 6 month old healthy controls (HC) and organoids with genetic defects (PRNPE200K1, PRNPE200K2,T21, LRRK2G2019S) as indicated. Scale bar is 500 µm. Corresponding plots (right) show pixel intensity quantification in DAPI-positive cells. (e) Western blot analysis of APP/amyloid beta in lysates from all the organoids and a brain homogenate from a terminal Alzheimer’s disease (AD) brain (antibody: 6E10). The arrows depict bands corresponding to the molecular mobility of APP and Aβ42. The right panel shows the total protein loaded for each sample. (f) A western blot analysis of insoluble Tau (solubilized by sarkosyl and pelleted by ultracentrifugation) in all the organoid lines and a brain homogenate from a terminal Alzheimer’s disease (AD) brain (antibody: T22). The right panel shows the total protein loaded for each sample. Bars and error denote mean and SEM. The levels of amyloid beta, alpha-synuclein, PrP, and THT were compared between organoid lines by One-way ANOVA on ranks with Dunnett’s correction for multiple comparisons. (g) Primary ThT fluorescence data of K12 Tau RT-QuIC. Representative traces from T21 tau-free mouse brain (KO), cerebrovascular disease (CVD), and Alzheimer disease (AD) brain homogenates, and Down syndrome (DS) organoid homogenates at 10–3 dilutions are displayed. Samples were also tested at 10–3-10–6 for CVD, 10–3-10–10 for AD, and 10–2-10–5 for DS. For the control brain homogenates, the curves are displayed as the average of 4 replicate wells. For the DS organoid homogenates, the curve is displayed as an average ± standard deviation of 5 independent organoids run in quadruplicate. (h) Primary ThT fluorescence data of α-synuclein RT-QuIC. Representative traces from Corticobasal degeneration (CBD) and Parkinson’s Disease (PD) brain homogenates, and organoid homogenates with (PD) or without (Control) the LRRK2G2019S mutation at 10–4 dilutions are displayed. For the control brain homogenates, the curves are displayed as the average of 4 replicate wells. For the organoid homogenates, the curve is displayed as an average ± standard deviation from the indicated number of independent organoids run in quadruplicate. The dashed line indicates the ThT fluorescence threshold for a sample to be considered positive (see methods). (i) Immunofluorescence images of GFAP (active astrocytes) in 6-month-old HC and dCOs. The scale bar is 100 μm.