Fig. 3

Chronic Aβ stimulation in hiNS(−) triggers a sequence of neurotoxic events and phenotypes in the absence of hiMG. A hiNS(−) cell culture media was supplemented with ~ 0.24 µg/mL oligomeric Aβ 42 for up to 35 days. Similar to the schematic shown in Fig. 1B, hiNS were transduced with AAVs at DIV 40 and tissue was imaged and collected at multiple time points. B Treatment results in the formation of plaque-like Aβ aggregates in hiNS(−) which can be stained with different Aβ antibodies (here 6E10 and mOC87). C Immunostaining for neurons (MAP2) and astrocytes/NPCs (GFAP) after 35 days of Aβ exposure reveals dystrophic appearing neuronal and astrocytic processes (see white arrows) D roGFP1 based redox imaging in neurospheres reveals significant oxidation indicating oxidative stress in neurons following chronic Aβ treatment (N = 4 hiNS each). E Calcium wave frequencies (quantified by GCaMP6f live imaging) gradually decline with Aβ treatment resulting in dysfunctional neuronal activity (N = 4 hiNS each). F: Immunostaining for cleaved Caspase- 3 (c-Cas- 3) indicates apoptosis in hiNS(−) after chronic Aβ exposure. Quantification of DAPI positive nuclei in hiNS(−) suggests neuronal death as the ultimate fate following 35 days of chronic Aβ treatment. G Co-staining for c-Cas- 3/MAP2 indicates neurons undergoing apoptosis and loss of nuclei density (N = 4 hiNS each). H Apoptosis after 35 days of Aβ exposure was further confirmed by a significant increase in TUNEL fluorescence (control: N = 4 hiNS; Aβ: N = 5 hiNS). All statistical testing was performed using unpaired student’s t-tests