Fig. 5

Functional neuroprotection by hiMG during chronic amyloidosis is limited to 3w Aβ hiNS(+). For schematic illustration of the chronic amyloidosis protocol see Fig. 4A. hiNS were transduced at DIV 39 to induce roGFP1 and GCaMP6f expression. Tissue was live imaged at multiple time points and collected at DIV 60. A Immunofluorescence staining for MAP2, GFAP and IBA1 reveals a larger hiMG population in 5w Aβ hiNS(+) compared to 3w Aβ hiNS(+) or controls (ctrl hiNS(+)) as well as an increase in GFAP immunoreactivity in 3w/5w Aβ hiNS(+) (N = 6 hiNS each). B Redox imaging using roGFP1 indicates mild oxidative stress in 3w Aβ hiNS(−) with a full rescue in 3w Aβ hiNS(+). Severe oxidative stress in 5w Aβ hiNS(−) was not ameliorated by hiMG (N = 6 hiNS each). At DIV 54, oxidative stress had not yet manifested in 3w Aβ hiNS(−), in contrast to significantly increased redox ratios at DIV 59. C Functional recovery in neuronal calcium wave activity by hiMG in 3w Aβ hiNS(+) quantified using GCaMP6f. Similar to our redox imaging, neural calcium wave activity was not perturbed at DIV 50 in 3w Aβ hiNS(−), in contrast to significantly reduced wave activity at DIV 58 (3w Aβ hiNS(−/+): N = 6 each). D Calcium waves in 5w Aβ hiNS were completely absent and hiMG did not result in a functional recovery (ctrl hiNS(−): N = 10; ctrl hiNS(+): N = 12; 5w Aβ hiNS(−/+): N = 6 each). E: Quantification of calcium wave frequencies of all experimental groups at DIV 50 and DIV 58. F: Paired comparison of calcium wave frequencies between DIV 50 and DIV 58 in ctrl and 3w Aβ hiNS (−/+) illustrating the reduction in 3w Aβ hiNS(−) wave frequencies between the two time points. G: At both time points, hiMG resulted in a reduction in c-Cas- 3 immunoreactivity and ameliorated cell death (ctrl hiNS(−): N = 9; ctrl hiNS(+): N = 11; 3w Aβ hiNS(-/+): N = 10 each; 5w Aβ hiNS(−): N = 8; 5w Aβ hiNS(+): N = 11). Statistical testing performed using two-way ANOVA followed by Holm-Sidak post-hoc tests on logarithmically transformed data, except for a one-way ANOVA followed Holm-Sidak post-hoc test for microglia quantification