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[Preprint]. 2023 Mar 14:rs.3.rs-2617979. [Version 1] doi: 10.21203/rs.3.rs-2617979/v1

Figure 4. Cardiac glycosides upregulate miR-132 to downregulate miR-132 targets and provide neuroprotection.

Figure 4

a-c, 100 nM oleandrin upregulated pre- and mature miR-132/212 and downregulated their mRNA targets over time (RT-qPCR analysis). d-h, Oleandrin downregulated total Tau, pTau (AT8 and S396), and FOXO3a protein after 72h treatment (Western blot analysis, unpaired two-tailed Student’s t-test, N= 4-8, error bars represent SD). i-k, Less mature neurons were more susceptible to cardiac glycoside toxicity, whereas more mature neurons were resistant. Primary rat neurons were treated with various doses of digoxin, oleandrin, and proscillaridin A for 96h before viability was measured using WST-1. Cells treated at DIV7 showed a dose-dependent reduction in viability. In contrast, cells treated at DIV14, 21, or 28 showed little loss of viability, particularly at EC100 for miR-132 upregulation (unpaired t-test comparing to DMSO condition for each dose, N=4-8 per dose, error bars represent SD.). l-o, For DIV21 neurons, proscillaridin A and oleandrin were not toxic at baseline and fully rescued viability loss due to glutamate or Aβ oligomer treatment (2-way ANOVA, followed by Šídák’s multiple comparisons test, N=8-16 per condition, error bars represent SD). h-j, For DIV7 neurons, proscillaridin A was mildly toxic at baseline. However, both proscillaridin A and oleandrin fully rescued viability loss and partially rescued neurite loss due to glutamate treatment (2-way ANOVA, followed by Šídák’s multiple comparisons test, N=8-16 per condition, error bars represent SD).