PMC7553085_01
Characterization of PS19 mice lacking the miR-132/212 cluster. (A) Kaplan-Meier survival curves, (B) clasping scores, and (C) body weight of PS19 and PS19-KO mice that are deficient for the miR-132/212 cluster. PS19 mice (JAX No. 008169) were bred with full miR-132/212 KO mice as before. Graphs were divided by sex. No significant changes were observed between mouse models. Kaplan-Meier and one-way ANOVA. N = 6-16 mice per group. (D) Western blot quantifications of cortical total tau expression and phosphorylation (Ser422 and PHF1 epitopes) (E) at different ages (3-12 months). N = 12-16 mice per group, mixed sex. Unpaired t-test, where *P < 0.05, ***P < 0.001. (F) Rectal temperature at sacrifice. # denotes significant changes (multiple t-tests, P < 0.05) between PS19 males and females at 12 months. (G) Western blot quantifications of cortical sarkosyl-insoluble tau (Tau total and CP27) at different ages. N = 9-16 mice per group, mixed sex. Unpaired t-test. (H) Western blot quantifications of cortical endogenous NeuN, Snap25, PSD95, GFAP, and Iba1 in 12-month-old PS19 and PS19-KO mice. N = 9-10 mice per group, divided by sex. One-way ANOVA with multiple comparison, where *P < 0.05. Error bars represent SEM. In (D-G), the groups of mice were analyzed separately per age.
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A trend for increased body weight was seen in 11-12-month-old PS19-KO females but not males (Figure 1C). A small but significant increase in tau expression was seen in pre-symptomatic (3 months) PS19-KO mice with a trend in aged (12 months) mice (Figure 1D). In contrast, tau phosphorylation at Ser422 and PHF1 (Ser396/Ser404) epitopes was reduced in aged PS19-KO mice (Figure 1E). This was not attributed to changes in body temperature known to influence tau phosphorylation (Figure 1F). Non-significant trends were noticed in tau aggregation (sarkosyl-insoluble tau) in PS19-KO mice, owing to ~20% of KO mice with more aggregates (Figure 1G and not shown). In addition, no or minimal effects were seen on different markers of brain integrity, including NeuN (neuron), Snap25 (presynaptic), PSD95 (postsynaptic), GFAP (astrocyte), and Iba1 (microglia) (Figure 1H). This cannot be underestimated since miR-132/212 deletion promoted tau aggregation in 3xTg-AD but not (robustly) in PS19 mice (Figure 1G). While some of these markers seem unaffected in PS19-KO mice (Figure 1H), a detailed analysis remains to be done.
Analysis of miR-132 targets in vitro and in vivo. (A) Western blot quantifications of endogenous miR-132 targets (Gsk-3beta, Foxo1, Foxo3a, Mapk1/Erk2, Mapk3/Erk1, Calpain2, p300) in PS19 and PS19-KO mice at different ages in the cortex. All significant trends and changes indicate lower expression in KO mice. N = 6-15 per group, mixed sex. Unpaired t-test, where *P < 0.05. (B) Western blot quantification of endogenous miR-132 targets, including tau, in native human SHSY-5Y, human HEK293, and murine Neuro2a cells. Cells were treated with 50 nM miR-132 mimics or scrambled control for 48 h prior to protein extraction. N = 2-4 experiments performed in triplicate. Results are shown as ratios between miR-132 over scrambled mimics (normalized to 1). Unpaired t-test, where *P < 0.05, **P < 0.01, ***P < 0.001. (C) Upper panel: Ectopic miR-132 levels measured by qRT-PCR in each cell line. Relative to scrambled mimic control. Lower panel. Representative western blot analyses. (D) Western blot quantifications of endogenous miR-132 targets. Here, cells were treated with 50 nM miR-132 inhibitors or scrambled control for 48 h. Unpaired t-test, where *P < 0.05, **P < 0.01, ***P < 0.001. (E) Upper panel: Endogenous miR-132 levels measured by qRT-PCR in each cell line following treatment. Relative to scrambled inhibitor control (normalized to 1). Lower panel. Representative western blot analyses. miRNA qRT-PCR were normalized using RNU19 as before. Blots were normalized to all proteins using Ponceau or Stain-Free technology. # denotes possible alternate splicing or phosphorylated form of targets. N.A., not available; N.D., not detected; GOF, gain-of-function; LOF, loss-of-function. Error bars represent SEM. In A, the groups of mice were analyzed separately per age. See Supplementary Methods for additional details.
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We observed no significant differences caused by miR-132/212 deficiency on endogenous GSK-3beta, Mapk1, and Foxo3a in vivo at all ages of study (Figure 2A). On the other hand, and unexpectedly, Mapk3, p300, Calpain2, and Foxo1a levels were reduced in PS19-KO mice, albeit at different ages (Figure 2A). Of note, we could confirm the regulatory effects of ectopic miR-132 on all genes in cells (Figure 2B). Representative miRNA qRT-PCR quantification and Western blots of targets are shown in Figure 2C. Inversely, inhibition of endogenous miR-132 in cells had only modest and sporadic effects on a subset of target genes (Figure 2D). Representative results are shown in Figure 2E. The results obtained herein in vivo (Figure 2A), often in theoretical disagreement with the mode of miRNA function, is somewhat in agreement with this hypothesis, more so given the subtle or negligible effects on tau aggregation and mouse phenotypes in PS19-KO mice.