Advances and Challenges in Understanding MicroRNA Function in Tauopathies: A Case Study of miR-132/212

Abstract

In the past decade, several groups have reported that microRNAs (miRNAs) can participate in the regulation of tau protein at different levels, including its expression, alternative splicing, phosphorylation, and aggregation. These observations are significant, since the abnormal regulation and deposition of tau is associated with nearly 30 neurodegenerative disorders. Interestingly, miRNA profiles go awry in tauopathies such as Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. Understanding the role and impact of miRNAs on tau biology could therefore provide important insights into disease risk, diagnostics, and perhaps therapeutics. In this Perspective article, we discuss recent advances in miRNA research related to tau. While proof-of-principle studies hold promise, physiological validation remains limited. To help fill this gap, we describe herein a pure tauopathy mouse model deficient for the miR-132/212 cluster. This miRNA family is strongly downregulated in human tauopathies and shown to regulate tau in vitro and in vivo. No significant differences in survival, motor deficits or body weight were observed in PS19 mice lacking miR-132/212. Age-specific effects were seen on tau expression and phosphorylation but not aggregation. Moreover, various miR-132/212 targets previously implicated in tau modulation were unaffected (GSK-3β, Foxo3a, Mapk1, p300) or, unexpectedly, reduced (Mapk3, Foxo1, p300, Calpain 2) in miR-132/212-deficient PS19 mice. These observations highlight the challenges of miRNA research in living models, and current limitations of transgenic tau mouse models lacking functional miRNA binding sites. Based on these findings, we finally recommend different strategies to better understand the role of miRNAs in tau physiology and pathology.

Keywords: PS19; miR-132; microRNA; tau; tauopathies.

Figure 1

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 (61). 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.

Figure 2

Analysis of miR-132 targets in vitro and in vivo. (A) Western blot quantifications of endogenous miR-132 targets (Gsk-3β, 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 (61, 64). Blots were normalized to all proteins using Ponceau or Stain-Free technology. # denotes possible alternate splicing (65) 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.