miR-132/212 deficiency impairs tau metabolism and promotes pathological aggregation in vivo

Abstract

Alzheimer's disease (AD) and related tauopathies comprise a large group of neurodegenerative diseases associated with the pathological aggregation of tau protein. While much effort has focused on understanding the function of tau, little is known about the endogenous mechanisms regulating tau metabolism in vivo and how these contribute to disease. Previously, we have shown that the microRNA (miRNA) cluster miR-132/212 is downregulated in tauopathies such as AD. Here, we report that miR-132/212 deficiency in mice leads to increased tau expression, phosphorylation and aggregation. Using reporter assays and cell-based studies, we demonstrate that miR-132 directly targets tau mRNA to regulate its expression. We identified GSK-3β and PP2B as effectors of abnormal tau phosphorylation in vivo. Deletion of miR-132/212 induced tau aggregation in mice expressing endogenous or human mutant tau, an effect associated with autophagy dysfunction. Conversely, treatment of AD mice with miR-132 mimics restored in part memory function and tau metabolism. Finally, miR-132 and miR-212 levels correlated with insoluble tau and cognitive impairment in humans. These findings support a role for miR-132/212 in the regulation of tau pathology in mice and humans and provide new alternatives for therapeutic development.

Figure 1.

miR-132/212 deficiency alters tau expression and phosphorylation in mice. (A, B, C) Representative western blot of endogenous tau and phospho-tau in 6-month-old miR-132/212 KO mice and wild-type littermate controls (n = 8/group, cortex, mixed gender). Total tau or Gapdh was used as normalization control, and quantifications are shown. (D) Real-time quantitative RT-PCR of endogenous tau mRNA in WT and KO mice aged 6 months (n = 5/group, mixed gender). Gapdh mRNA was used as normalization control. (E, F, G) Representative western blot of endogenous GSK-3β, phospho-GSK-3β S9 (inactivated form) and PP2B (n = 8/group, cortex, mixed gender). Gapdh was used as normalization control, and relative quantifications are shown. Statistical significance was determined by Student's unpaired t-test, where *P < 0.05 and ***P < 0.001

Figure 2.

Tau is a direct target of miR-132. (A) Sequence of the tau 3′ UTR showing the predicted miR-132/212-binding site. This region is highly conserved (shown here is human, monkey and mouse sequences). The miR-132 site mutation is shown in bold. (B) Left panel: Neuro2a cells were transfected with 50 nm final concentration of miR-132 or miR-195 mimics. Twenty-four hours post-transfection, luciferase signal was measured. Signals were normalized to Renilla luminescence for transfection efficiency, and graph represents the relative luciferase signals compared with a scrambled mimic control (SCR). Right panel: luciferase assays were performed using a mutant 3′ UTR construct for tau. Here, cells were treated with and 10 nm of miR-132 or miR-195 mimics. The tau mutation completely blocked the effects of miR-132. (C, D) Representative western blot of naive Neuro2a cells treated with 50 nm final concentration of miR-132 or SCR control. Cells were lysed 24 h post-transfection. Gapdh was used as normalization control, and quantifications are shown (n = 3 in triplicate). (E) Tau mRNA quantification after miR-132 overexpression (50 nm) in Neuro2a cells compared with SCR control. Gapdh served as normalization control (n = 2 in triplicate). Statistical significance was assessed by one-way ANOVA with Bonferroni multiple comparison test, where ***P < 0.001 and by Student's unpaired t-test, where ***P < 0.001.

Figure 3.

miR-132/212 deletion promotes tau phosphorylation and aggregation. (A, B, C) Representative western blot of endogenous tau, human tau (CP27) and phospho-tau (PHF1, S422, AT8) in 6-month-old 3xTg-AD^KO and 3xTg-AD^WT mice (n = 8/group, cortex, mixed gender). Gapdh and total tau were used as normalization control, and quantifications are shown. (D, E, F) Immunoblot analysis of sarkosyl-insoluble total tau, human tau and phospho-tau in the same samples shown in A. Total tau was used as normalization, and quantifications are shown. Heat shock protein of 40 kDa (Hsp40/DnaJ) was used as internal loading control. (F) Quantifications of total tau and human-specific tau. (G) Representative images of tau MC1 immunostainings (red) of 18-month-old 3xTg-AD^WT and 3xTg-AD^KO mice in the subiculum dorsal brain region (n = 4/genotype, mixed gender). Tau tangles are zoomed. Scale bars are 100 and 10 μm (zoomed panels). Statistical significances were assessed by Student's unpaired t-test, where *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4.

miR-132/212 knockout is prone to autophagy alteration in the brain. (A, B) Western blot analysis of autophagic component proteins (ATG5–12, ATG9a, Beclin1, P62) and TMEM106b in 6-month-old miR-132/212 KO mice (n = 8/group, cortex, mixed gender). Gapdh served as normalization control, and relative quantifications are shown. (C) (right panel) Representative EM images of 12-month-old miR-132/212 KO CA1 brain region (n = 3/genotype, mixed gender). Autophagic vacuoles (AVs) are zoomed. Scale bar are 500 and 100 nm (zoomed) respectively. EM images of control from the same brain region are shown in left panel. (D) Distribution number organized by AVs size is shown. Size (E) and number in the field (F) are measured from EM images. Statistical significances were assessed by Student's unpaired t-test, where *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5.

Long-term memory is affected in diseased mice lacking miR-132/212. Learning paradigm representations of naive 6-month-old (A–D) and 12-month-old (E–H) 3xTg-AD^WT mice and 3xTg-AD^KO mice (n = 12/group, mixed gender). (I–L) Learning phase representations of experienced 12-month-old 3xTg-AD^WT mice and 3xTg-AD^KO mice (n = 12/group, mixed gender). The ‘naive’ mice had no prior experience in the Barnes maze or any other behavioral tasks. The ‘experienced’ mice performed the Barnes maze test (learning phase) 6 months earlier. Statistical significances were assessed by two-way ANOVA (repeated measures) with Bonferroni multiple comparison test, where *P < 0.05, **P < 0.01 and ***P < 0.001. D, day.

Figure 6.

miR-132 brain injections partially rescue memory deficits in 3xTg-AD. (A) Time scale of our experimental paradigms, including the learning and probe phases in the Barnes maze. (B, D) Learning phase of 12-month-old 3xTg-AD^WT mice brain treated with miR-132 mimics compared with 3xTg-AD^WT controls (n = 11/group, mixed gender). No significant changes were observed during this period. (C, E) Probe trials were done at Day 1, 1 week, 2 weeks and 3 weeks after the learning phase. Here, a significant difference was observed in mobility and path efficiency at 3 weeks. Other behavioral parameters were unchanged (data not shown). (F–H) Western blot analysis of tau expression and phosphorylation after sacrifice. Quantifications are shown, where Gapdh and T-tau were used as normalization controls. Statistical significances were assessed by Student's unpaired t-test, where *P < 0.05, **P < 0.01 and ns = P > 0.05. D, day.

Figure 7.

miR-132 correlates with cognitive decline in AD patients. (A) Relative miR-132 expression levels in various brain regions of postmortem tissues from the Douglas Bell Canada Brain Bank (frontal lobe: n = 5 Ctrl, 7 = AD; temporal lobe: n = 8 Ctrl, 8 = AD; hippocampus: n = 13 Ctrl, 10 = AD). The miRNA let-7a was used as normalization control (using the average of controls as 1-fold). (B) Relative miR-132 expression levels in temporal lobe region of non-demented (n = 11), MCI (n = 10) and AD (n = 11) groups from the Religious Orders Study patients. Statistical significances were obtained with a Mann–Whitney test, and exact P-values are shown. The miRNA let-7a was used as normalization control (using the average of controls as 1-fold). Correlation between temporal lobe miR-132 levels and detergent-insoluble tau (C), MMSE (D), working memory (E), perceptual speed (F), episodic memory (G), semantic memory (H) visuospatial ability (I) or global cognitive scores (J) in the Religious Orders Study cohort. Statistical significances were determined using a linear regression analysis, and exact R² or P-values are given. Ctrl: non-demented controls, AD: Alzheimer disease, MCI: mild cognitive impairment.