Over-expression of miR-34a induces rapid cognitive impairment and Alzheimer's disease-like pathology

Abstract

Autosomal dominant Alzheimer disease (AD) is caused by rare mutations in one of three specific genes. This is in contrast to idiopathic, late-onset AD (LOAD), which has a more polygenetic risk profile and represents more than 95% of cases. Previously, we have demonstrated that increased expression of microRNA (miRNA)-34a (miR-34a) in AD brain targets genes linked to synaptic plasticity, energy metabolism, and resting state network activity. Here we report the generation of a heterozygous, conditional miR-34a overexpression mouse (miR-34a^+/-(TetR-TetO-miR-34a) Transgenic Mice). Doxycycline-treated mice of either sex exhibited profound behavioral impairment compared to untreated groups with only 1-2 months of over-expression of miR-34a. Cognitive impairment of individual mice in T- and Y-maze tasks correlated with elevated miR-34a expression in many parts of the brain including the hippocampus and prefrontal cortex, regions which are known to be involved in this task and implicated in LOAD dysfunction. Immunocytochemistry of brain sections from mice show high amyloid β and phosphorylated tau-specific staining in the hippocampus and cortex. Analysis of protein samples from these mice revealed that miR-34a targets specific genes involved in memory formation, amyloid precursor protein (APP) metabolism and phosphorylation-dephosphorylation of tau. Thus, our results suggest that the polygenetic dysfunction caused by miR-34a may occur in LOAD and disclose miR-34a as a potential therapeutic target. SIGNIFICANCE STATEMENT: Late-onset Alzheimer disease (LOAD) is associated with multiple gene alleles, a polygenetic profile of risk factors that is difficult to model in animals. Our approach to modeling LOAD was to produce a conditional over-expressing, miR-34a mouse using doxycycline-induction to activate expression. We observed that miR-34a over-expression results in a rapid cognitive impairment, associated with accumulation of intracellular Aβ and tau hyperphosphorylation in multiple brain regions. Targets for miR-34a, including ADAM10, NMDAR 2B, and SIRT1 RNAs, were profoundly reduced by miR-34a over-expression. Collectively, these results indicate that a rapid, profound cognitive decline and Alzheimer's disease neuropathology can be induced with miR-34a over-expression, suggesting that this animal model may represent a polygenetic risk factor model for LOAD.

Keywords: ADAM10; Late-onset Alzheimer’s disease; Memory-related behaviors; NMDAR 2B; SIRT1; Tau phosphorylation; miR-34a; microRNA; β amyloid.

Fig. 1.

Generation of transgenic mice. The mouse lines were generated cloning miR-34a gene ahead of a promoter containing tetracycline response element plasmid (PmRi-mCherry). This construct was injected into the pronucleus of mouse zygotes along with the Tet repressor expression plasmid (pTet-On Advanced) to achieve inducibility of the miR-34a expression in response to Doxy exposure. The correct genotype of offspring was verified by PCR-based genotyping of tail biopsied DNA as well as microscopic identification of red colored m-cherry positive expression in their respective skin cells.

Fig. 2.

Locomotor and behavioral phenotyping of transgenic mice with or without doxy exposure. A. Doxy exposure had no impact on either open field spontaneous locomotion (upper panel) or anxiety-like behavior (lower panel). B. Y-maze total distance moved [t(13) = 2.47, p < 0.05] (upper panel) and speed [t(13) = 2.47, p < 0.05] (lower panel) were reduced among mice given Doxy vs No Doxy controls. C. Hot plate total nociceptive behaviors (lower panel) tended to be reduced among mice given Doxy vs No Doxy controls [t(13) = 2.06, p < 0.07] but latency to first nociceptive behavior (upper panel) was not altered between the groups. D. Rotarod coordination was not different between the groups. * p < 0.05. N = 7–8 mice/group.

Fig. 3.

Cognitive impairment following doxycycline-induced miR-34a expression. A. On the forced alternation T-maze, a measure of working and reference memory, mice given Doxy were significantly impaired in their ability to successfully win-shift alternate (mean ± SEM percent successful alternations) [t-test: t(14) = 4.719, p < 0.0005]. B. On the spontaneous alternation Y-maze, a measure of working memory, mice given Doxy were significantly impaired in their ability to successfully undergo spontaneous alternation (mean ± SEM percent successful alternations) [t-test: t(12) = 5.845, p < 0.0001]. N = 7–8 mice/group. * p < 0.05.

Fig. 4.

Suppression of miR-34a target proteins in doxycycline-induced miR-34a expression in brain regions of transgenic mice. A. Representative western blots of NMDAR-2B expression (GRIN2B antibody) among mice given Doxy (N = 3) vs No Doxy controls (N = 3) in the entorhinal cortex (EC), hippocampus (HI), prefrontal cortex (PFC), and thalamus (TH). B. Densitometric quantification of NMDAR-2B protein levels (normalized to GAPDH levels) is shown in the bar graph. Data are mean ± SEM, and the results shown are representative of three animals in each group. The significant Treatment main effect [F(1,4) = 2381, p < 0.0001] was followed up by taking a conservative approach to multiple comparison analyses within each brain region using the Bonferroni correction, **** p < 0.0001. C. Representative western blots of SIRT1 expression among mice given Doxy (N = 3) vs No Doxy controls (N = 3). D. Densitometric quantification of SIRT1 protein levels (normalized to GAPDH levels) is shown in the bar graph. Data are mean ± SEM, and the results shown are representative of three animals in each group. The significant Treatment main effect [F(1,4) = 1003, p < 0.0001] was followed up by taking a conservative approach to multiple comparison analyses within each brain region using the Bonferroni correction, **** p < 0.0001

Fig. 5.

Upregulation of phosphorylation of Tau protein in doxycycline-induced miR-34a expression in brain regions of transgenic mice. A. Representative western blots of phospho-tau (AT8 antibody) expression among mice given Doxy vs No Doxy controls in the entorhinal cortex (EC), hippocampus (HI), prefrontal cortex (PFC), and thalamus (TH). B. Densitometric quantification of relative protein levels (p-Tau/total Tau) is shown in the bar graph. Data are mean ± SEM, and the results shown are representative of three animals in each group. The significant Treatment × Region interaction [F(3,12) = 8.33, p < 0.005] was followed up with Bonferroni-corrected multiple comparisons of the effect of Treatment in each brain region, * p < 0.05, **** p < 0.0001. C. Representative western blots of phospho-tau (Tau46 antibody) expression among mice given Doxy vs No Doxy controls. D. Densitometric quantification of respective protein levels (p-Tau/total Tau) is shown in the bar graph. Data are mean ± SEM, and the results shown are representative of three animals in each group. The significant Treatment main effect [F (1,4) = 660.2, p < 0.0001] was followed up with Bonferroni-corrected multiple comparisons of the effect of Treatment in each brain region, **** p < 0.0001. E. Expression of phosphorylated tau in the hippocampus of without (No (Doxy) over expressions of mir-34a, at low magnification (upper two panels; scale bars 200 μm), and high magnification with Doxy (lower panel; scale bar 100 μm).

Fig. 6.

Suppression of PTPA protein in doxycycline-induced miR-34a expression in brain regions of transgenic mice. A. Representative western blots of PTPA expression among mice given Doxy vs No Doxy controls in the entorhinal cortex (EC), hippocampus (HI), prefrontal cortex (PFC), and thalamus (TH). B. Densitometric quantification of relative protein levels (PTPA/actin) is shown in the bar graph. Data are mean ± SEM, and the results shown are representative of three animals in each group. The significant Treatment × Region interaction [F(3,12) = 12.51, p < 0.005] was followed up with Bonferroni-corrected multiple comparisons of the effect of Treatment in each brain region, **** p < 0.0001.

Fig. 7.

Downregulation of ADAM-10 in doxycycline-induced miR-34a expression in brain regions of transgenic mice. A. Representative western blots of Aβ expression (ADAM-10 antibody) among mice given Doxy vs No Doxy controls in the entorhinal cortex (EC), hippocampus (HI), prefrontal cortex (PFC), and thalamus (TH). B. Densitometric quantification of relative protein levels (ADAM10/GAPDH) is shown in the bar graph. Data are mean ± SD, and the results shown are representative of three animals in each group. The significant Treatment main effect [F(1,4) = 820.6, p < 0.0001] was followed up with Bonferroni-corrected multiple comparisons of the effect of Treatment in each brain region, **** p < 0.0001. Immunohistology of anti-β-amyloid antibody clone 6E10 in the cortex (C) and hippocampus (D) of mice given Doxy vs No Doxy controls. Scale bars 200 μm. E. High magnification of intracellular localization of anti-β-amyloid antibody clone 6E10 staining in the hippocampus. Scale bar 100 μm.