MicroRNA-138 Overexpression Alters Aβ42 Levels and Behavior in Wildtype Mice

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by changes in cognitive and behavioral functions. With the exception or rare mutations in PSEN and APP genes causing early-onset autosomal dominant AD (EOADAD), little is known about the genetic factors that underlie the vast majority (>95%) of early onset AD (EOAD) cases. We have previously identified copy number variations (CNVs) in microRNA genes in patients with EOAD, including a duplication of the MIR-138-2 gene. Overexpression of miR-138 in cultured cells increased Aβ production and tau phosphorylation, similar to what is seen in AD brain. In this study, we sought to determine if miR-138 overexpression could recapitulate certain features of disease in vivo in non-transgenic mice. A mild overexpression of pre-miR-138 in the brain of C57BL/6J wildtype mice altered learning and memory in a novel object recognition test and in the Barnes Maze. Increased levels of anxiety were also observed in the open-field test. MiR-138 upregulation in vivo caused an increase in endogenous Aβ42 production as well as changes in synaptic and inflammation markers. Tau expression was significantly lower with no overt effects on phosphorylation. We finally observed that Sirt1, a direct target of miR-138 involved in Aβ production, learning and memory as well as anxiety, is decreased following miR-138 overexpression. In sum, this study further strengthens a role for increased gene dosage of MIR-138-2 gene in modulating AD risk, possibly by acting on different biological pathways. Further studies will be required to better understand the role of CNVs in microRNA genes in AD and related neurodegenerative disorders.

Keywords: Alzheimer’s disease; MiR-138; adeno-associated virus; anxiety; memory; microRNA.

FIGURE 1

Expression and localization of AAV2 in the brain. (A) Characterization of AAV2/DJ8-CAG-eGFP in wild-type mice at 2 months of age: eGFP labeling in green; Neuronal marker: NeuN or Glial marker: GFAP or Iba1, in red. Nuclei marker: DAPI, in blue. Merge of green, red, and blue fluorescence. Immunofluorescence performed in half-brain of 2 months-old mice, 2 months after injections (n = 3). Note that most, if not all, co-stainings with eGFP are with the neuronal NeuN marker. Scale: 250 nm. (B) Expression of pre-miR-138 in hippocampus (Hipp.) and frontal cortex (Cortex) of mice at 4 months of age, injected with AAV2/DJ8-CAG-eGFP-MIR-138-2 (miR-138; n = 14) or AAV2/DJ8-CAG-eGFP (Ctrl; n = 16). (C) Expression of mature miR-138-5p in hippocampus (Hipp.) and frontal cortex (Cortex) of mice at 4 months of age injected with AAV2/DJ8-CAG-eGFP-MIR-138-2 (miR-138; n = 14) or AAV2/DJ8-CAG-eGFP (Ctrl; n = 16). (D) Expression of mature miR-138-5p in HEK293 and HT-22 cell lines (n = 3 in triplicate). Data presented as a mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t-test).

FIGURE 2

Alterations in learning, memory, and anxiety in miR-138 mice. (A) Experimental outline of the study herein. Open field: (B) travel distance, (C) speed, (D) entry number, and (E) exploration time into the center and the corners by miR-138 (n = 11) or Ctrl mice (n = 13). Data show with mean ± SEM: *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t-test). Novel object recognition: (F) recognition index and (G) exploration time of the novel object by miR-138 and Ctrl mice. Data show difference from 50% with mean ± SEM: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (column statistics test). Barnes: (H) primary error and (I) primary time for miR-138 mice and Ctrl mice during the 4 days of testing (learning). Data shows in mean ± SEM: ns, non-significant; *P < 0.05, **P < 0.01 (two-way ANOVA). (J) Entry number in exit (Ex) versus mean of other (O) quadrants during the probe test at day 5 and 12 for Ctrl mice and miR-138 mice. All tests were performed on 30 mice in total (n = 13 males and n = 17 females). Data shows in mean ± SEM: ns, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Kruskal-Wallis test).

FIGURE 3

Increase of Aβ42 and decrease of total tau in miR-138 mice. ELISA of (A) endogenous murine Aβ40, (B) Aβ42, and (C) Aβ42/40 ratio from hippocampus (Hipp.) and frontal cortex (Cortex), in miR-138 mice (n = 11, sex mixed) compared to Ctrl mice (n = 13, sex mixed), 4 months after injection. (D) Representative Western blot of endogenous (E) total Tau and (F) PHF1, AT8, and pS422 quantifications in hippocampus (Hipp.). (G) Representative Western blot of endogenous (H) total Tau and (I) PHF1, AT8, and pS422 quantifications in frontal cortex (Cortex). Total tau bands were normalized to total proteins and phospho-tau bands were normalized to total tau. All analyses were performed on 30 mice in total (n = 13 males and n = 17 females). Data are presented as ±SEM: *P < 0.05 (unpaired student t-test).

FIGURE 4

Decrease of Sirt1 in miR-138 mice. (A) Representative Western blot of endogenous (B) Sirt1, Fertm2, APP, GSK-3β, and GSK-3S9 quantification in hippocampus (Hipp.) of miR-138 mice compared to Ctrl mice, at 4 months of age. (C) Representative Western blot of endogenous (D) Bace1 Aph1b/c, PSEN1, Nicastrin (Ncstn), and Adam10 quantification in hippocampus (Hipp.) of miR-138 mice compared to Ctrl mice. (E) Representative Western blot of endogenous (F) Sirt1, Fertm2, APP GSK-3β, and GSK-3S9 quantification in frontal cortex (Cortex) of miR-138 mice compared to Ctrl mice, at 4 months of age. (G) Representative Western blot of endogenous (H) Bace1 Aph1b/c, PSEN1, Nicastrin (Ncstn), and Adam10 quantification in frontal cortex (Cortex) of miR-138 mice compared to Ctrl mice. Bands were normalized to total proteins. All analyses were performed on 30 mice in total (n = 13 males and n = 17 females). Data are presented as ±SEM: *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t-test).

FIGURE 5

Abnormal brain integrity markers in 138 mice. (A) Representative western blot of endogenous (B) PSD95, synaptophysin (Syp), and SNAP25 quantification in hippocampus (Hipp.) of miR-138 mice compared to Ctrl mice, at 4 months of age. (C) Representative Western blot of endogenous (D) NeuN, Iba1, and GFAP quantification in hippocampus (Hipp.) of miR-138 mice compared to Ctrl mice. (E) Representative western blot of endogenous (F) PSD95, synaptophysin (Syp), and SNAP25 quantification in frontal cortex (Cortex) of miR-138 mice compared to Ctrl mice. (G) Representative Western blot of endogenous (H) NeuN, Iba1 and GFAP quantification in frontal cortex (Cortex) of miR-138 mice compared to Ctrl mice. Bands were normalized to total proteins. All analyses were performed on 30 mice in total (n = 13 males and n = 17 females). Data are presented as ±SEM: *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t-test).