Opposing actions of environmental enrichment and Alzheimer's disease on the expression of hippocampal microRNAs in mouse models

Abstract

Alzheimer's disease (AD) is the most common form of dementia in the elderly. Although there are no drugs that modify the disease process, exposure to an enriched environment (EE) can slow the disease progression. Here, we characterize the effects of AD and EE on the post-transcriptional regulators, microRNAs (miRNAs), which may contribute to the detrimental and beneficial effects of AD and EE, respectively, on synaptic plasticity-related proteins and AD pathology. We found for the first time miRNAs that were inversely regulated in AD and EE, and may affect synaptic proteins and modulators, molecular factors associated with AD pathology, and survival and neuroprotective factors. MiRNAs that were upregulated only in 3xTgAD mice model of AD compared with their control mice were localized to synapses, predicted to downregulate essential synaptic proteins and are highly associated with regulating apoptosis, AD-associated processes and axon guidance. Studying the progressive change in miRNAs modulation during aging of 3xTgAD mice, we identified miRNAs that were regulated in earlier stages of AD, suggesting them as potential AD biomarkers. Last, we characterized AD- and EE-related effects in the mouse hippocampus on tomosyn protein levels, an inhibitor of the synaptic transmission machinery. While EE reduced tomosyn levels, tomosyn levels were increased in old 3xTgAD mice, suggesting a role for tomosyn in the impairment of synaptic transmission in AD. Interestingly, we found that miR-325 regulates the expression levels of tomosyn as demonstrated by a luciferase reporter assay, and that miR-325 was downregulated in AD and upregulated following EE. These findings improve our understanding of the molecular and cellular processes in AD pathology, following EE, and the interplay between the two processes, and open new avenues for the studies of understanding and controlling AD.

Figure 1

Hippocampal microRNAs (miRNAs) global expression signature in young 3xTgAD mice model of Alzheimer's disease (AD). General distribution of miRNAs modulation derived from young (4-month-old) mice model of AD compared with their age-matched wild-type (WT) C57BL/6J mice. (a) Distribution chart of miRNA changes showing miRNAs that were upregulated (Up), downregulated (Down) or unchanged (Unchanged). (b) Scatter-plot representation of miRNA expression changes. While most of the miRNAs were not modulated following exposure to enriched environment (EE) (presented around and on the regression line), some of the miRNAs were upregulated (below the regression line), and some were downregulated (above the regression line), compared with control mice. Values are presented as 1/threshold cycle (1/Ct). Properly detected miRNAs with Ct<40 and fold change >2 or <0.5 are presented. (c) Relative quantification (RQ) of miRNAs that were upregulated with RQ>2 or downregulated with RQ<−2.

Figure 2

Hippocampal microRNAs (miRNAs) global expression signature in very old 3xTgAD mice model of Alzheimer's disease (AD). General distribution of miRNAs modulation derived from very old (16-month-old) mice model of AD compared with their age-matched wild-type (WT) C57BL/6J mice. (a) Distribution chart of miRNA changes showing miRNAs that were upregulated (Up), downregulated (Down) or unchanged (Unchanged). (b) Scatter-plot representation of miRNA expression changes. While most of the miRNAs were not modulated following exposure to enriched environment (EE) (presented around and on the regression line), some of the miRNAs were upregulated (below the regression line), and some were downregulated (above the regression line), compared with control mice. Values are presented as 1/threshold cycle (1/Ct). Properly detected miRNAs with Ct<40 and fold change>2 or <0.5 are presented. (c) Relative quantification (RQ) of miRNAs that were upregulated with RQ>2 or downregulated with RQ<−2.

Figure 3

Common and differential changes in microRNAs (miRNAs) in the hippocampi of young and very old 3xTgAD mice model of Alzheimer's disease (AD) and their control age-matched mice. Venn diagrams presenting the number of miRNAs that their expression levels were (a) upregulated or (b) downregulated in each group of comparison: Blue—AD in very old mice, Yellow—AD in young mice, Green—aging in wild-type (WT) mice and Red—aging in AD mice model. OAD, Old AD; OC, Old Control; YAD, Young AD; YC, Young Control. The intersections between groups result with definition of subgroups. Several subgroup titles are indicated where relevant.

Figure 4

microRNAs (miRNAs) global expression signature in the hippocampus of wild-type (WT) C57BL/6J mice following environmental enrichment. General distribution of miRNAs modulation derived from WT C57BL/6J mice that were environmentally enriched, as compared with WT C57BL/6J control mice that were exposed to regular environment. (a) Distribution chart of miRNA changes showing miRNAs that were upregulated (Up), downregulated (Down) or unchanged (Unchanged). (b) Scatter-plot representation of miRNA expression changes. While most of the miRNAs were not modulated following exposure to enriched environment (EE) (presented around and on the regression line), some of the miRNAs were upregulated (below the regression line), and some were downregulated (above the regression line), compared with control mice. Values are presented as 1/threshold cycle (1/Ct). Properly detected miRNAs with Ct<40 and fold change>2 or <0.5 are presented. (c) Relative quantification (RQ) of miRNAs that were upregulated with RQ>2 or downregulated with RQ<−2.

Figure 5

Tomosyn protein expression levels increase in the mouse hippocampus as Alzheimer's disease (AD) pathology progresses. Analysis of immunofluorescence staining of tomosyn protein in coronal brain slices derived from hippocampi of 3xTgAD mice and their wild-type (WT) C57BL/6J age-matched control mice. (a) In the hippocampus of 3xTgAD mice, the expression levels of tomosyn protein were significantly increased along AD pathology compared with their levels in young 3xTgAD mice. (b) No significant difference in the expression levels of tomosyn protein in the hippocampus of young WT C57BL/6J mice compared with old and very old WT C57BL/6J mice. All values presented are normalized so that young AD (for a) or young WT (for b) values are 100 a.u. **P<0.005.

Figure 6

Effects of enriched environment (EE) on levels of synaptic proteins in the mouse hippocampus. Immunofluorescence staining of coronal slices of hippocampi from wild-type (WT) C57BL/6J mice exposed to regular (Control) or EE. In WT C57BL/6J mice subjected to EE (a) synaptophysin protein levels are significantly higher in the hippocampus, while (b) tomosyn protein levels are significantly lower. (c) Representative immunofluorescence images of synaptophysin protein labeling (upper row) and tomosyn protein labeling (lower row) in the hilus of WT C57BL/6J mice that were exposed to EE (left column) and their control (right column). MolDG, molecular layer of the dentate gyrus; GrDG, granular layer of the dentate gyrus; Hi, hilus. *P<0.05, **P<0.005.