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Targeted deletion of miR-132/-212 impairs memory and alters the hippocampal transcriptome

Hansen, Katelin F., Sakamoto, Kensuke, Aten, Sydney, Price, Kaiden H., Loeser, Jacob, Hesse, Andrea M., Page, Chloe E., Pelz, Carl, Arthur, J. Simon C., Impey, Soren, Obrietan, Karl·2016·Learning & Memory
DOI 10.1101/lm.039578.115PMC PMC4749831
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Abstract

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in a complex, nonredundant manner to shape the transcriptional profile of the CNS. The dysregulation of miR-132/-212 expression could contribute to signaling mechanisms that are involved in an array of cognitive disorders.

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Figure 1. Conditional deletion of miR-132/-212 in excitatory forebrain neurons. (A) miR-132/-212f/f animals were crossed with a line expressing Cre recombinase driven by a CaMKII promoter to yield mice with miR-132/-212 selectively knocked out in forebrain neurons (CaMKII-Cre::miR-132/-212f/f). (B) The addition of loxP sites in the CaMKII-Cre::miR-132/-212f/f line was confirmed by PCR amplification of the miR-132/-212 locus. Similarly, the presence of the CamKII-Cre driver was identified by PCR. Animal 1 represents a conditional knockout animal with both the CaMKII-Cre driver and the miR-132/-212 locus with flanking loxP sites. In contrast, Animal 2 is negative for the CaMKII-Cre driver and is positive for wild-type (WT) miR-132/-212. (C) Effective CRE recombinase excision within hippocampal neurons was confirmed by crossing CaMKII-Cre mice to a ROSA 26 β-galactosidase reporter line. β-Galactosidase expression was detected in forebrain regions, including the CA1 cell layer of the hippocampus (scale bar = 40 µm). (D) Deletion of the miR-132/-212 locus was confirmed by fluorescent in situ hybridization within the CA1 cell layer. SR: stratium radiatum; SO: stratum oriens. Scale bar = 40 µm.
Figure 1. Conditional deletion of miR-132/-212 in excitatory forebrain neurons. (A) miR-132/-212f/f animals were crossed with a line expressing Cre recombinase driven by a CaMKII promoter to yield mice with miR-132/-212 selectively knocked out in forebrain neurons (CaMKII-Cre::miR-132/-212f/f). (B) The addition of loxP sites in the CaMKII-Cre::miR-132/-212f/f line was confirmed by PCR amplification of the miR-132/-212 locus. Similarly, the presence of the CamKII-Cre driver was identified by PCR. Animal 1 represents a conditional knockout animal with both the CaMKII-Cre driver and the miR-132/-212 locus with flanking loxP sites. In contrast, Animal 2 is negative for the CaMKII-Cre driver and is positive for wild-type (WT) miR-132/-212. (C) Effective CRE recombinase excision within hippocampal neurons was confirmed by crossing CaMKII-Cre mice to a ROSA 26 β-galactosidase reporter line. β-Galactosidase expression was detected in forebrain regions, including the CA1 cell layer of the hippocampus (scale bar = 40 µm). (D) Deletion of the miR-132/-212 locus was confirmed by fluorescent in situ hybridization within the CA1 cell layer. SR: stratium radiatum; SO: stratum oriens. Scale bar = 40 µm.CC BY-NC · PubMed Central
Figure 2. Deletion of miR-132/-212 within hippocampal excitatory neurons impairs learning and memory. (A) miR-132/-212 conditional knockout (cKO) animals exhibited impaired recognition memory on the novel object recognition task. Discrimination ratio = (exploration time with novel object)/(total exploration time). Data are presented as mean ± SEM, n = 14/group, (*) P < 0.05. (B) cKO animals showed a significant decreases in freezing behavior after contextual fear conditioning compared to control mice. Data presented as mean ± SEM, n = 14/group, (*) P < 0.05. cKO animals also demonstrated impaired spatial learning capacity with increases in both latency to escape (C) and training errors (D) compared to controls. n = 13/group, Latency: F(1,24) = 5.44, P = 0.028, Error: F(1,24) = 7.32, P < 0.012).
Figure 2. Deletion of miR-132/-212 within hippocampal excitatory neurons impairs learning and memory. (A) miR-132/-212 conditional knockout (cKO) animals exhibited impaired recognition memory on the novel object recognition task. Discrimination ratio = (exploration time with novel object)/(total exploration time). Data are presented as mean ± SEM, n = 14/group, (*) P < 0.05. (B) cKO animals showed a significant decreases in freezing behavior after contextual fear conditioning compared to control mice. Data presented as mean ± SEM, n = 14/group, (*) P < 0.05. cKO animals also demonstrated impaired spatial learning capacity with increases in both latency to escape (C) and training errors (D) compared to controls. n = 13/group, Latency: F(1,24) = 5.44, P = 0.028, Error: F(1,24) = 7.32, P < 0.012).CC BY-NC · PubMed Central
Figure 3. High-throughput sequencing of the hippocampal transcriptome in tTA::miR-132, tTA::miR-212, and CaMKII-Cre::miR-132/-212f/f animals. (A) Hippocampal mRNAs were sequenced from tTA::miR-132, tTA::miR-212, and CaMKII-Cre::miR-132/-212f/f animals using the Illumina approach, followed by functional gene ontology analysis on the intersection of direction-specific gene alterations to determine the molecular effects of altered miR-132 and miR-212. Of note, for the sake of clarity, the schematic omits the intersection analysis for the tTA::miR-212 mouse line. (B) Representative hippocampal images from the three noted mouse lines. Hoechst labeling was used to delineate the hippocampal sublayers of the CaMKII-Cre::miR-132/-212f/f line. The miR-132 and miR-212 transgenic lines express cyan fluorescent protein (CFP) or RsRED, respectively, from bidirectional TRE transgenic promoter constructs (refer to the Methods section for a description of the transgenes).
Figure 3. High-throughput sequencing of the hippocampal transcriptome in tTA::miR-132, tTA::miR-212, and CaMKII-Cre::miR-132/-212f/f animals. (A) Hippocampal mRNAs were sequenced from tTA::miR-132, tTA::miR-212, and CaMKII-Cre::miR-132/-212f/f animals using the Illumina approach, followed by functional gene ontology analysis on the intersection of direction-specific gene alterations to determine the molecular effects of altered miR-132 and miR-212. Of note, for the sake of clarity, the schematic omits the intersection analysis for the tTA::miR-212 mouse line. (B) Representative hippocampal images from the three noted mouse lines. Hoechst labeling was used to delineate the hippocampal sublayers of the CaMKII-Cre::miR-132/-212f/f line. The miR-132 and miR-212 transgenic lines express cyan fluorescent protein (CFP) or RsRED, respectively, from bidirectional TRE transgenic promoter constructs (refer to the Methods section for a description of the transgenes).CC BY-NC · PubMed Central
Figure 4. miR-212/dsRED transgene expression. (A) Polymerase chain reaction (PCR)-based genotyping results for miR-212 and tTA. PCRs were run in a 1% agarose gel and visualized using ethidium bromide. Animal 1 represents an animal transgenically expressing miR-212 under the control of the CamKII-tTA driver, whereas Animal 2 has the miR-212 transgene, but lacks the driver and thus does not express the miR-212 transgene. (B) qPCR was performed to profile levels of miR-212 within the hippocampus; both the miR-212 sense strand (miR-212-3p) and anti-sense strand (miR-212-5p) were profiled. Mature miR-212 levels are shown as mean ± SEM, compared with nontransgenic controls (Ctrl: n = 4 animals per condition). Data were normalized to the control conditions which were set to a value of 1. (C) Coronal brains sections show fluorescence from the dsRED transgene marker within the CA1, CA3, and granule cell layer (GCL) of the hippocampus. (D) The novel object recognition task revealed an impaired recognition memory phenotype in tTA::miR-212 animals. Discrimination ratio = (exploration time with novel object)/(total exploration time). Data are presented as mean ± SEM, n = 6/group, (*) P < 0.05, Student's t-test.
Figure 4. miR-212/dsRED transgene expression. (A) Polymerase chain reaction (PCR)-based genotyping results for miR-212 and tTA. PCRs were run in a 1% agarose gel and visualized using ethidium bromide. Animal 1 represents an animal transgenically expressing miR-212 under the control of the CamKII-tTA driver, whereas Animal 2 has the miR-212 transgene, but lacks the driver and thus does not express the miR-212 transgene. (B) qPCR was performed to profile levels of miR-212 within the hippocampus; both the miR-212 sense strand (miR-212-3p) and anti-sense strand (miR-212-5p) were profiled. Mature miR-212 levels are shown as mean ± SEM, compared with nontransgenic controls (Ctrl: n = 4 animals per condition). Data were normalized to the control conditions which were set to a value of 1. (C) Coronal brains sections show fluorescence from the dsRED transgene marker within the CA1, CA3, and granule cell layer (GCL) of the hippocampus. (D) The novel object recognition task revealed an impaired recognition memory phenotype in tTA::miR-212 animals. Discrimination ratio = (exploration time with novel object)/(total exploration time). Data are presented as mean ± SEM, n = 6/group, (*) P < 0.05, Student's t-test.CC BY-NC · PubMed Central
Figure 5. Enriched ontological clusters of altered mRNA in miR-132 and miR-212 transgenic mice. (A,B) Using a ranked list of genes downregulated following miRNA overexpression, the top 10 clusters of DAVID categories were sorted by enrichment score and presented for each of the transgenic animal lines (tTA::miR-132 and tTA::miR-212). The number of genes within each cluster is reported as a bar corresponding to the lower axis. In addition, EASE scores (DAVID's modified Fischer exact P-value) for each cluster are depicted as a measure of the association strength of genes within each cluster with a line graph corresponding to the upper axis. (C,D) Upregulated genes from CaMKII-Cre::miR-132/-212f/f animals were compared with genes downregulated in tTA::miR-132 and tTA::miR-212 animals, respectively, as well as with algorithmic target predictions from microRNA.org. This intersection yielded a limited pool of potential targets for each miRNA.
Figure 5. Enriched ontological clusters of altered mRNA in miR-132 and miR-212 transgenic mice. (A,B) Using a ranked list of genes downregulated following miRNA overexpression, the top 10 clusters of DAVID categories were sorted by enrichment score and presented for each of the transgenic animal lines (tTA::miR-132 and tTA::miR-212). The number of genes within each cluster is reported as a bar corresponding to the lower axis. In addition, EASE scores (DAVID's modified Fischer exact P-value) for each cluster are depicted as a measure of the association strength of genes within each cluster with a line graph corresponding to the upper axis. (C,D) Upregulated genes from CaMKII-Cre::miR-132/-212f/f animals were compared with genes downregulated in tTA::miR-132 and tTA::miR-212 animals, respectively, as well as with algorithmic target predictions from microRNA.org. This intersection yielded a limited pool of potential targets for each miRNA.CC BY-NC · PubMed Central
Figure 6. Increased MASH1 and STX1A expression following deletion of miR-132/-212. (A) CaMKII-Cre::miR-132/-212f/f (cKO) animals exhibit an increase in MASH1-positive cells within the subgranular zone of the hippocampus, as demonstrated by DAB-based immunolabeling. Arrows indicate MASH1-positive cells, scale bar = 25 µm; GCL = granule cell layer, SGZ = subgranular zone. (B) Quantification of MASH1-positive cells in cKO tissue relative to control animals with intact miR-132/-212 ((*) P < 0.05); n = 6 animals per group. (C) cKO animals also showed an increase in STX1A expression in the stratum oriens (S. Oriens), and then stratum radiatum (S. Radiatum) relative to control animals. Tissue that was processed without the addition of primary antibody is provided as a negative control (NC) in the inset. Scale bar = 150 µm. (D) Quantification of STX1A staining intensity within the S. Radiatum, S. Oriens, and molecular layers in cKO animals relative to controls ((*) P < 0.05), Student's t-test; n = 7 animals per group. Values were normalized to labeling in the control S. Oriens, which was set equal to a value of 1.
Figure 6. Increased MASH1 and STX1A expression following deletion of miR-132/-212. (A) CaMKII-Cre::miR-132/-212f/f (cKO) animals exhibit an increase in MASH1-positive cells within the subgranular zone of the hippocampus, as demonstrated by DAB-based immunolabeling. Arrows indicate MASH1-positive cells, scale bar = 25 µm; GCL = granule cell layer, SGZ = subgranular zone. (B) Quantification of MASH1-positive cells in cKO tissue relative to control animals with intact miR-132/-212 ((*) P < 0.05); n = 6 animals per group. (C) cKO animals also showed an increase in STX1A expression in the stratum oriens (S. Oriens), and then stratum radiatum (S. Radiatum) relative to control animals. Tissue that was processed without the addition of primary antibody is provided as a negative control (NC) in the inset. Scale bar = 150 µm. (D) Quantification of STX1A staining intensity within the S. Radiatum, S. Oriens, and molecular layers in cKO animals relative to controls ((*) P < 0.05), Student's t-test; n = 7 animals per group. Values were normalized to labeling in the control S. Oriens, which was set equal to a value of 1.CC BY-NC · PubMed Central

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Metadata

Authors
Hansen, K. F., Sakamoto, K., Aten, S., Price, K. H., Loeser, J., Hesse, A. M., Page, C. E., Pelz, C., Arthur, J. S. C., Impey, S., & Obrietan, K.
Venue
Learning & Memory
Publisher
Cold Spring Harbor Laboratory
Published
2016-01-15
Volume
23
Issue
2
Pages
61-71
ISSN
1549-5485
DOI
10.1101/lm.039578.115
PMC
PMC4749831
Cited by
100
References
10
Language
en
Funders
  • National Institutes of Health (F31-MH096460, NS066345, NS091302, NS045758, MH103361)
  • National Science Foundation (1354612)

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