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Cited by 217MicroRNA in disease regulationNeuroinflammation and Neurodegeneration MechanismsAcute Ischemic Stroke Management

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An Antagomir to MicroRNA Let7f Promotes Neuroprotection in an Ischemic Stroke Model

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Selvamani, Amutha, Sathyan, Pratheesh, Miranda, Rajesh C., Sohrabji, Farida·2012·PLoS ONE
DOI 10.1371/journal.pone.0032662PMC PMC3290559
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Abstract

We previously showed that middle-aged female rats sustain a larger infarct following experimental stroke as compared to younger female rats, and paradoxically, estrogen treatment to the older group is neurotoxic. Plasma and brain insulin-like growth factor-1 (IGF-1) levels decrease with age. However, IGF-1 infusion following stroke, prevents estrogen neurotoxicity in middle-aged female rats. IGF1 is neuroprotective and well tolerated, but also has potentially undesirable side effects. We hypothesized that microRNAs (miRNAs) that target the IGF-1 signaling family for translation repression could be alternatively suppressed to promote IGF-1-like neuroprotection. Here, we report that two conserved IGF pathway regulatory microRNAs, Let7f and miR1, can be inhibited to mimic and even extend the neuroprotection afforded by IGF-1. Anti-mir1 treatment, as late as 4 hours following ischemia, significantly reduced cortical infarct volume in adult female rats, while anti-Let7 robustly reduced both cortical and striatal infarcts, and preserved sensorimotor function and interhemispheric neural integration. No neuroprotection was observed in animals treated with a brain specific miRNA unrelated to IGF-1 (anti-miR124). Remarkably, anti-Let7f was only effective in intact females but not males or ovariectomized females indicating that the gonadal steroid environment critically modifies miRNA action. Let7f is preferentially expressed in microglia in the ischemic hemisphere and confirmed in ex vivo cultures of microglia obtained from the cortex. While IGF-1 was undetectable in microglia harvested from the non-ischemic hemisphere, IGF-1 was expressed by microglia obtained from the ischemic cortex and was further elevated by anti-Let7f treatment. Collectively these data support a novel miRNA-based therapeutic strategy for neuroprotection following stroke.

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Figures

Figure 1 Age-related changes in the expression of selected miRNA associated with the IGF-1 gene. A. Schematic depiction of the 3′UTR of the IGF-1 gene: miR1 and Let7 have preferentially conserved, 7–8 mer and 8 mer binding sites respectively on the 3′UTR region of IGF-1 gene. Nucleotides (nn) indicate the seed region for each microRNA. B. QPCR analyses of microRNA in the cerebral cortex of adult and middle aged females. Expression of miR1 and Let7f is elevated in middle-aged (10–12 month) females as compared to adults females (5–7 month), while age does not affect the expression of miR124, a brain-specific miRNA not associated with IGF-1. *: p<0.05.
Figure 1 Age-related changes in the expression of selected miRNA associated with the IGF-1 gene. A. Schematic depiction of the 3′UTR of the IGF-1 gene: miR1 and Let7 have preferentially conserved, 7–8 mer and 8 mer binding sites respectively on the 3′UTR region of IGF-1 gene. Nucleotides (nn) indicate the seed region for each microRNA. B. QPCR analyses of microRNA in the cerebral cortex of adult and middle aged females. Expression of miR1 and Let7f is elevated in middle-aged (10–12 month) females as compared to adults females (5–7 month), while age does not affect the expression of miR124, a brain-specific miRNA not associated with IGF-1. *: p<0.05.CC BY · PubMed Central
Figure 2 Effect of anti-sense miRNA on infarct volume. Female rats were injected with either anti-sense miRNA or scrambled oligonucleotides (control) 4 h after ET-1 induced middle cerebral artery occlusion (MCAo). Infarct volume was assessed using TTC-stained brain sections and quantitative morphometry. A. Animals injected with anti-miR1 had significantly lower cortical infarct volume as compared to controls injected with a scrambled oligo. Striatal infarct was not significantly different between the two groups. B. Animals injected with anti-Let7f had significantly reduced cortical and striatal infarct volumes as compared to controls. C. Animals injected with anti-miR124 had infarct volumes that were similar to the control group. Histograms depict mean ± SEM. *: p<0.05, n = 5 per group.
Figure 2 Effect of anti-sense miRNA on infarct volume. Female rats were injected with either anti-sense miRNA or scrambled oligonucleotides (control) 4 h after ET-1 induced middle cerebral artery occlusion (MCAo). Infarct volume was assessed using TTC-stained brain sections and quantitative morphometry. A. Animals injected with anti-miR1 had significantly lower cortical infarct volume as compared to controls injected with a scrambled oligo. Striatal infarct was not significantly different between the two groups. B. Animals injected with anti-Let7f had significantly reduced cortical and striatal infarct volumes as compared to controls. C. Animals injected with anti-miR124 had infarct volumes that were similar to the control group. Histograms depict mean ± SEM. *: p<0.05, n = 5 per group.CC BY · PubMed Central
Figure 3 Sex difference in anti-Let7f treatment on infarct volume. A. Male rats received ICV injections of anti-Let7f 4 h post MCAo. Infarct volumes assessed using TTC stained brain sections indicated that anti-Let7f treatment had no effect on the severity of cortical or striatal infarction. B. Ovariectomized female rats received ICV injections of anti-Let7f 4 h post MCAo. Infarct volumes assessed using TTC-stained brain sections indicated that anti-Let7f treatment had no effect on cortical or striatal infarction. Histograms depict mean ± SEM, n = 4–6 per group.
Figure 3 Sex difference in anti-Let7f treatment on infarct volume. A. Male rats received ICV injections of anti-Let7f 4 h post MCAo. Infarct volumes assessed using TTC stained brain sections indicated that anti-Let7f treatment had no effect on the severity of cortical or striatal infarction. B. Ovariectomized female rats received ICV injections of anti-Let7f 4 h post MCAo. Infarct volumes assessed using TTC-stained brain sections indicated that anti-Let7f treatment had no effect on cortical or striatal infarction. Histograms depict mean ± SEM, n = 4–6 per group.CC BY · PubMed Central
Figure 4 Behavioral assessment of post-stroke anti-Let7f treatment. Animals injected with anti-Let7f or scrambled oligonucleotides 4 h post MCAo were assessed on the vibrissae evoked forelimb placement task and the rotarod task. A. Control animals were impaired post stroke (blue bars) on the cross midline task when the left vibrissa was stimulated (and required right paw placement), as compared to their pre-stroke (green bars) performance. Anti-Let7f treated animals performed no differently from their pre-stroke performance indicating significant functional neuroprotection. Histogram depicts percent correct responses+SEM, N = 4–5/group, *: p<0.05. B. Animals were placed on a slowly-accelerating rod and the amount of time spent on the rod was recorded. Post-stroke, anti-Let7f treated animals were able to maintain their balance on the rotating rod for significantly longer than control treated animals. Histogram depicts time in seconds+SEM, N = 5/group, *: p<0.05.
Figure 4 Behavioral assessment of post-stroke anti-Let7f treatment. Animals injected with anti-Let7f or scrambled oligonucleotides 4 h post MCAo were assessed on the vibrissae evoked forelimb placement task and the rotarod task. A. Control animals were impaired post stroke (blue bars) on the cross midline task when the left vibrissa was stimulated (and required right paw placement), as compared to their pre-stroke (green bars) performance. Anti-Let7f treated animals performed no differently from their pre-stroke performance indicating significant functional neuroprotection. Histogram depicts percent correct responses+SEM, N = 4–5/group, *: p<0.05. B. Animals were placed on a slowly-accelerating rod and the amount of time spent on the rod was recorded. Post-stroke, anti-Let7f treated animals were able to maintain their balance on the rotating rod for significantly longer than control treated animals. Histogram depicts time in seconds+SEM, N = 5/group, *: p<0.05.CC BY · PubMed Central
Figure 5 Anti-Let7f affects gene expression in the ischemic brain. RNA from the ischemic cortex and striatum was subject to quantitative PCR for a subset of genes with validated 3′ UTR binding sites for Let7f. Histograms represent gene expression normalized to cyclophilin (control) expression in the cortex (A) and striatum (B). Approximately 10 genes were upregulated in animals that received anti-Let7f as compared to the scrambled controls, indicating that the antagomir inhibited the actions of the endogenous miRNA. An asterisk (located by the gene name) indicates genes that were significantly upregulated by anti-Let7f. A large subset (7/10) of these genes were upregulated by anti-Let7f in both the cortex and striatum, while the remainder (3/10 in each case) were specific to either the striatum or cortex. (C) QPCR analysis of mRNA from the non-ischemic cortex of anti-Let7 and control treated ischemic animals showed no changes in any of the target genes. (D) The same set of genes was also assessed in males treated with anti-Let7f. None of these genes were significantly altered as compared to animals that received scrambled oligonucleotides, consistent with the data that anti-Let7f was not neuroprotective in males. Primers and protein names can be found in Table S2. *: p<0.05.
Figure 5 Anti-Let7f affects gene expression in the ischemic brain. RNA from the ischemic cortex and striatum was subject to quantitative PCR for a subset of genes with validated 3′ UTR binding sites for Let7f. Histograms represent gene expression normalized to cyclophilin (control) expression in the cortex (A) and striatum (B). Approximately 10 genes were upregulated in animals that received anti-Let7f as compared to the scrambled controls, indicating that the antagomir inhibited the actions of the endogenous miRNA. An asterisk (located by the gene name) indicates genes that were significantly upregulated by anti-Let7f. A large subset (7/10) of these genes were upregulated by anti-Let7f in both the cortex and striatum, while the remainder (3/10 in each case) were specific to either the striatum or cortex. (C) QPCR analysis of mRNA from the non-ischemic cortex of anti-Let7 and control treated ischemic animals showed no changes in any of the target genes. (D) The same set of genes was also assessed in males treated with anti-Let7f. None of these genes were significantly altered as compared to animals that received scrambled oligonucleotides, consistent with the data that anti-Let7f was not neuroprotective in males. Primers and protein names can be found in Table S2. *: p<0.05.CC BY · PubMed Central
Figure 6 Cellular localization of Let7f in vivo and ex vivo. A. Sections (40 um) from the ischemic cortex were probed for Let7f by in situ hybridization (red) and either GFAP (green, Ai) or CD11b (green, Aii) immunohistochemistry. While Let7f was occasionally colocalized to GFAP-positive cells, virtually all CD11b-positive cells were also Let7f labeled. In a parallel analysis, Let7f (green) was also localized to cells expressing moderate levels of Iba1 (red; red arrow), but not in cells expressing high levels of Iba1 (white arrow). Bar: 200 µm. B. Microglial cells were harvested from the ischemic cortex and cultured ex vivo for 2–4 days. (Bi) Combined in situ hybridization and immunohistochemistry indicated that all CD11b-positive cells were also positively labeled with Let7f probes. (Bii) Microglial cultures exposed to FAM-labeled anti-Let7f in media readily incorporated the antagomir. Shown here are confocal images of microglia (DIC illumination) with FAM-labeled antagomirs (green) Bar: 30 µm.
Figure 6 Cellular localization of Let7f in vivo and ex vivo. A. Sections (40 um) from the ischemic cortex were probed for Let7f by in situ hybridization (red) and either GFAP (green, Ai) or CD11b (green, Aii) immunohistochemistry. While Let7f was occasionally colocalized to GFAP-positive cells, virtually all CD11b-positive cells were also Let7f labeled. In a parallel analysis, Let7f (green) was also localized to cells expressing moderate levels of Iba1 (red; red arrow), but not in cells expressing high levels of Iba1 (white arrow). Bar: 200 µm. B. Microglial cells were harvested from the ischemic cortex and cultured ex vivo for 2–4 days. (Bi) Combined in situ hybridization and immunohistochemistry indicated that all CD11b-positive cells were also positively labeled with Let7f probes. (Bii) Microglial cultures exposed to FAM-labeled anti-Let7f in media readily incorporated the antagomir. Shown here are confocal images of microglia (DIC illumination) with FAM-labeled antagomirs (green) Bar: 30 µm.CC BY · PubMed Central
Figure 7 Anti-Let7f regulates IGF-1 in microglia. A. IGF-1 levels in vivo: Cortical lysates from the ischemic and non ischemic hemisphere of scrambled and anti-Let7f treated animals were analyzed for IGF-1 by ELISA. Ischemia induced a 4-fold elevation of IGF-1 in both scrambled and anti-Let7f treated animals. However, IGF-1 levels in the anti-Let7f treated group was no different from that of the scramble controls. Histogram depicts mean ± SEM of 4–5 animals per group. B. IGF-1 levels in microglia: Microglia were harvested from the ischemic cortex of adult rats and treated in culture with anti-Let7f or scrambled oligonucleotides for 48 h. Anti-Let7f treatment significantly increased IGF-1 levels in microglial media. Ex vivo experiments were performed 6 times using cells harvested from 1–2 animals for each run; histogram depicts mean ± SEM of all experiments. *: p<0.05.
Figure 7 Anti-Let7f regulates IGF-1 in microglia. A. IGF-1 levels in vivo: Cortical lysates from the ischemic and non ischemic hemisphere of scrambled and anti-Let7f treated animals were analyzed for IGF-1 by ELISA. Ischemia induced a 4-fold elevation of IGF-1 in both scrambled and anti-Let7f treated animals. However, IGF-1 levels in the anti-Let7f treated group was no different from that of the scramble controls. Histogram depicts mean ± SEM of 4–5 animals per group. B. IGF-1 levels in microglia: Microglia were harvested from the ischemic cortex of adult rats and treated in culture with anti-Let7f or scrambled oligonucleotides for 48 h. Anti-Let7f treatment significantly increased IGF-1 levels in microglial media. Ex vivo experiments were performed 6 times using cells harvested from 1–2 animals for each run; histogram depicts mean ± SEM of all experiments. *: p<0.05.CC BY · PubMed Central

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Metadata

Authors
Selvamani, A., Sathyan, P., Miranda, R. C., & Sohrabji, F.
Affiliations
Texas A&M Health Science Center (US)
Corresponding author
Sohrabji, Farida
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2012-02-29
Volume
7
Issue
2
Pages
e32662
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0032662
PMC
PMC3290559
OpenAlex
W2071432376
Cited by
217
References
79
Citations by year
2026: 1 · 2025: 4 · 2024: 6 · 2023: 7 · 2022: 11 · 2021: 12 · 2020: 16 · 2019: 19 · 2018: 18 · 2017: 26 · 2016: 20 · 2015: 23 · 2014: 27 · 2013: 22 · 2012: 4
FWCI
8.02
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • OakTrust (Texas A&M University Libraries) (submittedVersion)
Funders
  • National Institutes of Health
  • National Institute on Aging (R01AG027684, AG027684, AG028303, R01AG028303)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Acute Ischemic Stroke Management
Keywords
ischemic stroke, neuroprotection, microRNA, let-7f, miR-1, insulin-like growth factor I, IGF-1 signaling, microglia, sex differences, infarct size, sensorimotor function, hemispheric integration, sex steroids
MeSH terms
Animals, Behavior, Animal, Disease Models, Animal, Female, Gene Expression Regulation, Insulin-Like Growth Factor I, Ischemia, Male, Sex Factors, Reperfusion Injury, Microglia, Neuroprotective Agents, Stroke, MicroRNAs, Rats

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