REV-ERB Agonist SR9009 Regulates the Proliferation and Neurite Outgrowth/Suppression of Cultured Rat Adult Hippocampal Neural Stem/Progenitor Cells in a Concentration-Dependent Manner

Abstract

REV-ERBs are heme-binding nuclear receptors that regulate the circadian rhythm and play important roles in the regulation of proliferation and the neuronal differentiation process in neuronal stem/progenitor cells in the adult brain. However, the effects of REV-ERB activation in the adult brain remain unclear. In this study, SR9009, a synthetic REV-ERB agonist that produces anxiolytic effects in mice, was used to treat undifferentiated and neuronally differentiated cultured rat adult hippocampal neural stem/progenitor cells (AHPs). The expression of Rev-erbβ was upregulated during neurogenesis in cultured rat AHPs, and Rev-erbβ knockdown analysis indicated that REV-ERBβ regulates the proliferation and neurite outgrowth of cultured rat AHPs. The application of a low concentration (0.1 µM) of the REV-ERB agonist SR9009 enhanced neurite outgrowth during neurogenesis in cultured rat AHPs, whereas the addition of a high concentration (2.5 µM) of SR9009 suppressed neurite outgrowth. Further examination of the SR9009 regulatory mechanism showed that the expressions of downstream target genes of REV-ERBβ, including Ccna2 and Sez6, were modulated by SR9009. The results of this study indicated that REV-ERBβ activity in cultured rat AHPs was regulated by SR9009 in a concentration-dependent manner. Furthermore, SR9009 inhibited the growth of cultured rat AHPs through various pathways, which may provide insight into the multifunctional mechanisms of action associated with SR9009. The findings of this study may provide an improved understanding of proliferation and neuronal maturation mechanisms in cultured rat AHPs through SR9009-regulated REV-ERBβ signaling pathways.

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Data Availability

The data that support the findings of this study are available from the corresponding author, K. Shimozaki, upon reasonable request.

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Acknowledgements

I thank H. Kitagawa, D. Teraoka, and the members of the Life Science Support Center at Nagasaki University for technical assistance. I thank Lisa Kreiner, PhD, and Lisa Giles, PhD, from Edanz Group (https://en-author-services.edanz.com/ac) for editing a draft of this manuscript.

Funding

This work was supported by a Grant-in-Aid from the Alumni Association of Nagasaki University School of Medicine and followed the Uniform Requirements for Manuscripts Submitted to Biomedical Journals developed by the International Committee of Medical Journal Editors.

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Supplementary Material 1

Supplementary file 1 (DOCX 375 KB) The Vehicle solution used in the control experiments did not affect proliferation or neurite outgrowth in rat AHPs. a. Rat AHPs cultured in undifferentiated maintenance medium without (−) and with Vehicle solution were labeled with BrdU, after 24 hours of culture. BrdU-positive cells were detected and measured using an anti-BrdU antibody. Data in the graph are expressed as the mean ± s. e. m. Statistical comparisons were performed by two-tailed unpaired t-tests followed by Welch’s correction (df = 3.478; t = 0.2286; P = 0.8320; n = 3). b. Rat AHPs were cultured for 7 days in a neural differentiation medium without (−) and with Vehicle solution, which was added immediately after the induction of neural differentiation. Differentiated cells were detected with an antibody against βIII-tubulin, and neurite lengths were measured. Data in the graph are expressed as the mean ± s. e. m. Statistical comparisons were performed by two-tailed unpaired t-tests followed by Welch’s correction (df = 8.874; t = 0.6697; P = 0.0831; n = 6)

Supplementary Material 2

Supplementary file 1 (DOCX 747 KB) Gene knockdown vector plasmid expressing scrambled (a, c, e, and g; white bars) or Rev-erbβ-targeting (b, d, f, and h; gray bars) shRNA sequences, along with GFP, was transfected into rat AHPs. Subsequently, Vehicle, low- (0.1 µM) or high-concentration (2.5 µM) SR9009 solutions was added to induce neural differentiation for 7 days. The numbers of neurite branches (a and b, 0.1 µM; c and d, 2.5 µM) and primary neurites (e and f, 0.1 µM; g and h, 2.5 µM) in GFP and anti-βIII-tubulin double-positive cells were counted. Data for 18 differentiated cells are plotted on the graph (Vehicle, white circles; SR9009 addition, triangles). Data are represented as the median and interquartile range (***P < 0.001). Statistical comparisons were performed by two-tailed unpaired t-tests followed by Welch’s correction (a. df = 33.48, t = 0.7473, P = 0.4601; b. df = 23.75, t = 2.22, P = 0.0362; c. df = 27.73, t = 3.735, P = 0.0009; d. df = 24.68, t = 1.597, P = 0.1229; e. df = 27.37, t = 1.75, P = 0.0913; f. df = 32.65, t = 1.788, P = 0.0831; g. df = 33.18, t = 2.003, P = 0.0534; h. df = 33.44, t = 0.7786, P = 0.4417)

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Shimozaki, K. REV-ERB Agonist SR9009 Regulates the Proliferation and Neurite Outgrowth/Suppression of Cultured Rat Adult Hippocampal Neural Stem/Progenitor Cells in a Concentration-Dependent Manner. Cell Mol Neurobiol (2021). https://doi.org/10.1007/s10571-021-01053-y

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Keywords

  • Adult neurogenesis
  • Cultured neural stem/progenitor cells
  • REV-ERBβ
  • SR9009
  • Proliferation
  • Neurite outgrowth/suppression