Main Research Findings

1) SR-9009 regulates proliferation, outgrowth, and suppression of neurons in a concentration-dependent manner.

2) REV-ERB agonists like SR-9009 are capable of regulating circadian rhythm and metabolic activity.

Selected Data

1) Researcher Koli Shimozaki examined the effects elicited by SR-9009 stimulation of rat adult hippocampal neural stem/progenitor cells (AHPs). The rat AHPs were obtained from the Salk Institute and maintained in an undifferentiated state by culturing in an N2-medium containing DMEM, F12 medium supplemented with N2, and Invitrogen, as well as 20 ng/ml of FGF2. After 24 hours the undifferentiated maintenance medium was replaced with a neural differentiation medium containing 1 uM retinoic acid and 0.5% FBS, and was cultured for 7 days in order to induce neurogenesis in the AHPs. When inducing neural differentiation in the gene knockdown systems, the rat AHPs were transfected with knockdown plasmid vectors followed by washing three times with N2-medium and resuspension in neural differentiation medium before culturing on pO/L plates for 7 days [1].

DMSO was used to prepare the SR-9009 stock solutions; various concentrations of the SARM were used while the ratio of 1% DMSO and 50% ethanol was adjusted accordingly. The vehicle solution used as a control treatment was composed of 1% DMSO and 50% ethanol. Both the experimental and control treatments were diluted 200-fold and added into each culture system in a single administration. For the proliferation assay, the rat AHPs were left to incubate in the treatments for 24 hours and for the neurite outgrowth assay the samples were left for 7 days. Additionally, SR-9009 was added to the medium the day after the rat AHPs were transfected with plasmid DNA expressing shRNA targeting Rev-erb-beta. The cells were then cultured for an additional 24 hours to measure cell proliferation [1].

The next step of the study was to determine the functional relationship between SR-9009 and REV-ERB-beta. Rev-erb-beta mRNA was knocked down using a shRNA expression system while enhanced green fluorescent protein (GFP) was co-expressed under the control of the PGK promoter in order to successfully label transfected knockdown cells. The rats’ AHPs HCN1706 subline was transfected with plasmid DNA expressing scrambled shRNA or shRNA targeted against the Rev-erb-beta sequence through the use of the Nucleofector system. An RNeasy kit was used in order to extract total RNA from HCN1706 cells, while cDNA was transcribed using the ReverTra Ace Kit. An SYBR Green Q-PCR analysis kit allowed Shimozaki to conduct a qRT-PCR which was further examined with a QuantStudio 12 K Flex quantitative PCR system.

Both undifferentiated and differentiated rat AHPs were fixed in a 4% PFA solution and blocked using PBS containing 0.1% Triton X-100 and 1% donkey serum. Immunostaining was then performed using the primary antibodies: anti-NR1D2, anti-Nestin, aunty-Ki67, and anti-beta-III tubulin; as well as the secondary antibodies anti-rat, rabbit, or chicken DyLight 488, 549, and 649. Furthermore, DNA was stained using 4′-6-diamidino-2-phenylindole to identify cell nuclei. BrdU solution was added to rat AHPs in order to label proliferating cells. Prior to immunofluorescent staining with anti-BrdU antibody, the BrdU-treated cells were fixed with 4% PFA following treatment with hydrochloric acid. Detecting cells co-stained with anti-GFP and anti-BrdU antibodies were also used by Shimozaki to observe and measure proliferation rate of the cells [1].

Immunostaining images were taken using a confocal laser microscope and a fluorescence microscope while proliferation rate of the knockdown cells was measured by calculating the number of GFP-positive and GFP- and anti-BrdU double-positive cells stained with DAPI. This same procedure was followed in the samples treated with SR-9009. When observing the effects elicited by SR-9009 on neurite outgrowth, HC1706 cells were treated with varying concentrations of SR-9009, followed by immunostaining with anti beta-III tubulin antibodies and DAPI. Beta-III-tubulin-positive dendrites were measured using a BZ-X Analyzed and DP2-BSW application software. After determining the number of DAPI-positive cells, the average neurite length was found by dividing the total traced neurite length by the number of DAPI-positive cells. Additionally, Shimozaki counted the number of all neurite branching points and primary neurites that were formed directly from the cell bodies [1].

2) Researchers Solt et. Al examined how the popular SARM and REV-ERB agonist, SR-9009, assists in the regulation of circadian rhythm and metabolic action in mice. Mammalian circadian rhythm is promoted by the suprachiasmatic nucleus (SCN) of the hypothalamus as well as a transcriptional feedback loop. The negative feedback loops is primarily composed of heterodimers of the transcription factors BMAL1 (brain and muscle ARNT-like protein 1), CLOCK (circadian locomotor output cycles kaput), or NPAS2 (Neuronal PAS domain-containing protein 2). These heterodimers activate the transcription of Per 1, Per 2, Per 3, Cry1, and Cry2 (PER/CRY) genes. PER/CRY proteins inhibit the activity of BMAL1/CLOCK which results in a rhythmic, circadian pattern of gene expression.

REV-ERB receptors like SR-9009 play a crucial role in feedback regulation of the circadian rhythm since both Bmal1 and Clock genes are direct targets of the receptors. Current research has found that loss of the REV-ERB-alpha protein leads to altered circadian behavior. Additionally, the physiological ligand for both REV-ERB-alpha and -beta was identified as a heme; the next step of experimentation allowed researchers to observe how a small molecule ligand regulated REV-ERB activity, as well as identify and characterize various synthetically developed ligands [2].

The research team developed two REV-ERB agonists, labeled as alpha and beta, in order to track receptor activity in HEK293 cells expressing a chimeric Gal4 DNA Binding Domain (DBD) – REV-ERB ligand binding domain (LBD) alpha or beta, as well as a Gal4-responsive luciferase reporter. Circular dichroism analysis was used to confirm direct binding of SR-9009 to REV-ERB-alpha. Following these initial procedures, the next step was to examine the expression of REV-ERB responsive genes in mice liver. Over the course of six days the animals were treated with various doses of both SR-9011 and SR-9009.

When identifying how the REV-ERB agonist modulated gene expression and circadian rhythm in mice, the research team began by predicting how administration of the SARMs would alter circadian rhythm. Mice were released into constant dark conditions (D:D) after one week of housing in a standard 12 hour light, 12 hour dark schedule. A single dose of SR-9011 or SR-9009 was administered to the mice after 12 days in D:D conditions. Expression analysis of core clock genes in the hypothalami from mice in D:D conditions were assessed by a single subcutaneous injection of SR-9011 and SR-9009. Additionally, the researchers observed the effects of both REV-ERB ligands in the mice that experienced 12 hour light and 12 hour dark conditions (L:D) [2].

Since REV-ERB compounds have the potential to regulate genes involved in lipid and glucose metabolism, SR-9009 was administered to C57B16 mice while SR-9011 was administered to Balb/c mice in order to see effects on weight loss and fat mass. Due to the reported effects of the REV-ERB agonists on the C57BL6 and Balb/c mice, the researcher team conducted a secondary study utilizing 20-week-old C57BL6 mice maintained on a high fat diet for 14 weeks. Following 14 weeks of a high fat diet the same eating pattern was maintained with the addition of twice-daily administration of SR-9009 [2].

Discussion

1) The first portion of the study was to analyze the expression and subcellular localization of REV-ERB-beta in rat AHP. Shimozaki was able to confirm that the REV-ERB-beta protein is expressed and localized in the nucleus and cytoplasm of all cultured rat AHPs. The cultured rat AHPs uniformly expressed the intermediate filament Nestin; Nestin acts as an undifferentiated neural stem/progenitor cell marker protein. Ki67, the nuclear protein and cell proliferation maker, was also detected in cultured AHPs. A similar pattern of expression is identified in cultured neural stem/progenitor cells derived from adult mouse brains. These results suggest that there is a common expression pattern for REV-ERB-beta, specifically in rodents. This data indicates the cell-cycle dynamics are not linked to the localization of REV-ERB-beta.

Neuronal and glial cell differentiation was induced in cultured rat AHPs, in order to analyze the expression and subcellular localization of REV-ERB-beta during adult brain neurogenesis. The rate of neurogenesis in the hippocampal dentate gyrus of the adult mouse brain was reported to be approximately 70%, while the HCN1706 cell line used in this study exhibited a neuronal differentiation rate of more than 90% [1].

Furthermore, the reported results of immunostaining the cultured rat AHPs suggested that the REV-ERB-beta protein was more strongly localized in the nucleus under neural differentiation conditions rather than non-differentiating conditions. Rev-erb-beta expression in cultured rat AHPs was also compared in neural differentiation versus non-differentiating conditions. Results reported that under neuronal differentiation conditions Rev-erb-beta mRNA expression was more than two-fold that of the samples under non-differentiating conditions. Overall, these research findings suggest that REV-ERB-beta accumulated in the nucleus and expression of the protein was enhanced by the induction of neurogenesis [1].

Figure 1: changes in C) Rev-erb-beta expression levels, D) percent of BrdU/GFP expression, E) total length of neurites.

Proliferation in Rev-erb-beta specific knockout samples was measured by adding BrdU to the medium. The collected data found that the knockdown of Rev-erb-beta in AHPs significantly reduced BrdU uptake in comparison to the control group. This indicates that proliferation in rat AHPs is dependent on the presence of Rev-erb-beta.

When investigating the relationship between neurite outgrowth and REV-ERB-beta, the medium was replaced with a neural differentiation medium following electroporation followed by the induction of neural differentiation for 7 days. Anti-beta-III-tubulin antibody immunostaining revealed the molecular and physical properties of the differentiated AHPs by measuring the neurite projection lengths of GFP-positive cells. Results of this analysis found that neurite growth was significantly diminished in Rev-erb-beta knockdown cells in comparison to control cells. These findings suggest that REV-ERB-beta participates in the process of proliferation and neurite outgrowth in cultured rat AHPs in a manner similar to the reported results for the cultured adult mouse neural stem/progenitor cells [1].

Shimozaki observed changes in proliferation and neuronal differentiation in cultured rat AHPs after adding various concentrations of SR-9009 to the medium in order to further characterize the cellular properties associated with enhancement of REV-ERBs in response to activation with SR-9009. Proliferation was measured through the BrdU uptake method; treatment with a vehicle compound did not affect proliferation in rat AHPs. There were no significant changes in BrdU uptake when SR-9009 was administered at concentrations between 0 and 1 uM, however, when SR-9009 was added at concentrations about 1.5 uM, decreased proliferation was detected; equilibrium was reached at 2.5 uM. This data suggests that the growth arrest signal could potentially be activated by SR-9009 when the concentration exceeded a specific amount.

The next step of the study was to examine how knock down Rev-erb-beta in cultured rat AHPs determine the REV-ERB-beta dependence of effects mediated by SR-9009. When the cultured AHPs were transfected with a control vector, a reduced rate in BrdU uptake was observed after 2.5 uM of SR-9009 was added to the samples. It is important to note that BrdU uptake was significantly reduced after SR-9009 was added, even with the Rev-erb-beta gene being knocked down. These findings allowed Shimozaki to conclude that both REV-ERB-beta-dependent and -independent pathways are involved in the SR-9009-mediated inhibition of AHP proliferation [1].

Figure 2: Changes in BrdU uptake in response to varying concentrations of SR-9009.

When examining neurite outgrowth during neurogenesis, dendritic outgrowth was induced when concentrations between 0 and 1 uM of SR-9009 were added to the medium. Maximum growth was observed at a concentration of 0.1 um of SR-9009. However, dendritic growth was suppressed in concentrations higher than 2.5 uM, indicating that SR-9009 induces positive and negative effects on neurite outgrowth during neurogenesis. Cultured cells were fixed with 4% PFA and immunostained to reveal that cells transfected with control shRNA resulted in increased neurite length in comparison to the untreated cells. When the Rev-erb-beta gene was knocked down, the average neurite length of differentiated AHPs did not increase with the addition of low-concentrations (0.1 uM) of SR-9009. Addition of high-concentration SR-9009 (2.5 uM) to control cells resulted in a decrease in the mean neurite length of differentiated AHPs. However when SR-9009 was added to cells with a knocked down Rev-erb-beta gene, no suppressive activity was identified [1].

Figure 3: Changes in dendritic length in response to varying concentrations of SR-9009

The results of the study solidified the hypothesis that SR-9009 reduces proliferation in rat AHPs and induces positive and negative effects on neurite outgrowth. Shimozaki attempted to identify the underlying mechanism of action, by examining whether the expression levels of the Rev-erb-beta gene and REV-ERB-beta target genes were affected by SR-9009. The qRT-PCR conducted found that adding high-concentration of SR-9009 did not lead to any dramatic chances in Rev-erb-beta mRNA expression, however, expression of the REV-ERB-beta target gene Ccna2 was significantly reduced by high concentrations of SR-9009. After 7 days of culturing under neural differentiation conditions after treatment with low and high concentrations of SR-9009, there were no significant changes observed in the expression of Rev-erb-beta or the REV-ERB-beta target gene, Sez6, by either dose of the SARM. This indicates that SR-9009 did not affect the expression of Rev-erb-beta but rather the expression of various REV-ERB-beta target genes [1].

Figure 4: relative mRNA expression in Rev-erb-beta gene and REV-ERB-beta target genes, Ccna2 and Sez6

2) When examining the effects of the REV-ERB agonists on REV-ERB-dependent repressor activity the results showed that both SR-9011 and SR-9009 dose-dependently increased repressor activity in the HEK293 cells that expressed a chimeric Gal4 DNA DBD-LBD as well as a Gal4-responsive luciferase reporter. The use of cotransfection assay using full-length REV-ERB-alpha and a luciferase reporter driven by the Bmal1 promoter confirmed that SR-9009 was capable of effectively suppressing transcription. In a Rev-Erb-alpha/beta-dependent manner, SR-9009 was also able to suppress the expression of BMAL1 mRNA in HepG2 cells. Additionally, the research team noted that both SARMs increased recruitment of the CoRNR box peptide fragment using a biochemical assay. The use of circular dichroism analysis confirmed that there was direct binding of SR-9009 to REV-ERB-alpha. However, neither SR-9001 nor SR-9011 exhibited any activity at other nuclear receptors [2].

Figure 5: Relative luciferase activity reported after a dose of SR-9009 or SR-9011 in HEK293 cells

In terms of the ability of SR-9009 to alter circadian rhythm, the mice were kept in D:D conditions for 12 days before receiving a single 100 mg/kg dose of either SR-9011, SR-9009, or a vehicle. The vehicle injections resulted in no significant changes in circadian locomotor activity while a dose of either SR-9011 or SR-9009 led to a loss of locomotor activity during the subject dark phase. Since the SARM typically has a short half-life, normal locomotor activity returned less than 24 hours later before the start of the next circadian cycle. When comparing the D:D conditions to L:D conditions, loss of locomotor activity was not observed in the experiment conducted under standard conditions. This result allowed the researchers to conclude that effects on locomotor activity were not due to toxicity [2].

When assessing the expression of core clock genes in the hypothalami of test subject mice, there was a range of results of reported following treatment with a SR-9009. In regards to the expression of PER/CRY genes, researchers observed an enhancement in Per2 expression while Cry2 was suppressed. Expression of the Bmal1 gene was subtly affected; the researchers observed a left shift in the phase of the circadian pattern. Additionally, the circadian pattern Npas2 gene expression was eliminated completely while the expression pattern of Clock experienced an enhancement in oscillation amplitude so that Clock oscillation was in phase with the oscillation of the Per2 expression pattern.

The effects elicited by both REV-ERB ligands in the mice housed in D:D conditions were compared to the subjects maintained under L:D conditions. Instead of complete loss of locomotor activity, the L:D test subjects experienced a 1-3 hour delay in the onset of nocturnal locomotor activity. The researchers also observed that similar to the subtle effects of the SARMS on circadian rhythm in L:D conditions, SR-9009 elicited less severe effects on core clock gene expression. When the core clock genes are genetically altered, a range of metabolic phenotypes are developed while evidence suggests that REV-ERB compounds are directly involved in the metabolism of lipids and glucose [2].

Examination of metabolic regulations promoted by REV-ERBagonists revealed that administration of SR-9009 to C57B16 mice resulted in weight loss and decreased fat mass without an increase in food intake. These results that were observed led the research team to examine how SR-9009 affects obesity in rodents caused by maintenance of a high fat diet for 14 weeks. The researchers thought it was important to note that the slight decrease in weight experienced by the control group was due to the stress of handling and twice-daily injections. However, when treated with SR-9009, the rate of weight loss increased by 60% with no significant changes in food intake when comparing the treatment and control groups.

Figure 6: Change in fat mass in control group versus SR-9011 treatment group (similar effects were reported in the SR-9009 treatment group)

Additionally, the mice treated with SR-9009 experienced a drastic reduction of adiposity, as well as a 12% decrease in plasma triglycerides and 47% decrease in plasma total cholesterol. Plasma glucose and plasma non-esterified fatty acids were also reduced by 19% and 23%, respectively, with a notable downward trend in plasma insulin levels. Treatment with SR-9009 led to decreased total cholesterol and plasma triglyceride. These observations coincided with a dramatic decrease in the expressions of Fasn and Scd1 genes that encode lipogenic enzymes as well as a decrease in cholesterologenic regulatory proteins, Hmgcr and Srebf2. After 12 days of SR-9009 administration in a genetic model of obesity, the SARM was able to suppress the degree of weight gain in a manner similar to leptin-deficient mice, without drastically changing tolerance levels to insulin and glucose [2].

When observing the effects of SR-9009 in white adipose tissue (WAT), the research team identified a decrease in gene expression encoding enzymes involved in the synthesis of triglycerides. SR-9009 also induced expression of the Cpt1b, Ucp3, Ppargc1b, Pkm2, and Hk1 genes, all of which are involved in fatty acid and glucose oxidation. These results suggest that the REV-ERB agonists increase energy expenditure by inducing fatty acid and glucose oxidation through gene expression. This data also correlated with a decrease in triglyceride synthesis in the liver and WAT, in addition to reduced synthesis of hepatic cholesterol [2].

Figure 7: changes in plasma triglyceride, leptin, cholesterol, body weight, and fat mass

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] 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. 2022 Aug;42(6):1765-1776. doi: 10.1007/s10571-021-01053-y. Epub 2021 Feb 18. PMID: 33599915.

[2] Solt LA, Wang Y, Banerjee S, Hughes T, Kojetin DJ, Lundasen T, Shin Y, Liu J, Cameron MD, Noel R, Yoo SH, Takahashi JS, Butler AA, Kamenecka TM, Burris TP. Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists. Nature. 2012 Mar 29;485(7396):62-8. doi: 10.1038/nature11030. PMID: 22460951; PMCID: PMC3343186.

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