Lovastatin alters neurotrophin expression in rat hippocampus-derived neural stem cells in vitro

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VOLUME 79 , ISSUE 4 (December 2019) > List of articles

Lovastatin alters neurotrophin expression in rat hippocampus-derived neural stem cells in vitro

Farzaneh Fakheri / Alireza Abdanipour * / Kazem Parivar / Iraj Jafari Anarkooli / Hossein Rastegar

Keywords : lovastatin, neural stem cells, neurotrophins

Citation Information : Acta Neurobiologiae Experimentalis. Volume 79, Issue 4, Pages 413-420, DOI: https://doi.org/10.21307/ane-2019-038

License : (CC-BY-4.0)

Received Date : 17-March-2019 / Accepted: 17-September-2019 / Published Online: 10-January-2020

ARTICLE

ABSTRACT

Neural stem/progenitor cells hold valuable potential for the treatment of neurodegenerative disorders. The modulation of intrinsic growth factor expression, such as neurotrophins and their receptors, is a necessary step in achieving neural stem cells (NSCs) therapy. The statins have recently been reported to provide both anti-inflammatory and neuroprotective effects. In the developing and mature nervous systems, neurotrophic factors are known to impact neuronal growth and survival. In this study, we investigated for a positive effect of lovastatin on the expression of neurotrophins in the neonatal rat hippocampus-derived NSCs. NSCs were isolated and cultured up to passage three. To confirm cellular identity, immunocytochemical evaluation and flow cytometry analysis were performed using specific antibodies. To determine the optimum concentration of lovastatin, the MTT assay was used. Neurotrophin expression was evaluated using quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR). Flow cytometry results demonstrated that NSCs were positive for nestin, a marker for neural progenitor cells. An increase in cellular viability was observed with a 24 h exposure of lovastatin. Moreover, results showed an increase in mRNA expression for all neurotrophins compared to the control group. Taken together, the results of this study add to the growing body of literature on the neuroprotective effects of statins in neurological disorders. Lovastatin is a promising therapeutic agent for the treatment of neurodegenerative disorders.

Graphical ABSTRACT

INTRODUCTION

Neurotrophins are comprised of a small family of dimeric proteins, which assist in differentiation and survival of peripheral and central neurons. They regulate neurogenesis, synaptic strength, and plasticity (Ivanisevic and Saragovi, 2013), as well as neurons survival, development, and function in the vertebrate nervous system (Vilar and Mira, 2016). Nerve growth factor (NGF), the first described member of the neurotrophin family, is best known for its trophic role in the prevention of programmed cell death in the neuron populations of the peripheral nervous system (Frade and Barde, 1998). In 1982, researchers reported that a second member of the family of neurotrophic factors, brain-derived neurotrophic factor (BDNF), exerted a protective effect on the survival of specific dorsal root ganglion neurons, purified from pig brain (Barde et al., 1982). Other members of the neurotrophin family, including ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), glial cell line–derived neurotrophic factor (GDNF), and neurotrophin-4 (NT-4), have also been described with distinct trophic effects on different neuronal populations of the peripheral and central nervous system. CNTF is broadly studied and known to promote the survival of all classes of neurons (Dubovy et al., 2011). Statins are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are used to treat dyslipidemia and reduce the risks associated with atherosclerosis. Statins have recently been considered as potential remedies for the treatment of various neurological disorders and increasing clinical studies are underway (Malfitano et al., 2014). According to recent studies, the use of biomolecules with a positive impact on neurotrophic factors may reduce neuronal damage in neurodegenerative disorders (Lin et al., 2015; Razavi et al., 2015). In this study, we evaluated the effects of lovastatin on the expression of neurotrophins in neural stem cells and found the optimal concentration to induce such effects.

METHODS

Isolation and expansion of NSCs

NSCs were isolated from the hippocampus of three neonatal Wistar rats (5–10 days old) purchased from the Razi Vaccine and Serum Research Institute (Karaj, Iran). Briefly, under deep anesthesia by intraperitoneal injection of ketamine/xylazine (100 mg/kg and 10 mg/kg, respectively), the brains were removed and the dissected hippocampi washed in the ice-cold phosphate-buffered saline (PBS, Gibco), supplemented with 4.5 g/L glucose. The collected tissues were homogenized with a scalpel and then dissociated using a digestion mixture of papain [2.5 U/ml] (Sigma-Aldrich; Germany), dispase II [40 U/ml] (Sigma-Aldrich; Germany) and accutase [1 ml] (Invitrogen; Thermo Fisher Scientific, USA) for 30 min at the room temperature. The cell mixture was passed through a 70 μm cell strainer (Falcon) and centrifuged for 10 min, at 1,000 g and 4°C. Then, the pellets were washed in phosphate-buffered saline. The cells were seeded in non-adherent T25 flasks in NSCs medium containing Dulbecco modified eagle medium/F-12 (DMEM/F12) supplemented with 2% B27 (Gibco; Thermo Fisher Scientific Inc.), 20 ng/ml basic fibroblast growth factor (bFGF; Invitrogen; Thermo Fisher Scientific Inc.), 20 ng/ml epidermal growth factor (EGF; Invitrogen; Thermo Fisher Scientific Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA), and then incubated at 37°C in 5% CO2 to form neurospheres for six days (Pall et al., 2017). The medium was supplemented every two days. In the next step, the cells were dissociated enzymatically using trypsin-EDTA (0.25%; Sigma-Aldrich; Merck KGaA) and mechanically by pipetting to single cells. NSCs (105 cells/well) were suspended in DMEM/F12 supplemented with 2% B27, 20 ng/ml bFGF, 20 ng/ml EGF and 3% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) for one week (at 37˚C in 5% CO2) in 6-well adherent plates coated with poly-l-lysine and cultured up to passage number three. For identification of these cells, immunocytochemical evaluation, and flow cytometry analysis were performed using anti-nestin monoclonal antibody (ab6142; 1:300; Abcam), followed by incubation with a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse antibody 1/300 (Millipore, Billerica, MA, USA, AP307F). Cells were cultured on cover slides and fixed in 3% paraformaldehyde for 20 minutes, followed by a permeabilization step in 100% methanol for 30 min at room temperature. Then cells were incubated with the primary antibody at 4°C overnight, and the next day secondary FITC-conjugated for four hours. Ethidium bromide was used for 30 secs for nuclei counterstaining at room temperature. Images were captured with an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan). All experimental protocols were approved by the Zanjan University of Medical Sciences Ethics Committee.

Lovastatin dose-response

The third-passage of NSCs was cultured in 96-well plates (5×104 cells/well) in DMEM/F12 medium supplemented with 2% B27, 20 ng/ml bFGF and 20 ng/ml EGF for 24 h. The cells were treated with different concentrations of lovastatin (C24H36O5 – PubChem) (2, 4, 6, 8 and 10 μM) for the next 24 h, which was repeated every 12 h. The NSCs without lovastatin treatment were used as the control group. Then, the cells were incubated with 1 mg/ml MTT (Sigma Aldrich, Germany) solution for four hours. The culture medium was removed and 100 μl dimethyl sulfoxide added to each well to dissolve the formazan crystals. The amount of formazan was quantified at 570 nm using a microplate ELISA reader. The relative cell viability in percentage (Han et al., 2009) was calculated as: Relative cell viability = (A570 of treated samples / A570 of untreated samples) × 100.

Cresyl violet staining

The ability of NSCs to differentiate into neuron-like cells was evaluated by Nissl staining (0.1% cresyl violet). Briefly, the medium was aspirated and the cells were gently washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the fixation solution was aspirated and the cell monolayer was gently washed with PBS twice. In the next step, PBS was removed from the cells and staining solution (0.5% cresyl violet) was added, followed by incubation at room temperature for 30 min. After washing with PBS, the cells were observed under light microscope (BX61; Olympus, Tokyo, Japan) and the blue/violet colored cells were considered positive.

Real-time RT-qPCR

RT-qPCR was carried out using extracted cDNA from control and treatment groups. Total RNA of hippocampal tissues was isolated by TRIzol® (Invitrogen/Life Technologies). We used 1,000 ng of purified RNA to synthesize 20 μl of cDNA according to a Revert Aid™ First Strand cDNA Synthesis Kit (Fermentas, Germany). The cDNA was then used to quantify mRNA levels of the neurotrophins Bdnf, Gdnf, Cntf, Ngf, NT-3 and NT-4. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as the internal control for normalization. RT-qPCR was conducted using primers shown in Table I. The PCR solution contained forward and reverse primers (200 nM each), cDNA (0.5 μl), SYBR® Green I (6.5 μl; Fermentas; Thermo Fisher Scientific, Inc.) and nuclease-free water up to the final volume of 12.5 μl. The PCR reaction was repeated for 40 cycles, each cycle including 15 s in 95˚C followed by 1 min in 60˚C. Relative changes in target mRNA levels were determined using the ΔΔCq method (Livak and Schmittgen, 2001; Mosley and HogenEsch, 2017). The product size of the PCR was later verified by 2% agarose gel electrophoresis. The experiments were repeated three times.

Table I.

Primer sequences and PCR parameters. Primers for amplifcation of target sequences and their Gen Bank accession number.

10.21307_ane-2019-038-tbl1.jpg

Statistics

Statistical analyses were performed using SPSS software version 15 (IBM; Armonk, New York, United States). All data are presented as the mean ± standard error of mean (SEM) from independent experiments that were repeated three times. One-way ANOVA and Tukey’s post hoc test were used for data comparisons between the groups. P values less than 0.05 were considered significant.

RESULTS

Neural stem cells culture

The results of the primary culture of NSCs are presented in Fig. 1A-C. The initial culture of NSCs isolated from the neonatal rat hippocampus appeared single and round with clear boundaries in the first 24 h (Fig. 1A). After 3 days, self-renewing neuron-like cells with multipolar processes and growth cone-like features were identified (Fig. 1B). After 3 passages (Fig. 1C) the cells were placed into uncoated 6-well plates to allow neurosphere formation. After 24 h, small spheroids were observed (Fig. 1D). There was a statistically significant difference between groups as determined by one-way ANOVA (F2,27=97.26, p<0.001). A Tukey’s post hoc test revealed that the average spheroid diameter significantly increased after 3 days (136.90 ± 9.94 μm, p<0.001) and 6 days (224.30 ± 11.61 μm, p<0.001) compared to day one (47.40 ± 2.70 μm). There was also a significant difference between day 3 and day 6 (p<0.001) as shown in figures 1D-F and L. Moreover, with higher magnification it was observed that the cells separated from primary spheroids can replicate and form new spheroids (Fig. 1F). The NSCs produced from spheroids were strongly positive for nestin (Fig. 1G and 1H) immunostaining. Flow cytometry confirmed that 77.50% NSCs were positive for nestin (Fig. 1N).

Fig. 1.

Representative photomicrograph of hippocampus-derived NSCs, neurosphere diameters and dose-response assay. Cell attachments of the freshly extracted NSCs (A), cells at passage 1 (day 7) (B), and passage 3 (day of 16) (C). (D), (E) and (F) represent floating neurospheres derived from neonate rat hippocampus (rosette like structures) after 1, 3 and 6 days, respectively. (G) represents immunostaining of Nestin (specific markers for neural stem/progenitor cells); the cells were immunostained with relevant primary antibodies and labeled with FITC-conjugated secondary antibody (green color shows positive cells) and the red colors are ethidium bromide counterstaining of the nuclei. (H) represents phase contrast micrographs at the same field. (L) Histogram of neurosphere diameters at different time points; *represents the significant difference with day 6 (P<0.001, post-hoc Tukey’s test); # represents the significance difference with day 3 (P<0.001, post-hoc Tukey’s test). (M) MTT assay graphs represent dose-response NSC viability at different concentrations of lovastatin (results show the mean % viability relative to 0 μM treated NSCs); *represents the significant difference with the 10 μM experimental group (p<0.002, post-hoc Tukey’s test). (N) represents detection of nestin using flow cytometry analysis. Scale bars 200 μm at × 400 magnification. Each bar represents the average measurement from 5 replicates. The bars indicate the mean ± SEM.

10.21307_ane-2019-038-f001.jpg

MTT assay

The MTT assay was used to determine the highest tolerated dose of lovastatin for hippocampal derived NSCs. As shown in figure 1M, a gradual increase in concentrations of lovastatin from 6 to 10 μM subsequently decreased cell numbers in the cultures. The highest rate of cell proliferation was observed with 6 μM of lovastatin and a 24 h exposure (81.59% ± 2.44). There was a statistically significant difference between groups as determined by one-way ANOVA (F4,35=4.38, p<0.006). A Tukey’s post hoc test revealed there is significant difference between optimum (6 μM) and 10 μM concentration of lovastatin (63.89% ± 3.09, p<0.002). Lovastatin affected cell viability in a dose-dependent manner, thus we chose 6 μM lovastatin as the optimum dose for further studies to avoid neurotoxic effects of higher concentrations (10 μM). In the time course of the experiment, incubation of NSCs with 6 μM lovastatin for 24 h resulted in the retraction of the cell bodies and processes (Fig. 2A and 2B). Also, the treated cells were positive for Nissl bodies determined by cresyl violet dye (34.66% ± 5.61) (Fig. 2D). As shown in Fig. 2C, no cells were positive for Nissl staining in the control group.

Fig. 2.

Phase contrast images of the neuron-like cells differentiation of NSCs using lovastatin. (A) and (B) indicate 0 μM and 6 μM lovastatin-treated NSCs for 12 hours of incubation, respectively. Retraction of cell body and process formation are evident in the 6 μM lovastatin-treated cells. (C) and (D) indicate the Nissl body staining using cresyl violet. (C) negative control (0 μM lovastatin-treated NSCs), (D) positive control (6 μM lovastatin-treated NSCs). Dark blue particles in the cytoplasm show Nissl bodies and the red and black arrows show positive and negative cells, respectively. Scale bars 200 μm at × 400 magnification.

10.21307_ane-2019-038-f002.jpg

Gene expression

The changes in the mRNA levels of Bdnf, Gdnf, Cntf, Ngf, NT-3 and NT-4 were examined using RT-qPCR. The results of our study showed that lovastatin increased the expression of neurotrophins relative to the control group with a statistically significant difference between mRNA level-based gene expressions as determined by one-way ANOVA (F5,30=22.36, p<0.001) (Fig. 3A). A Tukey’s post hoc test revealed that mRNA expression levels for GDNF significantly increased after lovastatin treatment (128.85 ± 16.71, p<0.001) compared to other genes. The mean fold-changes for Bdnf, Gdnf, Cntf, Ngf, NT-3 and NT-4 genes were: 34.80 ± 3.23, 128.85 ± 16.71, 30.14 ± 3.55, 52.21 ± 6.74, 26.05 ± 3.56 and 42.29 ± 6.31, respectively. The induced NSCs expressed all assessed neurotrophins as detected by gel electrophoresis (Fig. 3B).

Fig. 3.

(A) Quantitative real-time RT-PCR results. All mRNA expression is presented as lovastatin-treated NSCs relative to the control group (non-treated NSCs) normalized to Gapdh mRNA amplification. (B) Expression of neurotrophic genes. L, Ct, T and NTC indicate the ladder (100 bp), NSCs treated with 0 μM lovastatin, NSCs treated with 6 μM lovastatin and negative control (without cDNA), respectively. This is a pairwise comparison and the bars indicate the mean ± SEM. *represents the significant difference versus Gdnf genes (p<0.001, post-hoc Tukey’s test).

10.21307_ane-2019-038-f003.jpg

DISCUSSION

The results of the present study showed that lovastatin induced expression of neurotrophic factors in the treated NSCs as compared to the control group. To our knowledge, this is the first study to demonstrate the increased release of neurotrophic factors from neural stem cells following lovastatin treatment in-vitro. Neurotrophic factors have been broadly investigated for their roles in supporting survival, proliferation and maturation of neural populations, leading to improved neural regeneration in neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases (Lin et al., 2015; Xiao and Le, 2016). The effects of neurotrophins are not limited to neurogenesis and axonal sprouting. It has also been shown that neurotrophins have distinct regulatory effects at various excitatory/inhibitory synapses and for survival of cells in the central nervous system (Quiroz et al., 2010).

Lovastatin, an HMG-CoA reductase inhibitor, has various pharmacological actions, including lowering cholesterol and reducing inflammation, as well as anticancer, antioxidant and, in particular, neuroprotective effects (Nasiri et al., 2016; Yan et al., 2015). This drug has shown neuroprotective activity and easily permeates the blood-brain barrier because of its lipophilicity (Lin et al., 2015). Statins alter the fate of neural stem-progenitor cells (NSPCs) in different ways and during differentiation may lead to the expression of mRNAs through a non-CBP pathway (Carson et al., 2018). Statins also have positive effects on NSPCs by increasing neurogenesis through the Wnt/beta-catenin signaling pathway (Robin et al., 2014). As demonstrated in animals, statins can produce significant toxicity at high doses (Hajar, 2011). In the present study, we demonstrated that increased concentrations of lovastatin lead to decreased cell viability. Other studies have reported that statins affect the G1 phase and alter neural precursor cell divisions in a dose-dependent manner (Carson et al., 2018). Statins are presumed to exert their neuroprotective effects by promoting the release of neurotrophic factors and inducing neurotrophic factor gene expression, such as the BDNF (Roy et al., 2015).

The results of this study simply and clearly add to the understanding of the mechanisms underlying the therapeutic potential of statins in neurological disorders found in previous studies (Rajanikant et al., 2007; Malfitano et al., 2014). Our results showed that the expression of BDNF in the treated group increased by approximately 34 fold in comparison with the control group. Most studies have investigated the effect of statins on Bdnf expression under in vivo condition. Roy et al. (2015) demonstrated that different statins up-regulate Bdnf and NT-3 in neurons, microglia and astrocytes. It has also been reported that treatment of rat primary microglial cultures with 20 μM simvastatin increases Bdnf expression (Churchward and Todd, 2014). BDNF supports the survival of existing neurons and encourages the growth and differentiation of new neurons and synapses through axonal and dendritic sprouting (Kim et al., 2017). Chen et al. (2015) reported that neural plasticity was promoted by statin therapy through upregulation of Bdnf after stroke in mice. BDNF regulates neuronal survival, cell migration, and synaptic function. Thus, statins, along with increased Bdnf expression, can play an important role in improving and restoring brain tissue after stroke (Chen et al., 2005). Han et al. (2011) demonstrated the role of simvastatin on functional improvement after spinal cord injury in rat by upregulating the expression of Bdnf and Gdnf. It has been suggested that BDNF promotes the survival of subventricular zone neurons and differentiation of postnatal hippocampal stem cells. In our study, the results showed that lovastatin increased Bdnf mRNA levels relative to non-treated NSCs. An increase of Gdnf mRNA expression was seen in our study. It was reported that simvastatin significantly increased the expression of Bdnf and Gdnf in the rat model of spinal cord injury (Gao et al., 2015). GDNF plays an important role in the survival of neurons, in enhancing the remyelination of damaged axons and causes neuronal regeneration after spinal cord injury (SCI) (Razavi et al., 2017).

Several studies have shown therapeutic effects of GDNF in various diseases of the central nervous system, such as Parkinson’s disease, stroke and epilepsy (Koppula et al., 2012; Zilliox et al., 2016). In rat models of SCI, the use of statins was associated with improved locomotor activity through up-regulation of Gdnf, Bdnf and Vegf expression (Kahveci et al., 2014). In our study, treatment with lovastatin lead to a significant increase in Gdnf mRNA expression. Simvastatin has also recently been demonstrated to improve peripheral nerve regeneration and functional recovery in an experimental model of sciatic damage that involves elevation of levels of Gdnf and several other growth factors (Guo et al., 2018). Quantitative PCR results from the present study indicate that lovastatin also increased Cntf expression. CNTF is expressed mainly in glial cells as well as in neurons, and its cytoprotective effect appears to be exerted after stress or damage. CNTF has recently been used to treat patients with neurodegenerative diseases such as ALS (Purser et al., 2013). In this study, we found that treating NSCs with lovastatin led to an increase in Ngf mRNA expression. It has been reported that statins such as simvastatin and atorvastatin enhance expression of growth factors (BDNF, VEGF and NGF) and activate the PI3K/Akt-mediated signaling pathway after experimental intracerebral hemorrhage (Yang et al., 2012). NGF is critical for the survival and maintenance of sympathetic and sensory neurons. We observed that lovastatin led to an increase in the expression of NT-3 and NT-4, especially NT-4. Statins have been reported to dose-dependently up-regulate Bdnf and NT-3 mRNA expression in mouse primary astrocytes (Roy et al., 2015). Statins were also shown to enhance expression of growth factors (BDNF, VEGF, and NGF) and activate the PI3K/Akt-mediated signaling pathway after experimental intracerebral hemorrhage (Yang et al., 2012). NT-3 belongs to the NGF family; it plays an essential role in cell survival, axonal growth and neuronal plasticity (Xu et al., 2002). It helps to support the survival and differentiation of existing neurons and encourages the growth and differentiation of new neurons and synapses. In-vitro studies demonstrated that exogenous NT-3 increased proliferation of neural crest and somite-derived NSCs (Levenberg et al., 2005). Finally, there are limited studies on the role of statins in the alteration of CNTF, NT-3, and NT-4 expression, making it hard to draw conclusions from our results. The most recently discovered member of this family is NT-4, which plays a role in the survival and differentiation of vertebrate neurons (Proenca et al., 2016).

The mechanisms by which neurotrophins and growth factors determine cell fate is still not fully understood. In this study, the flow cytometry analyses showed 77.50% of cells expressing nestin. This result may indicate contamination of primary cells isolated from brain tissue (the possible presence of mature astrocytes and microglia). Astrocytes were cultured under adherent culture conditions and mature astrocytes do not show nestin expression (Cho et al., 2013). The downregulation of nestin expression in astrocytes parallels the increase of glial fibrillary acidic protein (GFAP) in differentiating astrocytes (Cho et al., 2013). One of the limitations of this study is that the cells were not purified but brain cells can release neurotrophins and are sensitive to statin treatment both in vitro and in vivo (McFarland et al., 2014). Statins have been reported to upregulate Bdnf and NT-3 in neurons, microglia and astrocytes (Roy et al., 2015). Additionally, due to scientific sanctions against Iran and limited financial resources to conduct this investigation, we did not perform post-transcriptional analysis of the gene products using ELISA with antibodies. As a result of the sanctions, antibodies are difficult to obtain and very expensive.

Encouragingly, statins have been broadly identified as possible preventative or treatment options in a number of neurological disorders, such as stroke, epilepsy, depression, cancers and brain and spinal cord injuries. Results from preclinical animal models suggest that statins may be neuroprotective in response to acute brain injury and chronic neurodegenerative disorders (Roy et al., 2015). Our results, in summary, importantly add to the growing body of evidence on the neuroprotective effects of statins for neurological disorders and support that lovastatin is a promising therapeutic agent for the treatment of neurodegenerative disorders. Future investigations are necessary to validate the conclusions drawn from this study examining the underlying cellular and molecular mechanisms of neurotrophin regulation by statins.

CONTRIBUTOR’S STATEMENTS

Farzaneh Fakheri: Executed the research project; Alireza Abdanipour: Designed the study, analyzed the data, wrote the manuscript and supervised experiments; Kazem Parivar: supervised experiments; Iraj Jafari Anarkooli: advisor; Hossein Rastegar: advisor.

ACKNOWLEDGMENTS

This study was partly funded by Zanjan University of Medical Sciences, Zanjan, Iran (Grant number: A-12-82-14).

References


  1. Barde YA, Edgar D, Thoenen H (1982) Purification of a new neurotrophic factor from mammalian brain. EMBO J 1: 549–553.
    [PUBMED] [CROSSREF]
  2. Carson RA, Rudine AC, Tally SJ, Franks AL, Frahm KA, Waldman JK, Silswal N, Burale S, Phan JV, Chandran UR, Monaghan AP, DeFranco DB (2018) Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms. PLoS One 13: e0196387.
    [PUBMED] [CROSSREF]
  3. Chen J, Zhang C, Jiang H, Li Y, Zhang L, Robin A, Katakowski M, Lu M, Chopp M (2005) Atorvastatin induction of VEGF and BDNF promotes brain plasticity after stroke in mice. J Cereb Blood Flow Metab 25: 281–290.
    [CROSSREF]
  4. Cho JM, Shin YJ, Park JM, Kim J, Lee MY (2013) Characterization of nestin expression in astrocytes in the rat hippocampal CA1 region following transient forebrain ischemia. Anat Cell Biol 46: 131–140.
    [PUBMED] [CROSSREF]
  5. Churchward MA, Todd KG (2014) Statin treatment affects cytokine release and phagocytic activity in primary cultured microglia through two separable mechanisms. Mol Brain 7: 85.
    [PUBMED] [CROSSREF]
  6. Dubovy P, Raska O, Klusakova I, Stejskal L, Celakovsky P, Haninec P (2011) Ciliary neurotrophic factor promotes motor reinnervation of the musculocutaneous nerve in an experimental model of end-to-side neurorrhaphy. BMC Neurosci 12: 58.
    [PUBMED] [CROSSREF]
  7. Frade JM, Barde YA (1998) Nerve growth factor: two receptors, multiple functions. Bioessays 20: 137–145.
    [PUBMED] [CROSSREF]
  8. Gao K, Wang G, Wang Y, Han D, Bi J, Yuan Y, Yao T, Wan Z, Li H, Mei X (2015) Neuroprotective effect of simvastatin via inducing the autophagy on spinal cord injury in the rat model. Biomed Res Int 2015: 260161.
  9. Guo Q, Liu C, Hai B, Ma T, Zhang W, Tan J, Fu X, Wang H, Xu Y, Song C (2018) Chitosan conduits filled with simvastatin/Pluronic F-127 hydrogel promote peripheral nerve regeneration in rats. J Biomed Mater Res B Appl Biomater, 106: 787–799.
    [PUBMED] [CROSSREF]
  10. Hajar R (2011) Statins: past and present. Heart Views 12: 121–127.
    [PUBMED] [CROSSREF]
  11. Han J, Talorete TP, Yamada P, Isoda H (2009) Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells. Cytotechnology 59: 45–53.
    [CROSSREF]
  12. Han X, Yang N, Xu Y, Zhu J, Chen Z, Liu Z, Dang G, Song C (2011) Simvastatin treatment improves functional recovery after experimental spinal cord injury by upregulating the expression of BDNF and GDNF. Neurosci Lett 487: 255–259.
    [CROSSREF]
  13. Ivanisevic L, Saragovi Uri H (2013) Neurotrophins. In: Handbook of Biologically Active Peptides (Abba J. Kastin, Ed.). (2nd Edition) Boston Academic Press, p. 1639–1646.
  14. Kahveci R, Gokce EC, Gurer B, Gokce A, Kisa U, Cemil DB, Sargon MF, Kahveci FO, Aksoy N, Erdoğan B (2014) Neuroprotective effects of rosuvastatin against traumatic spinal cord injury in rats. Eur J Pharmacol 741: 45–54.
    [PUBMED] [CROSSREF]
  15. Kim J, Lee S, Choi BR., Yang H, Hwang Y, Park JH, LaFerla FM, Han JS, Lee KW, Kim J (2017) Sulforaphane epigenetically enhances neuronal BDNF expression and TrkB signaling pathways. Mol Nutr Food Res 61 doi: 10.1002/mnfr.
  16. Koppula, S, Kumar H, More SV, Kim BW, Kim IS, Choi DK (2012) Recent advances on the neuroprotective potential of antioxidants in experimental models of Parkinson’s disease. Int J Mol Sci 13: 10608–10629.
    [PUBMED] [CROSSREF]
  17. Levenberg S, Burdick JA, Kraehenbuehl T, Langer R (2005) Neurotrophin-induced differentiation of human embryonic stem cells on three-dimensional polymeric scaffolds. Tissue Eng 11: 506–512.
    [PUBMED] [CROSSREF]
  18. Lin CM, Lin YT, Lin RD, Huang WJ, Lee MH (2015) Neurocytoprotective effects of aliphatic hydroxamates from lovastatin, a secondary metabolite from monascus-fermented red mold rice, in 6-hydroxydopamine (6-OHDA)-treated nerve growth factor (NGF)-differentiated PC12 Cells. ACS Chem Neurosci 6: 716–724.
    [PUBMED] [CROSSREF]
  19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402–408.
    [PUBMED] [CROSSREF]
  20. Malfitano AM, Marasco G, Proto MC, Laezza C, Gazzerro P, Bifulco M (2014) Statins in neurological disorders: an overview and update. Pharmacol Res 88: 74–83.
    [CROSSREF]
  21. McFarland AJ, Anoopkumar-Dukie S, Arora DS, Grant GD, McDermott CM, Perkins AV, Davey AK (2014) Molecular mechanisms underlying the effects of statins in the central nervous system. Int J Mol Sci 15: 20607–20637.
    [CROSSREF]
  22. Mosley YC, HogenEsch H (2017) Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination. BMC Res Notes 10: 689.
    [CROSSREF]
  23. Nasiri M, Etebari M, Jafarian-Dehkordi A, Moradi S (2016) Lovastatin prevents bleomycin-induced DNA damage to HepG2 cells. Res Pharm Sci 11: 470–475.
    [CROSSREF]
  24. Pall O, Varga B, Collart-Dutilleul PY, Gergely C, Cuisinier FJG (2017) Re: “An overview of protocols for the neural induction of dental and oral stem cells in vitro” by Heng et al. (Tissue Eng Part B 2016;22: 220–250). Tissue Eng Part B Rev 23: 570–576.
    [CROSSREF]
  25. Proenca CC, Song M, Lee FS (2016) Differential effects of BDNF and neurotrophin 4 (NT4) on endocytic sorting of TrkB receptors. J Neurochem 138: 397–406.
    [CROSSREF]
  26. Purser MJ, Dalvi PS, Wang ZC, Belsham DD (2013) The cytokine ciliary neurotrophic factor (CNTF) activates hypothalamic urocortin-expressing neurons both in vitro and in vivo. PLoS One 8: e61616.
    [CROSSREF]
  27. Quiroz JA, Machado-Vieira R, Zarate CA Jr, Manji HK (2010) Novel insights into lithium’s mechanism of action: neurotrophic and neuroprotective effects. Neuropsychobiology 62: 50–60.
    [PUBMED] [CROSSREF]
  28. Rajanikant GK, Zemke D, Kassab M, Majid A (2007) The therapeutic potential of statins in neurological disorders. Curr Med Chem 14: 103–112.
    [PUBMED] [CROSSREF]
  29. Razavi S, Ghasemi N, Mardani M, Salehi H (2017) Remyelination improvement after neurotrophic factors secreting cells transplantation in rat spinal cord injury. Iran J Basic Med Sci 20: 392–398.
  30. Razavi S, Nazem G, Mardani M, Esfandiari E, Salehi H, Esfahani SH (2015) Neurotrophic factors and their effects in the treatment of multiple sclerosis. Adv Biomed Res: 4: 53.
    [PUBMED] [CROSSREF]
  31. Robin NC, Agoston Z, Biechele TL, James RG, Berndt JD, Moon RT (2014) Simvastatin promotes adult hippocampal neurogenesis by enhancing Wnt/beta-catenin signaling. Stem Cell Reports 2: 9–17.
    [PUBMED] [CROSSREF]
  32. Roy A, Jana M, Kundu M, Corbett GT, Rangaswamy SB, Mishra RK, Luan CH, Gonzalez FJ, Pahan K (2015) HMG-CoA reductase inhibitors bind to pparalpha to upregulate neurotrophin expression in the brain and improve memory in mice. Cell Metab 22: 253–265.
    [PUBMED] [CROSSREF]
  33. Vilar M, Mira H (2016) Regulation of neurogenesis by neurotrophins during adulthood: expected and unexpected roles. Front Neurosci 10: 26.
    [PUBMED] [CROSSREF]
  34. Xiao N, Quynh-Thu L (2016) Neurotrophic factors and their potential applications in tissue regeneration. Arch Immunol Ther Exp 64: 89–99.
    [CROSSREF]
  35. Xu B, Michalski B, Racine RJ, Fahnestock M (2002) Continuous infusion of neurotrophin-3 triggers sprouting, decreases the levels of TrkA and TrkC, and inhibits epileptogenesis and activity-dependent axonal growth in adult rats. Neuroscience 115: 1295–1308.
    [PUBMED] [CROSSREF]
  36. Yan JQ, Sun JC, Zhai MM, Cheng LN, Bai XL, Feng CL (2015) Lovastatin induces neuroprotection by inhibiting inflammatory cytokines in 6-hydroxydopamine treated microglia cells. Int J Clin Exp Med 8: 9030–9037.
    [PUBMED]
  37. Yang D, Han Y, Zhang J, Chopp M, Seyfried DM (2012) Statins enhance expression of growth factors and activate the PI3K/Akt-mediated signaling pathway after experimental intracerebral hemorrhage. World J Neurosci 2: 74–80.
    [PUBMED] [CROSSREF]
  38. Zilliox LA, Chadrasekaran K, Kwan JY, Russell JW (2016) Diabetes and cognitive impairment. Curr Diab Rep 16: 87.
    [PUBMED] [CROSSREF]
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FIGURES & TABLES

Fig. 1.

Representative photomicrograph of hippocampus-derived NSCs, neurosphere diameters and dose-response assay. Cell attachments of the freshly extracted NSCs (A), cells at passage 1 (day 7) (B), and passage 3 (day of 16) (C). (D), (E) and (F) represent floating neurospheres derived from neonate rat hippocampus (rosette like structures) after 1, 3 and 6 days, respectively. (G) represents immunostaining of Nestin (specific markers for neural stem/progenitor cells); the cells were immunostained with relevant primary antibodies and labeled with FITC-conjugated secondary antibody (green color shows positive cells) and the red colors are ethidium bromide counterstaining of the nuclei. (H) represents phase contrast micrographs at the same field. (L) Histogram of neurosphere diameters at different time points; *represents the significant difference with day 6 (P<0.001, post-hoc Tukey’s test); # represents the significance difference with day 3 (P<0.001, post-hoc Tukey’s test). (M) MTT assay graphs represent dose-response NSC viability at different concentrations of lovastatin (results show the mean % viability relative to 0 μM treated NSCs); *represents the significant difference with the 10 μM experimental group (p<0.002, post-hoc Tukey’s test). (N) represents detection of nestin using flow cytometry analysis. Scale bars 200 μm at × 400 magnification. Each bar represents the average measurement from 5 replicates. The bars indicate the mean ± SEM.

Full Size   |   Slide (.pptx)

Fig. 2.

Phase contrast images of the neuron-like cells differentiation of NSCs using lovastatin. (A) and (B) indicate 0 μM and 6 μM lovastatin-treated NSCs for 12 hours of incubation, respectively. Retraction of cell body and process formation are evident in the 6 μM lovastatin-treated cells. (C) and (D) indicate the Nissl body staining using cresyl violet. (C) negative control (0 μM lovastatin-treated NSCs), (D) positive control (6 μM lovastatin-treated NSCs). Dark blue particles in the cytoplasm show Nissl bodies and the red and black arrows show positive and negative cells, respectively. Scale bars 200 μm at × 400 magnification.

Full Size   |   Slide (.pptx)

Fig. 3.

(A) Quantitative real-time RT-PCR results. All mRNA expression is presented as lovastatin-treated NSCs relative to the control group (non-treated NSCs) normalized to Gapdh mRNA amplification. (B) Expression of neurotrophic genes. L, Ct, T and NTC indicate the ladder (100 bp), NSCs treated with 0 μM lovastatin, NSCs treated with 6 μM lovastatin and negative control (without cDNA), respectively. This is a pairwise comparison and the bars indicate the mean ± SEM. *represents the significant difference versus Gdnf genes (p<0.001, post-hoc Tukey’s test).

Full Size   |   Slide (.pptx)

REFERENCES

  1. Barde YA, Edgar D, Thoenen H (1982) Purification of a new neurotrophic factor from mammalian brain. EMBO J 1: 549–553.
    [PUBMED] [CROSSREF]
  2. Carson RA, Rudine AC, Tally SJ, Franks AL, Frahm KA, Waldman JK, Silswal N, Burale S, Phan JV, Chandran UR, Monaghan AP, DeFranco DB (2018) Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms. PLoS One 13: e0196387.
    [PUBMED] [CROSSREF]
  3. Chen J, Zhang C, Jiang H, Li Y, Zhang L, Robin A, Katakowski M, Lu M, Chopp M (2005) Atorvastatin induction of VEGF and BDNF promotes brain plasticity after stroke in mice. J Cereb Blood Flow Metab 25: 281–290.
    [CROSSREF]
  4. Cho JM, Shin YJ, Park JM, Kim J, Lee MY (2013) Characterization of nestin expression in astrocytes in the rat hippocampal CA1 region following transient forebrain ischemia. Anat Cell Biol 46: 131–140.
    [PUBMED] [CROSSREF]
  5. Churchward MA, Todd KG (2014) Statin treatment affects cytokine release and phagocytic activity in primary cultured microglia through two separable mechanisms. Mol Brain 7: 85.
    [PUBMED] [CROSSREF]
  6. Dubovy P, Raska O, Klusakova I, Stejskal L, Celakovsky P, Haninec P (2011) Ciliary neurotrophic factor promotes motor reinnervation of the musculocutaneous nerve in an experimental model of end-to-side neurorrhaphy. BMC Neurosci 12: 58.
    [PUBMED] [CROSSREF]
  7. Frade JM, Barde YA (1998) Nerve growth factor: two receptors, multiple functions. Bioessays 20: 137–145.
    [PUBMED] [CROSSREF]
  8. Gao K, Wang G, Wang Y, Han D, Bi J, Yuan Y, Yao T, Wan Z, Li H, Mei X (2015) Neuroprotective effect of simvastatin via inducing the autophagy on spinal cord injury in the rat model. Biomed Res Int 2015: 260161.
  9. Guo Q, Liu C, Hai B, Ma T, Zhang W, Tan J, Fu X, Wang H, Xu Y, Song C (2018) Chitosan conduits filled with simvastatin/Pluronic F-127 hydrogel promote peripheral nerve regeneration in rats. J Biomed Mater Res B Appl Biomater, 106: 787–799.
    [PUBMED] [CROSSREF]
  10. Hajar R (2011) Statins: past and present. Heart Views 12: 121–127.
    [PUBMED] [CROSSREF]
  11. Han J, Talorete TP, Yamada P, Isoda H (2009) Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells. Cytotechnology 59: 45–53.
    [CROSSREF]
  12. Han X, Yang N, Xu Y, Zhu J, Chen Z, Liu Z, Dang G, Song C (2011) Simvastatin treatment improves functional recovery after experimental spinal cord injury by upregulating the expression of BDNF and GDNF. Neurosci Lett 487: 255–259.
    [CROSSREF]
  13. Ivanisevic L, Saragovi Uri H (2013) Neurotrophins. In: Handbook of Biologically Active Peptides (Abba J. Kastin, Ed.). (2nd Edition) Boston Academic Press, p. 1639–1646.
  14. Kahveci R, Gokce EC, Gurer B, Gokce A, Kisa U, Cemil DB, Sargon MF, Kahveci FO, Aksoy N, Erdoğan B (2014) Neuroprotective effects of rosuvastatin against traumatic spinal cord injury in rats. Eur J Pharmacol 741: 45–54.
    [PUBMED] [CROSSREF]
  15. Kim J, Lee S, Choi BR., Yang H, Hwang Y, Park JH, LaFerla FM, Han JS, Lee KW, Kim J (2017) Sulforaphane epigenetically enhances neuronal BDNF expression and TrkB signaling pathways. Mol Nutr Food Res 61 doi: 10.1002/mnfr.
  16. Koppula, S, Kumar H, More SV, Kim BW, Kim IS, Choi DK (2012) Recent advances on the neuroprotective potential of antioxidants in experimental models of Parkinson’s disease. Int J Mol Sci 13: 10608–10629.
    [PUBMED] [CROSSREF]
  17. Levenberg S, Burdick JA, Kraehenbuehl T, Langer R (2005) Neurotrophin-induced differentiation of human embryonic stem cells on three-dimensional polymeric scaffolds. Tissue Eng 11: 506–512.
    [PUBMED] [CROSSREF]
  18. Lin CM, Lin YT, Lin RD, Huang WJ, Lee MH (2015) Neurocytoprotective effects of aliphatic hydroxamates from lovastatin, a secondary metabolite from monascus-fermented red mold rice, in 6-hydroxydopamine (6-OHDA)-treated nerve growth factor (NGF)-differentiated PC12 Cells. ACS Chem Neurosci 6: 716–724.
    [PUBMED] [CROSSREF]
  19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402–408.
    [PUBMED] [CROSSREF]
  20. Malfitano AM, Marasco G, Proto MC, Laezza C, Gazzerro P, Bifulco M (2014) Statins in neurological disorders: an overview and update. Pharmacol Res 88: 74–83.
    [CROSSREF]
  21. McFarland AJ, Anoopkumar-Dukie S, Arora DS, Grant GD, McDermott CM, Perkins AV, Davey AK (2014) Molecular mechanisms underlying the effects of statins in the central nervous system. Int J Mol Sci 15: 20607–20637.
    [CROSSREF]
  22. Mosley YC, HogenEsch H (2017) Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination. BMC Res Notes 10: 689.
    [CROSSREF]
  23. Nasiri M, Etebari M, Jafarian-Dehkordi A, Moradi S (2016) Lovastatin prevents bleomycin-induced DNA damage to HepG2 cells. Res Pharm Sci 11: 470–475.
    [CROSSREF]
  24. Pall O, Varga B, Collart-Dutilleul PY, Gergely C, Cuisinier FJG (2017) Re: “An overview of protocols for the neural induction of dental and oral stem cells in vitro” by Heng et al. (Tissue Eng Part B 2016;22: 220–250). Tissue Eng Part B Rev 23: 570–576.
    [CROSSREF]
  25. Proenca CC, Song M, Lee FS (2016) Differential effects of BDNF and neurotrophin 4 (NT4) on endocytic sorting of TrkB receptors. J Neurochem 138: 397–406.
    [CROSSREF]
  26. Purser MJ, Dalvi PS, Wang ZC, Belsham DD (2013) The cytokine ciliary neurotrophic factor (CNTF) activates hypothalamic urocortin-expressing neurons both in vitro and in vivo. PLoS One 8: e61616.
    [CROSSREF]
  27. Quiroz JA, Machado-Vieira R, Zarate CA Jr, Manji HK (2010) Novel insights into lithium’s mechanism of action: neurotrophic and neuroprotective effects. Neuropsychobiology 62: 50–60.
    [PUBMED] [CROSSREF]
  28. Rajanikant GK, Zemke D, Kassab M, Majid A (2007) The therapeutic potential of statins in neurological disorders. Curr Med Chem 14: 103–112.
    [PUBMED] [CROSSREF]
  29. Razavi S, Ghasemi N, Mardani M, Salehi H (2017) Remyelination improvement after neurotrophic factors secreting cells transplantation in rat spinal cord injury. Iran J Basic Med Sci 20: 392–398.
  30. Razavi S, Nazem G, Mardani M, Esfandiari E, Salehi H, Esfahani SH (2015) Neurotrophic factors and their effects in the treatment of multiple sclerosis. Adv Biomed Res: 4: 53.
    [PUBMED] [CROSSREF]
  31. Robin NC, Agoston Z, Biechele TL, James RG, Berndt JD, Moon RT (2014) Simvastatin promotes adult hippocampal neurogenesis by enhancing Wnt/beta-catenin signaling. Stem Cell Reports 2: 9–17.
    [PUBMED] [CROSSREF]
  32. Roy A, Jana M, Kundu M, Corbett GT, Rangaswamy SB, Mishra RK, Luan CH, Gonzalez FJ, Pahan K (2015) HMG-CoA reductase inhibitors bind to pparalpha to upregulate neurotrophin expression in the brain and improve memory in mice. Cell Metab 22: 253–265.
    [PUBMED] [CROSSREF]
  33. Vilar M, Mira H (2016) Regulation of neurogenesis by neurotrophins during adulthood: expected and unexpected roles. Front Neurosci 10: 26.
    [PUBMED] [CROSSREF]
  34. Xiao N, Quynh-Thu L (2016) Neurotrophic factors and their potential applications in tissue regeneration. Arch Immunol Ther Exp 64: 89–99.
    [CROSSREF]
  35. Xu B, Michalski B, Racine RJ, Fahnestock M (2002) Continuous infusion of neurotrophin-3 triggers sprouting, decreases the levels of TrkA and TrkC, and inhibits epileptogenesis and activity-dependent axonal growth in adult rats. Neuroscience 115: 1295–1308.
    [PUBMED] [CROSSREF]
  36. Yan JQ, Sun JC, Zhai MM, Cheng LN, Bai XL, Feng CL (2015) Lovastatin induces neuroprotection by inhibiting inflammatory cytokines in 6-hydroxydopamine treated microglia cells. Int J Clin Exp Med 8: 9030–9037.
    [PUBMED]
  37. Yang D, Han Y, Zhang J, Chopp M, Seyfried DM (2012) Statins enhance expression of growth factors and activate the PI3K/Akt-mediated signaling pathway after experimental intracerebral hemorrhage. World J Neurosci 2: 74–80.
    [PUBMED] [CROSSREF]
  38. Zilliox LA, Chadrasekaran K, Kwan JY, Russell JW (2016) Diabetes and cognitive impairment. Curr Diab Rep 16: 87.
    [PUBMED] [CROSSREF]

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