Retinoic acid triazole promotes neurotrophin-mediated cell growth and proliferation in nerve cells

In the present study, fluorophenyl retinoic acid triazole, a retinoic acid derivative having more bioavailability compared to the parent retinoic acid was investigated against cell growth and proliferation. The results indicated that fluorophenyl retinoic acid triazole leads to a significant expression of TRK mRNA in time a dependent manner in NB1643 cells. There were about 10-, 14-, and 21-fold induction of expression after 1, 2, and 3 days of treatment, respectively. The results revealed that TRK and TRKB were induced at 6 and 12 hours afterthe treatment respectively and the expression of TRKB was maximum at day 3. Fluorophenyl retinoic acid triazole exerts neurite outgrowth activity by inducing TRKB expression, which is a receptor for brain -derived neurotrophic factor and a protein-tyrosine kinase. Fluorophenyl retinoic acid triazole accumulation generates many small vesiclesin the cell which assembles at the neck of each small prominence and lead to significant morphological changes after 24 hours of treatment.


Introduction
Binding of neurotrophins to membrane receptors of neurons initiates a cascade of intracellular processes which enhance survival and trophic effects. Neurotrophins are specific in binding to the receptors of tyrosine kinase receptor family (nerve growth factor binds to TrkA, brain-derived neurotrophic factor, brain-derived neurotrophic factor and neurotrophin-4, NT-4 bind to TrkB andneurotrophin-3, NT-3 binds to TrkC receptors with high affinity).
Down-regulation of brain-derived neurotrophic factor in dentate gyrus and hippocampus of chronic stress model of rats indicated potential involvement of it in depression (Smith et al., 1995). Latter brain-derived neurotrophic factor was found to be main agent in the depression and suicidal behaviours (Dwivedi, 2009;Lee and Kim, 2010). The postmortem brain samples from depressed humans (Tripp et al., 2012) and lymphocytes of depressed patientswere found to be deficient of brain -derived neurotrophic factor, brain-derived neurotrophic factor-regulated genes, and the receptor TrkB (Pandey et al., 2010). The brain-derived neurotrophic factor was found to be deficient in serum of MDD patients (Karege et al., 2002;Kim et al., 2007) and the extent of its polymorphism indicated chronicity level of the disease (Lee et al., 2013). The antidepressant treatments including electroconvulsive therapy and repetitive transcranial magnetic stimulation enhanced the expression of brain-derived neurotrophic factor (Chen et al., 2001;Altar et al., 2003;M¨uller et al., 2000M¨uller et al., , Gonul et al., 2005Lang et al., 2006) and produced antidepressant effect on behavioral models of depression (Siuciak et al., 1996;Shirayama et al., 2002) deficient of TrkB receptor (Saarelainen et al., 2003). All these reports suggest a potential involvement of brain-derived neuro-trophic factor in the genesis of MDD.
The role of all-trans retinoic acid as an additive for in vitro neuronal differentiation of stem cells is well known (Jiang et al., 20012;Nojehdehian et al., 2009;Guan et al., 2001;Xu et al., 2012;Christie et al., 2010). Retinoic acid regulates a cascade of changes during the development of central nervous system which result in neuronal differentiation (Maden, 2007;Maden, 2002). Neurodegenerative diseases like Parkinson's disease (Krezel et al., 1998), schizophrenia (Goodman, 1998), motor neuron diseases (Corcoran et al., 2002), and even the decline of mental abilities with age also involve deficiencyof retinoids and retinoid signalling loss. Development of neurons and maintenance of synaptic signal transmission also involve a potential role of brain -derived neurotrophic factor (Huang et al., 2001;Waterhouse and Xu, 2009;Cohen-Cory, 2010). It is reported that in vitro brain-derived neurotrophic factor supplementation mediates differentiation in NSC (Ahmed et al., 1995). It also mediates survival and maturation of neurons (Vutskits et al., 2001;Kirschenbaum et al., 1995). However, in vivo administration of brain-derived neurotrophic factor leads to differentiation of NSCs into new neurons (Pencea et al., 2001;Zigova et al., 1998). In the present study we studied the role of florophenyl retinoic acid triazole in differentiation and proliferation of NB1643 cells for therapeutic purposes.

Materials and Methods
Reagents and cell culture: Fluorophenyl retinoic acid triazole (Sigma) stocks were prepared by dissolving it in ethanol. Human brain-derived neurotrophic factor and NGF were purchased from Santa Cruz Biotechnology, USA. NB1643 cells were grown in 10% FBS/RPMI (RPMI 1640 medium containing 2 mM glutamine (BioWhittaker) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 50 units/mL penicillin, 50 mg/mL streptomycin) (Life Technologies, Inc.) in 5% CO2 atmosphere at 37 °C with tissue culture plates from Costar. For counting and splitting, cells were trypsinizedin trypsin versene mixture (BioWhittaker). Nuclei were prepared using the method of Butler (Butler, 1984) and counted using a Z2 counter equipped with a 256 channelyzer (Coulter) for both cell plating and for growth and survival assays. The tissue culture plateswere coatedwith poly-lysine by incubiting plates for 14 hour with 1 mL of 50 mg/mLpoly-lysine in H2O. The plates after washing with H2O were air dryed under UV light, and subsequently crosslinked using UV light supplied by a Stratalinker (Stratagene).
Differentiation of NB1643 cells: 2.5 x 10 5 NB1643 cells were plated per well in 6-well tissue culture dishes with or without 5 µM fluorophenyl retinoic acid triazole in 10% FBS/RPMI medium. The concentration of ethanol was 0.1% in all wells. Photographs at a magnification of x225 were taken at day 3 following the treatment.
Cell viability assays: Inmedium (10% FBS/RPMI) containing 5 µM fluorophenyl retinoic acid triazole on poly -lysine-coated 96-well platesNB1643 cells at a density of 2.5 x 10 4 cells per well were distributed. After 24 hours of fluorophenyl retinoic acid triazole treatment, the fresh medium containing brain-derived neurotrophic factor (50 ng/mL) and varying concentrations of FBS (0, 0.1, 0.2, and 0.5%) and 5 µM fluorophenyl retinoic acid triazole was added. After 24 hours, wells were aspirated and 1 µM Calcein AM (Molecular Probes) in Hanks' solution (Life Technologies, Inc.) was added for 50 min at 37°C. Quantization of fluorescence using a cytofluor fluorescent plate reader (Millipore) was performed. A linear fluorescent signal with respect to viable cell number was obtained. To establish linearity of detection, 5, 10, 25, 50, and 75 x 10 3 cells were plated in 96well plates and cultured overnight.Quadruplicate wells were aspirated, and 1 µM Calcein AM (Molecular Probes) in Hanks' solution (Life Technologies, Inc.) for 1 hour at 37°C was addedfor Calcein AM detection. Quantitated of fluorescence usinga cytofluor fluorescent plate reader was done. Triplicate wells were trypsinized, and nuclei were prepared and counted for direct assessment of cell number in 3.5-cm tissue culture wells (Costar) coated with poly-lysine with 5 µM fluorophenyl retinoic acid triazole in 10% FBS/ RPMI. Final ethanol concentration was 0.1% in all wells. Medium was changed 24 hours later to medium with or without 50 ng/Ml BDNF and 5 µM fluorophenyl retinoic acid triazole in 0.1% FBS/RPMI. Triplicate wells were trypsinized, and nuclei were counted as described above at various times subsequent to brainderived neurotrophic factor addition.
Growth assays: In 6-well tissue culture dishes (3.5-cmdiameter wells) were plated 5 x 10 5 NB1643 cells per well. After 12 hours medium was changedto medium with or without 5 µM fluorophenyl retinoic acid triazole.2.5 x 10 5 NB1643 cells were plated per well in 6well tissue culture dishes. For the brain-derived neurotrophic factor growth rate experiments, 2.5 x 10 5 NB1643 cells were plated per well in 6-well tissue culture dishes (3.5-cm-diameter wells) (Costar). Medium was changed 14 hours later to medium with or without 5 µM FPRT. After 24 hours, brain-derived neurotrophic factor was added (100 ng/mL). Final ethanol concentration was 0.1% in all wells. Triplicate wells were harvested by trypsinization and nuclei were counted as described above. Linear regression and statistical analysis to determine if the dependence of growth rate (slopes) of NB1643 cells differs significantly were performed using the method described by Zar.
Immunoblotting analysis of TRK and TRKB: The fluorophenyl retinoic acid triazole induced expression of TRK and TRKB was analysed by platting 4 x 10 6 NB1643 cells in dishes followed by treatment with 5 µM fluorophenyl retinoic acid triazole after 2 days or no treatment. Cell lysis in each dish was achieved in 1 Ml of RIPA with fresh 1 µM phenylmethylsulfonyl fluori-deat 4°C. The lysates were cleared by centrifugation at 10,000 xg for 45 min. The supernatantswere incubated with 10 mL of TRK C-14 antisera (Santa Cruz) followed by incubationon rotation with 20 mL of protein A-Sepharose (Repligen) at 4°C. The immune complexes were again suspended in 1 mL of RIPA, vortexed, and pelleted in a microcentrifuge. After 3-washes, RIPA buffer was removed. The samples were subjected to electrophoresis in 30 mL of 23 SDS-polyacrylamide. The gels were transferred to immobilon-P (Millipore) using the milliblot semidry apparatus (Millipore). Detection of TRK and TRKB was carried out by using immunoblot analysis. The procedure involves membrane blockage for 1 hour in 5% milk in TBST (10 µM Tris-HCl, pH 8.0, 150 µM NaCl, 0.05% Tween-20). Washing of blot first for 20 min and then 10 min twice was followed by incubation with TRK C-14 (1:200) in 1% milk/1% bovine serum albumin in TBST for 1 hour. Washing membrane as above was followed by incubation with anti-rabbit horse radish peroxidase in 1% milk/1% bovine serum albumin in TBST for 1 hour. The membrane was washed for 20 minand then four times for 10 min in TBST. Visualization of TRK and TRKB bands was achieved using chemiluminescent detection with the ECL kit (Amersham Pharmacia Biotech) and XAR 5 film (Eastman Kodak Co.). For analysis of TRK-or TRKB-specific expression, 2 x 10 6 NB1643 cells were plated with either 0 or 5 µM fluorophenyl retinoic acid triazole for 4 days. TRK was immunoprecipitated using 1 mg of TRK-specific antibodies (catalog No. 9142, New England Biolabs) followed by immunoblotting with TRK C-14 antisera using the methods described above. For TRKB expression, TRKs were immunoprecipitated with TRK C-14 antisera followed by immunoblotting with TRKB-specific antiserum 5050 (1:400) and TRKB antibodies (1:200) (Santa Cruz).

Results
The results demonstrated that fluorophenyl retinoic acid triazoleinducessignificant expression of TRK mRNA in time dependent manner in NB1643 cells (Figure 2A). There wereabout 10-, 14-, and 21-fold induction of expression after 1, 2, and 3 days of fluoro-phenyl retinoic acid triazole treatment, respectively ( Figure 2B). We used antibodies against the C-terminal 14 amino acids of TRK to determine the expression time interval of TRK and TRKB ( Figure 2C) on fluorophenyl retinoic acid triazole treatment. The results revealed that TRK and TRKB were induced at 6 and 12 hour afterfluorophenyl retinoic acid triazole treatment respectively and the expression of TRKB was maximum at day 3. The expression of FPRT induced-TRK was further confirmed by immunoprecipitation with a TRKspecific antibody and then immunoblotting with the C-14 TRK antibodies ( Figure 2D). The confirmation of fluorophenyl retinoic acid triazole induced-TRKB was performed by immunoprecipitation with a TRK C-14 antibodies followed by immunoblotting with TRKBspecific antibodies ( Figure 2E). Therefore, fluorophenyl retinoic acid triazole induces TRKB expression, which is a receptor for brain-derived neurotrophic factor and a protein-tyrosine kinase. It seemed likely that NB cell differentiation may be comparable to NGF-induced differentiation of rat PC12 cells, because the origins of these cell lines are similar.
We observed a decrease in brain-derived neurotrophic factor mRNA levels in NB1643 cells on fluorophenyl retinoic acid triazole treatment (Figure 3). The results demonstrated a correlation between intervals of expression of TRKB with the time duration of phenotypic differentiation. This supports the notion that differentiation may be the result of an autocrine mechanism involving brain-derived neurotrophic factor and TRKB.
In order to elucidate the mechanism for the neurite outgrowth activity of fluorophenyl retinoic acid triazole, the NB1643 cells were incubated with fluorophenyl retinoic acid triazole (15 µg/mL) in the presence of NGF (10 ng/mL) (Figure 5). We observed no alteration in cell morphology during the initial 6 hours period. Fluorophenyl retinoic acid triazole molecule dispersed uniformly into the cell to generate many small vesicles (Figure 5, left picture). After 12 hours, short prominences appeared at the outer edge of the cell. Intriguingly, the fluorescent vesicles assembled at the neck of each small prominence (Figure 5, middle picture). Significant morphological changes were observed after 24 hours. Elongation of the prominences, where the fluorophenyl retinoic acid triazole molecules accumulated, became particularly noticeable (Figure 5, right picture). These results show the fluorophenyl retinoic acid triazole accumulate in the prominences.

Discussion
The results indicated that fluorophenyl retinoic acid triazole induces expression of TRK, brain-derived A. Northern analysis on 20 mg of total RNA isolated from NB1643 cell lines grown in 0 or 5 mM FPRT for 3 days. B. Total RNA was isolated from NB1643 cells grown in 0 or 5 mM ATRA for 1, 2, and 3 days. B.RT-PCR analysisfor expression of TRKB and G3PDH are shown. Arrows indicate the predicted 371-bp TRKB and 983-bp G3PDH PCR products. C. Immunoprecipitation of TRKs followed by immunoblottinganalysis is depicted at 1, 2, and 3days after ATRA treatment. D. TRK was immunoprecipitated with TRK-specific antiserum followed by immunoblotting with TRK C-14 antisera. E. TRKs were immunoprecipitated with C-14 TRK antiserum followed by immunoblotting using TRKB-specific antibodies (794 and 5050) and generic TRK C-14 antibodies Figure 3: FPRT induces expression of TRKB, and BDNF and NGF in NB1643 cells Total RNA was isolated from NB1643cells grown in 0 or 5 mM FPRT for 1, 2, and 3 days. RT-PCRanalysis for expression of BDNF, NGF, and G3PDH are shown. NB1643cells were treated with 0 or 5 mM ATRA. Arrows indicate the predicted 492-bp BDNF, 432-bp NGF, and 1347-bp transferrin receptor PCR products. 33P-Labeled PCR products were analyzed using a PhosphorImager neurotrophic factor, NGF andcauses an increase in neurite outgrowth in NB1643 cells.
Fluorophenyl retinoic acid triazole treatment led to a significant expression of TRK mRNA in time dependent manner in NB1643 cells. There were about 10-, 14-, and 21-fold induction of expression after 1, 2, and 3 days of fluorophenyl retinoic acid triazole treatment, respecttively. The results revealed that TRK and TRKB were induced at 6 and 12 hours after fluorophenyl retinoic acid triazole treatment respectively and the expression of TRKB was maximum at day 3. Therefore, fluorophenyl retinoic acid triazoleinduces TRKB expression, which is a receptor for brain-derived neurotrophic factor and a protein-tyrosine kinase. There was a decrease in brain-derived neurotrophic factor mRNA level in NB1643 cells on fluorophenyl retinoic acid triazole treatment. Our results showed that fluorophenyl retinoic acid triazole significantly promote NGF (20 ng/mL)-mediated neurite outgrowth. In order to elucidate the mechanism for the neurite outgrowth activity of fluorophenyl retinoic acid triazole, the NB1643 cells were incubated with fluorophenyl retinoic acid triazole (20 µg/mL) in the presence of NGF (10 ng/ mL). Initially no alteration in cell morphology was observed for 6 hours period. During this period, fluorophenyl retinoic acid triazole molecule dispersed uniformly into the cell to generate many small vesicles. After 24 hours, short prominences appeared at the outer edge of the cell. Fluorophenyl retinoic acid triazole molecules then assembled at the neck of each small prominence which caused significant morphological changes after 24 hours.
The results of the study demonstrate that fluorophenyl retinoic acid triazole has a potent neurite outgrowth activity. Fluorophenyl retinoic acid triazole exerts neurite outgrowth activity by inducing TRKB expre- Left picture: NB1643 cell after 6 hours of the culture with 15 µg FPRTand 10 ng/mL NGF. FPRT was identified as small-blue particles. Middle picture: NB1643 cell after 12 hours. Right picture: NB1643 cell after 24 hours. Note that the FPRT (blue particles) has accumulated inside each prominence ssion, which is a receptor for brain-derived neurotrophic factor and a protein-tyrosine kinase. Fluorophenyl retinoic acid triazole accumulation generates many small vesiclesin the cell which assembles at the neck of each small prominence and lead to significant morphological changes after 24 hours.