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CART peptide promotes the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor
BBRC Biochemical and Biophysical Research Communications 347 (2006) 656–661 www.elsevier.com/locate/ybbrc
CART peptide promotes the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor Bin Wu, Shengdi Hu, Min Yang, Hui Pan, Shigong Zhu
*
Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, People’s Republic of China Received 17 June 2006 Available online 28 June 2006
Abstract The neuropeptide cocaine- and amphetamine-regulated transcript (CARTp) plays a role in various physiological processes. CARTp is highly expressed in rat hippocampus and can promote the survival and differentiation of neurons in primary hippocampal cell cultures. However, little is known about the neurotrophic mechanism of CARTp on the hippocampal neuron. We show that CARTp fragment 55–102 promoted the survival of cultured hippocampal neurons by increasing the number of surviving neurons and their viability. The tyrosine kinase B (TrkB) antibody, known to inhibit the activity of brain-derived neurotrophic factor (BDNF), blocked the survival-promoting effect of CARTp on hippocampal neurons. Further study by reverse-transcription PCR showed that BDNF mRNA expression significantly increased after CARTp treatment. The prepro BDNF and mature BDNF protein also increased in level as seen on Western blot analysis. Thus, the neurotrophic effects of CARTp on cultured hippocampal neurons are mediated through the upregulation of BDNF mRNA expression and protein synthesis. The results of the present study suggest the therapeutic efficacy of CARTp in neurodegenerative disorders. 2006 Elsevier Inc. All rights reserved. Keywords: Neuropeptide; Cocaine- and amphetamine-regulated transcript (CART); BDNF; Hippocampal neuron; Neurotrophy
Cocaine- and amphetamine-regulated transcript peptide (CARTp) is widely distributed in the brain and peripheral nervous system [1,2]. It has been implicated in the regulation of feeding and body weight, drug reward, and other biological processes [3], although no receptor for CARTp has yet been identified. It also has neuroprotective properties and promotes the survival and differentiation of hippocampal neurons in vitro [4]. However, little is known about the molecular mechanism by which CARTp exerts its effects on neurotrophy and neuronal survival. The hippocampus contains the highest expression of brain-derived neurotrophic factor (BDNF) mRNA and protein, as well as nerve growth factor (NGF) [5–7]. Both BDNF and NGF are major members in the neurotrophin (NT) gene family. The neuroprotective and neurotrophic effects of CARTp in the hippocampal neurons could be *
Corresponding author. Fax: +86 10 82801443. E-mail address: [email protected] (S. Zhu).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.117
mediated by NGF or especially BDNF because the cultured hippocampal neurons show no response to NGF [8]. BDNF regulates dendritic and axonal growth, as well as synaptic plasticity in the cortex and hippocampus [9–11]. Exogenous BDNF directly potentiates synaptic transmission in the hippocampus [12,13], and endogenous BDNF is necessary for the establishment of long-term potentiation (LTP) [14,15]. BDNF-knockout mice show abnormal neuronal phenotypes and impaired LTP, and recombinant BDNF rescues the deficits in synaptic transmission and LTP in the hippocampus [15–17]. Increasing evidence shows that BDNF mRNA and protein are reduced in level in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [18–20]. BDNF protects against damage in nigral dopaminergic neurons induced by the infusion of neurotoxins in monkeys [21] and hippocampal pyramidal neurons following transient forebrain ischemia in rats [22]. BDNF exerts its neurotrophic effects by binding to high-affinity tyrosine kinase receptors. The
B. Wu et al. / Biochemical and Biophysical Research Communications 347 (2006) 656–661
subtype of tyrosine kinase receptor (TrkB) is highly expressed in hippocampal neurons [23]. BDNF transcription is regulated by many neuropeptides such as vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide [24], and corticotropin-releasing hormone [25]. However, whether the neurotrophic effects of CARTp are related to the effects of BDNF is unknown. CARTp may exert its neurotrophic effects in the hippocampal neurons by regulating BDNF transcription. In the present study, we explored the effect of CARTp on the survival of cultured hippocampal neurons, the relation between CARTp effects and mRNA expression, and protein synthesis of BDNF and NGF in cultured hippocampal neurons. Materials and methods Materials. Reagents for cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from Gibco-BRL (Life Technologies, Inc., Gaithersburg, MD, USA). Fetal bovine serum (FBS) was purchased from Hyclone (UT, USA). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CARTp 55–102 was obtained from Phoenix Pharmaceuticals (Belmont, CA). The reagents of the BCA method for protein quantification and restoration of Western blot stripping buffer were purchased from Pierce (Pierce Co., Rockford, IL, USA). Cell Cultures. Pregnant Sprague–Dawley rats were supplied by the Experimental Animal Center of Peking University. Primary hippocampal cell cultures were established from embryonic rats (day 18 of gestation). Hippocampi were excised, minced, and digested in calcium- and magnesium-free Hanks’ balanced salt solution (HBSS) containing 0.25% trypsin (Difco, Detroit, MI) and 6 g/L glucose, then resuspended in DMEM, high glucose type (Gibco, Rockville, MD), supplemented with 10% FBS, 100 U/ml penicillin, 100 lg/ml streptomycin, 25 lg/ml insulin, and 6 g/L glucose. The tissues were dissociated mechanically with use of a pipette, and cells were put on plates with 18-mm glass coverslips for immunohistochemistry or inserted directly into 96-well plates (with a special marker line on the bottom) or 60-mm culture dishes at 71,000 cells/cm2, the substratum being previously coated with poly-L-lysine (0.1 mg/ml in 100 mM borate buffer, pH 8.4) overnight. Cultures were maintained at 37 C in a humidified atmosphere of 95% air and 5% CO2. On the third day in vitro, the cultures were transferred to DMEM containing 5% FBS and 10 lM cytosine arabinoside (AraC) for 2 days to halt the proliferation of non-neuronal cells. The cultures consisted of more than 92% neurons as identified by double immunostaining with antibodies against microtubuleassociated protein-2 (MAP2) and glial fibrillary acidic protein (GFAP) (Zymed, San Francisco, CA), which are markers for neurons and astrocytes, respectively. The culture with 92% neurons was used in the following experiments. The procedures in this study were approved by the Animal Care Committee of Peking University and followed the guidelines of the Guide for the Care and Use of Laboratory Animals.
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Drug application. CARTp 55–102, a biologically active form of rat CART [26], was used. Doses and treatment schedules of CARTp are presented in the results of each experiment. CARTp was maintained in the culture media throughout the full treatment interval. Anti-TrkB IgG (5 lg/ml) was used to inhibit the binding of BDNF to the TrkB receptors 4 h before treatment with CARTp. Assays for neuronal survival. Total neuron survival was quantified by observing the growth of neurons, and counting the number of neurons in the same groups and by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, the viable neurons in culture plates containing lines on the bottom in a 1 · 1 mm2 area were observed and counted. Neurons that died in the intervals between examination points were usually absent, and the viability of the remaining neurons was assessed by morphological criteria, whereby neurons with intact neuritis of uniform diameter and soma with a smooth round appearance were considered viable, and neurons with fragmented neuritis and vacuolated cell bodies were considered nonviable. For MTT assay, the cells were incubated with 0.5 mg/ml MTT for 4 h at 37 C. Living cells converted the yellow MTT dye into an insoluble blue formazan product. The dye was solubilized by DMSO, and the absorbance intensity (540 nm; A540) of each solution was measured in a 96-well plate reader. To normalize the differences in the absorbance values of the MTT color reaction product among the different culture assays following the exposure to the drug conditions, values were expressed relative to the treated vehicle controls maintained on each plate from the same cellular preparation. The proportion of cell viability was calculated by the following formula: survival % = test A540/mean control A540 · 100. Immunocytochemistry. Cultures were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.2) for 30 min at room temperature. Fixed cells were washed with PBS, and nonspecific antibody binding was blocked by incubation with 4% normal goat serum in 0.1 M PBS. The cells were then incubated overnight at 4 C with the primary antibodies against MAP2 (1:500), then washed and incubated for 2 h with FITC-conjugated anti-goat IgG secondary antibody (1:200). Finally, cells were washed 3 times at room temperature in 0.1 M PBS. Images were acquired by using a confocal laser scanning microscope with the same laser intensity and photodetector gain to allow comparisons of the relative levels of immunoreactivity among all groups. Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from hippocampal neurons with use of TRIzol reagent (Gibco-BRL, Life Technologies, MD). Single-stranded cDNA was synthesized from 1 lg total RNA by use of the first-strand cDNA Synthesis Kit (Promega Corp). The synthesized cDNA as a template in the PCRs was stored at 20 C until use. The PCRs were performed in 25 ll volume, which included 1 ll cDNA sample, 2.5 mM MgCl2, 0.25 mM dNTP, 1· PCR buffer, 1 ll of each specific primer, and 0.4 ll (2 units) Taq DNA polymerase (Gibco-BRL). The primers and PCR conditions are shown in Table 1. PCR samples were analyzed on an ethidium bromide-stained 1.5% agarose gel and visualized by ethidium bromide staining. The intensity of ethidium bromide fluorescence was measured by use of NIH Image Analysis Software version 1.61 (National Institutes of Health, Bethesda, MD) and determined relative to b-actin. Western blot analysis. Cells were washed twice with ice-cold PBS and harvested in a lysis buffer containing 20 mM Tris, pH 7.4, 140 mM NaCl, 1 mM EDTA, 1 mM sodium vanadate, 20 mM NaF, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 lg/ml
Table 1 PCR primers, amplification product length, and PCR conditions Primers sets BDNF NGF b-Actin
Nucleotide sequences of primers 0
0
5 -CGACGTCCCTGGCTGACACTTTT-3 5 0 -AGTAAGGGCCCGAACATACGATTGG-3 0 5 0 -CTGAACCAATAGCTGCCCGTGTGAC-3 0 5 0 -GGCAGCCTGTTTGTCGTCTGTTGTC-3 0 5 0 -ATCTGGACCACACCTTC-3 0 5 0 -AGCCAGGTCCAGACGCA-3 0
Product size (bp)
Annealing temperature (C)
Cycles
491
60
30
491
60
35
291
55
28
658
B. Wu et al. / Biochemical and Biophysical Research Communications 347 (2006) 656–661
leupeptin, and 10 lg/ml aprotinin. Extracts were then centrifuged at 12,000g for 15 min at 4 C, and the amount of protein in the supernatant was estimated by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). The samples were boiled for 5 min for denaturation. For immunoblot analysis, 50 lg of protein was loaded into each well. The samples were separated on 10% or 15% SDS–polyacrylamide gels (Bio-Rad equipment). After electrophoresis, the proteins were electrically transferred to nitrocellulose membranes (0.22 mm) in solution containing 49.6 mM Tris, 384 mM glycine, and 0.01% SDS at 200 mA for 2 h. After transfer, the blots were incubated in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% nonfat milk, then incubated with primary antibody, antiBDNF (1:500), anti-NGF (1:500), and anti-tubulin (1:10,000), for 16 h at 4 C in TBST containing 5% nonfat milk. After primary antibody incubation, the blots were washed 3 times for 15 min each in TBST at 25 C and incubated with secondary horseradish peroxidase-conjugated antibodies (1:2000) for 1 h. Then the membrane was rinsed with TBST 3 times, each time for 10 min. Immunoreactive bands were visualized by use of an enhanced chemiluminescence method. After immunoblotting, digitized images of the immunoreactive bands were quantitated by image analysis with use of the NIH Image software (version 1.62). Additional background measurements were taken from each film and subtracted from these values. The ratios of BDNF or NGF to tubulin were then determined. Statistical analysis. All values are presented as means ± SEM. Comparisons among multiple groups involved one-way ANOVA followed by the Student–Newman–Keuls test and Dunnett’s test (for comparison of multiple experimental treatments to a common control value). All analyses involved use of SPSS. A value of p < 0.05 was considered significant.
Vehicle CART 1nM CART 10nM
Number of neuron (%)
120
100
** *
80
** **
60
40 10 0 0
3
6
Results CARTp sustains survival of hippocampal neurons To examine the effect of CARTp on cell viability in the cultured hippocampal neuron, CARTp was applied at 0.01, 0.1, 1, 10, and 100 nM at 8 days in vitro for 24 h and analyzed by MTT assay. The CARTp at 1-, 10-, and 100-nM produced a significant 9%, 13%, and 15% increase in neuronal viability as compared with untreated controls (p < 0.05). To examine the effect of CARTp on growth, hippocampal neurons were assayed for cell viability by cell counting and MTT metobolism assay at days 0, 3, 6, and 9 after 1- and 10-nM CARTp treatment. Compared with untreated controls at days 6 and 9, the viability of treated neurons with 1-nM CARTp was approximately 7% and 13% increase, respectively (p < 0.05 and p < 0.01), and approximately 16% and 24% increase in 10-nM CARTp group (p < 0.01) (Fig. 1). MAP2 immunoreactivity analysis showed a decrease in number of neurons, with some neuron process degeneration in the vehicle group (Fig. 2A) but more integrative soma and processes with 1- and 10-nM CARTp treatment (Fig. 2B and C). Blocking of BDNF receptor TrkB inhibits CARTp-enhanced neuronal viability To understand whether BDNF receptors were involved in the neurotrophic effects of CARTp, anti-TrkB IgG (5 lg/ ml) was used to inhibit the binding of BDNF to TrkB receptors. Cultures were first pretreated for 4 h with the blocker in media and then with CARTp. TrkB IgG inhibited the survival-promoting actions induced by 10 nM CARTp (Fig. 3). The results suggest that neurotrophins were involved in the regulation of neuronal survival by CARTp.
9
Period of incubation (d) Fig. 1. Effect of CARTp on neuronal number. The number of neurons in the pre-marked fields was counted at days 0, 3, 6, and 9 after CARTp treatment, respectively. Each point represents the mean ± SEM of at least 40 fields. *p < 0.05, **p < 0.01 versus control.
Effect of CARTp on expression of BDNF and NGF mRNA in hippocampal cultures Because of the high expression of BDNF and NGF mRNA and protein in the hippocampal neurons, the
Fig. 2. Effect of CARTp on the morphology of hippocampal neurons. Fluorescence laser scanning photomicrographs show MAP2 immunoreactivity in hippocampal neurons. (A) Vehicle; (B) 1-nM CARTp and (C) 10-nM CARTp treatment. Arrow indicates the degenerated processes.
B. Wu et al. / Biochemical and Biophysical Research Communications 347 (2006) 656–661
treated with 10 nM CARTp for 24 h and the intracellular BDNF and NGF protein level was detected by Western blot analysis. The expression of BDNF showed 2 bands at 14 and 30 kDa (Fig. 5), which corresponded to the respective mature BDNF and precursor forms of BDNF (ppBDNF), both increased from 1 to 16 h as compared with untreated controls (p < 0.01). The expression of NGF showed only one band at 30 kDa, which corresponded to the precursor form of NGF protein (data not shown), but no mature NGF was detected (data not shown). These results suggest that CARTp enhanced the content of BDNF but not NGF protein in hippocampal neurons.
Neuronal survival % (MTT metobolism)
125
100
75 5 0 vehcle
CART
TrkBIgG TrkBIgG+CART
Fig. 3. TrkB-IgG blocks the CARTp-induced increase in neuronal viability. Hippocampal cells were incubated with TrkB-IgG (5 lg/ml) for 4 h before CARTp exposure. Cell viability was determined by MTT metobolism. Results show the percentage of the value in the treated vehicle control wells. *p < 0.05 versus control. Each bar gives the mean ± SEM of at least 8 culture wells.
Discussion In the present study, we observed that CARTp enhanced neuronal viability by increasing the number of surviving neurons and maintaining the integrity in primary hippocampal neurons in a time- and dose-dependent manner, respectively. The results agree strongly with the previous work reported by Louis [4]. Because BDNF regulates dendritic and axonal growth, as well as synaptic plasticity, in the cortex and hippocampus [9–11], we explored the mechanism of CARTp promotion of neuronal survival through its effect on mRNA expression and protein synthesis of BDNF in hippocampal neurons. Neuronal viability was reduced by blocking the BDNF receptor with TrkB IgG, which implied that the endogenous BDNF mediated the effect of CARTp on the survival of cultured hippocampal neurons.
transcripts of both BDNF and NGF were analyzed by semiquantitative RT-PCR. Hippocampal cells incubated with 10 nM CARTp for 24 h showed significantly increased BDNF mRNA level at 1, 2, 4, 8, and 16 h after treatment (p < 0.01) (Fig. 4). BDNF mRNA levels returned to baseline values by 24 h (Fig. 4). However, CARTp treatment had no effect on the expression of NGF mRNA levels (data not shown). Effect of CARTp on BDNF and NGF synthesis in hippocampal cultures To examine the effect of CARTp on BDNF and NGF in hippocampal cultures at the protein level, neurons were A
659
Vehicle
CART
BDNF 491bp
-actin 291bp 0
B
1 Relative BDNF mRNA level (%)
M
2
4
8
16
24
0
1
150
2
4
8
16
24 h
vehicle
* ** **
**
CART
*
120
90 5 0 0
4
8
12
16
20
24
28
Period of incubation (h) Fig. 4. Analysis of BDNF mRNA in hippocampal neurons by RT-PCR. (A) Total RNA was extracted from cells with or without (vehicle) CARTp (10 nM) treatment at 0, 1, 2, 4, 8, 16, and 24 h. (B) Intensity of the amplified bands of BDNF and b-actin was measured by use of NIH Image 1.61. Optical density values are expressed as a proportion of baseline values. Values with SEM were derived from 3 plates at each time point. *p < 0.05, **p < 0.01 versus control.
660
B. Wu et al. / Biochemical and Biophysical Research Communications 347 (2006) 656–661
A 30KD
ppBDNF
BDNF
14KD 60KD
-tubulin
10 8
1
2
4
8
16
24 h 2
** ** **
**
**
* 6
**
** 1
*
4
*
ppBDNF
2
BDNF
0
BDNF relative level
B ppBDNF relative level
0
0 0
4
8
12
16
20
24
Culture duration (h)
Fig. 5. Effect of CARTp on BDNF protein in hippocampal neurons. (A) Hippocampal neurons were treated with 10 nM CARTp for 0, 1, 2, 4, 8, 16, or 24 h. Enhanced chemiluminescence shows the mature form of BDNF protein at 14 kDa and the precursor form of BDNF (ppBDNF) at 30 kDa. (B) All values are expressed as means ± SEM of the optical density ratio of BDNF/tubulin from 3 independent experiments. *p < 0.05, **p < 0.01 versus control.
CARTp enhanced survival by upregulating BDNF and another neurotrophin, NGF. Subsequent study demonstrated that CARTp increases BDNF mRNA and ppBDNF and mature BDNF synthesis, but not NGF mRNA and NGF protein in cultured hippocampal neurons. Thus, CARTp could influence neuronal survival through the regulation of BDNF protein synthesis rather than NGF during neuronal development. BDNF plays an important role in neuronal survival and differentiation [27], promotes neuronal survival during development [28], and is involved in the control of dendritic morphology [29]. In the present study, the soma and process both grew well at 1- and 10-nM CARTp treatment, which implied that BDNF mediated the effects of CARTp. In summary, CARTp exerts neurotrophic actions by promoting BDNF mRNA expression and protein synthesis in cultured hippocampal neurons. To our knowledge, this is the first demonstration of the up-regulation of endogenous BDNF by CARTp. The results of the present study suggest the therapeutic efficacy of CARTp in neurodegenerative disorders. Acknowledgments This work was supported by the National Natural Science Foundation of China (30370557), the Ministry of Education for Research Program of China (20020001083), and in part by the National Major Basic Research Program of China (G2000056908).
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CART peptide promotes the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor Bin Wu, Shengdi Hu, Min Yang, Hui Pan, Shigong Zhu
*
Department of Physiology and Pathophysiology, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, People’s Republic of China Received 17 June 2006 Available online 28 June 2006
Abstract The neuropeptide cocaine- and amphetamine-regulated transcript (CARTp) plays a role in various physiological processes. CARTp is highly expressed in rat hippocampus and can promote the survival and differentiation of neurons in primary hippocampal cell cultures. However, little is known about the neurotrophic mechanism of CARTp on the hippocampal neuron. We show that CARTp fragment 55–102 promoted the survival of cultured hippocampal neurons by increasing the number of surviving neurons and their viability. The tyrosine kinase B (TrkB) antibody, known to inhibit the activity of brain-derived neurotrophic factor (BDNF), blocked the survival-promoting effect of CARTp on hippocampal neurons. Further study by reverse-transcription PCR showed that BDNF mRNA expression significantly increased after CARTp treatment. The prepro BDNF and mature BDNF protein also increased in level as seen on Western blot analysis. Thus, the neurotrophic effects of CARTp on cultured hippocampal neurons are mediated through the upregulation of BDNF mRNA expression and protein synthesis. The results of the present study suggest the therapeutic efficacy of CARTp in neurodegenerative disorders. 2006 Elsevier Inc. All rights reserved. Keywords: Neuropeptide; Cocaine- and amphetamine-regulated transcript (CART); BDNF; Hippocampal neuron; Neurotrophy
Cocaine- and amphetamine-regulated transcript peptide (CARTp) is widely distributed in the brain and peripheral nervous system [1,2]. It has been implicated in the regulation of feeding and body weight, drug reward, and other biological processes [3], although no receptor for CARTp has yet been identified. It also has neuroprotective properties and promotes the survival and differentiation of hippocampal neurons in vitro [4]. However, little is known about the molecular mechanism by which CARTp exerts its effects on neurotrophy and neuronal survival. The hippocampus contains the highest expression of brain-derived neurotrophic factor (BDNF) mRNA and protein, as well as nerve growth factor (NGF) [5–7]. Both BDNF and NGF are major members in the neurotrophin (NT) gene family. The neuroprotective and neurotrophic effects of CARTp in the hippocampal neurons could be *
Corresponding author. Fax: +86 10 82801443. E-mail address: [email protected] (S. Zhu).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.117
mediated by NGF or especially BDNF because the cultured hippocampal neurons show no response to NGF [8]. BDNF regulates dendritic and axonal growth, as well as synaptic plasticity in the cortex and hippocampus [9–11]. Exogenous BDNF directly potentiates synaptic transmission in the hippocampus [12,13], and endogenous BDNF is necessary for the establishment of long-term potentiation (LTP) [14,15]. BDNF-knockout mice show abnormal neuronal phenotypes and impaired LTP, and recombinant BDNF rescues the deficits in synaptic transmission and LTP in the hippocampus [15–17]. Increasing evidence shows that BDNF mRNA and protein are reduced in level in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease [18–20]. BDNF protects against damage in nigral dopaminergic neurons induced by the infusion of neurotoxins in monkeys [21] and hippocampal pyramidal neurons following transient forebrain ischemia in rats [22]. BDNF exerts its neurotrophic effects by binding to high-affinity tyrosine kinase receptors. The
B. Wu et al. / Biochemical and Biophysical Research Communications 347 (2006) 656–661
subtype of tyrosine kinase receptor (TrkB) is highly expressed in hippocampal neurons [23]. BDNF transcription is regulated by many neuropeptides such as vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide [24], and corticotropin-releasing hormone [25]. However, whether the neurotrophic effects of CARTp are related to the effects of BDNF is unknown. CARTp may exert its neurotrophic effects in the hippocampal neurons by regulating BDNF transcription. In the present study, we explored the effect of CARTp on the survival of cultured hippocampal neurons, the relation between CARTp effects and mRNA expression, and protein synthesis of BDNF and NGF in cultured hippocampal neurons. Materials and methods Materials. Reagents for cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from Gibco-BRL (Life Technologies, Inc., Gaithersburg, MD, USA). Fetal bovine serum (FBS) was purchased from Hyclone (UT, USA). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CARTp 55–102 was obtained from Phoenix Pharmaceuticals (Belmont, CA). The reagents of the BCA method for protein quantification and restoration of Western blot stripping buffer were purchased from Pierce (Pierce Co., Rockford, IL, USA). Cell Cultures. Pregnant Sprague–Dawley rats were supplied by the Experimental Animal Center of Peking University. Primary hippocampal cell cultures were established from embryonic rats (day 18 of gestation). Hippocampi were excised, minced, and digested in calcium- and magnesium-free Hanks’ balanced salt solution (HBSS) containing 0.25% trypsin (Difco, Detroit, MI) and 6 g/L glucose, then resuspended in DMEM, high glucose type (Gibco, Rockville, MD), supplemented with 10% FBS, 100 U/ml penicillin, 100 lg/ml streptomycin, 25 lg/ml insulin, and 6 g/L glucose. The tissues were dissociated mechanically with use of a pipette, and cells were put on plates with 18-mm glass coverslips for immunohistochemistry or inserted directly into 96-well plates (with a special marker line on the bottom) or 60-mm culture dishes at 71,000 cells/cm2, the substratum being previously coated with poly-L-lysine (0.1 mg/ml in 100 mM borate buffer, pH 8.4) overnight. Cultures were maintained at 37 C in a humidified atmosphere of 95% air and 5% CO2. On the third day in vitro, the cultures were transferred to DMEM containing 5% FBS and 10 lM cytosine arabinoside (AraC) for 2 days to halt the proliferation of non-neuronal cells. The cultures consisted of more than 92% neurons as identified by double immunostaining with antibodies against microtubuleassociated protein-2 (MAP2) and glial fibrillary acidic protein (GFAP) (Zymed, San Francisco, CA), which are markers for neurons and astrocytes, respectively. The culture with 92% neurons was used in the following experiments. The procedures in this study were approved by the Animal Care Committee of Peking University and followed the guidelines of the Guide for the Care and Use of Laboratory Animals.
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Drug application. CARTp 55–102, a biologically active form of rat CART [26], was used. Doses and treatment schedules of CARTp are presented in the results of each experiment. CARTp was maintained in the culture media throughout the full treatment interval. Anti-TrkB IgG (5 lg/ml) was used to inhibit the binding of BDNF to the TrkB receptors 4 h before treatment with CARTp. Assays for neuronal survival. Total neuron survival was quantified by observing the growth of neurons, and counting the number of neurons in the same groups and by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, the viable neurons in culture plates containing lines on the bottom in a 1 · 1 mm2 area were observed and counted. Neurons that died in the intervals between examination points were usually absent, and the viability of the remaining neurons was assessed by morphological criteria, whereby neurons with intact neuritis of uniform diameter and soma with a smooth round appearance were considered viable, and neurons with fragmented neuritis and vacuolated cell bodies were considered nonviable. For MTT assay, the cells were incubated with 0.5 mg/ml MTT for 4 h at 37 C. Living cells converted the yellow MTT dye into an insoluble blue formazan product. The dye was solubilized by DMSO, and the absorbance intensity (540 nm; A540) of each solution was measured in a 96-well plate reader. To normalize the differences in the absorbance values of the MTT color reaction product among the different culture assays following the exposure to the drug conditions, values were expressed relative to the treated vehicle controls maintained on each plate from the same cellular preparation. The proportion of cell viability was calculated by the following formula: survival % = test A540/mean control A540 · 100. Immunocytochemistry. Cultures were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.2) for 30 min at room temperature. Fixed cells were washed with PBS, and nonspecific antibody binding was blocked by incubation with 4% normal goat serum in 0.1 M PBS. The cells were then incubated overnight at 4 C with the primary antibodies against MAP2 (1:500), then washed and incubated for 2 h with FITC-conjugated anti-goat IgG secondary antibody (1:200). Finally, cells were washed 3 times at room temperature in 0.1 M PBS. Images were acquired by using a confocal laser scanning microscope with the same laser intensity and photodetector gain to allow comparisons of the relative levels of immunoreactivity among all groups. Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from hippocampal neurons with use of TRIzol reagent (Gibco-BRL, Life Technologies, MD). Single-stranded cDNA was synthesized from 1 lg total RNA by use of the first-strand cDNA Synthesis Kit (Promega Corp). The synthesized cDNA as a template in the PCRs was stored at 20 C until use. The PCRs were performed in 25 ll volume, which included 1 ll cDNA sample, 2.5 mM MgCl2, 0.25 mM dNTP, 1· PCR buffer, 1 ll of each specific primer, and 0.4 ll (2 units) Taq DNA polymerase (Gibco-BRL). The primers and PCR conditions are shown in Table 1. PCR samples were analyzed on an ethidium bromide-stained 1.5% agarose gel and visualized by ethidium bromide staining. The intensity of ethidium bromide fluorescence was measured by use of NIH Image Analysis Software version 1.61 (National Institutes of Health, Bethesda, MD) and determined relative to b-actin. Western blot analysis. Cells were washed twice with ice-cold PBS and harvested in a lysis buffer containing 20 mM Tris, pH 7.4, 140 mM NaCl, 1 mM EDTA, 1 mM sodium vanadate, 20 mM NaF, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 lg/ml
Table 1 PCR primers, amplification product length, and PCR conditions Primers sets BDNF NGF b-Actin
Nucleotide sequences of primers 0
0
5 -CGACGTCCCTGGCTGACACTTTT-3 5 0 -AGTAAGGGCCCGAACATACGATTGG-3 0 5 0 -CTGAACCAATAGCTGCCCGTGTGAC-3 0 5 0 -GGCAGCCTGTTTGTCGTCTGTTGTC-3 0 5 0 -ATCTGGACCACACCTTC-3 0 5 0 -AGCCAGGTCCAGACGCA-3 0
Product size (bp)
Annealing temperature (C)
Cycles
491
60
30
491
60
35
291
55
28
658
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leupeptin, and 10 lg/ml aprotinin. Extracts were then centrifuged at 12,000g for 15 min at 4 C, and the amount of protein in the supernatant was estimated by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). The samples were boiled for 5 min for denaturation. For immunoblot analysis, 50 lg of protein was loaded into each well. The samples were separated on 10% or 15% SDS–polyacrylamide gels (Bio-Rad equipment). After electrophoresis, the proteins were electrically transferred to nitrocellulose membranes (0.22 mm) in solution containing 49.6 mM Tris, 384 mM glycine, and 0.01% SDS at 200 mA for 2 h. After transfer, the blots were incubated in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% nonfat milk, then incubated with primary antibody, antiBDNF (1:500), anti-NGF (1:500), and anti-tubulin (1:10,000), for 16 h at 4 C in TBST containing 5% nonfat milk. After primary antibody incubation, the blots were washed 3 times for 15 min each in TBST at 25 C and incubated with secondary horseradish peroxidase-conjugated antibodies (1:2000) for 1 h. Then the membrane was rinsed with TBST 3 times, each time for 10 min. Immunoreactive bands were visualized by use of an enhanced chemiluminescence method. After immunoblotting, digitized images of the immunoreactive bands were quantitated by image analysis with use of the NIH Image software (version 1.62). Additional background measurements were taken from each film and subtracted from these values. The ratios of BDNF or NGF to tubulin were then determined. Statistical analysis. All values are presented as means ± SEM. Comparisons among multiple groups involved one-way ANOVA followed by the Student–Newman–Keuls test and Dunnett’s test (for comparison of multiple experimental treatments to a common control value). All analyses involved use of SPSS. A value of p < 0.05 was considered significant.
Vehicle CART 1nM CART 10nM
Number of neuron (%)
120
100
** *
80
** **
60
40 10 0 0
3
6
Results CARTp sustains survival of hippocampal neurons To examine the effect of CARTp on cell viability in the cultured hippocampal neuron, CARTp was applied at 0.01, 0.1, 1, 10, and 100 nM at 8 days in vitro for 24 h and analyzed by MTT assay. The CARTp at 1-, 10-, and 100-nM produced a significant 9%, 13%, and 15% increase in neuronal viability as compared with untreated controls (p < 0.05). To examine the effect of CARTp on growth, hippocampal neurons were assayed for cell viability by cell counting and MTT metobolism assay at days 0, 3, 6, and 9 after 1- and 10-nM CARTp treatment. Compared with untreated controls at days 6 and 9, the viability of treated neurons with 1-nM CARTp was approximately 7% and 13% increase, respectively (p < 0.05 and p < 0.01), and approximately 16% and 24% increase in 10-nM CARTp group (p < 0.01) (Fig. 1). MAP2 immunoreactivity analysis showed a decrease in number of neurons, with some neuron process degeneration in the vehicle group (Fig. 2A) but more integrative soma and processes with 1- and 10-nM CARTp treatment (Fig. 2B and C). Blocking of BDNF receptor TrkB inhibits CARTp-enhanced neuronal viability To understand whether BDNF receptors were involved in the neurotrophic effects of CARTp, anti-TrkB IgG (5 lg/ ml) was used to inhibit the binding of BDNF to TrkB receptors. Cultures were first pretreated for 4 h with the blocker in media and then with CARTp. TrkB IgG inhibited the survival-promoting actions induced by 10 nM CARTp (Fig. 3). The results suggest that neurotrophins were involved in the regulation of neuronal survival by CARTp.
9
Period of incubation (d) Fig. 1. Effect of CARTp on neuronal number. The number of neurons in the pre-marked fields was counted at days 0, 3, 6, and 9 after CARTp treatment, respectively. Each point represents the mean ± SEM of at least 40 fields. *p < 0.05, **p < 0.01 versus control.
Effect of CARTp on expression of BDNF and NGF mRNA in hippocampal cultures Because of the high expression of BDNF and NGF mRNA and protein in the hippocampal neurons, the
Fig. 2. Effect of CARTp on the morphology of hippocampal neurons. Fluorescence laser scanning photomicrographs show MAP2 immunoreactivity in hippocampal neurons. (A) Vehicle; (B) 1-nM CARTp and (C) 10-nM CARTp treatment. Arrow indicates the degenerated processes.
B. Wu et al. / Biochemical and Biophysical Research Communications 347 (2006) 656–661
treated with 10 nM CARTp for 24 h and the intracellular BDNF and NGF protein level was detected by Western blot analysis. The expression of BDNF showed 2 bands at 14 and 30 kDa (Fig. 5), which corresponded to the respective mature BDNF and precursor forms of BDNF (ppBDNF), both increased from 1 to 16 h as compared with untreated controls (p < 0.01). The expression of NGF showed only one band at 30 kDa, which corresponded to the precursor form of NGF protein (data not shown), but no mature NGF was detected (data not shown). These results suggest that CARTp enhanced the content of BDNF but not NGF protein in hippocampal neurons.
Neuronal survival % (MTT metobolism)
125
100
75 5 0 vehcle
CART
TrkBIgG TrkBIgG+CART
Fig. 3. TrkB-IgG blocks the CARTp-induced increase in neuronal viability. Hippocampal cells were incubated with TrkB-IgG (5 lg/ml) for 4 h before CARTp exposure. Cell viability was determined by MTT metobolism. Results show the percentage of the value in the treated vehicle control wells. *p < 0.05 versus control. Each bar gives the mean ± SEM of at least 8 culture wells.
Discussion In the present study, we observed that CARTp enhanced neuronal viability by increasing the number of surviving neurons and maintaining the integrity in primary hippocampal neurons in a time- and dose-dependent manner, respectively. The results agree strongly with the previous work reported by Louis [4]. Because BDNF regulates dendritic and axonal growth, as well as synaptic plasticity, in the cortex and hippocampus [9–11], we explored the mechanism of CARTp promotion of neuronal survival through its effect on mRNA expression and protein synthesis of BDNF in hippocampal neurons. Neuronal viability was reduced by blocking the BDNF receptor with TrkB IgG, which implied that the endogenous BDNF mediated the effect of CARTp on the survival of cultured hippocampal neurons.
transcripts of both BDNF and NGF were analyzed by semiquantitative RT-PCR. Hippocampal cells incubated with 10 nM CARTp for 24 h showed significantly increased BDNF mRNA level at 1, 2, 4, 8, and 16 h after treatment (p < 0.01) (Fig. 4). BDNF mRNA levels returned to baseline values by 24 h (Fig. 4). However, CARTp treatment had no effect on the expression of NGF mRNA levels (data not shown). Effect of CARTp on BDNF and NGF synthesis in hippocampal cultures To examine the effect of CARTp on BDNF and NGF in hippocampal cultures at the protein level, neurons were A
659
Vehicle
CART
BDNF 491bp
-actin 291bp 0
B
1 Relative BDNF mRNA level (%)
M
2
4
8
16
24
0
1
150
2
4
8
16
24 h
vehicle
* ** **
**
CART
*
120
90 5 0 0
4
8
12
16
20
24
28
Period of incubation (h) Fig. 4. Analysis of BDNF mRNA in hippocampal neurons by RT-PCR. (A) Total RNA was extracted from cells with or without (vehicle) CARTp (10 nM) treatment at 0, 1, 2, 4, 8, 16, and 24 h. (B) Intensity of the amplified bands of BDNF and b-actin was measured by use of NIH Image 1.61. Optical density values are expressed as a proportion of baseline values. Values with SEM were derived from 3 plates at each time point. *p < 0.05, **p < 0.01 versus control.
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A 30KD
ppBDNF
BDNF
14KD 60KD
-tubulin
10 8
1
2
4
8
16
24 h 2
** ** **
**
**
* 6
**
** 1
*
4
*
ppBDNF
2
BDNF
0
BDNF relative level
B ppBDNF relative level
0
0 0
4
8
12
16
20
24
Culture duration (h)
Fig. 5. Effect of CARTp on BDNF protein in hippocampal neurons. (A) Hippocampal neurons were treated with 10 nM CARTp for 0, 1, 2, 4, 8, 16, or 24 h. Enhanced chemiluminescence shows the mature form of BDNF protein at 14 kDa and the precursor form of BDNF (ppBDNF) at 30 kDa. (B) All values are expressed as means ± SEM of the optical density ratio of BDNF/tubulin from 3 independent experiments. *p < 0.05, **p < 0.01 versus control.
CARTp enhanced survival by upregulating BDNF and another neurotrophin, NGF. Subsequent study demonstrated that CARTp increases BDNF mRNA and ppBDNF and mature BDNF synthesis, but not NGF mRNA and NGF protein in cultured hippocampal neurons. Thus, CARTp could influence neuronal survival through the regulation of BDNF protein synthesis rather than NGF during neuronal development. BDNF plays an important role in neuronal survival and differentiation [27], promotes neuronal survival during development [28], and is involved in the control of dendritic morphology [29]. In the present study, the soma and process both grew well at 1- and 10-nM CARTp treatment, which implied that BDNF mediated the effects of CARTp. In summary, CARTp exerts neurotrophic actions by promoting BDNF mRNA expression and protein synthesis in cultured hippocampal neurons. To our knowledge, this is the first demonstration of the up-regulation of endogenous BDNF by CARTp. The results of the present study suggest the therapeutic efficacy of CARTp in neurodegenerative disorders. Acknowledgments This work was supported by the National Natural Science Foundation of China (30370557), the Ministry of Education for Research Program of China (20020001083), and in part by the National Major Basic Research Program of China (G2000056908).
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