Abstract
Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for substantia nigra dopaminergic (DA) neuronal cells. Recent studies have demonstrated that neural cell adhesion molecule functions as a signal transduction receptor for GDNF. The purpose of this study is to reveal whether neural cell adhesion molecule (NCAM) mediates the protective effects of GDNF on DA neuronal cells and further explore the mechanisms involved. We utilized SH-SY5Y cell line to establish a model of 6-hydroxydopamine (6-OHDA)-injured DA neuronal cells. Lentiviral vectors were constructed to knockdown or overexpress NCAM-140, and a density gradient centrifugation method was employed to separate membrane lipid rafts. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), flow cytometric analysis, and western blotting were used to evaluate the protective effects of GDNF. The results showed that GDNF could protect 6-OHDA-injured SH-SY5Y cells via improving cell viability and decreasing the cell death rate and cleaved caspase-3 expression. NCAM-140 knockdown decreased cell viability and increased the cell death rate and cleaved caspase-3 expression, while its overexpression had the opposite effects. Notably, the amount of NCAM-140 located in lipid rafts increased after GDNF treatment. Pretreatment with 2-bromopalmitate, a specific inhibitor of protein palmitoylation, suppressed NCAM-140 translocation to lipid rafts and reduced the NCAM-mediated protective effects of GDNF on injured DA neuronal cells. Our results suggest that GDNF have the protective effects on injured DA cells by influencing NCAM-140 translocation into lipid rafts.
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Introduction
Parkinson’s disease (PD) is a chronic neurodegenerative disease that affects millions of people worldwide. Glial cell line-derived neurotrophic factor (GDNF) exerts specific protective effects on the dopaminergic (DA) neurons that are damaged in PD pathogenesis; however, the signaling mechanisms underlying these effects remain obscure.
Many previous reports have shown that GDNF plays important biological roles, mainly via the RET-GFRα1-dependent signaling pathway [1–3]. However, GFRαs are much more widely distributed than RET in the central nervous system, which suggests the existence of RET-independent signaling pathways [4–6]. In 2003, Paratcha et al. reported that GDNF could promote axonal growth in hippocampal and cortical neurons through the neural cell adhesion molecule (NCAM) signaling pathway and proposed that NCAM was an alternative signaling receptor for GDNF [7].
NCAM was originally characterized as a homophilic cell adhesion molecule, but recent studies have shown that NCAM can function as a signaling receptor, as well as a cell–cell adhesion molecule [8]. NCAM is encoded by a single gene that undergoes alternative splicing to generate three major isoforms of different molecular weights, NCAM-180, NCAM-140, and NCAM-120. The extracellular domains of these three NCAM subtypes are completely conserved. NCAM-120 lacks transmembrane and cytoplasmic domains and is attached to plasma membrane with glycosylphosphatidylinositol (GPI) membrane anchor, while NCAM-180 and NCAM-140 are both transmembrane proteins and differ in the size of their cytoplasmic domain. There is increasing evidence indicating that NCAM could mediate multiple biological processes induced by GDNF including cell viability, differentiation, migration, and neurite outgrowth [9–11]. However, few investigations have been carried out to examine the potential roles of NCAM in mediating the protective effect of GDNF on DA neurons.
Lipid rafts are highly dynamic plasma membrane microdomains enriched in cholesterol and sphingolipids [12]. A growing body of evidence suggests that lipid rafts play an important role in compartmentalizing the components involved in NCAM signal transduction pathways. The various signaling molecules are differently distributed inside or outside lipid rafts, and NCAM in and out of rafts will activate different signaling pathways and induce diverse biological functions [13]. However, whether the raft distribution of NCAM is involved in the GDNF-NCAM-mediated protective effects on DA neurons is not clear.
The present study aimed to explore whether and how NCAM could mediate the protective effects of GDNF on DA neural cells. We utilized the human SH-SY5Y neuroblastoma cell line to establish 6-hydroxydopamine (6-OHDA)-injured DA neuronal cells. A short hairpin RNA (shRNA) lentiviral vector targeting NCAM-140 and a lentiviral vector expressing NCAM were used to knockdown or overexpress NCAM, respectively, and then tested the influences of NCAM knockdown or overexpression on the protective effects of GDNF. Finally, we employed a density gradient centrifugation to separate lipid rafts and explored weather lipid rafts were involved in the neuroprotective effects of GDNF-NCAM signaling pathway.
We found that GDNF could protect 6-OHDA-injured SH-SY5Y cells via improving cell viability, decreasing the apoptotic rate, and inhibiting cleaved caspase-3 expression. NCAM-140 knockdown weakened the protective effects of GDNF, while NCAM overexpression was accompanied by enhanced protective effects of GDNF on 6-OHDA-injured SH-SY5Y cells. In addition, our results showed that GDNF could induce NCAM translocation into lipid rafts, and inhibition of NCAM-140 translocation induced by 2-bromopalmitate (2-BP) could depress NCAM-mediated protective effect of GDNF. Collectively, these findings suggest that NCAM-140 could mediate the protective effects of GDNF on injured DA cells via its translocation into lipid rafts.
Materials and Methods
Cell Culture
The dopaminergic neuroblastoma SH-SY5Y cell line was obtained from the Shanghai Institutes for Biological Sciences (SIBS) of Chinese Academy of Sciences (CAS). The cells were plated at a density of 5 × 105 cells on 25-cm2 dishes (Corning, Corning, NY, USA). The cells were cultured in Dulbecco’s modified Eagle media, nutrient mixture F-12 (DMEM/F12; Sigma, St. Louis, MO, USA) supplemented with 15 % fetal bovine serum (Cambrex, East Rutherford, NJ, USA) in a water-jacketed incubator at 37 °C in a humidified incubator with 5 % CO2. The differentiation of SH-SY5Y cells was induced using 10 μM all transretinoic acid (ATRA) for 6 days before cells were used for experiments.
6-OHDA and GDNF Treatment
The neurotoxin 6-OHDA · HBr (Sigma, St. Louis, MO) was made fresh for each experiment in a non-oxidizing vehicle. Cultures were exposed to 40 μM 6-OHDA for 24 h (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay) or 10 h (western blot and flow cytometric), followed by two washes of serum-free media, and then were prepared for further testing. GDNF (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS). GDNF (50 ng/ml) was co-incubated with 6-OHDA in every experiments.
MTT Cell Viability Assay
Vybrant® MTT Cell Viability Assay Kit was used to measure cell viability. SH-SY5Y cells were seeded in 96-well plates at a density of 1 × 104 cells/well in 100-μl medium. The cultures were grown for 48 h, and then, 10 μM ATRA was added to differentiate cells for 6 days before cells were used for experiments (changed the medium every 3 days). After co-incubation with 6-OHDA (40 μM) and GDNF (50 ng/ml) for 24 h, the medium was removed and replaced with 100 μl of fresh culture medium. Of the 12 mM MTT reagent (0.5 mg/ml MTT in PBS containing 10 mM HEPES), 10 μl was added to each well and incubated in the CO2 incubator for 4 h. Of the sodium dodecyl sulfate (SDS)-HCl solution (add 10 ml of 0.01 M HCl to 1 g of SDS), 100 μl was added to each well and mixed thoroughly using the pipette. The microplate was incubated at 37 °C for 4 h in a humidified chamber. Each sample was mixed again using a pipette, and absorbance of the solution was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad, USA). Wells were added with 100 μl of medium, and 10 μl of the MTT reagent alone was used as a negative control.
Flow Cytometric Analysis of Cell Death Rate
The SH-SY5Y cells were plated at a density of 5 × 105 cells on 25-cm2 dishes. The cultures were grown for 48 h, and then, 10 μM ATRA was added to differentiate cells for 6 days before cells were used for experiments. After incubation with 6-OHDA (40 μM) and GDNF (50 ng/ml) for 10 h, death cells were quantified by cytofluorometric analysis using a FACSCalibur (Becton–Dickinson, Mountain View, CA). In order to quantify cell death rate induced by 6-OHDA, using Annexin V-APC Apoptosis Detection Kit (BD Pharmingen, USA) according to the manufacturer’s instructions, briefly, cells were incubated with 5-μl APC-conjugated Annexin-V and 10 μl PI staining solution for 15 min in the dark at room temperature and further screened by flow cytometry. Under these conditions, Annexin V+/PI+ cells correspond to either secondary necrotic or late apoptotic cell and Annexin V+/PI− cells correspond to early apoptotic cell. The cell death rate was estimated by the relative amount of Annexin V+/PI− and Annexin V+/PI+ cell populations.
Real-Time Polymerase Chain Reaction
Total RNA was extracted from cells with TRIzol Reagent (Invitrogen, USA). Then, 2 μg was used to synthesize complementary DNA (cDNA) with the Superscript First-Strand Synthesis Kit (Promega, USA) following the manufacturer’s protocols. The real-time polymerase chain reaction (PCR) reaction kit contained 0.2-μM sense primer, 0.2-μM anti-sense primer, 12.5-μl SYBR Green I (Toyobo, Osaka, Japan), and 5 μl of previously synthesized cDNA in a total volume of 25 μl. A pair of GAPDH primers in identical reactions was used to control the starting template. The sense and anti-sense primers were 5′-TTGGCTACAGCAACAGGGTG-3′ and 5′-TCTACATGGCAACTGTGAGGAG-3′, respectively. The target genes detected were human NCAM (forward 5′-CCGATTCATAGTCCTGTCCAA-3 and reverse 5′-ATAAGTGCCCTCATCTGTTTTCT-3′.
Western Blotting
The cells were lysed in a lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 % Triton X-100, 2 mM EDTA, 1 % deoxycholic acid (DOC), 0.1 % SDS, 1 mM NaVO3, 10 mM NaF, and 1 mM DTT) and centrifuged to yield whole-cell lysates. Bradford protein concentration assay was used to measure total protein concentration. Aliquots of the lysates (20 μg of protein) were separated on a 5–10 % SDS-polyacrylamide gel with a SDS-running buffer (25 mM Tris (pH 8.3), 250 mM glycocol, and 0.1 % SDS) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Invitrogen, Carlsbad, CA, USA) with a transfer buffer (25 mM Tris–HCl (pH 8.3), 192 mM glycocol, and 20 % methyl alcohol). After blocking the non-specific site with 5 % non-fat dry milk, the membrane was then incubated with specific primary antibody (in 3 % BSA at 4 °C for overnight). The membrane was washed with 1× PBS prior to incubating with IR Dye® 800 anti-rabbit or anti-mouse secondary antibodies (Li-Cor, Lincoln, NE), at room temperature. Immunoactive proteins were detected using Odyssey® Imaging Systems (Li-Cor, Lincoln, NE). Anti-NCAM antibody, anti-flotillin-1 antibody, and anti-CD71 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-cleaved caspase 3 antibody was obtained from Millipore Corporation (Bedford, MA, USA). Band intensities were quantified using Image J software.
Isolation of Detergent-Resistant and Detergent-Sensitive Fractions
For isolation of detergent-resistant (DR) and detergent-sensitive (DS) fractions, the cell sample was first incubated for 30 min on ice in TNET buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and protease inhibitor cocktail) containing 1 % Triton X-100 [14]. Following centrifugation at 100,000g at 4 °C for 1 h, the DR (pellet) and DS (supernatant) fractions were collected and analyzed for western blots.
Isolation of Lipid Raft Fractions by Non-Detergent Density Gradient Ultracentrifugation Using OptiPrep™
Isolation of lipid raft fractions by non-detergent density gradient ultracentrifugation using OptiPrep™ was prepared as previously described [15]. Briefly, cells were washed and scraped into 2-ml base buffer (20 mM Tris–HCl, pH 7.8, 250 mM, 1 mM CaCl2, 1 mM MgCl2, and protease inhibitor cocktail). After homogenizing the cells by 20 passages through a 22-g syringe needle, centrifuge the homogenate at 1000g for 10 min. The supernatant was adjusted to 50 % OptiPrep™ in base buffer (2 ml) and laid at the bottom of a 12-ml centrifuge tube. After which, 4 ml each of 20 and 0 % OptiPrep™ in base buffer were overlaid. Following centrifugation at 52,000×g at 4 °C for 90 min, a density gradient was formed. Of the fractions, ×12 1000 μl were collected from the top to the bottom of the gradient and analyzed for western blots for NCAM, flotillin, and CD71.
Plasmid and Transfection
According to full sequence of human NCAM1 (NM_000615.6), the NCAM expression vector LV5- green fluorescent protein (GFP)-NCAM lentivirus; control lentivirus LV5-GFP (named as NCAM and NCAM NC, respectively); and NCAM shRNA expression vector LV3-GFP-shRNA1, LV3-GFP-shRNA2, LV3-GFP-shRNA3, LV3-GFP-shRNA4 lentivirus, and control lentivirus LV3-GFP control shRNA (named as shRNA1, shRNA2, shRNA3, shRNA4, and shRNA NC, respectively) were designed and constructed by Genepharma Corporation (Shanghai, China). The target sequences of shRNAs were GCAGGAGATGCCAAAGATAAA (shRNA1), GGACTTCTACCCGGAACATCA (shRNA2), GCTGCTGCCAAGCTCCAATTA (shRNA3), GCAGTTGGTGAAGAAGTATGG (shRNA4), and TTCTCCGAACGTGTCACGT (shRNA NC), respectively. Sequencing analysis was performed to confirm the recombinant plasmid. SH-SY5Y cells were transfected with the lentiviral vectors for 72 h, using the Lipofectamine 2000 reagent (Invitrogen, CA, USA) according to the manufacturer’s recommendations. To assess the efficiency of lentiviral transduction, SH-SY5Y cells were plated at a density of 5 × 105 cells/well into 24-well plates and infected with lent-GFP at a dose of multiplicity of infection (MOI) 10. After 72 h, GFP-expressing cells were detected by fluorescence microscopy (Olympus, Japan). The percentage of GFP-positive cells in 10 fields was counted to evaluate the transfection efficiency. Western blot was performed to detect NCAM-140 protein, and RT-PCR was performed to detect the level of NCAM messenger RNA (mRNA).
Statistical Analysis
All data are reported as mean ± SEM. Statistical significance between multiple groups was performed using one-way analysis of variance (ANOVA), and differences between groups were determined with Scheffe’s t test. A value of P < 0.05 was considered significant.
Results
NCAM Expression in Human Neuroblastoma SH-SY5Y Cells
The NCAM H-300 antibody detects against amino acids 1–300 at the N-terminus of human NCAM and can detect all three NCAM isoforms. Western blotting revealed that the expression of NCAM-140 was much higher than that of NCAM-180 and NCAM-120 in SH-SY5Y cells (Fig. 1).
Identification of shRNA Lentiviral Vectors and Lentiviral Expression Vectors of NCAM-140
The sequencing results confirmed that the oligo DNA sequence of shRNA was correctly inserted into the former vector (Supplement 1), while the sequences of the NCAM gene carried in NCAM lentiviral vector were consistent with the NCAM gene sequences in the Genbank database (Supplement 2). Lentiviral vectors were used to transfect SH-SY5Y cells for 3 days. Then, the transfection efficiency of shRNA targeting NCAM and lentiviral vector overexpressing NCAM-140 were assessed by examining the expression of the GFP gene tag. More than 70 % cells were infected with lent-GFP at a dose of MOI 10 after 72-h treatment (Fig. 2).
After transfection, the mRNA and protein expression of NCAM-140 were examined by real-time PCR and western blot analysis, respectively, to assess the knockdown and overexpression effect of lentiviral vectors. Results showed that NCAM-140 mRNA levels were decreased to 41, 81, 34, and 58 % with shRNAs 1, 2, 3, and 4, respectively, and shRNA3 had the most prominent inhibitory effect on NCAM-140 mRNA expression (Fig. 3). Therefore, shRNA3 was used in the subsequent experiments and suppressed the expression of NCAM. Conversely, the lentiviral vector overexpressing NCAM-140 significantly increased NCAM-140 mRNA and protein levels (Figs. 3 and 4).
Effects of NCAM-140 Knockdown or Overexpression on Protective Effects of GDNF
To determine whether NCAM-140 mediated the protective effects of GDNF on 6-OHDA-injured SH-SY5Y cells, we performed MTT assays to assess cell viability, flow cytometry to examine cell death rate, and western blotting to measure cleaved caspase-3 expression.
The cells were divided into the following nine groups: control group, the cells were cultured under normal condition; 6-OHDA group, treated with 40 μM 6-OHDA for 24 h (MTT assays) or 10 h (flow cytometry and western blotting); NCAM + 6-OHDA group, after transfection with lentiviral vector overexpressing NCAM-140, treated with 40 μM 6-OHDA for 24 or 10 h; shRNA + 6-OHDA group, after transfection with shRNA 3, treated with 40 μM 6-OHDA for 24 or10 h; 6-OHDA + GDNF group, treated with 40 μM 6-OHDA and 50 ng/ml GDNF for 24 or 10 h; shRNA + 6-OHDA + GDNF group, after transfection with shRNA 3, treated with 40 μM 6-OHDA and 50 ng/ml GDNF for 24 or10 h; NCAM + 6-OHDA + GDNF group, after transfection with lentiviral vector overexpressing NCAM-140, treated with 40 μM 6-OHDA and 50 ng/ml GDNF for 24 or 10 h; shRNA NC + 6-OHDA + GDNF group, after transfection with shRNA NC, treated with 40 μM 6-OHDA and 50 ng/ml GDNF for 24 or 10 h; and NCAM NC + 6-OHDA + GDNF group, after transfection with NCAM NC, treated with 40 μM 6-OHDA and 50 ng/ml GDNF for 24 or 10 h.
MTT assays results showed that the cell viability in the 6-OHDA group was less than that in the group without 6-OHDA treatment (Fig. 5). Similarly, the cell death rate, and expression of cleaved caspase-3 with 6-OHDA treatment were increased in 6-OHDA group (Figs. 6 and 7). These results indicated that the 6-OHDA treatment significantly affected cell viability, cell death rate and expression of cleaved caspase-3. In 6-OHDA + GDNF group, cell viability was more while the cell death rate and expression of cleaved caspase-3 were less than that in 6-OHDA group. These results showed that GDNF could protect 6-OHDA-injured SH-SY5Y cells via improving cell viability and decreasing the cell death rate and cleaved caspase-3 expression.
Based on this experimental model describing the protective effects of GDNF on 6-OHDA-injured SH-SY5Y cells, shRNA 3 targeting NCAM-140 or lentiviral vector overexpressing NCAM-140 were transfected into cells to knockdown or overexpress NCAM-140, respectively. When shRNA was transfected into cells, the effects of GDNF to stimulate cell viability and to inhibit cell death rate and cleaved caspase-3 expression were antagonized. And, the overexpression of NCAM-140 enhanced the influences of GDNF on cell viability, cell death rate, and cleaved caspase-3 expression. These results demonstrated that NCAM-140 could mediate, at least partially, the protective effect of GDNF.
GDNF-Induced NCAM-140 Translocation into Lipid Rafts
Insolubility in cold non-ionic detergents and flotation on OptiPrep™ density gradients are well-established techniques for separating lipid rafts, and we employed these two methods to isolate lipid rafts. Our initial aim was to determine whether GDNF treatment could result in the NCAM-140 translocation to or from the lipid rafts. Using non-ionic detergents, we found that only a small number of NCAM-140 was identified in lipid raft microdomains without GDNF. After GDNF treatment, NCAM-140 located in lipid rafts gradually increased and reached a maximum at 15 min (Fig. 8).
To further confirm NCAM-140 translocation into lipid rafts after GDNF treatment, OptiPrep™ density gradient centrifugation was used to separate lipid rafts, and similar results were obtained (Fig. 9). As mentioned above, we observed that a small fraction of NCAM-140 localized to lipid raft microdomains in the absence of GDNF, confirming an association of NCAM-140 with rafts under basal conditions, and that GDNF treatment could further induce NCAM-140 translocation into lipid rafts. These results suggested that lipid rafts might be involved in GDNF-NCAM-140 signaling events.
Inhibition of NCAM-140 Translocation into Lipid Rafts Suppressed GDNF-NCAM-140-Mediated Protective Effects
To further investigate whether lipid rafts were involved in the protective effects of GDNF-NCAM-140 on injured DA neuronal cells, 2-BP pretreatment was administered to block NCAM-140 translocation into lipid rafts. NCAM palmitoylation is required for NCAM displacement to lipid rafts [13, 16]. 2-BP inhibits the activity of protein acyltransferase and protein palmitoylation modification, thus reducing NCAM in lipid rafts [17]. Our results also showed that the amount of NCAM located in lipid raft microdomains was significantly reduced after 2-BP pretreatment, confirming that 2-BP could inhibit GDNF-induced NCAM translocation into lipid rafts (Fig. 10). Then, we assessed cell viability, cell apoptosis rates, and the expression of cleaved caspase-3 to explore the influences of NCAM translocation into lipid rafts on the protective effects of GDNF-NCAM-140 pathway.
The cells were divided into the following seven groups: 6-OHDA group, treated with 40 μM 6-OHDA for 24 (MTT assays) or 10 h (flow cytometry and western blotting); 2BP + 6-OHDA group, pretreated with 20 μM 2-BP for 15 min followed by 6-OHDA and GDNF treatment for 24 or 10 h; NCAM + 6-OHDA + GDNF group, after transfection with lentiviral vector overexpressing NCAM-140, 6-OHDA, and GDNF treatment for 24 or 10 h; NCAM + 2-BP + 6-OHDA + GDNF group, after transfection with lentiviral vector overexpressing NCAM-140, pretreated with 20 μM 2-BP for 15 min followed by 6-OHDA and GDNF treatment for 24 or 10 h; shRNA + 6-OHDA + GDNF group, after transfection with shRNA 3, treated with 6-OHDA and GDNF for 24 or 10 h; 6-OHDA + GDNF group, treated with 6-OHDA and GDNF for 24 or 10 h; and 2-BP + 6-OHDA + GDNF group, pretreated with 20 μM 2-BP for 15 min followed by treatment with 6-OHDA and GDNF for 24 or 10 h.
In 2-BP + 6-OHDA + GDNF group, pretreatment with 2-BP counteracted GDNF-induced effects on cell viability, cell death rate, and expression of cleaved caspase-3 when compared with 6-OHDA + GDNF group. In contrast to NCAM + 6-OHDA + GDNF group, results in NCAM + 2-BP + 6-OHDA + GDNF group showed decreased cell viability and increased cell death rate and cleaved caspase-3 expression, and the results are similar to that in shRNA + 6-OHDA + GDNF group (Figs. 11, 12, and 13). These above results showed that 2-BP pretreatment inhibited the protective effects of GDNF-NCAM-140 on 6-OHDA-injured SH-SY5Y cells, indicating that NCAM displacement to lipid rafts played a key role in GDNF’s beneficial effect.
Discussion
The present study was conducted to determine whether NCAM could mediate the protective effects of GDNF and, if so, whether lipid raft translocation of NCAM was involved. To determine whether NCAM could mediate the protective effects of GDNF on 6-OHDA-injured SH-SY5Y cells, we constructed shRNA lentiviral vectors targeting NCAM-140 and expressing NCAM-140 to inhibit and increase the protein’s expression, respectively. The results showed that NCAM knockdown suppressed the protective effect of GDNF, while overexpression promoted protection of 6-OHDA-injured cells. In accordance with our findings, many other studies have shown that NCAM can mediate neuronal survival. For example, Ditlevsen et al. found that stimulation of NCAM with the synthetic NCAM mimetic peptide C3d could promote the survival of cerebellar and dopaminergic neurons induced to undergo apoptosis [18, 19]. In addition, Chao et al. reported that function blocking anti-NCAM antibodies antagonized the survival-promoting effect of GDNF in tyrosine hydroxylase-positive DA neurons [20].
NCAM can interact with several different transmembrane and intracellular signaling molecules to activate a complex network of signaling pathways. NCAM signaling is markedly affected by the protein’s compartmentalization into lipid rafts. These highly dynamic membrane microdomains enriched in cholesterol and sphingolipids are reported to be involved in important intracellular signaling events [12, 21, 22]. Among several signal molecules linked to NCAM, the Src kinase family, heteromeric G proteins, and the neurite outgrowth-associated protein growth-associated protein-43 (GAP-43) are enriched in lipid rafts; nevertheless, the fibroblast growth factor (FGF) receptor is out of lipid rafts [23–25]. NCAM-mediated signaling events via lipid rafts are distinct from its signaling outside of lipid rafts. Within lipid rafts, NCAM-140 associates with Fyn, leading to the recruitment of focal adhesion kinase and subsequently activating the Ras–Raf extracellular signal-related kinase ERK1/2 pathway [13, 26]. Outside of lipid rafts, NCAM-140 signaling is dependent on the FGF receptor to increase intracellular Ca2+ and activate phospholipase C, protein kinase C, and Ca2+/calmodulin-dependent protein kinase II [13, 27].
Because NCAM can activate different signaling molecule depending on its subcellular location, we first investigated the distributions of NCAM within cytoplasmic membranes. We separated lipid rafts and found that NCAM-140 was the main isoform expressed in differentiated SH-SY5Y cells and was found in both raft and non-raft fractions in unstimulated cells. This is consistent with previous researches [13, 28, 29]. In addition, our results showed a gradual increase in the amount of NCAM-140 in lipid rafts after GDNF treatment, with a peak at 15 min. This finding is in accordance with previous studies showing that dynamic regulation of NCAM-140 raft association with specific redistribution upon NCAM clustering [28, 29]. There is growing evidence that movement of cellular receptors into rafts can enhance downstream signaling pathways [30, 31]. Therefore, we asked whether NCAM raft localization was essential for the protective effect of GDNF. We employed the palmitoylation inhibitor 2-BP to inhibit NCAM translocation into lipid rafts. It has been suggested that NCAM-140 is targeted to lipid rafts by the palmitoylation of four residues in the NCAM intracellular juxtamembrane region and that 2-BP pretreatment could inhibit its translocation into lipid rafts [16, 17]. Our results showed that 2-BP pretreatment indeed attenuated GDNF-induced NCAM redistribution to lipid rafts. The protective effect of GDNF-NCAM-140 was also inhibited after 2-BP pretreatment. Collectively, these results suggest that NCAM translocation into lipid rafts is necessary for GDNF-NCAM-140-mediated protective effects on injured DA cells. Considering that a major downstream mediator of NCAM located in lipid rafts is Fyn, we hypothesize that the interplay among these proteins is important. However, elucidation of the exact signaling pathways activated downstream of NCAM translocation into lipid rafts will require further investigation.
In conclusion, the present results suggest that NCAM could mediate the protective effect of GDNF on DA neurons. Lipid rafts are involved in this process, and subcellular compartmentalization of NCAM into lipid raft microdomains is necessary for GDNF-NCAM-140-mediated protective effects.
References
Maeda K, Murakami H, Yoshida R, Ichihara M, Abe A, Hirai M, Murohara T, Takahashi M (2004) Biochemical and biological responses induced by coupling of Gab1 to phosphatidylinositol 3-kinase in RET-expressing cells. Biochem Biophys Res Commun 323:345–354
He Z, Jiang J, Kokkinaki M, Golestaneh N, Hofmann MC, Dym M (2008) Gdnf upregulates c-Fos transcription via the Ras/Erk1/2 pathway to promote mouse spermatogonial stem cell proliferation. Stem Cells 26:266–278
Paveliev M, Lume M, Velthut A, Phillips M, Arumäe U, Saarma M (2007) Neurotrophic factors switch between two signaling pathways that trigger axonal growth. J Cell Sci 120:2507–2516
Trupp M, Arenas E, Fainzilber M, Nilsson AS, Sieber BA, Grigoriou M, Kilkenny C, Salazar-Grueso E et al (1996) Functional receptor for GDNF encoded by the c-ret proto-oncogene. Nature 381:785–789
Poteryaev D, Titievsky A, Sun YF, Thomas-Crusells J, Lindahl M, Billaud M, Arumäe U, Saarma M (1999) GDNF triggers a novel ret-independent Src kinase family-coupled signaling via a GPI-linked GDNF receptor alpha1. FEBS Lett 463:63–66
Trupp M, Scott R, Whittemore SR, Ibáñez CF (1999) Ret-dependent and -independent mechanisms of glial cell line-derived neurotrophic factor signaling in neuronal cells. J Biol Chem 274:20885–20894
Paratcha G, Ledda F, Ibáñez CF (2003) The neural cell adhesion molecule NCAM is an alternative signaling receptor for GDNF family ligands. Cell 113:867–879
Kiss JZ, Muller D (2001) Contribution of the neural cell adhesion molecule to neuronal and synaptic plasticity. Rev Neurosci 12:297–310
Euteneuer S, Yang KH, Chavez E, Leichtle A, Loers G, Olshansky A, Pak K, Schachner M et al (2013) Glial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis in the cochlear spiral ganglion via neural cell adhesion molecule (NCAM). Mol Cell Neurosci 54:30–43
Charoy C, Nawabi H, Reynaud F, Derrington E, Bozon M, Wright K, Falk J, Helmbacher F et al (2012) GDNF activates midline repulsion by semaphorin3B via NCAM during commissural axon guidance. Neuron 75:1051–1066
Ledda F, Paratcha G, Sandoval-Guzmán T, Ibáñez CF (2007) GDNF and GFRalpha1 promote formation of neuronal synapses by ligand-induced cell adhesion. Nat Neurosci 10:293–300
Pike LJ (2009) The challenge of lipid rafts. J Lipid Res Suppl: S323-328
Niethammer P, Delling M, Sytnyk V, Dityatev A, Fukami K, Schachner M (2002) Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis. J Cell Biol 157:521–532
Janich P, Corbeil D (2007) GM1 and GM3 gangliosides highlight distinct lipid microdomains within the apical domain of epithelial cells. FEBS Lett 581:1783–1787
Macdonald JL, Pike LJ (2005) A simplified method for the preparation of detergent-free lipid rafts. J Lipid Res 46:1061–1067
Little EB, Edelman GM, Cunningham BA (1998) Palmitoylation of the cytoplasmic domain of the neural cell adhesion molecule N-CAM serves as an anchor to cellular membranes. Cell Adhes Commun 6:415–430
Webb Y, Hermida-Matsumoto L, Resh MD (2000) Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J Biol Chem 275:261–270
Ditlevsen DK, Køhler LB, Pedersen MV, Risell M, Kolkova K, Meyer M, Berezin V, Bock E (2003) The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival. J Neurochem 84:546–556
Ditlevsen DK, Berezin V, Bock E (2007) Signalling pathways underlying neural cell adhesion molecule-mediated survival of dopaminergic neurons. Eur J Neurosci 25:1678–1684
Chao CC, Ma YL, Chu KY, Lee EH (2003) Integrin alphav and NCAM mediate the effects of GDNF on DA neuron survival, outgrowth, DA turnover and motor activity in rats. Neurobiol Aging 24:105–116
Schley PD, Brindley DN, Field CJ (2007) (n-3) PUFA alter raft lipid composition and decrease epidermal growth factor receptor levels in lipid rafts of human breast cancer cells. J Nutr 137:548–553
Lucero HA, Robbins PW (2004) Lipid rafts-protein association and the regulation of protein activity. Arch Biochem Biophys 426:208–224
Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39
Arni S, Keilbaugh SA, Ostermeyer AG, Brown DA (1998) Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues. J Biol Chem 273:28478–28478
Aarts LH, Verkade P, Schrama LH, Oestreicher AB, Gispen WH, Schotman P (1999) Local accumulations of B-50/GAP-43 evoke excessive bleb formation in PC12 cells. Mol Neurobiol 20:17–28
Beggs HE, Baragona SC, Hemperly JJ, Maness PF (1997) NCAM140 interacts with the focal adhesion kinase p125 (fak) and the SRC-related tyrosine kinase p59 (fyn). J Biol Chem 272:8310–8319
Povlsen GK, Ditlevsen DK, Berezin V, Bock E (2003) Intracellular signaling by the neural cell adhesion molecule. Neurochem Res 28:127–141
Bodrikov V, Leshchyns’ka I, Sytnyk V, Overvoorde J, den Hertog J, Schachner M (2005) RPTPalpha is essential for NCAM-mediated p59fyn activation and neurite elongation. J Cell Biol 168:127–139
Leshchyns’ka I, Sytnyk V, Morrow JS, Schachner M (2003) Neural cell adhesion molecule (NCAM) association with PKCbeta2 via betaI spectrin is implicated in NCAM-mediated neurite outgrowth. J Cell Biol 161:625–639
Cinek T, Horejsí V (1992) The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases. J Immunol 149:2262–2270
Tansey MG, Baloh RH, Milbrandt J, Johnson EM Jr (2000) GFRalpha-mediated localization of RET to lipid rafts is required for effective downstream signaling, differentiation, and neuronal survival. Neuron 25:611–623
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grant numbers 81401056 and 31040035) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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Electronic Supplementary Material
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Supplement 1
Sequencing results of shRNA targeting NCAM. Four complementary shRNA oligonucleotides targeting the NCAM gene were designed, synthesized, and inserted into a linearized LV3 vector. The recombinant NCAM shRNA vectors were identified by DNA sequencing. The sequencing results confirmed successful shRNA sequence insertion into the vectors. (RAR 477 kb)
Supplement 2
Sequencing results of recombinant NCAM-140 vector plasmid. The NCAM gene was amplified and subcloned into an LV5 vector, and successful NCAM recombinant vector construction was confirmed by DNA sequencing. (SQD 94 kb)
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Li, L., Chen, H., Wang, M. et al. NCAM-140 Translocation into Lipid Rafts Mediates the Neuroprotective Effects of GDNF. Mol Neurobiol 54, 2739–2751 (2017). https://doi.org/10.1007/s12035-016-9749-x
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DOI: https://doi.org/10.1007/s12035-016-9749-x