Degeneration and repair 549
BMP2 shows neurotrophic effects including neuroprotection against neurodegeneration Aybike Saglama,*, Sukbum Kimb,*, Kwangwook Ahna, Insuk Ohc and Kwan-Hee Leea In this study, we found that BMP2 exerts neurotrophic effects, including a neuroprotective effect against nocodazole-induced neuritic degeneration, on neuronal cells. We also found that BMP2-induced neurotrophic effects are directly involved in Smad-dependent signaling as well as PI3K/PTEN-Akt-mTOR signaling. Moreover, BMP2-induced neurotrophic effects occur by stabilization of neuronal microtubules. Thus, these findings suggest that BMP2 can be a potential therapeutic target for nerve c 2014 Wolters injury treatment. NeuroReport 25:549–555 Kluwer Health | Lippincott Williams & Wilkins.
Keywords: bone morphogenetic protein 2, microtubule, neuroprotection, PI3K, PTEN a TissueGene Inc., Rockville, Maryland, USA, bDepartment of Orthopedic Surgery, Gil Medical Center, Gachon University, Incheon and cDepartment of Orthopedics, The Uniformed Force Capital Hospital, Kyunggido, Republic of Korea
Correspondence to Kwan-Hee Lee, MD, PhD, TissueGene Inc., 9605 Medical Center Drive, Suite 200, Rockville, MD 20850, USA Tel: + 1 301 921 6000 x101; fax: + 1 301 921 6011; e-mail: [email protected] *Aybike Saglam and Sukbum Kim contributed equally to the writing of this article. Received 20 December 2013 accepted 13 January 2014
NeuroReport 2014, 25:549–555
Introduction In an adult mammalian nerve system, functional recovery of injured neurons hardly occurs because of lack of intrinsic axonal regeneration ability and upregulation of extracellular inhibitory molecules [1,2]. Previous trials on restoration of nerve regeneration ability by blocking extracellular inhibitory factors have been unsuccessful [3]. Thus, restoration of intrinsic nerve regrowth ability is the current focus as a therapeutic target for treatment of central nervous system (CNS) injury in adults [4]. Bone morphogenetic proteins (BMPs) are a member of the transforming growth factor-b (TGF-b) superfamily and participate in many signaling pathways to regulate the proliferation, differentiation, and survival of many cell types [5,6]. Moreover, recent studies have shown that BMPs exert significant neuroprotection/nerve regeneration effects after adult CNS injury [7–9]. However, the effect of BMPs treatment after CNS injury is still controversial [10]. In this study, we have investigated the effect of BMP2 in an in-vitro neurodegenerative model. Delivery of BMP2 specifically exerts neurotrophic effects including neuroprotection against nocodazole-induced neuritic degeneration. Also, a BMP2-induced neuroprotective effect occurs through Smad-dependent signaling and PI3K-Akt-mTOR signaling by stabilization of neurite microtubules. Thus, our study suggests that BMP2 can be a successful target for therapeutic treatment for nerve injury as BMP2 can exert a neuroprotective effect in neurodegenerative Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.neuroreport.com). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
models through multiple signaling by enhancing neuronal system integrity.
Materials and methods Materials Cells
Both rat adrenal medullary PC12 pheochromocytoma neuronal cells and mouse N1E-115 neuroblastoma cells were purchased from ATCC (Manassas, Virginia, USA). Reagents
Cell culture materials including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and horse serum were purchased from Mediatech Inc. (Manassas, Virginia, USA). TUJ-1 monoclonal rabbit antibody against neuronal class III b-tubulin was purchased from Covance Inc. (Gaithersburg, Maryland, USA). Monoclonal mouse antibody against acetylated a-tubulin, Akt 1(B-1) mouse monoclonal antibody, phosphorylated-Akt 1/2/3 (Ser 473) rabbit polyclonal antibody, rabbit polyclonal phosphorylated-Smad1 (Ser 463/Ser 465) antibody, b-actin mouse monoclonal antibody, and goat anti-rabbit HRPcoupled IgG were purchased from Santa Cruz Biotech Inc. (Santa Cruz, California, USA). Rabbit polyclonal antibody against tyrosine hydroxylase was purchased from Chemicon International Inc. (Temecula, California, USA). Anti-mouse IgG, HRP-linked antibody was purchased from Cell Signaling Tech. (Danvers, Massachusetts, USA). Goat serum, Texas Red goat anti-rabbit IgG antibody, Alexa Fluor 488 goat anti-mouse IgG antibody, 40 ,6-diamidino-2phenylindole, dilactate (DAPI), and Alamar Blue were from Molecular Probes–Invitrogen Inc. (Eugene, Oregon, USA). 2.5S nerve growth factors (NGF) mouse natural and human DOI: 10.1097/WNR.0000000000000130
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recombinant BMP2 protein was purchased from R&D Systems (Minneapolis, Michigan, USA). Human recombinant noggin protein, DMH-1, PP-242, nocodazole, kainic acid, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and cresyl violet dye were purchased from Sigma-Aldrich (St Louis, Missouri, USA). Laminin, mouse, was purchased from BD Bioscience (Sparks Glencoe, Maryland, USA). Potassium bisperoxopicolinatooxovanadate [bpV(pic)] was purchased from Santa Cruz Biotech Inc. Biotinylated antirabbit IgG and avidin–biotin–peroxidase reagents (Elite Vectastain ABC kit) were purchased from Vector Labs (Burlingame, California, USA). Apparatus
Cellular apparatus, including T-75 cm2 culture flasks, a six-well cell culture multiwall plate, cell culture inserts for the six-well plate, and the neurite outgrowth assay kit, was purchased from EMD Millipore (Billerica, Massachusetts, USA). The BCA protein assay kit was purchased from Pierce–Thermo/Fisher Scientific (Rockford, Illinois, USA). Novex 10–20% Tris-glycine mini gels were purchased from Invitrogen Inc. Microscope (BX51) and digital microscope camera (DP72) were purchased from Olympus (Tokyo, Japan).
containing human recombinant BMP2 protein (50 ng/ml) or BMP2 protein plus signaling inhibitor (10 mM) and incubated for 3 additional days. Neurite status was monitored before nocodazole treatment, after nocodazole treatment, and every 24 h using a photomicroscope. After a 72-h incubation, PC12 cells were either fixed for immunofluorescence assay or analyzed using NIS-Element BR image software (Nikon, Linthicum, Maryland, USA) for total neurite quantification. For the neurite outgrowth assay, PC12 cells or N1E-115 cells were seeded into a six-well plate at a 1.0 105 cells/ well seeding density. After a cell confluency of 60–70% was reached, neuronal cell differentiation was initiated – with NGF (50 ng/ml) for PC12 cells and with differentiation media (DMEM, 2 mM L-glutamine, 1% penicillin-streptomycin, and 1 mM sodium pyruvate) for N1E-115 cells. After 24 h of incubation, human recombinant BMP2 protein (50 ng/ml) or BMP2 protein plus signaling inhibitor (10 mM) was added to the wells in six-well plates and incubated for two additional days. Neurite status was monitored every 24 h.
PC12/astrocyte coculture Methods Neuronal cell culture
Rat adrenal medullary PC12 rat pheochromocytoma neuronal cells were supplemented with 7.5% FBS, 7.5% horse serum (ES), and 0.5% penicillin-streptomycin in T-75 cm2 flasks that were maintained at 371C in a 5% CO2 incubator. Cells were split at 50% confluence by gently mechanically detaching them from the flask and propagated at a split ratio of 1 : 7. We also used the mouse N1E115 neuroblastoma cell line. This cell has 36 h of doubling time and a 1 : 3 subcultivation ratio. N1E-115 cells were grown in DMEM without sodium pyruvate and FBS was added at a final concentration of 10%. Rat brain cortex primary neurons were used (cat. no. R-Cx-500; Lonza, Walkersville, Maryland, USA) according to an established procedure [11]. Each vial was thawed and resuspended in 10 ml of growth medium according to the manufacturer’s recommendations, and 1 ml/well volume of cell suspension was seeded onto previously poly-D lysine-coated wells of 24-well plates. Medium was replenished every 3–4 days and cells were used for the experiment at day 21. At day 24, cells were fixed and immunostaining was performed. For the neurite protection assay, PC12 cells were seeded to six-well plates at a seeding density of 5.0 104 cells/ scaffold (empirically determined as the optimal seeding density) and incubated for 24–48 h until a cell confluency of 60–70% was reached. Then, PC12 cells were differentiated with NGF (50 ng/ml) for 72–120 h and treated with nocodazole (1 mM) for 1 h to induce neurite degeneration. After nocodazole treatment, the old media containing nocodazole were switched with fresh media
For coculture with rat PC12 cells, we chose CRL-2005 cells, type 1 phenotype astrocytes that are derived from rat brain. CRL-2005 cells were grown in T-75 cm2 flasks in DMEM supplemented with 10% FBS and 0.5% penicillinstreptomycin. The medium was changed every other day and cells were split at 80% confluence at a ratio of 1 : 6. As CRL-2005 cells grow easily in a variety of media, we chose to adapt the astrocytes to grow in the PC12 cell culture medium [7.5% FBS, 7.5% horse serum (ES), and 0.5% penicillin-streptomycin in DMEM]. Astrocyte-conditioned media (ACM) were prepared by incubating CRL-2005 cells with PC12 culture media for 24 h. For the coculture assay, we used six-well plates with cell culture inserts. In the neurite protection assay, PC12 cells were seeded on the well of a six-well plate first (1.0 105 cell/well) and differentiated with NGF (50 ng/ml) for 72 h, followed by nocodazole (1 mM) treatment for 1 h to induce neurite degeneration. After nocodazole treatment, CRL-2005 astrocyte cells were seeded (1.0 105 cell/well seeding density) on the cell culture insert and incubated with the PC12 cells in PC12 cell culture media for 3 additional days. Also, after nocodazole treatment, differentiated PC12 cells were cultured with ACM or with PC12 cell culture media for 3 additional days. In the neurite outgrowth assay, PC12 cells were seeded on a cell culture insert (1.0 105 cell/ well) and CRL-2005 cells were seeded at the bottom of the well and cultured together with NGF (50 ng/ml), or PC12 cells were cultured with ACM containing NGF or with PC12 cell culture media containing NGF. Neurite status was monitored per each day and after 72-h
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BMP2 showing neurotrophic effects Saglam et al. 551
of incubation, PC12 cells were either fixed for immunofluorescence assay or used for total neurite quantification. Cell proliferation
Cell proliferation was analyzed every second day over a period of up to 6 days using the Alamar Blue cell viability reagent (cat. no. DAL1100; Invitrogen, Carlsbad, California, USA). Briefly, the cells washed once in PBS and then incubated for 3 h with their respective growth medium supplemented with 5% Alamar Blue reagent. Supernatants were collected and their fluorescence intensity was evaluated by excitation at 560 nm and emission at 595 nm using an ELISA Synergy 4 reader (BioTek, Winooski, Vermont, USA). The data, expressed as mean±SD, are presented in arbitrary fluorescence units. Neurite quantification
For the quantification of total neurites, we used the neurite outgrowth assay kit (EMD Millipore) with a spectrophotometer. To induce neurite differentiation, PC12 cells were treated with NGF (50 ng/ml) for 72 h. For N1E-115 cells, the growth media were replaced with serum-free differentiation media (DMEM, 2 mM L-glutamine, 1% penicillin-streptomycin, and 1 mM sodium pyruvate) for 24 h. After the undersides of the Millicell inserts (EMD Millipore) were coated with fresh extracellular matrix protein (10 mg/ml collagen for PC12 cells and 10 mg/ml laminin for N1E-115 cells) for 2 h at 371C, 1.0 105 neuronal cells were seeded per insert, which were placed into each well of a 24-well plate. Cells were kept at room temperature for 15 min for attachment, and then a total of 700 ml differentiation medium was added per well (600 and 100 ml below and above the membrane, respectively). Neurites were allowed to extend for 3 days and then the inserts were fixed with – 2001C methanol for 20 min at room temperature, followed by fresh PBS rinse. Next, inserts were placed into 400 ml of neurite staining solution for 30 min at room temperature, and after cell bodies were removed by a moistened cotton swab, each insert was placed onto 100 ml neurite stain extraction buffer (EMD Millipore). Finally, the solutions were transferred into a 96-well plate and quantified on a spectrophotometer by reading absorbance at 562 nm. Immunofluorescence
After cell culture, growth media were removed and the cells were fixed with 10% formalin at room temperature for 15 min. Afterward, the cells were washed with a 0.5 M glycine solution in PBS and blocked overnight at 401C with 5% goat serum and 0.2% Triton-X solution in PBS. For immunostaining with primary antibodies, cells were incubated overnight at 401C with TUJ-1 monoclonal rabbit antibody against neuronal class III b-tubulin (1 : 200 dilution) for total neurite staining and with monoclonal mouse antibody against acetylated a-tubulin (1 : 100 dilution) for stable neurite staining. Once cells were washed
three times with 1 PBS buffer (10 min/wash), secondary antibodies – Texas Red goat anti-rabbit IgG (1 : 200 dilution) for TUJ-1 antibody and Alexa Fluor 488 goat anti-mouse IgG (1 : 200 dilution) for acetylated a-tubulin antibody – were added and incubated overnight at 401C. Subsequently, the cells were washed three times in 1 PBS buffer (10 min/wash) and 1 mg/ml 40 ,6-diamidino-2-phenylindole; DAPI was added after the second washing step for staining cell nuclei. After the final washing, cells were prepared for examination under a fluorescence microscope. The excitation and emission wavelengths are 488/519 nm for Alexa Fluor 488 IgG (green), 595/615 nm for Texas Red goat antirabbit IgG (red), and 405/461 nm for DAPI. Fluorescence images of the cells were acquired at different magnifications and analyzed by ‘ImageJ’ image processing and analysis program (NIH, Bethesda, Maryland, USA). Western blot
After cell culture, PC12 cells were collected into 1.5 ml eppendorf tubes, washed with 1 TBST buffer (1 TBS buffer with 0.1% Tween-20), and centrifuged to prepare cell pellets. RIPA buffer (1 ) (with 1 protein inhibitor cocktail + 0.1–1 mM phenylmethylsulfonyl fluoride) was added to each cell pellet for resuspension. For cell lysis, resuspended cell mixtures were frozen by liquid nitrogen for 1 min and thawed in a 371C water bath for 5 min. After performing a freeze–thaw cycle five times, the cell mixtures were transferred to a 1-ml syringe and sprayed repeatedly through a 27-G needle (3–5 times). Then, the cell mixtures were subjected to ultracentrifugation (15 000g for 20 min at 41C) and the supernatants were collected to determine their total protein concentration using the BCA protein assay kit. SDS-PAGE was carried out with a Novex 10–20% Trisglycine gradient gel, followed by a transfer process with a polyvinylidene fluoride (PVDF) membrane. After transfer, the PVDF membrane was blocked with 5% nonfat milk (in 1 TBST buffer) for either 1 h at room temperature or overnight at 41C. Incubation of primary antibodies [antiphosphorylated-Smad1 (Ser 463/Ser 465) antibody with a 1 : 500 dilution or antiphosphorylated-Akt 1/2/3 (Ser 473) antibody with 1 : 400 dilution or anti-b-actin antibody with 1 : 500 dilution] was performed for 1 h at room temperature. After washing with 1 TBST buffer, secondary antibodies – either anti-mouse or anti-rabbit HRP-conjugated IgG (1 : 8000 dilution) were incubated for another 1 h at room temperature. After final washing, manually prepared enhanced chemiluminescence was applied to the PVDF membrane for 5–10 min for film development.
Results BMP2 shows neurotrophic effects including neuroprotection in a neurodegenerative model
Microtubule polymerization is essential for axonal growth, and stabilization of neuronal microtubules facilitates axonal
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Fig. 1
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BMP2 exerts a neurotrophic effect on neuronal cells. (a) Quantification of total neurites remaining in differentiated PC12 cells after nocodazole treatment. Neurites produced by NGF (50 ng/ml) were first treated with nocodazole (1 mM) and incubated with fresh media containing BMP2 (50 ng/ml) for 3 days. Total neurites that survived were quantified on days 2 and 3 after nocodazole treatment by microscope images (NIS-Element BR software) and normalized (P < 0.001 vs. 0 h/media only = 100%, ANOVA). (b) Quantification of neurite outgrowth on neuronal cells. Rat PC12 cells and N1E-115 neuroblastoma cells were differentiated with NGF (50 ng/ml)±BMP2 (50 ng/ml). Quantification of total neurites outgrowth was performed spectrophotometrically using the neurite quantification kit (EMD Millipore) on day 3 and normalized (media only as 100%) (P < 0.005 vs. media only, ANOVA). (c) Quantification of neurite formation on the primary neuron. Primary cortical neurons were differentiated with or without BMP2 (50 ng/ml). Neurite/neuron status was qualified using immunofluorescence microscopic images using ImageJ software. All the quantifications were performed in triplicate. ANOVA, analysis of variance; NGF, nerve growth factor.
regeneration upon nerve injury [12]. Thus, we induced neurite degeneration in neuronal cells by interfering in neuritic microtubule dynamics with nocodazole. Differentiated rat PC12 cells were treated with nocodazole (1 mM) first and incubated with fresh media containing BMP2 (50 ng/ml) for 3 days. Quantification of remaining neurites showed that BMP2 significantly delayed nocodazoleinduced neurite degeneration (Fig. 1a). We further investigated how BMP2 affects neurite growth. The addition of BMP2 to the differentiation of neuronal cells (rat PC12 cells and N1E-115 neuroblastoma cells) actually promotes neurite development in both cells (60% increase for PC12 cells and 45% increase for N1E-115 cells, Fig. 1b). BMP2 also promotes differentiation of primary cortical neuron (Fig. 1c). Taken together, we suggest that BMP2 exerts a neurotrophic effect and also protects mature neurites from degeneration.
decreased the neurite outgrowth level with BMP2. However, when the cells were only treated with signaling inhibitors, the neurite outgrowth levels did not change much compared with the positive control (no inhibitor). Therefore, these data clearly showed that the BMP2induced neurotrophic effect is specifically related to Smad-dependent signaling and mTOR-related signaling.
BMP2-induced neurotrophic effects are involved in multiple signaling
BMP2 expresses neurite-protective effect by enhancing neurite microtubule structure
Next, we tested several cell signaling inhibitors to block the BMP2-induced effects on neuronal cells. In a neuroprotection assay (Fig. 2a), the level of total neurites in the presence of BMP2 was reduced to 46% with DMH1 (Smad inhibitor) and 41% with PP-242 (mTOR inhibitor). However, treatment with SB203850 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) showed almost no effect. Neurites treated with noggin, a known BMP2 inhibitor, also reduced the total neurite level down to 55%, proving that the protective effect of neurite was specifically induced by BMP2. Neurite quantification data from the neurite outgrowth assay (Fig. 2b) also showed that DMH-1 (48%) and PP-242 (49%) treatment
We investigated whether the BMP2-induced neuroprotection effect is directly related to microtubule stabilization. Immunofluorescence was performed using two different tubulin antibodies (acetylated a-tubulin antibody for stable neurites and TUJ-1 b-tubulin antibody for total neurites, Figure S1, Supplemental digital content, http://links.lww.com/WNR/A276). As shown in Fig. 3a, quantification of relative neurite stability indicated that BMP2 clearly stabilizes the neurite microtubule structure to delay nocodazole-induced neuritic degeneration. Also, treatment of different signaling inhibitors (Fig. 3b) showed similar results as in Fig. 2 – DMH-1 and PP-242 again specifically blocked the BMP2-induced neurite structure stabilization.
As PTEN acts as an antagonist against PI3K-Akt-mTOR signaling [13], we performed a neurite outgrowth assay with a PTEN inhibitor [bpV(pic), IC50 = 31 nM]. Treatment of bpV(pic) with BMP2 increased the neurite outgrowth level much higher than with treatment of bpV(pic) only (Fig. 2c). Western blot data (Fig. 2d) also confirmed that BMP2 clearly activates Akt protein as well as Smad1 protein. Taken together, we conclude that BMP2 exerts its neurotrophic effect through Smaddependent signaling and PI3K/PTEN-mTOR signaling.
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Fig. 2
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BMP2-induced neurotropic effect is involved in multiple signaling. (a) Quantification of total neurites on differentiated PC12 cells after nocodazole treatment. Neurites were first treated with nocodazole (1 mM) for 1 h and incubated with fresh media containing BMP2 (50 ng/ml) or BMP2 + different signaling inhibitors (10 mM/each) for 3 days. Quantification of total neurites was performed by measuring the total neurite length using the NIS-Element BR computer software with the microscope images each day after nocodazole treatment. DMH-1 was used as a Smad1 inhibitor, PP-242 was used as an mTOR inhibitor, SB203850 was used as a p38 MAPK inhibitor, and SP600125 was used as a JNK inhibitor. Noggin was used as a negative control (*P < 0.001 vs. no inhibitor, ANOVA). (b) Quantification of neurite outgrowth on differentiated N1E-115 cells. N1E-115 neuroblastoma cells were treated with differentiation medium first, followed by the addition of different signaling inhibitors (10 mM/each) and/or BMP2 (50 ng/ml) for 3 days. Neurite quantification was performed spectrophotometrically using the neurite quantification kit (EMD Millipore) on day 3 and normalized (no inhibitor/without BMP2 = 100%). DMH-1 was used as a Smad1 inhibitor, PP-242 was used as an mTOR inhibitor (*P < 0.001 vs. no inhibitor/without BMP2, ANOVA). (c) Quantification of neurite outgrowth on neuronal cells with potent PTEN inhibitor. Rat PC12 cells and N1E-115 neuroblastoma cells were differentiated using NGF (50 ng/ml) and/or potent PTEN inhibitor [bpV(pic), 10 mM] for 3 days. The effect of BMP2 (50 ng/ml) was also tested with each sample. Neurite quantification was performed spectrophotometrically using the neurite quantification kit (EMD Millipore) on day 3 and normalized (media only = 100%) (P < 0.001 vs. media only, ANOVA). All the quantifications were performed in triplicate. (d) BMP2 stimulated multiple cell signaling. Western blots were performed with the PC12 cell lysates using antiphosphorylated-Smad1 (Ser 463/Ser 465) antibody, followed by goat anti-rabbit HRP-coupled IgG. Lane 1, PC12 cells treated with BMP2 and DMH-1; lane 2, PC12 cells treated with BMP2 and noggin; lane 3, PC12 cells treated with BMP2 and PP-242; lane 4, PC12 cells treated with BMP2; lane 5, PC12 cells with growth media only. BMP2 stimulated PI3K-Akt-mTOR signaling. Western blot was performed with the PC12 cell lysates using antiphosphorylated-Akt 1/2/3 (Ser 473) antibody, followed by goat anti-rabbit HRP-coupled IgG. Lane 1, PC12 cells treated with BMP2 and PTEN inhibitor; lane 2, PC12 cells treated with PTEN inhibitor; lane 3, PC12 cells treated with BMP2; lane 4, PC12 cells treated with growth media only. Relative quantifications were measured as a ratio of phosphorylated-Smad1 or phosphorylated-Akt versus b-actin (ImageJ software). ANOVA, analysis of variance; NGF, nerve growth factor.
Thus, we confirmed that BMP2 exerts a neuroprotective effect through multiple signaling pathways by enhancing the neuronal microtubule structure.
Discussion In the nerve regeneration process, BMPs specifically stabilized damaged neurons from further degeneration and also promoted axonal regeneration after injury
[7–9,14]. However, as different studies also reported that administration of BMPs in CNS injury triggers reactive astrogliosis to form glial scar formation [15] or actually prevents nerve regeneration [10,16], the effect of BMP treatment after nerve injury is still controversial. As microtubule stabilization is considered critical for rescue of spinal cord injury by promoting axonal regeneration ability and neuronal polarization [17,18],
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Fig. 3
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BMP2 is effective in stabilization of neurite microtubule structure in a cell signaling-dependent manner. Differentiated PC12 cells were first treated with nocodazole (1 mM) for 1 h and incubated with (a) fresh media containing BMP2 (50 ng/ml) or (b) BMP2 + different signaling inhibitors (10 mM/each) for 3 days. DMH-1 was used as a Smad1 inhibitor, PP-242 as an mTOR inhibitor, and noggin as a BMP-BMPR binding inhibitor as a negative control. Relative neurite stability was calculated as the ratio of green/red fluorescence signal intensities from immunofluorescence images using ImageJ software. All fluorescence signal intensities were measured at least three times per each sample for green/red ratio calculation and normalized (no Nocodazole = 100%) (P < 0.0001 vs. No Nocodazole ANOVA).
we used nocodazole to induce neuritic degeneration and showed that BMP2 exerts neurotrophic effects including a neuroprotective effect against neurodegeneration in neuronal cells and primary neurons (Fig. 1). In the mechanism study, we found that BMP2-induced neurotrophic effects including neuroprotection were selectively reduced by blocking Smad-dependent signaling and mTOR-related signaling. It is likely that Smad-dependent signaling might be related to BMP2-induced neuroprotection because Smad-dependent signaling is directly related to TGF-b signaling [19]. Moreover, Parikh et al. [7] and Ueki et al. [20] have already reported that BMPs promotes axon regeneration after mouse spinal cord injury through reactivation of Smad-dependent signaling.
However, there has been no previous report showing that the neurotrophic effects induced by BMPs are directly related to mTOR-related signaling. The fact that treatment of PP-242 without BMP2 showed almost no effect on neurite outgrowth inhibition (Fig. 2b) indicated that the decrease in BMP2-induced neuroprotection by PP-242 is not because of endogenous mTOR activity congenitally effective in neurite outgrowth [21]. Also, neurite outgrowth assays using a potent PTEN inhibitor bpV(pic) clearly showed that the highest neurite outgrowth level was achieved on treatment of BMP2 and bpV(pic) (Fig. 2c). As PTEN inhibition promotes PI3KmTOR signaling [13], these data confirm that BMP2 is specifically related to PI3K/PTEN-mTOR signaling to exert its neurotrophic effects including neuroprotection. To our knowledge, this is the first case reporting the relationship between the BMP2-induced neuroprotection and PI3K/PTEN-mTOR signaling. Western blot data shown in Fig. 2d also confirmed that BMP2 activates PI3K-Akt-mTOR signaling. This finding is completely consistent with the recent finding that PI3K signaling regulates axon regeneration by inducing the expression of Smad1 [22]. Taken together, we now suggest that BMP2 exerts the neuroprotective effect through at least two cell signaling mechanisms – Smad-dependent signaling and PI3K-AKt-mTOR signaling. Considering the significance and complexity of the PI3K-Akt-mTOR signaling in cell growth, proliferation, and survival, we suspect that BMP2 may be related to multiple signaling networks, or may even have additional mechanism(s) for a neuroprotection effect. We also determined the effect of BMP2 on microtubule stability. As microtubule stability is closely related to the a-tubulin acetylation level [23], we immunostained stable neurites with antiacetylated a-tubulin antibody. Immunofluorescence data (Fig. 3) showed that BMP2 actually stabilized the neurite microtubule structure to delay neurite degeneration and that the BMP2-induced microtubule stabilization effect can be reduced by blocking Smad-dependent signaling and mTOR-related signaling. These findings are consistent with previous studies reporting that TGF-b1 induces stable microtubule in 3T3 fibroblasts [24] or that BMP signaling is required for a normal axonal and neuromuscular junction microtubule cytoskeleton and stabilization [25,26].
Conclusion We investigated whether BMP2 is effective in neurodegenerative conditions using an in-vitro neurodegenerative model. BMP2 successfully exerted neurotrophic effects including neuroprotection against neurodegeneration. The mechanism study also showed that BMP2-induced neuroprotection effects occur by multiple signaling, including Smad-dependent signaling and PI3K/PTEN-mTOR signaling, by directly enhancing the neuron microtubule structure. This is the first case to report that BMP2 can
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BMP2 showing neurotrophic effects Saglam et al. 555
exert a neuroprotective effect against nerve degeneration by stabilizing neuronal system structure integrity. Currently, microtubule stabilization is focused on rescuing spinal cord injury by reducing fibrotic scarring and promoting axonal regeneration ability [12,17,18]. Thus, BMP2 can be a potential therapeutic target for nerve regeneration after nerve injury including CNS injury as well as for delaying neurodegenerative progress.
Acknowledgements
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Conflicts of interest
There are no conflicts of interest.
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Barghorn S, Nimmrich V, Striebinger A, Krantz C, Keller P, Janson B, et al. Globular amyloid b-peptide 1–42 oligomer – a homogenous and stable neuropathlological protein in Alzheimer’s disease. J Neurochem 2005; 95:834–847. Sengottuvel V, Fischer D. Facilitating axon regeneration in the injured CNS by microtubules stabilization. Commun Integr Biol 2011; 4:391–393. Zhang S, Yu D. PI(3)king apart PTEN’s role in cancer. Clin Cancer Res 2010; 16:4325–4330. Wu G, Ju L, Jin T, Chen L, Shao L, Wang Y, et al. Local delivery of recombinant human bone morphogenetic protein-2 increases axonal regeneration and the expression of tau protein after facial nerve injury. J Int Med Res 2010; 38:1682–1688. Sahni V, Mukhopadhyay A, Tysseling V, Hebert A, Birch D, Mcguire TL, et al. BMPR1a and BMPR1b signaling exert opposing effects on gliosis after spinal cord injury. J Neurosci 2010; 30:1839–1855. Dmitriev AE, Farhang S, Lehman RA Jr, Ling GS, Symes AJ. Bone morphogenetic protein-2 used in spinal fusion with spinal cord injury penetrates intrathecally and elicits a functional signaling cascade. Spine J 2010; 10:16–25. Hellal F, Hurtado A, Ruschel J, Flynn KC, Laskowski CJ, Umlauf M, et al. Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury. Science 2011; 331:928–931. Witte H, Neukirchen D, Bradke F. Microtubule stabilization specifies initial neuronal polarization. J Cell Biol 2008; 180:619–632. Zi Z, Chapnick DA, Liu X. Dynamics of TGF-b/Smad signaling. FEBS Lett 2012; 586:1921–1928. Ueki Y, Reh TA. Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo. PLoS One 2012; 7:e38690. Park KK, Liu K, Hu Y, Smith PD, Cai B, Xu B, et al. Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 2008; 322:963–966. Saijilafu, Hur EM, Liu CM, Jiao Z, Xu WN, Zhou FQ. PI3K–GSK3 signaling regulates mammalian axon regeneration by inducing the expression of Smad1. Nat Commun 2013; 4:2690. Takemura R, Okabe S, Umeyama T, Kanai Y, Cowan NJ, Hirokawa N. Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau. J Cell Sci 1992; 103:953–964. Gundersen GG, Kim I, Chapin CJ. Induction of stable microtubules in 3T3 fibroblasts by TGF-beta and serum. J Cell Sci 1994; 107:645–659. Wang X, Shaw WR, Tsang HT, Reid E, O’Kane CJ. Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules. Nat Neurosci 2007; 10:177–185. Barres BA. The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 2008; 60:430–440.
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BMP2 shows neurotrophic effects including neuroprotection against neurodegeneration Aybike Saglama,*, Sukbum Kimb,*, Kwangwook Ahna, Insuk Ohc and Kwan-Hee Leea In this study, we found that BMP2 exerts neurotrophic effects, including a neuroprotective effect against nocodazole-induced neuritic degeneration, on neuronal cells. We also found that BMP2-induced neurotrophic effects are directly involved in Smad-dependent signaling as well as PI3K/PTEN-Akt-mTOR signaling. Moreover, BMP2-induced neurotrophic effects occur by stabilization of neuronal microtubules. Thus, these findings suggest that BMP2 can be a potential therapeutic target for nerve c 2014 Wolters injury treatment. NeuroReport 25:549–555 Kluwer Health | Lippincott Williams & Wilkins.
Keywords: bone morphogenetic protein 2, microtubule, neuroprotection, PI3K, PTEN a TissueGene Inc., Rockville, Maryland, USA, bDepartment of Orthopedic Surgery, Gil Medical Center, Gachon University, Incheon and cDepartment of Orthopedics, The Uniformed Force Capital Hospital, Kyunggido, Republic of Korea
Correspondence to Kwan-Hee Lee, MD, PhD, TissueGene Inc., 9605 Medical Center Drive, Suite 200, Rockville, MD 20850, USA Tel: + 1 301 921 6000 x101; fax: + 1 301 921 6011; e-mail: [email protected] *Aybike Saglam and Sukbum Kim contributed equally to the writing of this article. Received 20 December 2013 accepted 13 January 2014
NeuroReport 2014, 25:549–555
Introduction In an adult mammalian nerve system, functional recovery of injured neurons hardly occurs because of lack of intrinsic axonal regeneration ability and upregulation of extracellular inhibitory molecules [1,2]. Previous trials on restoration of nerve regeneration ability by blocking extracellular inhibitory factors have been unsuccessful [3]. Thus, restoration of intrinsic nerve regrowth ability is the current focus as a therapeutic target for treatment of central nervous system (CNS) injury in adults [4]. Bone morphogenetic proteins (BMPs) are a member of the transforming growth factor-b (TGF-b) superfamily and participate in many signaling pathways to regulate the proliferation, differentiation, and survival of many cell types [5,6]. Moreover, recent studies have shown that BMPs exert significant neuroprotection/nerve regeneration effects after adult CNS injury [7–9]. However, the effect of BMPs treatment after CNS injury is still controversial [10]. In this study, we have investigated the effect of BMP2 in an in-vitro neurodegenerative model. Delivery of BMP2 specifically exerts neurotrophic effects including neuroprotection against nocodazole-induced neuritic degeneration. Also, a BMP2-induced neuroprotective effect occurs through Smad-dependent signaling and PI3K-Akt-mTOR signaling by stabilization of neurite microtubules. Thus, our study suggests that BMP2 can be a successful target for therapeutic treatment for nerve injury as BMP2 can exert a neuroprotective effect in neurodegenerative Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.neuroreport.com). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4965
models through multiple signaling by enhancing neuronal system integrity.
Materials and methods Materials Cells
Both rat adrenal medullary PC12 pheochromocytoma neuronal cells and mouse N1E-115 neuroblastoma cells were purchased from ATCC (Manassas, Virginia, USA). Reagents
Cell culture materials including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and horse serum were purchased from Mediatech Inc. (Manassas, Virginia, USA). TUJ-1 monoclonal rabbit antibody against neuronal class III b-tubulin was purchased from Covance Inc. (Gaithersburg, Maryland, USA). Monoclonal mouse antibody against acetylated a-tubulin, Akt 1(B-1) mouse monoclonal antibody, phosphorylated-Akt 1/2/3 (Ser 473) rabbit polyclonal antibody, rabbit polyclonal phosphorylated-Smad1 (Ser 463/Ser 465) antibody, b-actin mouse monoclonal antibody, and goat anti-rabbit HRPcoupled IgG were purchased from Santa Cruz Biotech Inc. (Santa Cruz, California, USA). Rabbit polyclonal antibody against tyrosine hydroxylase was purchased from Chemicon International Inc. (Temecula, California, USA). Anti-mouse IgG, HRP-linked antibody was purchased from Cell Signaling Tech. (Danvers, Massachusetts, USA). Goat serum, Texas Red goat anti-rabbit IgG antibody, Alexa Fluor 488 goat anti-mouse IgG antibody, 40 ,6-diamidino-2phenylindole, dilactate (DAPI), and Alamar Blue were from Molecular Probes–Invitrogen Inc. (Eugene, Oregon, USA). 2.5S nerve growth factors (NGF) mouse natural and human DOI: 10.1097/WNR.0000000000000130
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recombinant BMP2 protein was purchased from R&D Systems (Minneapolis, Michigan, USA). Human recombinant noggin protein, DMH-1, PP-242, nocodazole, kainic acid, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and cresyl violet dye were purchased from Sigma-Aldrich (St Louis, Missouri, USA). Laminin, mouse, was purchased from BD Bioscience (Sparks Glencoe, Maryland, USA). Potassium bisperoxopicolinatooxovanadate [bpV(pic)] was purchased from Santa Cruz Biotech Inc. Biotinylated antirabbit IgG and avidin–biotin–peroxidase reagents (Elite Vectastain ABC kit) were purchased from Vector Labs (Burlingame, California, USA). Apparatus
Cellular apparatus, including T-75 cm2 culture flasks, a six-well cell culture multiwall plate, cell culture inserts for the six-well plate, and the neurite outgrowth assay kit, was purchased from EMD Millipore (Billerica, Massachusetts, USA). The BCA protein assay kit was purchased from Pierce–Thermo/Fisher Scientific (Rockford, Illinois, USA). Novex 10–20% Tris-glycine mini gels were purchased from Invitrogen Inc. Microscope (BX51) and digital microscope camera (DP72) were purchased from Olympus (Tokyo, Japan).
containing human recombinant BMP2 protein (50 ng/ml) or BMP2 protein plus signaling inhibitor (10 mM) and incubated for 3 additional days. Neurite status was monitored before nocodazole treatment, after nocodazole treatment, and every 24 h using a photomicroscope. After a 72-h incubation, PC12 cells were either fixed for immunofluorescence assay or analyzed using NIS-Element BR image software (Nikon, Linthicum, Maryland, USA) for total neurite quantification. For the neurite outgrowth assay, PC12 cells or N1E-115 cells were seeded into a six-well plate at a 1.0 105 cells/ well seeding density. After a cell confluency of 60–70% was reached, neuronal cell differentiation was initiated – with NGF (50 ng/ml) for PC12 cells and with differentiation media (DMEM, 2 mM L-glutamine, 1% penicillin-streptomycin, and 1 mM sodium pyruvate) for N1E-115 cells. After 24 h of incubation, human recombinant BMP2 protein (50 ng/ml) or BMP2 protein plus signaling inhibitor (10 mM) was added to the wells in six-well plates and incubated for two additional days. Neurite status was monitored every 24 h.
PC12/astrocyte coculture Methods Neuronal cell culture
Rat adrenal medullary PC12 rat pheochromocytoma neuronal cells were supplemented with 7.5% FBS, 7.5% horse serum (ES), and 0.5% penicillin-streptomycin in T-75 cm2 flasks that were maintained at 371C in a 5% CO2 incubator. Cells were split at 50% confluence by gently mechanically detaching them from the flask and propagated at a split ratio of 1 : 7. We also used the mouse N1E115 neuroblastoma cell line. This cell has 36 h of doubling time and a 1 : 3 subcultivation ratio. N1E-115 cells were grown in DMEM without sodium pyruvate and FBS was added at a final concentration of 10%. Rat brain cortex primary neurons were used (cat. no. R-Cx-500; Lonza, Walkersville, Maryland, USA) according to an established procedure [11]. Each vial was thawed and resuspended in 10 ml of growth medium according to the manufacturer’s recommendations, and 1 ml/well volume of cell suspension was seeded onto previously poly-D lysine-coated wells of 24-well plates. Medium was replenished every 3–4 days and cells were used for the experiment at day 21. At day 24, cells were fixed and immunostaining was performed. For the neurite protection assay, PC12 cells were seeded to six-well plates at a seeding density of 5.0 104 cells/ scaffold (empirically determined as the optimal seeding density) and incubated for 24–48 h until a cell confluency of 60–70% was reached. Then, PC12 cells were differentiated with NGF (50 ng/ml) for 72–120 h and treated with nocodazole (1 mM) for 1 h to induce neurite degeneration. After nocodazole treatment, the old media containing nocodazole were switched with fresh media
For coculture with rat PC12 cells, we chose CRL-2005 cells, type 1 phenotype astrocytes that are derived from rat brain. CRL-2005 cells were grown in T-75 cm2 flasks in DMEM supplemented with 10% FBS and 0.5% penicillinstreptomycin. The medium was changed every other day and cells were split at 80% confluence at a ratio of 1 : 6. As CRL-2005 cells grow easily in a variety of media, we chose to adapt the astrocytes to grow in the PC12 cell culture medium [7.5% FBS, 7.5% horse serum (ES), and 0.5% penicillin-streptomycin in DMEM]. Astrocyte-conditioned media (ACM) were prepared by incubating CRL-2005 cells with PC12 culture media for 24 h. For the coculture assay, we used six-well plates with cell culture inserts. In the neurite protection assay, PC12 cells were seeded on the well of a six-well plate first (1.0 105 cell/well) and differentiated with NGF (50 ng/ml) for 72 h, followed by nocodazole (1 mM) treatment for 1 h to induce neurite degeneration. After nocodazole treatment, CRL-2005 astrocyte cells were seeded (1.0 105 cell/well seeding density) on the cell culture insert and incubated with the PC12 cells in PC12 cell culture media for 3 additional days. Also, after nocodazole treatment, differentiated PC12 cells were cultured with ACM or with PC12 cell culture media for 3 additional days. In the neurite outgrowth assay, PC12 cells were seeded on a cell culture insert (1.0 105 cell/ well) and CRL-2005 cells were seeded at the bottom of the well and cultured together with NGF (50 ng/ml), or PC12 cells were cultured with ACM containing NGF or with PC12 cell culture media containing NGF. Neurite status was monitored per each day and after 72-h
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BMP2 showing neurotrophic effects Saglam et al. 551
of incubation, PC12 cells were either fixed for immunofluorescence assay or used for total neurite quantification. Cell proliferation
Cell proliferation was analyzed every second day over a period of up to 6 days using the Alamar Blue cell viability reagent (cat. no. DAL1100; Invitrogen, Carlsbad, California, USA). Briefly, the cells washed once in PBS and then incubated for 3 h with their respective growth medium supplemented with 5% Alamar Blue reagent. Supernatants were collected and their fluorescence intensity was evaluated by excitation at 560 nm and emission at 595 nm using an ELISA Synergy 4 reader (BioTek, Winooski, Vermont, USA). The data, expressed as mean±SD, are presented in arbitrary fluorescence units. Neurite quantification
For the quantification of total neurites, we used the neurite outgrowth assay kit (EMD Millipore) with a spectrophotometer. To induce neurite differentiation, PC12 cells were treated with NGF (50 ng/ml) for 72 h. For N1E-115 cells, the growth media were replaced with serum-free differentiation media (DMEM, 2 mM L-glutamine, 1% penicillin-streptomycin, and 1 mM sodium pyruvate) for 24 h. After the undersides of the Millicell inserts (EMD Millipore) were coated with fresh extracellular matrix protein (10 mg/ml collagen for PC12 cells and 10 mg/ml laminin for N1E-115 cells) for 2 h at 371C, 1.0 105 neuronal cells were seeded per insert, which were placed into each well of a 24-well plate. Cells were kept at room temperature for 15 min for attachment, and then a total of 700 ml differentiation medium was added per well (600 and 100 ml below and above the membrane, respectively). Neurites were allowed to extend for 3 days and then the inserts were fixed with – 2001C methanol for 20 min at room temperature, followed by fresh PBS rinse. Next, inserts were placed into 400 ml of neurite staining solution for 30 min at room temperature, and after cell bodies were removed by a moistened cotton swab, each insert was placed onto 100 ml neurite stain extraction buffer (EMD Millipore). Finally, the solutions were transferred into a 96-well plate and quantified on a spectrophotometer by reading absorbance at 562 nm. Immunofluorescence
After cell culture, growth media were removed and the cells were fixed with 10% formalin at room temperature for 15 min. Afterward, the cells were washed with a 0.5 M glycine solution in PBS and blocked overnight at 401C with 5% goat serum and 0.2% Triton-X solution in PBS. For immunostaining with primary antibodies, cells were incubated overnight at 401C with TUJ-1 monoclonal rabbit antibody against neuronal class III b-tubulin (1 : 200 dilution) for total neurite staining and with monoclonal mouse antibody against acetylated a-tubulin (1 : 100 dilution) for stable neurite staining. Once cells were washed
three times with 1 PBS buffer (10 min/wash), secondary antibodies – Texas Red goat anti-rabbit IgG (1 : 200 dilution) for TUJ-1 antibody and Alexa Fluor 488 goat anti-mouse IgG (1 : 200 dilution) for acetylated a-tubulin antibody – were added and incubated overnight at 401C. Subsequently, the cells were washed three times in 1 PBS buffer (10 min/wash) and 1 mg/ml 40 ,6-diamidino-2-phenylindole; DAPI was added after the second washing step for staining cell nuclei. After the final washing, cells were prepared for examination under a fluorescence microscope. The excitation and emission wavelengths are 488/519 nm for Alexa Fluor 488 IgG (green), 595/615 nm for Texas Red goat antirabbit IgG (red), and 405/461 nm for DAPI. Fluorescence images of the cells were acquired at different magnifications and analyzed by ‘ImageJ’ image processing and analysis program (NIH, Bethesda, Maryland, USA). Western blot
After cell culture, PC12 cells were collected into 1.5 ml eppendorf tubes, washed with 1 TBST buffer (1 TBS buffer with 0.1% Tween-20), and centrifuged to prepare cell pellets. RIPA buffer (1 ) (with 1 protein inhibitor cocktail + 0.1–1 mM phenylmethylsulfonyl fluoride) was added to each cell pellet for resuspension. For cell lysis, resuspended cell mixtures were frozen by liquid nitrogen for 1 min and thawed in a 371C water bath for 5 min. After performing a freeze–thaw cycle five times, the cell mixtures were transferred to a 1-ml syringe and sprayed repeatedly through a 27-G needle (3–5 times). Then, the cell mixtures were subjected to ultracentrifugation (15 000g for 20 min at 41C) and the supernatants were collected to determine their total protein concentration using the BCA protein assay kit. SDS-PAGE was carried out with a Novex 10–20% Trisglycine gradient gel, followed by a transfer process with a polyvinylidene fluoride (PVDF) membrane. After transfer, the PVDF membrane was blocked with 5% nonfat milk (in 1 TBST buffer) for either 1 h at room temperature or overnight at 41C. Incubation of primary antibodies [antiphosphorylated-Smad1 (Ser 463/Ser 465) antibody with a 1 : 500 dilution or antiphosphorylated-Akt 1/2/3 (Ser 473) antibody with 1 : 400 dilution or anti-b-actin antibody with 1 : 500 dilution] was performed for 1 h at room temperature. After washing with 1 TBST buffer, secondary antibodies – either anti-mouse or anti-rabbit HRP-conjugated IgG (1 : 8000 dilution) were incubated for another 1 h at room temperature. After final washing, manually prepared enhanced chemiluminescence was applied to the PVDF membrane for 5–10 min for film development.
Results BMP2 shows neurotrophic effects including neuroprotection in a neurodegenerative model
Microtubule polymerization is essential for axonal growth, and stabilization of neuronal microtubules facilitates axonal
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Fig. 1
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BMP2 exerts a neurotrophic effect on neuronal cells. (a) Quantification of total neurites remaining in differentiated PC12 cells after nocodazole treatment. Neurites produced by NGF (50 ng/ml) were first treated with nocodazole (1 mM) and incubated with fresh media containing BMP2 (50 ng/ml) for 3 days. Total neurites that survived were quantified on days 2 and 3 after nocodazole treatment by microscope images (NIS-Element BR software) and normalized (P < 0.001 vs. 0 h/media only = 100%, ANOVA). (b) Quantification of neurite outgrowth on neuronal cells. Rat PC12 cells and N1E-115 neuroblastoma cells were differentiated with NGF (50 ng/ml)±BMP2 (50 ng/ml). Quantification of total neurites outgrowth was performed spectrophotometrically using the neurite quantification kit (EMD Millipore) on day 3 and normalized (media only as 100%) (P < 0.005 vs. media only, ANOVA). (c) Quantification of neurite formation on the primary neuron. Primary cortical neurons were differentiated with or without BMP2 (50 ng/ml). Neurite/neuron status was qualified using immunofluorescence microscopic images using ImageJ software. All the quantifications were performed in triplicate. ANOVA, analysis of variance; NGF, nerve growth factor.
regeneration upon nerve injury [12]. Thus, we induced neurite degeneration in neuronal cells by interfering in neuritic microtubule dynamics with nocodazole. Differentiated rat PC12 cells were treated with nocodazole (1 mM) first and incubated with fresh media containing BMP2 (50 ng/ml) for 3 days. Quantification of remaining neurites showed that BMP2 significantly delayed nocodazoleinduced neurite degeneration (Fig. 1a). We further investigated how BMP2 affects neurite growth. The addition of BMP2 to the differentiation of neuronal cells (rat PC12 cells and N1E-115 neuroblastoma cells) actually promotes neurite development in both cells (60% increase for PC12 cells and 45% increase for N1E-115 cells, Fig. 1b). BMP2 also promotes differentiation of primary cortical neuron (Fig. 1c). Taken together, we suggest that BMP2 exerts a neurotrophic effect and also protects mature neurites from degeneration.
decreased the neurite outgrowth level with BMP2. However, when the cells were only treated with signaling inhibitors, the neurite outgrowth levels did not change much compared with the positive control (no inhibitor). Therefore, these data clearly showed that the BMP2induced neurotrophic effect is specifically related to Smad-dependent signaling and mTOR-related signaling.
BMP2-induced neurotrophic effects are involved in multiple signaling
BMP2 expresses neurite-protective effect by enhancing neurite microtubule structure
Next, we tested several cell signaling inhibitors to block the BMP2-induced effects on neuronal cells. In a neuroprotection assay (Fig. 2a), the level of total neurites in the presence of BMP2 was reduced to 46% with DMH1 (Smad inhibitor) and 41% with PP-242 (mTOR inhibitor). However, treatment with SB203850 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) showed almost no effect. Neurites treated with noggin, a known BMP2 inhibitor, also reduced the total neurite level down to 55%, proving that the protective effect of neurite was specifically induced by BMP2. Neurite quantification data from the neurite outgrowth assay (Fig. 2b) also showed that DMH-1 (48%) and PP-242 (49%) treatment
We investigated whether the BMP2-induced neuroprotection effect is directly related to microtubule stabilization. Immunofluorescence was performed using two different tubulin antibodies (acetylated a-tubulin antibody for stable neurites and TUJ-1 b-tubulin antibody for total neurites, Figure S1, Supplemental digital content, http://links.lww.com/WNR/A276). As shown in Fig. 3a, quantification of relative neurite stability indicated that BMP2 clearly stabilizes the neurite microtubule structure to delay nocodazole-induced neuritic degeneration. Also, treatment of different signaling inhibitors (Fig. 3b) showed similar results as in Fig. 2 – DMH-1 and PP-242 again specifically blocked the BMP2-induced neurite structure stabilization.
As PTEN acts as an antagonist against PI3K-Akt-mTOR signaling [13], we performed a neurite outgrowth assay with a PTEN inhibitor [bpV(pic), IC50 = 31 nM]. Treatment of bpV(pic) with BMP2 increased the neurite outgrowth level much higher than with treatment of bpV(pic) only (Fig. 2c). Western blot data (Fig. 2d) also confirmed that BMP2 clearly activates Akt protein as well as Smad1 protein. Taken together, we conclude that BMP2 exerts its neurotrophic effect through Smaddependent signaling and PI3K/PTEN-mTOR signaling.
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BMP2 showing neurotrophic effects Saglam et al. 553
Fig. 2
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BMP2-induced neurotropic effect is involved in multiple signaling. (a) Quantification of total neurites on differentiated PC12 cells after nocodazole treatment. Neurites were first treated with nocodazole (1 mM) for 1 h and incubated with fresh media containing BMP2 (50 ng/ml) or BMP2 + different signaling inhibitors (10 mM/each) for 3 days. Quantification of total neurites was performed by measuring the total neurite length using the NIS-Element BR computer software with the microscope images each day after nocodazole treatment. DMH-1 was used as a Smad1 inhibitor, PP-242 was used as an mTOR inhibitor, SB203850 was used as a p38 MAPK inhibitor, and SP600125 was used as a JNK inhibitor. Noggin was used as a negative control (*P < 0.001 vs. no inhibitor, ANOVA). (b) Quantification of neurite outgrowth on differentiated N1E-115 cells. N1E-115 neuroblastoma cells were treated with differentiation medium first, followed by the addition of different signaling inhibitors (10 mM/each) and/or BMP2 (50 ng/ml) for 3 days. Neurite quantification was performed spectrophotometrically using the neurite quantification kit (EMD Millipore) on day 3 and normalized (no inhibitor/without BMP2 = 100%). DMH-1 was used as a Smad1 inhibitor, PP-242 was used as an mTOR inhibitor (*P < 0.001 vs. no inhibitor/without BMP2, ANOVA). (c) Quantification of neurite outgrowth on neuronal cells with potent PTEN inhibitor. Rat PC12 cells and N1E-115 neuroblastoma cells were differentiated using NGF (50 ng/ml) and/or potent PTEN inhibitor [bpV(pic), 10 mM] for 3 days. The effect of BMP2 (50 ng/ml) was also tested with each sample. Neurite quantification was performed spectrophotometrically using the neurite quantification kit (EMD Millipore) on day 3 and normalized (media only = 100%) (P < 0.001 vs. media only, ANOVA). All the quantifications were performed in triplicate. (d) BMP2 stimulated multiple cell signaling. Western blots were performed with the PC12 cell lysates using antiphosphorylated-Smad1 (Ser 463/Ser 465) antibody, followed by goat anti-rabbit HRP-coupled IgG. Lane 1, PC12 cells treated with BMP2 and DMH-1; lane 2, PC12 cells treated with BMP2 and noggin; lane 3, PC12 cells treated with BMP2 and PP-242; lane 4, PC12 cells treated with BMP2; lane 5, PC12 cells with growth media only. BMP2 stimulated PI3K-Akt-mTOR signaling. Western blot was performed with the PC12 cell lysates using antiphosphorylated-Akt 1/2/3 (Ser 473) antibody, followed by goat anti-rabbit HRP-coupled IgG. Lane 1, PC12 cells treated with BMP2 and PTEN inhibitor; lane 2, PC12 cells treated with PTEN inhibitor; lane 3, PC12 cells treated with BMP2; lane 4, PC12 cells treated with growth media only. Relative quantifications were measured as a ratio of phosphorylated-Smad1 or phosphorylated-Akt versus b-actin (ImageJ software). ANOVA, analysis of variance; NGF, nerve growth factor.
Thus, we confirmed that BMP2 exerts a neuroprotective effect through multiple signaling pathways by enhancing the neuronal microtubule structure.
Discussion In the nerve regeneration process, BMPs specifically stabilized damaged neurons from further degeneration and also promoted axonal regeneration after injury
[7–9,14]. However, as different studies also reported that administration of BMPs in CNS injury triggers reactive astrogliosis to form glial scar formation [15] or actually prevents nerve regeneration [10,16], the effect of BMP treatment after nerve injury is still controversial. As microtubule stabilization is considered critical for rescue of spinal cord injury by promoting axonal regeneration ability and neuronal polarization [17,18],
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Fig. 3
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BMP2 is effective in stabilization of neurite microtubule structure in a cell signaling-dependent manner. Differentiated PC12 cells were first treated with nocodazole (1 mM) for 1 h and incubated with (a) fresh media containing BMP2 (50 ng/ml) or (b) BMP2 + different signaling inhibitors (10 mM/each) for 3 days. DMH-1 was used as a Smad1 inhibitor, PP-242 as an mTOR inhibitor, and noggin as a BMP-BMPR binding inhibitor as a negative control. Relative neurite stability was calculated as the ratio of green/red fluorescence signal intensities from immunofluorescence images using ImageJ software. All fluorescence signal intensities were measured at least three times per each sample for green/red ratio calculation and normalized (no Nocodazole = 100%) (P < 0.0001 vs. No Nocodazole ANOVA).
we used nocodazole to induce neuritic degeneration and showed that BMP2 exerts neurotrophic effects including a neuroprotective effect against neurodegeneration in neuronal cells and primary neurons (Fig. 1). In the mechanism study, we found that BMP2-induced neurotrophic effects including neuroprotection were selectively reduced by blocking Smad-dependent signaling and mTOR-related signaling. It is likely that Smad-dependent signaling might be related to BMP2-induced neuroprotection because Smad-dependent signaling is directly related to TGF-b signaling [19]. Moreover, Parikh et al. [7] and Ueki et al. [20] have already reported that BMPs promotes axon regeneration after mouse spinal cord injury through reactivation of Smad-dependent signaling.
However, there has been no previous report showing that the neurotrophic effects induced by BMPs are directly related to mTOR-related signaling. The fact that treatment of PP-242 without BMP2 showed almost no effect on neurite outgrowth inhibition (Fig. 2b) indicated that the decrease in BMP2-induced neuroprotection by PP-242 is not because of endogenous mTOR activity congenitally effective in neurite outgrowth [21]. Also, neurite outgrowth assays using a potent PTEN inhibitor bpV(pic) clearly showed that the highest neurite outgrowth level was achieved on treatment of BMP2 and bpV(pic) (Fig. 2c). As PTEN inhibition promotes PI3KmTOR signaling [13], these data confirm that BMP2 is specifically related to PI3K/PTEN-mTOR signaling to exert its neurotrophic effects including neuroprotection. To our knowledge, this is the first case reporting the relationship between the BMP2-induced neuroprotection and PI3K/PTEN-mTOR signaling. Western blot data shown in Fig. 2d also confirmed that BMP2 activates PI3K-Akt-mTOR signaling. This finding is completely consistent with the recent finding that PI3K signaling regulates axon regeneration by inducing the expression of Smad1 [22]. Taken together, we now suggest that BMP2 exerts the neuroprotective effect through at least two cell signaling mechanisms – Smad-dependent signaling and PI3K-AKt-mTOR signaling. Considering the significance and complexity of the PI3K-Akt-mTOR signaling in cell growth, proliferation, and survival, we suspect that BMP2 may be related to multiple signaling networks, or may even have additional mechanism(s) for a neuroprotection effect. We also determined the effect of BMP2 on microtubule stability. As microtubule stability is closely related to the a-tubulin acetylation level [23], we immunostained stable neurites with antiacetylated a-tubulin antibody. Immunofluorescence data (Fig. 3) showed that BMP2 actually stabilized the neurite microtubule structure to delay neurite degeneration and that the BMP2-induced microtubule stabilization effect can be reduced by blocking Smad-dependent signaling and mTOR-related signaling. These findings are consistent with previous studies reporting that TGF-b1 induces stable microtubule in 3T3 fibroblasts [24] or that BMP signaling is required for a normal axonal and neuromuscular junction microtubule cytoskeleton and stabilization [25,26].
Conclusion We investigated whether BMP2 is effective in neurodegenerative conditions using an in-vitro neurodegenerative model. BMP2 successfully exerted neurotrophic effects including neuroprotection against neurodegeneration. The mechanism study also showed that BMP2-induced neuroprotection effects occur by multiple signaling, including Smad-dependent signaling and PI3K/PTEN-mTOR signaling, by directly enhancing the neuron microtubule structure. This is the first case to report that BMP2 can
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BMP2 showing neurotrophic effects Saglam et al. 555
exert a neuroprotective effect against nerve degeneration by stabilizing neuronal system structure integrity. Currently, microtubule stabilization is focused on rescuing spinal cord injury by reducing fibrotic scarring and promoting axonal regeneration ability [12,17,18]. Thus, BMP2 can be a potential therapeutic target for nerve regeneration after nerve injury including CNS injury as well as for delaying neurodegenerative progress.
Acknowledgements
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Conflicts of interest
There are no conflicts of interest.
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