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ProBDNF inhibits proliferation, migration and differentiation of mouse neural stem cells
Accepted Manuscript Research report ProBDNF inhibits proliferation, migration and differentiation of mouse neural stem cells Jia-yi Li, Jia Liu, Nimshitha Pavathuparambil Abdul Manaph, Larisa Bobrovskaya, Xin-Fu Zhou PII: DOI: Reference:
S0006-8993(17)30209-3 http://dx.doi.org/10.1016/j.brainres.2017.05.013 BRES 45362
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
18 December 2016 19 April 2017 12 May 2017
Please cite this article as: J-y. Li, J. Liu, N.P.A. Manaph, L. Bobrovskaya, X-F. Zhou, ProBDNF inhibits proliferation, migration and differentiation of mouse neural stem cells, Brain Research (2017), doi: http://dx.doi.org/ 10.1016/j.brainres.2017.05.013
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ProBDNF inhibits proliferation, migration and differentiation of mouse neural stem cells
Jia-yi Li1*, Jia Liu2, Nimshitha Pavathuparambil Abdul Manaph1, Larisa Bobrovskaya1*, Xin-Fu Zhou1 1
School of Pharmacy and Medical Sciences, Division of Health Sciences, Sansom Institute for Health
Research, University of South Australia, Adelaide, SA 5000 2
Animal Research Centre, Kunming Medical University, Kunming, China
*
To whom the correspondence should be addressed
Ms Jia-yi Li School of Pharmacy and Medical Sciences, Division of Health Sciences, Sansom Institute for Health Research, University of South Australia, Adelaide, SA 5000. Email: [email protected] Telephone: +61 8 830 21807
Dr Larisa Bobrovskaya School of Pharmacy and Medical Sciences, University of South Australia, Adelaide SA 5000. Email: [email protected] Telephone: +61 8 830 21218, Fax: +61 8 830 22389
Keywords: Neural stem cell, proBDNF, migration, proliferation, differentiation
1
Abstract ProBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is an important regulator of neurodegeneration, hippocampal long-term depression, and synaptic plasticity. ProBDNF and its receptors pan-neurotrophin receptor p75 (p75NTR), vps10p domain-containing receptor Sortilin and tropomyosin receptor kinase B (TrkB) are expressed in neuronal and glial cells. The role of proBDNF in regulation of neurogenesis is not fully defined. This study aims to uncover the function of proBDNF in regulating the differentiation, migration and proliferation of mouse neural stem cells (NSCs) in vitro. We have found that proBDNF and its receptors are constitutively expressed in NSCs when assessed by immunocytochemistry and western blotting. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay showed that exogenous proBDNF treatment reduced mouse NSCs viability by 38% at 10 ng/mL. The migration of NSCs was also reduced by exogenous proBDNF treatment in a concentration-dependent manner (by 90% at 10 ng/mL) but increased by anti-proBDNF antibody treatment (by 50%). BrdU (5-Bromo-2´-Deoxyuridine) incorporation was performed for detection of newborn cells. We have found that proBDNF significantly inhibited proliferation of NSCs and reduced the number of differentiated neurons, oligodendrocytes and astrocytes, while anti-proBDNF antibody treatment promoted proliferation and differentiation of NSCs. In conclusion, proBDNF may oppose the functions of mature BDNF by inhibiting the proliferation, differentiation and migration of NSCs during development. Conversely, anti-proBDNF antibody
treatment
promoted
proliferation
and
differentiation
of
NSCs.
2
1.
Introduction
Neural stem cells (NSCs) are multipotent cells able to self-renew and proliferate (Temple, 2001). NSCs have the capacity to differentiate into sub-lineage neural cells: neurons, astrocytes and oligodendrocytes. Due to these characteristics, it is proposed that NSCs can be used for treatment of many neurodegenerative diseases and psychiatric disorders, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and bipolar depression (Taupin, 2011). NSCs derived from mouse embryonic brains have been broadly studied. Transplantation of NSCs was effective in recovering the CNS and brain functions after trauma and diseases in animal models (Vandenbosch et al., 2009) and is a promising strategy for replacement and regeneration of specific neuronal cells. In the embryonic period of rodents, NSCs develop into neuroblasts and glioblasts which migrate and differentiate into mature neurons and glia. Migration and differentiation of NSCs are crucial steps for the CNS formation (Preedy et al., 2011). Given the fact that embryonic neurogenesis highly requires the existence of neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 and neurotrophin 4 are very important regulators in embryogenesis. Since BDNF was purified about 35 years ago (Barde et al., 1982) its pleiotropic functions have been studied for decades. It has been identified to be important for neuronal cell survival, differentiation and synaptic plasticity (Maisonpierre et al., 1990). BDNF is firstly synthesized as a precursor protein termed proBDNF (Koshimizu et al., 2010). ProBDNF is a key neurotrophic factor regulating CNS development and hippocampal neurogenesis (Koshimizu et al., 2009; Yang et al., 2009). Mature BDNF (mBDNF) has the high affinity of binding to the tropomyosin receptor kinase B (TrkB, also known as NTRK2) while proBDNF preferentially binds to p75 neurotrophin receptor (p75NTR) combined with the co-receptor sortilin (also known as NTR3) (Nykjaer et al., 2004; Teng et al., 2005). P75NTR is a cell surface receptor with intracellular death domain belonging to the tumour necrosis factor family (Bassili et al., 2010). Researchers have found that in comparison to mBDNF, proBDNF functions adversely in neural system via activation of the proBDNF-p75NTR-sortilin complex and causes neuronal cell apoptosis, long-term depression and neurite outgrowth collapse (Sun et al., 2012; Teng et al., 2005). The ratio of TrkB/p75NTR and the balance of mBDNF/proBDNF can also regulate neurodegeneration and neuro-regeneration (Je et al., 2012; Kotlyanskaya et al., 2013). 3
Interestingly, proBDNF and p75NTR are found most highly expressed in perinatal stage of mice and then reduced but still detectable in adulthood. Neurons are a crucial source of proBDNF and mBDNF secretion (Yang et al., 2009) while astrocytes take part in cleaning up, recycling and rerelease of proBDNF (Bergami et al., 2008). In embryonic neuronal development and maturation, astrocytic differentiation occurs following neuronal differentiation (English et al., 2013). During the progression of embryogenesis, newly formed astrocytes might reduce proBDNF levels which may improve the NSCs differentiation, neuronal cell survival and proliferation. Despite that BDNF is reported to regulate proliferation and differentiation of NSCs (Chen et al., 2013) and promote survival of postmitotic neurons (Davies, 1994), no specific study has addressed the role of proBDNF in behaviors of NSCs. Whether proBDNF has any physiological functions in the early neurogenesis is still not clear. In this study, we hypothesized that proBDNF may negatively regulate proliferation, migration and differentiation during development. We confirmed that p75NTR and sortilin were strongly expressed and colocalized in NSCs, and proBDNF acted as an effective factor inhibiting proliferation, migration and differentiation of NSCs derived from mouse embryos.
2.
Results
2.1.
Neural stem cells express proBDNF and its receptors p75NTR, TrkB and Sortilin
By immunofluorescence staining with specific markers, we found proBDNF, p75NTR, Sortilin and TrkB were constitutively expressed in cultured mouse NSCs. In addition, we double-stained NSCs with p75NTR and Sortilin to verify the colocalization of these two co-effected receptors. Pearson’s correlation coefficient (PCC) measures r=0.89 in the image of double-staining by p75NTR and Sortilin (Fig. 1A). For the two co-receptors of proBDNF, 89% of the variability of p75NTR expression can be explained by the variability of Sortilin expression. Statistical analysis with PCC showed colocalization of p75NTR and Sortilin that further indicated the proBDNF-p75NTR-Sortilin complex was exerted in NSCs. ProBDNF, p75NTR, Sortilin and TrkB expressions in mouse NSCs were further confirmed by western blotting, with spinal cord tissue of wild type mice as a positive control (Fig. 1B). These findings demonstrate that proBDNF and its receptors p75NTR, Sortilin and
4
TrkB are highly expressed in NSCs, which indicates that proBDNF can regulate physiological functions of these cells.
2.2.
ProBDNF reduces the viability of neural stem cells
We performed MTT assay to further identify whether proBDNF would reduce viability of NSCs within 24 h treatment at the following concentrations; 0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL. As shown in Fig. 2, the percentage of viable NSCs was significantly decreased by proBDNF treatment compared with the control group (p<0.0001). Mature BDNF (30 ng/mL) or anti-proBDNF antibody (10 µg/mL) did not significantly enhance NSCs survival.
2.3.
ProBDNF reduces the migration of neural stem cells
In a Transwell migration assay, proBDNF showed significant inhibition of NSCs migration from top to the bottom of the wells (Fig. 3) compared with the control wells which were cultured in normal neural stem cell medium. All the proBDNF wells displayed a significant decrease in migrated NSCs numbers; while mBDNF (30 ng/mL) and anti-proBDNF antibody (10 µg/mL) displayed significant increases in migrated NSCs numbers (p<0.01, Fig. 3B). These results indicate that mBDNF and antiproBDNF antibody significantly improved NSCs migration.
2.4.
ProBDNF reduces the radial migration of neurospheres
For investigation of neurosphere radial migration, we recorded the radial migrated distances of each neurosphere cultured in different medium. We found that proBDNF at all doses (0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL) significantly inhibited the neurosphere migration after 48 h of culture (p<0.0001). In contrast, mBDNF (30 ng/mL) and anti-proBDNF antibody (10 µg/mL) showed a prominent increase in the neurosphere migration (1.5-fold) in the first 24 h (Fig. 4B). The increase of migration was maintained up to 48 h after anti-proBDNF antibody treatment but was not significant in the mBDNF- treated wells (Fig. 4D).
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2.5.
ProBDNF inhibits neural stem cell proliferation
Based on the results of BrdU incorporation assay, we found that proBDNF functioned as an inhibitor of NSCs proliferation in a dose-dependent manner in the concentration range between 0.1-10 ng/ml. Increasing concentrations of proBDNF in the cell culture medium produced statistically significant inhibition of NSCs proliferation at 1 ng/mL, 3 ng/mL, and 10 ng/ml as counted by the percentage of BrdU+/DAPI+ staining (Fig. 5C). Mature BDNF and anti-proBDNF antibody improved the proliferation of NSCs (p<0.05, Fig. 5B).
2.6.
ProBDNF regulates neural stem cell differentiation
To investigate the percentage of different lineages of neural differentiation, specific cell markers were investigated by immunostaining the differentiated cells. Tuj1 was used for neuron staining; GFAP for astrocyte staining and Olig2 for oligodendrocyte staining (Fig. 6). In the neuronal differentiation detection, we found that 10 ng/mL proBDNF in the culture medium reduced the number of Tuj1positive cells (p<0.05) and the negative effects of proBDNF on neuronal differentiation showed dosedependent responses (Fig. 6C). On contrary, mature BDNF in culture medium promoted the neuronal differentiation (p<0.0001) while anti-proBDNF antibody did not affect the number of neurons compared with the control group (Fig. 6B). In case of astrocytic differentiation, 10 ng/mL of proBDNF added to the culture medium significantly reduced the number of astrocytes compared with control wells which were cultured in the normal differentiation medium (p<0.05). The decreases in astrocytic differentiation were also dependent on the proBDNF added to the cell culture medium (Fig. 6F). Mature BDNF and anti-proBDNF antibodies did not appear as strong enhancers of astrocytic differentiation compared with the control wells in our experiments (Fig. 6E). In oligodendrocyte differentiation test, we found Olig2-posive cells were significantly decreased in the proBDNF-treated wells (Fig. 6G). 10 ng/mL proBDNF in the cell culture medium inhibited the oligodendrocyticdifferentiation of NSCs (p<0.0001). The decrease of Olig2-positive cells in response to proBDNF treatment was dose-dependent. The number of positively stained cells was calculated and analyzed with ImageJ and GraphPad Prism 6.
6
3.
Discussion
In this research, we have shown: 1) NSCs express proBDNF and its receptors p75NTR and Sortilin; 2) anti-proBDNF antibodies added to the cell culture promote the proliferation, migration and differentiation, indicating proBDNF may act in an autocrine and/or paracrine manner; 3) in all our experiments, mBDNF was administered as a positive control, which confirmed its enhancing role in the proliferation, migration and differentiation of NSCs. Thus, our study has contributed to the field by providing another evidence that mBDNF and proBDNF act as opposing factors regulating functions of the nervous system. Mature BDNF has been verified to regulate the differentiation of embryonic stem cells and neuronal precursors (Jung and Kang, 2001) and to stimulate the proliferation of NSCs (Chen et al., 2013) and migration of cerebellar granule cells during development of the nervous system (Borghesani et al., 2002). ProBDNF is reported to exhibit an inhibitory role in cerebellar granule cells migration that counterbalances the functions of mBDNF (Xu et al., 2011). It is known that proBDNF regulates the gradual formation of neuronal cells (Swistowski et al., 2009) and the apoptosis of Schwann cells (Teng et al., 2005), oligodendrocytes (Beattie et al., 2002) and sympathetic neurons (Harrington et al., 2004). Given the fact that mBDNF has a decisive role in regulating perinatal brain development (Liu and Graybiel, 1998) while proBDNF has the opposing biological functions in CNS (Lu et al., 2005), it is intriguing for researchers to investigate the role of proBDNF in early neural development and embryogenesis. Furthermore, abnormalities in the CNS maturation during embryogenesis may underlie neurodevelopmental disorders. Perinatal mBDNF abnormalities are believed to program many postnatal neurodegeneration events and early mBDNF dysfunction might participate in the development of chronic neurological diseases (Preedy et al., 2011). Our present study might provide additional information on the influences of proBDNF on NSCs which possibly contribute to the development of CNS and neurological disorders. The migration characteristics of NSCs are a critical step in the CNS development and momentous for the repair of neurodegenerative diseases (Feng et al., 2012). For mimicking the embryonic neural development in vitro, we performed the assays with embryo-derived NSCs which demonstrated the negative role of proBDNF in NSCs and neurosphere migration. Like another proneurotrophin proNGF, 7
relatively low concentrations of exogenous proBDNF (0.1 ng/mL) could significantly inhibit the migration of multipotent neural cells. After 48 h treatment with proBDNF, continuous inhibitory effects on migration were displayed in NSCs. Mature BDNF improved the radial migration of neurospheres in the first 24 h but did not show further influences in the following 24 h. Interestingly, anti-proBDNF antibody showed similar functions as mature BDNF which significantly enhanced the migration of neurospheres in the 24 h and 48 h assays. It is proposed that the mechanisms of proBDNF and mature BDNF in cellular biology would be a ‘yin and yang’ model (Lu et al., 2005). Some pharmacological studies have demonstrated that proBDNF and mature BDNF act as punishment and reward signals, respectively, and the conversion of proBDNF to mature BDNF is critical for many biological processes (Je et al., 2013; Lu et al., 2005). Therefore, the results from our assays that anti-proBDNF antibody extended the migration of NSCs in Figure 4 are not unexpected, and consistent with our previous study which shows that anti-proBDNF promotes the migration of cerebella granule neurons (Xu et al, 2010). While the function of proBDNF is blocked by antibodies, the signaling by mature BDNF dominates displaying the enhancement in NSCs migration. It has been demonstrated that NSCs migration is an essential process for the development of CNS as well as the ongoing neurogenesis that occurs in the mature CNS of most vertebrate species, including mammals (Imitola et al., 2004). Also, NSCs migrate to the sites of pathological insult in various types of brain injury (i.e., ischemia and blunt trauma) and tumours (Aboody et al., 2000; Sun et al., 2004). The migration toward damaged CNS tissue may represent an adaptive response with the purpose of limiting and/or repairing the damage, although to date there are limited data to definitively support or refute this hypothesis. Remarkably, the aberrant migration of neurospheres is a sign of many neurodegenerative or neural system diseases, such as brain tumours (Aboody et al., 2000), demyelination and multiple sclerosis (Park et al., 2002a), ischemia (Imitola et al., 2004; Park et al., 2002b), Alzheimer’s disease (Park et al., 2002a) and schizophrenia (Patruno et al., 2014). Thus, the microenvironmental conditions and factors that regulate NSCs migration are vitally important. In our study, proBDNF is found to significantly reduce neurospheres and NSCs migration, absolute numbers and proliferation. Regardless of the physiological role of NSCs in neural injury, the migratory
8
properties of NSCs may theoretically be exploited for cell-based therapeutics of neurodegenerative diseases in the future. CNS development is confirmed highly dependent on proliferation and differentiation of NSCs resulting in increasing cellular complexity over time. NSCs differentiate into innumerable types of neurons and glial cells which are progressively and temporally restricted during embryogenesis. In our BrdU incorporation assay, proBDNF effectively acted as an inhibitor of NSCs proliferation while mBDNF and anti-proBDNF antibody, on the contrary, acted as promoters of NSCs proliferation. Additionally, we found that exogenous proBDNF significantly decreased the viability of NSCs suggesting that proBDNF inhibits the proliferative process. BDNF has been reported to be able to elevate the number of newly generated neurons in the adult olfactory bulb (Zigova et al., 1998) and act as a neuronal differentiation factor to promote the neuronal differentiation of stem cells (Zhang et al., 2009). In our study, we obtained similar results that administration of mBDNF facilitated the neuronal differentiation by up to 4-fold. BDNF has been proved to enhance neurogenesis while the enhancement could be potentiated by concurrent inhibition of glial differentiation (Chmielnicki et al., 2004). Studies of cultured embryo-derived progenitor cells have elucidated that differentiation of embryonic stem cells increased the TrkB expression which was further upregulated in the presence of BDNF (Jung and Kang, 2001). TrkB expression is found restricted to the neuronal cells which may complementarily explain the enhancement of neuronal differentiation induced by mBDNF. Thus, the results described here may be interpreted such that mBDNF facilitates the neuronal differentiation or modulates the fate of uncommitted NSCs with neuronal potential. In the case of astrocyte and oligodendrocyte detection, a considerable decrease in the number of astrocytes and oligodendrocytes was observed in the proBDNF-treated cells while antiproBDNF antibody slightly enhanced the glial differentiation. Previous studies have shown that addition of BDNF in the microenvironment of neurospheres has no significant effect on the differentiation of astrocytes or oligodendrocytes (Ahmed et al., 1995). In our NSCs experiment, data is consistent with those published reports that mBDNF did not obviously improve the differentiation of astrocytes or oligodendrocytes.
9
In contrast to mBDNF, proBDNF inhibited the spontaneous differentiation of NSCs. After 7-day exposure to proBDNF, the numbers of Tuj1-positive, GFAP-positive and Olig2-positive cells were decreased compared with the control group. Furthermore, the tendency of decreased differentiation in proBDNF-treated NSCs occurred in a dose-dependent manner. To understand the possible role played by proBDNF in neural differentiation of NSCs, the expression of proBDNF corresponding receptors were examined by immunoreaction and western blot in NSCs. Mouse embryonic cells have been previously found to express p75NTR (Moscatelli et al., 2009). p75NTR was also proved to be constitutively expressed in NSCs in our study. Given further identification that NCSs possess both p75NTR and Sortilin receptors, it is conceivable that the activation of p75NTR-sortilin complex by exogenous proBDNF could lead to the retarded lineage differentiation in NSCs. To confirm this hypothesis, we blocked the action of endogenous proBDNF by anti-proBDNF antibody and found that the administration of anti-proBDNF antibody resulted in a significant increase of neuronal differentiation in NSCs suggesting the negative role of endogenous proBDNF in regulating neuronal differentiation. A notable observation made in this study is that functional blocking of endogenous proBDNF did not significantly elevate gliogenesis of NSCs similar to the effects of mBDNF observed in this study. This result is rarely addressed in other studies and may suggest that mBDNF and proBDNF may not be the decisive factors in the NSCs gliogenesis; alternatively, mBDNF and/or proBDNF might exert their actions on neurogenesis and gliogenesis differentially depending on the developmental stages. The precise role of proBDNF in neural development and mechanisms behind the phenomena obtained in our study need further investigation in the future.
4.
Experimental procedure
4.1.
Primary neural stem cells derivation and culture
The primary NSCs were obtained from the C57/BL6 strain mice following previously published work on primary embryonic stem cell culture with some adjustments (Wu et al., 2002). The C57 strain mice were bred by our own animal facility with the accordance of approved animal ethics. The primary NSCs were dissociated from the 14-day-old (E14) embryos and gently washed with phosphatebuffered saline (PBS) for three times and plated in the T-75 flasks for suspension cell culture. The 10
detailed procedures for E14 NSCs primary cell culture can be found in literature elsewhere (AyusoSacido, 2012). Primary NSCs were cultured in the basic NSC culture medium according to the published NSC culture medium recipes (Pollard et al., 2006), which included the B27 (2%) and basic fibroblast growth factor (bFGF, 20 ng/mL) and epidermal growth factor (EGF, 20 ng/mL). Half amount of the cell culture medium was changed every 3-4 days depending on the confluence of cells. Neurospheres were observed forming in 6-7 days and after the first passage (P1) of neurospheres they were expanded at the ratio of 1:2. The P2 NSCs were harvested and applied for experiments.
4.2.
Passaging and expansion of neurospheres
When the primary neurospheres reached 150-200 µm in diameter, they were subcultured according to the published protocols (Azari et al., 2011) with minor modifications. Generally, the medium with suspended spheres was transferred into a conical tube and centrifuged at 700 rpm (110 g) for 5 min at RT. The supernatant was discarded and neurospheres were resuspended in 0.05% trypsin (Sigma, Australia). The cell suspension was then incubated in a 37°C water bath for 2-3 min and added an equal volume of fetal bovine serum (Life Technology, Australia) to stop the trypsin activity. After gently mix-up the cell suspension was centrifuged at 700 rpm (110 g) for 5 min and the supernatant was removed. Cells were resuspended in complete NSC medium and plated at a concentration of 5104 cells/ mL in flasks. Secondary neurospheres were formed in 5-7 days when incubated at 37° C in a humidified incubator with 5% CO2.
4.3.
Cell viability assay
MTT assay was applied to investigate the cell viability according to different treatments added to the culture medium. Primary cultured NSCs were plated in 96-well plates with the density of 3,000-4,000 cells/well and incubated at 37˚C, 5% CO2 for 48 h. After 48 h, the NSCs were processed following the MTT assay protocol for adherent cells published previously (van Meerloo et al., 2001). Cell viability was calculated by measuring the absorbance at 490 nm with the Wallac Station (PerkinElmer, San Diego, CA, USA).
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4.4. 4.4.1.
Cell migration assay Analysis of neural stem cell migration with Millicell-insert
We investigated the migration of dissociated NSCs using 24-well Millicell-inserts (PIRP12R48, Merck Millipore, Australia). NSCs were dissociated to single cell suspension according to the manufacturer’s instructions and by nuclear staining with DAPI, and the number of cells migrated across the mesh from the wells was counted. The upper inserts were filled with 1×105 cells/mL suspended NSCs and cultured overnight at 37˚C, 5% CO2. After 24 h incubation, the cells on the upper surface were removed from the filter with a cotton swab and washed three times with PBS. The cells remaining on the bottom surface were fixed in ice-cold 4% paraformaldehyde (PF)/PBS solution and stained with DAPI for 20 min at room temperature (RT) and examined under a fluorescence microscope. Migrated NSCs were imaged with Olympus fluorescence microscope (Olympus, SA, Australia) and counted by ImageJ (National Institute of Health, USA). 10-15 views of migrated NSCs on filters were counted (20× objective) to obtain the number of migrating cells. We performed the migration assay as per the published protocol of migration assay by using Transwell inserts (Dufour et al., 2008) with some adjustments.
4.4.2.
Analysis of neurosphere radial migration
Primarily cultured neurospheres were obtained from C57BL/6 embryonic day 14 (E14) and kept as neurosphere cell lines stably. Prior to seeding neurospheres for the migration assay, 24-well plates were coated with Matrigel (BD Matrigel™, BD Biosciences, Australia). Single neurospheres from P2 onwards in the diameter of 500-600 µ m with good refraction were selected to perform migration assay. Each single neurosphere was seeded in the middle of each well and cultured with different culture medium in accordance to the previous studies with some adjustments (Wang et al., 2006). Eight groups of neurospheres were cultured in different culture medium based on published studies (Sun, 2012), including a control group (basic neural stem cell culture medium), a mBDNF (30 ng/mL) group, an anti-proBDNF antibody (10 µg/mL) group and five proBDNF groups with different concentrations of proBDNF (0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL). Neurospheres were attached to the coating and cultured for 48 h, at 37˚C, 5% CO2, for observation. The images of 12
neurospheres were taken immediately after seeding as the original start point of record and subsequently taken at the time point of 24 h and 48 h by phase-contrast microscopy. All the cell culture medium was serum free to optimally inhibit neurospheres differentiation during our observed period (48 h). Single neurospheres migrated radiantly and formed typical neural stem cell colonies. The images of migrated distances were recorded by Olympus Phase-contrast Microscope (Olympus, SA, Australia). The migrated distances were calculated omitting the radius of neurospheres selected and measured with Image-Pro Plus software and statistical analysis was completed with GraphPad Prism 6 (GraphPad Software, Inc., CA, USA).
4.5.
BrdU incorporation assay
Dissociated NSCs was plated onto the 24-well adhesion cell culture plates. All the 24-well plates were prepared with Matrigel-coated coverslips. 5-bromo-2'-deoxyuridine (BrdU, 10 µM, Sigma, Australia) was added to the cell cultures medium 48 h before fixation of the cells. The number of NSCs nuclei that incorporated with BrdU was detected by immunofluorescence. The capability of cell proliferation was quantified by measurement of the percentage of BrdU-positive (BrdU+) cells to the total number of cells. All the NSCs were counterstained with DAPI and the total cell number was counted by DAPI-positive (DAPI+) immunostaining.
4.6.
Neural stem cell spontaneous differentiation
Mouse neural stem cells were incubated at 37˚C, 5% CO2 for over 2-3 passages. Monolayer cultured cells were then trypsinized with TrypLE™ Express (Life Technology, Australia) and reseeded at a density of 2.5×104 cells/cm2 for specific lineage differentiation. After 2-day recovery, the cell culture medium was replaced with differentiation medium. Depending on the conditions of cell growth, differentiation medium was changed every 3 days.
4.7.
Immunocytochemistry
Cells on coverslips were processed for immunocytochemistry as previously described. Primary antibodies against BrdU (5-bromo-2'-deoxyuridine) (mouse, 1:1000, Abcam), Nestin (rabbit, 1:500, 13
Osenses), β-III Tubulin (chicken, 1:1000, Millipore), glial fibrillary acidic protein (GFAP, mouse, 1:1000, DAKO), Olig2 (rabbit, 1:1000, Osenses), p75NTR (mouse, 1:1000, Osenses), TrkB (rabbit, 1:500, Osenses), Sortilin (rabbit, 1:500, Osenses) were used. Alexa Fluro 488, Cy2 and Cy3conjugated secondary antibodies (1:1000, Santa Cruz) were used as the secondary antibodies. DAPI (Sigma, Australia) was used to counterstain the cell nuclei of all the samples. After incubation, coverslips were mounted with mounting medium and kept in dark. For the BrdU incorporation assay, cells were denatured by 2N HCl for 30 min at 37˚C and then treated with Borate buffer (100 mM borate, 150 mM NaCl, pH 9) for 5 min at RT before blocking by following the published protocols of BrdU incorporation assay (Wojtowicz and Kee, 2006). All the fluorescence images were taken by Confocal and Olympus fluorescence microscopes (Olympus, SA, Australia). At least 10-15 fields of views were taken of each group for calculation and statistical analysis.
4.8.
Western blotting
Protein samples from cells and tissues were extracted using RIPA buffer (25 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1%Triton X-100) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Equal amounts of extracts from different preparations were performed using SDS-polyacrylamide gels and subjected to immunoblotting. Primary antibodies against p75NTR (mouse, 1:1000, Osenses), TrkB (rabbit, 1:500, Osenses), Sortilin (rabbit, 1:500, Osenses), anti-proBDNF antibody (rabbit, 1:500, Osenses), GAPDH (sheep, 1:1000, Abcam) were used. Immunoreactive bands were visualized by incubation with Horseradish peroxidase (HPR)conjugated secondary antibodies and detected by using ECL detection kit (Pierce, Rockford, IL, USA). On the basis of published studies (Garcia et al., 2011; Je et al., 2013; Koliatsos et al., 1993; Yan and Johnson, 1988), spinal cord from the same mice were collected and used as the control group.
4.9.
Statistical analysis
Statistical analysis was carried out with GraphPad Prism 6 software. Data were presented as a mean ± SEM of at least three independent experiments. For experiments with more than two conditions, results were compared using one-way analysis of variance (ANOVA) with post-hoc Tukey’s test. For 14
two group-designed experiments, comparisons were performed using unpaired student’s t-test. A pvalue of p<0.05 was considered statistically significant.
Disclosure of potential conflicts of interests The authors declare no potential conflicts of interest.
Acknowledgment This work was supported by NHMRC grants and a grant from Sansom Institute of the University of South Australia. JYL is supported by the president scholarship from University of South Australia. The generous contribution of the antibodies from Osenses is appreciated. Thank Dr. Jia Liu for assistance with project design and Dr. Larisa Bobrovskaya and Profesor Xin-Fu Zhou for comments that greatly improved the manuscript.
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Lu, B., Pang, P.T., Woo, N.H., 2005. The yin and yang of neurotrophin action. Nature Reviews Neuroscience. 6, 603. Maisonpierre, P.C., Belluscio L Fau - Squinto, S., Squinto S Fau - Ip, N.Y., Ip Ny Fau - Furth, M.E., Furth Me Fau - Lindsay, R.M., Lindsay Rm Fau - Yancopoulos, G.D., Yancopoulos, G.D., 1990. Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Moscatelli, I., Pierantozzi E Fau - Camaioni, A., Camaioni A Fau - Siracusa, G., Siracusa G Fau Campagnolo, L., Campagnolo, L., 2009. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells. Nykjaer, A., Lee, R., Teng, K.K., Jansen, P., Madsen, P., Nielsen, M.S., Jacobsen, C., Kliemannel, M., Schwarz, E., Willnow, T.E., Hempstead, B.L., Petersen, C.M., 2004. Sortilin is essential for proNGF-induced neuronal cell death. Nature. 427, 843. Park, K.I., Ourednik J Fau - Ourednik, V., Ourednik V Fau - Taylor, R.M., Taylor Rm Fau - Aboody, K.S., Aboody Ks Fau - Auguste, K.I., Auguste Ki Fau - Lachyankar, M.B., Lachyankar Mb Fau Redmond, D.E., Redmond De Fau - Snyder, E.Y., Snyder, E.Y., 2002a. Global gene and cell replacement strategies via stem cells. Park, K.I., Teng Yd Fau - Snyder, E.Y., Snyder, E.Y., 2002b. The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Patruno, A., Tran, N., Abdelrahim, M., Brennand, K., 2014. investigating the mechanisms involved in aberrant neurosphere migration in schizophrenia. Faseb Journal. 28. Pollard, S.M., Conti L Fau - Sun, Y., Sun Y Fau - Goffredo, D., Goffredo D Fau - Smith, A., Smith, A., 2006. Adherent neural stem (NS) cells from fetal and adult forebrain. Preedy, V.R., Watson, R.R., Martin, C.R., 2011. Handbook of behavior, food and nutrition. Vol., Springer, New York. Sun, L., Lee, J., Fine, H., 2004. Neuronally expressed stem cell factor induces neural stem cell migration to areas of brain injury. Journal of Clinical Investigation. 113, 1364-74. Sun, Y., Lim, Y., Li, F., Liu, S., Lu, J.J., Haberberger, R., Zhong, J.H., Zhou, X.F., 2012. ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA. PLoS One. 7, e35883. Sun, Y., Lim, Y. Li, F. Liu, S. Lu, J. J. Haberberger, R. Zhong, J. H. Zhou, X. F., 2012. ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA. PLoS One. 7, e35883. Swistowski, A., Peng J Fau - Han, Y., Han Y Fau - Swistowska, A.M., Swistowska Am Fau - Rao, M.S., Rao Ms Fau - Zeng, X., Zeng, X., 2009. Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them. Taupin, P., 2011. Neurogenesis, NSCs, pathogenesis and therapies for Alzheimer's disease. Front Biosci (Schol Ed). 3, 178-90. Temple, S., 2001. The development of neural stem cells. Nature. 414, 112-7. Teng, H.K., Teng Kk Fau - Lee, R., Lee R Fau - Wright, S., Wright S Fau - Tevar, S., Tevar S Fau Almeida, R.D., Almeida Rd Fau - Kermani, P., Kermani P Fau - Torkin, R., Torkin R Fau - Chen, Z.-Y., Chen Zy Fau - Lee, F.S., Lee Fs Fau - Kraemer, R.T., Kraemer Rt Fau - Nykjaer, A., Nykjaer A Fau - Hempstead, B.L., Hempstead, B.L., 2005. ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. van Meerloo, J., Kaspers Gj Fau - Cloos, J., Cloos, J., 2001. Cell sensitivity assays: the MTT assay. Vandenbosch, R., Borgs L Fau - Beukelaers, P., Beukelaers P Fau - Belachew, S., Belachew S Fau Moonen, G., Moonen G Fau - Nguyen, L., Nguyen L Fau - Malgrange, B., Malgrange, B., 2009. Adult neurogenesis and the diseased brain. Wang, T.Y., Sen A Fau - Behie, L.A., Behie La Fau - Kallos, M.S., Kallos, M.S., 2006. Dynamic behavior of cells within neurospheres in expanding populations of neural precursors. Wojtowicz, J.M., Kee, N., 2006. BrdU assay for neurogenesis in rodents. Nature Protocols. 1, 1399. Wu, Y.Y., Mujtaba, T., Rao, M.S., 2002. Isolation of Stem and Precursor Cells from Fetal Tissue. In Neural Stem Cells: Methods and Protocols. Vol., T. Zigova, P.R. Sanberg, J.R. Sanchez-Ramos, ed.^eds. Humana Press, Totowa, NJ, pp. 29-39.
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Xu, Z.Q., Sun Y Fau - Li, H.-Y., Li Hy Fau - Lim, Y., Lim Y Fau - Zhong, J.-H., Zhong Jh Fau - Zhou, X.-F., Zhou, X.F., 2011. Endogenous proBDNF is a negative regulator of migration of cerebellar granule cells in neonatal mice. Yan, Q., Johnson, E.M., Jr., 1988. An immunohistochemical study of the nerve growth factor receptor in developing rats. J Neurosci. 8, 3481-98. Yang, J., Siao, C.-J., Nagappan, G., Marinic, T., Jing, D., Mcgrath, K., Chen, Z.-Y., Mark, W., Tessarollo, L., Lee, F.S., Lu, B., Hempstead, B.L., 2009. Neuronal release of proBDNF. Nature Neuroscience. 12, 113. Zhang, S., Liu Xz Fau - Liu, Z.-l., Liu Zl Fau - Wang, Y.-m., Wang Ym Fau - Hu, Q.-l., Hu Ql Fau - Ma, T.-z., Ma Tz Fau - Sun, S.-z., Sun, S.Z., 2009. Stem cells modified by brain-derived neurotrophic factor to promote stem cells differentiation into neurons and enhance neuromotor function after brain injury. Zigova, T., Pencea, V., Wiegand, S.J., Luskin, M.B., 1998. Intraventricular Administration of BDNF Increases the Number of Newly Generated Neurons in the Adult Olfactory Bulb. Molecular and Cellular Neuroscience. 11, 234-245.
Figure legends Fig. 1 Immunofluorescence staining and western blotting of NSCs. (A) ProBDNF (green), p75NTR (red), Sortilin (green), TrkB (green) were detected by specific antibodies in NSCs. Double-staining with anti-p75NTR antibody and anti-Sortilin antibody were performed in NSCs and the colocalization of these two receptors was quantified with ImageJ. DAPI (blue) staining was used as the reference of nuclei. P75NTR (red) and Sortilin (green) were co-localized in NSCs and the scatterplot of red and green pixel intensities of the image was shown in Fig. 1A. Co-localization analysis was performed using an Olympus confocal microscope (10x objective) and indicated in merged panels (yellow). Snapshots of co-localization are shown in the right panels. PCC: r=0.89; Overlap Coefficient: r=0.9. Scale bar: 100 µm. (B) Magnified images of immunofluorescence staining were taken by higher power objective lens of Olympus confocal microscope (63x objective). Scale bar: 10 µm. (C) Immunoblot of proBDNF, p75NTR, Sortilin and TrkB in NSCs. Spinal cord of the same mice was used as the control group.
Fig. 2 MTT assay of neural stem cells treated with different concentrations of proBDNF, mature BDNF and anti-proBDNF antibody. (A-B) proBDNF-treated group displayed reduced cell viability compared with the control group. (****p<0.0001)
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Fig. 3 NSC migration assay using Millicell-insert. (A) NSCs were cultured in normal cell culture medium, normal medium plus mBDNF (30 ng/mL), normal medium plus anti-proBDNF antibody (10 µg/mL), normal medium plus different concentrations of proBDNF (0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL). DAPI was used for nucleus staining. (B) Significant differences were observed in the wells treated with mBDNF, anti-proBDNF antibody and proBDNF compared with control. (C) Among different concentrations of proBDNF treated cells and control cells, the reduced migration was displayed in a dose-dependent manner. *p<0.05, ****p<0.0001, Scale bar: 100 µm.
Fig. 4 In vitro neurosphere radiant migration. (A) Neurospheres were cultured in different medium for 48 h and imaged by phase-contrast microscopy. Distances of migration were measured individually. Red arrows indicated the radial migration outward from the core of the neurosphere and the migrated cells have the same spindle-shaped NSC morphology. (B-C) Migration distances were measured at 24 h after neurospheres were cultured on Matrigel-coated plates. (D-E) After 48 h treatment, migrated distances were recorded again. Neurospheres treated with different concentrations of proBDNF (0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL) showed dramatic decreases in migration compared with control group with no morphological changes of NSCs (****p<0.0001, n=5). Neurospheres treated with mBDNF (30 ng/mL) and anti-proBDNF antibody (10 µg/mL) displayed improved migration capability compared with control. Scale bar: 100 µm.
Fig. 5 BrdU incorporation to investigate the proliferation of neural stem cells. (A) Immunofluorescence staining with anti-BrdU antibody (red) indicated the newly born neural stem cells over 24 h. DAPI (blue) was used as the reference of nucleus. (B) Neural stem cells treated with mBDNF (30 ng/mL) and anti-proBDNF antibody (10 µg/mL) showed increased proliferation compared with control. proBDNF-treated NSCs showed significantly reduced proliferation (***p<0.001). (C) Cells treated with different concentrations of proBDNF displayed reduction in the proliferation and this effect was dose-dependent. Scale bar: 100 µm.
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Fig. 6 NSCs spontaneous differentiation. (A) Tuj1 (red) was used for neuron staining and DAPI (blue) was applied for nucleus location. (B) Significant higher percentage of neurons was found in the mBDNF-treated group (****p<0.0001). (C) proBDNF-treated groups showed reduced number of neurons compared with control (*p<0.05). (D) Immunofluorescent staining of differentiatedastrocytes. GFAP (green) was applied to indicate astrocytes and DAPI (blue) was applied for nucleus staining. (E) The ratio of GFAP-positive cells to the DAPI-positive cells. proBDNF-treated cells displayed decreased number of astrocytes compared with control cells (**p<0.01). (f) proBDNFtreated cells all showed decreases in the number of GFAP-positive cells and these effects were dosedependent. (G) Olig2 (red) was used to label oligodendrocytes and co-stained with DAPI (blue) to label nucleus. (H) Significant difference was observed between control and proBDNF treated cells (****p<0.0001). No statistical significant differences were found among control cells and mBDNF or anti-proBDNF treated cells. (I) Cells treated with proBDNF all showed decreased number of oligodendrocytes compared with control cells. 10 ng/mL proBDNF in cell culture medium displayed significant reduction in oligodendrocytes. Scale bar: 100 µm.
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Highlights:
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Mouse neural stem cells constitutively expresses proBDNF, p75NTR and Sortilin.
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proBDNF inhibits mouse neural stem cells proliferation, migration and differentiation in vitro.
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Anti-proBDNF antibody functions conversely to proBDNF in regulating proliferation, migration and differentiation of mouse neural stem cells.
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proBDNF is a factor which influences the decision of neural stem cell’s fate into neurons and glia.
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S0006-8993(17)30209-3 http://dx.doi.org/10.1016/j.brainres.2017.05.013 BRES 45362
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
18 December 2016 19 April 2017 12 May 2017
Please cite this article as: J-y. Li, J. Liu, N.P.A. Manaph, L. Bobrovskaya, X-F. Zhou, ProBDNF inhibits proliferation, migration and differentiation of mouse neural stem cells, Brain Research (2017), doi: http://dx.doi.org/ 10.1016/j.brainres.2017.05.013
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ProBDNF inhibits proliferation, migration and differentiation of mouse neural stem cells
Jia-yi Li1*, Jia Liu2, Nimshitha Pavathuparambil Abdul Manaph1, Larisa Bobrovskaya1*, Xin-Fu Zhou1 1
School of Pharmacy and Medical Sciences, Division of Health Sciences, Sansom Institute for Health
Research, University of South Australia, Adelaide, SA 5000 2
Animal Research Centre, Kunming Medical University, Kunming, China
*
To whom the correspondence should be addressed
Ms Jia-yi Li School of Pharmacy and Medical Sciences, Division of Health Sciences, Sansom Institute for Health Research, University of South Australia, Adelaide, SA 5000. Email: [email protected] Telephone: +61 8 830 21807
Dr Larisa Bobrovskaya School of Pharmacy and Medical Sciences, University of South Australia, Adelaide SA 5000. Email: [email protected] Telephone: +61 8 830 21218, Fax: +61 8 830 22389
Keywords: Neural stem cell, proBDNF, migration, proliferation, differentiation
1
Abstract ProBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is an important regulator of neurodegeneration, hippocampal long-term depression, and synaptic plasticity. ProBDNF and its receptors pan-neurotrophin receptor p75 (p75NTR), vps10p domain-containing receptor Sortilin and tropomyosin receptor kinase B (TrkB) are expressed in neuronal and glial cells. The role of proBDNF in regulation of neurogenesis is not fully defined. This study aims to uncover the function of proBDNF in regulating the differentiation, migration and proliferation of mouse neural stem cells (NSCs) in vitro. We have found that proBDNF and its receptors are constitutively expressed in NSCs when assessed by immunocytochemistry and western blotting. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay showed that exogenous proBDNF treatment reduced mouse NSCs viability by 38% at 10 ng/mL. The migration of NSCs was also reduced by exogenous proBDNF treatment in a concentration-dependent manner (by 90% at 10 ng/mL) but increased by anti-proBDNF antibody treatment (by 50%). BrdU (5-Bromo-2´-Deoxyuridine) incorporation was performed for detection of newborn cells. We have found that proBDNF significantly inhibited proliferation of NSCs and reduced the number of differentiated neurons, oligodendrocytes and astrocytes, while anti-proBDNF antibody treatment promoted proliferation and differentiation of NSCs. In conclusion, proBDNF may oppose the functions of mature BDNF by inhibiting the proliferation, differentiation and migration of NSCs during development. Conversely, anti-proBDNF antibody
treatment
promoted
proliferation
and
differentiation
of
NSCs.
2
1.
Introduction
Neural stem cells (NSCs) are multipotent cells able to self-renew and proliferate (Temple, 2001). NSCs have the capacity to differentiate into sub-lineage neural cells: neurons, astrocytes and oligodendrocytes. Due to these characteristics, it is proposed that NSCs can be used for treatment of many neurodegenerative diseases and psychiatric disorders, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and bipolar depression (Taupin, 2011). NSCs derived from mouse embryonic brains have been broadly studied. Transplantation of NSCs was effective in recovering the CNS and brain functions after trauma and diseases in animal models (Vandenbosch et al., 2009) and is a promising strategy for replacement and regeneration of specific neuronal cells. In the embryonic period of rodents, NSCs develop into neuroblasts and glioblasts which migrate and differentiate into mature neurons and glia. Migration and differentiation of NSCs are crucial steps for the CNS formation (Preedy et al., 2011). Given the fact that embryonic neurogenesis highly requires the existence of neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 and neurotrophin 4 are very important regulators in embryogenesis. Since BDNF was purified about 35 years ago (Barde et al., 1982) its pleiotropic functions have been studied for decades. It has been identified to be important for neuronal cell survival, differentiation and synaptic plasticity (Maisonpierre et al., 1990). BDNF is firstly synthesized as a precursor protein termed proBDNF (Koshimizu et al., 2010). ProBDNF is a key neurotrophic factor regulating CNS development and hippocampal neurogenesis (Koshimizu et al., 2009; Yang et al., 2009). Mature BDNF (mBDNF) has the high affinity of binding to the tropomyosin receptor kinase B (TrkB, also known as NTRK2) while proBDNF preferentially binds to p75 neurotrophin receptor (p75NTR) combined with the co-receptor sortilin (also known as NTR3) (Nykjaer et al., 2004; Teng et al., 2005). P75NTR is a cell surface receptor with intracellular death domain belonging to the tumour necrosis factor family (Bassili et al., 2010). Researchers have found that in comparison to mBDNF, proBDNF functions adversely in neural system via activation of the proBDNF-p75NTR-sortilin complex and causes neuronal cell apoptosis, long-term depression and neurite outgrowth collapse (Sun et al., 2012; Teng et al., 2005). The ratio of TrkB/p75NTR and the balance of mBDNF/proBDNF can also regulate neurodegeneration and neuro-regeneration (Je et al., 2012; Kotlyanskaya et al., 2013). 3
Interestingly, proBDNF and p75NTR are found most highly expressed in perinatal stage of mice and then reduced but still detectable in adulthood. Neurons are a crucial source of proBDNF and mBDNF secretion (Yang et al., 2009) while astrocytes take part in cleaning up, recycling and rerelease of proBDNF (Bergami et al., 2008). In embryonic neuronal development and maturation, astrocytic differentiation occurs following neuronal differentiation (English et al., 2013). During the progression of embryogenesis, newly formed astrocytes might reduce proBDNF levels which may improve the NSCs differentiation, neuronal cell survival and proliferation. Despite that BDNF is reported to regulate proliferation and differentiation of NSCs (Chen et al., 2013) and promote survival of postmitotic neurons (Davies, 1994), no specific study has addressed the role of proBDNF in behaviors of NSCs. Whether proBDNF has any physiological functions in the early neurogenesis is still not clear. In this study, we hypothesized that proBDNF may negatively regulate proliferation, migration and differentiation during development. We confirmed that p75NTR and sortilin were strongly expressed and colocalized in NSCs, and proBDNF acted as an effective factor inhibiting proliferation, migration and differentiation of NSCs derived from mouse embryos.
2.
Results
2.1.
Neural stem cells express proBDNF and its receptors p75NTR, TrkB and Sortilin
By immunofluorescence staining with specific markers, we found proBDNF, p75NTR, Sortilin and TrkB were constitutively expressed in cultured mouse NSCs. In addition, we double-stained NSCs with p75NTR and Sortilin to verify the colocalization of these two co-effected receptors. Pearson’s correlation coefficient (PCC) measures r=0.89 in the image of double-staining by p75NTR and Sortilin (Fig. 1A). For the two co-receptors of proBDNF, 89% of the variability of p75NTR expression can be explained by the variability of Sortilin expression. Statistical analysis with PCC showed colocalization of p75NTR and Sortilin that further indicated the proBDNF-p75NTR-Sortilin complex was exerted in NSCs. ProBDNF, p75NTR, Sortilin and TrkB expressions in mouse NSCs were further confirmed by western blotting, with spinal cord tissue of wild type mice as a positive control (Fig. 1B). These findings demonstrate that proBDNF and its receptors p75NTR, Sortilin and
4
TrkB are highly expressed in NSCs, which indicates that proBDNF can regulate physiological functions of these cells.
2.2.
ProBDNF reduces the viability of neural stem cells
We performed MTT assay to further identify whether proBDNF would reduce viability of NSCs within 24 h treatment at the following concentrations; 0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL. As shown in Fig. 2, the percentage of viable NSCs was significantly decreased by proBDNF treatment compared with the control group (p<0.0001). Mature BDNF (30 ng/mL) or anti-proBDNF antibody (10 µg/mL) did not significantly enhance NSCs survival.
2.3.
ProBDNF reduces the migration of neural stem cells
In a Transwell migration assay, proBDNF showed significant inhibition of NSCs migration from top to the bottom of the wells (Fig. 3) compared with the control wells which were cultured in normal neural stem cell medium. All the proBDNF wells displayed a significant decrease in migrated NSCs numbers; while mBDNF (30 ng/mL) and anti-proBDNF antibody (10 µg/mL) displayed significant increases in migrated NSCs numbers (p<0.01, Fig. 3B). These results indicate that mBDNF and antiproBDNF antibody significantly improved NSCs migration.
2.4.
ProBDNF reduces the radial migration of neurospheres
For investigation of neurosphere radial migration, we recorded the radial migrated distances of each neurosphere cultured in different medium. We found that proBDNF at all doses (0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL) significantly inhibited the neurosphere migration after 48 h of culture (p<0.0001). In contrast, mBDNF (30 ng/mL) and anti-proBDNF antibody (10 µg/mL) showed a prominent increase in the neurosphere migration (1.5-fold) in the first 24 h (Fig. 4B). The increase of migration was maintained up to 48 h after anti-proBDNF antibody treatment but was not significant in the mBDNF- treated wells (Fig. 4D).
5
2.5.
ProBDNF inhibits neural stem cell proliferation
Based on the results of BrdU incorporation assay, we found that proBDNF functioned as an inhibitor of NSCs proliferation in a dose-dependent manner in the concentration range between 0.1-10 ng/ml. Increasing concentrations of proBDNF in the cell culture medium produced statistically significant inhibition of NSCs proliferation at 1 ng/mL, 3 ng/mL, and 10 ng/ml as counted by the percentage of BrdU+/DAPI+ staining (Fig. 5C). Mature BDNF and anti-proBDNF antibody improved the proliferation of NSCs (p<0.05, Fig. 5B).
2.6.
ProBDNF regulates neural stem cell differentiation
To investigate the percentage of different lineages of neural differentiation, specific cell markers were investigated by immunostaining the differentiated cells. Tuj1 was used for neuron staining; GFAP for astrocyte staining and Olig2 for oligodendrocyte staining (Fig. 6). In the neuronal differentiation detection, we found that 10 ng/mL proBDNF in the culture medium reduced the number of Tuj1positive cells (p<0.05) and the negative effects of proBDNF on neuronal differentiation showed dosedependent responses (Fig. 6C). On contrary, mature BDNF in culture medium promoted the neuronal differentiation (p<0.0001) while anti-proBDNF antibody did not affect the number of neurons compared with the control group (Fig. 6B). In case of astrocytic differentiation, 10 ng/mL of proBDNF added to the culture medium significantly reduced the number of astrocytes compared with control wells which were cultured in the normal differentiation medium (p<0.05). The decreases in astrocytic differentiation were also dependent on the proBDNF added to the cell culture medium (Fig. 6F). Mature BDNF and anti-proBDNF antibodies did not appear as strong enhancers of astrocytic differentiation compared with the control wells in our experiments (Fig. 6E). In oligodendrocyte differentiation test, we found Olig2-posive cells were significantly decreased in the proBDNF-treated wells (Fig. 6G). 10 ng/mL proBDNF in the cell culture medium inhibited the oligodendrocyticdifferentiation of NSCs (p<0.0001). The decrease of Olig2-positive cells in response to proBDNF treatment was dose-dependent. The number of positively stained cells was calculated and analyzed with ImageJ and GraphPad Prism 6.
6
3.
Discussion
In this research, we have shown: 1) NSCs express proBDNF and its receptors p75NTR and Sortilin; 2) anti-proBDNF antibodies added to the cell culture promote the proliferation, migration and differentiation, indicating proBDNF may act in an autocrine and/or paracrine manner; 3) in all our experiments, mBDNF was administered as a positive control, which confirmed its enhancing role in the proliferation, migration and differentiation of NSCs. Thus, our study has contributed to the field by providing another evidence that mBDNF and proBDNF act as opposing factors regulating functions of the nervous system. Mature BDNF has been verified to regulate the differentiation of embryonic stem cells and neuronal precursors (Jung and Kang, 2001) and to stimulate the proliferation of NSCs (Chen et al., 2013) and migration of cerebellar granule cells during development of the nervous system (Borghesani et al., 2002). ProBDNF is reported to exhibit an inhibitory role in cerebellar granule cells migration that counterbalances the functions of mBDNF (Xu et al., 2011). It is known that proBDNF regulates the gradual formation of neuronal cells (Swistowski et al., 2009) and the apoptosis of Schwann cells (Teng et al., 2005), oligodendrocytes (Beattie et al., 2002) and sympathetic neurons (Harrington et al., 2004). Given the fact that mBDNF has a decisive role in regulating perinatal brain development (Liu and Graybiel, 1998) while proBDNF has the opposing biological functions in CNS (Lu et al., 2005), it is intriguing for researchers to investigate the role of proBDNF in early neural development and embryogenesis. Furthermore, abnormalities in the CNS maturation during embryogenesis may underlie neurodevelopmental disorders. Perinatal mBDNF abnormalities are believed to program many postnatal neurodegeneration events and early mBDNF dysfunction might participate in the development of chronic neurological diseases (Preedy et al., 2011). Our present study might provide additional information on the influences of proBDNF on NSCs which possibly contribute to the development of CNS and neurological disorders. The migration characteristics of NSCs are a critical step in the CNS development and momentous for the repair of neurodegenerative diseases (Feng et al., 2012). For mimicking the embryonic neural development in vitro, we performed the assays with embryo-derived NSCs which demonstrated the negative role of proBDNF in NSCs and neurosphere migration. Like another proneurotrophin proNGF, 7
relatively low concentrations of exogenous proBDNF (0.1 ng/mL) could significantly inhibit the migration of multipotent neural cells. After 48 h treatment with proBDNF, continuous inhibitory effects on migration were displayed in NSCs. Mature BDNF improved the radial migration of neurospheres in the first 24 h but did not show further influences in the following 24 h. Interestingly, anti-proBDNF antibody showed similar functions as mature BDNF which significantly enhanced the migration of neurospheres in the 24 h and 48 h assays. It is proposed that the mechanisms of proBDNF and mature BDNF in cellular biology would be a ‘yin and yang’ model (Lu et al., 2005). Some pharmacological studies have demonstrated that proBDNF and mature BDNF act as punishment and reward signals, respectively, and the conversion of proBDNF to mature BDNF is critical for many biological processes (Je et al., 2013; Lu et al., 2005). Therefore, the results from our assays that anti-proBDNF antibody extended the migration of NSCs in Figure 4 are not unexpected, and consistent with our previous study which shows that anti-proBDNF promotes the migration of cerebella granule neurons (Xu et al, 2010). While the function of proBDNF is blocked by antibodies, the signaling by mature BDNF dominates displaying the enhancement in NSCs migration. It has been demonstrated that NSCs migration is an essential process for the development of CNS as well as the ongoing neurogenesis that occurs in the mature CNS of most vertebrate species, including mammals (Imitola et al., 2004). Also, NSCs migrate to the sites of pathological insult in various types of brain injury (i.e., ischemia and blunt trauma) and tumours (Aboody et al., 2000; Sun et al., 2004). The migration toward damaged CNS tissue may represent an adaptive response with the purpose of limiting and/or repairing the damage, although to date there are limited data to definitively support or refute this hypothesis. Remarkably, the aberrant migration of neurospheres is a sign of many neurodegenerative or neural system diseases, such as brain tumours (Aboody et al., 2000), demyelination and multiple sclerosis (Park et al., 2002a), ischemia (Imitola et al., 2004; Park et al., 2002b), Alzheimer’s disease (Park et al., 2002a) and schizophrenia (Patruno et al., 2014). Thus, the microenvironmental conditions and factors that regulate NSCs migration are vitally important. In our study, proBDNF is found to significantly reduce neurospheres and NSCs migration, absolute numbers and proliferation. Regardless of the physiological role of NSCs in neural injury, the migratory
8
properties of NSCs may theoretically be exploited for cell-based therapeutics of neurodegenerative diseases in the future. CNS development is confirmed highly dependent on proliferation and differentiation of NSCs resulting in increasing cellular complexity over time. NSCs differentiate into innumerable types of neurons and glial cells which are progressively and temporally restricted during embryogenesis. In our BrdU incorporation assay, proBDNF effectively acted as an inhibitor of NSCs proliferation while mBDNF and anti-proBDNF antibody, on the contrary, acted as promoters of NSCs proliferation. Additionally, we found that exogenous proBDNF significantly decreased the viability of NSCs suggesting that proBDNF inhibits the proliferative process. BDNF has been reported to be able to elevate the number of newly generated neurons in the adult olfactory bulb (Zigova et al., 1998) and act as a neuronal differentiation factor to promote the neuronal differentiation of stem cells (Zhang et al., 2009). In our study, we obtained similar results that administration of mBDNF facilitated the neuronal differentiation by up to 4-fold. BDNF has been proved to enhance neurogenesis while the enhancement could be potentiated by concurrent inhibition of glial differentiation (Chmielnicki et al., 2004). Studies of cultured embryo-derived progenitor cells have elucidated that differentiation of embryonic stem cells increased the TrkB expression which was further upregulated in the presence of BDNF (Jung and Kang, 2001). TrkB expression is found restricted to the neuronal cells which may complementarily explain the enhancement of neuronal differentiation induced by mBDNF. Thus, the results described here may be interpreted such that mBDNF facilitates the neuronal differentiation or modulates the fate of uncommitted NSCs with neuronal potential. In the case of astrocyte and oligodendrocyte detection, a considerable decrease in the number of astrocytes and oligodendrocytes was observed in the proBDNF-treated cells while antiproBDNF antibody slightly enhanced the glial differentiation. Previous studies have shown that addition of BDNF in the microenvironment of neurospheres has no significant effect on the differentiation of astrocytes or oligodendrocytes (Ahmed et al., 1995). In our NSCs experiment, data is consistent with those published reports that mBDNF did not obviously improve the differentiation of astrocytes or oligodendrocytes.
9
In contrast to mBDNF, proBDNF inhibited the spontaneous differentiation of NSCs. After 7-day exposure to proBDNF, the numbers of Tuj1-positive, GFAP-positive and Olig2-positive cells were decreased compared with the control group. Furthermore, the tendency of decreased differentiation in proBDNF-treated NSCs occurred in a dose-dependent manner. To understand the possible role played by proBDNF in neural differentiation of NSCs, the expression of proBDNF corresponding receptors were examined by immunoreaction and western blot in NSCs. Mouse embryonic cells have been previously found to express p75NTR (Moscatelli et al., 2009). p75NTR was also proved to be constitutively expressed in NSCs in our study. Given further identification that NCSs possess both p75NTR and Sortilin receptors, it is conceivable that the activation of p75NTR-sortilin complex by exogenous proBDNF could lead to the retarded lineage differentiation in NSCs. To confirm this hypothesis, we blocked the action of endogenous proBDNF by anti-proBDNF antibody and found that the administration of anti-proBDNF antibody resulted in a significant increase of neuronal differentiation in NSCs suggesting the negative role of endogenous proBDNF in regulating neuronal differentiation. A notable observation made in this study is that functional blocking of endogenous proBDNF did not significantly elevate gliogenesis of NSCs similar to the effects of mBDNF observed in this study. This result is rarely addressed in other studies and may suggest that mBDNF and proBDNF may not be the decisive factors in the NSCs gliogenesis; alternatively, mBDNF and/or proBDNF might exert their actions on neurogenesis and gliogenesis differentially depending on the developmental stages. The precise role of proBDNF in neural development and mechanisms behind the phenomena obtained in our study need further investigation in the future.
4.
Experimental procedure
4.1.
Primary neural stem cells derivation and culture
The primary NSCs were obtained from the C57/BL6 strain mice following previously published work on primary embryonic stem cell culture with some adjustments (Wu et al., 2002). The C57 strain mice were bred by our own animal facility with the accordance of approved animal ethics. The primary NSCs were dissociated from the 14-day-old (E14) embryos and gently washed with phosphatebuffered saline (PBS) for three times and plated in the T-75 flasks for suspension cell culture. The 10
detailed procedures for E14 NSCs primary cell culture can be found in literature elsewhere (AyusoSacido, 2012). Primary NSCs were cultured in the basic NSC culture medium according to the published NSC culture medium recipes (Pollard et al., 2006), which included the B27 (2%) and basic fibroblast growth factor (bFGF, 20 ng/mL) and epidermal growth factor (EGF, 20 ng/mL). Half amount of the cell culture medium was changed every 3-4 days depending on the confluence of cells. Neurospheres were observed forming in 6-7 days and after the first passage (P1) of neurospheres they were expanded at the ratio of 1:2. The P2 NSCs were harvested and applied for experiments.
4.2.
Passaging and expansion of neurospheres
When the primary neurospheres reached 150-200 µm in diameter, they were subcultured according to the published protocols (Azari et al., 2011) with minor modifications. Generally, the medium with suspended spheres was transferred into a conical tube and centrifuged at 700 rpm (110 g) for 5 min at RT. The supernatant was discarded and neurospheres were resuspended in 0.05% trypsin (Sigma, Australia). The cell suspension was then incubated in a 37°C water bath for 2-3 min and added an equal volume of fetal bovine serum (Life Technology, Australia) to stop the trypsin activity. After gently mix-up the cell suspension was centrifuged at 700 rpm (110 g) for 5 min and the supernatant was removed. Cells were resuspended in complete NSC medium and plated at a concentration of 5104 cells/ mL in flasks. Secondary neurospheres were formed in 5-7 days when incubated at 37° C in a humidified incubator with 5% CO2.
4.3.
Cell viability assay
MTT assay was applied to investigate the cell viability according to different treatments added to the culture medium. Primary cultured NSCs were plated in 96-well plates with the density of 3,000-4,000 cells/well and incubated at 37˚C, 5% CO2 for 48 h. After 48 h, the NSCs were processed following the MTT assay protocol for adherent cells published previously (van Meerloo et al., 2001). Cell viability was calculated by measuring the absorbance at 490 nm with the Wallac Station (PerkinElmer, San Diego, CA, USA).
11
4.4. 4.4.1.
Cell migration assay Analysis of neural stem cell migration with Millicell-insert
We investigated the migration of dissociated NSCs using 24-well Millicell-inserts (PIRP12R48, Merck Millipore, Australia). NSCs were dissociated to single cell suspension according to the manufacturer’s instructions and by nuclear staining with DAPI, and the number of cells migrated across the mesh from the wells was counted. The upper inserts were filled with 1×105 cells/mL suspended NSCs and cultured overnight at 37˚C, 5% CO2. After 24 h incubation, the cells on the upper surface were removed from the filter with a cotton swab and washed three times with PBS. The cells remaining on the bottom surface were fixed in ice-cold 4% paraformaldehyde (PF)/PBS solution and stained with DAPI for 20 min at room temperature (RT) and examined under a fluorescence microscope. Migrated NSCs were imaged with Olympus fluorescence microscope (Olympus, SA, Australia) and counted by ImageJ (National Institute of Health, USA). 10-15 views of migrated NSCs on filters were counted (20× objective) to obtain the number of migrating cells. We performed the migration assay as per the published protocol of migration assay by using Transwell inserts (Dufour et al., 2008) with some adjustments.
4.4.2.
Analysis of neurosphere radial migration
Primarily cultured neurospheres were obtained from C57BL/6 embryonic day 14 (E14) and kept as neurosphere cell lines stably. Prior to seeding neurospheres for the migration assay, 24-well plates were coated with Matrigel (BD Matrigel™, BD Biosciences, Australia). Single neurospheres from P2 onwards in the diameter of 500-600 µ m with good refraction were selected to perform migration assay. Each single neurosphere was seeded in the middle of each well and cultured with different culture medium in accordance to the previous studies with some adjustments (Wang et al., 2006). Eight groups of neurospheres were cultured in different culture medium based on published studies (Sun, 2012), including a control group (basic neural stem cell culture medium), a mBDNF (30 ng/mL) group, an anti-proBDNF antibody (10 µg/mL) group and five proBDNF groups with different concentrations of proBDNF (0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL). Neurospheres were attached to the coating and cultured for 48 h, at 37˚C, 5% CO2, for observation. The images of 12
neurospheres were taken immediately after seeding as the original start point of record and subsequently taken at the time point of 24 h and 48 h by phase-contrast microscopy. All the cell culture medium was serum free to optimally inhibit neurospheres differentiation during our observed period (48 h). Single neurospheres migrated radiantly and formed typical neural stem cell colonies. The images of migrated distances were recorded by Olympus Phase-contrast Microscope (Olympus, SA, Australia). The migrated distances were calculated omitting the radius of neurospheres selected and measured with Image-Pro Plus software and statistical analysis was completed with GraphPad Prism 6 (GraphPad Software, Inc., CA, USA).
4.5.
BrdU incorporation assay
Dissociated NSCs was plated onto the 24-well adhesion cell culture plates. All the 24-well plates were prepared with Matrigel-coated coverslips. 5-bromo-2'-deoxyuridine (BrdU, 10 µM, Sigma, Australia) was added to the cell cultures medium 48 h before fixation of the cells. The number of NSCs nuclei that incorporated with BrdU was detected by immunofluorescence. The capability of cell proliferation was quantified by measurement of the percentage of BrdU-positive (BrdU+) cells to the total number of cells. All the NSCs were counterstained with DAPI and the total cell number was counted by DAPI-positive (DAPI+) immunostaining.
4.6.
Neural stem cell spontaneous differentiation
Mouse neural stem cells were incubated at 37˚C, 5% CO2 for over 2-3 passages. Monolayer cultured cells were then trypsinized with TrypLE™ Express (Life Technology, Australia) and reseeded at a density of 2.5×104 cells/cm2 for specific lineage differentiation. After 2-day recovery, the cell culture medium was replaced with differentiation medium. Depending on the conditions of cell growth, differentiation medium was changed every 3 days.
4.7.
Immunocytochemistry
Cells on coverslips were processed for immunocytochemistry as previously described. Primary antibodies against BrdU (5-bromo-2'-deoxyuridine) (mouse, 1:1000, Abcam), Nestin (rabbit, 1:500, 13
Osenses), β-III Tubulin (chicken, 1:1000, Millipore), glial fibrillary acidic protein (GFAP, mouse, 1:1000, DAKO), Olig2 (rabbit, 1:1000, Osenses), p75NTR (mouse, 1:1000, Osenses), TrkB (rabbit, 1:500, Osenses), Sortilin (rabbit, 1:500, Osenses) were used. Alexa Fluro 488, Cy2 and Cy3conjugated secondary antibodies (1:1000, Santa Cruz) were used as the secondary antibodies. DAPI (Sigma, Australia) was used to counterstain the cell nuclei of all the samples. After incubation, coverslips were mounted with mounting medium and kept in dark. For the BrdU incorporation assay, cells were denatured by 2N HCl for 30 min at 37˚C and then treated with Borate buffer (100 mM borate, 150 mM NaCl, pH 9) for 5 min at RT before blocking by following the published protocols of BrdU incorporation assay (Wojtowicz and Kee, 2006). All the fluorescence images were taken by Confocal and Olympus fluorescence microscopes (Olympus, SA, Australia). At least 10-15 fields of views were taken of each group for calculation and statistical analysis.
4.8.
Western blotting
Protein samples from cells and tissues were extracted using RIPA buffer (25 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1%Triton X-100) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Equal amounts of extracts from different preparations were performed using SDS-polyacrylamide gels and subjected to immunoblotting. Primary antibodies against p75NTR (mouse, 1:1000, Osenses), TrkB (rabbit, 1:500, Osenses), Sortilin (rabbit, 1:500, Osenses), anti-proBDNF antibody (rabbit, 1:500, Osenses), GAPDH (sheep, 1:1000, Abcam) were used. Immunoreactive bands were visualized by incubation with Horseradish peroxidase (HPR)conjugated secondary antibodies and detected by using ECL detection kit (Pierce, Rockford, IL, USA). On the basis of published studies (Garcia et al., 2011; Je et al., 2013; Koliatsos et al., 1993; Yan and Johnson, 1988), spinal cord from the same mice were collected and used as the control group.
4.9.
Statistical analysis
Statistical analysis was carried out with GraphPad Prism 6 software. Data were presented as a mean ± SEM of at least three independent experiments. For experiments with more than two conditions, results were compared using one-way analysis of variance (ANOVA) with post-hoc Tukey’s test. For 14
two group-designed experiments, comparisons were performed using unpaired student’s t-test. A pvalue of p<0.05 was considered statistically significant.
Disclosure of potential conflicts of interests The authors declare no potential conflicts of interest.
Acknowledgment This work was supported by NHMRC grants and a grant from Sansom Institute of the University of South Australia. JYL is supported by the president scholarship from University of South Australia. The generous contribution of the antibodies from Osenses is appreciated. Thank Dr. Jia Liu for assistance with project design and Dr. Larisa Bobrovskaya and Profesor Xin-Fu Zhou for comments that greatly improved the manuscript.
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Figure legends Fig. 1 Immunofluorescence staining and western blotting of NSCs. (A) ProBDNF (green), p75NTR (red), Sortilin (green), TrkB (green) were detected by specific antibodies in NSCs. Double-staining with anti-p75NTR antibody and anti-Sortilin antibody were performed in NSCs and the colocalization of these two receptors was quantified with ImageJ. DAPI (blue) staining was used as the reference of nuclei. P75NTR (red) and Sortilin (green) were co-localized in NSCs and the scatterplot of red and green pixel intensities of the image was shown in Fig. 1A. Co-localization analysis was performed using an Olympus confocal microscope (10x objective) and indicated in merged panels (yellow). Snapshots of co-localization are shown in the right panels. PCC: r=0.89; Overlap Coefficient: r=0.9. Scale bar: 100 µm. (B) Magnified images of immunofluorescence staining were taken by higher power objective lens of Olympus confocal microscope (63x objective). Scale bar: 10 µm. (C) Immunoblot of proBDNF, p75NTR, Sortilin and TrkB in NSCs. Spinal cord of the same mice was used as the control group.
Fig. 2 MTT assay of neural stem cells treated with different concentrations of proBDNF, mature BDNF and anti-proBDNF antibody. (A-B) proBDNF-treated group displayed reduced cell viability compared with the control group. (****p<0.0001)
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Fig. 3 NSC migration assay using Millicell-insert. (A) NSCs were cultured in normal cell culture medium, normal medium plus mBDNF (30 ng/mL), normal medium plus anti-proBDNF antibody (10 µg/mL), normal medium plus different concentrations of proBDNF (0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL). DAPI was used for nucleus staining. (B) Significant differences were observed in the wells treated with mBDNF, anti-proBDNF antibody and proBDNF compared with control. (C) Among different concentrations of proBDNF treated cells and control cells, the reduced migration was displayed in a dose-dependent manner. *p<0.05, ****p<0.0001, Scale bar: 100 µm.
Fig. 4 In vitro neurosphere radiant migration. (A) Neurospheres were cultured in different medium for 48 h and imaged by phase-contrast microscopy. Distances of migration were measured individually. Red arrows indicated the radial migration outward from the core of the neurosphere and the migrated cells have the same spindle-shaped NSC morphology. (B-C) Migration distances were measured at 24 h after neurospheres were cultured on Matrigel-coated plates. (D-E) After 48 h treatment, migrated distances were recorded again. Neurospheres treated with different concentrations of proBDNF (0.1 ng/mL, 0.3 ng/mL, 1 ng/mL, 3 ng/mL, 10 ng/mL) showed dramatic decreases in migration compared with control group with no morphological changes of NSCs (****p<0.0001, n=5). Neurospheres treated with mBDNF (30 ng/mL) and anti-proBDNF antibody (10 µg/mL) displayed improved migration capability compared with control. Scale bar: 100 µm.
Fig. 5 BrdU incorporation to investigate the proliferation of neural stem cells. (A) Immunofluorescence staining with anti-BrdU antibody (red) indicated the newly born neural stem cells over 24 h. DAPI (blue) was used as the reference of nucleus. (B) Neural stem cells treated with mBDNF (30 ng/mL) and anti-proBDNF antibody (10 µg/mL) showed increased proliferation compared with control. proBDNF-treated NSCs showed significantly reduced proliferation (***p<0.001). (C) Cells treated with different concentrations of proBDNF displayed reduction in the proliferation and this effect was dose-dependent. Scale bar: 100 µm.
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Fig. 6 NSCs spontaneous differentiation. (A) Tuj1 (red) was used for neuron staining and DAPI (blue) was applied for nucleus location. (B) Significant higher percentage of neurons was found in the mBDNF-treated group (****p<0.0001). (C) proBDNF-treated groups showed reduced number of neurons compared with control (*p<0.05). (D) Immunofluorescent staining of differentiatedastrocytes. GFAP (green) was applied to indicate astrocytes and DAPI (blue) was applied for nucleus staining. (E) The ratio of GFAP-positive cells to the DAPI-positive cells. proBDNF-treated cells displayed decreased number of astrocytes compared with control cells (**p<0.01). (f) proBDNFtreated cells all showed decreases in the number of GFAP-positive cells and these effects were dosedependent. (G) Olig2 (red) was used to label oligodendrocytes and co-stained with DAPI (blue) to label nucleus. (H) Significant difference was observed between control and proBDNF treated cells (****p<0.0001). No statistical significant differences were found among control cells and mBDNF or anti-proBDNF treated cells. (I) Cells treated with proBDNF all showed decreased number of oligodendrocytes compared with control cells. 10 ng/mL proBDNF in cell culture medium displayed significant reduction in oligodendrocytes. Scale bar: 100 µm.
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Highlights:
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Mouse neural stem cells constitutively expresses proBDNF, p75NTR and Sortilin.
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proBDNF inhibits mouse neural stem cells proliferation, migration and differentiation in vitro.
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Anti-proBDNF antibody functions conversely to proBDNF in regulating proliferation, migration and differentiation of mouse neural stem cells.
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proBDNF is a factor which influences the decision of neural stem cell’s fate into neurons and glia.
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