The Mental Retardation-Associated Protein srGAP3 Regulates Survival,Proliferation,and Differentiation of Rat Embryonic Neural Stem/Progenitor Cells

Abstract

The mental retardation-associated protein, srGAP3 is highly expressed in neurogenic sites. It is thought to regulate the key aspects of neuronal development and functions. Little is known about the interaction between srGAP3 and immature neural stem cells/neural progenitor cells (NSCs/NPCs). In the current study, the expression of srGAP3 in NSCs/NPCs was detected. Then, survival, proliferation, differentiation, and morphological alteration of NSCs/NPCs were assessed after a lentivirus-mediated knockdown of srGAP3. The results showed that srGAP3 is highly expressed in NSCs/NPCs both in vitro and in vivo. After knockdown of srGAP3 (LV3-srGAP3 infection), viability and proliferation of NSCs/NPCs dramatically decreased, approximately 85% displayed a similar morphology with type I cells that have no or only few indistinguishable processes. After 7 days culture in a differentiation medium, 62.5%±8.3% of cells in the srGAP3 knockdown group were nestin-positive and 24.8%±5.8% of them were β-tubulin III-positive, which are significantly higher (30.2%±9.9% and 14.6%±2.7%) than in the control group (LV3-NC infection). In addition, cells in the knockdown group had significantly fewer, but longer processes. Our results demonstrate that srGAP3 knockdown negatively regulates NSCs/NPCs survival, proliferation, differentiation, and morphological alteration, particularly, process formation. Taken together, our results provide strong evidence that srGAP3 is involved in the regulation of biological behavior and the morphological features in rat NSCs/NPCs in vitro.

Introduction

A s one of the 4 members in slit-robo guanosine triphosphatase (GTPase) activating proteins (srGAPs) family [1], srGAP3 (also named as MEGAP) attenuates the activity of Rho GTPases and consequently affects cell morphology, bio-behavior, and function through altering the cytoskeleton protein actin in mammals [2 –5]. It also has been linked to severe mental retardation in humans [6,7]. The inverse F-BAR domain of srGAP3 regulates the key aspects of synapse development and function, and plays crucial role in long-term memory [8]. In addition, srGAP3 is critical to neural development acting as a downstream effector of neurogenin 2, participating in the specification of neural fates [9]. The nuclear-localized srGAP3 has also been shown to play a crucial role in the neuronal differentiation of Neuro2A [10]. During neuronal development, srGAP3 has been implicated in axon guidance, neuronal migration, neurite outgrowth, and dendritic morphogenesis [7,11 –15]. The in vivo detection of srGAP3 has shown that it is highly expressed in the neurogenic sites such as the ventricular and subventricular zones (VZ/SVZ), and that haploinsufficiency of srGAP3 leads to the abnormal development of neuronal structures [6,13,16]. However, no direct correlation has yet been made between the expression of srGAP3 and the overall survival and morphological differentiation of neural stem cells (NSCs) and neural progenitor cells (NPCs).

NSCs and NPCs are self-renewing and multipotent cells [17 –19]. They exist in neurogenic sites in the brain such as the VZ/SVZ and subgranular zone in the dentate gyrus of the hippocampus in both the developing brain and adult brain. They are responsible for generating the 3 main cell types of the nervous system [19,20]. During development, NSCs/NPCs assume different cellular morphologies and reside within changing microenvironments (the stem cell niche), while retaining their basic properties. NSCs/NPCs behave differently when exposed to various niche signals [21 –23]. Since srGAP3 is highly expressed in VZ/SVZ and influences neuronal differentiation and cell morphology, there must be some direct interaction between NSCs/NPCs and srGAP3. In other words, the influence of srGAP3 on neural development might be attributed to the regulation of NSCs/NPCs biological behavior.

To evaluate the effect of srGAP3 on the NSCs/NPCs, the expression of endogenous srGAP3 in NSCs/NPCs was first detected. Then, the survival, proliferation, differentiation, and morphological alteration of srGAP3 knockdown NSCs/NPCs were analyzed in vitro.

Materials and Methods

Culture and identification of rat embryonic NSCs/NPCs

NSCs/NPCs were isolated from the cerebral cortex of Sprague Dawley (SD) rat embryos on embryonic day 14 (E14) and cultured in a serum-free growth medium that contained the DMEM/F12 (the Dulbecco's modified Eagle's medium and Ham's F12, 1:1), 10 ng/mL of the basic fibroblast growth factor, 20 ng/mL of the epidermal growth factor, 1% penicillin, 1% streptomycin, 1% N2, and 2% B27 supplement (all from Invitrogen) following the protocol of Gage et al. [24] and optimized in the authors' laboratory [25]. The primary neurospheres were subcultured after 5 to 7 days in vitro (DIV). Upon passaging, the neurospheres were trypsinized and mechanically triturated into single cells. For observation of spontaneous differentiation, NSCs/NPCs were cultured on poly-l-lysine-coated coverslips in 24-well plates in the differentiation medium that contained the DMEM/F12 (1:1) with 1% fetal bovine serum (GIBCO). Immunocytochemistry staining and mouse anti-nestin (monoclonal antibody, Chemicon, 1:200), mouse anti-β-tubulin III (monoclonal antibody, Chemicon, 1:200), mouse anti-glial fibrillary acidic protein (GFAP) (monoclonal antibody, Chemicon, 1:500), and mouse anti-O4 (monoclonal antibody, Milipore, 1:1000) were used to identify NSCs/NPCs, neurons, astrocytes, and oligodendrocytes, respectively.

Construction of lentiviral vector and infection of NSCs/NPCs

The LV3 lentiviral vector carries a U6 promoter followed by a CMV-WPRE cassette that controls enhanced green fluorescent protein expression. The lentivirus encoding shRNA for srGAP3, LV3-srGAP3 was prepared by GenePharma. The shRNA sequence is as follows: 5′-GAATATGAAGCCCAAATAA-3′, which was demonstrated to knockdown srGAP3 expression dramatically [10]. The sequence for control shRNA (LV3-NC) was 5′-TTCTCCGAAC GTGTCACGT-3′. The viral solutions (5–10×10⁸ TU/mL) were stored in aliquots at −80°C for later using.

NSCs/NPCs in passage 2 to 3 were processed for lentivirus infection. Neurospheres were trypsinized into single cells and were replaced at a density of 1×10⁵ cells/mL 24 h before infection. LV3-NC and LV3-srGAP3 were added to the culture system at an multiplicity of infection of 20. The total protein was subjected for immunoblotting analysis to confirm the knockdown efficiency of LV3-srGAP3, which followed Chen's protocol [10]. The immunolabeled protein was detected by BM Chemiluminescence Western Blotting kit (Roche). Densitometric quantitation was acquired with Gel Doc 1000 system and analyzed using Quantity One software (BioRad). The antibodies, including srGAP3-3A1 (polyclonal, 1:4000) and α-tubulin (Santa Cruz, 1:1000) were used.

Assessment of NSCs/NPCs survival and proliferation

The survival and growth of NSCs/NPCs were analyzed by using a cell counting kit-8 (CCK-8, Dojindo). Cells at a concentration of 1×10⁴/100 μL growth medium were added to each well of a 96-well plate. Cell viability in 3 wells was observed every day by using an UV-1750 spectrometer reader (Shimadzu) at 450 nm for 6 days. At the same time, the diameters of neurospheres obtained from 3 individual wells of the culture plate were measured using an eyepiece micrometer to reveal the survival and the proliferation of NSCs/NPCs. All spheroids were measured and the top 30 largest diameters were selected and averaged. The apoptosis of NSCs/NPCs was also examined by Hoechst 33342 (5 μg/mL, Sigma) staining. The percentage of apoptotic cells was compared. In addition, ki-67 expression and cell cycle were analyzed to further clarify the proliferation of NSCs/NPCs. NSCs/NPCs were cultured in 6-well plates at 5×10⁵ cells/mL. Five days after infection, cell aggregates were dissociated into single-cell suspensions. Some of these cells were fixed with 4% paraformaldehyde (PFA) in a 0.1 M sodium phosphate buffer [phosphate-buffered saline (PBS, pH 7.4)] at room temperature for 30 min and plated onto the poly-l-lysine-coated coverslips for Hoechst 33342 staining. The other cells were fixed with 75% ice-cold ethanol overnight at 4°C and followed by staining with 50 μg/mL propidium iodide containing 50 μg/mL RNase A (DNase free) for 30 min at 37°C in the dark for cell cycle analysis. Totally, 10,000 cells were analyzed by fluorescence activated cell sorting (FACS) and Modfit LT software (BD Biosciences). Cell cycle distribution was evaluated by calculating the proportion of cells in phase G0/G1, S, and G2/M, respectively. In each independent experiment, 3 parallel wells were made, and the procedures were carried out in triplicate.

Morphological evaluation of NSCs/NPCs

The morphology of NSCs/NPCs cultured on coverslips in the differentiation medium was observed under a phase-contract microscope every day. Based on the various characteristics of cell morphology (numbers of processes), the attached cells were classified into 3 different types. Cells without or with few processes (<3) were identified as type I. Cells with 4 to 6 processes were identified as type II, and the cells with more than 6 processes were identified as type III. The proportion of the different types of cells in 5 individual coverslips was quantified by an observer blind to the treatment groups. Meanwhile, the number of processes of each cell was counted and the length of the longest process was measured using an eyepiece micrometer at different time points.

Tissue preparation and immunochemistry staining

Three embryonic brains were submersion fixed in freshly prepared 4% PFA in 0.1 M PBS at 4°C. Three adult SD rats were fixed by transcardiac perfusion with 4% PFA under terminal anesthetized by intraperitoneal injection of a 10% (W/V) chloral hydrate solution (300 mg/kg). Whole brains were dissected and postfixed in 4% PFA at 4°C overnight and cryoprotected in sucrose 30%, in a sodium phosphate buffer (pH 7.4). The cryosections containing VZ/SVZ were cut at 16 μm before immune-staining. Cells were cultured on poly-l-lysine-coated coverslips for 7 days, and then fixed in 4% PFA for 30 min at room temperature before immune-staining.

Immunochemistry staining was performed following the standard protocol and optimized in the authors' laboratory [25]. Primary antibodies, including polyclonal rabbit anti-srGAP3-3A1 antibody [10], polyclonal mouse anti-ki-67 (Chemicon, 1:300), and mouse anti-nestin, mouse anti-β-tubulin III, mouse anti-GFAP, and mouse anti-O4 (described as above) were diluted in PBS with 2% normal goat serum (NGS). The blocking solution contained 5% NGS and 0.25% Triton X-100 in PBS. Tetramethyl rhodamine isothiocyanate- and fluorescein isothiocyanate-conjugated goat anti-mouse IgG/anti-rabbit IgG (1: 400; CoWin Biosciences) were used as secondary antibodies. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole-containing mounting media (Vector Labs) and visualized under a fluorescent microscope (Olympus BX57) equipped with a DP70 digital camera and the DPManager (DPController, Olympus) software. For the negative control, the primary antibody was replaced by the blocking buffer.

Statistic analysis

All the quantitative data were obtained from 3 independent experiments. They were presented as mean±SE, and statistical analysis was performed with software Prism 4.0. The T-test and one-way ANOVA were used and a P value <0.05 was considered significant.

Results

SrGAP3 is highly expressed in NSCs/NPCs

NSCs/NPCs were isolated from the cerebral cortex of SD rat embryos and cultured in the growth medium. Neurospheres of different sizes developed over a period of 5 to 7 DIV and the majority of cells in neurospheres were found to be nestin-positive (Fig. 1A, B). After 7 days culture in the differentiation medium, β-tubulin III-positive neuronal progenitors/neurons, GFAP-positive astrocyte progenitors/astrocytes, and O4-positive oligodendrocytes were observed (Fig. 1C–E). SrGAP3 expressed in almost 100% of cultured cells, including nestin-positive NSCs/NPCs (Fig. 1F, srGAP3/nestin double labeled), and in differentiated neuronal progenitors and neurons (Fig. 1G, srGAP3/β-tubulin III double labeled) or astrocyte progenitors and astrocytes (Fig. 1H, srGAP3/GFAP double labeled). The expression persisted for periods of up to 14 days in vitro (data not shown). Expression of endogenous srGAP3 was also detected in brain tissue, results showed that, both in developing (Fig.1I, J) and adult brain (Fig. 1K, L), srGAP3 expressed predominantly in VZ/SVZ and colocalized with nestin-positive NSCs/NPCs.

FIG. 1.

Expression of srGAP3 in neural stem cells/neural progenitor cells (NSCs/NPCs). NSCs/NPCs isolated from the rat embryo cortex were cultured in the Dulbecco's modified Eagle's medium/F12 serum-free medium. (A) Neurospheres of different sizes developed at 7 DIV. (B) Nestin-positive MSCs/NPCs in monolayer culture at 7 DIV. (C) β-tubulin III-positive neuronal progenitors and neurons. (D) Glial fibrillary acidic protein (GFAP)-positive astrocyte progenitors and astrocytes. (E) O4-positive oligodendrocytes. (F) Expression of srGAP3 in NSCs/NPCs, (nestin/srGAP3 double-labeled cells) at 2 DIV. (G) Expression of srGAP3 in neuronal progenitors and neurons (β-tubulin III/srGAP3 double-labeled cells) at 7 DIV. (H) Expression of srGAP3 in astrocyte progenitors and astrocytes (GFAP/srGAP3 double-labeled cells) at 7 DIV. (I, J) Expression of srGAP3 in the ventricular zone (VZ) of the embryonic brain and colocalized with nestin-positive NSCs/NPCs. (K, L) Expression of srGAP3 in the subventricular zone (SVZ) of the adult brain and colocalized with nestin-positive NSCs/NPCs. Nestin, β-tubulin III, and GFAP are in green and srGAP3 is in red. 4′,6-diamidino-2-phenylindole for nuclei in B–D and I-L is in blue. DIV: days in vitro. Scale bar=50 μm.

NSCs/NPCs viability and proliferation dramatically decreased after srGAP3 knockdown

Expression of srGAP3 in cultured NSCs/NPCs significantly decreased after the infection of LV3-srGAP3 (Fig. 2A). Shortly after infection, some of NSCs/NPCs started to attach to the surface of tissue culture flasks and the cell debris was clearly observed on the second day, especially in the knockdown group (LV3-srGAP3 infection). Although cells in both groups proliferated and grew into neurospheres at 7 DIV, the diameters of neurospheres in the knockdown group were significantly smaller than that in the control group (Fig. 2B, P<0.001). There were significantly more apoptotic cells with condensed chromatin and stained more heavily (Fig. 2C, arrow, P<0.001) in the knockdown group. The CCK-8 assay further confirmed that the number of viable cells dramatically decreased after LV3-srGAP3 infection (Fig. 2D). Approximately 58.7%±1.8% of cells in the knockdown group were ki-76-positive, which is significantly less than that in the control group (83.2%±2.1%, P<0.001, Fig. 2E). Cell cycle analysis by FACS similarly showed a significantly lower percentage of cells in phases G2/M and S in the srGAP3 knockdown group (5.1%±2.0%, 15.5%±3.1%) as compared to the control group (13.0%±3.4% and 26.9%±5.7%) (Fig. 2F, P<0.01).

FIG. 2.

Assessment of survival and proliferation of NSCs/NPCs after SrGAP3 knockdown. (A) Expression of srGAP3 dramatically decreased after infection of LV3-srGAP3. (B) Cells in the srGAP3 knockdown group developed significantly smaller neurospheres. (C) There were significantly more apoptotic cells (arrows) in the srGAP3 knockdown group. (D) Cell viability tested by the cell counting kit-8 dramatically decreased after srGAP3 knockdown. (E) Proportion of ki-67-positive cells (arrows) in the srGAP3 knockdown group significantly decreased. (F) In the srGAP3 knockdown group, significantly lower percentage of cells in phases G2/M and S. DAPI for nuclei in C and E is in blue. Ki-67 was labeled with tetramethyl rhodamine isothiocyanate in red and ki-67-positive cells were showed in purple after merged with DAPI. Scale bar=50 μm, **P< 0.01, ***P< 0.001.

Most of cells in the srGAP3 knockdown group appeared as type I cells

The number of cells showing different morphological features (Fig. 3A) was counted at different time points. In the knockdown group, with the culturing in the differentiation medium, the percentage of type I cells increased (Fig. 3B), whereas the proportion of type II cells slightly decreased (Fig. 3C). There was no significant alteration in regarding type III cells (Fig. 3D). In comparison with the control group, there was significantly more type I cells, but less type III cells in the srGAP3 knockdown group (Fig. 3B, D, P<0.05).

FIG. 3.

Morphological observation of NSCs/NPCs after srGAP3 knockdown. (A) Based on different morphological features, along the differentiation path, the attached cells were classified into 3 different cell types. (B) With the culturing in the differentiation medium, the percentage of type I cells in the knockdown group was increased and significantly higher than that in the control group. (C) The percentage of type II cells slightly decreased with respect to time in culture, but no significant difference was observed between the knockdown group and the control. (D) No significant alteration was found in regarding the population of type III cells with the culturing in the differentiation medium in the knockdown group, but it was significantly less than in the control group. *P<0.05, **P<0.01, ***P<0.001.

Cells in the srGAP3 knockdown group predominantly remained as immature cells

After 7 days culture in the differentiation medium, in the knockdown group, 62.5%±8.3% of the cells were nestin-positive undifferentiated cells and 24.8%±5.8% were β-tubulin III-positive neurons. This represented a significantly higher percentage than in the control group (30.2%±9.9% and 14.6%±2.7%, respectively). However, the percentage of GFAP-positive astrocytes was significantly lower in the knockdown group (26.4%±1.6%) than in the control group (40.3%±9.7%, Fig. 4A, P<0.05). No significant difference was noticed between these 2 groups in regarding to the ratio of O4-positive cells. In addition, a small proportion of these cells were nestin/β-tubulin III or nestin/GFAP double-positive progenitors (data not shown). Interestingly, in the srGAP3 knockdown group, cells displayed a similar morphology having less than 3 processes per cell, which differed from the control group and was independent of the specific phenotypes (Fig. 4B).

FIG. 4.

Differentiation of NSCs/NPCs after srGAP3 knockdown. (A) Population of different phenotypes after 7 days culture in the differentiation medium. (B) Morphology of nestin-, β-tubulin III-, and GFAP-positive cells in the control group (NC) and the srGAP3 knockdown group. Nestin, β-tubulin III, and GFAP are in red and srGAP3 is in green, DAPI for nuclei is in blue. Scale bar=50 μm, *P<0.05, **P<0.01.

Cells in the SrGAP3 knockdown group had significantly fewer, but longer processes

To further clarify the effect of srGAP3 on the morphological features of NSCs/NPCs during spontaneous differentiation, the average number of processes per each cell and the length of the longest process were quantified. Result showed that the density of cell processes in the control group increased with respect to time in culture, a similar pattern was not found in the srGAP3 knockdown group (Fig. 5A). Quantification of cell processes showed that after 3 days, each cell in the knockdown group had significantly less processes than those of the control group (Fig. 5B, P<0.01). Nevertheless, we noticed that with the increasing culture time, the length of the longest processes gradually increased, particularly in the srGAP3 knockdown group and were found to be significantly longer than those of the control group (Fig. 5C, P<0.01). No difference was found in the cell soma size.

FIG. 5.

Quantification of the number of processes and the length of axons. (A) Morphological features of cells in the control group (NC) and the srGAP3 knockdown group during spontaneous differentiation. (B) Cells in the srGAP3 knockdown group had significantly lower number of processes after 3 DIV. (C) The length of the longest process in the srGAP3 knockdown group is significantly higher than that in the control group. Scale bar=50 μm, *P<0.05, **P<0.01, ***P<0.001.

Discussion

The results presented here shown that srGAP3 is expressed predominately in NSCs/NPCs both in vitro and in vivo. After srGAP3 knockdown, NSCs/NPCs viability and proliferation is dramatically reduced, the differentiation of NSCs/NPCs is delayed, cell morphology altered, and the genesis of process was attenuated, but the extension was significantly enhanced.

It has been shown that srGAP3 is highly expressed in the neurogenic areas [8,16,26,27]. It is possible that it functions as a niche signal to regulate NSCs/NPCs biological behavior and fate decisions. Here we found that endogenous srGAP3 is not only widely expressed in the NSCs/NPCs in neurogenic sites in vivo, but also in cultured NSCs/NPCs. Interestingly, it was also detectable in β-tubulin III-positive and GFAP-positive cells. It may be because some of these cells were neuronal progenitors or glial progenitors. It also suggests that srGAP3 might have an effect on the neuron and glia.

In mammals, srGAP3 attenuates the activity of Rho GTPases resulting in the alteration of the cytoskeleton proteins. It therefore plays important roles in cell surviving and proliferation [2 –5]. We found here, after knockdown of srGAP3, more NSCs/NPCs went into apoptosis and less than a quarter of cells were in active mitosis, which further resulted in the dramatically decreased cell viability and reduced size of neurospheres. This result further confirms that srGAP3 is essential in regulating NSCs/NPCs survival and proliferation. The Slit-Robo signaling pathway and Rac1 may be involved [2,10,28]. In addition, we noticed that approximately two thirds of cells in the srGAP3 knockdown group were nestin-positive progenitors after 7 days culturing in the differentiation medium. In other words, differentiation of NSCs/NPCs was attenuated after srGAP3 knockdown. This result suggests that srGAP3 is also crucial for NSCs/NPCs differentiation. The mechanism regarding how srGAP3 is critically needed for maintaining the NSCs/NPCs population and also plays crucial roles in NSCs/NPCs differentiation still remain unknown.

Our previous studies observed the expression of endogenous srGAP3 was upregulated in the early stages of neuronal differentiation of Neuro2A cells, however, low levels of srGAP3 enhanced the Neuro2A neuronal differentiation. This paradoxically phenomenon may due to the membrane relocalization of srGAP3 [10]. Similarly, we found that srGAP3 is detectable in β-tubulin III-positive neuronal progenitors/neurons, and there were more β-tubulin III-positive, but less GFAP-positive cells in the srGAP3 knockdown group compared to the control group. Combining these results, it suggests that the effect of srGAP3 on neuronal differentiation might lie in a spatiotemporal sequence. It is relevant therefore that we should not ignore that some of the β-tubulin III-positive cells that we have counted as neurons as being neuronal progenitors (nestin/β-tubulin III double-positive), as neuronal differentiation precedes glial differentiation during development. However, the significant reduction of GFAP-positive cells in the srGAP3 knockdown group, including the nestin/GFAP double-positive glial progenitors, indicates that glial differentiation of NSCs/NPCs was attenuated dramatically by low levels of srGAP3. No significant effect was observed on oligodendrocyte differentiation. This phenomenon has not been reported previously, and it therefore suggests that srGAP3 may also play important roles in gliogenesis. Whether this is related to the activity of the Rho GTPase activity or neurogenin 2 needs to be further studied.

Rho proteins are key regulators of the cellular cytoskeleton. Different extracellular and intracellular signals converge on the regulation of the cytoskeleton. Rho GTPase is one of the major intracellular regulators [2,10,29 –33]. Immature NSCs/NPCs are normally round and symmetrical without any process in the undifferentiated state. The appearance of processes sprouting from the cell body indicates the start of differentiation/maturation. As development proceeds, the symmetry is broken, and cell polarity is established alongside the NSCs switch to lineage restricted progenitors. Finally, mature neurons and glia acquire their specific morphological features [32,33]. Breaking the symmetry, in particular axon specification depends on cytoskeletal rearrangements [32]. Cells with low levels of srGAP3 might have difficulties in the cytoskeletal remodeling that resulted in the arrest of morphological changes. This suggestion is confirmed by our results and showed that a high percentage of the cells in the srGAP3 knockdown group were of type I cells, which have no or very few indistinguishable processes. Furthermore, cell differentiation always accompany with morphological alteration. Therefore, the type I cells in the srGAP3 knockdown group might be undifferentiated progenitors or in a very early stage of differentiation. This is consistent with our immunochemistry staining.

SrGAP3 has also been implicated in axon guidance, neuronal migration, neurite outgrowth, and dendritic morphogenesis during neuronal development [11 –15]. In the current study, we found that the cell process development was attenuated by low levels of srGAP3. However, no corresponding changes in the process outgrowth were observed. The average length of the longest processes increased gradually with increasing time in culture and was significantly higher in the knockdown group compared to the control group. This is consistent with existed data [2,10], and indicates that srGAP3 may have a different influence on process genesis and extension. In addition, due to the distinct cytoskeletal reorganization, the morphology of neurons and astrocytes altered differently in response to insults. After stimulation, the processes of astrocytes retract as the cell soma enlarges and progresses to cell division. On the contrary, processes of neurons, particularly axons extend primarily to form synapses. In the current study, a dramatic decrease in the number, but a significant increase in the length of cell processes was observed. It should be pointed out that some astrocytic processes might have also been included in the evaluation, especially in the earlier period. However, after 3 days in culture, a significantly higher percentage of cells in the srGAP3 knockdown group could be identified as neurons as seen under the phase-contrast microscope and displayed typical neuronal morphology having 1 axon and few dendrites. The number of neurites dramatically decreased and the length of the longest neurite (axon) significantly increased. The question regarding the difference of the srGAP3 effect on the cytoskeletal reorganization of neurons and astrocytes needs to be further studied.

In conclusion, we found that knockdown of srGAP3 expression attenuated NSCs/NPCs survival, proliferation, and differentiation and arrested their morphological alteration. It suggested that srGAP3 may play an important role in the biological behavior and fate decision of rat NSCs/NPCs. It may also in some way direct the polarity formation of NSCs/NPCs. SrGAP3 is closely related to mental retardation. It might have a significant bearing on the ability of neurons to make appropriate synaptic contacts in the formation of neural networks. Failure to achieve this would lead to inappropriate or missing neural circuitry. Further in vivo studies regarding the relationship between srGAP3 and neural development as well as the cellular molecular mechanism are needed.

Footnotes

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (31070943, 81070998, and 31171033).

Author Disclosure Statement

The authors indicate there is no conflict of interest in connection with this article.

References

Coutinho-Budd

, Ghukasyan

, Zylka

, Polleux

. 2012. The F-BAR domains from srGAP1, srGAP2, and srGAP3 differentially regulate membrane deformation. J Cell Sci, 125:3390–3401.

Yang

, Marcello

, Endris

, Saffrich

, Fischer

, Trendelenburg

, Sprengel

, Rappold

. 2006. MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation. Exp Cell Res, 312:2379–2393.

Endris

, Haussmann

, Buss

, Bacon

, Bartsch

, Rappold

. 2011. SrGAP3 interacts with lamellipodin at the cell membrane and regulates Rac-dependent cellular protrusions. J Cell Sci, 124:3941–3955.

Lavelin

, Geiger

. 2005. Characterization of a novel GTPase-activating protein associated with focal adhesions and the actin cytoskeleton. J Biol Chem, 280:7178–7185.

Raftopoulou

, Hall

. 2004. Cell migration: Rho GTPases lead the way. Dev Biol, 265:23–32.

Endris

, Wogatzky

, Leimer

, Bartsch

, Zatyka

, Latif

, Maher

, Tariverdian

, Kirsch

, Karch

, Rappold

. 2002. The novel Rho-GTPase activating gene MEGAP/srGAP3 has a putative role in severe mental retardation. Proc Natl Acad Sci U S A, 99:11754–11759.

Newey

, Velamoor

, Govek

, Van Aelst

. 2005. Rho GTPases, dendritic structure, and mental retardation. J Neurobiol, 64:58–74.

Carlson

, Lloyd

, Kruszewski

, Kim

, Rodriguiz

, Heindel

, Faytell

, Dudek

, Wetsel

, Soderling

. 2011. WRP/srGAP3 facilitates the initiation of spine development by an inverse F-BAR domain, and its loss impairs long-term memory. J Neurosci, 31:2447–2460.

Mattar

, Britz

, Johannes

, Nieto

, Ma

, Rebeyka

, Klenin

, Polleux

, Guillemot

, Schuurmans

. 2004. A screen for downstream effectors of Neurogenin2 in the embryonic neocortex. Dev Biol, 273:373–389.

10.

Chen

, Mi

, Ma

, Fu

, Jin

. 2011. The mental retardation associated protein, srGAP3 negatively regulates VPA-induced neuronal differentiation of Neuro2A cells. Cell Mol Neurobiol, 31:675–686.

11.

Bacon

, Endris

, Andermatt

, Niederkofler

, Waltereit

, Bartsch

, Stoeckli

, Rappold

. 2011. Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord. PLoS One, 6:1–9.

12.

Fukata

, Nakagawa

, Kaibuchi

. 2003. Roles of Rho-family GTPases in cell polarisation and directional migration. Curr Opin Cell Biol, 15:590–597.

13.

Wong

, Ren

, Huang

, Xie

, Liu

, Saito

, Tang

, Wen

, Brady-Kalnay

et al. 2001. Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell, 107:209–221.

14.

Iden

, Collard

. 2008. Crosstalk between small GTPases and polarity proteins in cell polarization. Nat Rev Mol Cell Biol, 9:846–859.

15.

Arimura

, Kaibuchi

. 2007. Neuronal polarity: from extracellular signals to intracellular mechanisms. Nat Rev Neurosci, 8:194–205.

16.

Bacon

, Endris

, Rappold

. 2009. Dynamic expression of the Slit-Robo GTPase activating protein genes during development of the murine nervous system. J Comp Neurol, 513:224–236.

17.

Sommer

, Rao

. 2002. Neural stem cells and regulation of cell number. Prog Neurobiol, 66:1–18.

18.

Parati

, Pozzi

, Ottolina

, Onofrj

, Bez

, Pagano

. 2004. Neural stem cells: an overview. J Endocrinol Invest, 27:64–67.

19.

Price

, Williams

. 2001. Neural stem cells. Curr Opin Neurobiol, 11:564–567.

20.

Bottai

, Fiocco

, Gelain

, Defilippis

, Galli

, Gritti

, Vescovi

. 2003. Neural stem cells in the adult nervous system. J Hematother Stem Cell Res, 12:655–670.

21.

Fuentealba

, Obernier

, Alvarez-Buylla

. 2012. Adult neural stem cells bridge their niche. Cell Stem Cell, 10:698–708.

22.

Moyse

, Segura

, Liard

, Mahaut

, Mechawar

. 2008. Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system. Curr Stem Cell Res Ther, 3:163–184.

23.

Jordan

, Ma

, Ming

G-l

, Song

. 2007. Cellular niches for endogenous neural stem cells in the adult brain. CNS Neurol Disord Drug Targets, 6:336–341.

24.

Gage

, Coates

, Palmer

, Kuhn

, Fisher

, Suhonen

, Peterson

, Suhr

, Ray

. 1995. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci U S A, 92:11879–11883.

25.

, Hao

, Jiao

, Xie

, Zhang

, Lu

, Cai

, Wang

, Yang

, Parker

, Liu

. 2011. Neurotrophin-3 gene transduction of mouse neural stem cells promotes proliferation and neuronal differentiation in organotypic hippocampal slice cultures. Med Sci Monit, 17:BR305–BR311.

26.

Yao

, Jin

W-L

, Wang

, Ju

. 2008. Regulated shuttling of Slit-Robo-GTPase activating proteins between nucleus and cytoplasm during brain development. Cell Mol Neurobiol, 28:205–221.

27.

Waltereit

, Kautt

, Bartsch

. 2008. Expression of MEGAP mRNA during embryonic development. Gene Expr Patterns, 8:307–310.

28.

Dickinson

, Duncan

. 2010. The SLIT-ROBO pathway: a regulator of cell function with implications for the reproductive system. Reproduction, 139:697–704.

29.

Hall

, Lalli

. 2010. Rho and Ras GTPases in axon growth, guidance, and branching. Cold Spring Harb Perspect Biol, 2:a001818.

30.

Luo

, Shan

, Guo

, Smrt

, Johnson

, Li

, Pfeiffer

, Szulwach

, Duan

et al. 2010. Fragile X mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells. Plos Genet, 6:e1000898.

31.

Callan

, Cabernard

, Heck

, Luois

, Doe

, Zarnescu

. 2010. Fragile X protein controls neural stem cell proliferation in the Drosophila brain. Hum Mol Genet, 19:3068–3079.

32.

Tahirovic

, Bradke

. 2009. Neuronal polarity. Cold Spring Harb Perspect Biol, 1:a001644.

33.

Zhao

, Teng

, Summers

Jr. , Ming

, Gage

. 2006. Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J Neurosci, 26:3–11.