Antibody to collapsin response mediator protein 1 promotes neurite outgrowth from rat hippocampal neurons＊＊＊＊★

Hongsheng Lin, Jing Chen, Wenbin Zhang, , Xiaobing Gong, , Biao Chen, Guoqing Guo

1Department of Orthopedics, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China

2Department of Anatomy, Medical College of Jinan University, Guangzhou 510630, Guangdong Province, China

lNTRODUCTlON

Although much has yet to be learned about how neurites integrate guidance signals to generate the appropriate cellular response,the activation of surface membrane receptors leads to intracellular signaling cascades that ultimately lead to cytoskeletal changes[1]. Collapsin response mediator proteins (CRMPs; also known as TOAD-64, Ulip, and DRP) are a family of cytosolic phosphoproteins that are expressed exclusively in the nervous system[2]. Based on sequence similarity, five members of the protein family, CRMP-1, 2,3, 4 and 5, have been identified. CRMPs act as downstream effectors of extracellular signals and associate with lipid rafts to regulate cell morphological remodeling[3].

They are potent regulators of the cytoskeleton, transforming extracellular guidance signals into cytoskeletal changes,and they regulate axon outgrowth by modulating actin and microtubule dynamics, in response to neurite growth cues[4].

Distinct from the other CRMPs, CRMP-1 induces collapse of neuronal growth cones to inhibit neurite outgrowth[5-6]. Moreover,expression of CRMP-1 peaks from the late embryonic stage to neurogenesis. After this peak in developmental expression,CRMP-1 is constitutively expressed at lower levels throughout adulthood, reaching the highest levels in the elderly[7-9]. The persistent expression of CRMP-1 implies that it may play a role in neurodegenerative disease. Thus, the inhibition of CRMP-1 may accelerate neurite outgrowth and impede neural degeneration.

In the present study, we propose that blocking the function of CRMP-1 with antibody should promote neurite outgrowth of hippocampal neurons. In order to verify this hypothesis, we blocked CRMP-1 protein with a polyclonal antibody in cultured hippocampal neurons. Neurite outgrowth and cytoskeletal changes were captured with atomic force microscopy and laser confocal microscopy.

RESULTS

ldentification of hippocampal neurons

To determine whether cells isolated from rat hippocampus were neurons, we examined cell morphology with immunofluorescence staining. Isolated cells initially had a spherical appearance and began to extend processes within hours after adhesion. Almost all cells exhibited a characteristic neuron-like morphology after 8 days in culture. Many had a pyramidal shape with active growth cones seeking to expand the neuronal network (Figure 1A). We used tubulin and glial fibrillary acidic protein(GFAP) antibodies to identify neurons and astroglia, respectively. Neurons were more abundant than astroglia and exhibited tapered processes with multiple branches and several shorter, tapering dendrites, and represented,on average, 66.7% of the total cell population after 8 days in culture. Astroglia were smoother with few branches and were present at low levels, accounting for the remaining 33.3% of cells. Typical morphologies obtained with immunostaining are presented in Figures 1B,C.

Figure 1 Culture and identification of primary hippocampal neurons in rats. Scale bars: 100 μm.

Hippocampal neurons under atomic force microscopy

Isolated hippocampal neurons were seeded onto poly-L-lysine-coated 6-well plates prior to the following treatments: for the control group, cells were cultured with neurobasal medium alone; for the CRMP-1 antibody blocking group, rabbit anti-CRMP-1 polyclonal antibody was added to the medium; for the CRMP-1 antibody blocking control group, cells were treated with CRMP-1 antibody for 72 hours, then the medium was replenished;for the CRMP-1 antibody blocking and Y27632 intervention group, cells were treated with CRMP-1 antibody for 72 hours, then the medium was replenished and cell culture was continued in the presence of Y27632. Cells were observed with atomic force microscopy.

Scanning images at 90 μm × 90 μm were able to capture the full morphology of the cells. After 6 days of culture in control medium, neurons established their characteristic morphology and had a clearly distinguishable major process. They were asymmetric in shape, having a single, long major process (axon) and several minor processes (dendrites) of much shorter length. Approximately four processes emerged from the soma of each cell.

Typical morphology as revealed by atomic force microscopy is shown in Figure 2A. A scanning image at 40 μm × 40 μm revealed many smaller and shorter, tapering branches extending perpendicular to the axons and dendrites.

After blocking with CRMP-1 antibody, the number of processes emerging from each cell soma was approximately 5, similar to cells cultured in control medium.

However, branches emerging from the axons and dendrites were much longer, and some were as long as the parent process (Figure 2B). Images captured by the 40 μm scanner reveal that many shorter, tapering branches extended perpendicular to the main processes. Figure 2C shows one control neuron that was previously blocked with CRMP-1 antibody for 72 hours, but without Y27632. Images indicate that the branches were similar to those of neurons cultured in the presence of CRMP-1 antibody. These branches formed an extensive neurite network with neighboring cells.

In cells treated with Y27632 after blocking with CRMP-1 antibody, branches were more abundant, as judged by their morphology, compared with control cells. The cells had numerous neurites emerging from the soma, and despite the lack of neighboring cells, branches still formed an elaborate network (Figure 2D). The right panel is an image of the frame captured by the 40 μm scanner.It shows that abundant shorter, tapering branches emerged from the main branches, and formed an elaborate network among themselves in a single cell.

Quantification of neurite outgrowth from hippocampal neurons

Quantification of neurite outgrowth was performed to evaluate neurite outgrowth of treated hippocampal neurons at the same time as neurite outgrowth quantification assays. There was an increase in the absorbance of neurite extracts from blocked cells, and this increase was significantly greater than that of control cells (P ＜ 0.01).

When blocked cells were incubated with Y27632, the absorbance of the neurite extracts was the highest,compared with blocked control cells not treated with Y27632 (P ＜ 0.01; Figure 3).

Figure 2 Neurites of hippocampal neurons examined by atomic force microscopy. The left panel shows a neuron in an area of 90 μm × 90 μm. Scale bars: 30 μm. The right panel is an enlargement of the photo in the square frame in an area of 40 μm × 40 μm. Scale bars: 10 μm.

Figure 3 Quantification of hippocampal neuron neurite extracts. Data are expressed as mean ± SD. Two sample-t test was used to examine differences between control and treatment groups. aP ＜ 0.01, vs. control cells; bP ＜ 0.01, vs.blocked cells and cells treated without Y27632.

Microtubule rearrangement in hippocampal neurons under laser confocal microscopy

To examine neurite outgrowth in hippocampal neurons blocked with CRMP-1 antibody, we evaluated microtubule dynamics by laser confocal microscopy. After 8 days of culture in control medium, microtubules were clearly visible in the soma of neurons by red fluorescent dye labeling. Microtubules formed an elaborate network surrounding the nucleus and were aligned in parallel arrays to form bundles that projected into the base of neurites (Figure 4A). When cells were cultured in medium containing CRMP-1 antibody, small microtubule seeds were visible in cell bodies. However, the cellular microtubule network nearly disappeared (Figure 4B).

In cells previously blocked with CRMP-1 antibody for 72 hours, but without Y27632 treatment, the network formed by microtubules was similar to that of cells cultured in medium containing CRMP-1 antibody alone (Figure 4C).

Although the microtubules were not as clearly visible compared with neurons cultured in control medium, the microtubule network recovered in cells treated with Y27632. A greater number of microtubules were labeled by fluorescent dye and microtubule bundles extended into neurites (Figure 4D).

Figure 4 Microtubule reorganization of hippocampal neurons under laser confocal microscopy. Microtubules were labeled with a red fluorescent Cy3 dye and nuclei were stained with a green fluorescent Hoechst 33258 dye.Scale bars: 10 μm.

DlSCUSSlON

Although much has yet to be learned about how CRMPs mediate their crucial role in axon elongation, they are known to be substrates for Rho-associated protein kinase and relay neurite outgrowth signals[10-11]. Instead of binding to the microtubule polymer, CRMP-2 binds free tubulin subunits and promotes microtubule assembly resulting in neurite elongation[12-13]. However, the individual members of the CRMP family play different roles in neurite elongation. For example, CRMP-2, 3, 4 and 5 appear to induce axon outgrowth by promoting microtubule assembly, but CRMP-1 is thought to induce axon shortening by inhibiting microtubule assembly in cultured neurons[14-17]. In the present study, when cells were blocked with CRMP-1 antibody, branches emerging from axons and dendrites were elongated and some were as long as the parent process. Quantification of neurite outgrowth showed that the increase in optical density of neurite extracts was significantly greater than that of control cells. These results indicate that blocking CRMP-1 function promotes neurite outgrowth and branching of hippocampal neurons; similar to results obtained with DRG sensory neurons[5].

Microtubules are crucial for determining the fate of neurites (i.e., axon vs. dendrite), thereby establishing and maintaining neuronal polarity[18-19]. In addition to a crucial role in guidance, the dynamic regulation of microtubules has been found to be essential for neurite elongation as well, and was previously shown to be a prerequisite for continued growth cone migration[20-21].

CRMPs play key roles in axon guidance and elongation by regulating interactions between microtubules and actin filaments[22-24]. These roles are important for the formation and extension of both elongating axons and their growth cones. No evidence was found to indicate that CRMP1 could initiate and change microtubule polymerization in the cell body, axon or growth cone. During metaphase, the majority of CRMP-1 is strongly associated with the mitotic spindle and concentrated near centrosomes during very late telophase[25]. In neurons,CRMP-1 is diffusely distributed in the cytoplasm, but is concentrated at the end of each axon and branch site[7].

When neurons overexpress CRMP-1, neurite outgrowth and branching are inhibited in vitro[5-6]. In the present study, when cells were cultured in medium containing CRMP-1 antibody, small microtubule seeds were still visible in cell body, but the network formed by microtubules almost vanished and only a vestige of the network was visible. These results imply that the active polymerization and depolymerization of microtubules was at play during this treatment.

The actin and microtubule cytoskeleton of neuronal growth cones and their interactions play a central role in cellular shape changes and guidance during axon elongation. At first, filopodia and thin lamellipodia consisting of filamentous actin rapidly protrude from the leading edge of the growth cone. Subsequently, microtubules invade into these projections, enlarging them. When the majority of F-actin in the neck of the growth cone depolymerizes, the protrusions allow the membrane to shrink around the microtubule bundles, forming a cylindrical axon shaft or a new axon[26]. F-actin depolymerization is critical for axon guidance, elongation, advancement and retraction, while the microtubule array within the growth cone mainly reorganizes and reorients toward the future direction of axon outgrowth[27]. Because blocking CRMP-1 promoted neurite outgrowth by inducing cytoskeletal reorganization, our results suggest that the microtubule arrays were responsible for the neurite outgrowth evoked by CRMP-1 antibody blocking.

The selective Rho inhibitor, Y-27632, inhibits the kinase activity of the enzyme by competing with its ATP binding site, and has been widely used to examine the function of the kinase and related cellular factors. Y-27632 has been shown to obstruct the axon growth inhibition cascade,and induces neurite outgrowth in cultured neurons[28-29].

We investigated how Y27632 might affect neurite growth responses evoked by CRMP-1 blocking. Treatment with Y27632, after blocking with CRMP-1 antibody, led to the emergence of numerous neurites from the soma. In addition, branches were more abundant and the length of neurites was also significantly increased compared with control neurons. Furthermore, the microtubule network appeared to have recovered in cells treated with Y27632.

These results suggest that Y27632 not only promotes neurite outgrowth, but also helps form and stabilize cytoskeletal networks by promoting the polymerization of microtubules. The fact that Y27632 promotes neurite outgrowth provides further evidence that CRMP-1 and Rho kinase play critical roles in regulating microtubule dynamics.

MATERlALS AND METHODS

Design

A comparative in vitro cell culture experiment.

Time and setting

The experiments were conducted at the Medical College of Jinan University, China, from July 2006 to July 2007.

Materials

Thirty-five one-day-old Sprague-Dawley rats, SPF grade,weighing 15 ± 5 g, were purchased from Medical Laboratory Animal Center of Southern Medical University,China, with permission No. SCXK(Guangdong)2006-0015. All experimental protocols were approved by the Local Experimental Ethics Committee and were in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, formulated by the Ministry of Science and Technology of China[30].

Methods

Isolation and culture of primary hippocampal neurons

Hippocampal neurons were obtained by mechanical or enzymatic dissociation from 1-day-old Sprague-Dawley rats as previously described by Brewer[31]. Briefly, dissected tissue was digested for 30 minutes at 37°C in Dulbecco’s modified Eagle’s medium containing Nutrient Mixture F12 (Gibco, Grand Island, NY, USA) and 0.25%(w/v) Trypsin (Gibco). Subsequently, the tissue was gently triturated through a fire-polished Pasteur pipette in neurobasal medium (Invitrogen, Carlsbad, CA, USA)supplemented with 2% B27 (Gibco). After dissociation into a single cell suspension, cells were recovered by centrifugation, resuspended and cultured in neurobasal medium supplemented with 2% B27. Cultures were initiated in 6-well plates (Falcon, Suwanee, GA, USA) precoated (overnight at 4°C) with 20 mg/mL poly-L-lysine(Sigma, St. Louis, MO, USA) in Hank’s balanced salt solution (Gibco) at a seeding density of 2 × 105cells/dish.

Cultures were maintained in a humidified 37°C atmosphere containing 5% CO2. After 3 days in culture, all medium was replenished, and half of the growth medium was replenished every 3 days thereafter. Cells were visualized using an inverted microscope. Mouse anti-α-tubulin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GFAP antibodies(Chemicon, Billerica, MA, USA) were used to identify hippocampal primary neurons.

Grouping and treatment of primary hippocampal neurons

Cells were routinely plated onto 6-well plates containing slides coated with poly-L-lysine. Control medium contained neurobasal medium supplemented with 2% B27 and 0.2% rabbit anti-IgG (Serotec, Kidlington, UK). Antibody blocking cultures contained control medium and 0.2% rabbit anti-rat CRMP-1 polyclonal antibody(Chemicon). Rabbit anti-IgG and CRMP-1 antibody were diluted in control medium and added to cultures 12 hours after plating. Rho kinase inhibitor Y27632 (Sigma) cultures contained control medium, 0.2% CRMP-1 polyclonal antibody and 200 ng/mL Y27632 as previously described by Quach et al[32]and Nakayama et al[33]. After culture in control medium containing 0.2% CRMP-1 polyclonal antibody for 72 hours, all medium was replenished. Then Rho kinase inhibitor Y27632 was diluted in control medium and added to cultures. As a control, no Y27632 was added to cultures after all medium was replenished. After 8 days of culture, cells were fixed in 4%paraformaldehyde for 1 hour at room temperature for fluorescence labeling and atomic force microscopy.

Atomic force microscopy

Cells were seeded onto a 6-well culture plate with poly-L-lysine-coated glass coverslips inside at a seeding density of 2 × 105cells/dish. After treatment for 6 days,they were fixed with 4% paraformaldehyde for 1 hour at room temperature. After two washes in water, at least five samples were placed under the atomic force microscope (Thermo, West Chester, Pennsylvania, USA). The atomic force microscope was operated in tapping mode for improved imaging of soft samples in air (relative humidity of 40-50%). The two scanners used in the present study had a maximum scan range of 90 μm × 90 μm and 40 μm × 40 μm (x, y direction). The images were obtained over two different areas. An area of 90 μm × 90 μm was imaged to display the complete morphology of neurons,while the 40 μm × 40 μm images were used to capture neurites. They were equipped with V-shaped silicon nitride cantilevers (Thermo) with a spring constant of 2.5 N/m. The tip radius was shorter than 10 nm. All atomic force microscopy data were composed of 256 scan lines and 256 pixels per line, and were acquired only by one-direction scanning.

Neurite outgrowth quantification assays

To evaluate neurite outgrowth of hippocampal neurons,neurite outgrowth quantification assays (Chemicon) were performed according to the manufacturer’s instructions.

Prior to the neurite outgrowth assay, the membrane surface previously coated with poly-L-lysine was prepared specifically for primary culture. Then the single cell suspension in a volume of 100 μL was added on top of the membrane (upper chamber) at 1 × 104cells/well in 12-well plates, for both control and experimental conditions (supplementary Figure 1 online). For neurite extension to occur, cells were cultured for 6 days in five replicates. Following the neurite extension period, membrane inserts were removed, gently rinsed in excess PBS and fixed in 100% ice-cold methanol for 20 minutes at room temperature. Each insert was then placed into a well containing 500 μL of neurite stain solution for 15 minutes at room temperature. After briefly rinsing each insert, cell bodies were removed from the upper membrane surface by wiping with a cotton swab. For quantification, a 200 μL drop of neurite stain extraction buffer was placed on a flat piece of parafilm and positioned on the underside of the membrane such that the buffer and membrane were in contact. The underside of the membrane was incubated with the extraction buffer for 5 minutes at room temperature and then quantified on a spectrophotometer (Bio-Rad, Hercules, CA, USA) at 562 nm.

Immunofluorescence staining

Immunofluorescence staining was performed according to a standard protocol as described by Fukata et al[12].

Hippocampal neurons on coverslips in 6-well plates were fixed with 4% paraformaldehyde for 30 minutes at 37°C.

The cells were permeabilized with 0.1% Triton X-100 for 5 minutes at 37°C. After permeabilization, the cells were blocked with goat serum for 30 minutes and then incubated with 1: 1 000 mouse anti-α-tubulin and anti-GFAP monoclonal antibodies (Santa Cruz Biotechnology)overnight at 4°C, followed by 1: 200 Cy3-conjugated goat anti-mouse IgG (Amersham Pharmacia, Piscataway, NJ,USA) in the dark for 1 hour at room temperature. Cell nuclei were stained with 5 μg/mL Hoechst 33258 (Sigma)in the dark for 30 minutes at room temperature. Each step was followed by three sequential washes in PBS except incubation of primary antibody. Immunofluorescence staining was observed using fluorescence microscopy (Leica, Wetzlar, Germany) and laser confocal microscopy (Leica). Negative controls were performed by replacing the primary antibody with serum corresponding to the primary antibodies.

Statistical analyses

All values are presented as mean ± SD. Two sample t-test was used to examine differences between control and treatment groups. Alpha was set at 0.01, and less than 0.01 was considered statistically significant. All tests were two-tailed and all analyses were performed using SPSS for windows 11.0 software (SPSS, Chicago, IL,USA).

Author contributions:All authors participated in designing,conducting and analyzing the experiments.

Conflicts of interest:None declared.

Funding:This study was financially sponsored by Guangdong Science and Technology Foundation, No. 2010B031600102,2010-170-1; Guangdong Medical Science Foundation, No.A2008344; Macau Science and Technology Foundation, No.026-2010-A.

Ethical approval:Experiments were performed with the approval of the Animal Ethics Committee of Jinan University of Traditional Chinese Medicine in China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 20, 2011 after selecting the “NRR Current Issue” button on the page.

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