Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture

Abstract

As one major component of extracellular matrix (ECM) in the central nervous system, chondroitin sulfate proteoglycans (CSPGs) have long been known as inhibitors enriched in the glial scar that prevent axon regeneration after injury. Although many studies have shown that CSPGs inhibited neurite outgrowth in vitro using different types of neurons, the mechanism by which CSPGs inhibit axonal growth remains poorly understood. Using cerebellar granule neuron (CGN) culture, in this study, we evaluated the effects of different concentrations of both immobilized and soluble CSPGs on neuronal growth, including cell adhesion, spreading and neurite growth. Neurite length decreased while CSPGs concentration arised, meanwhile, a decrease in cell density accompanied by an increase in cell aggregates formation was observed. Soluble CSPGs also showed an inhibition on neurite outgrowth, but it required a higher concentration to induce cell aggregates formation than coated CSPGs. We also found that growth cone size was significantly reduced on CSPGs and neuronal cell spreading was restrained by CSPGs, attributing to an inhibition on lamellipodial extension. The effect of CSPGs on neuron adhesion was further evidenced by interference reflection microscopy (IRM) which directly demonstrated that both CGNs and cerebral cortical neurons were more loosely adherent to a CSPG substrate. These data demonstrate that CSPGs have an effect on cell adhesion and spreading in addition to neurite outgrowth.

Keywords: cell adhesion; chondroitin sulfate proteoglycans; interference reflection microscopy; neural regeneration; neurite growth.

Figure 1

Effects of CSPGs coating on the growth of CGNs. CGNs were fixed after 2 days of culture (DIV2) and stained with β-tubulin III (green) and 4',6-diamidino-2-phenylindole (DAPI; blue). (A) Representative images of CGNs growing on increasing concentrations (0, 1, 2, 4 μg/mL) of CSPG substrates. The bottom row images are the respective higher magnification images of the top row. The inserts inside the bottom row of images are DAPI-stained nucleus, showing multiple cells aggregated together in 2 μg/mL and 4 μg/mL CSPGs groups. Scale bars: 50 μm. (B) Quantification of relative neurite length. Data are normalized to no CSPGs (0 μg/mL) control. (C) Quantification of the cell density in each field. Data are normalized to no CSPGs (0 μg/mL) control. (D) Quantification of the percentage of the aggregated cells in each field. Data are presented as the mean ± SEM; *P < 0.05, ***P < 0.001; one-way analysis of variance with a Tukey's post hoc test. CSPGs: Chondroitin sulfate proteoglycans; CGN: cerebellar granule neuron.

Figure 2

Effects of soluble CSPGs on the growth of CGNs. (A) Representative images of CGNs growing on increasing concentrations of soluble CSPGs (0, 1, 2, 4 μg/mL) for 1 day (DIV1). Neurons are stained with β-tubulin III (green) and DAPI (blue). Scale bar: 100 μm. (B) Quantification of neurite length. (C) Quantification of the percentage of cells with neurites longer than the size of two cell bodies. (D) Representative images of CGNs growing on no CSPGs or 10 μg/mL soluble CSPGs for 1 (DIV1) and 3 days (DIV3). CGNs fixed for 1 day were stained with 488-Phalloidin (green) and DAPI (blue). CGNs fixed for 3 days were stained with β-tubulin III (green) and DAPI (blue). Scale bar: 50 μm. Data are presented as the mean ± SEM; **P < 0.01, vs. no CSPGs (0 μg/mL) group. One-way analysis of variance (ANOVA) with a Tukey's post hoc test was used. CSPGs: Chondroitin sulfate proteoglycans; CGN: cerebellar granule neuron.

Figure 3

Effect of CSPGs on the morphology of growth cone. (A) Representative images showing the morphology of growth cones of CGNs growing on PLL for 1 day (DIV1) with or without 1 μg/mL CSPGs. Neurons are stained with β-tubulin III (green) and Texas Red-Phalloidin (red). Scale bar: 10 μm. (B) Quantification of the growth cone size. Data are presented as the mean ± SEM; **P < 0.01. Two-tailed Student's t-test was used. CSPGs: Chondroitin sulfate proteoglycans; CGNs: cerebellar granule neurons; PLL: poly-L-lysine.

Figure 4

Effects of CSPGs on neuronal spreading and neurite initiation with or without the presence of LN. (A) Neuron spreading status at 30 minutes after plating on PLL, PLL + CSPGs (1 μg/mL), PLL + LN, or PLL + LN + CSPGs (1 μg/mL). Scale bar: 20 μm. (B) Three-dimensional demonstration of the overall morphology of neurons on PLL and PLL + CSPGs at 30 minutes after plating. (C) Quantification of neuronal spreading area. Data are presented as the mean ± SEM; ***P < 0.001, vs. PLL group; *P < 0.05 for comparison between PLL + CSPG and PLL + LN + CSPG; one-way analysis of variance with a Tukey post hoc test was used. (D) Neuronal morphology at 2 hours after plating on PLL, PLL + CSPGs, PLL + LN, or PLL + LN + CSPGs. Scale bar: 20 μm. (E) Three-dimensional manifestation of the overall morphology of neurons on PLL and PLL + CSPGs at 2 hours after plating. (F) Quantification of the percentage of neurons with neurite initiation. Data are presented as the mean ± SEM; **P < 0.01, vs. PLL, PLL + CSPG, or PLL + LN + CSPG; one-way analysis of variance with a Tukey's post hoc test was used. CGNs: Cerebellar granule neurons; CSPGs: chondroitin sulfate proteoglycans; LN: laminin; PLL: poly-L-lysine.

Figure 5

CSPGs showed a prominent inhibition on neurite outgrowth in the presence of LN. (A) Representative images of CGNs growing on PLL + LN and PLL + LN + CSPGs (1 μg/mL) for 1 day (DIV1). Neurons are stained with β-tubulin III (green) and DAPI (blue). Scale bar: 50 μm. (B) Quantification of neurite length. Data are presented as the mean ± SEM; ***P < 0.001; two-tailed Student's t-test. CGNs: Cerebellar granule neurons; CSPGs: chondroitin sulfate proteoglycans; LN: laminin; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 6

IRM results showing the anti-adhesive effect of CSPGs. (A) Representative IRM images and quantifications of CGNs and cerebral cortical neurons (CNs) growing on PLL, PLL + CSPGs (1 μg/mL), PLL + LN and PLL + LN + CSPGs (1 μg/mL) for 30 minutes. (B) Representative IRM images and quantifications of CGNs and cerebral cortical neurons (CNs) growing on the PLL, PLL + CSPGs (1 μg/mL), PLL + LN and PLL + LN + CSPGs (1 μg/mL) for 2 hours. Single-frame TIFF images of IRM were processed to display areas of minimum and maximum adhesion of the cells to the glass surface. Images were processed for background subtraction and color map named ‘Gem’ was assigned blue to higher intensity and orange to lower intensity pixels which represent stronger and weaker adhesions respectively. Area of the cell was traced using the bright field images to obtain “ROI”. The ROI was placed on the binary processed IRM image and the percentage of adhesion (relative adhesion area) was calculated by dividing the area of the cell by the area of adhesion. Cell adhesion of CGNs or cortical neurons was significantly reduced on the PLL + CSPG or PLL + LN + CSPG substrates compared to that on the PLL or PLL + LN substrate. Data are presented as the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, one-way analysis of variance with a Tukey's post hoc test was used. Scale bars: 20 μm.IMR: Interference reflection microscopy; CSPGs: chondroitin sulfate proteoglycans; CGNs: Cerebellar granule neurons; PLL: poly-L-lysine; LN: laminin.