Differential effects of neurotrophins and schwann cell-derived signals on neuronal survival/growth and synaptogenesis

Abstract

Recent studies have shown that the survival of mammalian motoneurons in vitro is promoted by neurotrophins (NTs) and cAMP. There is also evidence that neurotrophins enhance transmitter release. We thus investigated whether these agents also promote synaptogenesis. Cultured Xenopus spinal cord neurons were treated with a mixture of BDNF, glia-derived neurotrophic factor, NT-3, and NT-4, in addition to forskolin and IBMX or the cell-permeant form of cAMP, to elevate the cAMP level. The outgrowth and survival of neurons were dramatically increased by this trophic stimulation. However, when these neurons were cocultured with muscle cells, the trophic agents resulted in a failure of synaptogenesis. Specifically, the induction of ACh receptor (AChR) clustering in cultured muscle cells was inhibited at nerve-muscle contacts, in sharp contrast to control, untreated cocultures. Because AChR clustering induced by agrin or growth factor-coated beads in muscle cells was unaffected by trophic stimulation, its effect on synaptogenesis is presynaptic in origin. In the control, agrin was deposited along the neurite and at nerve-muscle contacts. This was significantly downregulated in cultures treated with trophic stimuli. Reverse transcriptase-PCR analyses showed that this decrease in agrin deposition was caused by an inhibition of agrin synthesis by trophic stimuli. Both agrin synthesis and induction of AChR clustering were restored under trophic stimulation when Schwann cell-conditioned medium was introduced. These results suggest that trophic stimulation maintains spinal neurons in the growth state, and Schwann cell-derived factors allow them to switch to the synaptogenic state.

Figure 1.

The effect of trophic stimuli on the survival and growth of spinal neurons. The number of neurites was scored as an indicator of neuronal survival and growth. Neurons cultured in the absence of trophic stimuli (control; filled circles) did not survive beyond day 5 in culture. The addition of a single neurotrophin (BDNF; open circles) or a combination of four neurotrophins(NTs; BDNF, NT-3, NT-4/5, and GDNF; open triangles) enhanced neuronal survival and growth. This effect was also seen when intracellular cAMP was elevated by forskolin and IBMX (cAMP; filled triangles). Combining neurotrophins and cAMP (NTs+cAMP; filled squares) further promoted survival and growth. For each point, triplicate cultures, each with at least 100 neurons at day 1, were scored. A survival of 100% refers to the number of neurites at day 1. SEs (error bars) were shown for the data points.

Figure 2.

AChR clustering at nerve—muscle contacts. GFP-synaptophysin was used as a presynaptic marker, and R-BTX was used to label postsynaptic AChR clusters. A, On day 1 of nerve—muscle coculture, punctate AChR clusters were seen along the contact area. B, These clusters were consolidated into larger AChR patches in day 3. Scale bar: (in B) A, B, 20 μm.

Figure 3.

The effect of trophic stimulation on NMJ formation. The formation of AChR clusters (right, white arrows) along nerve—muscle contacts (black arrows in phase images on the left) seen in the control (A, B) was abolished after NT plus cAMP treatment (C—F). However, preexistent AChR hot spots (open arrows) were dispersed in control cocultures but were left intact after trophic stimulation. Scale bar: (in E) A—F, 50 μm.

Figure 4.

Quantification of AChR clustering at nerve—muscle contacts in response to different stimuli (filled bars). Neurotrophins were used at 10 ng/ml either singly or in combination. NTs plus cAMP refers to neurotrophins plus forskolin, IBMX, and CPT-cAMP. The extent of AChR clustering was normalized to the control (open bar). Data were collected from three independent experiments with >160 nerve—muscle contacts scored. The p values for the difference between the test and the control were as follows: NTs, 0.0002, NTs plus K-252a, 0.71; NTs plus cAMP, 0.0007; NTs plus Rp-cAMP, 0.0003; CPT-cAMP, 0.001; BDNF, 0.001; GDNF, 0.15; NT-3, 0.05; and NT-4/5, 0.004. SEs (error bars) are shown.

Figure 5.

Trophic stimulation by neurotrophins and cAMP did not affect the induction of AChR clustering by HB-GAM-coated beads (A, B) or by agrin (C, D). A, B, Arrows point out correspondence in bead—muscle contacts and AChR clusters. D, Arrows point out several agrin-induced clusters. Scale bar: (in D) A—D, 10 μm. E, Quantification of AChR clustering in the presence of all four neurotrophins at different concentrations. For the bead experiments (filled bars), data from four independent experiments with 50 cells at each neurotrophin concentration were pooled. The p values for the difference between treated versus the control were 0.47 (for 10 ng/ml NT) and 0.41 (for 100 ng/ml NT). For the agrin experiment (open bars), 50 cells were scored for each concentration. The mean number of AChR clusters per 100 μm of muscle length was measured for each cell. The p values for the difference between treated versus the control were 0.63 (for 10 ng/ml NT) and 0.25 (for 100 ng/ml NT). SEs (error bars) are shown.

Figure 6.

The deposition of agrin along the neurite. A, B, Agrin was deposited along the neurite and its branches as revealed by Pab 36 labeling. C,D, Agrin deposition was much reduced after trophic stimulation. In this example, there was no association of agrin with the neurites. In the presence of K-252a, neurotrophins did not inhibit agrin deposition (E, F). Agrin deposition at nerve—muscle contacts was suppressed by trophic stimulation (G—I). However, an AChR hot spot that was not induced by the nerve was not affected (H, arrowhead). In contrast, nerve—muscle contacts in control cultures were associated with both AChR clusters and agrin deposition(J,K, arrows). Scalebars:(inF)A—F, 30 μm; (in I) G—I, 20 μm; (in K) J, K, 5 μm.

Figure 7.

Quantification of agrin deposition in control (open bar) and trophic factor-stimulated spinal neuron cultures (filled bars). Data from three independent experiments (except BDNF, NT-3, and NT-4/5) were pooled with >110 neurites scored for each treatment. They were normalized to the control. The p values for the difference between the test and the control were as follows: NTs, 0.006, NTs plus K-252a, 0.54; NTs plus cAMP, 0.01; NTs plus Rp-cAMP, 0.0003; cAMP, 0.003; and GDNF, 0.001. Data for BDNF, NT-3, and NT-4/5 were from one experiment only. SEs (error bars) are shown.

Figure 8.

RT-PCR analyses of agrin transcripts. Agrin isoforms at the B splicing site were investigated by RT-PCR analysis in nerve—muscle cocultures (A) and pure neuron culture (B). The nerve—muscle coculture in normal medium at 2 d showed all four isoforms (inactive B0 and active B8, B11, and B19)(A,lane1). The active isoforms were greatly reduced in cultures with NT plus cAMP treatment (A, lane 2). The SCCM treatment was able to restore the expression of active isoforms (A, lane 3). The B0 isoform observed in the NT plus cAMP treatment probably reflected the muscle agrin expression, as found in pure muscle culture (A, lane 4). No signal was found in RT-PCR reactions containing no RNA sample (A, lane 5). In pure neurons cultured in normal medium for 2 d, all four agrin isoforms were observed (B₁, lane1). SCCM treatment alone in this coculture had no effect on agrin expression (B₁, lane 2). However, all of these agrin isoforms were barely detectable after NT plus cAMP treatment (B₁, lane 3). These agrin isoforms were restored to near-normal level after SCCM treatment (B₁, lane 4). During all of these treatments, the general gene expression in neurons was not affected, as shown by GAPDH gene expression (B₁, bottom panel). Agrin expression in pure neurons was quantified by densitometric scanning and normalized for GAPDH expression (B₂)(n = 3 experiments; data are means ± SEM). Control, Open bar; experimental, filled bars. These results suggest that the stimulation by neurotrophins can downregulate the agrin expression in neurons, and Schwann cell-conditioned medium can reverse this process. They further indicate that the effects of NT plus cAMP and SCCM treatment are presynaptic in origin.

Figure 9.

SCCM reversed the effects of trophic stimuli on synaptogenesis. A, B, Nerve—muscle cocultures were subject to trophic stimulus (NT+cAMP) only. In this example, no R-BTX staining (B) was found on the muscle at the nerve—muscle contacts (A, B, arrowheads). Scale bar: (in B) A—D, 20 μm. C, D, Nerve—muscle cultures treated with NT plus cAMP plus SCCM showed AChR clusters as revealed by R-BTX staining (D, arrows) at >50% nerve—muscle contacts (C, arrows). The asterisk in D marks an AChR hotspot not directly associated with nerve—muscle contacts. E, The effect of SCCM was quantified. In control nerve—muscle coculture without any treatment, 84.2 ± 9.8% of contacts were associated with AChR clusters (5 experiments and 130 nerve—muscle contacts counted), and SCCM treatment alone did not change this ratio significantly (90.5 ± 8.2%; 5 experiments and 125 nerve—muscle contacts counted). However, in NT plus cAMP-treated cultures, only 10.1 ± 6.5% of contacts showed AChR clusters (5 experiments and 160 nerve—muscle contacts counted). In NT plus cAMP plus SCCM cultures, there was a significant increase in the percentage (54.2 ± 7.1%) of nerve—muscle contacts that showed AChR clusters (5 experiments and 154 nerve—muscle contacts counted) (Student's t test; ^*p < 0.001). In contrast, neither muscle-conditioned medium (MCM) nor fibroblast-conditioned medium (FCM) reversed the NT plus cAMP effect. Data are means ± SEM. Openbar, Control; filled bars, experimental.

Figure 10.

Restoration of agrin deposition and protein level by SCCM. A, B, Nerve—muscle cocultures were subject to trophic stimulus (NT+cAMP) only. In this example, no anti-agrin staining (B) was found along neurites (A, B, arrowheads). C, D, Nerve—muscle cultures treated with NT plus cAMP plus SCCM showed clear agrin deposition along the neurites (arrowheads). Scale bar: (in D) A—D, 40 μm. These results are quantified in E. Only 20.1 ± 0.9% of neurites in cocultures with trophic stimulus showed agrin deposition (open bar; 5 experiments). In contrast, SCCM was able to significantly restore this ratio to 56.2 ± 1.3% of neurites (filled bar; 5 experiments) (Student's t test; ^*p < 0.001). F, To further confirm this result, agrin protein level in pure neuron cultures was examined by Western blot. In control culture without any treatment, agrin immunoreactivity was detected at above 200 kDa (lane 1, arrowhead). After trophic stimulus, no agrin protein could be detected (lane 2). In contrast, pure neurons treated with NT plus cAMP plus SCCM showed clear agrin immunoreactivity (lane 3). These results suggest that SCCM was able to restore neural agrin expression that was downregulated by NT plus cAMP trophic stimulus, and this regulation was presynaptic.