Feng Z , Ko CP .

NS17954 NINDS NIH HHS , R01 NS017954-21 NINDS NIH HHS , R01 NS017954 NINDS NIH HHS

	Figure 1. Schwann cell-conditioned medium promotes synapse formation in vitro. A , B , Xenopus nerve–muscle cocultures were treated with neurotrophins plus cAMP elevation (trophic stimulation). In this example, no α-BTX-positive staining was detected at the nerve–muscle contacts (arrowheads). In this figure and the following figures, the top panels depict α-BTX staining for AChRs seen with fluorescence microscopy; the bottom panels combine fluorescence microscopy of α-BTX and synapsin staining for neurites with phase-contrast microscopy. Note that yolk granules, which are autofluorescent, can be seen in some muscle fibers. C , D , Nerve–muscle cocultures with trophic stimulation were also treated with SC-CM. Positive α-BTX staining at nerve–muscle contacts is labeled with arrowheads. Scale bar in D applies to A–C . E , Quantification of AChR cluster formation at nerve–muscle contacts under various treatments. Nerve–muscle cocultures with trophic stimulation alone showed AChR clusters only at 20.4 ± 3.5% of nerve–muscle contacts [control (open bar), 625 contacts]. SC-CM and the fraction of SC-CM containing molecules between 5 and 30 kDa increased the percentage of nerve–muscle contacts associated with AChR clusters to 52.7 ± 3.3% (359 contacts) and 51.1 ± 2.5% (360 contacts), respectively. Neither the fraction of SC-CM containing molecules smaller than 5 kDa (23.7 ± 2.3%; 216 contacts) nor the fraction containing molecules larger than 30 kDa (32.2 ± 2.1%; 397 contacts) increased significantly, compared with control, the percentage of nerve–muscle contacts associated with AChR clusters. Thus, molecules derived from Schwann cells that are active in increasing synaptic number are within the molecular weight range of 5–30 kDa. Data are mean ± SEM. *p < 0.05; two-tailed, unequal variance Student's t test.
	Figure 2. TGF-β1 mimics the synaptogenesis effect of SC-CM. A , B , Control nerve–muscle cocultures were treated with trophic stimulation alone. No α-BTX staining was observed at the nerve–muscle contacts (arrowheads) in this example. C , D , Nerve–muscle cocultures were treated with TGF-β1 (10 ng/ml) in addition to trophic stimulation. Note that α-BTX staining was present at nerve–muscle contacts (arrowheads). Scale bar in D applies to A–C . E , The effect of TGF-β1 (10 ng/ml) on the formation of AChR clusters was quantified. Whereas AChR clusters were formed only at 20.3 ± 1.9% of nerve–muscle contacts (n = 985), in cocultures with trophic stimulation only (control, open bar), both TGF-β1 (10 ng/ml) and SC-CM significantly increased the percentage of nerve–muscle contacts associated with AChR clusters (TGF-β1, 59.8 ± 3.3%, 1013 contacts; and SC-CM, 62.3 ± 2.8%, 901 contacts). Altogether, TGF-β1, similar to SC-CM, increased the formation of AChR clusters in nerve–muscle cocultures. F , The dose–response curve of TGF-β1 in the formation of AChR clusters at nerve–muscle contacts. The synaptogenic effect of TGF-β1 ranged from 38.8 ± 1.8% (n = 300 contacts) at 5 ng/ml to 61.9 ± 1.3% (n = 213 contacts) at 20 ng/ml, and it reached a plateau at the concentration of 10 ng/ml (63.2 ± 2.6%, n = 501 contacts). G , The effects of TGF-β1 and SC-CM on the size of AChR clusters at nerve–muscle contacts in nerve–muscle cocultures were quantified. The average size of each AChR cluster formed along nerve–muscle contacts with trophic stimulation alone was 50.5 ± 5.5 μm2 (n = 40 contacts) (open bar). The addition of TGF-β1 or SC-CM doubled the size of AChR clusters formed per unit length of nerve–muscle contacts (TGF-β1, 95.4 ± 8.6 μm2, n = 121 contacts; and SC-CM, 106.1 ± 7.6 μm2, n = 83 contacts). Data are mean ± SEM. Two-tailed, unequal variance Student's t test was used to determine statistical difference. Significance was defined as *p < 0.05.
	Figure 3. SC-CM contains TGF-β1. A , The expressions of TGF-β1 mRNA were examined using RT-PCR. Both Xenopus Schwann cells from sciatic nerves and cultured Xenopus Schwann cells, but not cultured Xenopus neurons or muscle cells, expressed TGF-β1. B , The presence of TGF-β1 protein in SC-CM was detected by Western blot. TGF-β1 protein, molecular weight of 25 kDa in nonreducing condition, was detected in SC-CM. Human recombinant TGF-β1 was used as a positive control and culture medium was used as a negative control for Western blot. C , The expression of TβR-II in cultured Xenopus neurons and muscle cells. TβR-II mRNA was expressed in cultured neurons but not in muscle cells. The expression of general gene GAPDH was used as a positive control for the sample of total cDNA in RT-PCR ( A , C ).
	Figure 4. TGF-β1 mediates SC-CM-promoted synaptogenesis. A , B , Control nerve–muscle cocultures after trophic stimulation. No AChR clusters, as indicated by the absence of α-BTX staining, were formed along nerve–muscle contacts (arrowheads) in this example. C , D , Nerve–muscle cocultures were treated with TGF-β1 in addition to trophic stimulation. More α-BTX staining was observed at nerve–muscle contacts (arrowheads). E , F , Nerve–muscle cocultures were treated with SC-CM in the presence of trophic stimulation. In this example, note that AChR clusters were formed at nerve–muscle contacts (arrowheads). G , H , One example of nerve–muscle cocultures treated with SC-CM that was immunodepleted of TGF-β1. No α-BTX staining was detected at nerve–muscle contacts when SC-CM was depleted of TGF-β1. Scale bar in H applies to A–G . I , The formation of AChR clusters at nerve–muscle contacts under different treatments was quantified. Data from 5–10 independent experiments were combined. Only 19.7 ± 2.1% of nerve–muscle contacts (n = 687) were associated with AChR clusters in control cultures with trophic stimulation only, whereas both TGF-β1 and SC-CM increased the percentage of nerve–muscle contacts associated with AChR clusters (TGF-β1, 60.9 ± 3.5%, n = 809 contacts; and SC-CM, 62.9 ± 3.1%, n = 519 contacts). SC-CM immunodepleted of TGF-β1 lost its synaptogenic effect: only 19.8 ± 2.5% of contacts (n = 742) were associated with AChR clusters. LAP, which prevents TGF-β1 from binding to its receptors, blocked the synaptogenic effect of TGF-β1 and SC-CM (TGF-β1 + LAP, 17.5 ± 2.6%, n = 556 contacts; SC-CM + LAP, 21.4 ± 2.5%, n = 624 contacts). Additionally, blockade of TβR-I kinase ALK-5 abolished the synaptogenic effect of TGF-β1 and SC-CM (TGF-β1 + TβR-I inhibitor, 20.0 ± 2.3%, n = 251 contacts; SC-CM + TβR-I, 25.8 ± 1.7%, n = 284 contacts). Thus, TGF-β1 is required for the synaptogenic effect of SC-CM. Data are mean ± SEM, *p < 0.05; two-tailed, unequal variance Student's t test. J , TGF-β1 protein was detected in SC-CM (lane 1), as well as in SC-CM with mock immunoprecipitation (IP) (lane 2) (beads without antibodies against TGF-β1, lane 4). After immunoprecipitation with antibodies against TGF-β1, TGF-β1 protein was absent from SC-CM (lane 3) but was associated with protein G-Sepharose beads (lane 5).
	Figure 5. Mammalian Schwann cells promote synapse formation in vitro. A , The expression of TGF-β1 mRNA in mammalian tissues was examined using RT-PCR. Cultured rat Schwann cells, as well as sciatic nerves from neonatal rats, expressed TGF-β1 mRNA. B , The presence of TGF-β1 protein was investigated by Western blot. TGF-β1 protein was detected in mammalian SC-CM. Human recombinant TGF-β1 was used as a positive control and culture medium (DMEM) was used as a negative control for Western blot. C , D , Xenopus nerve–muscle cocultures were treated with trophic stimulation only. The formation of AChR clusters was reduced along the nerve–muscle contacts (arrowheads). E , F , Nerve–muscle cocultures with trophic stimulation were treated with TGF-β1. The formation of AChR clusters was restored at nerve–muscle contacts (arrowheads), as demonstrated by the positive staining of α-BTX. G , H , Nerve–muscle cocultures were treated with mammalian SC-CM in the presence of trophic stimulation. The addition of mammalian SC-CM increased the formation of AChR clusters at nerve–muscle contacts (arrowheads). I , J , Nerve–muscle cocultures were treated with mammalian SC-CM that was immunodepleted of TGF-β1 protein. In this example, note that no α-BTX staining was observed at nerve–muscle contacts (arrowheads). Scale bar in J applies to C–I . K , Quantification of the formation of AChR clusters at nerve–muscle contacts. Data from five independent experiments were combined. In control nerve–muscle cocultures with trophic stimulation alone, only 18.3 ± 2.5% of nerve–muscle contacts (n = 242) were associated with AChR clusters. The addition of mammalian SC-CM increased the percentage of nerve–muscle contacts associated with AChR clusters to 66.8 ± 4.5% (mSC-CM, 247 contacts), which is similar to the effect produced by treatment with TGF-β1 (TGF-β1, 68.1 ± 2.5%, 279 contacts). When TGF-β1 protein was immunodepleted from SC-CM, SC-CM was no longer effective in promoting the formation of AChR clusters at nerve–muscle contacts (mSC-CM after immunoprecipitation with mAb TGF-β1, 18.6 ± 2.2, n = 311 contacts]. Because mammalian Schwann cells were cultured in DMEM, DMEM was used as a control. DMEM alone had no effect on the formation of AChR clusters at nerve–muscle contacts (DMEM, 17.4 ± 1.3%, n = 238 contacts). Data are mean ± SEM, *p < 0.05; two-tailed, unequal variance Student's t test.
	Figure 6. TGF-β1 upregulates neuronal agrin expression. A , Expression of agrin isoforms at C-terminal B splicing site in pure neuron cultures was investigated using RT-PCR. Two-day-old pure neuron cultures without any trophic stimulation expressed all four isoforms of agrin (top, lane 1). Trophic stimulation reduced the expression of all isoforms of agrin (top, lane 2). The addition of TGF-β1 and SC-CM each restored agrin expression in neurons (top, lanes 3 and 4, respectively). Depleting TGF-β1 from SC-CM abolished the effect of SC-CM in promoting agrin expression (top, lane 5). GAPDH gene expression was not affected by any of the treatments (bottom). B , Quantification of active agrin (B8, B11, and B19) expression in pure neurons. Both TGF-β1 and SC-CM were able to significantly (*p < 0.05) increase the expression of active agrin in pure neuron cultures. Additionally, TGF-β1 was required for the upregulation of agrin by SC-CM. C , Only the inactive isoform of agrin (B0) was expressed in muscle cells under the various treatments indicated. D , Quantification of synapsin-1 expression in pure neurons with the different treatments indicated. Similar levels of synapsin-1 were expressed in pure neuron cultures with these treatments.
	Figure S1 - We tested whether TGF-β1 or SC-CM influences neuronal survival and neurite outgrowth in pure Xenopus neuron cultures, and whether TGF-β1 or SC-CM affects AChR cluster formation in pure Xenopus muscle cultures. A: Quantification of the effects of TGF-β1 and SC-CM on neuronal survival in the presence of trophic stimulation. The percentage of neurons that survived in 4-day-old pure neuron cultures as compared to 1- day-old pure neuron cultures was used as an indicator of neuronal survival. Neither TGF-β1 nor SC-CM affected the survival of spinal neurons in the presence of trophic stimulation. B: Quantification of the effects of TGF-β1 and SC-CM on the number of neurites in 4-day-old pure neuron cultures in the presence of trophic stimulation. The number of neurites per neuron was not affected by TGF-β1 or SC-CM. C: Quantification of the effects of TGF-β1 and SC-CM on neurite outgrowth in 4-day-old pure neuron cultures in the presence of trophic stimulation. TGF-β1 or SC-CM did not affect the total length of neurites per neuron. D: Quantification of the effects of TGF-β1 and SC-CM on the formation of AChR clusters in 4-day-old pure muscle cultures with trophic stimulation. Neither TGF-β1 nor SC-CM had an effect on the formation of AChR clusters in pure muscle cultures. E: Quantification of the effects of TGF-β1 and SC-CM on the size of AChR clusters in 4-day-old pure muscle cultures with trophic stimulation. Neither TGF-β1 nor SC-CM affected the size of AChR clusters formed in pure muscle cultures. Two-tailed, unequal variance Student's t test was used to determine statistical difference. Significance was defined as p < 0.05.

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