Neuroprotection and Neuronal Differentiation Studies Using Substantia Nigra Dopaminergic Cells Derived from Transgenic Mouse Embryos

TH and SV40Tag immunostaining of SN DA cell line SN4741. For the establishment of DA cell lines, mesencephalic SN regions from E13.5 embryos from TA58-#8 were surgically removed under sterile conditions. The collected SN tissues were dissociated and cultured at 33°C with 5% CO₂ in RF medium. The dispersed primary neuronal cell lines from the transgenic embryos were grown through repeated passages for 3–4 months to establish a pure cell population. The clonal origin of a colony was monitored and confirmed by microscopic observation during its colony formation.A, The morphology of an early SN DA cell colony (arrow) named SN-47. After the third cloning step, the DA cell line became morphologically flatter compared with the early clones. A representative SN DA cell line SN4741 was immunostained with TH polyclonal antibody (B) and more intensely with SV40Tag monoclonal antibody (C), respectively. The TH antibody stained the cytoplasm, whereas SV40Tag antibody stained exclusively the nucleus. Scale bars: B,C, 100 μm.

Detection of TH expression and DA neuronal markers by Western blot analysis and RT-PCR. A, The representative DA marker, TH expression, was confirmed by Western blot analysis after maintaining the SN4741 cells for a week in high-density culture at 37°C (lane 4) and 33°C (lane 5). The 62 kDa TH band in the SN4741 cells was consistent with the molecular weight of TH isolated from mouse adrenal gland (lane 1) and similarly derived locus coeruleus noradrenergic cell line, grown at 37°C (lane 2) and 33°C (lane 3), which were used as positive controls for the Western blot analysis. Each lane contained 15 μg of protein except lane 1 (5 μg) and lane 2 (30 μg). The specific amount of TH protein in the SN4741 cells grown at 37°C was higher than the culture at 33°C. As demonstrated in a TH-positive pheochromocytoma cell, PC12, culture (Kim et al., 1995), our SN DA cell line also exhibited the increase in TH expression in the high cell density during a prolonged culture (7–10 d). B, The expression of some DA neuron-specific markers, such as NT-3, BDNF, DA-T, and D₂R were detected by RT-PCR in the SN4741 cell line. After Southern blotting of both 1 and 5 μl of each RT-PCR sample, the presence of each specific size band (the strongest band) was detected and confirmed with radiolabeled human BDNF cDNA, rat NT-3 cDNA, mouse DA-T cDNA, or mouse D₂R oligonucleotide probe, respectively. Each filter was hybridized with each specific probe and exposed separately. The expected major band sizes were 430 bp for NT-3, 407 bp for BDNF, 553 bp for DA-T, and 490 bp for D₂R. β represents the β-actin PCR product of 289 bp as an internal standard.

Morphological and phenotypic differentiation of the SN DA cell line SN4741 under various culture conditions. The morphological differentiation of the SN4741 cells was induced by culturing at the nonpermissive temperature (39°C) with a minimal serum concentration and/or retinoic acid. A, When grown at 33°C, the SN4741 cells have a fibroblast-like flat morphology with less prominent neurite growth. B, Under differentiation condition (i.e., nonpermissive temperature 39°C and reduction of FCS to 0.5%), the SN4741 cell line ceased proliferation and, after 2 d, started to display a neuronal morphology with extensive neurite outgrowth and, after 4 d, long bipolar or multipolar processes.C, Retinoic acid caused much longer and bipolar neurite extensions at 39°C. In contrast to the control SN4741 culture (D), the TH immunoreactivity of most SN4741 cell lines was greatly enhanced in the presence of the mesencephalic culture (E). In the coculture slides, the SN4741 cells can be distinguished from the primary DA neurons by their distinct morphology and size. The primary DA neurons (arrow) were morphologically multipolar or bipolar and much smaller than the SN4741 cells. F, MAP2 expression became consistently higher in the presence of retinoic acid at 39°C. Scale bars:A–C, 280 μm;D–F, 140 μm.

BDNF protected the SN4741 cell line against various neurotoxic stresses. To measure BDNF-mediated neuroprotection against MPP⁺-, SNP-, glutamate-, or dopamine-mediated neurotoxicity, the SN4741 cells were pretreated with BDNF (50 ng/ml) for 10 min (open bars) and exposed to either 10 μm MPP⁺ for 15 hr, 0.5 mm glutamate for 10 min, 0.5 mm SNP for 5 min, or 5–15 nm dopamine for 18 hr in the presence of BDNF (open bars). Solid bars represents each control group. BDNF protected against MPP⁺-, glutamate-, or NO-induced neurotoxicity, but not against dopamine-induced neurotoxicity. All values are the mean ± SEM; *p < 0.05.

BDNF but not NGF protects against MPP⁺-induced neurotoxicity. The neuroprotective functions of BDNF were further tested in comparison with NGF against MPP⁺-induced neurotoxicity in the SN DA cell culture. Only BDNF exerted its neuroprotective function against MPP⁺-neurotoxicity. All values are the mean ± SEM; *p < 0.05.

BDNF secretion from the SN DA cell line under normal and stress conditions. The five representative classes of known neurotoxic insults, such as mitochondrial electron transport inhibitor (MPP⁺), free radical generator (H₂O₂), NO producer (SNP), excitatory amino acid (glutamate), and proinflammatory cytokine (TNF-α) were tested. After treatment with sublethal doses of MPP⁺(5 μm), SNP (0.05 mm), H₂O₂ (0.05 mm), glutamate (0.1 mm), or TNF-α (50 ng/ml), the amount of BDNF in the cell-conditioned medium was quantified by ELISA. Glutamate and TNF-α increased the release of BDNF by 30–50%. Of interest, two potential free radical generators, NO (SNP) and H₂O₂, but not MPP⁺, significantly suppressed the amount of BDNF release by ∼30–40%. All values are the mean ± SEM; *p < 0.05.