Quantification of NGF-dependent neuronal differentiation of PC-12 cells by means of neurofilament-L mRNA expression and neuronal outgrowth
Introduction
The PC12 cell line was derived from rat pheochromocytoma, a tumour arising from chromaffin cells of the adrenal medulla. It is a useful and widespread model for studying neuronal differentiation into sympathetic neurons and other neuro-biochemical and -biological events (Guroff, 1985). Differentiation of PC12 cells, cessation of proliferation and neurite outgrowth, induced by NGF, was first described by Greene and Tischler (1976). Several marker proteins for PC12’s differentiation into sympathetic neuron-like cells are known (e.g. Fujita et al., 1989). These authors stated, that, although direct and detailed studies of the synthesis of various mRNAs in PC12 cells treated with NGF seem imminent, only a few would have appeared so far. This is still up-to-date in view of the huge number of events at the mRNA level (Angelastro et al., 2002, Lee et al., 1995); so far, only a few NGF induced mRNAs such as NF-L have been identified in structure and function.
The differentiation of PC12 cells is typically assessed by evaluating the amount of neuronal process formation, as done by counting the number of processes per cell (Greene and Tischler, 1976), by direct measurement of neurite length (e.g. Fujii et al., 1982) or by counting the number of cells extending neurites of length more than once or twice the cell diameter (e.g. Blackman et al., 1993). The neurite response has been adapted to serve as a quantitative assay for NGF (Greene, 1977). Nevertheless, it is tedious to use these procedures for assessing differentiation in a large number of samples, because it requires a lot of images to be analysed visually. Moreover, the procedure is difficult to automate.
Because PC12 cells must be exposed at least 5–7 days to NGF with additional changes of medium every second day to reach a certain degree of differentiation, some researchers shortened the priming procedure by producing “pre-primed” PC12 cells according to Blackman et al., 1993, Blackman et al., 1994. Primed cells are those, which upon re-stimulation with NGF, develop neurites more rapidly than cells not previously treated with NGF. Blackman et al. primed PC12 cells for 6 days with NGF, collected and then stored them at −80 °C in order to use them for further experiments. Sano et al. (1988) alternatively published investigations with the derivative cell line PC-12D, which in contrast to PC12 cells did not require priming for 6 days with NGF before use in standard NGF assays. PC-12D cells produce neurites within 22 h in response to NGF in a dose-dependent manner similarly to that elicited by primed PC-12 cells (Blackman et al., 1993).
Another way of studying neuronal differentiation is to investigate the formation of neurofilaments, the major structural components of neurons, both at the level of mRNA and protein expression. The three mammalian neurofilament subunits, NF-L (∼68 kDa), NF-M (∼145 kDa) and NF-H (∼200 kDa), are believed to form heterodimers consisting of NF-L in combination with either NF-M or NF-H (Lee and Cleveland, 1996). With reference to the 68 kDa neurofilament (NF-L) subunit, Levi et al. (1988) and Sawada et al. (2000) reported an increase in protein formation at day 7 of NGF exposure. Ohuchi et al. (2002) measured the degree of PC12 differentiation via the amount of neurofilament proteins (NF triplet-proteins), β-tubulin III and cell proliferation. Due to the 96-well format used and the sensitivity of response, this assay system is well suited for high-throughput analysis of PC12 cell differentiation after 7 days in cell culture (5 days NGF) which, however, represents a rather late time point of differentiation.
One of the major focus of the present investigation is the earliest time point where statistically significant responses can be detected. Since the production of NF-L mRNA occurs prior to the synthesis of the respective protein, it was considered an interesting candidate for reliably detecting differentiation at earlier times. Using the NF-L subunit as specific marker, we present data on the expression of its mRNA levels. In parallel, the neurite outgrowth is quantified and compared to the time course of the NF-L mRNA marker for 5 and 50 ng NGF/ml in order to identify possible dose–effect relationships.
Section snippets
Cell culture
Rat adrenal pheochromocytoma cells (PC-12, ECACC 88022401, UK) were described by Greene and Tischler (1976). Note, that different spellings of these cells are encountered (PC12 and PC-12). In our results we shall keep to the notion PC-12, according to the source of origin (ECACC). Cells were grown at 37 °C, 5% CO2 in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml of streptomycin (all reagents from Invitrogen, Groningen, Netherlands).
For treatment
Cell culture conditions and cell proliferation
Greene and Tischler (1976) stated, that the apparent doubling time of the PC12 cells is long (about 92 h), and that cell growth was slightly less satisfactory when RPMI 1640 medium was replaced, or when horse serum was omitted. Our cell cultures were grown in medium with 10% fetal calf serum (FCS), although this medium without horse serum was used by a minority of researchers only. In our hands, cell population doubling time in suspension with 5% FCS and 10% horse serum was 56 h, and was the same
Discussion
We have shown that both NO and NF-L mRNA expression in PC-12 cells increase monotonously with time when the cells were incubated with NGF at a concentration of 50 ng/ml, leading to a good correlation between the two endpoints. It should be noted, that this correlation also holds for the expression of NF-L protein at 50 ng/ml NGF, whose steady increase with time is evident from Fig. 7.
However, at sub-optimal NGF concentration (5 ng/ml), marked differences in the kinetics of NO and NF-L mRNA
Acknowledgements
We are indebted to Dr. Eric Gottwald for guiding the initial RT-PCR measurements, and gratefully acknowledge the excellent technical assistance by Evelin Krüger and Mechthild Herschbach in the starting phase of this work.
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