Firma VB Asesor

A primary neuronal cell culture model was employed to test the hypothesis that T3 directly regulates early neuronal differentiation. An important advantage of the cell culture model is that it is free from confounding factors such as placental dysfunction and maternal metabolic compromise, which may complicate the interpretation of findings in the hypothyroid dam model. Cells from 15 dg fetal brain were cultured for 20 - 40 h in order to model neuronal development during the period prior to the onset of fetal TH secretion in vivo. The culture conditions favoured the development and survival of neurons but not astrocytes, as indicated by extensive neurite outgrowth. Furthermore, cells in culture expressed a-IN protein and exhibited neuronal morphology, whereas GFAP protein was almost undetectable up to 94 h, confirming that the model comprised predominantly differentiating neurons, with minimal astrocytic contamination. The lack of significant astrocytic contamination allowed

investigation of the effects of T3 on neurons in isolation. The presence of glial progenitor cells cannot be ruled out however, since significant levels of GFAP protein where detectable after 150 h in culture.

Since the primary mechanism of TH action is the modulation of gene transcription via binding of T3 to TR (Section 1.5), it was important to compare TR expression in the cell culture model with that in fetal brain prior to the onset of fetal TH secretion. Levels of T R al, c-erbAa2 and c-erbAa3 mRNA were similar in neuronal cultures and 16 dg fetal brain. Nevertheless, the c-erbAa2:c-erbAa3 ratio was lower in neuronal cultures than in fetal brain. As mentioned previously however, the significance of this novel finding is unclear, due to the lack of information regarding the expression of these mRNAs in different cell types at different stages of brain development. However, since c-erbAa2 and c-erbAa3 transcripts are derived from alternate splicing of the same gene, the altered transcript ratio may represent different splicing mechanisms in the cell cultures compared with fetal brain. The lower abundance of TR pl mRNA in cell culture compared with fetal brain was expected (Castiglia et al. 1992), since TRpl mRNA expression is more closely associated with proliferative zones in fetal brain (Bradley et al. 1992), whereas the culture model comprised differentiating neurons. In agreement with previous studies, T3 was without effect on the expression of transcripts for TR or non-T3-binding c-erb A variants (Castiglia et al. 1992, Puymirat et al. 1992).

Nestin mRNA levels in the neuronal cultures appeared to be lower than in fetal brain, and whereas levels declined between 16 and 19 dg in fetal brain, they remained stable between 20 and 40 h in the neuronal cultures. Lower levels of nestin mRNA were expected in cell culture, since nestin expression is associated with neural precursors whereas the majority of neurons in culture were differentiating, as evidenced by widespread a-IN immunoreactivity, together with extensive neurite outgrowth. It is possible that a decline in nestin mRNA abundance occurred in the cell cultures prior to 20 h. Alternatively, the decline in nestin mRNA abundance in fetal brain in vivo may occur due to glial cell differentiation, thus a similar decline would not be expected in these predominantly neuronal cultures. T3 had no effect on nestin mRNA levels or on DNA content per well in cell culture, suggesting that neuronal proliferation was unaffected by T3 in this model. These findings are in agreement with the observation that nestin mRNA was normal in fetal brain from hypothyroid dams in the in vivo study, and support the notion that maternal hypothyroidism does not affect the acquisition of neuronal precursors in fetal brain prior to the onset of fetal TH secretion. Furthermore, the findings in cell culture confirm the redundancy of the putative TRE in nestin, during this stage of development.

The lack of effect of T3 on NF-L mRNA abundance in neuronal cultures was in agreement with findings in the in vivo model, but contrasts with a recent report in which T3 increased NF-L mRNA expression in cultures of neurons derived from 16-17 dg fetal rat brain (Rahaman et al. 2000), This discrepancy is probably related to differences in the two cell culture models. Firstly, the medium used by Rahaman et al. contained fetal calf serum, a poorly defined and batch variable supplement that contains numerous growth factors and hormones, such as platelet-derived growth factor, which are capable of influencing neuronal development (Bottenstein 1989, Butler 1992). Secondly, Rahaman et al. treated neurons in culture with 5 nM T3, a concentration 5-fold greater than that employed in the present study and supraphysiological relative to the concentration of T3 in fetal brain prior to the onset of fetal TH secretion. Furthermore, T3 treatment was performed for 7 - 2 5 days, thus the neurons would have reached an age far exceeding that equivalent to the period prior to the onset of fetal TH secretion in vivo (Rahaman et al. 2000).

In control cultures, a-IN protein, but not mRNA, abundance increased between 20 and 40 h, suggesting that a-IN is subject to post-transcriptional regulation during this period. A T3 concentration of 0.1 nM, which approximates the T3 level in rat fetal brain prior to the onset of fetal TH secretion (Ruiz de Ona et al. 1988), positively stimulated a-IN protein abundance at 20 h. At 40 h however, a significant increase in a-IN protein was only seen in cells treated with 1 nM T3, albeit a similar trend was seen with 0.1 nM T3. This trend perhaps failed to reach significance because the 0.1 nM T3 declined during the 40 h period due to its metabolism by the neurons, coupled with a relatively rapid protein turnover rate. Nevertheless, when taken together with the findings in vivo, demonstrating higher a-IN protein abundance in early fetal brain from euthyroid relative to hypothyroid dams, the findings in cell culture support a direct role for maternal TH in the regulation of early neuronal differentiation.

6.3.1 L im itations o f the cell culture m odel

Findings in the neuronal culture model must be interpreted cautiously due to the difficulties associated with extrapolating from cell culture models to the brain. The complex 3-dimensional structure of the brain cannot be reliably replicated in vitro, especially when cells are grown in monolayer, as in the present study. Consequently, important cell-cell contacts and paracrine interactions may be absent in culture models, possibly resulting in aberrant cell behaviour. Furthermore, commonly used cell culture substrata do not effectively mimic the complex nature of the ECM that is present in vivo. Since the ECM influences neuronal migration (Sobeih & Corfas 2002), the latter is unlikely to be modelled accurately in cell culture. The presence of cell clusters in the cultures employed here suggests however, that the PDL substrate facilitated some degree of migration, albeit only in 2-dimensions. Indeed, PDL has been favoured as the

standard substrate for primary neuronal cultures (Hertz et al. 1989). The presence of cell clusters may be also due to neurons migrating over each other.

As mentioned previously, neuronal-glial interactions influence neuronal maturation (Section 6.2.3). The present culture system aimed to model neuronal differentiation prior to the onset of fetal TH secretion. Although the abundance of mature astrocytes is minimal during this period, as evidenced by undetectable GFAP protein in fetal brain at 16 dg, interactions between neurons and glial progenitors/early differentiating glia may nevertheless be important for early neuronal migration/differentiation in vivo. Thus, the limited presence of glial cells in the cell culture model may have altered the characteristics of neuronal development compared with that in vivo. Indeed, NF-L protein was far lower in neuronal cell cultures than in fetal brain at 16 dg, despite NF-L mRNA abundance in the cultures being markedly higher than in fetal brain. As discussed previously (Section 4.4), this may have been due to impaired local translation of NF-L mRNA in dendrites, possibly because of inadequate dendritic development as a consequence of the absence of neuronal-glial interactions. This cell culture model may therefore be of limited relevance to later stages of fetal brain development when neuronal-glial interactions may be important. It is however thought to effectively model neuronal development prior to the onset of fetal TH synthesis, since cultures expressed T R al, c-erbAa2 and c-erbAa3 mRNA at similar levels to 16 dg fetal brain, and the up- regulation of a-IN protein by T3 mirrored the sensitivity of this protein to maternal thyroid status in early fetal brain. This cell culture model is therefore likely to serve as a useful model for future work aimed at studying the molecular mechanisms by which maternal TH regulates early neuronal differentiation.

6.4 Transcriptional versus post-transcriptional regulation of