Effect of thyroid hormone depletion on cultured murine cerebral cortex astrocytes

Abstract

Astrocytes play a crucial role in several steps of brain development, such as the proliferation of neural precursors, neuronal migration and differentiation, axonal growth, and synaptogenesis. Astrocyte generation and maturation is dramatically modulated by thyroid hormones (THs). Here, we propose a modified model for studying THs action on astroglial cells, in vitro. We investigated the effect of depletion of THs from fetal bovine serum (FBS) on the expression of the astrocyte maturation markers, GFAP (glial fibrillary acidic protein) and S100β, and the extracellular matrix (ECM) proteins laminin and fibronectin in cultured astrocytes. To accomplish this, murine cortical astrocytes were cultured in medium supplemented with THs-depleted serum, in contrast to the traditional techniques that use normal FBS which contains considerable amounts of THs. Immunostaining revealed that depletion of THs from FBS did not affect astrocyte proliferation, as observed by the number of astrocytes labeled for the proliferation antigen, Ki67. Surprisingly, western blot and RT-PCR assays revealed decreased levels of GFAP and S100β in astrocytes cultured with depleted serum. These events were reversed by addition of THs to the medium. Immunostaining and western blot assays did not reveal any difference in the organization and synthesis of the ECM protein, laminin; whereas the levels of fibronectin were decreased by 50% in THs-depleted serum. The fact that decreased expression of GFAP and fibronectin is associated with hypothyroidism in vivo suggests that our work might provide a useful model to assess in vitro the molecular mechanism underlying astrocytic maturation under conditions of THs deficiency.