Abstract
Thyroid hormone (T3) plays a crucial role in several steps of cerebellar ontogenesis. By using a neuron-astrocyte coculture model, we have investigated the effects of T3-treated astrocytes on cerebellar neuronal differentiation in vitro. Neurons plated onto T3-astrocytes presented a 40-60% increase on the total neurite length and an increment in the number of neurites. Treatment of astrocytes with epidermal growth factor (EGF) yielded similar results, suggesting that this growth factor might mediate T3-induced neuritogenesis. EGF and T3 treatment increased fibronectin and laminin expression by astrocytes, suggesting that astrocyte neurite permissiveness induced by these treatments is mostly due to modulation of extracellular matrix (ECM) components. Such increase in ECM protein expression as well as astrocyte permissiveness to neurite outgrowth was reversed by the specific EGF receptor tyrosine kinase inhibitor, tyrphostin. Moreover, studies using selective inhibitors of several transduction-signaling cascades indicated that modulation of ECM proteins by EGF is mainly through a synergistic activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. In this work, we provide evidence of a novel role of EGF as an intermediary factor of T3 action on cerebellar ontogenesis. By modulating the content of ECM proteins, EGF increases neurite outgrowth. Our data reveal an important role of astrocytes as mediators of T3-induced cerebellar development and partially elucidate the role of EGF and mitogen-activated protein kinase/phosphatidylinositol 3-kinase pathways on this process.
Highlights
Thyroid hormone (3,5,3Ј-triiodothyronine, T3)1 is essential for normal development of the vertebrate nervous system (NS), in
T3-astrocyte-induced Neuritogenesis Is Indirectly Mediated by epidermal growth factor (EGF)—We previously described that astrocytes treated by thyroid hormone modulate neuronal proliferation by secreting growth factors, one of them identified as EGF [19]
We provide the first evidence that EGF secreted by T3-treated astrocytes induces external granular cell layer (EGL) neurons to undergo differentiation initiated by outgrowth of neurites
Summary
T3, triiodothyronine; NS, nervous system; CNS, central nervous system; ECM, extracellular matrix protein; EGF, epidermal growth factor; EGFR, EGF receptor; EGL, external granular layer; MAPK, mitogen-activated protein (MAP) kinase; MEK, fluencing diverse processes of brain development such as neuronal migration, neurite outgrowth, synapse formation, myelination, and glial cell differentiation [1,2,3,4,5]. Cerebellar histogenesis is well studied, the molecular mechanisms that control proliferation and differentiation of granular cells are still unknown These processes have been shown to undergo dramatic modulation by thyroid hormone [6, 10, 11]. We used an in vitro system of neuronastrocyte coculture to assess the effects of T3 mediated by MAPK/extracellular signal regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; DMEM, Dulbecco’s modified Eagle’s medium; CM, conditioned medium. We provide evidence that EGF secreted by astrocytes in response to T3 presents a binary role in cerebellar ontogenesis; acting directly on neurons, EGF promotes proliferation of granular cell precursors, and indirectly, EGF increases neuronal morphological differentiation by modulating the content of two astrocytic extracellular matrix (ECM) proteins, laminin and fibronectin. Our work gives glial cells a novel attribute as mediators of the endocrineregulated cerebellar development and describes an additional role for EGF on brain morphogenesis
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