Proliferation of cerebellar neurons induced by astrocytes treated with thyroid hormone is mediated by a cooperation between cell contact and soluble factors and involves the epidermal growth factor-protein kinase a pathway.
Cerebellar development is fully dependent on thyroid hormone (T3) levels. We have previously demonstrated a glia-mediated effect of T3 on cerebellar neurons. We have reported that cerebellar astrocytes treated with thyroid hormone secrete epidermal growth factor (EGF), which directly induces neuronal proliferation and, indirectly, by increasing synthesis of extracellular matrix proteins, induces neurite outgrowth in vitro. Here, by using a neuron-astrocyte coculture model, we investigated the involvement of cell contact on neuronal proliferation. Culturing of cerebellar neurons on T3-treated astrocyte carpets or conditioned medium derived from them (T3CM) yielded similar results, revealed by a 60% increase in cell population. However, the absolute number of neurons in coculture assays was greatly enhanced in comparison with that in CM assays (3.5-4-fold). Bromodeoxyuridine (BrdU) incorporation assays revealed that such an increase was due mainly to proliferation of precursors cells. BrdU incorporation was three times higher in cell carpet (31%) than in CM (13%). Treatment of astrocytes by T3 increased neuronal proliferation either by T3CM (2.5 times) or by contact with T3-treated astrocytes (1.5 times). Neuronal death was not affected by T3 treatment of astrocytes as revealed by either trypan blue viability assays or active caspase-3 labeling. Treatment of astrocytes by EGF mimicked T3 effects on neuronal proliferation. Addition of the EGF receptor tyrosine kinase inhibitor genistein and the protein kinase A (PKA) inhibitor KT5720 to cocultutres and T3CM completely reversed neuronal proliferation. Our results implicate EGF and the PKA pathway in the proliferation induced by T3-treated astrocytes. Furthermore, the fact that cocultures potentiate the effect of T3 on neuronal proliferation suggests that neuron-astrocyte contact may cooperate with astrocyte soluble factors to enhance neuronal population. Our data reveal an important role of astrocytes as mediators of T3-induced cerebellar development and partially elucidate the role of cell contact and soluble factors on this process.
Martinez
Rodrigo
R
Instituto de Ciências Biomédicas, Departamento de Anatomia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Gomes
Flávia Carvalho Alcantara
FC
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001973:Bromodeoxyuridine; D002450:Cell Communication / Q000502:physiology; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000196:embryology* / Q000254:growth & development; D018920:Coculture Techniques; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D014284:Triiodothyronine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D017868:Cyclic AMP-Dependent Protein Kinases; D001973:Bromodeoxyuridine