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1.
J Cell Biol ; 212(7): 789-801, 2016 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-27022091

RESUMO

Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin-proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses.


Assuntos
Diferenciação Celular , Hipocampo/enzimologia , Poliubiquitina/metabolismo , Terminações Pré-Sinápticas/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ubiquitinadas/metabolismo , Animais , Axônios/enzimologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/embriologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Terminações Pré-Sinápticas/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Proteólise , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Vesículas Sinápticas/enzimologia , Fatores de Tempo , Imagem com Lapso de Tempo , Transfecção , Ubiquitinação
2.
Integr Biol (Camb) ; 6(6): 586-95, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24675920

RESUMO

Innervation has proven to be critical in bone homeostasis/regeneration due to the effect of soluble factors, produced by nerve fibers, associated with changes in the activity of bone cells. Thus, in this study, we have established and characterized a coculture system comprising sensory neurons and osteoblasts to mimic the in vivo scenario where nerve fibers can be found in a bone microenvironment. Embryonic or adult primary dorsal root ganglion (DRG) and MC3T3-E1 osteoblastic cells were cocultured in compartmentalized microfluidic platforms and morphological and functional tests were performed. The time of adhesion and readout of axonal outgrowth were improved by the alignment of DRG with the axis of microgrooves, which showed to be a crucial step for the designed experiments. Cocultures of entire DRG from adult origin with osteoblasts were performed, showing extended DRG projections towards the axonal compartment, reaching osteoblastic cells. Immunocytochemistry showed that the neurites present within the osteoblastic compartment were immunoreactive to synapsin and calcitonin gene-related peptide suggesting the presence of specialized structures involved in this crosstalk. This evidence was further confirmed by electron microscopy where varicosities were detected as well as electron dense structures in neurite membranes. Aiming to mimic the properties of tissue extracellular matrices, MC3T3-E1 cells were seeded in the axonal side upon laminin, collagen or within 3D functionalized alginate matrices and axonal outgrowth was clearly observed. In order to analyze and quantify data with reproducible image analysis, a semi-automated algorithm was also developed. The collagen and laminin substrates displayed a higher amount of axons reaching the axonal side. Overall, the established method revealed to be a suitable tool to study the interaction between the peripheral nervous system and bone cells in different contexts mimicking the in vivo scenario.


Assuntos
Osso e Ossos/inervação , Gânglios Espinais/metabolismo , Microfluídica/métodos , Neuritos/metabolismo , Osteoblastos/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura , Gânglios Espinais/citologia , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Neuritos/ultraestrutura , Ratos , Ratos Wistar
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