Neuritic Outgrowth Associated with Astroglial Phenotypic Changes Induced by Antisense Glial Fibrillary Acidic Protein (GFAP) mRNA in Injured Neuron–Astrocyte Cocultures

Abstract

In the adult CNS, axons fail to regenerate after injury. Among the cell interactions that lead to this failure are those developed with astrocytes. In an effort to elucidate the mechanisms underlying these negative interactions, we have used astrocytes treated with antisense glial fibrillary acidic protein (GFAP) mRNA to inhibit the formation of gliofilaments, indispensable for the astroglial morphological response to injury, and have studied their permissivity for neuritic outgrowth. In a neuronastrocyte coculture, a mechanical lesion led to hypertrophy of astrocytes neighboring the lesion. Neuronal cell bodies and neurites were absent both from the area of lesion and from its surroundings. Reactive astrocytes appeared, therefore, to be a nonpermissive substrate. Transfection that used antisense GFAP mRNA blocked astroglial morphological changes and was characterized by both a persistence of neuronal cell bodies in the vicinity of the lesion site and a growth of neurites into the same region. These morphological differences were associated with a 46% decrease in the GFAP translation capacity and a 50% increase in the concentration of GAP-43 in the treated cultures. Neurons were associated mainly with an extracellular laminin network, which was predominant at the lesion site in treated cocultures. In contrast, those astrocytes highly lamininimmunoreactive appeared to be a nonpermissive substrate for neurons. These results show that inhibition in GFAP synthesis, leading to a reduction of astroglial hypertrophy, relieves the blockade of neuritic outgrowth that normally is observed after a lesion. The mechanisms may involve changes in the secretion of extracellular matrix molecules by astrocytes.

Role of laminin bioavailability in the astroglial permissivity for neuritic outgrowth

“…Mechanical lesion modified the labeling distribution in the monolayer, where it significantly decreases, out of a population of large and flat astrocytes around the lesion border which remained labeled with punctate intracellular spots (Lefrançois et al 1997). The laminin labeled network, overlaying the astroglial lesioned monolayer, appeared unstructured and a dense labeled network was concentrated along the lesion border.…”

“…These reactive, hypertrophic astrocytes appeared, therefore, as a non-permissive substrate and the coculture model used could be considered valid to study the consequences of astroglial reactivity on axonal growth, in spite of the absence of the three dimensions of a tissue. Transfection of an antisense for GFAP-mRNA in the lesioned coculture, in order to modulate the astrocytic response to the injury, reduced cell hypertrophy and decreased the capacity of the cells to translate GFAP-mRNA (Lefrançois et al 1997). …”

Modulators of axonal growth and guidance at the brain midline with special reference to glial heparan sulfate proteoglycans

“…To our knowledge, there are no other examples on functionally-related patterns of lamininimmunoreactive structures at the midline. It should be noted, in passing, that punctate and fibrillary patterns of laminin immunoreactivity have also been observed in reactive astrocytes that express GFAP (laminin punctate pattern) and GFAP-null mutants (laminin fibrillary pattern) in correlation with their permissive and non-permissive nature, respectively (Lefrançois et al 1997, Tardy 2002.…”

Estudo da distribuição da proteína S100b em encéfalo de ratos.

“…A GFAP é a maior proteína de filamentos intermediários encontrada no citoplasma de astrócitos e é um marcador de diferenciação astrocitária (Eng, 1985;Kimelberg et al, 1989;Lefrançois et al, 1997;Menet, et al, 2001).…”

“…Durante o desenvolvimento do SNC de mamíferos, as células da glia, mais especificamente os astrócitos, constituem o melhor substrato para a migração neuronal e o crescimento de axônios (Rakic, 2003;Lefrançois et al, 1997). As interações entre neurônios e astrócitos podem ocorrer através da liberação de fatores solúveis, moléculas de sinalização celular e neurotransmissores, ou através de junções celulares do tipo "gap" (Haydon, 2000;Barres et al, 2000).…”