Astroglial permissivity for neuritic outgrowth in neuron–astrocyte cocultures depends on regulation of laminin bioavailability

Abstract

The molecular determinants underlying the failure of axons to regenerate in the CNS after injury were studied in an in vitro model of astrogliosis and neuronal coculture. Mechanically lesioned neuron-astrocyte mouse cortical cocultures were treated with antisense glial fibrillary acidic protein (GFAP)-mRNA in order to inhibit the formation of gliofilaments that occurs in response to injury. This inhibition relieves the blockage of neuron migration and neuritic outgrowth observed after lesion, and migrating neurons reappeared, supported by a laminin-labeled extracellular network (permissive conditions). We then questioned the relationship between this permissivity and laminin production. Follow-up studies on the concentration of laminin indicated that, after antisense treatment, the laminin level was increased in the cocultures and was under the control of astrocyte-neuron interactions. The addition of exogenous laminin favored neuronal migration and neurite outgrowth, whereas neutralizing laminin bioavailability with antibodies recognizing the astroglial laminin resulted in an inhibition of both neuronal access to the lesion site and neurite outgrowth, suggesting an active role for laminin in the permissive process. This permissive process could be associated with modulation of extracellular matrix (ECM) molecule degradation by proteinases. Among the latter, matrix metalloproteinases (MMPs) are involved in the breakdown of the ECM component. Our investigation showed a net decrease of the matrix metalloproteinase MMP-2 expression and activity and an increase of its endogenous inhibitor TIMP-2 expression. Both proteins associated with permissivity should be involved in the laminin stabilization and cell-matrix interactions. High levels of laminin and laminin bioavailability, consequent to a reduction in astrogliosis, may be important permissive elements for neuronal migration and neurite outgrowth postlesion.

Role of laminin bioavailability in the astroglial permissivity for neuritic outgrowth

“…A dynamic role for the overlaying laminin in neuronal migration and neurite outgrowth was confirmed when permissive cocultures were treated with anti-laminin antibodies (Costa et al 2002). These antibodies which recognize the astroglial laminin blocked a large part of neurite outgrowth and inhibition of neuronal migration to the lesion site, whereas an antibody against tenascin C, another component of the extracellular matrix (Faissner and Steindler 1995), did not.…”

“…In addition, we found that an enhanced laminin level was under neuronal control and consequent to either secreted factors or to cell interactions. Such signals may regulate both, laminin synthesis and extracellular availability, in order to improve neurite outgrowth (Costa et al 2002).…”

“…The laminin labeled network, overlaying the astroglial lesioned monolayer, appeared unstructured and a dense labeled network was concentrated along the lesion border. Antisense treatment restructured the labeled network, underlining that permissivity involves particular patterns of laminin expression (Costa et al 2002).…”

Cardosins improve neuronal regeneration after cell disruption: a comparative expression study

“…In fact, the protein expression for both gelatinases is much higher for neuron isolated with cardosins after the first 24 h. Laminin is an important ECM protein that has been found to be involved in cellular activities such as neuronal migration and neurite outgrowth. A high level of laminin constitutes a key permissive element that can be modulated by proteolytic activity (Costa et al 2002). The immunoblotting analysis detected a 200-to 220-kDa protein band expected for the size of laminin β/γ chains.…”

“…This treatment was reported by Costa et al 2002 to block neurite outgrowth and to induce an apparent inhibition of neuronal migration toward the lesion site. This is corroborated by our results (Fig.…”