The neural cell adhesion molecule (N-CAM) inhibits proliferation in primary cultures of rat astrocytes.

Abstract

Cell proliferation is a key primary process during neural development and also plays an important role in the regenerative response of neural tissue to injury. It has been reported that glial cell proliferation is, at least in part, controlled by a neuronal signal, possibly involving cell surface molecules. We report here that the addition of purified rat neural cell adhesion molecule (N-CAM) to primary cultures of rat forebrain astrocytes inhibits their proliferation. This inhibitory effect can be elicited in cultures grown in chemically defined serum-free medium or in medium that had been supplemented with growth factors. Polyclonal antibodies to N-CAM or their Fab' fragments elicited a similar inhibitory effect. The magnitude of the inhibitory effect of N-CAM was dependent on cell density: it was maximal at low cell densities and weakened progressively as cells approached confluency. Synthetic peptides with sequences identical to a putative homophilic binding region of N-CAM mimicked the effect of purified N.CAM, while peptides of the same length and amino acid composition but with a randomized sequence did not. The addition of N-CAM antisense oligonucleotides to primary astrocyte cultures for 48 h resulted in reduced levels of N-CAM expression. After N-CAM levels on astrocytes were diminished by this treatment, the antiproliferative effect of N-CAM added to the medium was significantly reduced. The combined results suggest that N-CAM homophilic binding may be involved in the control of glial cell proliferation.

Angiotensin II and aldosterone-induced neuronal damage in neurons through an astrocyte-dependent mechanism

“…23 Briefly, newborn mice were decapitated and the cerebral cortices were removed. After removal of the meninges, the cerebral cortices were digested with 0.4 mg ml -1 dispase (Invitrogen) for 15 min.…”

Cultured rat hippocampal neural progenitors generate spontaneously active neural networks

“…N-CAM influences cell proliferation, differentiation, migration, neurite outgrowth, and fasciculation during development and in the adult (8). Our previous studies indicated that N-CAM binding inhibited proliferation of rat hippocampal progenitor cells (9) in culture similar to its effect on astrocytes (10,11). Moreover, N-CAM also promoted progenitor differentiation toward a neuronal phenotype in the presence or absence of FGF2 (9), indicating that this CAM exhibited neurotrophin-like activity.…”

A short segment within the cytoplasmic domain of the neural cell adhesion molecule (N-CAM) is essential for N-CAM-induced NF-κB activity in astrocytes

“…The medium was changed to serum-free DMEM for 24 h before treatment. Cells were treated with a number of different reagents: 100 g͞ml purified N-CAM (19), or 100 g͞ml recombinant IgIII (5), or 1 ng͞ml IL-1␤, or 1 ng͞ml TNF-␣, or 20 g͞ml lipopolysaccharide (LPS) for 7 hr. The cells were harvested and assayed for luciferase activity (9).…”

“…Primary cultures of astrocytes were obtained from the forebrains of postnatal day 3-4 rats as described (19) and from the forebrains of postnatal day 2-3 N-CAM-null mice (37). Primary astrocytes were transfected via electroporation after 7-10 days in vitro as previously described (9) with either 5 g of pcDNA3.1 ϩ or 5 g of the cytoplasmic domain construct being tested, as well as 5 g of NF-B-luc, and 5 g of CMV-␤-gal by using a Gene Pulser electroporator (Bio-Rad).…”

“…2 for review). In particular, N-CAM binding promotes neurite outgrowth (16,17), inhibits astrocyte proliferation in vitro and in vivo (18,19), and activates the NF-B transcription factor and the glucocorticoid receptor (7,9,20). Several models have been proposed describing possible mechanisms of N-CAM signaling.…”