Astroglia‐released neurite growth‐inducing activity for embryonic hippocampal neurons is associated with laminin bound in a sulfated complex and free fibronectin

Abstract

Neurons from embryonic (E18) rat hippocampus were chosen to identify and characterize neurite growth-stimulating proteins accumulating in serum-free conditioned media (CM) obtained from primary or secondary cultures of cerebral astrocytes (less than 5% nonglial cells) using a quantitative cell culture bioassay. CM were fractionated by FPLC on an anion exchange column (Mono Q) and by gel filtration (Superose 6). Column fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and enzyme-linked immunosorbent assay (ELISA) using antibodies to laminin (LN) and fibronectin (FN). The neurite-promoting activity (NPA) was tested by incubating aliquots of the eluted fractions with poly-L-lysine precoated glass coverslips prior to addition of neurons suspended in chemically defined medium. We provide evidence that the NPA in astroglial CM could be assigned mainly to a negatively charged, highly sulfated LN complex consisting predominantly of the B-chains of LN and presumably a sulfated proteoglycan that was sensitive for chondroitinase and to a lower degree to heparinase degradation. In addition, a smaller proportion of the NPA was associated with uncomplexed LN and free FN. FN reached approximately 10 times the concentration of LN in astroglial CM. As revealed by immunofluorescence microscopy, both LN and FN are simultaneously expressed by cultured astrocytes; however, only the production of FN, measured by ELISA, increased during the time astrocytes were in culture, whereas the release of LN remained unchanged. We conclude that, besides the most active LN complex, FN bound to a polycationic matrix is able to induce neurite growth in hippocampal neurons in vitro.

Modulation of Neuritogenesis by Astrocyte Muscarinic Receptors

“…The levels of fibronectin and laminin-1 present in astrocyte-conditioned medium were normalized for biglycan, a proteoglycan whose release by astrocytes is not affected by carbachol treatment. 4 It should be noticed that fibronectin levels were considerably higher than those of laminin, in agreement with a previous report (61).…”

“…Although laminin is generally recognized to be highly involved in neuritogenesis, the levels of laminin-1 released by astrocytes in our cultures were considerably lower than the levels of fibronectin (Figs. 4, A and B and 5, A and B), as also reported by others (61), and carbachol-induced up-regulation of laminin-1 was considerably lower than the up-regulation observed for fibronectin (Figs. 4 and 5).…”

Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation

“…4). Several of these proteins are involved in neuritogenesis including prosaposin (O'Brien et al 1994), fibronectin (Gundersen 1987;Matthiessen et al 1989), fibulin-1 (Ohsawa et al 2001), thrombospondin 1 (Adams 1997), PAI-1 (Soeda et al 2006), galectin-3 (Pesheva et al 1998), monocyte chemoattractant protein-1 (Bianchi et al 2005), neural cell adhesion molecule 1 (Ronn et al 1998), and Plau (Pittman et al 1989). …”

Relationship between sprouting axons, proteoglycans and glial cells following unilateral nigrostriatal axotomy in the adult rat

“…For example, addition of soluble HS disturbs growth of cockroach pioneer axons (Wang and Denburg, 1992) while scission with HS lyases a¡ects retinotectal path¢nding in Xenopus embryos (Walz et al, 1997). HSPG^laminin complexes also modulate neurite outgrowth in vitro including growth promotion and growth inhibition (Lander et al, 1985a,b;Chiu et al, 1986;Hantaz-Ambroise et al, 1987;Mathiessen et al, 1989). For example, whereas monoclonal antibodies to HSPG reduce neurite outgrowth on sciatic nerve in vitro (Sandrock and Matthew, 1985) a Schwannoma-derived HSPG also blocks the neurite-promoting activity of laminin (Muir et al, 1989).…”