Distribution and substrate properties of agrin, a heparan sulfate proteoglycan of developing axonal pathways

Abstract

The distribution and substrate properties of agrin, an extracellular matrix heparan sulfate proteoglycan (HSPG), was investigated in the developing chick nervous system by immunocytochemistry, Western blotting, and in neurite outgrowth assays. By comparing the distribution of agrin with that of laminin-1, merosin (laminin-2), neurofilament, and neural cell adhesion molecule (NCAM), it was found that throughout development, agrin is a constituent of all basal laminae. From embryonic day (E) 4 onwards, agrin is also abundant in axonal pathways of the central nervous system, such as the optic nerve, the tectobulbar pathway, the white matter of the spinal cord, and the marginal and the molecular layers of the forebrain and the cerebellum. The abundance of agrin in brain decreases from E13 onwards. In the peripheral nervous system, agrin is present throughout development as a constituent of the Schwann cell basal laminae. Western blots confirmed the immunocytochemical data, showing maximum expression of agrin occurs during the early to medium stages of brain development. Western blots also showed that in mouse and human brain, agrin exists as an HSPG. Purified agrin did not support neurite outgrowth, rather it inhibited retinal neurite extension on mixed agrin/merosin substrates. Despite the fact that agrin, when used as a substrate inhibited neurite outgrowth, its temporal and spatial overlap with growing axons suggests that agrin has a supportive role in the development of axonal pathways, possibly as a binding component for growth factors and cell adhesion proteins.

Synapse Loss in Cortex of Agrin-Deficient Mice after Genetic Rescue of Perinatal Death

“…Agrin staining in the brain is most prominent in the blood vessels and in pial basement membranes (Barber and Lieth, 1997;Halfter et al, 1997). Moreover, neurons in the CNS have been shown to express mRNA encoding agrin isoforms that contain amino acid inserts at the B/z site (O'Connor et al, 1994;Stone and Nikolics, 1995).…”

Behavior of in vitro cultured ameboid microglial cells migrating on Müller cell end‐feet in the quail embryo retina

“…The 39.4D5 and H5 mAbs, which recognize antigens islet-1 and vimentin, respectively, were used to reveal retinal ganglion cells (Fischer et al, 2002) and M€ uller cells (Fischer et al, 2004), which were present in low numbers in some ILM/ MCEF sheets. Laminin and agrin, two components of the ILM, were recognized with 3H11 and 6D2 mAbs (Halfter et al, 1997), respectively. MCEF were labeled with JG22 mAb, which was raised against the b1 subunit of avian integrins (Hofer et al, 1990;Hynes, 2004); in the retina, this mAb specifically labels the surface of developing and mature M€ uller cells and is highly concentrated at their end-feet (Hering et al, 2000).…”

Agrin Is a Chimeric Proteoglycan with the Attachment Sites for Heparan Sulfate/Chondroitin Sulfate Located in Two Multiple Serine-Glycine Clusters

“…In the case of PF7, the size of glycosylation is reduced in the double and triple mutations, suggesting that more than one GAG chain could be attached to the peptide. This is consistent with the fact that the size difference between glycosylated agrin and its core protein suggests 3-4 HS side chains (14,18).…”

“…We hypothesize that the GAG chains of agrin shield agrin and its associated proteins from proteolytic degradation and may be responsible for the slow turnover of amyloid peptide aggregates (11). Tissue culture studies have shown that agrin, when used as a substrate, inhibits neurite outgrowth (13,16,18). It is conceivable that the inhibitory activity of agrin contributes to the localization and termination of axons at particular sites of the muscle basement membrane by slowing the rate of axon outgrowth down.…”