Outgrowth by neuronal axons is a major feature of development and regeneration in the nervous system. In peripheral nerve, Schwann cells can promote axonal outgrowth by depositing stimulatory cues on their cell surface and into the surrounding basal lamina. Laminin (LN) is known to be one of the most potent of these promoters. Recent evidence suggests that inhibitory cues play an important role in restricting and directing axonal growth. The PI has found that rat Schwann cells secrete a proteoglycan (Neurite-Inhibiting Factor or NIF) which complexes with laminin and blocks its neurite-promoting activity in vitro. In addition, when the NIF-LN complex is exposed to proteases produced by neurons or Schwann cells, the inhibitory proteoglycan component is abolished resulting in the unmasking of LN's stimulatory activity. Based on these findings we propose the following thesis for the regulation of axonal outgrowth: i) Schwann cells present to neurons a matrix of stimulatory and inhibitory components; ii) the stimulatory component LN can exist in an inactive complex with NIF; iii) proteolysis is required to unmask the stimulatory potential of the NIF-LN complex and; iv) proteases are released by neurons and Schwann cells which degrade NIF and in effect activate the neurite-promoting ability of the LN.
The aims of this project are: (1) to determine the homogeneity of our NIF preparation by electrophoretic analysis, characterizations of the glycosaminoglycan side-chains and amino acid sequencing of the core protein. Synthetic peptides corresponding to unique amino acid sequences will be used to produce monospecific antibodies against the core protein of NIF; (2) to determine if NIF binds to LN through side-chain or core protein interactions. Defined fragments of LN which contain neurite- promoting or cell-binding sites will be used to determine which regions of LN NIF interacts with and whether both the cell attachment and neurite-promoting activities of LN are inhibited by NIF. In addition, we will attempt to describe how NIF inhibits LN's activity by determining the affect that NIF binding to LN has on LN binding to neuronal surface receptors; (3) to determine if stromelysin/transin and other neural proteases can degrade NIF and de-inhibit the neurite-promoting potential of the NIF-LN complex and if the release of proteases by neurons can de- inhibit NIF-LN complexes during neurite outgrowth. Similarly, we will determine if neurons secrete heparan sulfate degrading endoglycosidases (heparanases) that can de-inhibit NIF-LN substrates; and (4) to determine by immunolocalizations and direct interventions if the inhibitory proteoglycan and proteoglycanases actively regulate axonal outgrowth in normal nerve and in models of neural regeneration.
| Zuo, J; Ferguson, T A; Hernandez, Y J et al. (1998) Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan. J Neurosci 18:5203-11 |
| Zuo, J; Hernandez, Y J; Muir, D (1998) Chondroitin sulfate proteoglycan with neurite-inhibiting activity is up-regulated following peripheral nerve injury. J Neurobiol 34:41-54 |
