Integrins and cAMP mediate netrin-induced growth cone collapse.

Growth cones integrate a remarkably complex concert of chemical cues to guide axons to their appropriate destinations. Recent work suggests that integrins contribute to axon guidance by interacting with a wide range of extracellular molecules including axon guidance molecules, by mechanisms that are not fully understood. Here, we describe an interaction between integrins and netrin-1 in growth cones that contributes to growth cone collapse. Our data show that netrin-1 causes growth cone collapse in a substratum-specific manner and is integrin-dependent. Netrin-1 causes collapse of cultured chick dorsal root ganglion (DRG) growth cones extending on high levels of laminin-1 (LN) but not growth cones extending on low levels of LN or on fibronectin. Blocking integrin function significantly decreases netrin-induced growth cone collapse on high LN. Netrin-1 and integrins interact on growth cones; netrin-1 causes integrin activation, a conformational shift to a high ligand-affinity state. Netrin-1 directly binds to integrin α3 and α6 peptides, further suggesting a netrin-integrin interaction. Interestingly, our data reveal that netrin-1 increases growth cone levels of cAMP in a substratum-specific manner and that netrin-induced growth cone collapse requires increased cAMP in combination with integrin activation. Manipulations that either decrease cAMP levels or integrin activation block netrin-induced collapse. These results imply a common mechanism for growth cone collapse and novel interactions between integrins, netrin-1 and cAMP that contribute to growth cone guidance.

Combined integrin activation and intracellular cAMP cause Rho GTPase dependent growth cone collapse on laminin-1.

Cyclic nucleotides regulate the response of both developing and regenerating growth cones to a wide range of guidance molecules through poorly understood mechanisms. It is not clear how cAMP levels are regulated or how they translate into altered growth cone behavior. Here, we show that intracellular cAMP levels are influenced by substrata and integrin receptors. We also show that growth cones require a substratum-specific balance between cAMP levels, integrin function and Rho GTPases to maintain motility and prevent collapse. Embryonic chick dorsal root ganglion neurons plated on different concentrations of laminin extend growth cones at similar speeds, yet have distinct levels of integrin expression, integrin activation and intracellular cAMP levels. Either increasing cAMP signaling or activating integrins enhances the rate of growth cone motility, but only on substrata where these two factors are endogenously low (i.e. low concentrations of laminin). Surprisingly, combining these two positive manipulations induces growth cone collapse and retraction on laminin but not on fibronectin. Collapse and retraction on laminin are Rho and Rac1 GTPase dependent and are associated with internalization of integrins, the primary receptors responsible for adhesion. These observations define a novel pathway through which cAMP influences growth cone motility and establish a link between integrins, cAMP and Rho GTPases in growth cones.

Intact aggrecan and fragments generated by both aggrecanse and metalloproteinase-like activities are present in the developing and adult rat spinal cord and their relative abundance is altered by injury.

Aggrecan is a large proteoglycan (PG) that has been grouped with different PG families on the basis of its physical characteristics. These families include the chondroitin sulfate PGs, which appear to inhibit the migration of cells and axons during development. Although aggrecan has been studied primarily in cartilage, in the present study, tissue samples from developing, mature, and injured-adult rat spinal cords were used to determine whether aggrecan is present in the mammalian spinal cord. By the use of Western blot analysis, tissues were probed with aggrecan-specific antibodies (ATEGQV, TYKHRL, and LEC-7) and aggrecan-specific neoepitope antibodies (NITEGE, FVDIPEN, and TFKEEE) to identify full-length aggrecan and several fragments. Unlike many other aggrecan gene family members, aggrecan species were similar in embryonic day 14, postnatal day 1, and adult spinal cords. Spinal cord injury caused significant decreases in aggrecan. Partial recovery in some aggrecan species was evident by 2 weeks after injury. The presence of specific aggrecan neoepitopes suggested that aggrecan is cleaved in the spinal cord by both a disintegrin and metalloproteinase thrombospondin (also known as aggrecanase) and metalloproteinase-like activities. Many aggrecan species found in the spinal cord were similar to species in cartilage. Additional antibodies were used to identify two other aggrecan gene family members, neurocan and brevican, in the adult spinal cord. These studies present novel information on the aggrecan core protein species and enzymes involved in aggrecan cleavage in vivo in the rat spinal cord throughout development and after injury. They also provide the basis for investigating the function of aggrecan in the spinal cord.

Chondroitin sulfate proteoglycan immunoreactivity increases following spinal cord injury and transplantation.

Extrinsic factors appear to contribute to the lack of regeneration in the injured adult spinal cord. It is likely that these extrinsic factors include a group of putative growth inhibitory molecules known as chondroitin sulfate proteoglycans (CSPGs). The aims of this study were to determine: (1) the consequences of spinal cord contusion injury on CSPG expression, (2) if CSPGs can be degraded in vivo by exogenous enzyme application, and (3) the effects of intraspinal transplantation on the expression of CSPGs. Chondroitin 6-sulfate proteoglycan immunoreactivity (CSPG-IR) dramatically increased following spinal cord contusion injury both at and adjacent to the injury site compared to normal controls (no surgical procedure) and laminectomy-only controls by 4 days postinjury. The dramatic increase in CSPG-IR persisted around the lesion and in the dorsal one-half to two-thirds of the spinal cord for at least 40 days postinjury. Glial fibrillary acidic protein (GFAP)-IR patterns were similarly intensified and spatially restricted as CSPG-IR patterns. These results suggest that: (1) CSPGs may contribute to the lack of regeneration following spinal cord injury and (2) astrocytes may contribute to the production of CSPGs. In addition, our results show that CSPGs could be cleaved in vivo with exogenous chondroitinase ABC application. This demonstration of cleavage may the basis for a model to directly assess CSPGs' role in growth inhibition in vivo (studies in progress) and hold potential as a therapeutic approach to enhance growth. Interestingly, the robust, injury-induced CSPG-IR patterns were not altered by intraspinal grafts of fetal spinal cord. The CSPG expression profile in the host spinal cord was similar to time-matched contusion-only animals. This was also true of GFAP-IR patterns. Furthermore, the fetal spinal cord tissue, which was generally CSPG negative at the time of transplantation, developed robust CSPG expression by 30 days posttransplantation. This increase in CSPG expression in the graft was paired with a moderate increase in GFAP-IR. CSPG-IR patterns suggest that these molecules may contribute to the limited regeneration seen following intraspinal transplantation. In addition, it suggests that the growth permissiveness of the graft may change overtime as CSPG expression develops within the graft. These correlations in the injured and transplanted spinal cord support CSPGs' putative growth inhibitory effect in the adult spinal cord.