Application of Neutralizing Antibodies against NI-35/250 Myelin-Associated Neurite Growth Inhibitory Proteins to the Adult Rat Cerebellum Induces Sprouting of Uninjured Purkinje Cell Axons

Abstract

The myelin-associated proteins NI-35/250 exert a powerful inhibition on axon regeneration, but their function exerted on intact neurons is still unclear. In the adult CNS these proteins are thought to regulate axon growth processes to confine plasticity within restricted regions and to prevent the formation of aberrant connections. We have recently shown that application of neutralizing IN-1 antibody Fab fragment against NI-35/250 proteins to the adult cerebellum induces the expression of injury/growth-associated markers in intact Purkinje cells. Here, we asked whether these cellular modifications are accompanied by growth phenomena of Purkinje neurites. A single intraparenchymal application of IN-1 Fab fragment to the adult cerebellum induces a profuse sprouting of Purkinje axons along their intracortical course. The newly formed processes spread to cover most of the granular layer depth. A significant axon outgrowth is evident 2 d after injection; it tends to increase at 5 and 7 d, but it is almost completely reversed after 1 month. No axonal modifications occur in control Fab-treated cerebella. The IN-1 Fab fragment-induced cellular changes and axon remodeling are essentially reproduced by applying affinity-purified antibody 472 raised against a peptide sequence of the recombinant protein NI-220, thus confirming the specificity of the applied treatments on these myelin-associated molecules. Functional neutralization of NI-35/250 proteins induces outgrowth from uninjured Purkinje neurites in the adult cerebellum. Together with previous observations, this suggests that these molecules regulate axonal plasticity to maintain the proper targeting of terminal arbors within specific gray matter regions.

Degradation of Chondroitin Sulfate Proteoglycans Induces Sprouting of Intact Purkinje Axons in the Cerebellum of the Adult Rat

“…Injection of anti-Nogo-A antibodies in the cerebellar cortex induces sprouting from Purkinje axons in the granular layer (Buffo et al, 2000), and a similar phenomenon is observed after injury in GAP-43 (growthassociated protein-43)-overexpressing Purkinje cells (Buffo et al, 1997;Gianola and Rossi, 2004). In both conditions, in addition to the outgrowth of terminal fibers from the infraganglionic plexus, sprouting also occurs along the corticofugal neurite, and this phenomenon may be accompanied by disruption of the normal axon-myelin relationship (Gianola and Rossi, 2004).…”

“…In addition, numerous terminal branches, with no preferential direction or orientation, covered the extent of the granular layer, although they appeared less dense in the deeper portions. The corticofugal axons, which could be followed in confocal stacks, did not show any dystrophic alterations such as torpedoes or thickened segments (Dusart and Sotelo, 1994;Rossi et al, 1994Rossi et al, , 1995a, nor did they display the "hot-spots" with multiple sprouts, observed after application of anti-Nogo-A antibodies (Buffo et al, 2000).…”

“…Quantitative morphometric evaluations were made according to previously established procedures (Buffo et al, 2000), using Neurolucida software (MicroBrightField, Colchester, VT) connected to a Nikon E-800 microscope via a color CCD camera. Purkinje axons were evaluated at different survival times after enzyme or vehicle application (see above) on DAB-processed calbindin-immunolabeled cerebellar sections (see examples in Fig.…”

“…Procedures that disrupt myelino-genesis or application of neutralizing antibodies against Nogo-A induce aberrant sprouting from Purkinje axons that invade the deep portions of the granular layer. This indicates that the peculiar distribution pattern of the intracortical plexus is maintained, at least in part, by inhibitory signals issued by myelin and/or associated proteins (Buffo et al, 2000;Gianola et al, 2003). Because different types of CSPGs are strongly expressed in the adult cerebellum (Rauch et al, 1991;Yamada et al, 1997), here we asked whether these molecules also participate in this regulation.…”

White matter inhibitors in CNS axon regeneration failure

“…For example, the infusion of a recombinant, humanized IN-1 antibody Fab fragment (rIN-1 Fab) into a spinal cord injury site was able to promote long-distance regeneration of injured axons in the spinal cord of adult rats (Brosamle et al, 2000). Application of IN-1 in adult cerebellum resulted in the sprouting of uninjured Purkinje cell axon, suggesting that a normal function for such an inhibitor is to maintain the proper targeting by axonal terminals (Buffo et al, 2000). Behavior outcome such as locomotor recovery also demonstrated improvement after IN-1 application (Merkler et al, 2001).…”