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Regulation of neuritic growth is crucial for neural development, adaptation and repair. The intrinsic growth potential of nerve cells is determined by the activity of specific molecular sets, which sense environmental signals and sustain structural extension of neurites. The expression and function of these molecules are dynamically regulated by multiple mechanisms, which adjust the actual growth properties of each neuron population at different ontogenetic stages or in specific conditions. The neuronal potential for axon elongation and regeneration are restricted at the end of development by the concurrent action of several factors associated with the final maturation of neurons and of the surrounding tissue. In the adult, neuronal growth properties can be significantly modulated by injury, but they are also continuously tuned in everyday life to sustain physiological plasticity. Strict regulation of structural remodelling and neuritic elongation is thought to be required to maintain specific patterns of connectivity in the highly complex mammalian CNS. Accordingly, procedures that neutralize such mechanisms effectively boost axon growth in both intact and injured nervous system. Even in these conditions, however, aberrant connections are only formed in the presence of unusual external stimuli or experience. Therefore, growth regulatory mechanisms play an essentially permissive role by setting the responsiveness of neural circuits to environmental stimuli. The latter exert an instructive action and determine the actual shape of newly formed connections. In the light of this notion, efficient therapeutic interventions in the injured CNS should combine targeted manipulations of growth control mechanisms with task-specific training and rehabilitation paradigms.

The limited regenerative capability of adult central neurons results from an interplay between the intrinsic growth potential of injured nerve cells and environmental growth-inhibitory signals, such as myelin-associated molecules. These molecules, which are known to inhibit axonal regeneration, are expressed in the adult intact central nervous system, suggesting that they also exert a constitutive regulatory function on uninjured neurites. However, most of the mechanisms underlying this function of myelin-associated factors are still unclear. To address this issue, we examined Purkinje cells, which show extremely poor regenerative capabilities. Such a weak intrinsic growth potential is attributed, at least in part, to inhibitory activity exerted by the myelin-associated molecule Nogo-A on growth-associated gene expression and axon plasticity of adult Purkinje cells. Indeed, the regenerative potential of these neurons declines during postnatal development in parallel with myelin deposition. In addition, myelin-associated factors regulate the development of the Purkinje axon intracortical plexus, contributing to the shaping of mature cerebellar connectivity. The action of environmental inhibitory factors can be partially counteracted by overexpression of neuronal growth-associated molecules. In fact, following axotomy, transgenic Purkinje cells that overexpress GAP-43 are able to sprout their axons into white matter and show a pronounced tendency to lose myelin. Altogether, these results suggest that the control exerted by myelin on Purkinje axon plasticity plays an important role in preventing aberrant growth and in stabilizing intracortical connectivity. Such a constitutive regulation may eventually hamper compensatory mechanisms set up by the same neurons in response to injury and thus determine their weak regenerative abilities.

Up-regulation of growth-associated proteins in adult neurons promotes axon regeneration and neuritic elongation onto nonpermissive substrates. To investigate the interaction between these molecules and myelin-related inhibitory factors, we examined transgenic mice in which overexpression of the growth-associated protein GAP-43 is driven by the Purkinje cell-specific promoter L7. Contrary to their wild-type counterparts, which have extremely poor regenerative capabilities, axotomized transgenic Purkinje cells exhibit profuse sprouting along the intracortical neurite and at the severed stump [Buffo et al. (1997) J. Neurosci., 17, 8778-8791]. Here, we investigated the relationship between such sprouting axons and oligodendroglia to ask whether GAP-43 overexpression enables Purkinje neurites to overcome myelin-derived inhibition. Intact transgenic Purkinje axons display normal morphology and myelination. Following injury, however, many GAP-43-overexpressing neurite stumps are devoid of myelin cover and sprout into white matter regions containing densely packed myelin and Nogo-A- or MAG-immunopositive oligodendrocytes. The intracortical segments of these neurites show focal accumulations of GAP-43, which are associated with disrupted or retracted myelin sheaths. Numerous sprouts originate from such demyelinated segments and spread into the granular layer. Some myelin loss, though not axon sprouting, is also evident in wild-type mice, but this phenomenon is definitely more rapid and extensive in transgenic cerebella. Thus, GAP-43-overexpressing Purkinje axons are endowed with enhanced capabilities for growing into nonpermissive territories and show a pronounced tendency to lose myelin. Our observations suggest that accumulation of GAP-43 along precise axon segments disrupts the normal axon-glia interaction and enhances the retraction of oligodendrocytic processes to facilitate the outgrowth of neuritic sprouts.

Neurons with enhanced intrinsic growth capabilities can elongate their axons into non-permissive territories, but the mechanisms that enable the outgrowing processes to overcome environmental inhibition are largely unknown. To address this issue, we examined adult mouse Purkinje cells that overexpress the axonal growth-associated protein GAP-43. After injury, these neurons exhibit sprouting along the intracortical neuritic course and at the severed stump in the white matter. To determine whether GAP-43-overexpressing Purkinje cells are responsive to extrinsic inhibitory cues, we investigated the content and subcellular localization of major receptors for myelin-associated inhibitory proteins, PlexinB1 and the Nogo receptor (NgR) with the related co-receptors LINGO-1 and p75. Expression of these molecules, estimated by measuring perikaryal immunostaining intensity and Western blot, was not different in wild-type or transgenic mice, and it was not overtly modified after axotomy. Following injury, however, the content of PlexinB1 was significantly reduced in GAP-43-overexpressing neurites. Furthermore, in the same axons the distribution of both PlexinB1 and NgR was altered, being inverse to that of GAP-43. Labelling for the two receptors was conspicuously reduced on the axonal surface and it was almost undetectable in the outgrowing sprouts, which showed strong GAP-43 immunoreactivity. These observations indicate that although GAP-43 overexpression does not modify the expression of receptors for myelin-associated inhibitory factors, it interferes with their subcellular localization and exposure on the neuritic membrane. Therefore, GAP-43 promotes axon growth by multiple synergistic mechanisms that potentiate the intrinsic motility of the elongating processes, while reducing their sensitivity to environmental inhibition.

In the last few years Purkinje cells have become a most interesting model to investigate cellular/molecular mechanisms of axon regeneration and plasticity. Adult Purkinje cells are most peculiar for their weak cell body response to axotomy, which is accompanied by a strong resistance to injury and a virtually absolute inability to regenerate severed neurites, even in the presence of favourable environmental conditions. The same neurons show a vigorous intrinsic inclination toward axonal sprouting and structural plasticity, which can be elicited by removing extrinsic growth-inhibitory cues. These features gradually develop during early postnatal life, but the underlying mechanisms and biological significance remain unclear. This article reviews recent studies aimed at addressing these questions with respect to the general issue of brain repair. Indeed, understanding the reasons for the extremely poor regenerative capacity of Purkinje cells will be most important to elucidate basic biological mechanisms of axon regeneration and plasticity, and to promote circuit rewiring in the adult CNS.

The highly specific connection patterns of the mature CNS are shaped through finely regulated processes of axon growth and retraction. To investigate the relative contribution of cell-autonomous mechanisms and extrinsic cues in these events, we examined the development of Purkinje axon intracortical plexus in the rat cerebellum. During the first postnatal week, several new processes sprout from focal swellings along the initial portion of the Purkinje neurite and spread in the granular layer. Intense structural plasticity occurs during the following week, with pruning of collateral branches and remodeling of terminal arbors. The mature distribution of the Purkinje infraganglionic plexus, confined within the most superficial portion of the granular layer, is attained at approximately postnatal day 15. A similar neuritic branching pattern is also developed by Purkinje cells grown in cultures of dissociated cerebellar cells or transplanted to extracerebellar CNS regions, suggesting that cell-autonomous mechanisms contribute to determining the Purkinje axon phenotype.The structural remodeling of Purkinje intracortical plexus is concomitant with the development of cerebellar myelin. To ask whether myelin-associated factors contribute to the morphological maturation of Purkinje neurites, we prevented normal myelinogenesis by killing oligodendrocyte precursors with 5′-azacytidine or by applying neutralizing antibodies against the myelin-associated neurite growth inhibitor Nogo-A. In both conditions, Purkinje axons retained exuberant branches, and the terminal plexus spanned the entire extent of the granular layer. Thus, the formation of Purkinje axon collaterals is, in part, controlled by intrinsic determinants, but their growth and distribution are regulated by environmental signals, among which are myelin-derived cues.

Long-distance axon regeneration requires the activation of a specific set of neuronal growth-associated genes. Adult Purkinje cells fail to upregulate these molecules in response to axotomy and show extremely weak regenerative properties. Nevertheless, starting from several months after injury, transected Purkinje axons undergo spontaneous sprouting. Here, we asked whether long-term injured Purkinje cells acquire novel intrinsic growth properties that enable them to upregulate growth-associated genes and sustain axon regeneration. To test this hypothesis, we examined axon growth and cell body changes in adult rat Purkinje neurons following axotomy and implantation of embryonic neocortical tissue or Schwann cells into the injury track. Purkinje cells that survived over 6 months after injury/transplantation displayed profuse sprouting in the injured cerebellum and developed extensive networks of terminal branches into embryonic neocortical grafts. In addition, severed Purkinje axons exposed to these transplants 6 months after injury grew faster than their counterparts confronted with the same environment immediately after axotomy. Nevertheless, long-term injured Purkinje cells failed to regenerate stem neurites into Schwann cell grafts, and, under all experimental conditions, they did not upregulate growth-associated molecules, including c-Jun, GAP-43, SNAP-25, and NADPH-diaphorase. These results indicate that the long-term injured Purkinje cells remain unable to activate the gene program required to sustain axon regeneration and their plasticity is restricted to terminal arbor remodeling. We propose that the delayed growth of injured Purkinje cells reflects an adaptive phenomenon by which the severed axon stump develops a new terminal arbor searching for alternative connections with local partners.

To understand the mechanisms leading to the progressive loss of intrinsic neuronal growth properties during central nervous system development, we have investigated the evolution of the response to injury and regenerative potential of immature Purkinje cells, axotomised at different postnatal ages from postnatal day (P)3 to P12. In adult rodents, these neurons are characterised by a weak cell body response to axotomy, which is associated with a remarkable resistance to injury and a poor regenerative capability. During the first postnatal week, Purkinje cells are strongly sensitive to injury and massively degenerate within a few days. Immature Purkinje cells react to neurite transection by a strong upregulation of c-Jun, accompanied by a moderate, but consistent, expression of the growth-associated protein (GAP)-43. In contrast, nicotinamide adenine dinucleotide monophosphate (NADPH)-diaphorase reactivity, which can be activated by adult Purkinje neurons, is not modified in their juvenile counterparts. The severed Purkinje axons show a vigorous regenerative sprouting both into the lesioned cerebellar environment and into embryonic neocortical tissue transplanted into the injury site. The typical adult features of the response to injury progressively develop during the second postnatal week, when the injured neurons acquire resistance, cell body changes become milder, the regenerative potential declines, and the severed axons undergo characteristic morphological modifications, including torpedoes and the hypertrophy of recurrent collateral branches. This complete reversal of the features and the outcome of the Purkinje cell reaction to axotomy likely results from the profound changes that occur in the maturing Purkinje cells and/or in their microenvironment during this phase of cerebellar development. J. Comp. Neurol. 430:101–117, 2001. © 2000 Wiley-Liss, Inc.