Robust Axonal Growth Research Articles

Selective Targeting of ER Exit Sites Supports Axon Development

During neuron development, the biosynthetic needs of the axon initially outweigh those of dendrites. However, although a localized role for the early secretory pathway in dendrite development has been observed, such a role in axon growth remains undefined. We therefore studied the localization of Sar1, a small GTPase that controls ER export, during early stages of neuronal development that are characterized by selective and robust axon growth. At these early stages, Sar1 was selectively targeted to the axon where it gradually concentrated within varicosities in which additional proteins that function in the early secretory pathway were detected. Sar1 targeting to the axon followed axon specification and was dependent on localized actin instability. Changes in Sar1 expression levels at these early development stages modulated axon growth. Specifically, reduced expression of Sar1, which was initially only detectable in the axon, correlated with reduced axon growth, where as overexpression of Sar1 supported the growth of longer axons. In support of the former finding, expression of dominant negative Sar1 inhibited axon growth. Thus, as observed in lower organisms, mammalian cells use temporal and spatial regulation of endoplasmic reticulum exit site (ERES) to address developmental biosynthetic demands. Furthermore, axons, such as dendrites, rely on ERES targeting and assembly for growth.

Robust axonal growth and a blunted macrophage response are associated with impaired functional recovery after spinal cord injury in the MRL/MpJ mouse

Spinal cord injury (SCI) in mammals leads to a robust inflammatory response followed by the formation of a glial and connective tissue scar that comprises a barrier to axonal regeneration. The inbred MRL/MpJ mouse strain exhibits reduced inflammation after peripheral injury and shows true regeneration without tissue scar formation following an ear punch wound. We hypothesized that following SCI, the unique genetic wound healing traits of this strain would result in reduced glial and connective tissue scar formation, increased axonal growth, and improved functional recovery. Adult MRL/MpJ and C57BL/6J mice were subjected to a mid-thoracic spinal contusion and the distribution of axon profiles and selected cellular and extracellular matrix components was compared at 1, 2, 4 and 6 weeks post-injury. Recovery of hind-limb locomotor function was assessed over the same time period. The MRL/MpJ mice exhibited robust axon growth within the lesion, beginning at 4 weeks post-injury. This growth was accompanied by reduced macrophage staining at 1, 2, 4 and 6 weeks post-injury, decreased chondroitin sulfate proteoglycan staining at 1–2 weeks and increased laminin staining throughout the lesion at 2–6 weeks post-injury. Paradoxically, the extent of locomotor recovery was impaired in the MRL/MpJ mice. Close examination of the chronic lesion site revealed evidence of ongoing degeneration both within and surrounding the lesion site. Thus, the regenerative genetic wound healing traits of the MRL/MpJ mice contribute to the evolution of a lesion environment that supports enhanced axon growth after SCI. However, this response occurs at the expense of meaningful functional recovery.

Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons

Inhibitory chondroitin sulfate proteoglycans (CSPGs) and myelin-associated molecules are major impediments to axon regeneration within the adult central nervous system (CNS). Decorin infusion can however suppress the levels of multiple inhibitory CSPGs and promote axon growth across spinal cord injuries [Davies, J.E., Tang, X., Denning, J.W., Archibald, S.J., and Davies, S.J., 2004. Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries. Eur. J. Neurosci. 19, 1226–1242]. A question remained as to whether decorin can also increase axon growth on inhibitory CSPGs and myelin via a direct effect on neurons. We have therefore conducted an in vitro analysis of neurite extension by decorin-treated adult dorsal root ganglion (DRG) neurons cultured on substrates of inhibitory CSPGs or myelin membranes mixed with laminin. Decorin treatment promoted 14.5 and 5-fold increases in average neurite length/neuron over untreated controls on CSPGs or myelin membranes respectively. In addition to suppressing inhibitory scar formation, our present data shows that decorin can directly boost the ability of neurons to extend axons within CSPG or myelin rich environments.

Axon growth across a lesion site along a preformed guidance pathway in the brain

Our previous studies showed that axonal outgrowth from dorsal root ganglia (DRG) transplants in the adult rat brain could be directed toward a specific target location using a preformed growth-supportive pathway. This pathway induced axon growth within the corpus callosum across the midline to the opposite hemisphere. In this study, we examined whether such pathways would also support axon growth either through or around a lesion of the corpus callosum. Pathways expressing GFP, NGF, or FGF2/NGF were set up by multiple injections of adenovirus along the corpus callosum. Each pathway included the transplantation site in the left corpus callosum, 2.8 mm away from the midline, and a target site in the right corpus callosum, 2.5 mm from the midline. At the same time, a 1 mm lesion was made through the corpus callosum at the midline in an anteroposterior direction. A group of control animals received lesions and Ad-NGF injections only at the transplant and target sites, without a bridging pathway. DRG cell suspensions from postnatal day 1 or 2 rats were injected at the transplantation site three to four days later. Two weeks after transplantation, brain sections were stained using an anti-CGRP antibody. The CGRP+ axons were counted at 0.5 mm and 1.5 mm from the lesion site in both hemispheres. Few axons grew past the lesion in animals with control pathways, but there was robust axon growth across the lesion site in the FGF2/NGF and NGF-expressing pathways. This study indicated that preformed NGF and combination guidance pathways support more axon growth past a lesion in the adult mammalian brain.

Neurotrophic activity of proNGF in vivo

Ectopic expression of nerve growth factor (NGF) in transgenic mice results in the directional growth of sympathetic and/or sensory fibers. For instance, mice that over-express NGF under the control of the glial fibrillary acidic protein (GFAP) promoter exhibit robust axonal sprouting into the cerebellum, with no apparent loss of neurons in peripheral ganglia. Given the disagreement in the literature over whether pro-NGF exerts neurotrophic or apoptotic effects, we assessed the relative levels of proNGF and mature NGF in the cerebella of these transgenic mice. Blinded western blot analyses revealed that proNGF was the major species in both transgenic and wild type mice, with very low levels of mature NGF expression. While transgenic mice displayed significantly higher levels of cerebellar proNGF protein as compared to wild type mice, both strains possessed comparable levels of mature NGF. These data reveal that the ectopic expression of NGF in the cerebellum results in an increase in proNGF rather than mature NGF levels. Together with the robust axonal growth and lack of neuronal death in the ganglia in these animals, our results are clearly consistent with proNGF exhibiting neurotrophic activity in vivo.

A role for neurotrophin-3 in targeting developing cholinergic axon projections to cerebral cortex

This study examined the relationship between expression of neurotrophin-3 (NT-3) and the ingrowth of cholinergic axonal projections in cerebral cortex. Patterns of expression of NT-3 (defined by β-galactosidase reporter expression in heterozygous offspring of transgenic NT-3lacZneo/+ mice) revealed that limbic cortical regions (including frontal, cingulate, and insular cortex, as well as the dentate gyrus) express NT-3 and that these cortical regions receive early and relatively dense cholinergic axons (stained for acetylcholinesterase, AChE). Using the dentate gyrus as a model system, studies revealed that expression of the NT-3 reporter parallels, and precedes by approximately 2 days, the ingrowth of AChE positive cholinergic axons. Studies of forebrain organotypic slice cultures demonstrate that basal forebrain-derived cholinergic axons extend into cortical regions in a pattern that mimics the pattern of expression of the NT-3 reporter. Similarly, chimeric co-cultures, combining wild type septum with a slice of hippocampus from heterozygous NT-3lacZneo/+ mice, demonstrate that cholinergic axons grow into regions of the dentate gyrus that express the NT-3 reporter. Hemisphere slice cultures made from NT-3 knockout mice reveal cholinergic axonal growth into cortex, but these axons do not form the regional pattern characteristic of slice cultures made from wild type or heterozygous NT-3lacZneo/+ mice. Further, chimeric co-cultures made using slices of wild type septum combined with slices of hippocampus from NT-3 knockout mice demonstrate robust cholinergic axonal growth into the hippocampus, but the cholinergic axons do not form the characteristic preterminal pattern associated with the dentate gyrus. Slice cultures from limbic cortical tissue from the NT-3 null mice do not display exaggerated levels of cell death. In aggregate, these data support the hypothesis that expression of NT-3 by cortical neurons serves to attract basal forebrain cholinergic projections to their target cells in cerebral cortex.

Axon regeneration through scars and into sites of chronic spinal cord injury

Cellular and extracellular inhibitors are thought to restrict axon growth after chronic spinal cord injury (SCI), confronting the axon with a combination of chronic astrocytosis and extracellular matrix-associated inhibitors that collectively constitute the chronic “scar.” To examine whether the chronically injured environment is strongly inhibitory to axonal regeneration, we grafted permissive autologous bone marrow stromal cells (MSCs) into mid-cervical SCI sites of adult rats, 6 weeks post-injury without resection of the “chronic scar.” Additional subjects received MSCs genetically modified to express neurotrophin-3 (NT-3), providing a further local stimulus to axon growth. Anatomical analysis 3 months post-injury revealed extensive astrocytosis surrounding the lesion site, together with dense deposition of the inhibitory extracellular matrix molecule NG2. Despite this inhibitory environment, axons penetrated the lesion site through the chronic scar. Robust axonal regeneration occurred into chronic lesion cavities expressing NT-3. Notably, chronically regenerating axons preferentially associated with Schwann cell surfaces expressing both inhibitory NG2 substrates and the permissive substrates L1 and NCAM in the lesion site. Collectively, these findings indicate that inhibitory factors deposited at sites of chronic SCI do not create impenetrable boundaries and that inhibition can be balanced by local and diffusible signals to generate robust axonal growth even without resecting chronic scar tissue.

Localized gene expression of axon guidance molecules in neuronal co-cultures

Axonal growth cones are guided to their targets by contact-dependent mechanisms or by diffusible chemotropic factors. Axon guidance by these factors typically involves culturing neurons on an acellular substrate which may not represent the in vivo biological environment. We developed two novel in vitro methods to create patterned gene expression of guidance molecules in a physiologically-relevant cellular environment. In the Matrigel™ assay, a droplet of adenovirus-Matrigel™ suspension was placed on astrocytes grown in Matrigel™. The adenovirus diffused through the gel and transduced underlying astrocytes, creating a radial infection gradient within a localized area. In the second model, recombinant adenovirus was bound to an anti-hexon antibody adsorbed onto stripe patterns of nitrocellulose. Once the cells were added, only those contacting the adenovirus were infected. The outgrowth pattern of chick DRG neurons on NGF, semaphorin 3A and brevican were studied. As expected, results showed robust axonal growth toward NGF as opposed to either secreted Sema 3A or membrane bound brevican, however subtle differences in axonal growth responses were observed in comparison to those obtained with less physiologically-relevant methods. Novel to this technology, the location and area of molecule expression can be controlled and manipulated in an intricate cellular environment.

Extracellular regulators of axonal growth in the adult central nervous system.

Robust axonal growth is required during development to establish neuronal connectivity. However, stable fibre patterns are necessary to maintain adult mammalian central nervous system (CNS) function. After adult CNS injury, factors that maintain axonal stability limit the recovery of function. Extracellular molecules play an important role in preserving the stability of the adult CNS axons and in restricting recovery from pathological damage. Adult axonal growth inhibitors include a group of proteins on the oligodendrocyte, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein and ephrin-B3, which interact with axonal receptors, such as NgR1 and EphA4. Extracellular proteoglycans containing chondroitin sulphates also inhibit axonal sprouting in the adult CNS, particularly at the sites of astroglial scar formation. Therapeutic perturbations of these extracellular axonal growth inhibitors and their receptors or signalling mechanisms provide a degree of axonal sprouting and regeneration in the adult CNS. After CNS injury, such interventions support a partial return of neurological function.

Neurotrophins support regenerative axon assembly over CSPGs by an ECM-integrin-independent mechanism

Chondroitin sulfate proteoglycans (CSPGs) and myelin-based inhibitors are the most studied inhibitory molecules in the adult central nervous system. Unlike myelin-based inhibitors, few studies have reported ways to overcome the inhibitory effect of CSPGs. Here, by using regenerating adult dorsal root ganglion (DRG) neurons, we show that chondroitin sulfate proteoglycans inhibit axon assembly by a different mechanism from myelin-based inhibitors. Furthermore, we show that neither Rho inhibition nor cAMP elevation rescues extracellular factor-induced axon assembly inhibited by CSPGs. Instead, our data suggest that CSPGs block axon assembly by interfering with integrin signaling. Surprisingly, we find that nerve growth factor (NGF) promotes robust axon growth of regenerating DRG neurons over CSPGs. We have found that, unlike naive neurons that require simultaneous activation of neurotrophin and integrin pathways for axon assembly, either neurotrophin or integrin signaling alone is sufficient to induce axon assembly of regenerating neurons. Thus, our results suggest that the ability of NGF to overcome CSPG inhibition in regenerating neurons is probably due to the ability of regenerating neurons to assemble axons using an integrin-independent pathway. Finally, our data show that the GSK-3beta-APC pathway, previously shown to mediate developing axon growth, is also necessary for axon regeneration.

NG2 Colocalizes With Axons and Is Expressed by a Mixed Cell Population in Spinal Cord Lesions

The NG2 proteoglycan is of general interest after spinal cord injury because it is expressed by oligodendrocyte progenitors (OPCs), which contribute to central nervous system remyelination; however, NG2 may inhibit axon regeneration. We and others have examined the spatiotemporal expression of NG2 after spinal cord injury (SCI). Here, we extend those observations and provide a comprehensive analysis of the distribution, phenotype, and colocalization of NG2 cells with axons in a clinically relevant model of spinal contusion. Because contusion models mimic the majority of human SCI, this information is important for understanding endogenous processes that promote and/or prevent repair. The data demonstrate that NG2 levels rise significantly between 3 and 7 days postinjury (dpi) and remain elevated chronically throughout the lesions. NG2 within the lesions could be derived from an array of infiltrating cells; thus, a panel of antibodies was used to investigate NG2 cell phenotypes. First, platelet-derived growth factor-alpha receptor (PDGFalphaR) colocalization was examined because OPCs normally express both markers. PDGFalphaR cells were present in lesions at all times examined. However, only 37% of NG2 cells coexpressed PDGFalphaR at 14 dpi, which dropped to <1% by 70 dpi. This contrasts with the nearly complete overlap in spared tissue surrounding the lesion. In contrast, 40% to 60% of NG2 cells expressed p75 and approximately 84% expressed Sox10, suggesting that many NG2 cells were nonmyelinating Schwann cells. Despite rising levels of NG2, we noted robust and sustained axon growth into the lesions, many of which were located along NG2 profiles. Thus, spinal contusion produces an NG2-rich environment into which axons grow and in which the source of NG2 appears considerably different from that in surrounding spared tissue.

Astrocytes derived from glial-restricted precursors promote spinal cord repair

BackgroundTransplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair.ResultsTransplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery.ConclusionPre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries.

Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations

Bone marrow stromal cells (MSC) are non-hematopoietic support cells that can be easily derived from bone marrow aspirates. Human MSC are clinically attractive because they can be expanded to large numbers in culture and reintroduced into patients as autografts or allografts. We grafted human MSC derived from aspirates of four different donors into a subtotal cervical hemisection in adult female rats and found that cells integrated well into the injury site, with little migration away from the graft. Immunocytochemical analysis demonstrated robust axonal growth through the grafts of animals treated with MSC, suggesting that MSC support axonal growth after spinal cord injury (SCI). However, the amount of axon growth through the graft site varied considerably between groups of animals treated with different MSC lots, suggesting that efficacy may be donor-dependent. Similarly, a battery of behavioral tests showed partial recovery in some treatment groups but not others. Using ELISA, we found variations in secretion patterns of selected growth factors and cytokines between different MSC lots. In a dorsal root ganglion explant culture system, we tested efficacy of conditioned medium from three donors and found that average axon lengths increased for all groups compared to control. These results suggest that human MSC produce factors important for mediating axon outgrowth and recovery after SCI but that MSC lots from different donors vary considerably. To qualify MSC lots for future clinical application, such notable differences in donor or lot–lot efficacy highlight the need for establishing adequate characterization, including the development of relevant efficacy assays.

Degeneration and regeneration of perivascular innervation in arterial grafts.

Because the understanding of postoperative changes in arterial graft innervation is limited, this study was performed to characterize neuronal degeneration and regeneration events immunohistochemically in femoral arterial grafts transplanted to carotid arteries in rats. Specimens taken 1 day, 3 days, 7 days, 1 month, 3 months, and 5 months after surgery were assessed for vasoactive intestinal peptide, neurofilaments, growth-associated protein 43, tyrosine hydroxylase, and nitric oxide synthase isoenzymes. During neuronal degeneration, vasoactive intestinal peptide disappeared within 1 day, transmitter-synthesizing enzymes (nitric oxide synthase and tyrosine hydroxylase) had vanished by day 7, and neurofilaments (cytoskeletal markers) had essentially disappeared after 1 week. In the regeneration phase, the most robust axonal growth, as visualized by growth-associated protein 43, was observed at 1 month, followed by a gradual increase in neurotransmitter markers at 1 and 3 months, whereas the neurofilaments increased gradually up to the end of the 5-month observation period. Reinnervation proceeded from the proximal carotid (host) trunk distally to the graft. Axonal re-growth occurred mainly in arterial adventitia. Innervation density, as visually assessed, was denser in the graft than in the host. These findings suggest that 1) the main sequence of degeneration and regeneration follows that reported in other models of neuronal degeneration; 2) reinnervation of the arterial grafts comes mainly from the host arteries; and 3) the innervation density in the graft may differ from that in the host, which may suggest target-derived regulation of innervation. The latter finding may have clinical implications. It suggests that for a good outcome it would be beneficial to choose a sparsely innervated graft rather than a densely innervated one.

Acute inactivation of MAP1b in growing sympathetic neurons destabilizes axonal microtubules

Microtubule-associated-protein 1b (MAP1b) is abundant in neurons actively extending axons. MAP1b is present on microtubules throughout growing axons, but is preferentially concentrated on microtubule polymer in the distal axon and growth cone. Although MAP1b has been implicated in axon growth and pathfinding, its specific functions are not well understood. Biochemical and transfection studies suggest that MAP1b has microtubule-stabilizing activity, but recent studies with neurons genetically deficient in MAP1b have not confirmed this. We have explored MAP1b functions in growing sympathetic neurons using an acute inactivation approach. Neurons without axons were injected with polyclonal MAP1b antibodies and then stimulated to extend axons. Injected cells were compared to controls in terms of axon growth behavior and several properties of axonal microtubules. The injected antibodies rapidly and quantitatively sequestered MAP1b in the cell body, making it unavailable to perform its normal functions. This immunodepletion of MAP1b had no statistically significant effect on axon growth, the amount of microtubule polymer in the axon, and the relative tyrosinated tubulin content of this polymer, and this was true in sympathetic neurons from rat, wild type mice, and tau knockout mice. Thus, robust axon growth can occur in the absence of MAP1b alone or both MAP1b and tau. However, immunodepletion of MAP1b significantly increased the sensitivity of microtubules in the distal axon and growth cone to nocodazole-induced depolymerization. These results indicate that MAP1b has microtubule-stabilizing activity in growing axons. This stabilizing activity may be required for some axonal functions, but it is not necessary for axon growth.

Olfactory ensheathing cells: bridging the gap in spinal cord injury.

Spinal cord injury (SCI) continues to be an insidious and challenging problem for scientists and clinicians. Recent neuroscientific advances have changed the pessimistic notion that axons are not capable of significant extension after transection. The challenges of recovering from SCI have been broadly divided into four areas: 1) cell survival; 2) axon regeneration (growth); 3) correct targeting by growing axons; and 4) establishment of correct and functional synaptic appositions. After acute SCI, there seems to be a therapeutic window of opportunity within which the devastating consequences of the secondary injury can be ameliorated. This is supported by several observations in which apoptotic glial cells have been identified up to 1 week after acute SCI. Moreover, autopsy studies have identified anatomically preserved but unmyelinated axons that could potentially subserve normal physiological properties. These observations suggest that therapeutic strategies after SCI can be directed into two broad modalities: 1) prevention or amelioration of the secondary injury, and 2) restorative or regenerative interventions. Intraspinal transplants have been used after SCI as a means for restoring the severed neuraxis. Fetal cell transplants and, more recently, progenitor cells have been used to restore intraspinal circuitry or to serve as relay for damaged axons. In an attempt to remyelinate anatomically preserved but physiologically disrupted axons, newer therapeutic interventions have incorporated the transplantation of myelinating cells, such as Schwann cells, oligodendrocytes, and olfactory ensheathing cells. Of these cells, the olfactory ensheathing cells have become a more favorable candidate for extensive remyelination and axonal regeneration. Olfactory ensheathing cells are found along the full length of the olfactory nerve, from the basal lamina of the epithelium to the olfactory bulb, crossing the peripheral nervous system-central nervous system junction. In vitro, these cells promote robust axonal growth, in part through cell adhesion molecules and possibly by secretion of neurotrophic growth factors that support axonal elongation and extension. In animal models of SCI, transplantation of ensheathing cells supports axonal remyelination and extensive migration throughout the length of the spinal cord. Although the specific properties of these cells that govern enhanced axon regeneration remain to be elucidated, it seems certain that they will contribute to the establishment of new horizons in SCI research.