Trauma to the adult CNS results in devastating clinical consequences because of the failure of neurons to regenerate their injured axons. This contrasts with what is observed in the adult PNS where regeneration is possible, and even in more extreme cases where surgical intervention is required, much functional recovery can occur and clinical outcomes are more positive. Studies done over 25 years ago demonstrated that adult CNS neurons show a similar intrinsic capacity for axon outgrowth following injury as do those in the PNS, and ultimately implicated factors present in the CNS microenvironment responsible for regeneration failure (Aguayo et al. 1981, 1983). Since then, many factors, including those associated with CNS myelin, have been identified as being inhibitory to axon outgrowth and as potential targets for therapies designed to promote recovery via regeneration and/or plasticity of the injured CNS.
Several investigators have confirmed the inhibitor potential of isolated myelin membrane preparations on axonal outgrowth in vitro. Major inhibitory molecules identified in CNS myelin include myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte myelin glycoprotein (OMgp) (for reviews see Filbin 2003; Liu et al. 2006; Schwab 1996). These molecules were thought to inhibit axon growth by binding to a common cell-surface receptor, Nogo-66 receptor (NgR), localized on the growth cones of injured axons (Yiu and He 2006), and acting primarily on growth cone collapse (Chivatakarn et al. 2007). Through the action of NgR co-receptors, which include p75, TROY, and LINGO1, this binding leads to the activation of downstream neural signaling pathways that inhibit axon growth (Yiu and He 2006). The best characterized of these pathways is the RhoA/RhoA-associated kinase pathway that when activated leads to stabilization in the growth cone cytoskeleton which restricts outgrowth of injured axons (He and Koprivica 2004). However, mutant mice deficient in these myelin proteins or NgR show only limited axon regeneration and functional recovery (Zheng et al.2006); suggesting that other myelin components and receptors on neurons play role in this inhibition. In this regard, the paired immunoglobulin-like receptor B (PirB) that also binds Nogo, MAG, and OMgp has been shown recently to inhibit axon regrowth, and blocking both PirB and NgR leads to axon regeneration on myelin (Atwal et al. 2008). Another study has shown that the mammalian target of rapamycin (mTOR) signaling pathway is down-regulated following axonal injury by the tumor suppressor PTEN (phosphatase and tensin homolog), whose deletion allows mTOR activity on downstream targets including ribosomal S6 kinase and eukaryotic initiation factor4E to promote axon regeneration in an inhibitory environment (Park et al. 2008) Further understanding of the complex mechanisms by which myelin-associated inhibitors impede axon outgrowth will aid in developing treatments to counter the inhibitory CNS environment and facilitate regeneration.
One potential means by which neurons might counteract the inhibitory factors present in CNS myelin would be to degrade these factors or interfere with their receptors in ways that neutralize their inhibitory effects on axon growth. Extracellular proteolysis is a strategic mechanism employed by many cell types to disrupt or modify both cell–cell and cell–extracellular matrix (ECM) contacts thereby altering cellular responses to environmental cues (Werb 1997). During many physiological and pathological events, this function is achieved through the expression of several different proteolytic enzymes, including members of the plasminogen activator/plasmin system and matrix metalloproteases (MMPs). Tissue plasminogen activator (tPA) (EC# 184.108.40.206) is a serine protease that converts the inactive precursor plasminogen into its active counterpart, plasmin (EC# 220.127.116.11). Plasmin is a broad-spectrum protease with a long list of extracellular targets including cellular receptors, growth factors, and other ECM components (Tsirka 2002). In the circulatory system, the primary function of plasmin is to degrade fibrin clots, and as such tPA is most recognized for its role in thrombolysis and vascular homeostasis. However, recent years have seen a growing appreciation for tPAs potential roles in the nervous system, with the enzyme being linked to neural mechanisms such as plasticity, synaptic remodeling, and axonal regeneration (for review see Tsirka 2002).
Tissue plasminogen activator is widely expressed in the nervous system by neurons and some glial cells. tPA activity is up-regulated by neurons during both normal and pathological events that lead to neural plasticity. tPA is focally concentrated at axonal growth cones, where it can facilitate axonal growth in vivo and in 3-D cell cultures (Krystosek and Seeds 1981a,b; Pittman and DiBenedetto 1995). In vivo, tPA is up-regulated in the brain during motor learning and promotes a form of plasticity known as long-term potentiation (LTP) (Seeds et al. 1995, 2003; Baranes et al. 1998). tPA has been shown to play a role in experience-dependent plasticity of the visual cortex during the developmental critical period (Mataga et al. 2002, 2004), and tPA induces mossy fiber sprouting and reorganization during seizures (Wu et al. 2000). Peripheral nerve injury leads to an up-regulation of tPA by injured neurons where tPA promotes axon regeneration and functional recovery (Siconolfi and Seeds 2001a,b).
The following study was designed to test the potential role that tPA plays in promoting nerve regeneration in the presence of myelin-associated molecules which represent key barriers to axonal outgrowth following CNS injury. As injured neurons show limited axon growth in the presence of myelin inhibitors, we utilized an experimental model known as the conditioning-injury paradigm that enhances the ability of dorsal root ganglia (DRG) sensory neurons to grow axons in the presence of these inhibitors in culture and more importantly to regenerate CNS axons following a dorsal column spinal cord injury. In this paradigm, axonal growth is stimulated via a pre-conditioning lesion made to the peripheral branch of DRG neurons (Richardson and Issa 1984; Richardson and Verge 1986). Following a conditioning period of 1–2 weeks, injured neurons display a greater propensity for neurite outgrowth in vitro when neurons are explanted and cultured on either growth-permissive substrates or inhibitory myelin (Neumann and Woolf 1999; Qiu et al. 2002). Interestingly, our prior study showed that the peak of tPA up-regulation in the DRG and regenerating axons was also at 1 week following sciatic nerve injury (Siconolfi and Seeds 2001a); suggesting that tPA may play a role in the conditioning-injury paradigm. To test this possibility, mice deficient for either tPA or plasminogen genes were used in the conditioning-injury model of axonal growth on myelin. Our experiments show that tPA is important for conditioned axonal outgrowth and may represent a potential treatment in promoting regenerative growth and plasticity in the injured CNS.
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- Materials and methods
To assess the potential role of tPA in the conditioning-injury model of axonal growth on CNS myelin, sciatic nerves of adult wild-type, tPA−/−, and Plgn−/− mice were injured as described in the Materials and methods. One week after injury, L4/L5 DRG neurons were isolated and cultured on a substrate of either growth-permissive fibronectin or inhibitory myelin membranes purified from mouse brain and spinal cord. DRG neurons were cultured over a period of 40 h. As shown in Fig. 1(a and c), neurite outgrowth by non-conditioned wild-type or tPA−/− DRG neurons is inhibited in the presence of myelin membranes. Pre-conditioned wild-type neurons (Figs 1b and 2c) display robust neurite outgrowth with complex arborization patterns on myelin membranes demonstrating the conditioning-injury response of axonal regeneration. In contrast, conditioned DRG neurons from tPA−/− mice (Fig. 1d) fail to display extensive neurite outgrowth and growth is visibly more like that of non-conditioned neurons. As seen in Fig. 2(a), many tPA−/− neurons fail to produce neurites and show signs of abortive outgrowth with broad lamellapodia-like structures on the myelin substrate, in marked contrast to the dramatic neurite outgrowth seen with conditioned wild-type neurons on myelin (Fig. 2c). Fewer tPA−/− neurons are able to initiate outgrowth, but many of those display abnormal processes that are in some cases swollen and not well-defined, while others appear to be blebbing (Fig. 2b), a morphological indicator of neurite degeneration (Ivins et al. 1998).
Figure 1. Mice deficient for tPA fail to display conditioned-injury induced neurite outgrowth on myelin membranes. Photomicrographs of (a) non-conditioned wild-type neurons, (b) conditioned wild-type neurons, (c) non-conditioned tPA−/− neurons, and (d) conditioned tPA−/− neurons cultured on myelin membranes. Neurons were stained using rabbit anti-β tubulin III. All bars are 50 μm.
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Figure 2. Sensory DRG neurons from conditioned-injury tPA−/− mice show signs of abortive neurite outgrowth and blebbing on myelin when compared to conditioned wild-type neurons. Photomicrographs of representative neurons from tPA−/− mice grown on myelin show signs of (a) abortive neurite outgrowth and broad lamellapodia-like structures and (b) swelling and blebbing of neurites, compared to the conditioned-injury wild-type neurons (c). All bars equal 25 μm.
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Quantitative analysis of neurite outgrowth was carried out to obtain measurements of both neurite initiation and elongation. Initiation analysis was done by counting the number of neurons possessing at least one neurite > 25 μm in length and comparing this value to the total number of neurons randomly sampled. Results are presented as % total number neurons assayed and provided as a bar graph in Fig. 3(a). As expected, injury-conditioned wild-type neurons display enhanced neurite initiation over that of non-conditioned neurons (35.0 ± 3.1% vs. 21.7 ± 2.0%). Injury-conditioned neurons from tPA−/− mice show no increase in neurite initiation over their non-injured counterparts, and display significantly fewer (18.8 ± 3.3%) neurite-bearing cells comparable to that observed for non-conditioned wild-type neurons. Interestingly, addition of 140 nM recombinant tPA to the culture medium of tPA−/− neurons was able to induce neurite initiation in these neurons to levels (30.0 ± 5%) that are on average not significantly different from those of conditioned wild-type neurons. However, addition of tPA to non-conditioned wild-type neurons has no enhancement effect. These results demonstrate the importance of tPA as one of the initiating factors for conditioning-induced neurite outgrowth on the inhibitory myelin membrane substrate.
Figure 3. Quantitatively tPA−/− neurons fail to display conditioning-injury induced neurite initiation and elongation on myelin. (a) Neurite initiation was measured by calculating the average number of neurons that displayed at least one neurite of 25 μm or longer in length. Results are represented as the percent of neurite-bearing neurons out of the total number of neurons counted. (b) Average lengths for each type of neuron assayed. Gray bars indicate neurons from wild-type mice, and Black bars indicated neurons from tPA−/− mice. Error bars represent standard errors, and asterisks indicate p < 0.01 compared to other cultures from the same genotype mice.
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For quantitative analysis of neurite elongation, all observed neurites for each neuron type or condition were measured for length and averaged. Results are presented in Fig. 3(b). As expected conditioned wild-type neurons display on average significantly greater (3.1-fold increase) neurite lengths than that of non-conditioned neurons (187.4 ± 12.4 μm vs. 60.3 ± 10.0 μm), again confirming the conditioning-injury effect on axon regeneration. Conditioned neurons from tPA−/− mice show only a slight increase in neurite length over neurons from non-conditioned tPA−/− mice, but display a 45% reduction in average neurite length (102.9 ± 6.4 μm) compared to conditioned wild-type neurons. However, tPA−/− neurons still are able to grow neurites on average that are longer than those of non-conditioned wild-type neurons, demonstrating the possibility of compensatory molecules that can partially recover the effects of a tPA deficiency. Addition of recombinant tPA to conditioned tPA−/− cultures did induce neurite elongation (140.1 ± 9.1 μm) that is statistically significant to the non-conditioned tPA−/− neurons, but not quite to the levels observed for conditioned wild-type neurons; however, tPA addition had no effect on neurite length when added to non-conditioned wild-type neurons.
Further analysis of neurite elongation by conditioned wild-type and tPA−/− neurons in the presence of myelin membranes was carried out to obtain distribution patterns on the percentage of neurites that fell between various length ranges (≤ 50 μm, 51–99 μm, 100–149 μm, 150-199 μm, 200–249 μm, and ≥ 250 μm). Neurite lengths were binned and tabulated for the various length ranges and presented as a bar graph in Fig. 4(a). The majority (67.7%) of conditioned tPA−/− neurites fail to grow to lengths greater that 100 μm. In contrast, only 24.3% of conditioned wild-type neurites fail to grow to lengths greater that 100 μm. In fact, 35.7% of conditioned wild-type neurites grow to lengths equal to or greater that 200 μm, compared to only 11.5% of conditioned tPA−/− neurites falling into this range. Clearly, tPA-deficiency drastically limits the ability of conditioned neurons to grow long neurites in the presence of myelin membranes. Finally, it is important to note that when grown in the presence of the permissive substrate fibronectin, injury-induced conditioning stimulated neurite growth by both wild-type and tPA−/− neurons, but to similar lengths that are not significantly different between genotypes (Fig. 4b). This finding indicates that tPA has a important role in conditioning-induced outgrowth only in the presence of inhibitory myelin membranes, but doesn’t necessarily play a part in the general enhancement of growth potential that occurs as a result of a conditioning injury.
Figure 4. The majority of neurons from conditioned-injury tPA−/− mice display shorter processes relative to those from wild-type mice. (a) Distribution patterns on the percentage of neurites that fell between various length ranges (≤ 50, 51–99, 100–149, 150–199, 200–249, and ≥ 250 μm). Neurite lengths were binned and tabulated for the various length ranges and presented as a bar graph. The majority (67.7%) of conditioned tPA−/− neurites fail to grow to lengths > 100 μm. (b) No difference was observed in the conditioning stimulated average neurite lengths for wild-type and tPA−/− neurons grown on fibronectin. Error bars represent standard errors, and asterisks indicate p < 0.01 compared to unconditioned neurons of the same genotype.
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Since our prior studies (Siconolfi and Seeds 2001a) showed that sciatic nerve injury led to an induction of tPA in DRG neurons and their regenerating axons, we compared the tPA activity in culture media collected from non-injured and injury-conditioned DRG neurons of wild-type mice. The media were collected at 17hr. when axon outgrowth is in its earliest stages on the myelin substratum. Zymography of equivalent amounts of media from cultures containing the same number of plated neurons shows that the conditioning-injury leads to a 60% greater secretion of tPA by these neurons, when analyzed densitometrically under conditions of linear activity (Fig. 5). There was no difference in the urokinase plasminogen activator (uPA) activity with a conditioning-injury.
Figure 5. Tissue plasminogen activator activity is increased in cultures of injury-conditioned wild-type DRG neurons. Culture media was removed from cultures of dissociated DRG neurons from injury-conditioned and non-injured wild-type mice at 17 h, when axon outgrowth is just beginning on myelin. The media was subjected to zymography to assess relative amounts of secreted tPA activity between these different cultures. tPA activity is 60% greater in the media from injury-conditioned wild-type DRG neurons (lane 4) compared to media from DRG neurons of non-injured wild-type mice (lane 3). Plasminogen activator standards for tPA (r-tPA in lane 1) top arrow, and urokinase plasminogen activator (uPA) (mouse urine in lane 2) thin lower arrow were included on this gel.
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In a separate analysis for neurite elongation, random DRG neurite-bearing neurons were measured for cellular diameter and were separated into groups defined as small (< 25 μm) or medium-to-large (≥ 25 μm) subtypes of neurons. As shown in Fig. 6, the greatest impact of tPA deficiency on neurite elongation is imparted on small caliper neurons. Injury-conditioned small caliper tPA−/− neurons grow neurites that are 2.5-fold shorter in length than those of corresponding wild-type neurons. For medium- to large-sized neurons, the difference is not so robust; conditioned tPA−/− neurons grow neurites that are only 1.3-fold shorter than those of wild-type neurons. Interestingly, the addition of recombinant tPA only improves the outgrowth of small neurons bringing the average neurite lengths to levels not different from those of wild-type neurons; whereas, addition of recombinant tPA fails to rescue the diminished outgrowth observed for medium–large tPA−/− neurons. This finding reflects a greater dependency by small DRG neurons for tPA in axonal elongation, which coincides with the earlier report from this laboratory showing that small neurons were the primary source of tPA in these DRG cultures (Hayden and Seeds 1996).
Figure 6. Mainly smaller neurons from tPA−/− mice show a failure for conditioning-injury induced neurite outgrowth on myelin. In a separate analysis for neurite elongation, random DRG neurite-bearing neurons were measured for cellular diameter and were separated into groups defined as small (< 25 μm) or medium-to-large (≥ 25 μm) subtypes of neurons. For each neuron type and condition, the longest neurite displayed was measured and these values were used to generate overall average lengths. Gray bars, conditioned wild-type neurons; black bars, conditioned tPA−/− neurons; striped bars, conditioned tPA−/− neurons with added rtPA (140 nM). Error bars represent standard errors, and asterisk indicates p < 0.01 for tPA−/− small neurons compared to wild-type small neurons.
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To begin investigating the mechanism(s) by which tPA can promote conditioning-induced neurite outgrowth in the presence of inhibitory myelin, we evaluated the importance of tPA action on the presence of its principal substrate plasminogen/plasmin. To test the potential role that this conversion might play in conditioning-induced outgrowth on myelin, injury-conditioned neurons were also generated from Plgn−/− mice. As shown in Fig. 7(a), conditioned Plgn−/− neurons were able to grow neurites that were visually comparable to those of conditioned wild-type neurons shown in Fig. 1(b). Quantitative analysis of neurite elongation demonstrated that conditioned Plgn−/− neurons grow neurites of lengths not different from corresponding wild-type neurons (194.3 ± 19.6 vs. 187.4 ± 12.4, respectively) (Fig. 7b); nor was any difference seen in neurite initiation. This finding points to a plasminogen/plasmin-independent role of tPA in conditioning-injury induced neurite outgrowth on myelin.
Figure 7. Neurons from Plgn−/− mice show robust conditioning-injury induced neurite outgrowth on myelin. (a) Photomicrograph of sensory neurons from Plgn−/− mice grown on myelin (bar: 50 μm). (b) Quantitative length analysis showed no difference in the conditioning response displayed by wild-type and Plgn−/− neurons. Error bars represent standard errors.
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