Neurotrophic factor gene delivery supports axonal regeneration after injury

A successful treatment for spinal cord injury (SCI) must include means to induce axonal regeneration and synaptogenesis. Though much research has demonstrated the effectiveness of neurotrophic factors (NFs) in supporting axonal regeneration, systemic delivery of doses sufficient to reach therapeutic concentrations and overcome their short half‐lives has caused adverse effects. Local expression of NFs would overcome these limitations. We tested whether local expression of NFs would induce axonal regeneration without adverse effects in two models of neural injury. In a chemical injury model the rat serotonergic system was lesioned with p‐chloroamphetamine. When an adenoviral vector carrying the gene for brain‐derived neurotrophic factor (BDNF) was injected into the denervated cortex BDNF expressed by the transfected cells induced serotonergic axon reinnervation only in area around the injection site. In a mechanical injury model the cortical spinal tract (CST) in rats was lesioned unilaterally at the level of the hindbrain. Neurotorphin‐3 (NT‐3) was expressed locally in the spinal cord either by direct injection of an adenoviral vector carrying the gene for NT‐3 or by retrograde delivery of the vector from the sciatic nerve. Axons were observed growing from the unlesioned CST across the midline to the denervated side. These data demonstrate that local expression of NFs will induce and support axonal regeneration in a circumscribed area after injury without adverse effects and suggest that a therapy for SCI based upon this strategy may include NF gene delivery. Acknowledgements: Supported by NIH grant NS35280 and Mission Connect of the TIRR Foundation.     

Rho-associated kinase (ROCK) inhibitor, Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurotrophins support neuronal survival and axonal regeneration after injury. To test whether local expression of Neurotrophin-3 (NT-3) would elicit axonal regeneration we lesioned the corticospinal tract (CST) at the level of the hindbrain and measured the number of axons that would grow from the unlesioned CST to the contralateral side where NT-3 was over expressed at the lumbar level of the spinal cord. An adenoviral vector that carried the rat NT-3 gene and the NGF signal peptide driven by the EF1α promoter (Adv.EF-NT-3) was used. This model enabled us to test the effects of NT-3 on axonal regeneration without confounding injury processes. Biotinylated dextran amine (BDA) was injected into the rat cortex on unlesioned side to mark CST axons 10 days postlesion. Adenoviral vectors (1 × 10⁹ pfu, Adv.EF-NT-3 or Adv.EF-LacZ) were delivered to lumbar spinal cord by retrograde transport from the sciatic nerve 4 days later. Histological examination 3 weeks later revealed that more BDA-labelled axons had grown from the unlesioned CST to the denervated side at the lumbar level. Morphometric measurements showed that a significantly larger number of BDA-labelled CST axons ( p  < 0.001) were present in the animals that were treated with Adv.EF-NT-3 than those treated with Adv.EF-LacZ. These data demonstrate that local expression of NT-3 will support axonal regeneration in the injured spinal cord without adverse effects and suggest that gene delivery of neurotrophins may be an effective strategy for nervous system repair after injury.
Acknowledgements:   Funded by NIH Grant NS35280 and by Mission Connect of the TIRR Foundation.     

Effects of retrograde gene transfer of brain-derived neurotrophic factor in the rostral spinal cord of a compression model in rat

Recovery after spinal cord injury (SCI) is rare in humans and experimental animals. Following SCI in adults, changes in gene expression and the regulation of these genes are associated with the pathological development of the injury. High levels of brain-derived neurotrophic factor (BDNF) in the injury area during the post-injury period contribute to enhanced neuroprotection and axonal regeneration. Intervention at the level of gene regulation has the potential to promote SCI repair. In this study, the injection of adenovirus-mediated BDNF in the lesion area (rostral spinal cord) up-regulated the expression of BDNF in the injury zone of a compression model in rat, thereby protecting neurons and enhancing behavioral function.     

Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in nonneuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neutrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.     

Neurotrophin expressions in neural stem cells grafted acutely to transected spinal cord of adult rats linked to functional improvement

It is well known that neural stem cells (NSC) could promote the repairment after spinal cord injury, but the underlying mechanism remains to be elucidated. This study showed that the transplantation of NSC significantly improved hindlimb locomotor functions in adult rats subjected to transection of the spinal cord. Biotin dextran amine tracing together with the stimulus experiment in motor sensory area showed that little CST regeneration existed and functional synaptic formation in the injury site. Immunocytochemistry and RT-PCR demonstrated the secretion of NGF, BDNF, and NT-3 by NSC in vitro and in vivo, respectively. However, only mRNA expression of BDNF and NT-3 but not NGF in injury segment following NSC transplantation was upregulated remarkably, while caspase-3, a crucial apoptosis gene, was downregulated simultaneously. These provided us a clue that the functional recovery was correlated with the regulation of BDNF, NT-3, and caspase-3 in spinal cord transected rats following NSC transplantation.     

Assessment of Glial Scar,Tissue Sparing,Behavioral Recovery and Axonal Regeneration following Acute Transplantation of Genetically Modified Human Umbilical Cord Blood Cells in a Rat Model of Spinal Cord Contusion

Objective and Methods

This study investigated the potential for protective effects of human umbilical cord blood mononuclear cells (UCB-MCs) genetically modified with the VEGF and GNDF genes on contusion spinal cord injury (SCI) in rats. An adenoviral vector was constructed for targeted delivery of VEGF and GDNF to UCB-MCs. Using a rat contusion SCI model we examined the efficacy of the construct on tissue sparing, glial scar severity, the extent of axonal regeneration, recovery of motor function, and analyzed the expression of the recombinant genes VEGF and GNDF in vitro and in vivo.

Results

Transplantation of UCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced tissue sparing, behavioral recovery and axonal regeneration comparing to the other constructs tested. The adenovirus encoding VEGF and GDNF for transduction of UCB-MCs was shown to be an effective and stable vehicle for these cells in vivo following the transplantation into the contused spinal cord.

Conclusion

Our results show that a gene delivery using UCB-MCs-expressing VEGF and GNDF genes improved both structural and functional parameters after SCI. Further histological and behavioral studies, especially at later time points, in animals with SCI after transplantation of genetically modified UCB-MCs (overexpressing VEGF and GDNF genes) will provide additional insight into therapeutic potential of such cells.     

Effects of Wnt5a overexpression in spinal cord injury

Accordingly to its known function in corticospinal tract (CST) developmental growth, previous reports have shown an inhibitory role of Wnt5a in CST regeneration after spinal cord injury (SCI). Interestingly, it has been subsequently demonstrated that Wnt5a also modulates the developmental growth of non-CST axons and that different Wnt5a receptors are expressed in neurons, oligodendrocytes, NG2+ glial precursors and reactive microglia/macrophages and astrocytes after SCI. However, the role of Wnt5a in the response of these cell types, in the regeneration of non-CST axons and in functional recovery after SCI is currently unknown. To evaluate this, rats were subjected to spinal cord contusion and injected with a lentiviral vector generated to overexpress Wnt5a. Histological analyses were performed in spinal cord sections processed for the visualization of myelin, oligodendrocytes, neurons, microglia/macrophages, astrocytes, NG2+ glial precursors and serotonergic axons. Motor and bladder function recovery were also assessed. Further advancing our knowledge on the role of Wnt5a in SCI, we found that, besides its previously reported functions, Wnt5a overexpression elicits a reduction on neuronal cell density, the accumulation of NG2+ glial precursors and the descending serotonergic innervation in the affected areas, along with impairment of motor and bladder function recovery after SCI.     

AAV-mediated inhibition of ULK1 promotes axonal regeneration in the central nervous system in vitro and in vivo

Axonal damage is an early step in traumatic and neurodegenerative disorders of the central nervous system (CNS). Damaged axons are not able to regenerate sufficiently in the adult mammalian CNS, leading to permanent neurological deficits. Recently, we showed that inhibition of the autophagic protein ULK1 promotes neuroprotection in different models of neurodegeneration. Moreover, we demonstrated previously that axonal protection improves regeneration of lesioned axons. However, whether axonal protection mediated by ULK1 inhibition could also improve axonal regeneration is unknown. Here, we used an adeno-associated viral (AAV) vector to express a dominant-negative form of ULK1 (AAV.ULK1.DN) and investigated its effects on axonal regeneration in the CNS. We show that AAV.ULK1.DN fosters axonal regeneration and enhances neurite outgrowth in vitro. In addition, AAV.ULK1.DN increases neuronal survival and enhances axonal regeneration after optic nerve lesion, and promotes long-term axonal protection after spinal cord injury (SCI) in vivo. Interestingly, AAV.ULK1.DN also increases serotonergic and dopaminergic axon sprouting after SCI. Mechanistically, AAV.ULK1.DN leads to increased ERK1 activation and reduced expression of RhoA and ROCK2. Our findings outline ULK1 as a key regulator of axonal degeneration and regeneration, and define ULK1 as a promising target to promote neuroprotection and regeneration in the CNS.Subject terms: Cell death in the nervous system, Neurodegeneration, Spinal cord injury     

Mimicking the neurotrophic factor profile of embryonic spinal cord controls the differentiation potential of spinal progenitors into neuronal cells

Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) mRNA before differentiation. BDNF gene expression significantly decreases with their differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic development, we applied BDNF or neurotrophin (NT)-3 exogenously in combination with the anti-CNTF antibody. The exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes, respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Sox11 promotes endogenous neurogenesis and locomotor recovery in mice spinal cord injury

We introduced a lentiviral vector containing the Sox11 gene into injured spinal cords of mice to evaluate the therapeutic potential of Sox11 in spinal cord injury. Sox11 markedly improved locomotor recovery after spinal cord injury and this recovery was accompanied by an up-regulation of Nestin/Doublecortin expression in the injured spinal cord. Sox11 was mainly located in endogenous neural stem cells lining the central canal and in newly-generated neurons in the spinal cord. In addition, Sox 11 significantly induced expressions of BDNF in the spinal cords of LV-Sox11-treated mice. We concluded that Sox11 induced activation of endogenous neural stem cells into neuronal determination and migration within the injured spinal cord. The resultant increase of BDNF at the injured site might form a distinct neurogenic niche which induces a final neuronal differentiation of these neural stem cells. Enhancing Sox11 expression to induce neurogenic differentiation of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after SCI in mammals.     

Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain-derived neurotrophic factor in motoneurons

Progesterone (PROG) provides neuroprotection to the injured central and peripheral nervous system. These effects may be due to regulation of myelin synthesis in glial cells and also to direct actions on neuronal function. Both types of cells express classical intracellular PROG receptors (PR), while neurons additionally express the PROG membrane-binding site called 25-Dx. In motoneurons from rats with spinal cord injury (SCI), PROG restores to normal the deficient levels of choline acetyl-transferase and of alpha3 subunit Na,K-ATPase mRNA, while levels of the growth associated protein GAP-43 mRNA are further stimulated. Recent studies suggest that neurotrophins are possible mediators of hormone action, and in agreement with this assumption, PROG treatment of rats with SCI increases the expression of brain-derived neurotrophic factor (BDNF) at both the mRNA and protein levels in ventral horn motoneurons. In situ hybridization (ISH) has shown that SCI reduces BDNF mRNA levels by 50% in spinal motoneurons, while PROG administration to injured rats (4mg/kg/day during 3 days, s.c.) elicits a three-fold increase in grain density. In addition to enhancement of mRNA levels, PROG increases BDNF immunoreactivity in perikaryon and cell processes of motoneurons of the lesioned spinal cord, and also prevents the lesion-induced chromatolytic degeneration of spinal cord motoneurons as determined by Nissl staining. Our findings strongly indicate that motoneurons of the spinal cord are targets of PROG, as confirmed by the expression of PR and the regulation of molecular parameters. PROG enhancement of endogenous neuronal BDNF could provide a trophic environment within the lesioned spinal cord and might be part of the PROG activated-pathways to provide neuroprotection. Thus, PROG treatment constitutes a new approach to sustain neuronal function after injury.     

Neurotrophic factors in combinatorial approaches for spinal cord regeneration

Axonal regeneration is inhibited by a plethora of different mechanisms in the adult central nervous system (CNS). While neurotrophic factors have been shown to stimulate axonal growth in numerous animal models of nervous system injury, a lack of suitable growth substrates, an insufficient activation of neuron-intrinsic regenerative programs, and extracellular inhibitors of regeneration limit the efficacy of neurotrophic factor delivery for anatomical and functional recovery after spinal cord injury. Thus, growth-stimulating factors will likely have to be combined with other treatment approaches to tap into the full potential of growth factor therapy for axonal regeneration. In addition, the temporal and spatial distribution of growth factors have to be tightly controlled to achieve biologically active concentrations, to allow for the chemotropic guidance of axons, and to prevent adverse effects related to the widespread distribution of neurotrophic factors. Here, we will review the rationale for combinatorial treatments in axonal regeneration and summarize some recent progress in promoting axonal regeneration in the injured CNS using such approaches.     

Effects of combining methylprednisolone with rolipram on functional recovery in adult rats following spinal cord injury

Methylprednisolone (MP) has been widely used as a standard therapeutic agent for the treatment of spinal cord injury (SCI). Because of its controversial beneficial effects, the combination of MP and other pharmacological agents aimed at enhancing functional recovery is desirable. The phosphodiesterase 4 (PDE4) inhibitor rolipram has been implicated in promotion of regeneration due to elevating cAMP. In the present study, we sought to determine the effects of MP and rolipram, administered in combination, after spinal cord injury (SCI) in adult rats. Here we show that in vitro administration of rolipram and MP significantly increased neuron survival and promoted neurite outgrowth of neurons on the inhibitory substrate CSPGs by upregulation of MMP-2 expression; in vivo administration of rolipram and MP inhibited CSPG expression and increase CSPG digestion after rat SCI. Rolipram and MP combining treatment promoted significant neuroprotection through reduced motoneuron death, minimized lesion cavity, and increased regeneration of lesioned corticospinal tract (CST) axons beyond the lesion site after SCI. Enhanced functional recovery was also observed. Overall, our study strongly suggested that the combination treatment of MP and rolipram may represent a promising strategy for clinically applicable pharmacological therapy for rapid initiation of neuroprotection after SCI.     

Integration and Long Distance Axonal Regeneration in the Central Nervous System from Transplanted Primitive Neural Stem Cells

Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.     

Low‐level laser therapy 810‐nm up‐regulates macrophage secretion of neurotrophic factors via PKA‐CREB and promotes neuronal axon regeneration in vitro

Macrophages play key roles in the secondary injury stage of spinal cord injury (SCI). M1 macrophages occupy the lesion area and secrete high levels of inflammatory factors that hinder lesion repair, and M2 macrophages can secrete neurotrophic factors and promote axonal regeneration. The regulation of macrophage secretion after SCI is critical for injury repair. Low‐level laser therapy (810‐nm) (LLLT) can boost functional rehabilitation in rats after SCI; however, the mechanisms remain unclear. To explore this issue, we established an in vitro model of low‐level laser irradiation of M1 macrophages, and the effects of LLLT on M1 macrophage polarization and neurotrophic factor secretion and the related mechanisms were investigated. The results showed that LLLT irradiation decreased the expression of M1 macrophage‐specific markers, and increased the expression of M2 macrophage‐specific markers. Through forward and reverse experiments, we verified that LLLT can promote the secretion of various neurotrophic factors by activating the PKA‐CREB pathway in macrophages and finally promote the regeneration of axons. Accordingly, LLLT may be an effective therapeutic approach for SCI with clinical application prospects.     

The Effects of Controlled Release of Neurotrophin-3 from PCLA Scaffolds on the Survival and Neuronal Differentiation of Transplanted Neural Stem Cells in a Rat Spinal Cord Injury Model

Neural stem cells (NSCs) have emerged as a potential source for cell replacement therapy following spinal cord injury (SCI). However, poor survival and low neuronal differentiation remain major obstacles to the use of NSCs. Biomaterials with neurotrophic factors are promising strategies for promoting the proliferation and differentiation of NSCs. Silk fibroin (SF) matrices were demonstrated to successfully deliver growth factors and preserve their potency. In this study, by incorporating NT-3 into a SF coating, we successfully developed NT-3-immobilized scaffolds (membranes and conduits). Sustained release of bioactive NT-3 from the conduits for up to 8 weeks was achieved. Cell viability was confirmed using live/dead staining after 14 days in culture. The efficacy of the immobilized NT-3 was confirmed by assessing NSC neuronal differentiation in vitro. NSC neuronal differentiation was 55.2±4.1% on the NT-3-immobilized membranes, which was significantly higher than that on the NT-3 free membrane. Furthermore, 8 weeks after the NSCs were seeded into conduits and implanted in rats with a transected SCI, the conduit+NT-3+NSCs group achieved higher NSC survival (75.8±15.1%) and neuronal differentiation (21.5±5.2%) compared with the conduit+NSCs group. The animals that received the conduit+NT-3+NSCs treatment also showed improved functional outcomes, as well as increased axonal regeneration. These results indicate the feasibility of fabricating NT-3-immobilized scaffolds using the adsorption of NT-3/SF coating method, as well as the potential of these scaffolds to induce SCI repair by promoting survival and neuronal differentiation of transplanted NSCs.     

Aquaporin-4 Mitigates Retrograde Degeneration of Rubrospinal Neurons by Facilitating Edema Clearance and Glial Scar Formation After Spinal Cord Injury in Mice

Atrophy of upper motor neurons hampers axonal regeneration and functional recovery following spinal cord injury (SCI). Apart from the severity of primary injury, a series of secondary pathological damages including spinal cord edema and glial scar formation affect the fate of injured upper motor neurons. The aquaporin-4 (AQP4) water channel plays a critical role in water homeostasis and migration of astrocytes in the central nervous system, probably offering a new therapeutic target for protecting against upper motor neuron degeneration after SCI. To test this hypothesis, we examined the effect of AQP4 deficiency on atrophy of rubrospinal neurons after unilateral rubrospinal tract transection at the fourth cervical level in mice. AQP4 gene knockout (AQP4^?/?) mice exhibited high extent of spinal cord edema at 72 h after lesion compared with wild-type littermates. AQP4^?/? mice showed impairments in astrocyte migration toward the transected site with a greater lesion volume at 1 week after surgery and glial scar formation with a larger cyst volume at 6 weeks. More severe atrophy and loss of axotomized rubrospinal neurons as well as axonal degeneration in the rubrospinal tract rostral to the lesion were observed in AQP4^?/? mice at 6 weeks after SCI. AQP4 expression was downregulated at the lesioned spinal segment at 3 days and 1 week after injury, but upregulated at 6 weeks. These results demonstrated that AQP4 not only mitigates spinal cord damage but also ameliorates retrograde degeneration of rubrospinal neurons by promoting edema clearance and glial scar formation after laceration SCI. This finding supports the notion that AQP4 may be a promising therapeutic target for SCI.