Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury

Background

The blood brain barrier (BBB) and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF) applied into the peripheral (PNS) and central nervous system (CNS) thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons.

Methodology/Principal Findings

The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG) neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions.

Conclusions/Significance

Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.     

Role of Thy-1 in in vivo and in vitro neural development and regeneration of dorsal root ganglionic neurons

We have examined the expression of Thy-1, an abundant glycosylphosphatidylinositol (GPI)-anchored glycoprotein, in dorsal root ganglia (DRG) and associated nerve fascicles, during postnatal development and following a nerve crush. The expression levels of Thy-1 in DRG neurons, dorsal roots, and central processes in spinal cord were rather low at postnatal day 2, and gradually increased as DRG neurons matured. During early development, the expression of Thy-1 within DRG neurons was low and equally distributed between plasma membrane and cytosol. With maturation, the staining intensities of Thy-1 in both the plasma membrane and the cytosol of DRG neurons became increased. We also studied Thy-1 expression in the regeneration of mature DRG neurons following the crush injury of sciatic nerve. Two days after the crush injury, Thy-1 expression dramatically decreased in the DRG neurons on the lesion side. Between 4 and 7 days after the injury, the expression of Thy-1 gradually increased and returned to a normal level 1 week after the sciatic nerve crush. The time course of the up-regulation of Thy-1 expression during regeneration matched that of the recovery of sensory functions, such as pain withdraw reflex, placing reflex, and the score of Basso-Beattie-Bresnahan Locomotor Rating Scale. Taken together, our results suggest that Thy-1 expression is developmentally regulated and is closely associated with the functional maturation of DRG neurons during both postnatal development and nerve regeneration. Furthermore, perturbation of Thy-1 function with anti-Thy-1 antibodies promoted neurite outgrowth from primary cultured DRG neurons, again confirming the inhibitory role of Thy-1 on neurite outgrowth.     

GAP-43 regulates NCAM-180-mediated neurite outgrowth

The neural cell adhesion molecule (NCAM), and the growth-associated protein (GAP-43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP-43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP-43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP-43. We demonstrate that phosphorylation of GAP-43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM-induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP-43, NCAM-induced neurite outgrowth requires functional association of NCAM-180/spectrin/GAP-43, whereas in the absence of GAP-43, the NCAM-140/non-receptor tyrosine kinase (Fyn)-associated signaling pathway is pivotal. Thus, expression of GAP-43 presumably acts as a functional switch for NCAM-180-induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP-43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM-180/spectrin-mediated modulation of the actin cytoskeleton, NCAM-140-mediated activation of Fyn, or both.     

Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.

Regeneration is abortive following adult mammalian CNS injury. We have investigated whether increasing the intrinsic growth state of primary sensory neurons by a conditioning peripheral nerve lesion increases regrowth of their central axons. After dorsal column lesions, all fibers stop at the injury site. Animals with a peripheral axotomy concomitant with the central lesion show axonal growth into the lesion but not into the spinal cord above the lesion. A preconditioning lesion 1 or 2 weeks prior to the dorsal column injury results in growth into the spinal cord above the lesion. In vitro, the growth capacity of DRG neurite is also increased following preconditioning lesions. The intrinsic growth state of injured neurons is, therefore, a key determinant for central regeneration.     

Alterations in growth-associated protein (GAP-43) expression in lower urinary tract pathways following chronic spinal cord injury

Alterations in the expression of growth-associated protein 43 (GAP-43) were examined in lower urinary tract micturition reflex pathways 6 or 8 weeks following complete spinal cord transection (approximately T9). In control animals, expression of GAP-43 was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the corticospinal tract; (3) the dorsal horn; and (4) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (5) in the lateral collateral pathway of Lissauer in L6-S1 spinal segments. Densitometry analysis has demonstrated significant increases (p < or =0.001; 1.3-6.4-fold increase) in GAP-43-immunoreactivity (IR) in these regions of the rostral lumbar (L1-L2) and caudal lumbosacral (L6-S1) spinal cord 6 weeks following spinal cord injury. Changes in GAP-43-IR were restricted to the L1-L2 and L6-S1 segments that are involved in lower urinary tract reflexes. Changes in GAP-43-IR were not observed at the L5 segmental level except for an increase in GAP-43-IR in the superficial, dorsal horn at 6 weeks post-injury. In all segments examined, GAP-43-IR was decreased (2-5-fold) in the corticospinal tract (dorsal division) 6 and 8 weeks following spinal cord injury. Eight weeks following spinal cord injury, changes in GAP-43-IR had returned to control levels except for the persistence of increased GAP-43-IR in the region of the sacral parasympathetic nucleus and the lateral collateral pathway in the S1 spinal segment. Alterations in GAP-43-IR following chronic spinal cord injury may suggest a reorganization of bladder afferent projections and spinal elements involved in urinary bladder reflexes consistent with alterations in urinary bladder function (hyperreflexia) observed in animals following spinal cord injury above the lumbosacral spinal cord.     

Alterations in growth-associated protein (GAP-43) expression in lower urinary tract pathways following chronic spinal cord injury

Alterations in the expression of growth-associated protein 43 (GAP-43) were examined in lower urinary tract micturition reflex pathways 6 or 8 weeks following complete spinal cord transection (~ T9). In control animals, expression of GAP-43 was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the corticospinal tract; (3) the dorsal horn; and (4) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (5) in the lateral collateral pathway of Lissauer in L6-S1 spinal segments. Densitometry analysis has demonstrated significant increases (p 0.001; 1.3-6.4-fold increase) in GAP-43-immunoreactivity (IR) in these regions of the rostral lumbar (L1-L2) and caudal lumbosacral (L6-S1) spinal cord 6 weeks following spinal cord injury. Changes in GAP-43-IR were restricted to the L1-L2 and L6-S1 segments that are involved in lower urinary tract reflexes. Changes in GAP-43-IR were not observed at the L5 segmental level except for an increase in GAP-43-IR in the superficial, dorsal horn at 6 weeks post-injury. In all segments examined, GAP-43-IR was decreased (2-5-fold) in the corticospinal tract (dorsal division) 6 and 8 weeks following spinal cord injury. Eight weeks following spinal cord injury, changes in GAP-43-IR had returned to control levels except for the persistence of increased GAP-43-IR in the region of the sacral parasympathetic nucleus and the lateral collateral pathway in the S1 spinal segment. Alterations in GAP-43-IR following chronic spinal cord injury may suggest a reorganization of bladder afferent projections and spinal elements involved in urinary bladder reflexes consistent with alterations in urinary bladder function (hyperreflexia) observed in animals following spinal cord injury above the lumbosacral spinal cord.     

Absence of persistent spreading, branching, and adhesion in GAP-43- depleted growth cones

The growth-associated protein GAP-43 is a major protein kinase C substrate of growth cones and developing nerve terminals. In the growth cone, it accumulates near the plasma membrane, where it associates with the cortical cytoskeleton and membranes. The role of GAP-43 in neurite outgrowth is not yet clear, but recent findings suggest that it may be a crucial competence factor in this process. To define the role of GAP- 43 in growth cone activity, we have analyzed neurite outgrowth and growth cone activity in primary sensory neurons depleted of GAP-43 by a specific antisense oligonucleotide procedure. Under optimal culture conditions, but in the absence of GAP-43, growth cones adhered poorly, displayed highly dynamic but unstable lamellar extensions, and were strikingly devoid of local f-actin concentrations. Upon stimulation, they failed to produce NGF-induced spreading or insulin-like growth factor-1-induced branching, whereas growth factor-induced phosphotyrosine immunoreactivity and acceleration of neurite elongation were not impaired. Unlike their GAP-43-expressing counterparts, they readily retracted when exposed to inhibitory central nervous system myelin-derived liposomes. Frequency and extent of induced retraction were attenuated by NGF. Our results indicate that GAP-43 can promote f- actin accumulation, evoked morphogenic activity, and resistance to retraction of the growth cone, suggesting that it may promote regulated neurite outgrowth during development and regeneration.     

Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons

Peripheral nerve injury results in the increased synthesis and axonal trasnport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine wheter the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se. © 1993 John Wiley & Sons, Inc.     

Increased expression of GAP-43 in small sensory neurons after stimulation by NGF indicative of neuroregeneration in capsaicin-treated rats.

Intraplantar injections of human recombinant nerve growth factor (rhNGF-beta) into the hind paw of capsaicin-treated adult rats are known to lead to a recovery of depleted peptide transmitter substances, to the immunohistochemical reappearance of peptidergic innervation in the skin and in the dorsal horn of the spinal cord, as well as to a recovery of the function of capsaicin-lesioned neurons. In the present study a marker peptide for neuronal regeneration and outgrowth, growth associated protein 43 (GAP-43), was investigated in lumbar dorsal root ganglia (DRGs) and in the hindpaw skin, in order to differentiate which population of the sensory neurons responds with a neuroregenerative behaviour. In situ hybridization histochemistry (ISH) revealed that at day 8 after the capsaicin treatment GAP-43 expression was significantly increased in small DRG cells as compared to control animals, and treatment with NGF in capsaicinized rats lead to an even more pronounced increase of GAP-43 expression in the small-sized cell population. Intraepidermal labelling of GAP-43 peptide was observed in the skin of control animals, but was markedly reduced in the animals that were treated with capsaicin alone. However, intraepidermal GAP-43 immunoreactive (GAP-43-IR) fibres nearly fully recovered in the capsaicin + NGF-treated group. These results indicate that the population of small DRG cells shows spontaneous regenerative activity after a capsaicin lesion which does not lead to a successful recovery of nerve terminals in the skin. Only after an additional NGF treatment small DRG cells show an even stronger regenerative response which now also involves structural reorganization of neuron membranes and axogenesis in the periphery.     

Netrin-1 signaling for sensory axons

During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a ‘waiting period’ of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.     

The Nogo receptor, its ligands and axonal regeneration in the spinal cord; A review

At least three proteins present in CNS myelin, Nogo, MAG and OMgp are capable of causing growth cone collapse and inhibiting neurite outgrowth in vitro. Surprisingly, Nogo and OMgp are also strongly expressed by many neurons (including neocortical projection cells). Nogo expression is increased by some cells at the borders of CNS lesion sites and by cells in injured peripheral nerves, but Nogo and CNS myelin are largely absent from spinal cord injury sites, which are none the less strongly inhibitory to axonal regeneration. Nogo is found on growing axons during development, suggesting possible functions for neuronal Nogo in axon guidance. Although Nogo, MAG and OMgp lack sequence homologies, they all bind to the Nogo receptor (NgR), a GPI-linked cell surface molecule which, in turn, binds p75 to activate RhoA. NgR is strongly expressed by cerebral cortical neurons but many other neurons express NgR weakly or not at all. Some neurons, such as DRG cells, respond to Nogo and CNS myelin in vitro although they express little or no NgR in vivo which, with other data, indicates that other receptors are available for NgR ligands. NgR expression is unaffected by injury to the nervous system, and there is no clear correlation between NgR expression by neurons and lack of regenerative ability. In the injured spinal cord, interactions between NgR and its ligands are most likely to be important for limiting regeneration of corticospinal and some other descending tracts; other receptors may be more important for ascending tracts. Antibodies to Nogo, mainly the poorly-characterised IN-1 or its derivatives, have been shown to enhance recovery from partial transections of the spinal cord. They induce considerable plasticity from the axons of corticospinal neurons, including sprouting across the midline and, to a limited extent, regeneration around the lesion. Regeneration of corticospinal axons induced by Nogo antibodies has not yet been demonstrated after complete transections or contusion injuries of the spinal cord. It is not clear whether antibodies against Nogo act on oligodendrocytes/myelin or by binding to neuronal Nogo, or whether they can stimulate regeneration of ascending axons in the spinal cord, most of which express little or no NgR. Despite these uncertainties, however, NgR and its ligands offer important new targets for enhancing plasticity and regeneration in the nervous system.     

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.     

microRNA-222 Targeting PTEN Promotes Neurite Outgrowth from Adult Dorsal Root Ganglion Neurons following Sciatic Nerve Transection

Dorsal root ganglia (DRG) neurons spontaneously undergo neurite growth after nerve injury. MicroRNAs (miRNAs), as small, non-coding RNAs, negatively regulate gene expression in a variety of biological processes. The roles of miRNAs in the regulation of responses of DRG neurons to injury stimuli, however, are not fully understood. Here, microarray analysis was performed to profile the miRNAs in L4-L6 DRGs following rat sciatic nerve transection. The 26 known miRNAs were differentially expressed at 0, 1, 4, 7, 14 d post injury, and the potential targets of the miRNAs were involved in nerve regeneration, as analyzed by bioinformatics. Among the 26 miRNAs, microRNA-222 (miR-222) was our research focus because its increased expression promoted neurite outgrowth while it silencing by miR-222 inhibitor reduced neurite outgrowth. Knockdown experiments confirmed that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major inhibitor of nerve regeneration, was a direct target of miR-222 in DRG neurons. In addition, we found that miR-222 might regulate the phosphorylation of cAMP response element binding protein (CREB) through PTEN, and c-Jun activation might enhance the miR-222 expression. Collectively, our data suggest that miR-222 could regulate neurite outgrowth from DRG neurons by targeting PTEN.     

Dose-dependent expression of neuronal injury markers during experimental osteoarthritis induced by monoiodoacetate in the rat

ABSTRACT: BACKGROUND: It was recently reported that the mono-iodoacetate (MIA) experimental model of osteoarthritis (OA) courses with changes of neurons innervating the affected joints that are commonly interpreted as a neuronal response to axonal injury. To better characterize these changes, we evaluated the expression of two markers of neuronal damage, ATF-3 and NPY, and the growth associated protein GAP-43, in primary afferent neurons of OA animals injected with three different doses of MIA (0.3, 1 or 2 mg). Measurements were performed at days 3, 7, 14, 21 and 31 post-MIA injection. RESULTS: OA animals showed the characteristic histopathological changes of the joints and the accompanying nociceptive behaviour, evaluated by the Knee-Bed and CatWalk tests. An increase of ATF-3 expression was detected in the DRG of OA animals as early as 3 days after the injection of 1 or 2 mg of MIA and 7 days after the injection of 0.3 mg. NPY expression was increased in animals injected with 1 or 2 mg of MIA, at day 3 or in all time-points, respectively. From day 7 onwards there was a massive increase of GAP-43 expression in ATF-3 cells. CONCLUSIONS: The expression of the neuronal injury markers ATF-3 and NPY as well as an up-regulation of GAP-43 expression, indicative of peripheral fibre regeneration, suggests that axonal injury and a regeneration response may be happening in this model of OA. This opens new perspectives in the unravelling of the physiopathology of the human disease.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.