Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.     

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     

Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals.

In adult mammals, the severing of the optic nerve near the eye is followed by a loss of retinal ganglion cells (RGCs) and a failure of axons to regrow into the brain. Experimental manipulations of the non-neuronal environment of injured RGCs enhance neuronal survival and make possible a lengthy axonal regeneration that restores functional connections with the superior colliculus. These effects suggest that injured nerve cells in the mature central nervous system (CNS) are strongly influenced by interactions with components of their immediate environment as well as their targets. Under these conditions, injured CNS neurons can express capacities for growth and differentiation that resemble those of normally developing neurons. An understanding of this regeneration in the context of the cellular and molecular events that influence the interactions of axonal growth cones with their non-neuronal substrates and neuronal targets should help in the further elucidation of the capacities of neuronal systems to recover from injury.     

Neuronal STAT3 activation is essential for CNTF- and inflammatory stimulation-induced CNS axon regeneration

CNS neurons, such as retinal ganglion cells (RGCs), do not normally regenerate injured axons, but instead undergo apoptotic cell death. Regenerative failure is due to inhibitory factors in the myelin and forming glial scar as well as due to an insufficient intrinsic capability of mature neurons to regrow axons. Nevertheless, RGCs can be transformed into an active regenerative state upon inflammatory stimulation (IS) in the inner eye, for instance by lens injury, enabling these RGCs to survive axotomy and to regenerate axons into the lesioned optic nerve. The beneficial effects of IS are mediated by various factors, including CNTF, LIF and IL-6. Consistently, IS activates various signaling pathways, such as JAK/STAT3 and PI3K/AKT/mTOR, in several retinal cell types. Using a conditional knockdown approach to specifically delete STAT3 in adult RGCs, we investigated the role of STAT3 in IS-induced neuroprotection and axon regeneration. Conditional STAT3 knockdown in RGCs did not affect the survival of RGCs after optic nerve injury compared with controls, but significantly reduced the neuroprotective effects of IS. STAT3 depletion significantly compromised CNTF-stimulated neurite growth in culture and IS-induced transformation of RGCs into an active regenerative state in vivo. As a consequence, IS-mediated axonal regeneration into the injured optic nerve was almost completely abolished in mice with STAT3 depleted in RGCs. In conclusion, STAT3 activation in RGCs is involved in neuroprotection and is a necessary prerequisite for optic nerve regeneration upon IS.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

Nogo and axon regeneration

Nogo-A is one of several neurite growth inhibitory components present in oligodendrocytes and CNS myelin membranes. Nogo has a crucial role in restricting axonal regeneration and compensatory fibre growth in the injured adult mammalian CNS. Recent studies have shown that in vivo applications of Nogo neutralizing antibodies, peptides blocking the Nogo receptor subunit NgR, or blockers of the postreceptor components Rho-A and ROCK induce long-distance axonal regeneration and compensatory sprouting, accompanied by an impressive enhancement of functional recovery, in the rat and mouse spinal cord.     

Postinjury Changes in Platelet-Derived Growth Factor-Like Activity in Fish and Rat Optic Nerves

The poor regenerative ability of the CNS of mammals has been attributed, at least in part, to the presence of mature oligodendrocytes, which have been shown to inhibit axonal growth. Proliferation of oligodendrocyte progenitor cells in the rat optic nerve during development, and thereby the timing of oligodendrocyte differentiation, has been shown to depend on a factor derived from type 1 astrocytes, later characterized as platelet-derived growth factor (PDGF). In the present study we examine whether injury to the optic nerve induces changes in the levels of PDGF in spontaneously regenerating systems, compared with nonregenerating systems. Soluble substances, derived from nonneuronal cells surrounding injured fish and rat optic nerves, were prepared and examined for the presence of PDGF immunoreactivity and biological mitogenic activity on PDGF-responsive cells. The results suggest that PDGF-like mitogenic activity and immunoreactivity are present in both fish and rat optic nerves. However, in the rat optic nerve PDGF levels increased after axonal injury, whereas in the fish optic nerve injury was accompanied by an apparent decrease in PDGF-like levels. The results are discussed with respect to the possible role of PDGF in regeneration.     

Macrophages can modify the nonpermissive nature of the adult mammalian central nervous system

Although astrocytic gliosis has been linked to failure of axonal regeneration in the adult mammalian CNS, its role is not fully established. We used an in vitro assay to investigate the role of reactive astrocytes and macrophages in influencing axonal growth in the lesioned adult rat optic nerve. Soon after optic nerve transection, the nonpermissive nature of the optic nerve is altered to a permissive state near the lesion. This may account for injury-induced axonal sprouting and may contribute to the failure of these sprouts to elongate beyond the site of the lesion in vivo. We provide evidence that this lesion-induced change in the axonal growth-promoting properties of the CNS near the lesion may be produced by mononuclear phagocytes. In addition, several months after optic nerve transection, the degenerated nerves, which consist mainly of astrocytes and lack myelin, i.e., astrocytic "scar" tissue, are a good substrate for neurite growth. Taken together, these results suggest that in this in vitro system, substantial inhibitory effects are not associated with regions of astrocytic gliosis and that the nonpermissive nature of the CNS white matter can be modified by macrophages.     

Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and axonal regeneration in vitro and in vivo

Hepatocyte growth factor (HGF) is known to promote the survival and foster neuritic outgrowth of different subpopulations of CNS neurons during development. Together with its corresponding receptor c-mesenchymal-epithelial transition factor (Met), it is expressed in the developing and the adult murine, rat and human CNS. We have studied the role of HGF in paradigms of retinal ganglion cell (RGC) regeneration and cell death in vitro and in vivo. After application of recombinant HGF in vitro, survival of serum-deprived RGC-5 cells and of growth factor-deprived primary RGC was significantly increased. This was shown to be correlated to the phosphorylation of c-Met and subsequent activation of serine/threonine protein kinase Akt and MAPK downstream signalling pathways involved in neuronal survival. Furthermore, neurite outgrowth of primary RGC was stimulated by HGF. In vivo, c-Met expression in RGC was up-regulated after optic nerve axotomy lesion. Here, treatment with HGF significantly improved survival of axotomized RGC and enhanced axonal regeneration after optic nerve crush. Our data demonstrates that exogenously applied HGF has a neuroprotective and regeneration-promoting function for lesioned CNS neurons. We provide strong evidence that HGF may represent a trophic factor for adult CNS neurons, which may play a role as therapeutic target in the treatment of neurotraumatic and neurodegenerative CNS disorders.     

Traumatology of the optic nerve and contribution of crystallins to axonal regeneration

Within a few decades, the repair of long neuronal pathways such as spinal cord tracts, the optic nerve or intracerebral tracts has gone from being strongly contested to being recognized as a potential clinical challenge. Cut axonal stumps within the optic nerve were originally thought to retract and become irreversibly necrotic within the injury zone. Optic nerve astrocytes were assumed to form a gliotic scar and remodelling of the extracellular matrix to result in a forbidden environment for re-growth of axons. Retrograde signals to the ganglion cell bodies were considered to prevent anabolism, thus also initiating apoptotic death and gliotic repair within the retina. However, increasing evidence suggests the reversibility of these regressive processes, as shown by the analysis of molecular events at the site of injury and within ganglion cells. We review optic nerve repair from the perspective of the proximal axon stump being a major player in determining the successful formation of a growth cone. The axonal stump and consequently the prospective growth cone, communicates with astrocytes, microglial cells and the extracellular matrix via a panoply of molecular tools. We initially highlight these aspects on the basis of recent data from numerous laboratories. Then, we examine the mechanisms by which an injury-induced growth cone can sense its surroundings within the area distal to the injury. Based on requirements for successful axonal elongation within the optic nerve, we explore the models employed to instigate successful growth cone formation by ganglion cell stimulation and optic nerve remodelling, which in turn accelerate growth. Ultimately, with regard to the proteomics of regenerating retinal tissue, we discuss the discovery of isoforms of crystallins, with crystallin beta-b2 (crybb2) being clearly upregulated in the regenerating retina. Crystallins are produced and used to promote the elongation of growth cones. In vivo and in vitro, crystallins beta and gamma additionally promote the growth of axons by enhancing the production of ciliary neurotrophic factor (CNTF), indicating that they also act on astrocytes to promote axonal regrowth synergistically. These are the first data showing that axonal regeneration is related to crybb2 movement within neurons and to additional stimulation of CNTF. We demonstrate that neuronal crystallins constitute a novel class of neurite-promoting factors that probably operate through an autocrine and paracrine mechanism and that they can be used in neurodegenerative diseases. Thus, the post-injury fate of neurons cannot be seen merely as inevitable but, instead, must be regarded as a challenge to shape conditions for initiating growth cone formation to repair the damaged optic nerve.     

Stimulating axonal regeneration of mature retinal ganglion cells and overcoming inhibitory signaling

Like other neurons of the central nervous system (CNS), retinal ganglion cells (RGCs) are normally unable to regenerate injured axons and instead undergo apoptotic cell death. This regenerative failure leads to lifelong visual deficits after optic nerve damage and is partially attributable to factors located in the inhibitory environment of the forming glial scar and myelin as well as to an insufficient intrinsic ability for axonal regrowth. In addition to its ophthalmological relevance, the optic nerve has long been used as a favorable paradigm for studying regenerative failure in the CNS as a whole. Findings over the last 15 years have shown that, under certain circumstances, mature RGCs can be transformed into an active regenerative state enabling these neurons to survive axotomy and to regenerate axons in the optic nerve. Moreover, combinatorial treatments overcoming the inhibitory environment of the glial scar and optic nerve myelin, together with approaches activating the intrinsic growth program, can further enhance the amount of regeneration in vivo. These findings are encouraging and open the possibility that clinically meaningful regenerationmay become achievable in the future.     

Long-term gene therapy causes transgene-specific changes in the morphology of regenerating retinal ganglion cells

Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5-8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.     

Attenuation of Axonal Degeneration by Calcium Channel Inhibitors Improves Retinal Ganglion Cell Survival and Regeneration After Optic Nerve Crush

Axonal degeneration is one of the initial steps in many traumatic and neurodegenerative central nervous system (CNS) disorders and thus a promising therapeutic target. A focal axonal lesion is followed by acute axonal degeneration (AAD) of both adjacent axon parts, before proximal and distal parts follow different degenerative fates at later time points. Blocking calcium influx by calcium channel inhibitors was previously shown to attenuate AAD after optic nerve crush (ONC). However, it remains unclear whether the attenuation of AAD also promotes consecutive axonal regeneration. Here, we used a rat ONC model to study the effects of calcium channel inhibitors on axonal degeneration, retinal ganglion cell (RGC) survival, and axonal regeneration, as well as the molecular mechanisms involved. Application of calcium channel inhibitors attenuated AAD after ONC and preserved axonal integrity as visualized by live imaging of optic nerve axons. Consecutively, this resulted in improved survival of RGCs and improved axonal regeneration at 28 days after ONC. We show further that calcium channel inhibition attenuated lesion-induced calpain activation in the proximity of the crush and inhibited the activation of the c-Jun N-terminal kinase pathway. Pro-survival signaling via Akt in the retina was also increased. Our data thus show that attenuation of AAD improves consecutive neuronal survival and axonal regeneration and that calcium channel inhibitors could be valuable tools for therapeutic interventions in traumatic and degenerative CNS disorders.     

CD82 protects against glaucomatous axonal transport deficits via mTORC1 activation in mice

Glaucoma is a leading cause of irreversible blindness worldwide and is characterized by progressive optic nerve degeneration and retinal ganglion cell loss. Axonal transport deficits have been demonstrated to be the earliest crucial pathophysiological changes underlying axonal degeneration in glaucoma. Here, we explored the role of the tetraspanin superfamily member CD82 in an acute ocular hypertension model. We found a transient downregulation of CD82 after acute IOP elevation, with parallel emergence of axonal transport deficits. The overexpression of CD82 with an AAV2/9 vector in the mouse retina improved optic nerve axonal transport and ameliorated subsequent axon degeneration. Moreover, the CD82 overexpression stimulated optic nerve regeneration and restored vision in a mouse optic nerve crush model. CD82 exerted a protective effect through the upregulation of TRAF2, which is an E3 ubiquitin ligase, and activated mTORC1 through K63-linked ubiquitylation and intracellular repositioning of Raptor. Therefore, our study offers deeper insight into the tetraspanin superfamily and demonstrates a potential neuroprotective strategy in glaucoma treatment.Subject terms: Molecular neuroscience, Neurodegeneration     

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre^+/−xRtn4/Nogo-A^flox/flox) and neuron-specific (Thy1-Cre^tg+xRtn4^flox/flox) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4^flox/flox animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.In the adult mammalian central nervous system (CNS), axons have a very limited capacity to regenerate after traumatic injury. This lack of axonal regeneration is thought to be mainly due to the presence of growth-inhibiting molecules in the injured CNS environment^{1, 2} and due to the low intrinsic growth capacity of mature neurons.³Nogo-A is a well-studied inhibitory protein for axonal growth, plasticity and regeneration after CNS injury.^{4, 5} Nogo-A is predominantly expressed in oligodendrocytes in the adult CNS, where it is thought to stabilize the neuronal circuits in healthy conditions and to inhibit neurite growth and plasticity after lesion.² Neutralizing Nogo-A by function-blocking antibodies or genetic knockout (KO) has been shown to improve axonal sprouting and regeneration in the injured spinal cord and brain.^{6, 7, 8, 9, 10, 11}In addition to oligodendrocytes and myelin, Nogo-A is expressed in growing and immature neurons, as well as in some adult neurons.^{12, 13} Neurons express Nogo-A receptors such as the Nogo-66 receptor 1 (NgR1)¹⁴ and the Nogo-A-Δ20-specific sphingosine 1-phosphate receptor 2 (S1PR2).¹⁵ They can co-express them along with Nogo-A,¹³ an observation that raises the possibility of cis-interactions between the ligand and its receptors within or at the cell surface of the same cell. This mechanism has previously been described for axonal guidance molecules such as Ephrins and Semaphorins, and could have a major role in the neuronal response to extracellular growth inhibitors during development.^{16, 17}In the adult CNS, the expression of neuronal Nogo-A remains elevated mainly in plastic regions such as in the hippocampus, olfactory bulb or neocortex, and in the dorsal root ganglia.¹² Nogo-A and NgR1 were shown to regulate synaptic plasticity, for example, long-term potentiation in the hippocampus and in the sensory-motor cortex,^{18, 19, 20, 21, 22} whereas the effects of neuronal Nogo-A after injury are not yet well understood. During development, neuronal Nogo-A influences neuronal migration,^{23, 24} survival,^{25, 26} cell spreading and neurite growth.^{27, 28} In injured adult retinal ganglion cells (RGCs), silencing neuronal Nogo-A resulted in a marked reduction of regenerative sprouting and decreased expression of growth-associated molecules.²⁹ Furthermore, in the optic nerve, axonal regeneration was not improved in conventional Nogo-A KO animals, in which both glial and neuronal Nogo-A were deleted.²⁹ The present study therefore aimed to investigate whether glial and neuronal Nogo-A differently influence axonal growth in vivo using cell type-specific Nogo-A KO mouse lines and adeno-associated virus (AAV)-mediated recombination of the Nogo-A gene in neurons. The results show that significantly more axons grew through the lesion site in the oligodendrocyte-specific Nogo-A KO mice. In contrast, neuron-specific ablation of Nogo-A in RGCs reduced the number of regenerating axons after optic nerve crush injury (ONC). In summary, we show that inactivating Nogo-A specifically in oligodendrocytes appears to be the most successful strategy to promote axonal regeneration in the adult optic nerve.     

Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons

Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and 15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity in DRG neurons in explant culture, disruption of cell–cell interactions, rather than the culture itself, may be an inducible factor for localization of CNTF in the neurons.     

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult.

Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-beta and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L-Cre; Stat3(flox/KO) mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.