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.     

Imaging of rat optic nerve axons in vivo

In this protocol, we describe the imaging of single axons in the rat optic nerve in vivo. Axons are labeled through the intravitreal injection of adeno-associated viral vectors (AAVs) expressing a fluorophore (duration of the procedure ～1 h). Two weeks after intravitreal injection, the optic nerve is surgically exposed (duration ～1 h) and labeled axons are imaged with an epifluorescence microscope either for up to 8 h or repetitively on the following days. Additionally, intravitreal injection of calcium-sensitive dyes allows for imaging of intra-axonal calcium kinetics. This procedure enables the analysis of the morphological changes of degenerating axons in the optic nerve in different lesion paradigms, such as optic nerve crush, axotomy or pin lesion. Furthermore, the effects of pharmacological manipulations on axonal stability and axonal calcium kinetics in axons of the central nervous system can be studied in vivo.     

Axonal degeneration as a therapeutic target in the CNS

Degeneration of the axon is an important step in the pathomechanism of traumatic, inflammatory and degenerative neurological diseases. Increasing evidence suggests that axonal degeneration occurs early in the course of these diseases and therefore represents a promising target for future therapeutic strategies. We review the evidence for axonal destruction from pathological findings and animal models with particular emphasis on neurodegenerative and neurotraumatic disorders. We discuss the basic morphological and temporal modalities of axonal degeneration (acute, chronic and focal axonal degeneration and Wallerian degeneration). Based on the mechanistic concepts, we then delineate in detail the major molecular mechanisms that underlie the degenerative cascade, such as calcium influx, axonal transport, protein aggregation and autophagy. We finally concentrate on putative therapeutic targets based on the mechanistic prerequisites.     

Exogenous laminin induces regenerative changes in traumatized sciatic and optic nerve

Laminin is an extracellular matrix component which can promote neuritic elongation in vitro and has been implicated in the promotion of nerve regeneration in vivo. The present study was undertaken to determine if implantation of Elvax pellets containing exogenous laminin distal to site of lesion could promote regenerative responses in vivo in the adult rat peripheral (sciatic) and central (optic) nerve. In peripheral nerve preparations, Elvax pellets containing laminin or collagen were assessed for their ability to "lure" transected axons into 5-mm-long silicone tubes. In optic nerve studies, laminin pellets were inserted distal to site of nerve crush, and the extent of axonal elongation 2.5 mm to the injury site was assessed. Laminin-containing pellets appeared to support appreciable axonal elongation in both systems. This effect was dose-dependent and not exerted by collagen pellets, substrate-free pellets, or pellets containing irradiated laminin. Collagen IV had some beneficial effect in peripheral, but not central, nerve preparations.     

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     

Changes in intra-axonal calcium distribution following nerve crush

We used the oxalate-pyroantimonate method to demonstrate the ultrastructural distribution of calcium within rat sciatic nerve 4 h after a crush injury. In normal nerve there are discrete gradients of axoplasmic calcium precipitate with the amount of precipitate decreasing in the axoplasm beneath the Schmidt Lantermann clefts and in the paranodal regions at the node of Ranvier. Near the crush site a marked increase in endoneurial and intra-axonal calcium precipitate correlated with morphologic evidence of axonal degeneration. More distant from the crush site, both in the distal segment destined to degenerate and in the proximal segment destined to regenerate, the most prominent finding was a loss of the normal gradient of precipitate beneath the Schmidt Lantermann clefts. The calcium influx at the crush site corresponds to the known role of calcium in triggering degeneration. The alterations in the distal axon may be an early stage leading to degeneration. Alteration in calcium distribution in the proximal nerve stump may play a role in the regulation of the response to injury.     

Axonal protection by Nmnat3 overexpression with involvement of autophagy in optic nerve degeneration

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the crucial role of nicotinamide mononucleotide adenylyltransferase (Nmnat) 1, 2, and 3 in axonal protection. In this study, Nmnat3 immunoreactivity was observed inside axons in the optic nerve. Overexpression of Nmnat3 exerts axonal protection against tumor necrosis factor-induced and intraocular pressure (IOP) elevation-induced optic nerve degeneration. Immunoblot analysis showed that both p62 and microtubule-associated protein light chain 3 (LC3)-II were upregulated in the optic nerve after IOP elevation. Nmnat3 transfection decreased p62 and increased LC3-II in the optic nerve both with and without experimental glaucoma. Electron microscopy showed the existence of autophagic vacuoles in optic nerve axons in the glaucoma, glaucoma+Nmnat3 transfection, and glaucoma+rapamycin groups, although preserved myelin and microtubule structures were noted in the glaucoma+Nmnat3 transfection and glaucoma+rapamycin groups. The axonal-protective effect of Nmnat3 was inhibited by 3-methyladenine, whereas rapamycin exerted axonal protection after IOP elevation. We found that p62 was present in the mitochondria and confirmed substantial colocalization of mitochondrial Nmnat3 and p62 in starved retinal ganglion cell (RGC)-5 cells. Nmnat3 transfection decreased p62 and increased autophagic flux in RGC-5 cells. These results suggest that the axonal-protective effect of Nmnat3 may be involved in autophagy machinery, and that modulation of Nmnat3 and autophagy may lead to potential strategies against degenerative optic nerve disease.     

Axonal damage, autophagy and neuronal survival

In recent years autophagy modulation has been shown to reduce or increase neuronal cell death in several models of neurodegeneration. How autophagy exerts these dual effects is currently unknown. Here we review recent evidence from our laboratory demonstrating that autophagy can protect the cell soma after axonal traumatic injury. Damage in the optic nerve induces retinal ganglion cell (RGC) death in glaucoma and other retinal diseases and is often modeled by axotomy of the optic nerve in laboratory animals. Using this well-known model of RGC degeneration we show that autophagy is strongly upregulated following the insult and before cell death. Enhancement of autophagy by pharmacological treatment with rapamycin decreases the number of degenerating neurons. Conversely, axotomy in Atg4B (-/-) mice increases the number of dying cells in the retinal ganglion cell layer. Similar findings were observed in Atg5 (flox/flox) mice following specific downregulation of the autophagy regulator ATG5 in RGCs, by intravitreal injection of a cre-expressing vector. Taken together, these findings point to a cytoprotective role of autophagy following axonal damage in vivo.     

Taurine and its Trophic Effects in the Retina Lucimey Lima

The sulphur amino acid taurine possesses variable functions during development and regeneration of the central nervous system. The retina synthesize and uptake taurine, which is the amino acid present in the highest concentration in this tissue. Deficiency of taurine alters the structure and the function of the cerebral and cerebelar cortex, as well as the retina. Taurine increases outgrowth of postcrush goldfish retina in culture, partially by elevating calcium influx, and also by the modulation of protein phosphorylation. Its concentration increases in the retina after the lesion of the optic nerve, and the intraocular injection of it, between the crush and the explantation, stimulates the outgrowth of neurites. Taken together, although there are a great number of unresolved questions on the mechanisms of action of this amino acid as atrophic substance, the results support the role of taurine during regeneration of the optic nerve.     

Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage

In glaucoma, harmful intraocular pressure often contributes to retinal ganglion cell death. It is not clear, however, if intraocular pressure directly insults the retinal ganglion cell axon, the soma, or both. The pathways that mediate pressure-induced retinal ganglion cell death are poorly defined, and no molecules are known to be required. DBA/2J mice deficient in the proapoptotic molecule BCL2-associated X protein (BAX) were used to investigate the roles of BAX-mediated cell death pathways in glaucoma. Both Bax+/- and Bax-/- mice were protected from retinal ganglion cell death. In contrast, axonal degeneration was not prevented in either Bax+/- or Bax-/- mice. While BAX deficiency did not prevent axonal degeneration, it did slow axonal loss. Additionally, we compared the effects of BAX deficiency on the glaucoma to its effects on retinal ganglion cell death due to two insults that are proposed to participate in glaucoma. As in the glaucoma, BAX deficiency protected retinal ganglion cells after axon injury by optic nerve crush. However, it did not protect retinal ganglion cells from N-methyl-D-aspartate (NMDA)-induced excitotoxicity. BAX is required for retinal ganglion cell death in an inherited glaucoma; however, it is not required for retinal ganglion cell axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma. Finally, our data support a role for optic nerve injury but not for NMDA receptor-mediated excitotoxicity in this glaucoma. These findings indicate a need to understand axon-specific degeneration pathways in glaucoma, and they suggest that distinct somal and axonal degeneration pathways may need to be targeted to save vision.     

The origin of the electroretinogram induced by structured stimuli in man

The Authors studied the checkerboard pattern electroretinogram (PERG) behaviour in normal subjects and in patients suffering from traumatic lesion of optic nerve, demyelinating optic neuritis, axonal optic neuritis, retinal vascular disorders and retinitis pigmentosa in order to verify the PERG origin. According to our data the PERG appears to originate from ganglion cells since the signal diminished or disappeared in axonal lesion of optic nerve, as a result of retrograde ganglion cell degeneration. Furthermore, the demyelinating lesions of optic nerve were not able to modify the PERG wave. In patients suffering from retinal receptor disturbances the flash electroretinogram (FERG), which is probably an expression of receptor activity, was greatly impaired, whereas PERGs were either normal or less impaired than FERGs. These data demonstrate the different origin of PERG and FERG.     

ROCK2 is a major regulator of axonal degeneration,neuronal death and axonal regeneration in the CNS

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.     

Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.     

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.     

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.Download video file.^{(53M, mov)}     

BCLXL gene therapy moderates neuropathology in the DBA/2J mouse model of inherited glaucoma

Axonal degeneration of retinal ganglion cells (RGCs) causes blindness in glaucoma. Currently, there are no therapies that target axons to prevent them from degenerating. Activation of the BAX protein has been shown to be the determining step in the intrinsic apoptotic pathway that causes RGCs to die in glaucoma. A putative role for BAX in axonal degeneration is less well elucidated. BCLX_L (BCL2L1) is the primary antagonist of BAX in RGCs. We developed a mCherry-BCLX_L fusion protein, which prevented BAX recruitment and activation to the mitochondria in tissue culture cells exposed to staurosporine. This fusion protein was then packaged into adeno-associated virus serotype 2, which was used to transduce RGCs after intravitreal injection and force its overexpression. Transduced RGCs express mCherry-BCLX_L throughout their somas and axons along the entire optic tract. In a model of acute optic nerve crush, the transgene prevented the recruitment of a GFP-BAX fusion protein to mitochondria and provided long-term somal protection up to 12 weeks post injury. To test the efficacy in glaucoma, DBA/2J mice were transduced at 5 months of age, just prior to the time they begin to exhibit ocular hypertension. Gene therapy with mCherry-BCLX_L did not affect the longitudinal history of intraocular pressure elevation compared to naive mice but did robustly attenuate both RGC soma pathology and axonal degeneration in the optic nerve at both 10.5 and 12 months of age. BCLX_L gene therapy is a promising candidate for glaucoma therapy.Subject terms: Cell death in the nervous system, Neurodegeneration     

Oxidative stress induces autophagy in response to multiple noxious stimuli in retinal ganglion cells

Retinal ganglion cells (RGCs) are the only afferent neurons that can transmit visual information to the brain. The death of RGCs occurs in the early stages of glaucoma, diabetic retinopathy, and many other retinal diseases. Autophagy is a highly conserved lysosomal pathway, which is crucial for maintaining cellular homeostasis and cell survival under stressful conditions. Research has established that autophagy exists in RGCs after increasing intraocular pressure (IOP), retinal ischemia, optic nerve transection (ONT), axotomy, or optic nerve crush. However, the mechanism responsible for defining how autophagy is induced in RGCs has not been elucidated. Accumulating data has pointed to an essential role of reactive oxygen species (ROS) in the activation of autophagy. RGCs have long axons with comparatively high densities of mitochondria. This makes them more sensitive to energy deficiency and vulnerable to oxidative stress. In this review, we explore the role of oxidative stress in the activation of autophagy in RGCs, and discuss the possible mechanisms that are involved in this process. We aim to provide a more theoretical basis of oxidative stress-induced autophagy, and provide innovative targets for therapeutic intervention in retinopathy.     

Oxidative stress induces autophagy in response to multiple noxious stimuli in retinal ganglion cells

Retinal ganglion cells (RGCs) are the only afferent neurons that can transmit visual information to the brain. The death of RGCs occurs in the early stages of glaucoma, diabetic retinopathy, and many other retinal diseases. Autophagy is a highly conserved lysosomal pathway, which is crucial for maintaining cellular homeostasis and cell survival under stressful conditions. Research has established that autophagy exists in RGCs after increasing intraocular pressure (IOP), retinal ischemia, optic nerve transection (ONT), axotomy, or optic nerve crush. However, the mechanism responsible for defining how autophagy is induced in RGCs has not been elucidated. Accumulating data has pointed to an essential role of reactive oxygen species (ROS) in the activation of autophagy. RGCs have long axons with comparatively high densities of mitochondria. This makes them more sensitive to energy deficiency and vulnerable to oxidative stress. In this review, we explore the role of oxidative stress in the activation of autophagy in RGCs, and discuss the possible mechanisms that are involved in this process. We aim to provide a more theoretical basis of oxidative stress-induced autophagy, and provide innovative targets for therapeutic intervention in retinopathy.     

Taurine trophic modulation of goldfish retinal outgrowth and its interaction with the optic tectum

Summary. Goldfish retinal explant outgrowth in the presence of fetal calf serum is stimulated by taurine. In the absence of it, but with glucose in the medium, length of neurites is still elevated by the amino acid. Using the medium in the presence of glucose, but in the absence of fetal calf serum, we explored the effect of optic tectum medium from cultures of them coming from goldfish without crush of the optic nerve or 3, 5, 10, 14 and 20 days after crush. Retinal explants, intact or from goldfish with crush of the optic nerve 10 days prior to starting the culture, were employed in order to measure the possible effect of optic tectum media and the inter action with taurine. In other type of experiments the optic nerve was crushed 1, 2, 4, 7 and 10 days before dissection of the optic tectum, and then co-cultured with intact or 10 days post-crush retinal explants. Optic tectum media produced a time-dependent effect on outgrowth in lesioned retinas with a maximum effect around 5 days after the lesion for the corresponding optic tectum. Taurine, 4 mM, did not further affect the outgrowth in the presence of optic tectum media, but did significantly increase length of neurites either in intact or in post-lesion retinas. Co-culture of optic tectum at different days post-lesion and retinas at 10 days post-lesion increased the outgrowth around 4 days post-lesion, in a preparation resulting in mutual effects of both types of tissues. The addition of taurine in these conditions did not further increase outgrowth, rather inhibited it according to the time after lesion of optic nerve corresponding to the co-cultured optic tectum. The effect of taurine was concentration-dependent, since 0.2 mM was more effective than 2 or 4 mM in the presence of optic tectum with lesion of 2 days. These results demonstrate the time-course of the regeneration processes in the visual system of goldfish, indicating the crucial periods after crush in which the tectum could produce stimulation and later decrease or no effect on outgrowth from the retina. In addition, they are evidences of the interaction between taurine and optic tectum production of time-produced specific agents. The mechanisms underlying these effects are closely related to calcium, as it was demonstrated by the addition of extracellular or intracellular chelators to the medium, which inhibited the effects of the optic tectum and the trophic properties of taurine in this system. The inhibitor of taurine transport, guanidoethylsulfonate, also decreased the stimulatory effects of the optic tectum and of taurine, indicating an interaction of substances produced by the tectum with taurine, and an effect of taurine mediated through its entrance to the cells. Overall, retinal explants outgrowth in the absence of fetal calf serum, the interaction of agents of the optic tectum and taurine modulates outgrowth from the retina, and these effects are mediated by calcium levels and by the levels of intracellular taurine.