Acute axonal degeneration in vivo is attenuated by inhibition of autophagy in a calcium-dependent manner

Axonal degeneration is a pathological hallmark of many traumatic and neurodegenerative neurological disorders. Although the underlying mechanisms remain largely unclear, increased autophagy and the influx of extracellular calcium have been implicated in the pathogenesis of axonal degeneration based on in vitro data. Using in vivo imaging of the rat optic nerve after crush lesion we could now show that both mechanisms are linked and play an important role in acute axonal degeneration in vivo. Our data suggest that crush lesion of the optic nerve induces a rapid calcium influx through calcium channels, which results in a secondary induction of autophagy that participates actively in axonal degradation. Therapeutic manipulation of both events could significantly alter the time course of acute axonal degeneration in vivo and may thus represent promising therapeutic targets for the future.     

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.     

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)}     

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     

Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush

Retinal ganglion cells (RGCs) are central nervous system (CNS) neurons that transmit visual information from the retina to the brain. Apoptotic RGC degeneration causes visual impairment that can be modeled by optic nerve crush. Neuronal apoptosis is also a salient feature of CNS trauma, ischemia (stroke), and diseases of the CNS such as Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis. Optic nerve crush induces the apoptotic cell death of ～ 70% of RGCs within the first 14 days after injury. This model is particularly attractive for studying adult neuron apoptosis because the time-course of RGC death is well established and axon regeneration within the myelinated optic nerve can be concurrently evaluated. Here, we performed a large scale iTRAQ proteomic study to identify and quantify proteins of the rat retina at 1, 3, 4, 7, 14, and 21 days after optic nerve crush. In total, 337 proteins were identified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated (>1.3 ×), 46 were downregulated (<0.7 ×), and 6 showed both positive and negative regulation over 21 days, relative to normal retinas. Among the differentially expressed proteins, Thymosin-β4 showed an early upregulation at 3 days, the time-point that immediately precedes the induction of RGC apoptosis after injury. We examined the effect of exogenous Thymosin-β4 administration on RGC death after optic nerve injury. Intraocular injections of Thymosin-β4 significantly increased RGC survival by ～ 3-fold compared to controls and enhanced axon regeneration after crush, demonstrating therapeutic potential for CNS insults. Overall, our study identified numerous proteins that are differentially regulated at key time-points after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the adult CNS.     

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.     

Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.     

Axon degeneration: molecular mechanisms of a self-destruction pathway

Axon degeneration is a characteristic event in many neurodegenerative conditions including stroke, glaucoma, and motor neuropathies. However, the molecular pathways that regulate this process remain unclear. Axon loss in chronic neurodegenerative diseases share many morphological features with those in acute injuries, and expression of the Wallerian degeneration slow (WldS) transgene delays nerve degeneration in both events, indicating a common mechanism of axonal self-destruction in traumatic injuries and degenerative diseases. A proposed model of axon degeneration is that nerve insults lead to impaired delivery or expression of a local axonal survival factor, which results in increased intra-axonal calcium levels and calcium-dependent cytoskeletal breakdown.     

Low Intensity Repetitive Transcranial Magnetic Stimulation Does Not Induce Cell Survival or Regeneration in a Mouse Optic Nerve Crush Model

Low intensity repetitive Transcranial Magnetic Stimulation (LI-rTMS), a non-invasive form of brain stimulation, has been shown to induce structural and functional brain plasticity, including short distance axonal sprouting. However, the potential for LI-rTMS to promote axonal regeneration following neurotrauma has not been investigated. This study examined the effect of LI-rTMS on retinal ganglion cell (RGC) survival, axon regeneration and levels of BDNF in an optic nerve crush neurotrauma model. Adult C57Bl/6J mice received a unilateral intraorbital optic nerve crush. Mice received 10 minutes of sham (handling control without stimulation) (n=6) or LI-rTMS (n = 8) daily stimulation for 14 days to the operated eye. Immunohistochemistry was used to assess RGC survival (β-3 Tubulin) and axon regeneration across the injury (GAP43). Additionally, BDNF expression was quantified in a separate cohort by ELISA in the retina and optic nerve of injured (optic nerve crush) (sham n = 5, LI-rTMS n = 5) and non-injured mice (sham n = 5, LI-rTMS n = 5) that received daily stimulation as above for 7 days. Following 14 days of LI-rTMS there was no significant difference in mean RGC survival between sham and treated animals (p>0.05). Also, neither sham nor LI-rTMS animals showed GAP43 positive labelling in the optic nerve, indicating that regeneration did not occur. At 1 week, there was no significant difference in BDNF levels in the retina or optic nerves between sham and LI-rTMS in injured or non-injured mice (p>0.05). Although LI-rTMS has been shown to induce structural and molecular plasticity in the visual system and cerebellum, our results suggest LI-rTMS does not induce neuroprotection or regeneration following a complete optic nerve crush. These results help define the therapeutic capacity and limitations of LI-rTMS in the treatment of neurotrauma.     

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     

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.     

The Role of Peroxisome Proliferator-Activated Receptor and Effects of Its Agonist,Pioglitazone, on a Rat Model of Optic Nerve Crush: PPARγ in Retinal Neuroprotection

It has been shown that peroxisome proliferators-activated receptor gamma (PPARγ) is beneficial for central nervous system injury. However its role on optic nerve injury remains unknown. In the present study, we examined the change of PPARγ expression in rat retina following optic nerve injury and investigated the effect of pioglitazone (Pio), a PPARγ agonist, on retinal ganglion cells (RGCs) neuroprotection using a rat optic nerve crush (ONC) model. Our results showed that PPARγ mRNA and protein levels were increased after ONC, and most of PPARγ-immunoreactive cells colocalized with Müller cells. Pio treatment significantly enhanced the number of surviving RGCs and inhibited RGCs apoptosis induced by ONC. However, when PPARγ antagonist GW9662 was used, these neuroprotective effects were abolished. In addition, pio attenuated Müller cell activation after ONC. These results indicate that PPARγ appears to protect RGCs from ONC possibly via the reduction of Müller glial activation. It provides evidence that activation of PPARγ may be a potential alternative treatment for RGCs neuroprotection.     

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.     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.     

Extrinsic cellular and molecular mediators of peripheral axonal regeneration

The ability of injured peripheral nerves to regenerate and reinnervate their original targets is a characteristic feature of the peripheral nervous system (PNS). On the other hand, neurons of the central nervous system (CNS), including retinal ganglion cell (RGC) axons, are incapable of spontaneous regeneration. In the adult PNS, axonal regeneration after injury depends on well-orchestrated cellular and molecular processes that comprise a highly reproducible series of degenerative reactions distal to the site of injury. During this fine-tuned process, named Wallerian degeneration, a remodeling of the distal nerve fragment prepares a permissive microenvironment that permits successful axonal regrowth originating from the proximal nerve fragment. Therefore, a multitude of adjusted intrinsic and extrinsic factors are important for surviving neurons, Schwann cells, macrophages and fibroblasts as well as endothelial cells in order to achieve successful regeneration. The aim of this review is to summarize relevant extrinsic cellular and molecular determinants of successful axonal regeneration in rodents that contribute to the regenerative microenvironment of the PNS.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Optic nerve input‐dependent regulation of neural stem cell proliferation in the optic tectum of adult zebrafish

Adult neurogenesis attracts broad attention as a possible cure for neurological disorders. However, its regulatory mechanism is still unclear. Therefore, they have been studying the cell proliferation mechanisms of neural stem cells (NSCs) using zebrafish, which have high regenerative potential in the adult brain. The presence of neuroepithelial‐type NSCs in the optic tectum of adult zebrafish has been previously reported. In the present study, it was first confirmed that NSCs in the optic tectum decrease or increase in proportion to projection of the optic nerves from the retina. At 4 days after optic nerve crush (ONC), BrdU‐positive cells decreased in the optic tectum's operation side. In contrast, at 3 weeks after ONC, BrdU‐positive cells increased in the optic tectum's operation side. To study the regulatory mechanisms, they focused on the BDNF/TrkB system as a regulatory factor in the ONC model. It was found that bdnf was mainly expressed in the periventricular gray zone (PGZ) of the optic tectum by using in situ hybridization. Interestingly, expression level of bdnf significantly decreased in the optic tectum at 4 days after ONC, and its expression level tended to increase at 3 weeks after ONC. They conducted rescue experiments using a TrkB agonist and confirmed that decrease of NSC proliferation in the optic tectum by ONC was rescued by TrkB signal activation, suggesting stimuli‐dependent regulation of NSC proliferation in the optic tectum of adult zebrafish. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Axonal transport of synaptic vesicle proteins in the rat optic nerve

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.