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.     

Apoptosis in adult retinal ganglion cells after axotomy

Lesions to the mature mammalian central nervous system cause irreversible degeneration, in which neurons have been previously thought to be passive victims. In this study, axon-lesioned adult rat nerons are shown instead to actively degrade themselves through the process of apoptosis: a programmed type of cell death in which the cellular apparatus is actively involved in the degradation process. To investigate whether retinal ganglion cells of an adult mammal follow an apoptotic type of death when their axons are severed, DNA breaks in nuclei were labeled in situ, using a method that specifically incorporates biotinylated deoxynucleotides by exogenous terminal deoxynucleotidyl transferase on the 3′-OH ends of DNA. The active nature of the death mechanism was demonstrated by the reduction in biotin-labeled nuclei after administering the protein synthesis inhibitor cycloheximide. Our results suggest that retinal ganglion cells of the adult rat die through apoptosis when axotomized. This raises new possibilities in the treatment of CNS injuries, by the potential interruptibility of a program for neuronal death. 1994 John Wiley & Sons, Inc.     

Formation of functional synapses by regenerating adult rat retinal ganglion cell axons in midbrain target regions in vitro

The ability of adult rat retinal ganglion cell (RGC) axons to reinnervate normal target regions was examined in vitro. In co-culture experiments, adult rat retinal explants were placed adjacent to fetal rat midbrain sections that contained the superior colliculus (SC) which is the main target for RGC axons. Adult rat RGCs regrew axons over more than 500 μm on a polylysine-laminin substrate to reach the co-cultured explants. By using neurofilament immunohistochemistry and the fluorescent dye Dil for anterograde and retrograde tracing, it was shown that (1) adult rat RGCs with a stereotyped morphology survived in explant cultures for more than 4 weeks in the presence of fetal midbrain explants, (2) regenerating RGC axons preferentially terminated within midbrain target regions, and (3) RGCs formed functional synapses. In addition, the maturation of the SC region in midbrain explants was examined histologically and ultrastructurally to demonstrate appropiate target development. © 1993 John Wiley & Sons, Inc.     

Growth preferences of adult rat retinal ganglion cell axons in retinotectal cocultures

We examined whether regenerating axons from adult rat ganglion cells are able to recognize their appropriate target region in vitro. Explants from adult rat retina were cocultured with embryonic sagittal midbrain slices in Matrigel®. The midbrain sections contained the superior colliculus, the main target for retinal ganglion cell axons in rats, and the inferior colliculus. We observed a statistically significant preference of both temporal and nasal retinal axons to grow toward their appropriate target region (anterior and posterior superior colliculus, respectively). No preferential growth of retinal ganglion cell axons was detected in controls, for which retinal explants were cultured on their own. When retinal ganglion cell axons were given a choice between superior colliculus and inferior colliculus, axons from nasal retina preferentially grew toward the posterior superior colliculus and avoided the inferior colliculus. In contrast, temporal axons in the same assay did not show preference for either of the colliculi. These findings suggest that regenerating axons from adult rat retina are able to recognize target-specific guidance cues released from embryonic midbrain targets in vitro. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 379–387, 1998     

Gap-43 immunoreactivity and axon regeneration in retinal ganglion cells of the rat

Retinal ganglion cells (RGCs) in rats were retrogradely labeled with the fluorescent tracer Fluorogold (FG) and subjected to GAP-43 and c-JUN immunocytochemistry to identify those RGSs that are capable of regenerating an axon. After optic nerve section (ONS) and simultaneous application of FG to the nerve stump (group 1 experiments), GAP-43 immunoreactive RGCs (between 2 and 21 days after ONS) always represented a subfraction of both FG-labeled (i.e., surviving) RGCs and RGCs exhibiting c-JUN. GAP-43 immunoreactive RGCs represented 22% of RGCs normally present in rat retinae and 25% of surviving RGCs at 5 days after ONS but were reduced to 2% and 1%, which is 6% and 5% of survivors at 14 and 21 days, respectively. In animals that received a peripheral nerve (PN) graft after ONS (group 2 experiments), RGCs with regenerating axons were identified by FG application to the graft at 14 and 21 days. When examined at 21 and 28 days, all FG-labeled RGCs exhibited GAP-43 immunoreactivity, and FG/GAP-43-labeled RGCs were 3% and 2% of those resent in normal rat retinae. In relation to surviving. RGCs GAP-43 immunoreactive RGCs represented 10% at both time points. FG-/GAP-43 labeled RGCs also exhibited c-JUN, but c-JUN immunoreactive RGCs were at both time points at least twice as numerous a FG-/GAP-43-labeled RGCs. These data suggest that regenerating axons in PN grafts derive specifically from GAP-43 reexpressing RGCs. Appearance of GAP-43 immunoreactivity may therefore identify those RGCs that are capable of axonal regeneration or sprouting. 1994 John Wiley & Sons, Inc.     

基于IPA分析自噬对大鼠肝再生中树突状细胞的调节作用

为探讨自噬对大鼠肝再生中树突状细胞(Dendritic cells, DCs)的调节作用,文章通过Percoll 密度梯度离心结合免疫磁珠分选分离大鼠DCs,Rat Genome 230 2.0芯片检测大鼠肝再生中自噬相关基因表达变化,利用IPA等软件分析自噬在DCs中的生理活动。结果表明,LC3、BECN1、ATG7和SQSTM1等关键基因在部分肝切除后不同恢复时间段有明显表达变化;芯片中对应的自噬相关基因为593个,其中210个基因发生了有意义的变化。比较分析自噬生理活动情况,发现自噬在再生早期和晚期阶段增强,增殖期减弱。与自噬相关的生理活动主要有RNA表达、RNA转录细胞分化和增殖,其中涉及的信号通路主要有PPARα/RXRα激活、急性期反应、TREM1 信号通路、IL-6 信号通路、IL-8 信号通路和IL-1 信号通路等,它们在肝再生阶段发生了不同程度的上调或下调。Cluster 分析还发现,P53和AMPK信号参与调控DCs的自噬活动,在肝再生早期主要是AMPK信号,在肝再生末期P53和AMPK信号共同参与自噬的调节。以上研究结果说明DCs自噬可能在肝再生早期激活细胞免疫反应和后期清除DCs等方面发挥着重要作用。     

Antibody that neutralizes myelin-associated inhibitors of axon growth does not interfere with recognition of target-specific guidance information by rat retinal axons

During development, many CNS projection neurons establish topographically ordered maps in their target regions. Myelin-associated inhibitors of neurite growth contribute to the confinement of fiber tracts during development and limit plastic changes after CNS projections have been formed. Neutralization of myelin-associated growth inhibitors leads to an expansion of the retinal innervation of the superior colliculus (SC). In the lesioned adult mammalian CNS, these long projection neurons are usually unable to regrow axons over long distances after lesion due to myelin-associated inhibitors, which interfere with axonal growth in vivo and in vitro. Application of a specific antibody directed against myelin-inhibitors (IN-1) promotes regrowth of corticospinal tract or retinal ganglion cell axons. In the present study, we asked whether application of an antibody to myelin-associated growth inhibitors would lead to disturbances of target-specific axon guidance. To examine this issue, we used an in vitro model, the “stripe assay,” to examine the behavior of rat retinal ganglion cell axons on membranes from embryonic and deafferented adult rat SC. On membrane preparations from embryonic rat SC, retinal fibers avoid posterior tectal membranes, possibly due to the presence of a repulsive factor. Nasal retinal axons show a random growth pattern. On membranes prepared from the deafferented adult rat SC, temporal and nasal axons prefer to grow on membranes prepared from their specific target region, which suggests the involvement of target-derived attractive guidance components. The results of the present study show that retinal axons grow significantly faster in the presence of IN-1 antibody that neutralizes myelin-associated growth inhibitors present in the membrane preparations from the adult rat SC. IN-1 antibody, however, does not interfere with specific axonal guidance. This suggests that axonal guidance and specific target finding are independently regulated in retinal axons. © 1996 John Wiley & Sons, Inc.     

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.     

JNK inhibitory kinase is up-regulated in retinal ganglion cells after axotomy and enhances BimEL expression level in neuronal cells

Optic nerve transection results in retinal ganglion cell (RGC) death in adult mammals, after the alteration of gene expression of RGCs. To elucidate the molecular mechanism by which axotomy induces RGC death, we isolated the molecules up-regulated after optic nerve transection. One of these, axotomy-related [corrected] gene (ARG)357, an 898-amino-acid [corrected] protein containing a complete serine-threonine kinase domain, was isolated from a subtraction library of the rat retina. The sequence showed that this gene was a rat homolog of human c-Jun N-terminal kinase (JNK) inhibitory kinase and so belonged to the germinal center kinase-VIII subfamily of Sterile20s protein kinase. We designated ARG357 as rat JNK inhibitory kinase (JIK). Rat JIK was expressed ubiquitously in various tissues and was highly expressed in the retina, with selective expression in RGCs. After axotomy, BimEL and Hrk, which are BH3-only proteins, and rat JIK were up-regulated in RGCs. Overexpression of rat JIK in neuronal cells up-regulated the expression of BimEL, but not that of Hrk. These results indicate that JIK may contribute to axotomy-induced RGC death by up-regulating the expression of BH3-only protein.     

Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays

One of the most striking features of neurons in the mature peripheral nervous system is their ability to survive and to regenerate their axons following axonal injury. To perform a comprehensive survey of the molecular mechanisms that underlie peripheral nerve regeneration, we analyzed a cDNA library derived from the distal stumps of post-injured sciatic nerve which was enriched in non-myelinating Schwann cells using cDNA microarrays. The number of up- and down-regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up-regulated group, the number of known genes was 216 and the number of expressed sequence tag (EST) sequences was 154. In the down-regulated group, the number of known genes was 103 and that of EST sequences was 54. We obtained several genes that were previously reported to be involved in regeneration of the injured neurons, such as cathepsin D, ninjurin 1, tenascin C, and co-receptor for glial cell line-derived neurotrophic factor family of trophic factors. In addition to unknown genes, there seemed to be a lot of annotated genes whose role in nerve regeneration remains unknown.     

Gene expression analysis of zebrafish retinal ganglion cells during optic nerve regeneration identifies KLF6a and KLF7a as important regulators of axon regeneration

Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.     

Temporal gene expression profile after acute electroconvulsive stimulation in the rat

Electroconvulsive therapy (ECT) remains one of the most effective treatments of major depression. It has been suggested that the mechanisms of action involve gene expression. In recent decades there have been several investigations of gene expression following both acute and chronic electroconvulsive stimulation (ECS). These studies have focused on several distinct gene targets but have generally included only few time points after ECS for measuring gene expression. Here we measured gene expression of three types of genes: Immediate early genes, synaptic proteins, and neuropeptides at six time points following an acute ECS. We find significant increases for c-Fos, Egr1, Neuritin 1 (Nrn 1), Bdnf, Snap29, Synaptotagmin III (Syt 3), Synapsin I (Syn 1), and Psd95 at differing time points after ECS. For some genes these changes are prolonged whereas for others they are transient. Npy expression significantly increases whereas the gene expression of its receptors Npy1r, Npy2r, and Npy5r initially decreases. These decreases are followed by a significant increase for Npy2r, suggesting anticonvulsive adaptations following seizures. In summary, we find distinct changes in mRNA quantities that are characteristic for each gene. Considering the observed transitory and inverse changes in expression patterns, these data underline the importance of conducting measurements at several time points post-ECS.     

Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish

Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response.     

Modulation of mitochondria in the axon and soma of retinal ganglion cells in a rat glaucoma model

Mitochondrial abnormality has been implicated in various models of retinal ganglion cell (RGC) degeneration. We investigated modulation of mitochondrial membrane permeability and apoptosis-inducing factor (AIF) translocation in a rat experimental glaucoma model. A decrease in MitoTracker-labeled mitochondria around the lamina area of the optic nerve was observed in the glaucomatous eye. Immunoblot analysis for axonal motor proteins showed that a significant decrease in kinesin 1 and myosin Va levels in the glaucomatous optic nerve. A significant decrease in mitochondrial thioredoxin 2 (Trx2) level was observed in the optic nerve after intraocular pressure (IOP) elevation. Translocation of AIF from the mitochondria to the axoplasm and nucleus was observed in the axon and cell body, respectively. Trx2 over-expression in the mitochondrial membrane of RGC-5 cells inhibited AIF translocation, resulting in cytoprotective effect against neurotoxicity induced by TNF-α/buthionine sulfoximine treatment. In vivo transfection was performed with EGFP-Trx2 plasmid and electroporation. Over-expression of Trx2 in the retina and optic nerve indicated the protective effect against high IOP induced axonal degeneration. Thus, the decreased mitochondrial membrane potential and subsequent AIF translocation were involved in the glaucomatous neurodegeneration. Furthermore, modulation of mitochondria through the inhibition of AIF translocation may become a new treatment strategy for neurodegenerative disease, such as glaucoma.