Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration

Microtubule dynamics are important for axon growth during development as well as axon regeneration after injury. We have previously identified HDAC5 as an injury-regulated tubulin deacetylase that functions at the injury site to promote axon regeneration. However, the mechanisms involved in the spatial control of HDAC5 activity remain poorly understood. Here we reveal that HDAC5 interacts with the actin binding protein filamin A via its C-terminal domain. Filamin A plays critical roles in HDAC5-dependent tubulin deacetylation because, in cells lacking filamin A, the levels of acetylated tubulin are elevated markedly. We found that nerve injury increases filamin A axonal expression in a protein synthesis-dependent manner. Reducing filamin A levels or interfering with the interaction between HDAC5 and filamin A prevents injury-induced tubulin deacetylation as well as HDAC5 localization at the injured axon tips. In addition, neurons lacking filamin A display reduced axon regeneration. Our findings suggest a model in which filamin A local translation following axon injury controls localized HDAC5 activity to promote axon regeneration.     

精氨酸酶Ⅰ参与环腺苷酸促进脑缺血再灌注大鼠的轴突再生

环腺苷酸（cAMP）作为细胞内的重要第二信使之一,主要通过激活下游cAMP依赖性蛋白激酶A（PKA）,进一步激活转录因子-cAMP效应元件结合蛋（CREB）,达到促进损伤轴突再生的作用.精氨酸酶Ⅰ主要是通过促进多胺的表达,从而克服髓鞘相关抑制因子对轴突再生的抑制作用,达到促进轴突再生的效果.在脑缺血中,cAMP促进轴突再生的过程是否有精氨酸酶Ⅰ的参与及其与RhoA信号通路的关系尚不清楚.本研究采用线栓法制备脑缺血再灌注模型（MACO）,采用Longa 5评分法对大鼠运动功能进行评分,利用逆转录聚合酶链反应（RT-PCR）和Western蛋白印迹方法分别检测缺血灶周边脑组织生长相关蛋白43（GAP-43）和RhoA的mRNA和蛋白表达,免疫组化法进行GAP-43的形态学检测,作为轴突再生的标志.通过尾静脉注射cAMP类似物db-cAMP增加脑缺血后大鼠脑组织内cAMP的浓度后发现：db-cAMP处理可明显降低MACO大鼠的运动功能评分,且可促进GAP-43 mRNA及蛋白的表达,抑制RhoA mRNA及蛋白的表达,由此可见db-cAMP处理可促进脑缺血后大鼠运动功能的恢复,且这一过程与抑制RhoA通路,进而促进轴突再生有关;通过在db-cAMP的基础上给予精氨酸酶Ⅰ拮抗剂NOHA来降低精氨酸酶Ⅰ的活性发现：给予NOHA的大鼠运动功能评分明显增加,这一变化趋势与RhoA mRNA及蛋白表达的变化趋势相一致,而与GAP-43 mRNA及蛋白表达的变化趋势相反. 因此可推断：精氨酸酶Ⅰ参与了db-cAMP促进轴突再生、改善脑缺血后大鼠运动功能的过程,且与钝化RhoA通路有关.     

The evolving roles of axonally synthesized proteins in regeneration

Work emerging during the past decade has shown that axons, similar to dendrites, are capable of autonomously generating new proteins through translation of localized mRNAs. Even in mammals, neurons maintain the ability to target mRNAs and translational machinery into the axonal compartment well into adulthood. The biological functions of axonal protein synthesis in adult neurons are just now being revealed, and recent studies indicate that locally synthesized proteins facilitate regeneration. Local translation, in addition to protein degradation, is needed for growth cone formation after axotomy, for generating a retrogradely transported injury signal, and then to help structurally maintain the growing axon. Regulation of axonal protein synthesis by exogenous stimuli might provide a means to facilitate regeneration for neuronal populations that normally show poor regenerative capacity in the adult nervous system.     

Syntaxin13 Expression Is Regulated by Mammalian Target of Rapamycin (mTOR) in Injured Neurons to Promote Axon Regeneration

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.     

A novel biological function for CD44 in axon growth of retinal ganglion cells identified by a bioinformatics approach

The failure of CNS regeneration and subsequent motor and sensory loss remain major unsolved questions despite massive accumulation of experimental observations and results. The sheer volume of data and the variety of resources from which these data are generated make it difficult to integrate prior work to build new hypotheses. To address these challenges we developed a prototypic suite of computer programs to extract protein names from relevant publications and databases and associated each of them with several general categories of biological functions in nerve regeneration. To illustrate the usefulness of our data mining approach, we utilized the program output to generate a hypothesis for a biological function of CD44 interaction with osteopontin (OPN) and laminin in axon outgrowth of CNS neurons. We identified CD44 expression in retinal ganglion cells and when these neurons were plated on poly- l -lysine 3% of them initiated axon growth, on OPN 15%, on laminin-111 (1×) 41%, on laminin-111 (0.5×) 56%, and on a mixture of OPN and laminin (1×) 67% of neurons generated axon growth. With the aid of a deoxyribozyme (DNA enzyme) to CD44 that digests the target mRNA, we demonstrated that a reduction of CD44 expression led to reduced axon initiation of retinal ganglion cells on all substrates. We suggest that such an integrative, applied systems biology approach to CNS trauma will be critical to understand and ultimately overcome the failure of CNS regeneration.     

Local protein synthesis in neuronal axons: why and how we study

Adaptive brain function and synaptic plasticity rely on dynamic regulation of local proteome. One way for the neuron to introduce new proteins to the axon terminal is to transport those from the cell body, which had long been thought as the only source of axonal proteins. Another way, which is the topic of this review, is synthesizing proteins on site by local mRNA translation. Recent evidence indicates that the axon stores a reservoir of translationally silent mRNAs and regulates their expression solely by translational control. Different stimuli to axons, such as guidance cues, growth factors, and nerve injury, promote translation of selective mRNAs, a process required for the axon’s ability to respond to these cues. One of the critical questions in the field of axonal protein synthesis is how mRNA-specific local translation is regulated by extracellular cues. Here, we review current experimental techniques that can be used to answer this question. Furthermore, we discuss how new technologies can help us understand what biological processes are regulated by axonal protein synthesis in vivo. [BMB Reports 2015; 48(3): 139-146]     

3.6 kb genomic sequence from Takifugu capable of promoting axon growth-associated gene expression in developing and regenerating zebrafish neurons

Unlike mammals, fish have the capacity for functional adult CNS regeneration, which is due, in part, to their ability to express axon growth-related genes in response to nerve injury. One such axon growth-associated gene is gap43, which is expressed during periods of developmental and regenerative axon growth, but is not expressed in CNS neurons that do not regenerate in adult mammals. We previously demonstrated that cis-regulatory elements of gap43 that are sufficient for developmental expression are not sufficient for regenerative expression in the zebrafish. Here we have identified a 3.6kb genomic sequence from Fugu rubripes that can promote reporter gene expression in the nervous system during both development and regeneration in zebrafish. This compact sequence is advantageous for functional dissection of regions important for axon growth-associated gene expression during development and/or regeneration. In addition, this sequence will also be useful for targeting gene expression to neurons during periods of growth and plasticity.     

Tenascin-R and axon growth-promoting molecules are up-regulated in the regenerating visual pathway of the lizard (Gallotia galloti)

It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down-regulation of axon growth-inhibitory proteins, and to what extent outgrowth-promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth-inhibiting extracellular matrix molecules tenascin-R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth-promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin-R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin-R and CSPG were expressed in an extracellular matrix-, oligodendrocyte/myelin- and neuron-associated pattern and up-regulated in the regenerating optic pathway. The expression pattern of tenascin-R was not indicative of a role in channeling or restriction of re-growing RGC axons. Up-regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin-R expression. Moreover, we analyzed the influence of substrates containing tenascin-R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin-R, and further improved on mixed substrates containing tenascin-R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down-regulation of tenascin-R or CSPG. Presence of tenascin-R is insufficient to prevent RGC axon growth, and concomitant up-regulation of axon growth-promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up-regulation of contactin in RGCs suggests that tenascin-R may have an instructive function during axon regeneration in the lizard optic pathway.     

The DLK signalling pathway—a double‐edged sword in neural development and regeneration

Dual leucine zipper kinase (DLK), a mitogen‐activated protein kinase kinase kinase, controls axon growth, apoptosis and neuron degeneration during neural development, as well as neurodegeneration after various insults to the adult nervous system. Interestingly, recent studies have also highlighted a role of DLK in promoting axon regeneration in diverse model systems. Invertebrates and vertebrates, cold‐ and warm‐blooded animals, as well as central and peripheral mammalian nervous systems all differ in their ability to regenerate injured axons. Here, we discuss how DLK‐dependent signalling regulates apparently contradictory functions during neural development and regeneration in different species. In addition, we outline strategies to fine‐tune DLK function, either alone or together with other approaches, to promote axon regeneration in the adult mammalian central nervous system.     

Substrate-cytoskeletal coupling as a mechanism for the regulation of growth cone motility and guidance

Growth cones are highly motile structures at the end of neuronal processes, capable of receiving multiple types of guidance cues and transducing them into directed axonal growth. Thus, to guide the axon toward the appropriate target cell, the growth cone carries out different functions: it acts as a sensor, signal transducer, and motility device. An increasing number of molecular components that mediate axon guidance have been characterized over the past years. The vast majority of these molecules include proteins that act as guidance cues and their respective receptors. In addition, more and more signaling and cytoskeleton-associated proteins have been localized to the growth cone. Furthermore, it has become evident that growth cone motility and guidance depends on a dynamic cytoskeleton that is regulated by incoming guidance information. Current and future research in the growth cone field will be focussed on how different guidance cues transmit their signals to the cytoskeleton and change its dynamic properties to affect the rate and direction of growth cone movement. In this review, we discuss recent evidence that cell adhesion molecules can regulate growth cone motility and guidance by a mechanism of substrate-cytoskeletal coupling.     

RSK1 promotes mammalian axon regeneration by inducing the synthesis of regeneration-related proteins

In contrast to the adult mammalian central nervous system (CNS), the neurons in the peripheral nervous system (PNS) can regenerate their axons. However, the underlying mechanism dictating the regeneration program after PNS injuries remains poorly understood. Combining chemical inhibitor screening with gain- and loss-of-function analyses, we identified p90 ribosomal S6 kinase 1 (RSK1) as a crucial regulator of axon regeneration in dorsal root ganglion (DRG) neurons after sciatic nerve injury (SNI). Mechanistically, RSK1 was found to preferentially regulate the synthesis of regeneration-related proteins using ribosomal profiling. Interestingly, RSK1 expression was up-regulated in injured DRG neurons, but not retinal ganglion cells (RGCs). Additionally, RSK1 overexpression enhanced phosphatase and tensin homolog (PTEN) deletion-induced axon regeneration in RGCs in the adult CNS. Our findings reveal a critical mechanism in inducing protein synthesis that promotes axon regeneration and further suggest RSK1 as a possible therapeutic target for neuronal injury repair.

This study shows that p90 ribosomal S6 kinase 1 (RSK1) responds differentially to nerve injury in the peripheral and central nervous systems, and identifies it as a crucial regulator of axonal regeneration; mechanistically, RSK1 preferentially induces the synthesis of regeneration-related proteins via the RSK1-eEF2K-eEF2 axis.     

NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC

Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. Here we demonstrate that a tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of glycogen synthase kinase 3beta (GSK-3beta). These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). Our results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway.     

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

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

Drug-delivering nerve conduit improves regeneration in a critical-sized gap

Autologous nerve grafts are the current “gold standard” for repairing large nerve gaps. However, they cause morbidity at the donor nerve site and only a limited amount of nerve can be harvested. Nerve conduits are a promising alternative to autografts and can act as guidance cues for the regenerating axons, without the need to harvest donor nerve. Separately, it has been shown that localized delivery of GDNF can enhance axon growth and motor recovery. FK506, an FDA approved small molecule, has also been shown to enhance peripheral nerve regeneration. This paper describes the design of a novel hole-based drug delivery apparatus integrated with a polytetrafluoroethylene (PTFE) nerve conduit for controlled local delivery of a protein such as GDNF or a small molecule such as FK506. The PTFE devices were tested in a diffusion chamber, and the bioactivity of the released media was evaluated by measuring neurite growth of dorsal root ganglions (DRGs) exposed to the released drugs. The drug delivering nerve guide was able to release bioactive concentrations of FK506 or GDNF. Following these tests, optimized drug releasing nerve conduits were implanted across 10 mm sciatic nerve gaps in a BL6 yellow fluorescent protein (YFP) mouse model, where they demonstrated significant improvement in muscle mass, compound muscle action potential, and axon myelination in vivo as compared with nerve conduits without the drug. The drug delivery nerve guide could release drug for extended periods of time and enhance axon growth in vitro and in vivo.     

Serotonin transporter-mediated molecular axis regulates regional retinal ganglion cell vulnerability and axon regeneration after nerve injury

Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin β3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin β3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein, Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin β3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin β3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.     

DSCR1 is required for both axonal growth cone extension and steering

Local information processing in the growth cone is essential for correct wiring of the nervous system. As an axon navigates through the developing nervous system, the growth cone responds to extrinsic guidance cues by coordinating axon outgrowth with growth cone steering. It has become increasingly clear that axon extension requires proper actin polymerization dynamics, whereas growth cone steering involves local protein synthesis. However, molecular components integrating these two processes have not been identified. Here, we show that Down syndrome critical region 1 protein (DSCR1) controls axon outgrowth by modulating growth cone actin dynamics through regulation of cofilin activity (phospho/dephospho-cofilin). Additionally, DSCR1 mediates brain-derived neurotrophic factor–induced local protein synthesis and growth cone turning. Our study identifies DSCR1 as a key protein that couples axon growth and pathfinding by dually regulating actin dynamics and local protein synthesis.     

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.     

Mechanisms of axon regeneration: The significance of proteoglycans

BackgroundTherapeutics specific to neural injury have long been anticipated but remain unavailable. Axons in the central nervous system do not readily regenerate after injury, leading to dysfunction of the nervous system. This failure of regeneration is due to both the low intrinsic capacity of axons for regeneration and the various inhibitors emerging upon injury. After many years of concerted efforts, however, these hurdles to axon regeneration have been partially overcome.Scope of reviewThis review summarizes the mechanisms regulating axon regeneration. We highlight proteoglycans, particularly because it has become increasingly clear that these proteins serve as critical regulators for axon regeneration.Major conclusionsStudies on proteoglycans have revealed that glycans not only assist in the modulation of protein functions but also act as main players—e.g., as functional ligands mediating intracellular signaling through specific receptors on the cell surface. By regulating clustering of the receptors, glycans in the proteoglycan moiety, i.e., glycosaminoglycans, promote or inhibit axon regeneration. In addition, proteoglycans are involved in various types of neural plasticity, ranging from synaptic plasticity to experience-dependent plasticity.General significanceAlthough studies on proteins have progressively facilitated our understanding of the nervous system, glycans constitute a new frontier for further research and development in this field. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.