Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity

Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of β-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of β-actin mRNA, and introducing GFP with the 3'UTR of β-actin mRNA depletes axons of endogenous β-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of β-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.     

Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration

Injury to the axons of facial motoneurons stimulates increases in the synthesis of actin, tubulins, and GAP-43, and decreases in the synthesis of neurofilament proteins: mRNA levels change correspondingly. In contrast to this robust response of peripheral neurons to axotomy, injured central nervous system neurons show either an attenuated response that is subsequently aborted (rubrospinal neurons) or overall decreases in cytoskeletal protein mRNA expression (corticospinal and retinal ganglion neurons). There is evidence that these changes in synthesis are regulated by a variety of factors, including loss of endoneurially or target-derived trophic factors, positive signals arising from the site of injury, changes in the intraaxonal turnover of proteins, and substitution of target-derived trophic support by factors produced by glial cells. It is concluded that there is, as yet, no coherent explanation for the upregulation or downregulation of any of the cytoskeletal proteins following axotomy or during regeneration. In considering the relevance of these changes in cytoskeletal protein synthesis to regeneration, it is emphasized that they are unlikely to be involved in the initial outgrowth of the injured axons, both because transit times between cell body and injury site are too long, and because sprouting can occur in isolated axons. Injuryinduced acceleration of the axonal transport of tubulin and actin in the proximal axon is likely to be more important in providing the cytoskeletal protein required for initial axonal outgrowth. Subsequently, the increased synthesis and transport velocity for actin and tubulin increase the delivery of these proteins to support the increased volume of the maturing regenerating axons. Reduction in neurofilament synthesis and changes in neurofilament phosphorylation may permit the increased transport velocity of the other cytoskeletal proteins. There is little direct evidence that alterations in cytoskeletal protein synthesis are necessary for successful regeneration, nor are they sufficient in the absence of a supportive environment. Nevertheless, the correlation that exists between a robust cell body response and successful regeneration suggests that an understanding of the regulation of cytoskeletal protein synthesis following axon injury must be a part of any successful strategy to improve the regenerative capacity of the central nervous system.     

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.     

The Role of the Axonal Cytoskeleton in Diabetic Neuropathy

The neuropathy associated with diabetes includes well documented impairment of axonal transport, a reduction in axon calibre and a reduced capacity for nerve regeneration. All of those aspects of nerve function rely on the integrity of the axonal cytoskeleton. Alterations in the axonal cytoskeleton in experimental diabetes include an insulin-dependent non-enzymatic glycation of actin that is reflected in increased glycation of platelet actin in the clinical situation. There is a reduced synthesis of mRNA for the isoforms of tubulin that are associated with nerve growth and regeneration and an elevated non-enzymatic glycation of peripheral nerve tubulin in both diabetic patients and diabetic animals. mRNAs for neurofilament proteins are selectively reduced in the diabetic rat and the post-translational modification of at least one of the neurofilament proteins is altered. There is some evidence that altered expression of isoforms of protein kinases may contribute to these changes.     

Axon Viability and Mitochondrial Function are Dependent on Local Protein Synthesis in Sympathetic Neurons

(1) Axons contain numerous mRNAs and a local protein synthetic system that can be regulated independently of the cell body. (2) In this study, cultured primary sympathetic neurons were employed, to assess the effect of local protein synthesis blockade on axon viability and mitochondrial function. (3) Inhibition of local protein synthesis reduced newly synthesized axonal proteins by 65% and resulted in axon retraction after 6 h. Acute inhibition of local protein synthesis also resulted in a significant decrease in the membrane potential of axonal mitochondria. Likewise, blockade of local protein transport into the mitochondria by transfection of the axons with Hsp90 C-terminal domain decreased the mitochondrial membrane potential by 65%. Moreover, inhibition of the local protein synthetic system also reduced the ability of mitochondria to restore axonal levels of ATP after KCl-induced depolarization. (4) Taken together, these results indicate that the local protein synthetic system plays an important role in mitochondrial function and the maintenance of the axon.     

Synthesis and functional integration of a neurotransmitter receptor in isolated invertebrate axons

Neurotransmitter receptors are considered an important class of membrane proteins that are involved in plasticity-induced changes underlying learning and memory. Recent studies, which demonstrated that the mRNAs encoding for various receptor proteins are localized to specific dendritic domains, allude toward the possibility that these membrane bound molecules may be synthesized locally. However, direct evidence for the local axonal or dendritic synthesis and functional integration of receptor proteins in either vertebrates or invertebrates is still lacking. In this study, using an invertebrate model system we provide the first direct evidence that isolated axons (in the absence of the soma) can intrinsically synthesize and functionally integrate a membrane-bound receptor protein from an axonally injected mRNA. Surgically isolated axons from identified neurons were injected with mRNA encoding a G-protein-coupled conopressin receptor. Immunocytochemical and electrophysiological techniques were used to demonstrate functional integration of the receptor protein into the membrane of the isolated axon. Ultrastructural analysis of axonal compartments revealed polyribosomes, suggesting that some components of the protein synthesizing machinery are indeed present in these extrasomal compartments. Such axonal propensity to locally synthesize and functionally insert transmitter receptors may be instrumental in plasticity induced changes, for instance those that underlie learning and memory.     

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.     

Extracellular regulators of axonal growth in the adult central nervous system

Robust axonal growth is required during development to establish neuronal connectivity. However, stable fibre patterns are necessary to maintain adult mammalian central nervous system (CNS) function. After adult CNS injury, factors that maintain axonal stability limit the recovery of function. Extracellular molecules play an important role in preserving the stability of the adult CNS axons and in restricting recovery from pathological damage. Adult axonal growth inhibitors include a group of proteins on the oligodendrocyte, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein and ephrin-B3, which interact with axonal receptors, such as NgR1 and EphA4. Extracellular proteoglycans containing chondroitin sulphates also inhibit axonal sprouting in the adult CNS, particularly at the sites of astroglial scar formation. Therapeutic perturbations of these extracellular axonal growth inhibitors and their receptors or signalling mechanisms provide a degree of axonal sprouting and regeneration in the adult CNS. After CNS injury, such interventions support a partial return of neurological function.     

Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.     

Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury

Axon regeneration after injury to the adult mammalian CNS is limited in part by three inhibitory proteins in CNS myelin: Nogo-A, MAG, and OMgp. All three of these proteins bind to a Nogo-66 receptor (NgR) to inhibit axonal outgrowth in vitro. To explore the necessity of NgR for responses to myelin inhibitors and for restriction of axonal growth in the adult CNS, we generated ngr(-/-) mice. Mice lacking NgR are viable but display hypoactivity and motor impairment. DRG neurons lacking NgR do not bind Nogo-66, and their growth cones are not collapsed by Nogo-66. Recovery of motor function after dorsal hemisection or complete transection of the spinal cord is improved in the ngr(-/-) mice. While corticospinal fibers do not regenerate in mice lacking NgR, regeneration of some raphespinal and rubrospinal fibers does occur. Thus, NgR is partially responsible for limiting the regeneration of certain fiber systems in the adult CNS.     

Proteins of fast axonal transport in the regenerating hypoglossal nerve of the rat

The composition of proteins conveyed by fast axonal transport in growing or regenerating axons is different from that of intact, mature axons. Consistent alterations have been observed in several different types of neurons, but adult peripheral axons (rabbit hypoglossal motoneurons) seemed to be exceptions because during their regeneration there was no increased labelling of a 23 kilodalton (kD) protein associated with the growth state. We examined the composition of fast-transported proteins, labelled by application of [35S]methionine to the hypoglossal nuclei, in intact and regenerating hypoglossal nerves of the rat. Using one- and two-dimensional electrophoresis we detected both increases and decreases in the labelling of specific polypeptides during regeneration. In particular, there was increased labelling of a 23 kD polypeptide. Changes were maximal 7 days after axotomy and subsided thereafter, coincident with reinnervation of the tongue. We conclude that hypoglossal axons show the same changes in transported protein composition which are characteristic of the growth state in other axons. Thus, we have strengthened the correlation between the growth state and changes in synthesis of a set of polypeptides of unknown function.     

Regeneration of an adult peripheral nerve preparation in culture

The methods used to maintain the vagus nerve from the adult rat in culture and how regeneration is studied in this preparation are described. A hypothesis is presented on the triggering of the cell body reaction. It is suggested that this reaction is initiated by proteins synthesized in nonneuronal cells at the site of a nerve lesion. These proteins, referred to as regenerins, reach the nerve cell body by retrograde axonal transport, where they initiate the regeneration process.     

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.     

Function and translational regulation of mRNA in developing axons

The capacity to synthesize proteins in axons is limited to early stages of neuronal development, while axons are undergoing elongation and pathfinding. Although the roles of local protein synthesis are not fully understood, it has been implicated in regulating the morphological plasticity of growth cones. Recent studies have identified specific mRNAs that are translated in growth cones in response to specific extracellular signals. In this review, we discuss the functional relevance of axonal protein translation for developing axons, the differences in translational capacity between developing and mature vertebrate axons, and possible pathways governing the specific translational activation of axonal mRNAs.     

Protein Synthesis and Rapid Axonal Transport During Regrowth of Dorsal Root Axons

Damage to the sciatic nerve produces significant changes in the relative synthesis rates of some proteins in dorsal root ganglia and in the amounts of some fast axonally transported proteins in both the sciatic nerve and dorsal roots. We have now analyzed protein synthesis and axonal transport after cutting the other branch of dorsal root ganglia neurons, the dorsal roots. Two to three weeks after cutting the dorsal roots, [35S]methionine was used to label proteins in the dorsal root ganglia in vitro. Proteins synthesized in the dorsal root ganglia and transported along the sciatic nerve were analyzed on two-dimensional gels. All of the proteins previously observed to change after sciatic nerve damage were included in this study. No significant changes in proteins synthesized in dorsal root ganglia or rapidly transported along the sciatic nerve were detected. Axon regrowth from cut dorsal roots was observed by light and electron microscopy. Either the response to dorsal root damage is too small to be detected by our methods or changes in protein synthesis and fast axonal transport are not necessary for axon regrowth. When such changes do occur they may still aid in regrowth or be necessary for later stages in regeneration.