Specificity of peripheral nerve regeneration: interactions at the axon level

Peripheral nerves injuries result in paralysis, anesthesia and lack of autonomic control of the affected body areas. After injury, axons distal to the lesion are disconnected from the neuronal body and degenerate, leading to denervation of the peripheral organs. Wallerian degeneration creates a microenvironment distal to the injury site that supports axonal regrowth, while the neuron body changes in phenotype to promote axonal regeneration. The significance of axonal regeneration is to replace the degenerated distal nerve segment, and achieve reinnervation of target organs and restitution of their functions. However, axonal regeneration does not always allows for adequate functional recovery, so that after a peripheral nerve injury, patients do not recover normal motor control and fine sensibility. The lack of specificity of nerve regeneration, in terms of motor and sensory axons regrowth, pathfinding and target reinnervation, is one the main shortcomings for recovery. Key factors for successful axonal regeneration include the intrinsic changes that neurons suffer to switch their transmitter state to a pro-regenerative state and the environment that the axons find distal to the lesion site. The molecular mechanisms implicated in axonal regeneration and pathfinding after injury are complex, and take into account the cross-talk between axons and glial cells, neurotrophic factors, extracellular matrix molecules and their receptors. The aim of this review is to look at those interactions, trying to understand if some of these molecular factors are specific for motor and sensory neuron growth, and provide the basic knowledge for potential strategies to enhance and guide axonal regeneration and reinnervation of adequate target organs.     

Specific activities of individual c-Jun N-terminal kinases in the brain

The c-Jun N-terminal kinases (JNKs) are multifunctional molecules which, on the one hand, regulate various processes in brain development, repair and memory formation. On the other hand, JNKs are potent effectors of neuronal death and neuroinflammation. This review summarizes recent findings on individual JNK functions in the nervous system under pathophysiological conditions and on their regulation by upstream kinases, phosphatases and formation of context-dependent signalosomes. By focusing on different aspects of JNK signaling, it becomes increasingly obvious that the JNK cascade is intricately regulated and intensely dependent on the availability and functionality of its single components and their intracellular localization. Our review also emphasizes, that JNKs are indispensable for neuronal cell death as well as many physiological functions in the brain. Finally, we discuss pharmacological strategies which target pathological JNK activities without affecting their physiological functions.     

Regulation of intrinsic neuronal properties for axon growth and regeneration

Regulation of neuritic growth is crucial for neural development, adaptation and repair. The intrinsic growth potential of nerve cells is determined by the activity of specific molecular sets, which sense environmental signals and sustain structural extension of neurites. The expression and function of these molecules are dynamically regulated by multiple mechanisms, which adjust the actual growth properties of each neuron population at different ontogenetic stages or in specific conditions. The neuronal potential for axon elongation and regeneration are restricted at the end of development by the concurrent action of several factors associated with the final maturation of neurons and of the surrounding tissue. In the adult, neuronal growth properties can be significantly modulated by injury, but they are also continuously tuned in everyday life to sustain physiological plasticity. Strict regulation of structural remodelling and neuritic elongation is thought to be required to maintain specific patterns of connectivity in the highly complex mammalian CNS. Accordingly, procedures that neutralize such mechanisms effectively boost axon growth in both intact and injured nervous system. Even in these conditions, however, aberrant connections are only formed in the presence of unusual external stimuli or experience. Therefore, growth regulatory mechanisms play an essentially permissive role by setting the responsiveness of neural circuits to environmental stimuli. The latter exert an instructive action and determine the actual shape of newly formed connections. In the light of this notion, efficient therapeutic interventions in the injured CNS should combine targeted manipulations of growth control mechanisms with task-specific training and rehabilitation paradigms.     

Neuroprotection against ischemic brain injury by a small peptide inhibitor of c-Jun N-terminal kinase (JNK) via nuclear and non-nuclear pathways

Our previous studies and the others have strongly suggested that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Here we reported that Tat-JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), a smaller 11-mer peptide corresponding to residues 153-163 of murine JIP-1 conjugated to Tat peptide, perturbed the assembly of JIP-1-JNK3 complexes, thus inhibiting the activation of JNK3 induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. As a result, Tat-JBD diminished the increased phosphorylation of c-Jun (a nuclear substrate of JNK) and the increased expression of Fas ligand induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and the release of Bax from Bcl-2/Bax dimers, Tat-JBD attenuated Bax translocation to mitochondria and the release of cytochrome c induced by ischemia/reperfusion. Furthermore, the activation of caspase3 and hydrolyzation of poly-ADP-ribose-polymerase induced by brain ischemia/reperfusion were also significantly suppressed by preinfusion of the peptide Tat-JBD. Importantly, Tat-JBD showed neuroprotective effects on ischemic brain damage in vivo, and administration of the peptide after ischemia also achieved the same effects as preinfusion of the peptide did. Thus, our findings imply that Tat-JBD induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via inhibiting nuclear and non-nuclear pathways of JNK signaling. Taken together, these results indicate that Tat-JBD peptide provides a promising therapeutic approach for ischemic brain injury.     

Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system

c-Jun N-terminal kinases (JNKs) are implicated in cell death in neurodegenerative disorders. Therefore, JNK inhibitors could act as neuroprotective agents. To evaluate potential candidates, reproducible and quantitative CNS in vivo models are required. To that end, a pentylenetetrazole-induced seizure model was explored. c-Jun phosphorylation was detected in hippocampal extracts by blotting c-Jun immunoprecipitates with phosphorylation-specific antibodies. Pentylenetetrazole administration induced rapid and reproducible increases in c-Jun phosphorylation. However, special attention had to be paid to the composition of the extraction buffer to ensure stabilization of protein phosphorylation, as demonstrated using internal standards of phosphorylated recombinant c-Jun. As JNK and its upstream activator MKK4 are activated by phosphorylation, these events were also evaluated. In principle, kinase inhibitors could act at the level of JNK or upstream kinases to inhibit c-Jun phosphorylation. MKK4 phosphorylation was dramatically increased in response to pentylenetetrazole but, again, only when appropriate phosphatase inhibitors were in the extraction buffer. In contrast, JNK was found to be constitutively phosphorylated and unaltered upon pentylenetetrazole treatment. The JNK inhibitor SP600125 was shown to inhibit c-Jun phosphorylation without affecting MKK4 phosphorylation. Our procedures enable analysis of JNK pathway signalling in a CNS model and, also, should be applicable to that of other protein phosphorylation events in vivo.     

Age-associated changes in SAPK/JNK and p38 MAPK signaling in response to the generation of ROS by 3-nitropropionic acid

Mitochondrial dysfunction has been identified as a major source of oxidative stress in aged tissues. In this study we asked whether activities of components of the SAPK/JNK and p38 MAPK stress response signaling pathways are indicative of oxidative stress in aged mouse livers and whether these pathways are responsive to oxidative stress generated by 3-nitropropionic acid (3-NPA), an inhibitor of complex II (succinic dehydrogenase). We asked whether (a) aging affects the basal activity of the SAPK/JNK stress signaling pathway; (b) specific isoforms of JNK, i.e. 46 or 54 kDa JNKs are activated by 3-NPA; (c) aging affects the response of this signaling pathway to 3-NPA; (d) there is a cross pathway activation of JNK or p38 MAPK by upstream activators. Our studies have shown that although their protein pool levels are not altered, the basal JNK activities using c-Jun as substrate is elevated. Furthermore, in aged livers, JNK activity is induced to a greater extent and takes longer to recover from 3-NPA treatment. The activities of the upstream activators of JNKs, MAP kinase kinase (MKK) 4 and 7, are also elevated in livers of aged C57BL/6 male mice. These activator kinases, which are induced (phosphorylated) by 3-NPA in young livers, are not inducible by this inhibitor in aged livers. In fact, these proteins are highly phosphorylated in the control aged livers and are dephosphorylated in response to 3-NPA. Finally, we demonstrate for the first time that MKK7 serves as an upstream activator of p38 MAPK and that MKK3 and MKK6 activates 54 kDa JNK2 in aged liver. Our studies suggest that failure to respond to 3-NPA may be indicative of the susceptibility of aged tissue to oxidative stress, supporting our hypothesis that aged tissues (especially liver) develop a state of chronic stress even in the absence of a challenge.     

c-Jun N-terminal kinase binding domain–dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions.     

Antioxidant marine algae phlorotannins and radioprotection: A review of experimental evidence

Radiation has been widely used for cancer therapy in human medicine. However, the side effects of radiation are problematic and can limit its application. Radiation generates reactive oxygen species, leading to cell death via multiple signaling pathways. The blocking of certain signaling cascades using antioxidants represents a compensatory therapy of radiation-induced tissue injury. Although synthetic chemicals have been investigated in recent decades, anti-oxidants from natural resources have been searched for continuously. Among them, phlorotannins from marine algae, including Ecklonia cava, have been shown to protect cells from radiation-induced injury as well as oxidative stress. In the present review, the radioprotective capacity of phlorotannins derived from marine algae and the mechanisms involved are discussed.     

Activation of JNK pathway in persistent pain

The c-Jun N-terminal kinase (JNK) is a stress-activated member of MAP kinase family. JNK activation has been strongly implicated in inflammatory responses, neurodegeneration, and apoptosis. Recent evidence shows that JNK pathway is also transiently activated in primary sensory neurons after tissue or nerve injury, which is required for the development of hyperalgesia and allodynia. In particular, JNK is persistently activated in astrocytes of the spinal cord after nerve injury, and this activation can maintain central sensitization and mechanical allodynia. In this mini-review, we will provide evidence for the involvement of JNK pathway in regulating persistent pain sensitization. We will also discuss possible upstream signaling mechanisms that cause JNK activation and downstream signaling mechanisms by which JNK modulates pain sensitivity. Thus, targeting JNK pathway might be a useful strategy to treat both neurodegeneration and chronic pain.     

Plasminogen activator inhibitor-1 aids nerve growth factor-induced differentiation and survival of pheochromocytoma cells by activating both the extracellular signal-regulated kinase and c-Jun pathways

Astrocytes are thought to be critical to neurons' surviving damage caused by ischemic stroke or other injury. Plasminogen activator inhibitor-1 is one of the active soluble factors released by astrocytes and regulates plasminogen activator-plasmin proteolytic sequence in the CNS as a serpin. In this study, we show that plasminogen activator inhibitor-1 can promote neurite outgrowth and survival of rat pheochromocytoma cells in serum-deprived conditions, and that this neuroprotective activity is correlated with enhanced activation of both extracellular signal-regulated kinases following a direct phosphorylation of nerve growth factor receptor, Trk A, and of c-Jun. Our results suggest that plasminogen activator inhibitor-1 can act as a neurotrophic factor, protecting neurons from serum deprivation-induced neuron death not only by compensating for nerve growth factor functions, but also by activating the c-Jun/activating protein-1 pathway.     

Activation of c-Jun N-terminal kinases by ribotoxic stresses

The c-Jun N-terminal kinases （JNKs） are classic stress-activated protein kinases. Many cellular stresses have been shown to stimulate JNK activation. In this review, we focus on ribotoxic stresses based on their multiple biological potencies including anti-HIV-1 activity. Some of the functions of ribotoxins and the signaling transduction pathway that mediated are mentioned. Different from other stimulators, ribotoxic stresses act on special motifs of 28S rRNA in translationally active mammal ribosomes. Binding and damaging on the motif leads to JNK activation and subsequently biological response to the signal initiator, which is named ribotoxic stress response. Cellular ＆ Molecular Immunology. 2005;2（6）：419-425.     

Neural plasticity after peripheral nerve injury and regeneration

Injuries to the peripheral nerves result in partial or total loss of motor, sensory and autonomic functions conveyed by the lesioned nerves to the denervated segments of the body, due to the interruption of axons continuity, degeneration of nerve fibers distal to the lesion and eventual death of axotomized neurons. Injuries to the peripheral nervous system may thus result in considerable disability. After axotomy, neuronal phenotype switches from a transmitter to a regenerative state, inducing the down- and up-regulation of numerous cellular components as well as the synthesis de novo of some molecules normally not expressed in adult neurons. These changes in gene expression activate and regulate the pathways responsible for neuronal survival and axonal regeneration. Functional deficits caused by nerve injuries can be compensated by three neural mechanisms: the reinnervation of denervated targets by regeneration of injured axons, the reinnervation by collateral branching of undamaged axons, and the remodeling of nervous system circuitry related to the lost functions. Plasticity of central connections may compensate functionally for the lack of specificity in target reinnervation; plasticity in human has, however, limited effects on disturbed sensory localization or fine motor control after injuries, and may even result in maladaptive changes, such as neuropathic pain, hyperreflexia and dystonia. Recent research has uncovered that peripheral nerve injuries induce a concurrent cascade of events, at the systemic, cellular and molecular levels, initiated by the nerve injury and progressing throughout plastic changes at the spinal cord, brainstem relay nuclei, thalamus and brain cortex. Mechanisms for these changes are ubiquitous in central substrates and include neurochemical changes, functional alterations of excitatory and inhibitory connections, atrophy and degeneration of normal substrates, sprouting of new connections, and reorganization of somatosensory and motor maps. An important direction for ongoing research is the development of therapeutic strategies that enhance axonal regeneration, promote selective target reinnervation, but are also able to modulate central nervous system reorganization, amplifying those positive adaptive changes that help to improve functional recovery but also diminishing undesirable consequences.