microRNA-124 is down regulated in nerve-injured motor neurons and it potentially targets mRNAs for KLF6 and STAT3.

MicroRNA (miRNA) is a small non-coding RNA that regulates gene expression by degrading target mRNAs or inhibiting translation. Although many miRNAs play important roles in various conditions, it is unclear whether miRNAs are involved in motor nerve regeneration. In this study, we identified the possible implication of miR-124 in nerve regeneration using a mouse hypoglossal nerve injury model. The significant down-regulation of miR-124 was observed in injured hypoglossal motor neurons after nerve injury, and this transient down-regulation showed a clear inverse correlation with the up-regulation of KLF6 and STAT3, known as axon elongation factor and regeneration-associated molecules, respectively. Furthermore, the luciferase assay and in vitro gain of function methods supported that both genes could be potent targets of miR-124. These results suggest that injury-induced repression of miR-124 may be implicated in the regulation of expression of several injury-associated transcription factors, which are crucial for appropriate nerve regeneration.

Localization and ontogeny of damage-induced neuronal endopeptidase mRNA-expressing neurons in the rat nervous system.

Neuropeptides are crucial mediators in nervous and endocrine systems. Processing and degradation, the major regulatory mechanisms, of enzymes are essential for the control of these peptidergic intercellular signaling systems. Damage-induced neuronal endopeptidase (or endothelin converting enzyme-like1), a member of the neprilysin family, has recently been identified as an M13 zinc metalloprotease. Damage-induced neuronal endopeptidase mRNA expression is strikingly restricted to neurons, and is remarkably induced in response to various types of neuronal injuries, although its function and substrate remain unknown. To clarify the role of damage-induced neuronal endopeptidase, we examined the localization and ontogeny of damage-induced neuronal endopeptidase mRNA expression in the rat nervous system using in situ hybridization. Damage-induced neuronal endopeptidase mRNA was detected at embryonic day 12, and its expression restricted to the ventral region of the neural tube. Subsequently, expression was also apparent in primordia of the striatum, hypothalamus, and cranial motor nuclei during neural development. This specific distribution was relatively maintained in the adult brain, although expression levels became weaker. Expression of damage-induced neuronal endopeptidase was absent in the cerebral cortex, hippocampus, and cerebellum. In addition to prominent expression in CNS, intestinal and sensory ganglia and retina demonstrated transient intense damage-induced neuronal endopeptidase mRNA expression during the embryonic period that then declined, and disappeared after birth. The results indicated that damage-induced neuronal endopeptidase might play an important role in embryonic neural development, in particular in peripheral ganglia derived from the neural crest, and in some neurons originating from the basal plate such as the hypothalamus and cranial motor neurons.

Neprilysin degrades both amyloid beta peptides 1-40 and 1-42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases.

To identify the amyloid beta peptide (Abeta) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha, NEPLP beta, and NEPLP gamma cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha, and NEPLP beta, as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta1-40 and Abeta1-42 most rapidly and efficiently. PEX degraded cold Abeta1-40 and NEPLP alpha degraded both cold Abeta1-40 and Abeta1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta-degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.

Endothelin-converting enzymes and endothelin receptor B messenger RNAs are expressed in different neural cell species and these messenger RNAs are coordinately induced in neurons and astrocytes respectively following nerve injury.

There is some evidence that endothelins may be a signal mediator between neuronal and glial cells, at least in some regions of the brain. To evaluate this possibility, the localization of messenger RNAs for endothelin-converting enzymes and endothelin receptor B in the rat brain were examined using in situ hybridization histochemistry. The messenger RNAs for endothelin-converting enzyme-1 and endothelin-converting enzyme-2 were expressed mainly in neurons located in various brain regions, whereas the messenger RNA for endothelin receptor B was mainly localized in the astrocytes located throughout the brainstem, Bergmann glia, choroid plexus and ependymal cells. The localization patterns of endothelin-converting enzyme and endothelin receptor B messenger RNAs were strikingly different. For instance, in the cerebellum, endothelin-converting enzyme-1 messenger RNA was localized in Purkinje cells, and endothelin-converting enzyme-2 mRNA was expressed in Purkinje cells and granule cells. On the other hand, endothelin receptor B messenger RNA was expressed in Bergmann glia and the astrocytes located in the granule cell layer. This suggests that final cleavages of big endothelins are performed on neuronal cells, and the major target of the processed endothelins could be astrocytes, which express endothelin receptor B most abundantly in the brain. Since evidence that endothelin is implicated in brain injury has also accumulated, we examined whether the expressions of endothelin-converting enzymes and endothelin receptor B are regulated by nerve injury. Following hypoglossal nerve injury, expression of messenger RNA for endothelin-converting enzymes-1 and -2 and endothelin receptor B was enhanced in the injured motor neurons and astrocytes respectively. The up-regulation of these messenger RNAs was also confirmed by a reverse transcription-polymerase chain reaction based strategyThese results lead us to suggest that endothelin can be an inducible intercellular mediator between injured neurons and astrocytes in response to nerve injury.

Damage-induced neuronal endopeptidase (DINE) is a unique metallopeptidase expressed in response to neuronal damage and activates superoxide scavengers.

We isolated a membrane-bound metallopeptidase, DINE (damage-induced neuronal endopeptidase), by differential display PCR using rat normal and axotomized hypoglossal nuclei. The most marked properties of DINE were neuron-specific expression and a striking response to axonal injury in both the central nervous system and peripheral nervous system. For instance, cranial and spinal nerve transection, ischemia, corpus callosum transection, and colchicine treatment increased DINE mRNA expression in the injured neurons, whereas kainate-induced hyperexcitation, immobilization, and osmotic stress failed to up-regulate DINE mRNA. Expression of DINE in COS cells partially inhibited C2-ceramide-induced apoptosis, probably because of the activation of antioxidant enzymes such as Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase through the proteolytic activity of DINE. These data provide insight into the mechanism of how injured neurons protect themselves against neuronal death.

Discordant expression of c-Ret and glial cell line-derived neurotrophic factor receptor alpha-1 mRNAs in response to motor nerve injury in neonate rats.

Adult motoneurons can survive following axotomy, whereas neonate motoneurons result in cell death. Following hypoglossal nerve axotomy in neonate rat, Glial cell line-Derived Neurotrophic Factor (GDNF) receptor alpha-1 (GFRalpha-1) mRNA expression was dramatically suppressed in the injured motoneurons, while a slight increase of c-Ret mRNA expression was observed. In adult, both GFRalpha-1 and c-Ret mRNAs increased substantially after axotomy. The present result suggests that the difference of motoneuron fate after axotomy may be partly due to the coordinate or discordant responses of GFRalpha-1 and c-Ret expression to nerve injury.

Dimethylarginine dimethylaminohydrolase (DDAH) as a nerve-injury-associated molecule: mRNA localization in the rat brain and its coincident up-regulation with neuronal NO synthase (nNOS) in axotomized motoneurons.

As part of a project to identify genes up-regulated by injury of the motor neuron, a clone encoding dimethylarginine dimethylaminohydrolase (DDAH) was isolated. This enzyme is known to metabolize methylarginines, which are endogenous inhibitors of NOS activity. DDAH may therefore contribute to the control of NO synthesis. The present study demonstrated that both DDAH and nNOS mRNAs are up-regulated after axotomy in injured hypoglossal motor neurons. The profile of DDAH mRNA up-regulation in the injured hypoglossal motor neurons paralleled that of NADPH diaphorase staining. While the expression of both DDAH and nNOS was upregulated in motor neurons following nerve injury, the normal distribution of DDAH and nNOS mRNAs in the noninjured central nervous system were distinctly different. We speculate that both genes are involved in the upregulation of NO production following nerve transection, although the role of NO in the process of nerve regeneration is so far unknown.

Expressed-sequence-tag approach to identify differentially expressed genes following peripheral nerve axotomy.

Gene expression profiles in the rat hypoglossal nucleus after axotomy were demonstrated using expressed-sequence-tag (EST) approach. To demonstrate the gene-expression profiles after axotomy, nerve-transected hypoglossal nuclei were dissected and collected from about 1000 rats, with which a cDNA library was constructed. More than 750 clones were sub-cloned and sequenced from the library. The clones which hit frequently are likely to be associated with mitochondrial respiratory chain, cytoskeletal protein and protein synthesis. One hundred three clones from among the sequenced clones were further processed for histological screening using unilateral-hypoglossal nerve-transected brain sections by in situ hybridization histochemistry. In situ hybridization study revealed that 26% of clones examined showed upregulated expression of mRNA in response to axotomy. They included genes encoding proteins associated with glucose, lipid and protein metabolism, cytoskeleton, neurotransmission and immune reaction. The present EST analysis may have an advantage in targeting genes which are associated with nerve injury with a good efficacy, as compared with other methods such as differential display and subtraction.

A sequence-specific splicing activator, tra2beta, is up-regulated in response to nerve injury.

Tra2beta is the first mammalian protein which is proved to activate mRNA splicing in sequence-specific manner. Following hypoglossal nerve injury, the expression of Tra2beta mRNA was elevated in injured motoneurons transiently. The up-regulation of Tra2beta mRNA was observed from post-operative day 3 to 21. In addition to the nerve injury in PNS, a brain lesion in CNS also enhanced the expression of Tra2beta mRNA. The present study could be the first observation showing that an expression of the sequence-specific splicing activator is enhanced in neuronal cells in response to nerve injury, and indicates that Tra2beta may participate in the control of injury-specific splicing patterns in order to express molecules which are necessary for regeneration.

Up-regulation of thioredoxin expression in motor neurons after nerve injury.

A substantial up-regulation of thioredoxin, a dithiol/disulfide oxido-reductase, in adult rat motoneurons following hypoglossal nerve axotomy, was demonstrated by using both in situ hybridization and immunohistochemistry. Although thioredoxin is normally accumulated more in the nucleus of a motoneuron rather than in the cytoplasm, a dramatic increase of thioredoxin in the cytoplasmic region after nerve injury was observed. The up-regulation of mRNA lasted more than 9 weeks, whereas, the detectable up-regulation of protein was observed for more than 5 weeks.

Enhancement of extracellular glutamate scavenge system in injured motoneurons.

An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve injury was demonstrated by differential display PCR using single arbitrary primer coupled with in situ hybridization screening called in situ display. Differential display PCR was carried out to compare differences in mRNA expression between axotomized (6 h after the transection) and normal hypoglossal nuclei in mice. Several gene fragments were increased after nerve injury; one was identified as GS. Subsequent emulsion autoradiography of hybridization tissue sections revealed that the increase in GS mRNA was observed in injured motoneurons. As GS is a key enzyme participating in the metabolism of the major excitatory neurotransmitter glutamate, we examined the significance of increased GS expression on glutamate-uptake kinetics. GS-transfected human embryonic kidney cells showed an up-regulation in glutamate-uptake kinetics. Therefore, newly expressed GS together with an increased expression of the neuronal glutamate transporter EAAC1 in the injured motoneurons accelerates glutamate uptake. The present results may suggest that the glutamate-uptake system involving the neuronal glutamate transporter and GS in injured neurons is enhanced so as to provide resistance against neurotoxic glutamate accumulation during the early process of nerve regeneration.

Enhanced expression of 14-3-3 family members in injured motoneurons.

An increase in 14-3-3 mRNA expression after hypoglossal nerve injury was demonstrated by RNA finger printing using the arbitrary primed polymerase chain reaction (RAP-PCR). RAP-PCR was carried out to compare differences in mRNA expression between axotomized (6 h after the transection) and normal hypoglossal nuclei in mice. The expression of several gene fragments was increased after nerve injury; one fragment was identified as 14-3-3 which is an activator of Raf-1. Since a family of 14-3-3 genes are identified in the rat, we examined the expression of five members of the rat 14-3-3 family after injury (beta, gamma, zeta, eta and theta). Among these family members, a substantial up-regulation in mRNA expression was observed for the zeta and θ forms. Subsequent emulsion autoradiography of hybridization tissue sections revealed an increase in zeta and theta mRNA in injured motoneurons. Since 14-3-3 has the ability to dimerize and activate Raf-1, the up-regulation of 14-3-3 expression would be expected to facilitate the Ras-Erk signal pathway by Raf-1 activation. Our previous results have demonstrated that Shc, Erk1 and Mek1 mRNAs are up-regulated during nerve regeneration, whereas PKA which inhibits the Ras-Erk pathway via Raf-1 was down-regulated. Taken together, the present results suggest that enhancement in expression of molecules involved in the Ras-Erk signaling is required for peripheral nerve regeneration.

Alternative expression of Shc family members in nerve-injured motoneurons.

Expression of Shc family protein (Shc/ShcA, SCK/ShcB and N-Shc/ShcC) and Grb2 mRNAs in the hypoglossal motoneurons after axotomy was examined by in situ hybridization. In normal hypoglossal motor neurons, N-Shc mRNA was expressed predominantly, whereas the Shc mRNA level is very low. Rat hypoglossal nerve injury reversed the expressions of these two molecules in hypoglossal motoneurons. Shc mRNA expression was up-regulated markedly whereas N-Shc was down-regulated after nerve injury. Expression levels of SCK, another Shc family member, and Grb2 were unaffected by nerve injury. These results suggest that, whereas the N-Shc-mediated pathway dominates under normal conditions, an alternative Shc-mediated pathway is utilized in the event of nerve injury. By changing the expression of the Shc family members, the signaling pathway can be altered and various responses induced for nerve regeneration.