Saya Nakagomi

Expression of the Activating Transcription Factor 3 Prevents c-Jun N-Terminal Kinase-Induced Neuronal Death by Promoting Heat Shock Protein 27 Expression and Akt Activation

Activating transcription factor 3 (ATF3) is induced and functions both as a cellular response to stress and to stimulate proliferation in multiple tissues. However, in the nervous system ATF3 is expressed only in injured neurons. Here we reveal a function of ATF3 in neurons under death stress. Overexpression of ATF3 by adenovirus inhibits the mitogen-activated kinase kinase kinase 1 (MEKK1)–c-Jun N-Terminal Kinase (JNK)-induced apoptosis and induces neurite elongation via Akt activation in PC12 cells and superior nerve ganglion neurons. A DNA microarray study reveals that ATF3 expression and JNK activation induce expression of the heat shock protein 27 (Hsp27). Immunoprecipitation analysis and promoter assay for Hsp27 expression suggest that both ATF3 and c-Jun are necessary for transcriptional activation of Hsp27. Hsp27 expression significantly inhibits JNK-induced apoptosis as well as Akt activation in PC12 cells and superior cervical ganglion neurons. We conclude that the combination of ATF3 and c-Jun induces the anti-apoptotic factor Hsp27, which directly or indirectly activates Akt, and thereby possibly inhibits apoptosis and induces nerve elongation. Our results suggest that ATF3- and c-Jun-induced Hsp27 expression is a novel survival response in neurons under death stress such as nerve injury.

Collapsin response mediator protein-2 accelerates axon regeneration of nerve-injured motor neurons of rat.

The rat collapsin response mediator protein‐2 (CRMP‐2) is a member of CRMP family (CRMP‐1–5). The functional consequence of CRMP‐2 during embryonic development, particularly in neurite elongation, is relatively understood; however, the role in nerve regeneration is unclear. Here we examined the role of CRMP‐2 during nerve regeneration using rat hypoglossal nerve injury model. Among the members, CRMP‐1, CRMP‐2, CRMP‐5 mRNA expressions increased after nerve injury, whereas CRMP‐3 and CRMP‐4 mRNA did not show any significant change. In the N1E‐115 cells, CRMP‐2 has the most potent neurite elongation activity among the CRMP family members. In dorsal root ganglion (DRG) organ culture, CRMP‐2 overexpression by adenoviral vector demonstrated substantial neurite elongation. On the other hand, CRMP‐2 (ΔC381), which acts as a dominant negative form of CRMP‐2, inhibited neurite formation. Collectively, it would be plausible that CRMP‐2 has potent nerve regeneration activity after nerve injury. We therefore examined whether CRMP‐2 overexpression in the injured hypoglossal motor neurons accelerates nerve regeneration. A retrograde‐tracer, Fluoro‐Gold (FG), was used to evaluate the number of reprojecting motor neurons after nerve injury. CRMP‐2‐overexpressing motor neurons demonstrated the accelerated reprojection. The present study suggests that CRMP‐2 has potent neurite elongation activity in nerve regeneration in vivo.

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.

Neuronal Injury-inducible Gene Is Synergistically Regulated by ATF3, c-Jun, and STAT3 through the Interaction with Sp1 in Damaged Neurons

Nerve injury requires the expression of large ensembles of genes. The key molecular mechanism for this gene transcription regulation in injured neurons is poorly understood. Among many nerve injury-inducible genes, the gene encoding damage-induced neuronal endopeptidase (DINE) showed most marked expression response to various kinds of nerve injuries in central and peripheral nervous system neurons. This unique feature led us to examine the promoter region of the DINE gene and clarify both the injury-responsive element within the promoter and its related transcriptional machinery. This study showed that DINE promoter was activated by leukemia inhibitory factor and nerve growth factor withdrawal, which were pivotal for the up-regulation of DINE mRNA after nerve injury. The injury-inducible transcription factors such as activating transcription factor 3 (ATF3), c-Jun, and STAT3, which were located at the downstream of leukemia inhibitory factor and nerve growth factor withdrawal, seemed to be involved in the activation of the DINE promoter. Surprisingly, these transcription factors did not bind to the DINE promoter directly. Instead, the general transcription factor, Sp1, bound to a GC box within the promoter. ATF3, c-Jun, and STAT3 interacted with Sp1 and are associated with the GC box region of the DINE gene in injured neurons. These findings suggested that Sp1 recruit ATF3, c-Jun, and STAT3 to obtain the requisite synergistic effect. Of these transcription factors, ATF3 may be the most critical, because ATF3 is specifically expressed after nerve injury.

Biphasic expression of activating transcription factor-3 in neurons after cerebral infarction.

It has been demonstrated that some of immediate early genes such as c-Jun are induced immediately and transiently following focal cerebral ischemia. Here we newly characterize the activating transcription factor (ATF)-3 as a focal ischemia associated immediate early gene. Using in situ hybridization and immunohistochemistry, we compared the expression profile of ATF-3 with those of ATF-2 and c-Jun after middle cerebral artery (MCA) occlusion. Focal cerebral ischemia induced two temporal and spatial patterns of ATF-3 expression. Early and transient induction of ATF-3 mRNA was observed in the core and margins of the cortex immediately after MCA occlusion. Late-onset and prolonged expression of ATF-3 mRNA and its protein were specifically identified in the peri-infarct cortex and thalamus where neurons survive at least 1 month. The expression profiles of ATF-3 and c-Jun were virtually similar, but c-Jun expression was also observed in other regions of the brain in control rats. Expression of ATF-2 was ubiquitously seen in neuronal cells throughout the brain in normal rats, but was suppressed in ischemic regions. Double immunohistochemical labeling revealed concurrent expression of ATF-3 and phospho-c-Jun in neurons. We conclude that the transcription factor ATF-3 is a suitable marker of neurons subjected to ischemic insult directly and indirectly, and that cooperative works of ATF-3 and c-Jun may be crucial triggers of various transcriptional responses to the ischemic insult.

Vesicular acetylcholine transporter can be a morphological marker for the reinnervation to muscle of regenerating motor axons.

This study was designed to evaluate whether the vesicular acetylcholine transporter (VAChT), which packages acetylcholine into synaptic vesicles, can be used as a marker for regenerating motor axon terminal. We examined motor axon regeneration in the tongue after hypoglossal nerve axotomy, using an anterograde tracer biotin-dextran (BD), retrograde tracer Fluoro-Gold (FG), electron microscopic (EM) observation, and VAChT immunocytochemistry. BD study demonstrated that outgrowth of thin regenerating axons into the frontal area of the tongue was firstly observed at 14 post-operative days, and presynaptic formation of neuromuscular junction (NMJ) was observed from 21 post-operative days. Under electron microscopic observation, reconstruction of new NMJs was observed within the interval between 21 and 28 days. VAChT-immunoreactive nerve terminals disappeared by 3 days after axotomy, slightly appeared at 14 post-operative days, and thereafter gradually increased in number from 21 to 28 post-operative days. The re-expression of VAChT positive presynaptic terminal was almost the same as those obtained in BD, FG and EM studies. Regenerating axons tip in the crush model of the hypoglossal nerve exhibited prominent VAChT immunoreactivity in growing tip of regenerating axons. These indicate that VAChT is an excellent morphological indicator for regenerating nerve terminals of motor neurons.

Id1, Id2 and Id3 are induced in rat melanotrophs of the pituitary gland by dopamine suppression under continuous stress

In rats under continuous stress (CS) there is decreased hypothalamic dopaminergic innervation to the intermediate lobe (IL) of the pituitary gland, which causes hyperactivation and subsequent degeneration of melanotrophs in the IL. In this study, we investigated the molecular basis for the changes that occur in melanotrophs during CS. Using microarray analysis, we identified several genes differentially expressed in the IL under CS conditions. Among the genes up-regulated under CS conditions, we focused on the inhibitor of DNA binding/differentiation (Id) family of dominant negative basic helix-loop-helix (bHLH) transcription factors. RT-PCR, Western blotting and in situ hybridization confirmed the significant inductions of Id1, Id2 and Id3 in the IL of CS rats. Administration of the dopamine D2 receptor agonist bromocriptine prevented the inductions of Id1-3 in the IL of CS rats, whereas application of the dopamine D2 antagonist sulpiride induced significant expressions of Id1-3 in the IL of normal rats. Moreover, an in vitro study using primary cultured melanotrophs demonstrated a direct effect on Id1-3 inductions by dopamine suppression. These results suggest that the decreased dopamine levels in the IL during CS induce Id1-3 expressions in melanotrophs. Because Id family members inhibit various bHLH transcription factors, it is conceivable that the induced Id1-3 would cooperatively modulate gene expressions in melanotrophs under CS conditions to induce hormone secretion.

Simultaneous expression of glutathione, thioredoxin-1, and their reductases in nerve transected hypoglossal motor neurons of rat

Anti-oxidative stress responses are crucial for the survival of nerve-injured motor neurons. Herein, we examined changes in expression of glutathione reductase (GSHr), thioredoxins (TRX1 and TRX2), and thioredoxin reductases (TRXr1 and TRXr2), important constituents of anti-oxidative pathways, following rat hypoglossal nerve transection. RT-PCR and in situ hybridization demonstrated that GSHr, TRX1, and TRXr1 mRNAs were significantly up-regulated during the first few weeks in nerve-injured motor neurons, while TRX2 and TRXr2 mRNAs were unchanged throughout 8 weeks after nerve transection. The up-regulation of GSH, GSHr, TRX1, and TRXr1 proteins in injured neurons was confirmed by immunohistochemical analysis. Western blotting also demonstrated up-regulation of GSHr, TRX1, and TRXr1 in injured neurons. These data suggest that the two major redox systems, GSH/GSHr and TRX1/TRXr1, are simultaneously activated in injured neurons, and likely provide protection of injured neurons against oxidative stress.

Accumulated fatigue induces over-activation and subsequent degeneration of melanotrophs in rat pituitary gland

Novel peptidergic neurons, metastin neurons, are thought to facilitate sexual maturation and ovulation by stimulating gonadotropin releasing hormone (GnRH) neurons, but their physiological functions are still largely unknown. Here we focused on the relationship between the metastin and hypothalamic/extrahypothalamic GnRH neurons. Taking advantage of well-developed teleost GnRH systems, we used medaka, which is a useful teleost for the application of various molecular genetic tools. First, we cloned and sequenced the metastin gene (Kiss1) in medaka for the first time in non-mammalian species. Then, Kiss1 expression in the brain was analyzed by in situ hybridization. We found two hypothalamic nuclei with Kiss1 expression and discovered prominent sexual difference in cell number (male female) in one of the two nuclei. On the other hand, we found no circadian variation in expression of Kiss1 by real-time PCR, although medaka shows daily ovulatory cyclicity.

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.