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.

We isolated a membrane-bound metallopeptidase, DINE (damageinduced 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 Cu͞Zn-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. P eripheral nerve regeneration entails sequential changes in the expression of thousands of genes, which are necessary to protect damaged neurons from death, activate surrounding glial cells, and accelerate neurite elongation. For the last few years, we have attempted to identify molecules involved in this process by using a technique known as differential display PCR (DD-PCR) and random cloning with a specific cDNA library derived from nerve-injured hypoglossal nuclei (1, 2).Among the molecules we have identified as being markedly up-regulated in response to nerve injury (3, 4), growth factors, cytokines, and neuropeptides are well established as survival factors for injured neurons (5). These molecules might participate in the protective process as intercellular signaling molecules via secretion in an autocrine or paracrine manner. Generally, secreted proteins such as neuropeptides and growth factors are biosynthesized as large precursor proteins, and processing occurs in the trans-Golgi network by endoproteolytic serine proteases, which are members of the proprotein convertase (PC) family (6). An increasing number of other secreted proteins now are recognized as being derived from integral plasma membrane proteins by hydrolysis (shedding) on the cell surface (7). Proteins secreted in this fashion include some membrane receptors, receptor ligands, ectoenzymes, and cell adhesion molecules. These ectodomain shedding events have been shown to be associated with metalloprotease inhibitors (8). Since identification of the ADAM (a disintegrin and metalloproteinase) family (9) and MMP (matrix metalloprotease) family, our understanding of the shedding events on the cell surface has greatly improved in recent years.As for nerve regeneration, the repertoire of proteases involved in the process is limited. Among regeneration processes, the roles of proteases in a process of axon elongation are relatively well studied both in vitro and in vivo. It has been assumed that this axonal behavior is, for instance, a consequence of the bala...

The Golgi apparatus processes intracellular proteins, but undergoes disassembly and fragmentation during apoptosis in several neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer's disease. It is well known that other cytoplasmic organelles play important roles in cell death pathways. Thus, we hypothesized that Golgi fragmentation might participate in transduction of cell death signals. Here, we found that Golgi fragmentation and dispersal precede neuronal cell death triggered by excitotoxins, oxidative/nitrosative insults, or ER stress. Pharmacological intervention or overexpression of the C-terminal fragment of Grasp65, a Golgi-associated protein, inhibits fragmentation and decreases or delays neuronal cell death. Inhibition of mitochondrial or ER cell death pathways also decreases Golgi fragmentation, indicating crosstalk between organelles and suggesting that the Golgi may be a common downstream-effector of cell death. Taken together, these findings implicate the Golgi as a sensor of stress signals in cell death pathways.

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 damageinduced 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 upregulation 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.

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b 0,+ AT/SLC7A9 and its auxiliary subunit rBAT/ SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b 0,+ AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.amino acid transporter | cystine reabsorption | cystinuria | kidney T he heteromeric amino acid transporter (HAT) family is one of the major amino acid transporter families responsible for cellular uptake and epithelial transport (1-3). HATs form heterodimers composed of a 12 membrane spanning light chain (SLC7) that catalyzes transport functions and a single membrane spanning heavy chain (SLC3) essential for plasma membrane localization and stabilization of the light chains. Two heavy chains, SLC3A1/ rBAT and SLC3A2/4F2hc/CD98hc, covalently bound to light chains via a disulfide bridge have been identified so far (4-6). 4F2hc interacts with most of the light chains in HATs whereas rBAT has been known to form a heterodimer only with b 0,+ AT/ SLC7A9. Because the rBAT-b 0,+ AT complex is presented on the apical membrane of proximal tubules in the kidney and involved in the reabsorption of cystine and dibasic amino acids, the mutations of either rBAT or b 0,+ AT cause cystinuria, a disorder of renal reabsorption of cystine and dibasic amino acids leading to serious renal lithiasis due to low solubility of cystine (7).An unsolved paradox on rBAT and b 0,+ AT has been the discrepancy between the distribution of rBAT and that of b 0,+ AT (5,(8)(9)(10). rBAT is the most abundant in the S3 segmen...

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 (DC381), 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.

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The layers of the epithelial syncytium, i.e., syncytiotrophoblasts, differentiate from chorionic trophoblasts via cell fusion and separate maternal and fetal circulations in hemochorial placentas. L-type amino acid transporter 1 (LAT1) and its covalently linked ancillary subunit 4F2hc are colocalized on both maternal and fetal surfaces of syncytiotrophoblasts, implying their roles in amino acid transfer through the placental barrier. In this study, LAT1 knockout, in addition, revealed a novel role of LAT1 in syncytiotrophoblast development. LAT1 at midgestation was selectively expressed in trophoblastic lineages in the placenta, exclusively as a LAT1-4F2hc heterodimer. In LAT1 homozygous knockout mice, chorionic trophoblasts remained largely mononucleated, and the layers of syncytiotrophoblasts were almost completely absent. The amount of 4F2hc protein, which possesses a fusogenic function in trophoblastic cells, as well as in virus-infected cells, was drastically reduced by LAT1 knockout, with less affecting the mRNA level. Knockdown of LAT1 in trophoblastic BeWo cells also reduced 4F2hc protein and suppressed forskolin-induced cell fusion. These results demonstrate a novel fundamental role of LAT1 to support the protein expression of 4F2hc via a chaperone-like function in chorionic trophoblasts and to promote syncytiotrophoblast formation by contributing to cell fusion in the developing placenta.