Joan Ribera

Naturally Occurring (Programmed) and Radiation‐induced Apoptosis are Associated with Selective c‐Jun Expression in the Developing Rat Brain

Expression of the different members of transcription factors Fos and Jun was examined in the developing rat brain. Constitutive expression of c‐Fos, Fos‐related antigens, Jun B and Jun D, as revealed with immunohistochemistry, is higher and more widely distributed in the developing rat brain than in the adult. Selective strong c‐Jun expression is observed in the cytoplasm and nuclei of apoptotic cells during the whole process of naturally occurring (programmed) cell death. Cells expressing strong c‐Jun immunoreactivity are undetermined cells, neurons and astrocytes. Selective c‐Jun expression is also observed following ionizing radiation in rats aged 3 days. Induction of c‐jun mRNA, as revealed with in situ hybridization, occurs between 5 and 15 min following gamma‐irradiation. Strong c‐Jun protein expression appears at 2 h, peaks at 6 h and decreases thereafter to reach normal levels 48 h after gamma‐ray exposure. Strong c‐Jun protein expression is coincidental with endonuclease activation, as revealed with the method of in situ labelling of nuclear DNA fragmentation, and is restricted to apoptotic cells. Cycloheximide injection at the time of irradiation blocks c‐Jun expression, indicating that c‐Jun immunoreactivity is attributable to de novo protein synthesis. These observations demonstrate in vivo selective strong c‐Jun expression associated with programmed cell death and ionizing radiation‐induced apoptosis in the developing rat brain.

Nitric oxide synthase in rat neuromuscular junctions and in nerve terminals of torpedo electric organ: Its role as regulator of acetylcholine release

The distribution of nitric oxide synthase on peripheral motor system was studied using a specific antibody against the neuronal isoform of nitric oxide synthase (nNOS). The immunoreactivity for nNOS was detected on the sarcolemmal surface of muscle cells, in intramuscular axons and in neuromuscular synapses. At the neuromuscular junctions, ultrastructural immunolabeling demonstrated that nNOS immunoreactivity was localized mainly into the presynaptic nerve terminals as well as adjacent postsynaptic muscle membrane. Similar immunostaining pattern was present in frog muscles and Torpedo electric organs. After chronic muscle denervation, nNOS immunoreactity at endplate level decreased during the first week but it was upregulated after 30 days of denervation. In denervated endplates, nNOS immunoreactivity was localized in the terminal Schwann cells covering the degenerated neuromuscular junctions whereas nNOS was not detected in Schwann cells under normal conditions. In Torpedo synaptosomes, acetylcholine (ACh) release elicited by potassium depolarization was inhibited by NO donors such as sodium nitroprusside. In contrast, application of inhibitors of NOS activity, aminoguanidine (AMG) and Nω‐Nitro‐L‐arginine methyl esther (L‐NAME) increased acetylcholine release. These results indicate that nNOS is present at the motor nerve terminals in a variety of vertebrates and that it may be involved in the physiological modulation of ACh release and in the regulation of muscle response to nerve injury.

Regulation of Motoneuronal Calcitonin Gene–related Peptide (CGRP) During Axonal Growth and Neuromuscular Synaptic Plasticity Induced by Botulinum Toxin in Rats

The aim of this study was to examine whether changes in rat motoneuronal calcitonin gene–related peptide (CGRP) can be correlated with axonal growth and plasticity of neuromuscular synapses. Nerve terminal outgrowth was induced by local paralysis with botulinum toxin. Normal adult soleus and tibialis anterior did not show detectable CGRP content at the motor endplates. Following botulinum toxin injection there was a progressive, transient and bimodal increase in CGRP in both motoneuron cell bodies which innervated poisoned muscles and their motor endplates. CGRP content was moderately increased 1 day after paralysis and, after an initial decline, reached a peak 20 days after injection. This was followed by a gradual decrease and a return to normal levels at the 200th day. CGRP changes in intoxicated endplates were less evident in the tibialis anterior than in the soleus muscle. The CGRP content in motoneurons was positively correlated with the degree of intramuscular nerve sprouting found by silver staining. In situ hybridization revealed an increase in CGRP mRNA in spinal cord motoneurons 20 days after toxin administration. We conclude that motoneurons regulate their CGRP in situations in which peripheral synapse remodelling and plasticity occur.

c-Jun regulation in rat neonatal motoneurons postaxotomy.

Motoneurons respond to peripheral nerve transection by either regenerative or degenerative events depending on their state of maturation. Since the expression of c‐Jun has been involved in the early signalling of the regenerative process that follows nerve transection in adults, we have investigated c‐Jun on rat neonatal axotomized motoneurons during the period in which neuronal death is induced. Changes in levels of c‐Jun protein and its mRNA were determined by means of quantitative immunocytochemistry and in situ hybridization. Three hours after nerve transection performed on postnatal day (P)3, c‐Jun protein and mRNA is induced in axotomized spinal cord motoneurons, and high levels were reached between 1 and 10 days after. This response is associated with a detectable c‐Jun activation by phosphorylation on serine 63. No changes were found in the levels of activating transcription factor ‐2. Most of dying motoneurons were not labelled by either a specific c‐Jun antibody or a c‐jun mRNA probe. However, dying motoneurons were specifically stained by a polyclonal anti c‐Jun antibody, indicating that some c‐Jun antibodies react with unknown epitopes, probably distinct from c‐Jun p39, that are specifically associated with apoptosis. J. Neurosci. Res. 63:469–479, 2001.

Induction of reactive astrocytosis and prevention of motoneuron cell death by the I2-imidazoline receptor ligand LSL 60101

I2‐imidazoline receptors are mainly expressed on glial cells in the rat brain. This study was designed to test the effect of treatment with the I2‐imidazoline selective receptor ligand LSL 60101 [2‐(2‐benzofuranyl)imidazole] on the morphology of astrocytes in the neonate and adult rat brain, and to explore the putative neuroprotective effects of this glial response. Short‐term (3 days) or chronic (7–10 days) treatment with LSL 60101 (1 mg kg−1, i.p. every 12 h) enhanced the area covered by astroglial cells in sections of facial motor nucleus from neonate rats processed for glial fibrillary acidic protein (GFAP) immunostaining. Facial motoneurons surrounded by positive glial cell processes were frequently observed in sections of LSL 60101‐treated rats. A similar glial response was observed in the parietal cortex of adult rats after chronic (10 days) treatment with LSL 60101 (10 mg kg−1, i.p. every 12 h). Western‐blot detection of the specific astroglial glutamate transporter GLT‐1, indicated increased immunoreactivity after LSL 60101 treatment in the pons of neonate and in the parietoccipital cortex of adult rats. In the facial motor nucleus of neonate rats, the glial response after LSL 60101 treatment was associated to a redistribution of the immunofluorescence of the basic fibroblast growth factor (FGF‐2) from the perinuclear area of motoneurons to cover most of their cytoplasm, suggesting a translocation of this mitogenic and neurotrophic factor towards secretion pathways. The neuroprotective potential of the above effects of LSL 60101 treatment was tested after neonatal axotomy of facial motor nucleus. Treatment with LSL 60101 (1 mg kg−1, i.p. every 12 h from day 0 to day 10 after birth) significantly reduced (38%) motoneuron death rate 7 days after facial nerve axotomy performed on day 3 after birth. It is concluded that treatment with the I2‐imidazoline selective receptor ligand LSL 60101 provokes morphological/biochemical changes in astroglia that are neuroprotective after neonatal axotomy.

Antibodies against c-Jun N-terminal peptide cross-react with neo-epitopes emerging after caspase-mediated proteolysis during apoptosis.

In previous studies it has been shown that neural cells undergoing programmed cell death display strongly positive cytoplasmic immunoreactivity to polyclonal antibodies directed against a c‐Jun N‐terminal peptide. It was later found that c‐Jun‐like immunoreactivity in apoptosis was due to cross‐reactivity with proteins other than c‐Jun. We have analysed the biochemical counterpart of this property in neuroblastoma cell lines treated to induce apoptosis. Using the c‐Jun/sc‐45 antibody, several bands with apparent molecular masses distinct from c‐Jun were detected in extracts in parallel with both the degree of apoptosis and the appearance of the cytoplasmic signal after immunostaining. c‐Jun/sc‐45 immunostaining was prevented by caspase inhibitors and did not require de novo protein synthesis. One of the antigens recognized by the c‐Jun/sc‐45 antibody was identified as seryl‐tRNA synthetase. We provide evidence that seryl‐tRNA synthetase is a substrate of caspase‐3 in vitro and that the digested form turns highly immunoreactive towards the antibody. A carboxy‐terminus epitope of the protein that constitutes a consensus site for caspase‐3 is involved in c‐Jun/sc‐45 recognition. This epitope shares some amino acids with the peptide used as the immunogen and this could explain the cross‐reactivity observed. In conclusion, we demonstrate here that cytoplasmic c‐Jun/sc‐45‐like immunoreactivity specific to apoptosis is due to post‐translational changes which occur in seryl‐tRNA synthetase and probably also in other proteins as a consequence of caspase mediated proteolysis.

Treatment with digestive agents reveals several glycoconjugates specifically associated with rat neuromuscular junction

SummaryThe lectins DBA, WGA, SBA, Con A, GS-1, LFA and PNA were used to characterize the carbohydrate domains of the rat neuromuscular junction. DBA stained only the synaptic domains of muscle surface. All other lectins stained the whole muscle surface but the intensity of staining was stronger at the synaptic regions. However, when sections were treated with several digestive agents prior to lectin application, the lectin staining pattern changed dramatically. Collagenase-sensitive GlcNac, Mannose, Sialic acid, and GalNac-containing glycoconjugates associated with synaptic regions but not present extrasynaptically were revealed after chemical treatment. On the basis of these modifications it is proposed that, apart from the synapse-specific Gal-Nac-containing glycoconjugate already described elsewhere, new carbohydrate-containing compounds are evidenced. These results provide a new insight into regional specialization of the extracellular matrix associated with the neuromuscular junctions and indicates that pretreatment with various agents, not necessary digestive substances, may alter molecular properties of muscle membrane and uncover previously unknown binding sites.

Phylogenetic polymorphism on lectin binding to junctional and non-junctional basal lamina at the vertebrate neuromuscular junction

SummaryThe histochemical binding of seven fluoreceinated lectins was comparatively studied in muscular tissue from twenty three different animal species including mamalians, amphibians, avians and fishes. Special interest was taken in the exploration of the differential lectin-binding properties at the neuromuscular synapse. Binding to synaptic sites was demonstrated using lectins that recognizes N-acetylgalactosamine and among of them, Dolichus biflorus agglutinin (DBA), was the most specific. Nevertheless, DBA fails to stain endplates in the muscle from most of the avians and the fishes (including the Torpedo electric organ) indicating that a polymorphic distribution of glycoconjugates exist at the vertebrate neuromuscular junction. Other lectins such as Concanavalin A (ConA) or Wheat germ agglutinin (WGA), share a similar staining properties in all animals that we examined making an intense label over the complete muscle surface. Although the species-related polymorphism on lectin binding does not reveal a clear relationship with the evolutionary tree, they give an evidence on the chemical heterogeneity of molecules specifically concentrated at the neuromuscular junction.

Receptors to agglutinin fromDolichus biflorus (DBA) at the synaptic basal lamina of rat neuromuscular junction

SummaryThe binding of agglutinin fromDolichus biflorus (DBA) and other lectins (Concanavalin A, agglutinin from wheat germ and lectin fromBandeiraea simplicifolid) to synaptic and extrasynaptic portions of the basal lamina of muscle fibers, was studied with histochemical methods. In rat muscle, DBA-binding is specifically detected at the basal lamina of neuromuscular junction. However, long-term (6 months) denervated end-plate in adult rat muscle failed to bind DBA. During normal development, synaptic DBA receptors appear later than acetylcholine receptors or acetylcholinesterase at the rat neuromuscular junction. Generalized DBA-binding to motor end-plates is first visualized in 3-day-old rats, but section of sciatic nerve in 1-day-old rats prevents the appearence of synaptic DBA-binding on the leg end-plates. It is suggested, therefore, that the synaptic DBA receptors could be related to the postnatal stabilization of rat neuromuscular synapses.

c-Jun-like Immunoreactivity in Apoptosis Is the Result of a Crossreaction with Neoantigenic Sites Exposed by Caspase-3-mediated Proteolysis

Previous reports in various cells and species have shown that apoptotic cells are specifically and strongly labeled by certain c-Jun/N-terminal antibodies, such as c-Jun/sc45. This kind of immunoreactivity is confined to the cytoplasm. It is not due to c-Jun but appears to be related to c-Jun-like neoepitopes generated during apoptosis. This study was planned to gain further information about c-Jun-like immunostaining during apoptosis and to evaluate these antibodies as possible tools for characterizing cell death. Most of the experiments were performed in chick embryo spinal cord. When the apoptotic c-Jun-like immunoreactivity and caspase-3 immunostaining patterns were compared, we found that both antibodies immunostained the same dying cells in a similar pattern. In contrast to TUNEL staining, which reveals a positive reaction in both apoptotic and necrotic dying cells, active caspase-3 and c-Jun/sc45 antibodies are more selective because they stained only apoptotic cells. When cytosolic extracts from normal tissues were digested in vitro with caspase-3, c-Jun/sc45 immunoreactivity was strongly induced in several proteins, as demonstrated by Western blotting. Similar results were found when normal tissue sections were treated with caspase-3. Our results show that c-Jun/sc45 antibodies react with neoepitopes generated from cell proteins cleaved by activated caspases during apoptosis. We conclude that c-Jun/sc45 antibodies may be useful for detecting apoptosis. They can even be used in archival paraffin-embedded tissue samples.