Strong c-Jun Immunoreactivity Research Articles

Co-expression of c-Jun and ATF-2 characterizes the surviving retinal ganglion cells which maintain axonal connections after partial optic nerve injury

The expression of c- fos, c- jun, jun-b, jun-d, srf and pc4 mRNA was examined after partial optic nerve crush in the adult rat retina by in situ hybridization. Optic nerve injury led exclusively to the upregulation of c- jun, with cellular label indicative for c- jun mRNA in the retinal ganglion cell layer after two days, three days and one week post-injury. This expression pattern was in accordance with the appearance of c-Jun immunoreactivity in retinal flat mounts. Injection of an antisense but not a missense oligonucleotide against c- jun after partial crush resulted in a reduced number of connected retinal ganglion cells (RGCs) as shown by retrograde labeling. Prelabeling of RGCs with fluorogold before optic nerve section and subsequent antisense targeting against c- jun, however, led to a slightly higher number of surviving but axotomized RGCs. C-Jun antibody staining of retinal whole mounts pre- or postlabeled after crush by intracollicular administration of fluorogold showed strong c-Jun immunoreactivity in connected RGCs and also in a population of disconnected RGCs. Double labeling with an antibody directed against the transcription factor ATF-2 revealed strong co-expression of c-Jun and ATF-2 in connected RGCs but not in axotomized cells. Taken together these data indicate that both RGCs in continuity and those in discontinuity with the superior colliculus respond both equally to the noxious stimulus with c-Jun expression. Moreover, the co-expression of c-Jun with high levels of ATF-2 appears to be essential for either the continuity or survival of RGCs which remain connected with their target. In disconnected RGCs, however, low levels of ATF-2 and the co-expression of c-Jun may be related to cell death.

Radiation-induced apoptosis in developing rats and kainic acid-induced excitotoxicity in adult rats are associated with distinctive morphological and biochemical c-Jun/AP-1 (N) expression

Ionizing radiation produces apoptosis in the developing rat brain. Strong c-Jun immunoreactivity, as revealed with the antibody c-Jun/AP-1 (N) which is raised against the amino acids 91–105 mapping with the amino terminal domain of mouse c-Jun p39, is simultaneously observed in the nucleus and cytoplasm of apoptotic cells. Western blotting of total brain homogenates, using the same antibody, shows a p39 band in control rats which is accompanied by a strong, phosphorylated p62 double-band in irradiated animals. In addition, increased c-Jun N-terminal kinase 1 expression, as found on western blots, is found in irradiated rats when compared with controls. Intraperitoneal injection of kainic acid at convulsant doses to the adult rat produces cell death with morphological features of necrosis, together with the appearance of cells with fine granular chromatin degeneration and small numbers of apoptotic-like cells, in the entorhinal and piriform cortices, basal amygdala, certain thalamic nuclei, and CA1 region of the hippocampus. c-Jun expression in kainic acid-treated rats, as revealed with the c-Jun/AP-1 (N) antibody, is found in the nuclei of a minority of cells in the same areas. The vast majority of c-Jun-immunoreactive cells have normal nuclear morphology, whereas necrotic cells are negative and only a few cells with fine granular chromatin condensation and apoptotic cells following kainic acid injection are stained with c-Jun antibodies. Western blotting, using the same antibody, shows a p39 band in control rats, which is accompanied by a band at about p26 from 6 h onwards following kainic acid injection. Decreased c-Jun N-terminal kinase 1 expression, as revealed on western blots, is observed in kainic acid-treated rats. These results show that the antibody c-Jun/AP-1 (N) recognizes three different forms of c-Jun-related immunoreactivity in normal and pathological states, which are associated with the different outcome of cells. These results stress the necessity of examining in detail the composition of c-Jun-immunoreactive bands and the metabolic state of c-Jun(s) in different paradigms of cell death and survival.

Strong c-Jun/AP-1 immunoreactivity is restricted to apoptotic cells following intracerebral ibotenic acid injection in developing rats

Strong c-Jun immunoreactivity, as revealed with the antibody c-Jun/activator protein 1 (AP-1) which is raised against the amino acids 91–105 mapping with the amino terminal domain of mouse c-Jun p39, is observed in apoptotic cells, but not in necrotic cells, following intracerebral injection of ibotenic acid in the developing rat brain processed for immunohistochemistry. Immunostaining occurs in the cytoplasm and dendrites, thus suggesting impaired nuclear translocation of c-Jun in apoptotic cells. Western blotting of total brain homogenates, using the same antibody, shows a band at p39 which is more marked in treated animals than in age-matched controls. In addition, increased c-Jun N-terminal kinase 1 (JNK-1) expression, as revealed on Western blots, is found in rats treated with ibotenic acid when compared with controls. In contrast, apoptotic cells are not stained with antibodies to Jun B and Jun D. These results give further support to previous studies showing strong c-Jun expression in apoptotic cells at determinate stages of development, and emphasize that intracellular distribution of c-Jun, possible post-translational modifications of c-Jun due to phosphorylation at specific transactivation sites, and lack of associated Jun B and Jun D expression may differentiate the Jun response in apoptotic cells from other forms of cellular response involving c-Jun which are not associated with cell death.

Spinal muscular atrophy in Holstein-Friesian calves.

The clinical and neuropathological findings of spinal muscular atrophy (SMA) in Holstein-Friesian calves are described in four females and one male from a dairy farm composed of 150 cows and 2 breeding bulls. Locomotion difficulties started at the age of 15 days, and progressed to paraparesis and tetraparesis in 2 weeks. Signs consistent with denervation were revealed with electromyography. The neuropathological examination showed degeneration and loss of motor neurons in the spinal cord, together with astrocytosis. Among the remaining motor neurons were ghost cells and neurons filled with accumulations of straight filaments measuring 10-12 nm in diameter, which were strongly immunoreactive with antibodies produced against phosphorylated neurofilaments. Degenerating cells in SMA did not stain with the method of in situ labelling of nuclear DNA fragmentation and did not show c-Jun immunoreactivity. This feature contrasts with the in situ labelling of DNA breaks of apoptotic cells and with the strong c-Jun immunoreactivity restricted to dying cells during the whole process of naturally occurring cell death in the developing central nervous system. These features suggest that cell death in SMA differs from programmed cell death during normal development, and that pathological cell death in SMA should not be considered as a mere persistence or reactivation of normally occurring developmental cell death.

Methylazoxymethanol acetate-induced apoptosis in the external granule cell layer of the developing cerebellum of the rat is associated with strong c-Jun expression and formation of high molecular weight c-Jun complexes.

Intraperitoneal administration of methylazoxymethanol (MAM) acetate (0.05 microl/g of body weight) in male Sprague-Dawley rats aged 3 days produced cell death in the external granule layer of the cerebellum which peaked at 48 hours (h) and was followed by removal of cellular debris at 72 h. Dying cells had the morphological features of apoptosis and were stained with the method of in situ labeling of nuclear DNA fragmentation. Strong c-Jun immunoreactivity was observed in apoptotic cells during the whole process of MAM-induced apoptosis. No differences of c-Fos immunoreactivity were observed between control and MAM-treated rats throughout the period studied. Western blotting of cerebellar homogenates in control rats disclosed two bands which reacted with both c-Jun antibodies, one located at p39 that corresponds to the molecular weight of c-Jun, and the other at about p62. MAM-treated rats showed a robust band at p62, together with a thinner band located immediately above it, which was accompanied by a reduction of the p39 band. The specificity of the immunoreaction was tested by incubating the antibodies with the appropriate control peptides. No difference between control and MAM-treatad rats was observed in Western blots processed with antibodies to c-Fos during this study. These results show that MAM-induced apoptosis in the external granule cell layer of the rat is associated with strong c-Jun expression, which is restricted to apoptotic cells, and with the formation of high-molecular-weight c-Jun complexes. Taken together, the present observations suggest that c-Jun may participate in the genetic cascade of events leading to apoptotic cell death in the developing cerebellum.

Cell death in the normal developing brain, and following ionizing radiation, methyl-azoxymethanol acetate, and hypoxia-ischaemia in the rat.

Naturally occurring (programmed) cell death in the developing brain has morphological characteristics of apoptosis and is associated with internucleosomal DNA fragmentation. Apoptosis also plays a role in cell death following hypoxia-ischaemia in the developing rat brain. Ionizing radiation-induced cell death in the brain of the young rat has morphological characteristics of apoptosis, is mediated by protein synthesis and is associated with internucleosomal DNA fragmentation. Methyl-azoxymethanol (MAM) acetate injection in the young rat produces apoptotic cell death in the external granule cell layer of the cerebellum. In addition, strong c-Jun immunore-activity is observed in apoptotic cells during normal development and following experimentally induced cell death. Moreover, c-Jun mRNA induction and de novo c-Jun protein synthesis, together with activation of c-Jun/AP-1, as revealed with gel mobility shift assay, occurs in irradiated animals. Western blotting of total brain homogenates shows a c-Jun-immunoreactive band at p39, which corresponds to the molecular weight of c-Jun, in control rats. However, a thick c-Jun-immunoreactive band at about p62, accompanied by a decrease of the p39 band, occurs in irradiated and MAM-treated rats. A thin band immediately above the thick p62 band, suggestive of c-Jun phosphorylation, is also observed in treated rats. Taken together, these observations indicate that c-Jun expression is associated with apoptotic cell death in the developing central nervous system.

Strong c-Jun immunoreactivity is associated with apoptotic cell death in human tumors of the central nervous system

Strong c-Jun immunoreactivity is associated with apoptotic cell death in human tumors of the central nervous system

Strong c-Jun immunoreactivity is associated with apoptotic cell death in human tumors of the central nervous system

Strong c-Jun immunoreactivity, but not c-Fos expression, was observed in typical apoptotic cells in medulloblastomas, central neuroblastomas, malignant astrocytomas and glioblastomas, and in lymphocyte-like cells bearing nuclear fragmented DNA in medulloblastomas. In contrast, only a few proliferating cells, as revealed with PCNA immunohistochemistry, colocalized c-Jun. These results support the concept that strong c-Jun expression is associated with certain forms of cell death in human tumors of the central nervous system.

Jun expression is found in neurons located in the vicinity of subacute plaques in patients with multiple sclerosis

The expression of members of the Fos and Jun families is examined by immunohistochemistry in the brains of two patients with multiple sclerosis (MS). Strong c-Jun immunoreactivity is observed in the cytoplasm of neurons located in the vicinity of subacute plaques, but not in neurons of brain compartments not compromised by MS and in the neighborhood of chronic plaques. Strong c-Jun immunoreactivity also contrast with weak c-Jun immunoreactivity of corresponding neurons in control brains. In addition, punctate Jun D immunoreactivity is observed in the neuropil of the same areas that express c-Jun. No immunoreaction is found to c-Fos, Fos-related antigens and Jun B in these areas. The present results suggest that selective Jun neuronal expression in the vicinity of subacute plaques is a consistent reaction to demyelination and axonal damage.

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.