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Biomedical subjects

J Hugon

Publications and source records attributed to J Hugon.

At least 19 recordsLinked to original sources

N-methyl-D-aspartate receptor blockade enhances neuronal apoptosis induced by serum deprivation.

Neuronal apoptosis a hallmark of brain development could also be involved in neurodegenerative diseases. Glutamate toxicity is widely proposed as an important factor in the pathogenesis of neurological disorders. We show here that, in rat primary cortical cultures, the blockade of N-methyl-D-aspartate (NMDA) glutamate receptors exacerbated neuronal apoptosis induced by serum deprivation. This effect is observed at early stage of cultures (9 days in vitro (DIV)) and mildly decreases in more mature cultures (13 and 15 DIV). At the opposite, low concentrations of NMDA (5 microM) or glutamate (5 microM) prevented the neuronal apoptosis induced by trophic support withdrawal. In primary cortical cultures, the proapoptotic effect of trophic support removal can be modulated by NMDA receptors depending upon the magnitude of these glutamate receptor activation.

Animals↗

The Guam cycad toxin methylazoxymethanol damages neuronal DNA and modulates tau mRNA expression and excitotoxicity.

As in Alzheimer's disease, brains of Guam Chamorros with amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia complex (PDC) contain intraneuronal-paired helical filaments composed of accumulated phosphorylated tau protein. Tau mRNA expression in rat neuronal cultures-normally modulated by glutamate-increases after treatment with the aglycone of cycasin, a cycad-derived toxin whose concentration in Chamorro food varies with disease incidence. Elevated Tau gene expression in vitro is coincident with increased cycasin-related DNA adducts and reduced DNA repair. Cycasin and endogenous glutamate may together promote the accumulation of tau protein and neuronal degeneration in Western Pacific ALS/PDC.

Animals↗

Damage and repair of nerve cell DNA in toxic stress.

It is generally agreed that ALS/PDC is triggered by a disappearing environmental factor peculiar to the lifestyle of people of the western Pacific (i.e., Guam, Irian Jaya, Indonesia, and the Kii Peninsula of Japan). A strong candidate is the cycad plant genotoxin cycasin, the beta-D-glucoside of methylazoxymethanol (MAM). We propose that prenatal or postnatal exposure to low levels of cycasin/MAM may damage neuronal DNA, compromise DNA repair, perturb neuronal gene expression, and irreversibly alter cell function to precipitate a slowly evolving disease ("slow-toxin" hypothesis). In support of our hypothesis, we have demonstrated the following: 1. DNA from postmitotic rodent central nervous system neurons is particularly sensitive to damage by MAM. 2. MAM reduces DNA repair in human and rodent neurons, whereas DNA-repair inhibitors potentiate MAM-induced DNA damage and toxicity in mature rodent nervous tissue. 3. Human neurons (SY5Y neuroblastoma) that are deficient in DNA repair are susceptible to MAM-induced cytotoxicity and DNA damage, whereas overexpression of DNA repair in similar cells is protective. 4. MAM alters gene expression in SY5Y human neuroblastoma cells and, in the presence of DNA damage and reduced DNA repair, enhances glutamate-modulated expression of tau mRNA in rat primary neurons; the corresponding protein (TAU) is elevated in ALS/PDC and Alzheimer's disease. These findings support a direct relationship between MAM-induced DNA damage and neurotoxicity and suggest the genotoxin may operate in a similar manner in vivo. More broadly, a combination of genotoxin-induced DNA damage (via exogenous and/or endogenous agents) and disturbed DNA repair may be important contributing factors in the slow and progressive degeneration of neurons that is characteristic of sporadic neurodegenerative disease. Preliminary studies demonstrate that DNA repair is reduced in the brain of subjects with western Pacific ALS/PDC, ALS, and Alzheimer's disease, which would increase the susceptibility of brain tissue to DNA damage by endogenous/exogenous genotoxins. Interindividual differences in the extent of prior exposure to DNA-damaging agents and/or the efficiency of its repair might produce population variety in the rate of damage accumulation and explain the susceptibility of certain individuals to sporadic neurodegenerative disease. Studies are underway using DNA-repair proficient and deficient neuronal cell cultures and mutant mice to explore gene-environment interplay with respect to MAM treatment, DNA damage, and DNA repair, and the age-related appearance of neurobehavioral and neuropathological compromise.

Animals↗

Toxic neuronal apoptosis and modifications of tau and APP gene and protein expressions.

The causes and the mechanisms of neuronal death in Alzheimer's disease are not elucidated, although some new insights have been proposed over the past years, including free-radical toxicity, beta-amyloid toxicity, excitotoxicity, and disturbed cellular calcium metabolism. Some authors have also pointed out that apoptosis could play a role in neuronal degeneration, but it is still largely debated. Here, we review some recent data linking the induction of experimental neuronal apoptosis in vitro and the molecular pathology of the tau protein and amyloid precursor protein (APP). In cultures exposed to mild glutamate toxicity, tau mRNA expression, not beta-actin, is enhanced in stressed neurons. The Guam cycad toxin metabolite methylazoxymethanol also produces an increase of tau gene transcription that exacerbates changes induced by glutamate. In serum-deprived cultures or glutamate-exposed cultures, neurons committed to apoptosis have a reduced tau gene expression, whereas resistant neurons display a stable or even augmented tau mRNA expression accompanied by a persistent tau phosphorylation near serine 202. In the same conditions, stressed neurons produce membrane blebbings strongly immunopositive for APP and putative amyloidogenic fragments that are subsequently released in the extracellular space. Experimental apoptosis in neurons can recapitulate tau and APP modifications that could be associated with a selective vulnerability and a progression of cellular degeneration along the neuronal network.

Amyloid beta-Protein Precursor↗

Mild kainate toxicity produces selective motoneuron death with marked activation of CA(2+)-permeable AMPA/kainate receptors.

Motoneuron death could be produced by higher sensitivity to excitoxicity during the development and pathological conditions. We report here that in ventral spinal cord cultures mild kainate exposure (12.5 microM, 20 min or 100 microM, 2.5 min) induced selective cobalt stain of motoneurons, indicating a specific Ca2+ entry through the Ca(2+)-permeable AMPA/kainate receptors. This result was associated with a selective motoneuron death as previously described. In these cultures, motoneuron immunoreactivity for the Ca2+ buffering protein, calretinin was negative. These findings suggest that the selective motoneuron death due to a mild excitotoxic insult could be linked to a marked Ca2+ influx associated with the lack of some Ca2+ buffering proteins.

Animals↗

Brefeldin A-induced apoptosis is expressed in rat neurons with dephosphorylated tau protein.

Brefeldin A (BFA) is a fungus metabolite (penicillum brefeldanum) that is known to produce the disintegration of the Golgi apparatus in exposed cells, and apoptosis in various cancer cells. This study reports that in rat primary cortical cell cultures BFA also produces apoptosis assessed by the TUNEL method and DAPI (4',6-diemidino-2-phenylindole) staining. The percentages of apoptotic neurons range from 26.9% +/- 8.3 to 43.2 +/- 2.5% in cultures exposed from 4 to 8 h to BFA (10 microg/ml). A double fluorescent staining, using AT8 antibody (phosphorylated tau) or tau1 antibody (dephosphorylated tau) associated with DAPI labeling reveals that tau1 positive neurons are more sensitive to BFA-induced apoptosis compared to AT8 positive neurons. This result and previous results using other apoptosis inducers suggest that tau phosphorylation in the vicinity of the AT8-tau1 epitopes is a marker of resistance or sensitivity to neuronal apoptosis in rat cortical cell cultures.

Adrenal Cortex↗

FK506 antagonizes apoptosis and c-jun protein expression in neuronal cultures.

FK506 is an immunosuppressive drug that binds to FK506 binding protein (FKBPs), a subgroup of cytosolic proteins called immunophillins. Previous works have revealed that FK506 protects neural cells from ischemia or excitotoxicity. Here we report that FK506 (10(-6) M) and not cyclosporine A (10(-6) M) blocks neuronal apoptosis induced by serum deprivation in rat neuronal cultures. In addition the immunohistochemical staining of C-jun protein in deprived cultures is markedly attenuated by FK506. The proportion of C-jun-positive neurons in control cultures, in serum-deprived cultures (48 h) and in serum-deprived cultures exposed to FK506 (10(-6) M) were 12.5%, 56.5% and 16.5%, respectively. The down-regulation of C-jun could play a major role in the anti-apoptotic action of FK506 in stressed neuronal cultures.

Animals↗

Neuronal apoptosis is associated with a decrease in tau mRNA expression.

Apoptosis is a programmed cell death that occurs during the development of the nervous system and in neurodegenerative disorders. Tau protein is a microtubule-associated protein which promotes microtubule polymerization and stabilization. Apoptosis was induced in primary neuronal cultures by glutamate exposure (200 microM, 15 min) or by serum deprivation, and tau mRNA levels were studied by quantitative in situ hybridization in apoptotic and non apoptotic neurons. Compared to controls, tau mRNA expression was decreased in apoptotic neurons produced by excitotoxicity or trophic support withdrawal. Under these conditions, resistant neurons to apoptosis display either increased tau mRNA levels after glutamate exposure or a stable tau mRNA expression after serum deprivation. In conclusion, in this in vitro model, neurons which are resistant and sensitive to apoptosis can be differentiated according to tau mRNA expression.

Animals↗

Characterization of human presenilin 1 transgenic rats: increased sensitivity to apoptosis in primary neuronal cultures.

Mutations in the gene for presenilin 1 are causative for the majority of cases of early onset familial Alzheimer's disease. Yet, the physiological function of presenilin 1 and the pathological mechanisms of the mutations leading to Alzheimer's disease are still unknown. To analyse potential pathological effects of presenilin 1 over-expression, we have generated transgenic rats which express high levels of human presenilin 1 protein in the brain. The over-expression of presenilin 1 leads to saturation of its normal processing and to the appearance of full-length protein in the transgenic rat brain. The transgenic protein is expressed throughout the brain and is predominantly found in neuronal cells. Cultured primary cortical neurons derived from these transgenic rats are significantly more sensitive than non-transgenic controls to apoptosis induced by standard culture conditions and to apoptosis induced by trophic factor withdrawal. Furthermore, the observed apoptosis is directly correlated with the expression of the transgenic protein. The results further emphasize the role of presenilin 1 in apoptotic cell death in native neuronal cultures.

Alzheimer Disease↗

Effects of depolarizing stimuli on calcium homeostasis in cultured rat motoneurones.

Intracellular calcium concentrations in individual rat motoneurones in enriched primary cultures were measured by Indo-1 fluorimetry. Motoneurones in the cultures were characterized morphometrically and by cholineacetyltransferase immunocytochemistry. Depolarization of the cells with glutamic acid or veratridine increased intracellular calcium levels, which returned to baseline only slowly after removal of the depolarizing agent. The use of selective agonists (N-methyl-D-aspartic acid, AMPA, kainic acid, quisqualic acid and 1R-3S-ACPD) and antagonists (MK 801 and CNQX) showed that the excitatory amino acid-evoked responses were mediated by AMPA/kainate receptors rather than by NMDA receptors. Depolarization-evoked calcium transients in motoneurones are blocked by the neuroprotective drug riluzole Calcium transients reflected entry of calcium from without the cell, and their blockade by nitrendipine and lanthanum chloride suggested that this entry took place primarily through voltage-dependent calcium channels. These findings may be relevant for understanding the selective vulnerability of motoneurones to excitotoxicity in amyotrophic lateral sclerosis, and the therapeutic activity of riluzole in the treatment of this disease.

Animals↗

Glutamate toxicity enhances tau gene expression in neuronal cultures.

Tau protein is a microtubule-associated protein normally expressed in neurons. In Alzheimer's disease (AD) brains, phosphorylated tau accumulates in paired helical filaments which form neurofibrillary tangles in affected neurons; moreover, tau mRNA expression is increased in affected regions of AD brains. Glutamate, an excitatory neurotransmitter but also a potent neurotoxin under pathologic conditions, is known to produce neuronal degeneration and death accompanied by an increase in tau immunoreactivity in primary neuronal cultures. The goal of the present study is to evaluate the effects of glutamate on tau gene expression in neuronal cultures. We report a delayed and long-lasting enhancement of tau mRNA expression after a 15 min exposure to toxic concentrations of glutamate: neuronal tau mRNA levels reach a peak after 3 hr and remain increased 6 and 12 hr after the end of glutamate exposure. Both NMDA and AMPA/ kainate receptors are involved in this tau gene overexpression. Actinomycin D prevents this tau mRNA induction indicating that transduction signals elicited by glutamate act at the transcriptional level. The role of this delayed tau overexpression is not elucided but could be linked to either a reactive survival process or to a programmed cellular degeneration.

Animals↗

NMDA induces apoptosis and necrosis in neuronal cultures. Increased APP immunoreactivity is linked to apoptotic cells.

In rat neuronal cultures exposed for a prolonged period (16 h) to a low concentration of N-methyl-D-aspartate (NMDA; 20 microM) two different types of neuronal death were observed, necrosis and apoptosis. The immunocytochemical study of necrotic neurons revealed the absence of amyloid precursor protein (APP) labeling in the cytoplasmic region. In contrast, neuronal apoptosis was associated with an increase in cytoplasmic APP immunoreactivity. In addition, the presence of extracellular APP was also detected around some apoptotic neurons, suggesting that apoptosis could contribute to the presence of extracellular APP.

Amyloid beta-Protein Precursor↗

Neuronal APP accumulates in toxic membrane blebbings.

The occurence of plasma membrane blebbings is an early cytotoxic event, associated with the reorganization of cytoskeletal proteins, the alteration of interactions between the plasma membrane and the underlying cytoskeleton. The blebbing formation remains poorly understood but the involvement of cytosolic Ca2+ and the production of free radicals may contribute to this cellular phenomenom. The amyloid precursor protein (APP), is a transmembrane protein that can be cleaved to produce the beta amyloid peptide (Abeta) which accumulates in brain senile plaques of Alzheimer's disease. Our study reveals that the exposure of rat and human (hNT) neuronal cultures to a mild concentration of the excitotoxin NMDA slowly induces perturbations of the neuronal cytoskeleton and the occurence of plasma membrane blebbings. An immunocytochemical study using four different APP antibodies demonstrates that these membrane blebs are also associated with a redistribution and an accumulation of cellular APP. This phenomenon is linked to a Ca2+-influx through NMDA-receptors since it is prevented by the NMDA antagonist MK801 or by Ca2+-depleted conditions. In conclusion this study shows that neuronal degeneration induced by slow excitotoxicity, is associated with the presence of APP-accumulating blebs, that can be secondly released in the extracellular region.

Amyloid beta-Protein Precursor↗

Therapeutic advances in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and rapidly fatal neurodegenerative disease in which both upper and lower motoneurones are involved. The recent discovery of mutations affecting the superoxide dismutase (SOD) gene has given impetus to research on the role of oxidative stress in the pathogenesis of familial ALS, while further evidence for a role of excitotoxicity in the disease process has arisen. In this review, Erik Louvel, Jacques Hugon and Adam Doble discuss these findings and, in addition, describe how a number of large, well-controlled clinical trials have taken place to test potential therapies suggested by different aetiological hypotheses, including immunosuppressive therapies, neurotrophic factors, antioxidants and anti-excitotoxic drugs. These trials have led to the first modest steps in the treatment of this devastating neurological disease.

Amyotrophic Lateral Sclerosis↗

Cultured neurons expressing phosphorylated tau are more resistant to apoptosis induced by NMDA or serum deprivation.

Apoptosis is a programmed cell death that occurs during the development of the nervous system and in neurodegenerative disorders. Tau protein is a cytoskeletal component that promotes microtubule polymerization and stabilization. Apoptosis was induced in primary neuronal cultures by a prolonged exposure (16 h) to the NMDA (N-methyl-D-aspartate 20 microM) or by serum deprivation. The percentages of apoptotic neurons expressing phosphorylated tau (AT8) immunoreactivity are comparable in control and NMDA-exposed cultures (7.5 +/- 1.9 and 6.9 +/- 1.9%, respectively). At the opposite, the percentage of apoptotic neurons expressing de-phosphorylated tau (tau 1) immunolabelings is dramatically increased in NMDA-treated cultures (X 2.3 of controls). Similar results were also observed 48 h after serum deprivation. These results demonstrate in vitro that under these conditions, resistant and sensitive cortical neurons to apoptosis can be partly differentiated according to their phosphorylated tau immunoreactivities.

Animals↗

Increased hypoxic tolerance by chemical inhibition of oxidative phosphorylation: "chemical preconditioning".

A short ischemic episode preceding sustained ischemia is known to increase tolerance against ischemic cell death. We report early-onset long-lasting neuroprotection against in vitro hypoxia by preceding selective chemical inhibition of oxidative phosphorylation: "chemical preconditioning." The amplitude of CA1 population spikes (psap) in hippocampal slices prepared from control animals (control slices) was 31 +/- 27% (mean +/- SD) upon 45-min recovery from 15-min in vitro hypoxia. In slices prepared from animals treated in vivo with 20 mg/kg 3-nitropropionate (3-np) 1-24 h prior to slice preparation (preconditioned slices), psap improved to 90 +/- 15% (p < 0.01). Posthypoxic oxygen free radicals were reduced to 65 +/- 10% (mean +/- SD) of control in preconditioned slices (p < 0.05). Posthypoxic neuronal density improved from 52 +/- 15% (mean +/- SD) in control slices to 97 +/- 23% in preconditioned slices (p < 0.001). Glibenclamide, an antagonist at KATP-channels, partly reversed increased hypoxic tolerance. We conclude that chemical preconditioning induces early-onset long-lasting tolerance against in vitro hypoxia. Ultimately, this strategy may be applicable as a neuroprotective strategy in humans.

Adenosine Triphosphate↗

Identical chromosome imbalance in two siblings born to a mother with a double reciprocal translocation.

We report the case of a woman who carried two reciprocal translocations. Her karyotype was 46,XX,t(3;12)(q12;q21)(4;17)(p14;p13). She had two children, a phenotypically normal daughter (karyotype (46,XX,t(3;12)(q12;q21)) and a son with partial 4p trisomy (karyotype 46,XY,t(3;12) (q12;q21),-17,+maternal der(17)). She was pregnant with a female fetus who had the same karyotype as her son. She also reported a history of two spontaneous abortions. This viable recurrent abnormality was due to the maternal (4;17) translocation with meiotic segregation type 2:2 adjacent 1. In this case of the two reciprocal translocations carried by the mother, one led to imbalances, whereas the other remained balanced in the viable offspring.

Adult↗