Biomedical subjects

J Mariani

Publications and source records attributed to J Mariani.

At least 37 records · Page 2Linked to original sources

PubMed2001

Ceramide-induced apoptosis in cortical neurons is mediated by an increase in p38 phosphorylation and not by the decrease in ERK phosphorylation.

Ceramide, the central molecule of the sphingomyelin pathway, serves as a second messenger for cellular functions ranging from proliferation and differentiation to growth arrest and apoptosis. In this study we show that c2-ceramide induces apoptosis in primary cortical neuron cultures and that this effect correlates with differential modulation of mitogen-activated protein kinase (MAPK) cascades. Phosphorylation of extracellular signal-regulated kinases (ERKs) and their upstream activators MAPK kinases (MEKs), as measured by immunoblotting is rapidly decreased by c2-ceramide. However, the MEK inhibitor PD98059 alone does not induce apoptosis and in combination with c2-ceramide it does not modify c2-ceramide-induced apoptosis. Treatment with c2-ceramide increases p38 and c-Jun N-terminal kinase (JNK) phosphorylation before and during caspase-3 activation. The p38 inhibitor SB203580 partially protects cortical neurons against c2-ceramide-induced apoptosis, implicating the p38 pathway in this process. The c2-ceramide treatment also increases levels of c-jun, c-fos and p53 mRNA in primary cortical neuron cultures, but this is independent of p38 activation. Our study further elucidates the time-courses of MAPK cascade modulation, and of c-jun, c-fos and p53 activation during c2-ceramide-induced neuronal apoptosis. It reveals that one of the activated kinases, p38, is necessary for this apoptosis.

Animals↗

PubMed2001

The orphan nuclear receptor ROR alpha is a negative regulator of the inflammatory response.

Retinoid-related orphan receptor alpha (ROR alpha) (NR1F1) is a member of the nuclear receptor superfamily whose biological functions are largely unknown. Since staggerer mice, which carry a deletion in the ROR alpha gene, suffer from immune abnormalities, we generated an adenovirus encoding ROR alpha1 to investigate its potential role in control of the inflammatory response. We demonstrated that ROR alpha is expressed in human primary smooth-muscle cells and that ectopic expression of ROR alpha1 inhibits TNFalpha-induced IL-6, IL-8 and COX-2 expression in these cells. ROR alpha1 negatively interferes with the NF-kappaB signalling pathway by reducing p65 translocation as demonstrated by western blotting, immunostaining and electrophoretic mobility shift assays. This action of ROR alpha1 on NF-kappaB is associated with the induction of IkappaB alpha, the major inhibitory protein of the NF-kappaB signalling pathway, whose expression was found to be transcriptionally upregulated by ROR alpha1 via a ROR response element in the IkappaB alpha promoter. Taken together, these data identify ROR alpha1 as a potential target in the treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis.

Adenoviridae↗

PubMed2000

Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha.

Triglyceride-rich remnant lipoproteins are considered as major risk factors contributing to the pathogenesis of atherosclerosis. Because apolipoprotein (apo) C-III is a major determinant of plasma triglyceride and remnant lipoprotein metabolism, it is important to understand how the expression of this gene is regulated. In the present study, we identified the orphan nuclear receptor RORalpha1 as a regulator of human and mouse apo C-III gene expression. Plasma triglyceride and apo C-III protein concentrations in staggerer (sg/sg) mice, homozygous for a deletion in the RORalpha gene, were significantly lower than in wild type littermates. The lowered plasma apo C-III levels were associated with reduced apo C-III mRNA levels in liver and intestine of sg/sg mice. Transient transfection experiments in human hepatoma HepG2, human colonic CaCO2, and rabbit kidney RK13 cells demonstrated that overexpression of the human RORalpha1 isoform specifically increases human apo C-III promoter activity, indicating that RORalpha1 enhances human apo C-III gene transcription. RORalpha1 response elements were mapped by promoter deletion analysis and gel shift experiments to two AGGTCA half-sites located at positions -83/-78 (within the C3P site) and -23/-18 (downstream of the TATA box) in the human apo C-III promoter, with the -23/-18 site exhibiting the highest binding affinity. Transfection of site-directed mutated constructs in HepG2 cells indicated that the RORalpha1 effect is predominantly mediated by the -23/-18 site. This site is conserved in the mouse apo C-III gene promoter. Moreover, RORalpha binds to the equivalent mouse site and activates constructs containing three copies of the mouse site cloned in front of an heterologous promoter. Taken together, our data identify RORalpha as a transcriptional regulator of apo C-III gene expression, providing a novel, physiological role for RORalpha1 in the regulation of genes controlling triglyceride metabolism.

Animals↗

PubMed2000

In vitro and in vivo evidence for orphan nuclear receptor RORalpha function in bone metabolism.

Bone is a major target site for steroid hormone action. Steroid hormones like cortisol, vitamin D, and estradiol are responsible for principal events associated with bone formation and resorption. Over the past decade, new members of the nuclear hormone gene family have been identified that lack known ligands. These orphan receptors can be used to uncover signaling molecules that regulate yet unidentified physiological networks. In the present study the function of retinoic acid receptor-related orphan receptor (ROR) alpha in bone metabolism has been examined. We showed that RORalpha and RORgamma, but not RORbeta, are expressed in mesenchymal stem cells derived from bone marrow. Interestingly, for RORalpha we observed an increased messenger signal expression between control cells and cells undergoing osteogenic differentiation. Furthermore, the direct activation of mouse bone sialoprotein by RORalpha, typically 7-fold, has been shown. In contrast, transient overexpression of RORalpha overrides the activation of the osteocalcin promoter by 1alpha,25-dihydroxyvitamin D(3). In addition, we have investigated bone mass parameters and bone geometry in the mouse mutant staggerer (sg/sg), a mouse strain that carries a deletion within the RORalpha gene. Homozygote mutants have thin long bones compared with the heterozygote animals and wild-type littermates. More interestingly, the bones of the sg/sg animals are osteopenic as indicated by the comparison of bone mineral contents of sg/sg animals to the heterozygote and wild-type animals. We conclude that these in vitro and in vivo results suggest a function for RORalpha in bone biology. RORalpha most likely acts by direct modulation of a bone matrix component.

Absorptiometry, Photon↗

PubMed2000

Bax inactivation in lurcher mutants rescues cerebellar granule cells but not purkinje cells or inferior olivary neurons.

Lurcher is a gain-of-function mutation in the delta2 glutamate receptor gene (Grid2) that turns the receptor into a leaky ion channel. The expression of the Lurcher gene in heterozygous (Grid2(Lc/+)) mutants induces the death of almost all Purkinje cells starting from the second postnatal week. Ninety percent of the granule cells and 60-75% of the inferior olivary neurons die because of the loss of their target neurons, the Purkinje cells. The apoptotic nature of the neurodegeneration has been demonstrated previously by the presence of activated caspase-3 and DNA fragmentation. Bax, a pro-apoptotic gene of the Bcl-2 family, has been shown to be involved in developmental neuronal death. To study the role of Bax in Grid2(Lc/+) neurodegeneration, double mutants with Grid2(Lc/)+ mice and Bax knock-out mice (Bax-/-) were generated. Bax deletion had no effect on the death of Purkinje cells and inferior olivary neurons, although a temporary rescue of some Purkinje cells could be detected in P15 Grid2(Lc/)+;Bax-/- animals. From postnatal day 15 (P15) to P60, the number of granule cells in Grid2(Lc/)+;Bax-/-mice did not significantly change and was significantly increased compared with the number found in Grid2(Lc/)+;Bax+/+ mice. Granule cell number in P60 Grid2(Lc/)+;Bax-/- mice corresponded to 70% of the number found in wild-type mice. Our results show that Bax inactivation in Grid2(Lc/+) mice does not rescue intrinsic Purkinje cell death or the target-related cell death of olivary neurons, but Bax inactivation does inhibit persistently target-related cell death in cerebellar granule cells.

Animals↗

PubMed2000

Synaptic prion protein immuno-reactivity in the rodent cerebellum.

The cellular prion protein PrP(c) is a neurolemmal glycoprotein essential for the development of the transmissible spongiform encephalopathies. In these neurodegenerative diseases, host PrP(c) is converted to infectious protease-resistant isoforms PrP(res) or prions. Prions provoque predictable and distinctive patterns of PrP(res) accumulation and neurodegeneration depending on the prion strain and on regional cell-specific properties modulating PrP(c) affinity for infectious PrP(res) in the host brain. Synaptolysis and synaptic accumulation of PrP(res) during PrP-related diseases suggests that the synapses could be primary sites able to propagate PrP(res) and neurodegeneration in the central nervous system. In the rodent cerebellum, the present light and electron microscopic immuno-cytochemical analysis shows that distinct types of synapses display differential expression of PrP(c), suggesting that synapse-specific parameters could influence neuroinvasion and neurodegeneration following cerebral infection by prions. Although the physiological functions of PrP(c) remain unknown, the concentration of PrP(c) almost exclusively at the Purkinje cell synapses in the cerebellum suggests its critical involvement in the synaptic relationships between cerebellar neurons in agreement with their known vulnerability to PrP deficiencies.

Animals↗

PubMed2000

Olivocerebellar climbing fibers in the granuloprival cerebellum: morphological study of individual axonal projections in the X-irradiated rat.

Elimination of cerebellar granule cells early during postnatal development produces abnormal neural organization that retains immature characteristics in the adult, including innervation of each Purkinje cell by multiple climbing fibers from the inferior olive. To elucidate mechanisms underlying development of the olivocerebellar projection, we studied light-microscopic morphology of single olivocerebellar axons labeled with biotinylated dextran amine in adult rats rendered agranular by a single postnatal X-irradiation. Each reconstructed olivocerebellar axon gave off approximately 12 climbing fibers, approximately twice as many as in normal rats. Terminal arborizations of climbing fibers made irregular tufts in most areas, whereas they were arranged vertically in a few mildly affected areas. Each climbing fiber terminal arborization innervated only part of the dendritic arbor of a Purkinje cell, and multiple climbing fibers innervated a single Purkinje cell. These climbing fibers originated either from the same olivocerebellar axon (pseudomultiple innervation) or from distinct axons (true multiple innervation). Abundant non-climbing fiber thin collaterals projected to all cortical layers. Although the longitudinal pattern of the zonal olivocerebellar projection was generally observed, lateral branching, including bilateral projections, was relatively frequent. These results suggest that the granule cell-parallel fiber system induces several important features of olivocerebellar projection: (1) organization of the climbing fiber terminal arborization tightly surrounding Purkinje cell dendrites, (2) elimination of pseudo- and true multiple innervations establishing one-to-one innervation, (3) retraction of non-climbing fiber thin collaterals from the molecular layer, and (4) probable refinement of the longitudinal projection domains by removing aberrant transverse branches.

Age Factors↗

PubMed2000

Central origin of IL-1beta produced during peripheral inflammation: role of meninges.

Brain expression of Interleukin-1beta (IL-1beta) and its modulation have been extensively documented in different animal models. The majority of the studies were based on techniques such as RT-PCR, RIA and ELISA using global extracts. Meningeal tissue or choroid plexus both belong to the peripheral compartment. Thus, their presence in nervous tissue extracts may lead to erroneous interpretations. We measured IL-1beta mRNA in the cerebellum, hippocampus and cerebral cortex collected with meninges and in the same structures collected without meninges after a peripheral lipopolysaccharide (LPS) stimulation (0.5 microgram/g body weight), using RT-PCR. The presence of meninges in the extracts dramatically increased IL-1beta mRNA levels after LPS treatment while basal levels (before LPS injection) were not affected. Indeed, two-thirds of the response originated from the meningeal tissue and choroid plexus. Immunohistochemical studies showed that IL-1beta labelled cells, identified as macrophages, were exclusively localized in the ventricular and meningeal spaces, in our LPS condition treatment. These results point out the need of integrated analyses of data obtained with different techniques to demonstrate the presence in the nervous tissue of a molecule which is also widely expressed in the peripheral compartment.

Animals↗

PubMed2000

Target-related and intrinsic neuronal death in Lurcher mutant mice are both mediated by caspase-3 activation.

The Lurcher (Lc) mutation in the delta2 glutamate receptor gene leads to the presence of a constitutive inward current in the cerebellar Purkinje cells of Lurcher heterozygous mice and to the postnatal degeneration of these neurons. In addition, cerebellar granule cells and olivary neurons of Lc/+ mice die as an indirect effect of the mutation after the loss of their target Purkinje cells. The apoptotic nature of Lc/+ Purkinje cell death remains controversial. To address this question, we studied the involvement of caspase-3, a key effector of apoptosis, in the neurodegenerative processes occurring in Lc/+ cerebellum. Several antibodies recognizing different regions of caspase-3 were used in immunoblotting and immunohistochemical experiments. We demonstrate that pro-caspase-3 is specifically upregulated in the dying Lc/+ Purkinje cells, but not in granule cells and olivary neurons, suggesting that different death-inducing signals trigger variant apoptotic pathways in the CNS. The subcellular localization of pro-caspase-3 was shown to be cytoplasmic and mitochondrial. Active caspase-3 as well as DNA fragmentation was found in numerous granule cells and some Purkinje cells of the Lc/+ cerebellum. Thus, caspase-3 activation is involved in both the direct and indirect neuronal death induced by the Lurcher mutation, strongly supporting the idea that the Lc/+ Purkinje cell dies by apoptosis.

Animals↗

PubMed2000

ATM is a cytoplasmic protein in mouse brain required to prevent lysosomal accumulation.

We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.

Animals↗

PubMed2000

Hemisynaptic distribution patterns of presenilins and beta-APP isoforms in the rodent cerebellum and hippocampus.

Healthy brain neurons co-express Alzheimer's disease (AD) related proteins presenilins (PS) and beta-amyloid precursor protein (beta-APP). Deposition of beta-amyloid and PS in the senile plaques of AD brain and their ability to interact in vitro suggest that AD pathology could arise from a defect in the physiological interactions between beta-APP and PS within and/or between neurons. The present study compares the immunocytochemical distribution of PS (1 and 2) and beta-APP major isoforms (695 and 751/770) in the synapses of the cerebellum and hippocampus of the adult rat and mouse. In the cerebellar cortex of both species, the four molecules are immunodetected in the presynaptic or the postsynaptic compartments of synapses, suggesting that they are involved in interneuronal relationships. In contrast, PS and beta-APP are postsynaptic in almost all the immunoreactive synapses of the hippocampus. The different distribution patterns of these proteins in cerebellar and hippocampal synapses may reflect specific physiological differences, responsible for differential vulnerability of neurons to AD synaptic pathology. Defective interactions between beta-APP and PS at the synapses could impede the synaptic functions of beta-APP, inducing the selective loss of synapses that accounts for cognitive impairment in AD.

Amyloid beta-Protein Precursor↗

PubMed2000

Bax and p53 are differentially involved in the regulation of caspase-3 expression and activation during neurodegeneration in Lurcher mice.

Intrinsic Purkinje cell death in heterozygous Lurcher (Grid2Lc/+) mice is accompanied by the target-related death of granule cells and olivary neurons. The expression of pro-caspase-3 is increased in Grid2Lc/+ Purkinje cells and activated caspase-3 is detected in all three cell types before their death. Bax inactivation in Grid2Lc/+ mutants rescues granule cells but not Purkinje cells. Here, we show that, while Bax inactivation inhibits caspase-3 activation in both cell types, p53 inactivation does not affect caspase-3 activation and neuronal loss in Grid2Lc/+ mice. The up-regulation of pro-caspase-3 in Grid2Lc/+ Purkinje cells is Bax and p53 independent. These results suggest that Grid2Lc/+ granule cell death is dependent on Bax and caspase-3 activation, whereas several pathways can mediate Grid2Lc/+ Purkinje cell death.

Animals↗

PubMed2000

Tolerance to ischemia and hypoxia is reduced in aged human myocardium.

BACKGROUND: Recovery of cardiac function after cardiac surgery and other interventional cardiac procedures in elderly patients is inferior to that in younger patients, suggesting that the aged myocardium is more sensitive to ischemia and other stresses. Although convincing data from animal studies of senescence now exist, there is a dearth of controlled in vitro studies that examine the specific response of aged human myocardium to the stress of hypoxia or ischemia. OBJECTIVE: We sought to determine the effect of age on the capacity of human atrial trabeculae to recover contractile function after in vitro hypoxic or ischemic stress. METHODS: Atrial pectinate trabeculae were dissected from the tip of 58 right atrial appendages harvested during an operation in patients aged between 34 and 89 years and electrically stimulated at 1 Hz in oxygenated Ringer's solution at 37 degrees C. Tissues experienced 30 minutes of either hypoxia (N(2) and perfusate) or simulated ischemia (humidified N(2) without perfusate) and were returned to normoxia for recovery of function for 30 minutes. Developed force and other contractile variables were determined during each period. RESULTS: Under normoxic conditions, no significant age difference was observed for any contractile function variable. However, after hypoxia, the old (70-89 years) and intermediate age groups (60-69 years) showed reduced recovery of developed force (48.5% +/- 22.2% [n = 11] and 44.9% +/- 19% [n = 12], respectively) compared with that found (66.4% +/- 19.7% [n = 15]) in the younger (34-59 years) group (mean +/- SD, P =.02). Similarly, after simulated ischemia, the groups of 70- to 89-year-old and 60- to 69-year-old subjects showed reduced recovery of developed force (35.7% +/- 17% [n = 5] and 51.1% +/- 11.8% [n = 9], respectively) compared with that found (68.2% +/- 10.4% [n = 6]) in the group of 34- to 59-year-old subjects (P =.01). Multivariable analysis, comparing 20 factors of surgical patient characteristics and recovery of developed force, found that only age (P =.01) and hypertension (P =.01) were predictors of reduced recovery of developed force after either hypoxia or simulated ischemia. CONCLUSIONS: In aged human atrial myocardium, the capacity to recover contractile function after in vitro hypoxia or simulated ischemia is reduced compared with the younger myocardium of mature adults. These findings suggest that enhanced myocardial protective strategies may be indicated for elderly patients undergoing cardiac surgery.

Adult↗

PubMed1999

Cerebellar Purkinje cell loss during life span of the heterozygous staggerer mouse (Rora(+)/Rora(sg)) is gender-related.

The staggerer mutation causes dysgenesis of the cerebellar cortex in the homozygous mutant (Rora(sg)/Rora(sg)). The mutation acts intrinsically within the Purkinje cells (PCs), leading to cytological abnormalities and a severe deficit in the number of these cells. In contrast, in the heterozygous staggerer (Rora(+)/Rora(sg)), the cytoarchitecture of the cerebellar cortex appears to be normal, but quantitative studies have revealed a significant loss of cerebellar neurons with advancing age. In the heterozygous reeler (+/rl), another mutant presenting a PC loss with age, we have found that only males were affected (Hadj-Sahraoui et al., 1996). In the present study, we have investigated whether a similar gender effect exists in the heterozygous staggerer during life span. PCs were counted on cerebellar sagittal sections in male and female Rora(+)/Rora(sg) and in their Rora(+)/Rora(+) littermates at 1, 3, 9, 13, 18, and 24 months of age. In the Rora(+)/Rora(+), the number of PCs remained stable until 18 months, but there was a 25% significant loss in 24- month-old mice of both genders. During life span, Rora(+)/Rora(+) males had slightly more PC than females. In the Rora(+)/Rora(sg) of both genders, the deficit in PC number was similar at 13 months but it appeared earlier in males, beginning between 1 and 3 months, and was aggravated regularly up to 13 months. By contrast, the decline was delayed and more abrupt in Rora(+)/Rora(sg) females, from a value still normal at 9 months to its maximal extent at 13 months. In view of these results, the heterozygous (Rora(+)/Rora(sg)) mouse offers an interesting model to test the interaction between sex, age, and genetic background on the development and maintenance of cerebellar neuronal populations.

Aging↗

PubMed1999

Afferent-target cell interactions in the cerebellum: negative effect of granule cells on Purkinje cell development in lurcher mice.

Lurcher (Lc) is a gain-of-function mutation in the delta2 glutamate receptor gene that results in a large, constitutive inward current in the cerebellar Purkinje cells of +/Lc mice. +/Lc Purkinje cells fail to differentiate fully and die during postnatal development. In normal mice, interactions with granule cells promote Purkinje cell dendritic differentiation. Partial destruction of the granule cell population in young +/Lc mice by x irradiation resulted in a significant increase in Purkinje cell dendritic growth and improved cytoplasmic structure but did not prevent Purkinje cell death. These results indicate two components to Purkinje cell abnormalities in +/Lc mice: a retardation/blockade of dendritic development that is mediated by interactions with granule cells and the death of the cell. Thus, the normal trophic effects of granule cell interaction on Purkinje cell development are absent in the +/Lc cerebellum, suggesting that granule cells are powerful regulators of Purkinje cell differentiation.

Afferent Pathways↗

PubMed1999

Role of an enriched environment on the restoration of behavioral deficits in Lurcher mutant mice.

Lurcher mutant mice, characterized by massive degeneration of the cerebellar cortex, and normal littermate controls were reared from birth either in standard conditions or in an enriched environment. The effects of this manipulation on motor functions, landmark water maze learning, exploration, and anxiety were evaluated at 3 months of age. Under standard conditions, Lurcher mutants were impaired in comparison to controls on tests of sensorimotor function and had altered exploratory tendencies. The enriched housing improved the motor coordination of Lurcher mutants and decreased the number of trials before reaching criterion in the landmark water maze. In addition to its effects in Lurcher mutants, enriched rearing also increased some behavioral abilities in normal mice. It is hypothesized that enriched housing altered brain morphology or neurochemistry in both normal and cerebellar-damaged animals.

Animals↗

PubMed1999

Hu-Bcl-2 transgenic mice with supernumerary neurons exhibit timing impairment in a complex motor task.

Programmed neuronal cell death is common during development, and is thought to be important in the elimination of errors in axonal projection, cell position and sculpting of neuronal circuits. However, the potential importance of programmed cell death for complex behaviour in the adult animal has never been addressed. We studied motor abilities in a strain of transgenic mice with neuronal overexpression of the human Bcl-2 protein, which have supernumerary neurons due to reduced developmental cell death. Our results show that these mice have a clear deficiency in fine timing of motor coordination without impairment of basic motor functions. This is the first indication that altered developmental cell death and the consequent neuronal surplus can impair complex behaviour in the adult animal.

Animals↗

PubMed1998

Severe atherosclerosis and hypoalphalipoproteinemia in the staggerer mouse, a mutant of the nuclear receptor RORalpha.

BACKGROUND: Hypoalphalipoproteinemia is the most common lipoprotein abnormality in patients with coronary artery disease, yet its causes are unknown. METHODS AND RESULTS: We show that the homozygous staggerer (sg/sg) mutant mouse, which carries a deletion within the nuclear receptor RORalpha gene, develops severe atherosclerosis when maintained on an atherogenic diet. In addition, sg/sg mice display a profound hypoalphalipoproteinemia, which is associated with decreased plasma levels of the major HDL proteins, apolipoprotein (apo) A-I and apoA-II. This decrease in HDL levels in sg/sg mice is due to lowered apoA-I gene expression in the intestine but not in the liver. ApoA-II gene expression is unaffected. CONCLUSIONS: These results suggest that the RORalpha gene contributes to the plasma HDL level and susceptibility to atherosclerosis.

Animals↗