Differences in protein quality control correlate with phenotype variability in 2 mouse models of familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a disease of variable severity in terms of speed of progression of the disease course. We found a similar variability in disease onset and progression of 2 familial ALS mouse strains, despite the fact that they carry the same transgene copy number and express the same amount of mutant SOD1G93A messenger RNA and protein in the central nervous system. Comparative analysis of 2 SOD1G93A mouse strains highlights differences associated with the disease severity that are unrelated to the degree of motor neuron loss but that appear to promote early dysfunction of these cells linked to protein aggregation.

CLIC1 function is required for beta-amyloid-induced generation of reactive oxygen species by microglia.

The Alzheimer's disease (AD) brain is characterized by plaques containing beta-amyloid (Abeta) protein surrounded by astrocytes and reactive microglia. Activation of microglia by Abeta initiates production of reactive oxygen species (ROS) by the plasmalemmal NADPH oxidase; the resultant oxidative stress is thought to contribute to neurodegeneration in AD. We have previously shown that Abeta upregulates a chloride current mediated by the chloride intracellular channel 1 (CLIC1) protein in microglia.

Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants.

The transcription factor Sox2 is active in neural stem cells, and Sox2 'knockdown' mice show defects in neural stem/progenitor cells in the hippocampus and eye, and possibly some neurons. In humans, heterozygous Sox2 deficiency is associated with eye abnormalities, hippocampal malformation and epilepsy. To better understand the role of Sox2, we performed in vitro differentiation studies on neural stem cells cultured from embryonic and adult brains of 'knockdown' mutants.

Accumulation of human SOD1 and ubiquitinated deposits in the spinal cord of SOD1G93A mice during motor neuron disease progression correlates with a decrease of proteasome.

Mutations in SOD1 cause selective motor neuron degeneration in familial amyotrophic lateral sclerosis patients and transgenic mice overexpressing the mutant enzyme. Formation and accumulation of ubiquitinated aggregates in motor neurons are thought to be involved in the toxic gain of function of mutant SOD1. The present study shows that the accumulation of soluble and detergent-insoluble mutant SOD1 in spinal cord of symptomatic SOD1G93A transgenic mice is due to impaired degradation of mutant SOD1 rather than to increased transcript levels.

Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain.

In many species, the Sox2 transcription factor is a marker of the nervous system from the beginning of its development, and we have previously shown that Sox2 is expressed in embryonic neural stem cells. It is also expressed in, and is essential for, totipotent inner cell mass stem cells and other multipotent cell lineages, and its ablation causes early embryonic lethality. To investigate the role of Sox2 in the nervous system, we generated different mouse mutant alleles: a null allele (Sox2beta-geo 'knock-in'), and a regulatory mutant allele (Sox2DeltaENH), in which a neural cell-specific enhancer is deleted.

Activated p38MAPK is a novel component of the intracellular inclusions found in human amyotrophic lateral sclerosis and mutant SOD1 transgenic mice.

Cytoskeletal abnormalities with accumulation of ubiquilated inclusions in the anterior horn cells are a pathological hallmark of both familial and sporadic amyotrophic lateral sclerosis (ALS) and of mouse models for ALS. Phosphorylated neurofilaments besides ubiquitin and dorfin have been identified as one of the major components of the abnormal intracellular perikaryal aggregates. As we recently found that p38 mitogen-activated protein kinase (p38MAPK) colocalized with phosphorylated neurofilaments in spinal motor neurons of SOD1 mutant mice, a model of familial ALS, we investigated whether this kinase also contributed to the inclusions found in ALS patients and SOD1 mutant mice.

Subcellular localization and axonal transport of the survival motor neuron (SMN) protein in the developing rat spinal cord.

The subcellular localization of the survival motor neuron (SMN) protein, encoded by the spinal muscular atrophy determining gene, was investigated in motor neurons of the developing and adult rat spinal cord by light and electron microscopy immunocytochemistry. The experiments were carried out with a panel of anti-SMN antibodies, all recognizing an SMN-specific protein band at 39 kDa in HeLa cells and rat spinal cord protein extracts. SMN protein expression decreased during postnatal spinal cord development, but it remained unchanged in distribution and intensity in motor neurons at all ages examined.

Prenatal methylazoxymethanol treatment in rats produces brain abnormalities with morphological similarities to human developmental brain dysgeneses.

A double methylazoxymethanol (MAM) intraperitoneal injection was prenatally administered to pregnant rats at gestational day 15 to induce developmental brain dysgeneses. Thirty adult rats from 8 different progenies were investigated with a combined electrophysiological and neuroanatomical analysis. The offspring of treated dams was characterized by extensive cortical layering abnormalities, subpial bands of heterotopic neurons in layer I, and subcortical nodules of heterotopic neurons extending from the periventricular region to the hippocampus and neocortex.

Free and total malondialdehyde assessment in biological matrices by gas chromatography-mass spectrometry: what is needed for an accurate detection.

A method to determine free and total (free and bound) malondialdehyde (MDA) in fresh human plasma, or in rat liver microsomes, using selected ion monitoring (SIM) gas chromatography-mass spectrometry in the electron impact mode was set up. The dideuterated internal standard, 3-hydroxy[1, 3-2H2]-2-propenal (dMDA), was added to the biological samples before their analytical manipulation. To detect free MDA the samples were reacted under mild conditions (25 degreesC, pH 4.

Beta-ethoxyacrolein contamination increases malondialdehyde inhibition of milk xanthine oxidase activity.

beta-Ethoxyacrolein (BEA), a side product that forms during the preparation of malondialdehyde (MDA) by acidic hydrolysis of tetraethoxypropane (TEP), has been found to be an inhibitor of milk xanthine oxidase (XO) several times more potent than pure MDA (NaMDA). The incubation of XO with 10 microM BEA abolished 50% of the enzyme activity within 1 min; the inhibited enzyme was totally regenerated by dialysis and filtration through Sephadex. The BEA inhibition mode of the enzyme was mixed-type with the apparent inhibition constants (Ki) of 2.

EAAC1, a high-affinity glutamate tranporter, is localized to astrocytes and gabaergic neurons besides pyramidal cells in the rat cerebral cortex.

High-affinity uptake of glutamate from the synaptic cleft plays a crucial role in regulating neuronal activity in physiological and pathological conditions. We have used affinity-purified specific polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of rabbit and rat EAAC1, a glutamate (Glu) transporter believed to be exclusively neuronal, to investigate its cellular and subcellular localization and whether it is expressed exclusively in glutamatergic cells of infragranular layers, as suggested by previous studies. Light microscopic studies revealed that EAAC1 immunoreactivity (ir) is localized to neurons and punctate elements in the neuropil.

Inhibition of in vitro lipid peroxidation by stable steroidic nitroxyl radicals.

4',4'-dimethylspiro (5 alpha-cholestane-3,2'-oxazolidin)-3'-yloxy (IK-1) and 7 alpha,12 alpha-dihydroxy-4',-4'-dimethylspiro (5 beta-cholan-24-oic-3,2'-oxazolidin)-3'-yloxy acid (IK-2), two stable steroidic nitroxyl radicals, were newly synthesized and tested as possible inhibitors of lipid peroxidation, induced by Fenton's reagent in both rat liver microsomes and egg phosphatidylcholine liposomes. The inhibitory activity, evaluated through the formation of thiobarbituric acid reactive substances (TBARS) and the conjugated diene, was compared with that of alpha-tocopherol and 2,2,6,6-tetramethylpiperidine-1-yloxy (TEMPO). In each model system IK-1 and IK-2 exhibited an IC50 of 8 microM and reduced the formation of TBARS and conjugated diene, showing IK-1 a potency comparable to alpha-tocopherol and higher than TEMPO.

Neuronal and glial localization of NMDA receptors in the cerebral cortex.

The crucial role of glutamate receptors of the N-methyl-D-aspartate (NMDA) type in many fundamental cortical functions has been firmly established, as has its involvement in several neuropsychiatric diseases, but until recently, very little was known of the anatomical localization of NMDA receptors in the cerebral cortex of mammals. The recent application of molecular biological techniques to the study of NMDA receptors has allowed the production of specific tools, the use of which has much increased our understanding of the localization of NMDA receptors in the cerebral cortex. In particular, immunocytochemical studies on the distribution of cortical NMDA receptors have: 1.

Presynaptic NMDA receptors in the neocortex are both auto- and heteroreceptors.

We used electron microscopic immunocytochemistry with antibodies against NR1 and NR2A and B subunits to study the distribution of N-methyl-D-aspartate (NMDA) receptors in presynaptic axon terminals in the rat cerebral cortex. In all sections examined, NR1 and NR2A/B immunoreactivities were observed in axon terminals: NR1- and NR2A/B-positive axon terminals made both symmetrical and asymmetrical synapses on unlabelled dendritic profiles. Combined pre- and postembedding studies showed that all NR1 and NR2A/B-positive axon terminals making symmetrical synapses were gamma-aminobutyric acid (GABA)-positive.

GAT-3, a high-affinity GABA plasma membrane transporter, is localized to astrocytic processes, and it is not confined to the vicinity of GABAergic synapses in the cerebral cortex.

The termination of GABA synaptic action by high-affinity, Na(+)-dependent, neuronal, and glial plasma membrane transporters plays an important role in regulating neuronal activity in physiological and pathological conditions. We have investigated the cellular localization and distribution in the cerebral cortex of adult rats of one GABA transporter (GAT), GAT-3, by immunocytochemistry with affinity-purified polyclonal antibodies directed to its predicted C terminus that react monospecifically with a protein of approximately 70 kDa. Light microscopic studies revealed specific GAT-3 immunoreactivity (ir) in small punctate structures, and it was never observed in fibers or cell bodies.

Expression of NR1 and NR2A/B subunits of the NMDA receptor in cortical astrocytes.

Ionotropic glutamate (Glu) receptors of the N-methyl-D-aspartate type (NMDA) play a fundamental role in many cortical functions. Native NMDA receptors are composed of a heteromeric assembly of different subunits belonging to two classes: NMDAR1 (NR1) and NMDAR2 (NR2). To date, NMDA receptors are believed to be expressed only in neurons, although electrophysiological and in situ hybridization studies have suggested that this class of Glu receptors might be also expressed by some astrocytes.

GAT-1, a high-affinity GABA plasma membrane transporter, is localized to neurons and astroglia in the cerebral cortex.

High affinity, GABA plasma membrane transporters influence the action of GABA, the main inhibitory neurotransmitter. The cellular expression of GAT-1, a prominent GABA transporter, has been investigated in the cerebral cortex of adult rats using in situ hybridization with 35S-labeled RNA probes and immunocytochemistry with affinity purified polyclonal antibodies directed to the C-terminus of rat GAT-1. GAT-1 mRNA was observed in numerous neurons and in some glial cells.

No documentable role for xanthine oxidase in the pathogenesis of hepatic in vivo ischaemia/reperfusion injury.

An investigation was made into the possible involvement of the enzyme xanthine oxidase (XO) (EC 1.1.3.

Metastasis-associated mts1 gene expression is down-regulated by heat shock in variant cell lines of the B16 murine melanoma.

Tumour cells are more heat sensitive than corresponding normal cells but the reasons for this are poorly understood. Here we report that induction of heat shock proteins was associated with a down-regulation of the metastasis associated mts1 gene in BL6-B16 murine melanoma cells, and the heat-resistant HTG variant of the BL6 line. Melanocyte stimulating hormone, which does not affect B16 cell proliferation but upregulates mts1 expression, only marginally enhanced heat shock protein expression in F1 cells as determined by immunohistochemical methods.

Calcitonin gene-related peptide (CGRP) in the cat neocortex: evidence for a sparse but widespread network of immunoreactive fibers.

The morphology, and laminar and topographic distribution of fibers containing calcitonin gene-related peptide (CGRP) immunoreactivity were studied by light and electron microscopic methods in the cerebral cortex of adult cats using a rabbit antiserum raised against the C-terminal region of the rat alpha-CGRP. At the light microscopic level, a sparse number of CGRP-positive fibers were observed in the frontal, parietal, and occipital cortices. They showed numerous irregularly spaced varicosities, were mostly oriented vertically, and in rare cases gave rise to boutons terminaux as they ascended toward the pial surface.

Effect of glutathione depletion on the conversion of xanthine dehydrogenase to oxidase in rat liver.

The ability of endogenous glutathione (GSH) to modify the activity of the enzyme xanthine oxidase (XO) in rat liver was investigated. The effect of hepatic GSH depletion on the conversion of xanthine dehydrogenase (XDH) (EC 1.1.

Presence of a smooth muscle system in aortic valve leaflets.

The location and the spatial arrangement of smooth muscle cells in aortic valves have been assessed by a systematic analysis of serial semithin sections of plastic embedded porcine and human aortic leaflets, combined with an electron microscope study. The investigation showed that smooth muscle cells, either single and arranged in thin bundles, and other cell types such as myofibroblasts are constantly present in the aortic valve leaflets. In addition, it was possible to devise a model of the three dimensional, specific organization of the smooth muscle bundles which can be interpreted as an intrinsic muscle system of the leaflets.