Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

Oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), and astrocytes constitute macroglia. This review deals with the recent progress related to the origin and differentiation of the oligodendrocytes, their relationships to other neural cells, and functional neuroglial interactions under physiological conditions and in demyelinating diseases. One of the problems in studies of the CNS is to find components, i.e., markers, for the identification of the different cells, in intact tissues or cultures. In recent years, specific biochemical, immunological, and molecular markers have been identified. Many components specific to differentiating oligodendrocytes and to myelin are now available to aid their study. Transgenic mice and spontaneous mutants have led to a better understanding of the targets of specific dys- or demyelinating diseases. The best examples are the studies concerning the effects of the mutations affecting the most abundant protein in the central nervous myelin, the p...

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Biology of Oligodendrocyte and Myelin in the Mammalian Central Nervous System

Remyelination involves reinvesting demyelinated axons with new myelin sheaths. In stark contrast to the situation that follows loss of neurons or axonal damage, remyelination in the CNS can be a highly effective regenerative process. It is mediated by a population of precursor cells called oligodendrocyte precursor cells (OPCs), which are widely distributed throughout the adult CNS. However, despite its efficiency in experimental models and in some clinical diseases, remyelination is often inadequate in demyelinating diseases such as multiple sclerosis (MS), the most common demyelinating disease and a cause of neurological disability in young adults. The failure of remyelination has profound consequences for the health of axons, the progressive and irreversible loss of which accounts for the progressive nature of these diseases. The mechanisms of remyelination therefore provide critical clues for regeneration biologists that help them to determine why remyelination fails in MS and in other demyelinating diseases and how it might be enhanced therapeutically.

Remyelination in the CNS: from biology to therapy

The proteasome is an abundant multi-enzyme complex that provides the main pathway for degradation of intracellular proteins in eukaryotic cells. As such, it controls the levels of proteins that are important for cell-cycle progression and apoptosis in normal and malignant cells; for example, cyclins, caspases, BCL2 and nuclear factor of κB. A proteasome inhibitor — bortezomib — has been developed that has shown efficacy as an anticancer agent in the clinic. How can targeting such a universal, broadly active cellular component provide the selectivity and specificity that are required for cancer therapeutics?

http://dipbsf.uninsubria.it/monti/BFPN%202009/proteasoma.pdf

The proteasome: a suitable antineoplastic target

Binding sites with high affinity and specificity for [3H]quinuclidinyl benzilate (QNB) are present in homogenates of rat brain. The characteristics of the binding sites resemble those of muscarinic cholinergic receptors. Specific binding is saturable with respect to [3H]QNB and tissue concentration and is time-, temperature-, and pH-dependent. The bimolecular rate of association (2.0 × 108 M-1 min-1) and dissociation (1.2 × 10-2 min-1) at 35° indicate a dissociation constant of 60 pM and a density of 65 pmol/g of brain. Muscarinic antagonists and agonists displace specific [3H]QNB binding, while nicotinic and non-cholinergic drugs possess little affinity for [3H]QNB-binding sites.

https://www.pnas.org/content/pnas/71/5/1725.full.pdf

Muscarinic Cholinergic Binding in Rat Brain

Gangliosides and acetylcholinesterase in isolated membranes of the rat-brain cortex

Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles (NFT), senile plaques, and cerebrovascular deposits of amyloid-beta. Ubiquitin has also been shown to be present in some of the inclusions characteristic of this disease. To obtain further insight into the role played by the ubiquitin pathway in AD, we investigated the capacity of postmortem samples of cerebral cortex from normal and AD patients to form high-molecular-weight ubiquitin-protein conjugates. Activity of the ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2) involved in the ubiquitin pathway was also determined. In normal samples, the amount of high-molecular-weight ubiquitin-protein conjugates (HMW-UbPC) in cytosol increased with incubation time, whereas, in samples of AD cases, these were almost undetectable. The addition of an adult rat fraction, enriched in ubiquitinating enzymes, restored the capacity of AD brain cytosolic fraction to form conjugates. The trypsin-like proteolytic activity of the 26S proteasome was found to be decreased in AD cytosol brain. Assay of the activity of E1 and E2 by thiol-ester formation revealed a significant decrease in AD samples. Moreover, Western blotting using a specific antibody against E1 showed a dramatic drop of this enzyme in the cytosolic fraction, whereas normal levels were found in the particulate fraction, suggesting a possible delocalization of the enzyme. Our results suggest that a failure in the ubiquitination enzymatic system in brain cytosol may contribute to fibrillar pathology in AD.

Defective ubiquitination of cerebral proteins in Alzheimer's disease

In order to further characterize the still unknown mechanism of cuprizone-induced demyelination, we investigated its effect on rat primary oligodendroglial cell cultures. Cell viability was not significantly affected by this treatment. However, when concentrations of IFNγ and/or TNFα having no deleterious effects per se on cell viability were added together with cuprizone, cell viability decreased significantly. In mitochondria isolated from cuprizone-treated glial cells, we observed a marked decrease in the activities of the various complexes of the respiratory chain, indicating a disruption of mitochondrial function. An enhancement in oxidant production was also observed in cuprizone and/or TNFα-treated oligodendroglial cells. In in vivo experiments, inhibition of microglial activation with minocycline prevented cuprizone-induced demyelination. Based on the above-mentioned results we suggest that these microglial cells appear to have a very active role in cuprizone-induced oligodendroglial cell death and demyelination, through the production and secretion of pro-inflammatory cytokines.

The Neurotoxic Effect of Cuprizone on Oligodendrocytes Depends on the Presence of Pro-inflammatory Cytokines Secreted by Microglia

Thyroid hormones promote differentiation of oligodendrocyte progenitor cells and improve remyelination after cuprizone-induced demyelination

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The cerebral cortex of the rat was submitted to an extensive cell fractionation schedule and in the various fractions, protein, proteolipid protein, total phospholipids, cholesterol, galactolipids, plasmalogens, and gangliosides were determined. With increasing purification the different isolated membranous structures: i.e. myelin, nerve ending membranes, synaptic vesicles, mitochondria, and microsomes, show a definite biochemical specialization reflected in their lipid composition. The presence of gangliosides in some nerve ending membranes is confirmed, and the possible functional role of these acid glyco-lipids is discussed.



The importance of proteolipids as structural components of membranes is recognized. The richness of these compounds in myelin is confirmed and a special localization in the nerve ending membranes is indicated.



Analysis of the molar ratios of the different lipids and proteins in the isolated membranes demonstrates that each one has a specific pattern of molecular organization. This pattern is discussed in relation to the macromolecular structures revealed by electronmicroscopy and some of the molecular models postulated for cell membranes.

Eduardo F. Soto

Papers

Gangliosides and acetylcholinesterase in isolated membranes of the rat-brain cortex

Defective ubiquitination of cerebral proteins in Alzheimer's disease

The Neurotoxic Effect of Cuprizone on Oligodendrocytes Depends on the Presence of Pro-inflammatory Cytokines Secreted by Microglia

Thyroid hormones promote differentiation of oligodendrocyte progenitor cells and improve remyelination after cuprizone-induced demyelination

Lipids and proteolipids in isolated subcellular membranes of rat brain cortex.