Eduardo F. Soto

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

Abstract The subcellular distribution of gangliosides was studied in the rat-brain cortex. In the primary fractions most of the gangliosides are in the microsomal and crude mitochondrial fractions, with the highest concentration in the light microsomes which contain mainly membranous components. After osmotic disruption of the synaptic complex, gradient techniques were used to purify the synaptic vesicles and to isolate the synaptic membranes. Two layers of synaptic membranes were found to contain the highest concentrations of gangliosides and acetylcholinesterase. Another layer of synaptic membranes poor in acetylcholinesterase had a low content of gangliosides. The lack of gangliosides and acetylcholinesterase in synaptic vesicles is emphasized, and the possible physiological significance of these complex acidic glycolipids in some neuronal membranes is discussed.

Defective ubiquitination of cerebral proteins in Alzheimer's disease

Alzheimers disease (AD) is characterized by the presence of neurofibrillary tangles (NFT), senile plaques, and cerebrovascular deposits of amyloid‐β. 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. J. Neurosci. Res. 62:302–310, 2000.

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

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

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.

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

In the present work we analyzed the capacity of thyroid hormones (THs) to improve remyelination using a rat model of cuprizone-induced demyelination previously described in our laboratories. Twenty one days old Wistar rats were fed a diet containing 0.6% cuprizone for two weeks to induce demyelination. After cuprizone withdrawal, rats were injected with triiodothyronine (T3). Histological studies carried out in these animals revealed that remyelination in the corpus callosum (CC) of T3-treated rats improved markedly when compared to saline treated animals. The cellular events occurring in the CC and in the subventricular zone (SVZ) during the first week of remyelination were analyzed using specific oligodendroglial cell (OLGc) markers. In the CC of saline treated demyelinated animals, mature OLGcs decreased and oligodendroglial precursor cells (OPCs) increased after one week of spontaneous remyelination. Furthermore, the SVZ of these animals showed an increase in early progenitor cell numbers, dispersion of OPCs and inhibition of Olig and Shh expression compared to non-demyelinated animals. The changes triggered by demyelination were reverted after T3 administration, suggesting that THs could be regulating the emergence of remyelinating oligodendrocytes from the pool of proliferating cells residing in the SVZ. Our results also suggest that THs receptor beta mediates T3 effects on remyelination. These results support a potential role for THs in the remyelination process that could be used to develop new therapeutic approaches for demyelinating diseases.

Relationship between β-amyloid degradation and the 26S proteasome in neural cells

Beta-amyloid peptide (Abeta) plays a central role in mediating neurotoxicity and in the formation of senile plaques in Alzheimers disease (AD). The investigation of the roles of ubiquitin (Ub) in the process underlying the association of abnormal protein with the inclusion bodies that characterize AD is of great importance for the further understanding of this disorder. We have used primary cultures of cortical neurons and astrocytes to investigate the participation of the Ub-proteasome pathway in the degradation of Abeta and the effect of Abeta(1-42) and of the fragment Abeta(25-35) upon neural cells. We have found that Abeta(25-35) and Abeta(1-42) produce a significant increase in Ub-protein conjugates and in the expression of the Ub-activating enzyme E1. On the other hand, beta peptides inhibited the proteolytic activities of the 26S proteasome. When the proteolytic activity of the 26S proteasome was inhibited with lactacystin, there was a marked decrease in Abeta(1-42) degradation, suggesting that the peptide, in both astrocytes and neurons, could be a possible substrate of this enzymatic complex. Treatment of the cultures with lactacystin prior to the exposure to Abeta produced a significant decrease in cell viability, possibly as a consequence of the inhibition of Abeta degradation leading to a persistent exposure of the cells to the amyloidogenic peptide which results in cell death. Alterations in the Ub-proteasome pathway in AD could affect the normal proteolytic removal of Abeta, leading to an abnormal accumulation of Abeta(1-42).

Cholinergic Binding Capacity of Proteolipids from Isolated Nerve-Ending Membranes

The capacity for binding dimethyl d-tubocurarine-C14 was studied in isolated nerve-ending membranes from cerebral cortex and myelin. After treatment of the membrane with organic solvents most of the radioactivity was recovered in the extract. Preliminary evidence indicates that dimethyl d-tubocurarine-C14 is not bound to lipids or glycolipids. While the proteolipids of myelin have a low binding capacity, the results obtained with the nerve-ending membranes rich in acetylcholinesterase suggest that the cholinergic receptor may be a special type of proteolipid.

Remyelination after cuprizone-induced demyelination in the rat is stimulated by apotransferrin.

Twenty-one-day-old Wistar rats were fed a diet containing 0.6% cuprizone for 2 weeks. Studies carried out after withdrawal of cuprizone showed histological evidences of marked demyelination in the corpus callosum. Biochemical studies of isolated myelin showed a marked decrease in myelin proteins, phospholipids, and galactocerebrosides as well as a marked decrease in myelin yield. Treatment of these animals with a single intracranial injection of 350 ng of apotransferrin at the time of withdrawal of cuprizone induced a marked increase in myelin deposition resulting in a significantly improved remyelination, evaluated by histological, immunocytochemical, and biochemical parameters, in comparison to what was observed in spontaneous recovery. Immunocytochemical studies of cryotome sections to analyze developmental parameters of the oligodendroglial cell population at the time of termination of cuprizone and at different times thereafter showed that in the untreated animals, there was a marked increase in the number of NG2-BrdU-positive precursor cells together with a marked decrease in MBP expression at the peak of cuprizone-induced demyelination. As expected, the amount of precursor cells decreased markedly during spontaneous remyelination and was accompanied by an increase in MBP reactivity. In the apotransferrin-treated animals, these phenomena occurred much faster, and remyelination was much more efficient than in the untreated controls. The results of this study suggest that apotransferrin is a very active promyelinating agent which could be important for the treatment of certain demyelinating conditions.

Sustained neonatal hyperthyroidism in the rat affects myelination in the central nervous system

We have carried out a study of the effects of sustained neonatal hyperthyroidism on myelin and on the oligodendroglial cells, in an effort to obtain further insight into the molecular mechanisms underlying the action of thyroid hormones on the central nervous system (CNS). Expression of the mRNAs of myelin basic protein (MBP) myelin proteolipid protein (PLP), 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase), transferrin, and c‐Jun was investigated in 10‐ and 17‐day‐old normal and hyperthyroid rats, using Northern blot analysis. At 10 days of age, the levels of all the explored mRNAs were markedly higher in the experimental animals. The mRNA of transferrin showed a ninefold increase over control values, suggesting the possibility that this putative trophic factor might act as one of the mediators in the action of thyroid hormones. At 17 days of age on the other hand, the levels of all the mRNAs decreased markedly, reaching values below control, except for c‐Jun, which remained higher than in normals. At 70 days of age, hyperthyroid rats showed clear evidence of myelin deficit, in agreement with previous results of our laboratories (Pasquini et al.: J Neurochem 57: Suppl S124, 1991). Immunocytochemistry of 70‐day‐old rat brain tissue sections showed a substantial reduction in the amount of MBP‐reacting structures and a marked decrease in the number of oligodendroglial cells. Although the above‐mentioned results could be the consequence, as proposed by Barres et al. (Development 120:1097–1108, 1994) and Baas et al. (Glia 19:324–332, 1997) of a premature arrest in oligodendroglial cell proliferation followed by early differentiation, the persistent high levels of expression of c‐Jun, together with the dramatic decrease in the number of oligodendrocytes, suggested the possibility that prolonged hyperthyroidism could activate apoptotic mechanisms in the myelin forming cells. Using propidium iodide‐labeled isolated oligodendroglial cells, we found, by flow cytometry, a significant increase in the number of apoptotic/hypo‐diploid propidium iodide‐positive cells. These results indicate that one of the actions of sustained levels of thyroid hormones in the neonate rat is to increase oligodendroglial cell death by apoptosis. J. Neurosci. Res. 53:251–259, 1998.

Rapid purification of proteolipids from rat brain subcellular fractions by chromatography on a lipophilic dextran gel

Proteolipids from adult rat brain subcellular fractions were purified by a one-step procedure involving chromatography through Sephadex LH-60 eluted with an acidified chloroform-methanol mixture. The protein peak was eluted with the void volume and was free of adventitious lipids. The degree of purification was similar to that attained with the neutral-acidified chloroform-methanol dialysis method with the advantage that this new procedure can be carried out in only 3 h, with a recovery of proteins of 95-100%. Samples containing different lipid/protein ratios passed through the gel gave similar elution profiles. When labeled amino acids or palmitic acid were added to myelin total lipid extracts, no radioactivity was eluted with the protein, indicating that the proteolipid apoproteins purified by this method do not adsorb hydrophobic low-molecular-weight compounds.