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Growth-associated protein-43 (GAP-43) is a phosphoprotein whose expression in neurons is related to the initial establishment and remodeling of neural connections. GAP-43 gene expression is known to be regulated at both the transcriptional and the postranscriptional levels. However, very little is known about the cellular mechanism involved in the degradation of this protein. Ubiquitin (Ub) is well known for its role in targeting cytoplasmic proteins for degradation by the 26S proteasome. The ubiquitin-proteasome system (UPS) consists of a conserved cascade of three enzymatic components that attach Ub covalently to various substrates and control the degradation of protein involved in several important cellular processes. In this study, we investigated the degradation of GAP-43 in transfected NIH 3T3 cells and neuronal cultures. We found that the proteasome inhibitors, lactacystin and MG132 increased the cellular GAP-43 level, leading to the accumulation of polyubiquitinated forms of this protein in transfected cells and that the Ub-proteasome pathway is also involved in the turnover of this protein in neurons. We conclude based on our findings that GAP-43 is a substrate of the UPS., (Copyright (c) 2005 Wiley-Liss, Inc.)

The multicatalytic protease complex or proteasome is a fundamental nonlysosomal tool that the cell uses to process or degrade proteins at a fast rate through the ubiquitin and ATP-dependent proteolytic pathway. Examples of these important proteins include the tumor suppressor protein p53, various cyclins, the cyclin-dependent kinase inhibitor p27, NFkappaB, IkappaB, c-fos, and c-jun. The activation of proteolytic enzymes, including certain cystein-proteases of the ced-3/ICE (interleukin-1beta-converting enzyme) family, is a characteristic feature of the apoptotic program. However, the role of the multicatalytic protease complex in apoptosis is not well known. In order to obtain further information regarding the participation of the ubiquitin-mediated pathway in the decision of the cell to execute the cell death program, we have used a specific inhibitor of the multicatalytic protease complex, lactacystin, in cultured cerebellar granule cells. Cells were obtained from the cerebellum of 6- to 8-day-old Wistar rats and cultured in Neurobasal medium supplemented with B-27. Addition of lactacystin to the cultures induced apoptosis of the granule cells in a time-dependent fashion. The morphological changes produced by the proteasome inhibitor included nuclear condensation and DNA fragmentation measured by the diphenylamine test, as well as a positive labeling by the TUNEL (terminal deoxynucleotidyltransferase mediated-dUTP nick end labeling) assay, all of them typical features of apoptosis. Concomitant with apoptosis, there were changes in the expression of the ubiquitin mRNA, a progressive depletion in the free ubiquitin pool, and an increase in the high molecular weight ubiquitin-protein conjugates. Caspase-3, a member of the ced-3/ICE family of cystein-proteases, showed a marked increase in activity in the lactacystin-treated cells. In flow cytometry studies, the amount of cells in the S phase of the cell cycle was smaller in the lactacystin-treated cells than in controls, suggesting that apoptosis could be due, in part, to an alteration of the cell cycle., (Copyright 2000 Wiley-Liss, Inc.)

Ubiquitin (Ub) modification of different proteins plays an important role in many cellular processes. However, the best studied function of Ub is the labeling of proteins committed to rapid degradation, by an ATP-dependent pathway. We previously found that this pathway is operative in the central nervous system (CNS) of adult rats (Adamo et al. [1994] J. Neurosci. Res. 38:358-364). In the present study, we examined the changes in the capacity to form high-molecular-weight Ub protein conjugates (UbPC) and the changes in the production of 2-thiobarbituric acid-reactive substances (TBARS), in the content of protein-associated carbonyl groups (PAC), and in the activity of glutamine synthetase produced by in vitro peroxidation of the cell cytosolic proteins and of the mitochondrial fraction isolated from rat brain. Under these experimental conditions, there was an increase in PAC and TBARS in the cytosol, indicating that damage to certain cellular components had occurred. Simultaneously there was a marked increase in UbPC in comparison with the nonoxidized controls. These conjugates showed an active turnover and accumulated when Ub-mediated proteolysis was inhibited. In vitro peroxidation of the mitochondrial fractions resulted in an increase in the production of PAC and in an enhancement in the formation of UbPC. These results demonstrate that the oxidized proteins can be recognized by the ubiquitylating system and that in the CNS the Ub-dependent proteolytic pathway is one of the possible mechanisms involved in the removal of cytosolic and mitochondrial fraction damaged proteins., (Copyright 1999 Wiley-Liss, Inc.)

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.

The capacity to form ubiquitin (Ub)-protein conjugates was investigated in the cytosol of different structures of the rat central nervous system (CNS) in order to confirm the presence of this extralysosomal, adenosine triphosphate (ATP)-dependent, protein degradation system as well as its structural localization. Using 125I-Ub, we found that in the presence of ATP, the cytosol obtained from whole brains was able to form high molecular weight Ub-protein conjugates. These conjugates could be detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. The formation of these conjugates was much higher in the cerebral cortex than in the brain stem, which is mainly constituted by white matter, being intermediate in the cytosol isolated from whole brain total homogenates. These results suggested to us that under normal conditions the capacity to form Ub-protein conjugates was mainly located in structures containing neuronal cell bodies. Strong support for this contention was obtained when the cytosol isolated from rat optic nerves or from oligodendroglial cells isolated from whole brain was found to be totally unable to form Ub-protein conjugates. The inability of certain CNS structures to form conjugates with Ub could be attributed, among other reasons, to the lack of enzymes catalyzing the various steps of the Ub degradation system, to the absence of short half-life (target) proteins in those structures, or to the lack of activity of the enzymes catalyzing the reaction due to regulatory control mechanisms operating under normal conditions.

The effect of neonatal hyperthyroidism produced by injection of tri-iodothyronine (T3) on myelination and on the microperoxisomal population of the brain was studied in young rats. Data on the lipid composition of myelin show that myelinogenesis starts earlier in treated animals. In coincidence with the early appearance of myelin, there is an increase in the population of brain microperoxisomes, indicated by the increase in the activity of two enzymes that have been shown to be located in these organelles: catalase and acyl CoA-dihydroxyacetone phosphate acyl transferase. Double-label experiments using (1,2,3-3H) and (2-3H) glycerol to study the synthesis of glycerophospholipids through the dihydroxyacetone phosphate (DHAP) pathway give further support to the above-mentioned findings and suggest that there is an active participation of microperoxisomes in the synthesis of myelin lipids during the period of myelin formation.