Oligodendrogenesis in iron-deficient rats: effect of apotransferrin.

In rats, iron deficiency produces an alteration in myelin formation. However, there is limited information on the effects of this condition on oligodendroglial cell (OLGc) proliferation and maturation. In the present study, we further analyzed the hypomyelination associated with iron deficiency by studying the dynamics of oligodendrogenesis. Rats were fed control (40 mg Fe/kg) or iron-deficient (4 mg Fe/kg) diets from gestation day 5 until postnatal day 3 (P3) or 11 (P11). OLGc proliferation, migration and differentiation were investigated before and after an intracranial injection of apotransferrin at 3 days of age (P3). The proliferating cell population was evaluated at P3. Iron-deficient (ID) animals showed an increase in the oligodendrocyte precursors cell (OPC) population in comparison with controls. The overall pattern of migration of cells labeled with BrdU was investigated at P11. Iron deficiency increased the amount of BrdU(+) cells in the corpus callosum (CC) and decreased OLGc maturation and myelin formation. Changes in nerve conduction were analyzed by measuring visual evoked potentials. Latency and amplitude were significantly disturbed in ID rats compared with controls. Both parameters were substantially normalized when animals were treated with a single intracranial injection of 350 ng apotransferrin (aTf). The current results give support to the idea that iron deficiency increases the number of proliferating and undifferentiated cells in the CC compared with the control. Treatment with aTf almost completely reverted the effects of iron deficiency, both changing the migration pattern and increasing the number of mature cells in the CC and myelin formation.

Partial inhibition of proteasome activity enhances remyelination after cuprizone-induced demyelination.

We have previously demonstrated that addition of low concentrations of lactacystin (a specific inhibitor of the proteasome) to oligodendroglial cell cultures containing a high percentage of precursor cells induces their exit from the cell cycle and their differentiation. On the other hand, we have recently shown that the mechanism of cuprizone toxicity on oligodendroglial cells involves the recruitment of microglia and their secretion of pro-inflammatory cytokines and in the increased production of oxidant species, which results in a decrease in the activities of the mitochondrial respiratory chain. In the present paper we investigated the effect of a decrease in proteasome activity induced by the injection of lactacystin in the corpus callosum in the remyelination process that normally occurs after cuprizone-induced demyelination. This treatment markedly improves the remyelination process that normally occurs in cuprizone-induced demyelination. It also attenuates the activation of NFkappaB and the recruitment of microglia and astrocytes, thus helping in the recovery of the mitochondrial respiratory chain activities that are affected by cuprizone treatment.

Partial inhibition of the proteasome enhances the activity of the myelin basic protein promoter.

We have previously shown that low concentrations of a specific proteasome inhibitor accelerate exit from the cell cycle and enhance oligodendroglial cell (OLGc) differentiation. To elucidate the mechanisms involved in this process, OLGcs of the N20.1 cell line, transfected with a reporter gene driven by the MBP promoter, were treated with proteasome inhibitors and/or inhibitors of different signaling pathways. Partial proteasome inhibition resulted in enhanced activation of the MBP promoter which involved the tyrosine kinase, PI3-Akt and PKC pathways, accompanied by an increase in the levels of p21(Cip1), p27(Kip1) and Sp1 and by a decrease in Nkx2.2. Binding of Sp1 to DNA was also increased. These results were not observed when the Sp1 binding site was mutated. We conclude that the enhanced activation of the MBP promoter induced by partial inhibition of the proteasome could be due, at least in part, to the stabilization of p27(Kip1) and Sp1.

Fyn kinase is involved in oligodendroglial cell differentiation induced by apotransferrin.

Mechanisms that regulate oligodendroglial cell (OLGc) differentiation are the focus of intensive research in the field of cellular and molecular neurobiology. We have previously shown that the addition of apotransferrin (aTf) to primary OLGc cultures accelerates their differentiation and induces an increase in the expression of different components of the myelin cytoskeleton (CSK) such as actin, tubulin, and some of the microtubule-associated proteins, particularly the stable tubulin only peptide (STOP). Fyn protein-tyrosine kinase (Fyn kinase), a member of the Src family, participates in signalling pathways that regulate OLGs/myelin cytoskeletal reorganization. It is essential for myelin development in the central nervous system (CNS), and its absence results in hypomyelination. In the present study, we used both primary cell and N19 cell line cultures to investigate further the mechanisms of action involved in the accelerated differentiation of OLGcs induced by aTf. In particular, we were interested in studying the participation of Fyn kinase in the different pathways involved in the reorganization of the OLGc/myelin cytoskeleton. In agreement with results already published, we found that in OLGcs, Fyn kinase is associated with Tau and tubulin. Using a dominant-negative of Tau in which the Fyn-Tau-microtubules (MTs) interaction is blocked, we found that aTf was unable to induce OLGc morphological differentiation. It was also observed that aTf decreases the activated RhoA content in coincidence with a redistribution of actin immunoreactivity. These results give support to our hypothesis that Fyn kinase plays a key role in the differentiation process of OLGcs promoted by aTf.

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.

Effect of transferrin on hypomyelination induced by iron deficiency.

We have used a model of iron deficiency in the rat to analyze the effects of a disruption in iron availability on oligodendroglial cell (OLGc) maturation and myelinogenesis and to explore the possible beneficial influence of an intracranial injection (ICI) of apotransferrin (aTf) at 3 days of age on this process. Studies carried out on postnatal days 17 and 24 showed that iron deficiency produced a decrease in myelin proteins and lipids at 24 days of age. Immunohistochemistry showed that in untreated iron-deficient (ID) rats, the immunoreactivity of anti-adenomatous polyposis coli (APC) and anti-MBP antibodies decreased markedly with reference to normal controls, whereas in ID rats treated with an ICI of aTf, the immunoreactivity of these markers increased. A similar situation occurred with the immunoreactivity of H-ferritin. In primary OLGc cultures from ID rats, there was a high number of cells positive to the antibody against the polysialylated form of the cell surface glycoprotein NCAM (PSA-NCAM) compared with in OLGc cultures prepared from normal controls or from ID animals treated with aTf. The number of MBP+ cells in cultures from ID rats increased after treatment with aTf. The presence of lipid rafts evaluated with a specific anti-protein prion cellular (PrPc) antibody showed a smaller number of PrPc-positive structures in ID rat cultures. Treatment of the ID animals with a single ICI of aTf stimulated myelination, producing a significant correction in the different biochemical parameters affected by ID.

The mRNA of transferrin is expressed in Schwann cells during their maturation and after nerve injury.

Transferrin, the iron carrier protein, has been shown to be involved in oligodendroglial cell differentiation in the central nervous system but little is known about its role in the peripheral nervous system. In the present work, we have studied the presence of transferrin and of its mRNA in rat sciatic nerves and in Schwann cells isolated at embryonic and adult ages as well as during the regeneration process that follows nerve crush. We have also studied the correlation between the expression of the mRNAs of transferrin and the expression of mature myelin markers in the PNS. We show that transferrin is present in whole sciatic nerves at late stages of embryonic life as well as at postnatal day 4 and in adult rats. We demonstrate for the first time, that in normal conditions, the transferrin mRNA is expressed in Schwann cells isolated from sciatic nerves between embryonic days 14 and 18, being absent at later stages of development and in adult animals. In adult rats, 3 days after sciatic nerve crushing, the mRNA of transferrin is expressed in the injured nerve, but 7 days after injury its expression disappears. Transferrin protein in the sciatic nerve closely follows the expression of its mRNA indicating that under these circumstances, it appears to be locally synthesized. Transferrin in the PNS could have a dual role. During late embryonic ages it could be locally synthesized by differentiating Schwann cells, acting as a pro-differentiating factor. A similar situation would occur during the regeneration that follows Wallerian degeneration. In the adult animals on the other hand, Schwann cells could pick up transferrin from the circulation or/and from the axons, sub serving possible trophic actions closely related to myelin maintenance.

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 IFNgamma and/or TNFalpha 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 TNFalpha-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.

Presence of alpha-globin mRNA and migration of bone marrow cells after sciatic nerve injury suggests their participation in the degeneration/regeneration process.

We have previously reported that in the distal stump of ligated sciatic nerves, there is a change in the distribution of myelin basic protein (MBP) and P0 protein immunoreactivities. These results agreed with the studies of myelin isolated from the distal stump of animals submitted to ligation of the sciatic nerve, showing a gradual increase in a 14 kDa band with an electrophoretic mobility similar to that of an MBP isoform, among other changes. This band, which was resolved into two bands of 14 and 15 kDa using a 16% gel, was found to contain a mixture of MBP fragments and peptides with great homology with alpha- and beta-globins. In agreement with these results, we have demonstrated that the mRNA of alpha-globin is present in the proximal and distal stumps of the ligated nerve. It is also detected at very low levels in Schwann cells isolated from normal nerves. These results could be due to the presence of alpha- and/or beta-globin arising from immature cells of the erythroid series. Also, they could be present in macrophages, which spontaneously migrate to the injured nerve to promote the degradation of myelin proteins. Cells isolated from normal adult rat bone marrow which were injected intraortically were found to migrate to the injured area. These cells could contribute to the remyelination of the damaged area participating in the removal of myelin debris, through their transdifferentiation into Schwann cells or through their fusion with preexisting Schwann cells in the distal stump of the injured sciatic nerve.

Apotransferrin decreases the response of oligodendrocyte progenitors to PDGF and inhibits the progression of the cell cycle.

In the CNS, transferrin (Tf) is expressed by the oligodendroglial cells (OLGcs) and is essential for their development. We have previously shown that apotransferrin (aTf) accelerates maturation of OLGcs in vivo as well as in vitro. The mechanisms involved in this action appear to be complex and have not been completely elucidated. The aim of this study was to investigate if Tf participates in the regulation of the cell cycle of oligodendroglial progenitor cells (OPcs). Primary cultures of OPcs were treated with aTf and/or with different combinations of mitogenic factors. Cell cycle progression was studied by BrdU incorporation, flow cytometry and by the expression of cell cycle regulatory proteins. Apotransferrin decreased the number of BrdU+ cells, increasing the cell cycle time and decreasing the number of cells in S phase. The cell cycle inhibitors p27kip1, p21cip1 and p53 were increased, and in agreement with these results, the activity of the complexes involved in G1-S progression (cyclin D/CDK4, cyclin E/CDK2), was dramatically decreased. Apotransferrin also inhibited the mitogenic effects of PDGF and PDGF/IGF on OPcs, but did not affect their proliferation rate in the presence of bFGF, bFGF/PDGF or bFGF/IGF. Our results indicate that inhibition of the progression of the cell cycle of OPcs by aTf, even in the presence of PDGF, leads to an early beginning of the differentiation program, evaluated by different maturation markers (O4, GC and MBP) and by morphological criteria. The modulation by aTf of the response of OPcs to PDGF supports the idea that this glycoprotein might act as a key regulator of the OLGc lineage progression.

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.

Involvement of the ubiquitin-mediated proteolytic system in the signaling pathway induced by ceramide in primary astrocyte cultures.

The selective degradation of abnormal or short half-life proteins in eukaryotic cells proceeds through the ubiquitin-mediated proteolytic system (UbPS). The signals that tag the proteins for their ubiquitination are well known. In the present study, our aim was to investigate the relationship between the action of ceramide and the changes in the expression of certain mRNAs of the Ub pathway and in the activation of the UbPS in cultured astrocytes (ASTs). Changes in the expression of components that are known to be substrates of the UbPS and that participate in the regulation of the cell death process were also studied. Addition of different concentrations of C2 ceramide to cultured ASTs produced an increase in the expression of the Ub gene and in the gene that encodes E1, one of the enzymes involved in the ubiquitination process, without any changes on cell viability. Immunocytochemical studies showed an increase in the expression of Bcl-2 with no changes in cytochrome c. Also, there was an increase in the nuclear reactivity of NFkappaB, suggesting a translocation of this factor towards the nucleus. Western blots showed a decrease in IkappaB and its phosphorylated form as well as an increase in Bcl-2 with no changes in cytochrome c. All of these compounds appear to be acting as possible modulators of AST responses to C2 ceramide. Our results suggest that in AST primary cultures, C2 ceramide, at the concentrations used in this study, does not produce apoptosis. However, it induces an activation of the UbPS, probably as a consequence of an activation of phosphatases and kinases, or through the generation of reactive oxygen species, which act as triggering signals of the UbPS. The fundamental role of NFkappaB and Bcl-2 as antiapoptotic factors is discussed.

Apotransferrin induces cAMP/CREB pathway and cell cycle exit in immature oligodendroglial cells.

We have demonstrated previously that a single intracranial injection of apotransferrin (aTf) in neonatal rats increases myelination and accelerates differentiation of oligodendroglial cells (OLGc). In addition, we have shown through in vitro experiments that OLGc isolated from 4-day-old rats (OLGc-4) treated with aTf were more differentiated than were controls although aTf had no effect upon OLGc isolated from 10-day-old animals (OLGc-10). In the present work, we analyzed the role of second messengers in the effect of aTf upon the maturation of OLGc at different stages of development. We isolated OLGc-4 and OLGc-10 from rat brain using a Percoll density gradient and briefly treated the cells with a pulse of aTf or kept them in culture during 2 days in the presence or absence of aTf. In OLGc-4, after a short pulse of aTf, there was an increase in the levels of cyclic AMP (cAMP), in the phosphorylation of cAMP response element-binding protein (CREB) and in the DNA-binding capacity of cAMP-responsive transcription factors. Treatment of OLGc-4 with aTf diminished bromodeoxyuridine (BrdU) incorporation and changed levels of p27 and cyclin D1. This glycoprotein seemed to act on OLGc through the cAMP pathway only at early stages of development and on a certain sensitive cell population, accelerating their differentiation, probably as a consequence of premature withdrawal from the cell cycle.

Differential effects of apotransferrin on two populations of oligodendroglial cells.

In the central nervous system (CNS), apotransferrin (aTf) is produced by oligodendroglial cells (OLGcs), and aTf is essential for cell survival. We previously demonstrated that a single intracranial injection of aTf in 3-day-old rats accelerates differentiation of OLGc and that aTf acts at early stages of development on certain populations of OLGcs, promoting accelerated maturation, with no effect on late markers of cell differentiation. The objective of the present study was to analyze OLGc maturation at two different stages of rat development, 4 and 10 days of age, in OLGcs isolated from the brain after intracranial injection of aTf at 3 days of age, and to explore the in vitro effect of aTf added to cultures of OLGc isolated from aTf-injected and control brains. The maturational cell stages were identified by immunocytochemistry with different OLGc markers and by analysis of their morphological complexity. The OLGcs isolated from 4- and 10-day-old animals intracranially injected with aTf were more differentiated than control cells. Treatment with aTf of the cultures of OLGcs that were isolated from 4-day-old saline-injected control animals induced their differentiation, while a similar treatment of the cultures of OLGcs that were isolated from 10-day-old animals did not induce further maturation of the cells. The results presented in the present report demonstrate that the in vivo effects of aTf on OLGc maturation can be reproduced in cultures and that the effects of aTf occur early in development during a narrow, transient "temporal window" within which OLGcs are sensitive to its action.

HoloTransferrin but not ApoTransferrin prevents Schwann cell de-differentiation in culture.

Schwann cells (SCs) in culture, without the presence of axons, become de-differentiated, reaching a condition similar to that of their precursor cells. The cytoplasmic accumulation of transferrin (Tf) in the myelinated peripheral nerve has been reported and data in the literature support a role for apoTf in myelination in the CNS. In the present report, we used SC cultures to evaluate the capacity of apoTf and holoTf to prevent cell de-differentiation promoted by fetal calf serum deprivation. SCs incubated in a serum-free medium showed a decrease in the expression of myelin basic protein (MBP) and P(0), markers of mature myelin-forming SCs, together with an increase in the levels of p75NTR and glial fibrillary acidic protein, markers of immature SCs. Treatment with holoTf prevented the decrease in expression of MBP and P(0) and the increase in p75NTR. ApoTf was unable to prevent these changes except when iron was added to the cultures. These results suggest a role for holoTf in the regulation of myelin formation by SCs.

Changes in the expression of cytoskeletal proteins in the CNS of transferrin transgenic mice.

Apotransferrin injected intracranially into young rats has been shown in our laboratories to induce an early differentiation of oligodendroglial cells and an increased deposition of myelin. The expression of some myelin-specific proteins such as myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and of their mRNAs were significantly increased in these animals. Also, in the cytoskeleton obtained from isolated myelin, it was found that several microtubule associated proteins (MAPs), particularly the stable tubule only peptide (STOP) and MAP 1B, as well as actin and tubulin were markedly increased. In the present paper, we compare the changes in expression of brain and myelin cytoskeletal proteins in a newly generated transferrin transgenic mouse (Tg), overexpressing the human transferrin gene, with the results obtained in aTf-injected rats. In the myelin cytoskeletal fraction of Tg mice there was a significant increase in the expression of MBP, tubulin, tau and STOP, similarly to what was previously found in the aTf-injected rats. Immunohistochemical studies showed that a variance with what occurs in the aTf-injected model, in which the above mentioned changes were limited to the corpus callosum, in the Tg mice the changes in expression of cytoskeletal proteins were observed in the various anatomical areas studied such as cerebral cortex, brain stem and cerebellum. There was also an increased expression of neurofilaments in the Tg animals, in contrast with results obtained in aTf-injected rats, suggesting that in the Tg mice, the continuous overexpression of Tf might also induce some neuronal changes. Changes in tau, total and acetylated tubulin and MAP 1B were observed in both neurons and OLGc. The increase in STOP was more significant in OLGc while the changes in MAP2 were exclusively found in neurons.

Apoptosis in Schwann cell cultures is closely interrelated with the activity of the ubiquitin-proteasome proteolytic pathway.

Although the participation of the ubiquitin-dependent pathway and of the proteasome in apoptosis has been proposed, its role in this process is not yet clearly defined. In previous studies, we have shown that in the central nervous system of the rat, programmed cell death and the ubiquitin-dependent proteolytic pathway are closely related to each other and that different types of neurons and of glial cells, shown different types of correlation between the two phenomena. In this work, we have used lactacystin, a highly specific inhibitor of the proteasome, to explore in Schwann cell cultures the relationship between the activity of the Ub-dependent pathway and apoptosis. Apoptosis was explored analyzing changes in nuclear morphology, using the Annexin V assay and by flow cytometry. Activity of caspase-3 was also measured. Changes in the levels of ubiquitin-protein conjugates and of the ubiquitin activating enzymes, E1, as well as expression of proteins that instruct the cells to apoptosis (p53, NFkappaB-IkappaB, Bcl2), or that participate in the control and regulation of the cell cycle, were also examined. Our results indicate that the decrease in the activity of the proteasome induced by lactacystin in Schwann cells, induces apoptotic cell death through changes in the concentration of certain key proteins that are involved in the apoptosis-signaling pathways.

Anxiolytic-like behavior in rats is induced by the neonatal intracranial injection of apotransferrin.

To determine whether neonatal intracranial injection of apotransferrin (aTf), which increases myelin deposition, has behavioral effects in rats, 3-day-old rats were intracranially injected with 350 ng of aTf and tested at 25 and 60 days of age. An anxiolytic-like behavior was observed in aTf-treated rats, evidenced by an increase in the exploration of open arms in the plus maze test without changes in the locomotor activity. This behavioral profile persists until adulthood. Intraperitoneal injection of 0.75 mg/kg of picrotoxin, a GABA(A) receptor channel antagonist, abolished this anxiolytic-like behavior, indicating that neonatal aTf induces a long-lasting increase in GABA(A) receptor functionality.

Defective ubiquitination of cerebral proteins in Alzheimer's disease.

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.

Relationship between the ubiquitin-dependent pathway and apoptosis in different cells of the central nervous system: effect of thyroid hormones.

We have recently shown that sustained neonatal hyperthyroidism in the rat activates apoptosis of oligodendroglial cells (OLGc) and that inhibition of the proteasome-ubiquitin (Ub) pathway by lactacystin produces increased apoptosis in cerebellar granule cells (CGC). In the present study we have analyzed the relationship between the activation of the Ub-dependent pathway, the expression of the Ub genes and programmed cell death in neurons of the rat cerebellum and cerebral cortex and in OLGc. This study was carried out in normal animals, in rats submitted to sustained neonatal hyperthyroidism and in cell cultures treated with an excess of thyroid hormones. In neurons of the cerebral cortex, thyroid hormone produces an increase of Ub-protein conjugates, an enhancement in the expression of the Ub genes and an increase in apoptosis, while the opposite results are obtained in CGC. These results indicate that in neurons, the changes in the cell death program produced by thyroid hormone run in parallel with those occurring in the Ub-dependent pathway. In OLGc, thyroid hormone increases apoptosis but does not produce changes in the Ub pathway. Preliminary studies indicate that in coincidence with what occurs in optic nerves, the sciatic nerves both in controls and in hyperthyroid animals are unable to form Ub-protein conjugates. These results indicate that in cells of the CNS such as neurons, in which the Ub-dependent pathway is actively expressed, it appears to be closely correlated with apoptosis.