Ana M. Adamo

Thyroid hormones and the central nervous system.

Thyroid hormones have a significant influence on the development and maturation of the central nervous system. Among their actions, T3 and T4 have effects on the differentiation of various cell types in the rat brain and cerebellum as well as on the process of myelination. Recently, several investigators have shown effects of thyroid hormones on myelin protein gene expression. Thyroid hormones seem to have a regulatory role with regard to life span. Hyperthyroid animals appear to have a shorter life and, at advanced age, show a myelin deficit. This may be due to the damage produced by the oxidative stress generated by an excess of thyroid hormones.

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.

The Role of Zinc in the Modulation of Neuronal Proliferation and Apoptosis

Although a requirement of zinc (Zn) for normal brain development is well documented, the extent to which Zn can modulate neuronal proliferation and apoptosis is not clear. Thus, we investigated the role of Zn in the regulation of these two critical events. A low Zn availability leads to decreased cell viability in human neuroblastoma IMR-32 cells and primary cultures of rat cortical neurons. This occurs in part as a consequence of decreased cell proliferation and increased apoptotic cell death. In IMR-32 cells, Zn deficiency led to the inhibition of cell proliferation through the arrest of the cell cycle at the G0/G1 phase. Zn deficiency induced apoptosis in both proliferating and quiescent neuronal cells via the intrinsic apoptotic pathway. Reductions in cellular Zn triggered a translocation of the pro-apoptotic protein Bad to the mitochondria, cytochrome c release, and caspase-3 activation. Apoptosis is the resultant of the inhibition of the prosurvival extracellular-signal-regulated kinase, the inhibition of nuclear factor-kappa B, and associated decreased expression of antiapoptotic proteins, and to a direct activation of caspase-3. A deficit of Zn during critical developmental periods can have persistent effects on brain function secondary to a deregulation of neuronal proliferation and apoptosis.

Dimeric procyanidin B2 inhibits constitutively active NF-κB in Hodgkin's lymphoma cells independently of the presence of IκB mutations

Due to long-term toxicity of current Hodgkins lymphoma (HL) treatment, the present challenge is to find new therapies that specifically target deregulated signaling cascades, including NF-kappaB, which are involved in Hodgkin (H) and Reed-Sternberg (RS) cell proliferation and resistance to apoptosis. We previously presented evidence that dimeric procyanidin B2 (B2) can interact with NF-kappaB proteins inhibiting the binding of NF-kappaB to DNA. Herein, we investigated if B2, acting at a late event in NF-kappaB signaling cascade, could be effective in inhibiting NF-kappaB in H-RS cells with different mechanisms of constitutive NF-kappaB activation. B2 caused a concentration-dependent inhibition of NF-kappaB-DNA binding to a similar extent (41-48% inhibition at 25 microM B2) in all the tested H-RS cell lines (L-428, KM-H2, L-540, L-1236 and HDML-2). This was associated with the inhibition of NF-kappaB-driven gene expression, including cytokines (IL-6, TNFalpha and RANTES) and anti-apoptotic proteins (Bcl-xL, Bcl-2, XIAP and cFLIP). The finding of similar amounts of RelA and p50 proteins in the nucleus, but decreased NF-kappaB-DNA binding, even in those H-RS cells characterized by mutations in the inhibitory IkappaB proteins, supports that B2 acts by preventing the binding of NF-kappaB to DNA. B2 did not inhibit AP-1 and STAT3 constitutive activation in H-RS cells, indicating that the moderate effects of B2 on cell viability are due to the complex signaling aberrations in HL. Thus, several signaling pathways should be targeted when designing therapeutics for HL. In this regard, the capacity of B2 to inhibit NF-kappaB could be valuable in a multi-drug approach.

Zinc deficiency and neurodevelopment: the case of neurons.

Zinc is essential for normal brain development. Gestational severe zinc deficiency can lead to overt fetal brain malformations. Although not teratogenic, suboptimal zinc nutrition during gestation can have long‐term effects on the offsprings nervous system. This article will review current knowledge on the role of zinc in modulating neurogenesis and neuronal apoptosis as well as the proposed underlying mechanisms. A decrease in neuronal zinc causes cell cycle arrest, which in part involves a deregulation of select signals (ERK1/2, p53, and NF‐κB). Zinc deficiency also induces apoptotic neuronal death through the intrinsic (mitochondrial) pathway, which can be triggered by the activation of the zinc‐regulated enzyme caspase‐3, and as a consequence of abnormal regulation of prosurvival signals (ERK1/2 and NF‐κB). Alterations in the finely tuned processes of neurogenesis, neuronal migration, differentiation, and apoptosis, which involve the developmental shaping of the nervous system, could have a long‐term impact on brain health. Zinc deficiency during gestation, even at the marginal levels observed in human populations, could increase the risk for behavioral/neurological disorders in infancy, adolescence, and adulthood.

Blood pressure-lowering effect of dietary (−)-epicatechin administration in L-NAME-treated rats is associated with restored nitric oxide levels

Epidemiological and intervention studies have shown that the intake of certain chocolates or cocoa products decreases blood pressure (BP) in humans. (-)-Epicatechin is the most abundant flavanol present in cocoa seeds and its derived foods. This work investigates the effects of dietary (-)-epicatechin on BP in rats that received N(ω)-nitro-l-arginine methyl ester (L-NAME) for 4 days. (-)-Epicatechin administration prevented the 42mm Hg increase in BP associated with the inhibition of NO production in a dose-dependent manner (0.2-4.0g/kg diet). This BP effect was associated with a reduction in L-NAME-mediated increase in the indexes of oxidative stress (plasma TBARS and GSSG/GSH(2) ratio) and with a restoration of the NO concentration. At the vascular level, none of the treatments modified NOS expression, but (-)-epicatechin administration avoided the L-NAME-mediated decrease in eNOS activity and increase in both superoxide anion production and NOX subunit p47(phox) expression. In summary, (-)-epicatechin was able to prevent the increase in BP and in oxidative stress and restored NO bioavailability. The fact that (-)-epicatechin is present in several plants usually consumed by humans gives the possibility of developing diets rich in those plants or pharmacological strategies using that flavonoid to diminish BP in hypertensive subjects.

The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expression in Xenopus primary neurogenesis

We analyzed the influence of presenilins on the genetic cascades that control neuronal differentiation in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduced the number of N-tubulin+ primary neurons and modulated Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increased shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduced shh expression. Both shh and presenilin promoted cell proliferation and apoptosis, but the effects of shh were widely distributed, while those resulting from presenilin injection coincided with the range of shh signaling. We suggest that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling.

Effect of oxidant systems on the ubiquitylation of proteins in the central nervous system

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. J. Neurosci. Res. 55:523–531, 1999.

Induction of Apoptosis in Cerebellar Granule Cells by 2,4-Dichlorophenoxyacetic Acid

Abstract2,4-Dichlorophenoxyacetic acid (2,4-D) and derivatives are herbicides widely used in Argentina and other parts of the world. Exposure to 2,4-D, its ester and salt formulations, have been associated with a range of adverse health effects in humans and different animal species, from embryotoxicity and teratogenicity to neurotoxicity. In this work, we demonstrate that after 24 hs of treatment with 1 and 2 mM 2,4-D there is an induction of apoptosis in cerebellar granule cells (CGC) in culture. However, with 2 mM 2,4-D one population of CGC developed features of apoptosis while another appeared to die by necrosis. This process is associated with an increase in caspase-3 activity after 12 hs of treatment with the herbicide, which is preceded by cytochrome c release from the mitochondria. Treatment of CGC with 2,4-D appears to induce apoptosis by a direct effect on mitochondria producing cytochrome c release and consequently activation of caspase-3, being mitochondrial damage sufficient for triggering the events that may cause apoptosis.

The notch signaling pathway: its role in focal CNS demyelination and apotransferrin-induced remyelination

Oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Identifying the signaling pathways involved in myelin repair through oligodendroglial progenitor maturation is essential for the development of new therapies. This article investigated the role of the Notch signaling pathway in CNS demyelination and apotransferrin‐induced remyelination in a focal lysolecithin‐induced demyelination model in rats. Notch was found activated in Nestin‐expressing neural progenitor cells and in NG2‐expressing oligodendroglial precursor cells in the subventricular zone and corpus callosum of lysolecithin‐demyelinated rats. Notch activation seemed to be driven by Jagged1, which led to a high expression of downstream gene Hes5 in the subventricular zone of demyelinated rats. Apotransferrin injection induced remyelination, while the injection of the γ‐secretase inhibitor reversed this effect. In addition, 24 h after apotransferrin injection, evidence showed Notch activation concomitantly with an increase in F3/contactin levels and the up‐regulation of the myelin‐associated glycoprotein gene in the subventricular zone and corpus callosum of demyelinated rats. Collected evidence supports the participation of both canonical and non‐canonical Notch signaling pathways in demyelination/remyelination. Notch activation was found to trigger Hes5 expression as a consequence of focal demyelination, which might promote oligodendroglial precursor cell proliferation. During apotransferrin‐induced remyelination, Notch activation seemed to be mediated by the expression of F3/contactin, which might induce apotransferrin‐mediated oligodendroglial maturation.