Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain

doi:10.1016/0009-2797(94)90020-5

Chemico-Biological Interactions

Volume 93, Issue 3, December 1994, Pages 197-219

https://doi.org/10.1016/0009-2797(94)90020-5 Get rights and content

Abstract

The knowledge of brain metallothionein (MT) regulation and especially of MT presence in specific cell types is scarce. Therefore, the effect of several well-known MT inducers, measured by radioimmunoassays using antibodies that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibitory factor (GIF or MT-III), has been studied in primary cultures of neurons or astrocytes obtained from rat cerebrum. MT-I levels in glial cells were about ten times higher than those in neuronal cells (538 ± 194 vs. 49 ± 16 pg MT-I/μg protein, mean ± SD from three separate cell preparations). Increasing the concentration of Zn in the bovine serum albumin (BSA)-containing culture medium up to 50 μM significantly increased MT-I levels by up to 3.5-fold in neurons and 2.5-fold in astrocytes. In contrast, Cu up to 50 μM increased MT-I levels in a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1.5-fold), the maximum effect occurring at 5 μM Cu. In general, the combination of Zn and Cu further increased MT-I levels. The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked when the BSA concentration in the medium was increased from 2 to 10 mg/ml. Dexamethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentration-dependent manner. Endotoxin, IL-1 and IL-6 did not have a significant effect on glial MT levels at the concentrations studied. The administration of dexamethasone to rats increased MT-I levels in non-frontal cortex, cerebellum, pons + medulla, midbrain and hippocampus, but not in hypothalamus, frontal cortex and striatum. Endotoxin increased liver but not brain MT-I levels. Immunocytochemical studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostaining was always nuclear in glial cells, whereas in neurons it was nuclear in the cerebral cortex, hippocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purkinje cells in the cerebellum, hypothalamic nuclei and gigantocellular reticular nucleus in the brain stem. Meninges, choroidal plexus, ependymal and endothelial cells were also MT-immunoreactive.

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Absence of metallothionein-3 produces changes on MT-1/2 regulation in basal conditions and alters hypothalamic-pituitary-adrenal (HPA) axis
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Citation Excerpt :
The response to stress or inflammation on corticosterone plasma levels is similar in wild type or Mt-3 KO mice. It is generally accepted that the expression of MT-1/2 is highly inducible in response to several conditions like stress and inflammation (Chua et al., 2012; Chung et al., 2008; Bremner et al., 1987; Sato and Bremner, 1993; Ghoshal et al., 1998) Glucocorticoids appear to have a role in brain MT regulation, at least in basal conditions as previously noted (Hidalgo et al., 1994a). In our laboratory, we have shown in earlier experiments that glucocorticoids appear to mediate brain MT-1 induction in some but not all brain areas (Hidalgo et al., 1997) indicating that other factors must be involved.
Metallothioneins (MTs) are multipurpose proteins with clear antioxidant, anti-inflammatory and metal homeostasis properties. The roles of brain MT-1 and MT-2 are similar to those described in the periphery, and are inducible by metals, inflammatory and stress stimuli. MT-3, originally named growth inhibitory factor, exists mainly in the central nervous system, is hardly ever inducible and its functional role and regulation are poorly understood and controversial. In the present study we examined how absence of MT-3 affects phenotypic characteristics and its effects on MT1/2 expression in basal situation and after induction. Hyperactive behavior was found only in young male Mt-3 KO mice and disappeared in the older ones. Absence of MT-3 was associated with a significant increase of MT-1/2 protein levels in several brain areas but decreased MT-1 mRNA levels, which might be related to lower corticosterone levels. The response to stress or inflammation on corticosterone plasma levels was similar in wild type and Mt-3 KO mice, suggesting that the relevant MT-3 role as MT-1/2 regulator in basal conditions is lost when other important regulatory factors such as glucocorticoids or cytokines appear.
Metabolism and functions of copper in brain
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There are three MT isoforms in the brain. MT1, MT2 and MT3 are expressed in the BBB and BCB as well as in astrocytes and neurons, whereas microglial cells and oligodendrocytes have been reported to be essentially devoid of MTs (Anezaki et al., 1995; Hidalgo et al., 1994, 2001; Uchida et al., 1991). Elevated copper levels in brain are accompanied by an increase in MT levels, most likely reflecting a compensatory response against copper-induced toxicity (Haywood et al., 2008; Haywood and Vaillant, in press; Ono et al., 1997; Zatta et al., 2008).
Copper is an important trace element that is required for essential enzymes. However, due to its redox activity, copper can also lead to the generation of toxic reactive oxygen species. Therefore, cellular uptake, storage as well as export of copper have to be tightly regulated in order to guarantee sufficient copper supply for the synthesis of copper-containing enzymes but also to prevent copper-induced oxidative stress. In brain, copper is of importance for normal development. In addition, both copper deficiency as well as excess of copper can seriously affect brain functions. Therefore, this organ possesses ample mechanisms to regulate its copper metabolism. In brain, astrocytes are considered as important regulators of copper homeostasis. Impairments of homeostatic mechanisms in brain copper metabolism have been associated with neurodegeneration in human disorders such as Menkes disease, Wilson's disease and Alzheimer's disease. This review article will summarize the biological functions of copper in the brain and will describe the current knowledge on the mechanisms involved in copper transport, storage and export of brain cells. The role of copper in diseases that have been connected with disturbances in brain copper homeostasis will also be discussed.
Basal metallothionein-I/II protects against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures
2011, Toxicology
Citation Excerpt :
Our results show that the MT-I/II expression was significantly increased following 24 h NMDA exposure in cortical neuron/astrocyte cocultures from MT+/+ neonatal mice but not in the cultures from MT−/− mice. Many studies have demonstrated only low level of MT-I/II was expressed in neurons, and MT-I/II was not induced following exposure to zinc or excitotoxic concentrations of glutamate (Hidalgo et al., 2001; Hidalgo et al., 1994; Taylor et al., 2004). Thus, the increased MT-I/II expression observed in the present study likely reflects increased MT-I/II in astrocytes.
N-methyl-d-aspartate (NMDA) receptor overactivation-mediated oxidative stress has been proposed to contribute to brain injury. Metallothionein-I/II (MT-I/II), a member of cysteine-rich metalloproteins, has been found to express in the central nervous system primarily in cortical tissues and be upregulated following brain injury. To address the role of MT-I/II on NMDA-mediated oxidative injury, we established primary cortical neuron/astrocyte cultures from neonatal MT-I/II deficient (MT^−/−) and wild type (MT^+/+) mice to test whether basal MT-I/II protects cortical cultures against NMDA-mediated injury. We found that MT-I/II expression was increased by NMDA in MT^+/+ cultures but was not detectable in MT^−/− cultures. NMDA concentration-dependently induced oxidative injury in both MT^+/+ and MT^−/− cultures as evidenced by decrease of cell viability, increases of lipid peroxidation and DNA damage. However, these toxic effects were greater in MT^−/− than MT^+/+ cultures. NMDA significantly increased reactive oxygen species (ROS) generation and disrupted mitochondrial membrane potential in neurons in MT^+/+ cultures, and these effects were exaggerated in MT^−/− cultures. Our findings clearly show that basal MT-I/II provides protection against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures, and suggest that the protective effects are possibly associated with inhibition of ROS generation and preservation of mitochondrial membrane potential.
Differential time-course of the increase of antioxidant thiol-defenses in the acute phase after spinal cord injury in rats
2009, Neuroscience Letters
Spinal cord injury (SCI) is a world-wide health problem. After traumatic injury, spinal cord tissue starts a series of self-destructive mechanisms, known as the secondary lesion. The leading mechanisms of damage after SCI are excitotoxicity, free radicals’ overproduction, inflammation and apoptosis. Metallothionein (MT) and reduced glutathione (GSH) are low-molecular-weight, cysteine-rich peptides able to scavenge free radicals. MT and GSH participation as neuroprotective molecules after SCI is unknown. The aim of the present study is to describe the changes of MT and GSH contents and GSH peroxidase (GPx) activity in the acute phase after SCI in rats. Female Wistar rats weighing 200–250 g were submitted to spinal cord contusion model, by means of a computer-controlled device (NYU impactor). Rats receiving laminectomy were used as a control group. Animals were killed 2, 4, 12 and 24 h after surgery. MT was quantified by the silver-saturation method, using atomic absorption spectrophotometry. GSH and GPx were assayed by spectrophotometry. Results indicate an increased MT content by effect of SCI, only at 4 and 24 h, as compared to sham group values. Meanwhile, GSH was found decreased at 4, 12 and 24 h after SCI. Interestingly, GPx activity was raised at all time points, indicating that this enzymatic defense is activated soon after SCI. Results suggest that thiol-based defenses, MT and GSH, are differentially expressed by spinal cord tissue to cope with the various processes of damage after lesion.
Redefining the role of metallothionein within the injured brain: Extracellular metallothioneins play an important role in the astrocyte-neuron response to injury
2008, Journal of Biological Chemistry
Citation Excerpt :
RGC counts and axon measurements were analyzed using analysis of variance (StatView, USA) and p values calculated using the Scheffe post hoc test. Detection of Extracellular MT-I/-II Released from Cultured Cortical Astrocytes—To test the hypothesis that astrocytes are capable of releasing MT-I/-II, primary cortical astrocytes were maintained in vitro, and the presence of MT-I/-II in the culture media was assessed using an MT-I/-II-specific radioimmunoassay (RIA) that is capable of detecting picogram amounts (16, 17). In confluent astrocyte cultures, MT-I/-II was detected in the medium at a range of 15.6 ± 0.49 pg of MT/μg of total protein (average over 12 different experiments).
A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.
Metallothionein in the central nervous system: Roles in protection, regeneration and cognition
2008, NeuroToxicology
Metallothionein (MT) is an enigmatic protein, and its physiological role remains a matter of intense study and debate 50 years after its discovery. This is particularly true of its function in the central nervous system (CNS), where the challenge remains to link its known biochemical properties of metal binding and free radical scavenging to the intricate workings of brain. In this compilation of four reports, first delivered at the 11th International Neurotoxicology Association (INA-11) Meeting, June 2007, the authors present the work of their laboratories, each of which gives an important insight into the actions of MT in the brain. What emerges is that MT has the potential to contribute to a variety of processes, including neuroprotection, regeneration, and even cognitive functions. In this article, the properties and CNS expression of MT are briefly reviewed before Dr Hidalgo describes his pioneering work using transgenic models of MT expression to demonstrate how this protein plays a major role in the defence of the CNS against neurodegenerative disorders and other CNS injuries. His group's work leads to two further questions, what are the mechanisms at the cellular level by which MT acts, and does this protein influence higher order issues of architecture and cognition? These topics are addressed in the second and third sections of this review by Dr West, and Dr Levin and Dr Eddins, respectively. Finally, Dr Aschner examines the ability of MT to protect against a specific toxicant, methylmercury, in the CNS.

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¹: Present address: Medical Academy of Wroclaw, Department of Toxicology, Traugutta 57/59, Wroclaw 50-417, Poland.

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Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain

Abstract

J. Nutr.

Brain Res.

Methods Enzymol.

Anal. Biochem.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Inorg. Biochem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Methods Enzymol.

Dev. Biol.

Neurosci. Lett.

J. Nutr.

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

Biochim. Biophys. Acta

Brain Res.

Life Sci.

Neuron

Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin

Physiol. Rev.

Metallothionein

Annu. Rev. Biochem.

Copper in brain

Neurochem. Res.

The Neurobiology of Zinc

Biochemical characterization of a metallothionein-like protein in rat brain

Biol. Trace Elem. Res.

Regulation of zinc metallothionein II mRNA level in rat rain

Neurochem. Int.