Impaired Inflammatory Response to Glial Cell Death in Genetically Metallothionein-I- and -II-Deficient Mice

doi:10.1006/exnr.1998.7009

Experimental Neurology

Volume 156, Issue 1, March 1999, Pages 149-164

https://doi.org/10.1006/exnr.1998.7009 Get rights and content

Abstract

Metallothionein I+II (MT-I+II) are acute-phase proteins which are upregulated during pathological conditions in the brain. To elucidate the neuropathological importance of MT-I+II, we have examined MT-I+II-deficient mice following ip injection with 6-aminonicotinamide (6-AN). 6-AN is antimetabolic and toxic for bone marrow cells and grey matter astrocytes. In MT+/+ mice, injection with 6-AN resulted in breakdown of the blood–brain barrier (BBB) and absence of GFAP-positive astrocytes in specific grey matter areas of the brain stem. Reactive astrocytosis encircled the damaged grey matter areas, which were heavily infiltrated by microglia/macrophages. The recruitment of hematogenous macrophages was accompanied by leakage of the BBB. The immunoreactivity (ir) of granulocyte–macrophage-colony-stimulating factor (GM-CSF) and the receptor for GM-CSF (GM-CSFrec) was significantly upregulated in astrocytes and microglia/macrophages, respectively. MT-I+IIir was also clearly increased in astrocytes surrounding the damaged areas, while that of the CNS-specific MT isoform, MT-III, was mildly increased in both astrocytes and microglia/macrophages. In MT−/− mice injected with 6-AN, the BBB remained almost intact. The damage to specific grey matter areas was similar to that observed in MT+/+ mice, but reactive astrocytosis, microglia/macrophages infiltration, and GM-CSFir and GM-CSFrecir were clearly reduced in MT−/− mice. In contrast, MT-IIIir was dramatically increased in MT−/− mice. Total zinc decreased and histochemically detectable zinc increased in the brain stem after 6-AN similarly in MT+/+ and MT−/− mice. Bone marrow myeloid monocytes and macrophages were increased as a reaction to 6-AN only in MT+/+ mice. The results demonstrate that the capability of MT−/− mice to mount a normal inflammatory response in the brain is severely attenuated, at least in part because of 6-AN-induced bone marrow affectation, involving MT-I+II for the first time as major factors during CNS tissue damage.

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Besides their roles in maintaining cellular Zn homeostasis, a growing body of work indicates that both MT1 and 2 play important roles in neuroprotection and neuroregeneration. MT1/2 KO mice exhibit impaired wound recovery following brain lesion, with increased apoptosis and increased inflammatory response (Penkowa et al., 1999). Concurrently, transgenic MT1 mice show reduced oxidative stress, neuronal damage, and inflammatory response (van Lookeren Campagne et al., 1999).
Zinc transporters (ZnTs) and metallothioneins (MT) are important in maintaining Zn homeostasis in the brain. The present study was designed to find out whether alterations in ZnTs and MTs are associated with the pathophysiology of depression and the mechanism of antidepressant action.
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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.
Obesity and downregulated hypothalamic leptin receptors in male metallothionein-3-null mice
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Mt1 and Mt2 are expressed ubiquitously (Palmiter, 1987; Palmiter et al., 1992), whereas Mt3 is expressed mainly in the central nervous system (CNS) (Palmiter et al., 1992) and Mt4 is expressed in stratified squamous epithelia (Quaife et al., 1994). Mt1/2-null mice are more vulnerable to insults (Carrasco et al., 2000; Penkowa et al., 1999a,b; Satoh et al., 1997b; Trendelenburg et al., 2002), indicating the cytoprotective roles of these proteins. Moreover, Mt1/2 ablation results in obesity, probably due to abnormal peripheral metallothionein function (Beattie et al., 1998).
In the present study, we examined whether metallothionein-3 (Mt3), a zinc-binding protein that is specifically enriched in a brain, plays a role in obesity and hypothalamic leptin signaling in mice. Upon aging, male Mt3-null mice gained more body weight than male wild-type mice; however, the daily amount of food intake was little different. Rather, the obesity in male Mt3-null mice was likely the result of decreased metabolic rates, as indicated by lower oxygen consumption and carbon dioxide production. Consistent with this, mRNA levels for the mitochondrial proton carrier UCP1 were reduced in brown adipose tissue of Mt3-null mice. Although Mt3-null mice showed increases in serum leptin levels, probably due to increased fat mass, the level of the leptin receptor (Lepr) in the hypothalamus of these mice was significantly reduced. In addition, levels of phosphorylated extracellular signal-regulated kinase (p-Erk-1/2) were also reduced in the hypothalamus of Mt3-null mice. Because zinc released from Mt3 may activate Erk-1/2, we examined whether zinc is involved in the upregulation of Lepr levels through the activation of Erk-1/2. Consistent with this possibility, exposure of hypothalamic cells to zinc activated Erk-1/2 and induced Lepr expression in an Erk-dependent manner. The present results demonstrate that Mt3 in the brain of male mice, particularly in the hypothalamus, may be involved in central leptin signaling and the consequent increase in peripheral energy expenditure. In addition to providing insight into the role of zinc and metallothioneins in the development of obesity, this new information may help design ways to overcome the pervasive problem of obesity.
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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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The lack of differences in disease progression and lesion profile observed between the MT-1 + 2 KO and WT mice suggests the presence of a compensatory mechanism. One obvious possibility, which has not been investigated in the present work, was that a compensatory up-regulation of MT3 compensated for the lack of the 1 and 2 isoforms (Carrasco et al., 2000; Penkowa et al., 1999b). Concerning the hippocampus, the differential up-regulation of HSP25 could be the compensatory mechanism for the lack of MT-1 + 2.
Metallothioneins (MT) are heavy metal-binding, antioxidant proteins with relevant roles described in many pathological conditions affecting the central nervous system (CNS). Regarding prion diseases, a number of publications demonstrate an up-regulation of MT-1 + 2 in the brains of TSE affected cattle, humans and experimentally inoculated rodents. Since the prion protein also binds copper, and oxidative stress is one of the events presumably triggered by PrPsc deposition, it seems plausible that MTs have a relevant role in the outcome of these neurodegenerative processes. To gain knowledge of the role of MTs in TSE pathogeny, and particularly of that of MT-1 + 2, a transgenic MT-1 + 2 knockout mouse model (MT-1 + 2 KO) was intracerebrally inoculated with the mouse-adapted Rocky Mountain Laboratory (RML) strain of scrapie; 129SvJ mice were used as controls (WT). Clinical signs were monitored and animals were humanely sacrificed when they scored positive clinically. Brains were fixed following intracardiac perfusion with 4% formaldehyde, paraffin embedded, and processed for histological, histochemical and immunohistochemical evaluation. The incubation period did not show significant differences between MT-1 + 2 KO and WT mice, nor did the evolution of neurological signs. Upon neuropathological characterisation of the brains, moderate differences were observed in astroglial and microglial response, spongiosis score and PrPsc deposition, particularly in brain regions to which the studied strain showed a stronger tropism (i.e. hippocampus). Results showed that the brain defence mechanisms against PrPsc deposition involve, aside from MT-1 + 2, other molecules, such as HSP25, which are capable of compensating for the lack of MT-1 + 2.
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Zinc is a relevant trace element for the efficiency of the entire immune system. The binding of zinc with some proteins, such as metallothioneins (MT) and α-2 macroglobulin (α-2M) is crucial for the immune efficiency during ageing and in age-related diseases, because these proteins may be involved in antagonistic pleiotropic effects. Indeed, the presence of chronic inflammation during ageing, generally, induces overexpression of these proteins that, due to their original biological function in fighting stressor agents, continuously sequester intracellular zinc. As a consequence, a low zinc ion availability may appear in aged organisms leading to impairments of the immune response at thymic and extrathymic levels with the risk of the appearance of age-related diseases. Therefore, MT and α-2M turn from protective in “young-adult age” to harmful agents in “ageing” following the basic assumption of an evolutionary theory of ageing, named the “antagonistic pleiotropy”, which suggests that a trade off between early beneficial effects and late negative outcomes can occur at a genetic and molecular level. On the other hand, some polymorphisms of MT (MT2A) and α-2M have been associated with atherosclerosis or Alzheimer disease, respectively. Physiological zinc supplementation in elderly restores the thymic endocrine activity and innate immune response (NK cell cytotoxicity) and increases the survival rate in old mice. Therefore, zinc supplementation is useful to achieve health longevity because these zinc-binding proteins may regain their original protective task against oxidative damage with, thus, a beneficial impact on immune response.

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Regular ArticleImpaired Inflammatory Response to Glial Cell Death in Genetically Metallothionein-I- and -II-Deficient Mice

Abstract

Neuroscience

Neuroscience

Neurochem. Int.

Exp. Neurol.

Exp. Neurol.

Neurochem. Int.

Brain. Res.

J. Neuroimmunol.

Mol. Brain Res.

Trends Neurosci.

Brain Res.

Brain Res.

Brain Res.

Mol. Brain Res.

Neuroscience

Trends Neurosci.

J. Neurosci. Methods

Toxicol. Appl. Pharmacol.

Brain Res.

Curr. Opinion Neurobiol.

Trends Neurosci.

Free Radic. Biol. Med.

Neurosci. Lett.

Neurosci. Lett.

J. Neurol. Sci.

Brain Res.

Biochim. Biophys. Acta

Neuron

Mol. Brain Res.

Pheotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds

Glia

The functional significance of brain metallothioneins

Faseb J

Reactive astrogliosis in the neonatal mouse brain and its modulation by cytokines

J. Neurosci.

Regular Article
Impaired Inflammatory Response to Glial Cell Death in Genetically Metallothionein-I- and -II-Deficient Mice