Latest in Metallothionein Research

Metallothionein induces a regenerative reactive astrocyte phenotype via JAK/STAT and RhoA signalling pathways
Y.K.J. Leung ^a, M. Pankhurst ^a, S.A. Dunlop ^b, S. Ray ^a, J. Dittmann ^a, E.D. Eaton ^a, P. Palumaa ^c, R. Sillard ^c, M.I. Chuah ^a, A.K. West ^a and R.S. Chung ^a.

a) Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, Tasmania 7001, Australia
b) School of Animal Biology, University of Western Australia, Nedlands, Western Australia 6907, Australia
c) Department of Gene Technology, Tallinn Technical University. Akadeemia tee 15, Tallinn 12618, Estonia

Journal: Experimental Neurology, Vol 221 (1), 98-106, 2010.

Abstract: Following central nervous system injury, astrocytes rapidly respond by undergoing a stereotypical pattern of molecular and morphological alterations termed “reactive” astrogliosis. We have reported previously that metallothioneins (MTs) are rapidly expressed by reactive astrocytes and that their secretion and subsequent interaction with injured neurons leads to improved neuroregeneration. We now demonstrate that exogenous MT induces a reactive morphology and elevated GFAP expression in cultured astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT and RhoA signalling pathways. However, rather than being inhibitory, MT induced a form of astrogliosis that was permissive to neurite outgrowth and which was associated with decreased chondroitin sulphate proteoglycan (CSPG) expression. The results suggest that MT has an important role in mediating permissive astrocytic responses to traumatic brain injury.

Keywords: Traumatic brain injury; Astrogliosis; Regeneration

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Metallothionein II-A improves healing after a burn wound

Morellini N.^1,2,3, Giles N. ^3,4, Rea S. ^3,5,6, King C. ^1,2, Dunlop S. ^1,2, Beazley L. ^1,2, West A. ⁷, Wood F. ^1,5,6, Fear M. ³

1School of Animal Biology, University of Western Australia,
2Western Australian Institute for Medical Research, Australia,
3The McComb Research Foundation, Perth, Australia,
4Department of Anatomy and Human Biology, University of Western Australia,
5Royal Perth Hospital, Perth, Australia,
6Princess Margaret Hospital for Children, Perth, Australia,
7NeuroRepair Group, Menzies Research Institute, University of Tasmania

The epidermal barrier prevents infection and dehydration. Its rapid repair is essential after injury. Severe injuries often result in scarring and life-long functional deficits, the outcome worsening with longer times to heal. We investigated the potential of Metallothionein II-A (Apo-MT-IIA: Bestenbalt, Tallinn, Estonia, rabbit-derived, >98% pure by HPLC in zinc sulphate solution, PBS, pH 7.4), a naturally occurring small cysteine-rich protein, to accelerate healing after burn wounds. In vitro assays of a human keratinocyte cell line (HaCaT) indicated that at 1 g/ml and 2 g/ml MT-IIA significantly increased cell proliferation (p<0.05). Annexin V and propidium iodide FACS analysis of keratinocytes with increasing amount of MTII-A reduced the percentage of cells undergoing apoptosis in response to both a UV insult and to Staurosporine in a dose-dependent manner with the effect at 1 g/ml reducing apoptosis by >50% p<0.05)). After a full thickness burn to the dorsal skin of adult mice, immunohistochemistry revealed that endogenous MT-I/II expression increased in basal keratinocytes during healing. Increases are seen in the wound margin at early stages (3 and 7 days; p<0.05) and in its centre by 11 days (p<0.05). Topical administration of exogenous MT-IIA immediately post-burn accelerated the return of MTI/II expression to normal values by day 14 in the wound margin (Normal: 39 ± 3% vs MT: 49 ± 7%, p>0.05; vs PBS: 68 ± 7%, p<0.05) and improved healing as assessed by reduced epidermal thickness (MTII-A: 45 ± 4 μm vs control: 101 ± 19 μm, p<0.05) and faster wound closure at Day 3 post-injury 8.9 mm ± 0.27 mm in controls compared to 7.1 mm ± 0.7 mm in treated wounds, by day 7 5.8 mm ± 0.98mm in controls versus 3.6 mm ± 1.0 mm in treated wounds, p<0.05).. Our data suggest that MT-IIA may prove a valuable therapeutic for patients with burns and other skin injuries.

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Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley grain tissues using SEC-ICP-MS and IP-ICP-MS

Daniel P. Persson, Thomas H. Hansen, Kristian H. Laursen, Jan K. Schjoerring and Søren Husted

Journal: Metallomics, 2009, 1, 418 - 426.

Abstract: The increasing prevalence of iron (Fe) and zinc (Zn) deficiencies in human populations worldwide has stressed the need for more information about the distribution and chemical speciation of these elements in cereal products. In order to investigate these aspects, barley grains were fractionated into awns, embryo, bran and endosperm and analysed for Fe and Zn. Simultaneously, phosphorus (P) and sulfur (S) were determined since these elements are major constituents of phytic acid and proteins, respectively, compounds which are potentially involved in Fe and Zn binding. A novel analytical method was developed in which oxygen was added to the octopole reaction cell of the ICP-MS. This approach greatly improved the sensitivity of sulfur, measured as 48SO+. Simultaneously, Fe was measured as 72FeO+, P as 47PO+, and Zn as 66Zn+, enabling sensitive and simultaneous analysis of these four elements. The highest concentrations of Zn, Fe, S and P were found in the bran and embryo fractions. Further analysis of the embryo using SEC-ICP-MS revealed that the speciation of Fe and Zn differed. The majority of Fe co-eluted with P as a species with the apparent mass of 12.3 kDa, whereas the majority of Zn co-eluted with S as a 3 kDa species, devoid of any co-eluting P. Subsequent ion pairing chromatography of the Fe/P peak showed that phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate: IP6) was the main Fe binding ligand, with the stoichiometry Fe4(IP6)18. When incubating the embryo tissue with phytase, the enzyme responsible for degradation of phytic acid, the extraction efficiency of both Fe and P was doubled, whereas that of Zn and S was unaffected. Protein degradation on the other hand, using protease XIV, boosted the extraction of Zn and S, but not that of Fe and P. It is concluded that Fe and Zn have a different speciation in cereal grain tissues; Zn appears to be mainly bound to peptides, while Fe is mainly associated with phytic acid.

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Metallothionein Treatment Attenuates Microglial Activation and Expression of Neurotoxic Quinolinic Acid Following Traumatic Brain Injury
R. S. Chung¹, Y. K. Leung¹, C. W. Butler¹, Y. Chen², E. D. Eaton¹, M. W. Pankhurst¹, A. K. West¹ and G. J. Guillemin²

(1) NeuroRepair Group, Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, TAS, 7001, Australia
(2) Centre for Immunology, University of New South Wales, Sydney, 2052, Australia

Journal: Neurotoxicity Research, Volume 15, Number 4 / May, 2009.

Abstract: The kynurenine pathway has been implicated as a major component of the neuroinflammatory response to brain injury and neurodegeneration. We found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN) is rapidly expressed, within 24 h, by reactive microglia following traumatic injury to the rodent neocortex. Furthermore, administration of the astrocytic protein metallothionein attenuated this neuroinflammatory response by reducing microglial activation (by approximately 30%) and QUIN expression. The suppressive effect of MT was confirmed upon cultured cortical microglia, with 1 μg/ml MT almost completely blocking interferon–gamma induced activation of microglia and QUIN expression. These results demonstrate the neuroimmunomodulatory properties of MT, which may have therapeutic applications for the treatment of traumatic brain injury.

Keywords: Traumatic brain injury - Neuroinflammation - Neuron-glia interactions

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Potential Role of a-Synuclein and Metallothionein in Lead-Induced Inclusion Body Formation
Peijun Zuo*, Wei Qu*, Ryan N. Cooper*, Robert A. Goyer*, Bhalchandra A. Diwan and Michael P. Waalkes*

* Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Basic Research Program, SAIC-Frederick, Inc., NCI at Frederick, Maryland 21702

Journal: Toxicological Sciences 2009 111(1):100-108;

Abstract: Lead (Pb) produces aggresome-like inclusion bodies (IBs) in target cells as a toxic response. Our prior work shows metallothionein (MT) is required for this process. We used MT-I/II double knockout (MT-null) and parental wild-type (WT) cell lines to further explore the formation process of Pb-induced IBs. Unlike WT cells, MT-null cells did not form IBs after Pb exposure. Western blot of cytosol showed soluble MT protein in WT cells was lost during Pb exposure as IBs formed. Transfection of MT-I into MT-null cells allowed IBs formation after Pb exposure. Considering Pb-induced IBs may be like disease-related aggresomes, which often contain alpha-synuclein (Scna), we investigated Scna expression in cells capable (WT) and incapable (MT-null) of producing IBs after Pb exposure. Scna protein showed poor basal expression in MT-null cells. Pb exposure increased Scna expression only in WT cells. MT transfection increased Scna transcript to WT levels. In WT or MT-transfected MT-null cells, Pb-induced Scna expression rapidly increased and then decreased over 48 h as Pb-induced IBs were formed. A direct interaction between Scna and MT was confirmed ex vivo by antibody pulldown assay where the proteins coprecipitated with an antibody to MT. Pb exposure caused increased colocalization of MT and Scna proteins with time only in WT cells. In WT mice after chronic Pb exposure Scna was localized in renal cells containing forming IBs, whereas MT-null mice did not form IBs. Thus, Scna could be component of Pb-induced IBs and, with MT, may play a role in IBs formation.
 

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Redefining the Role of Metallothionein within the Injured Brain
EXTRACELLULAR METALLOTHIONEINS PLAY AN IMPORTANT ROLE IN THE ASTROCYTE-NEURON RESPONSE TO INJURY.

Roger S. Chung¹, Milena Penkowa², Justin Dittmann¹, Carolyn E. King³, Carole Bartlett³, Johanne W. Asmussen², Juan Hidalgo⁴, Javier Carrasco⁴, Yee Kee J. Leung¹, Adam K. Walker¹, Samantha J. Fung¹, Sarah A. Dunlop³, Melinda Fitzgerald³, Lyn D. Beazley³, Meng I. Chuah¹, James C. Vickers¹, and Adrian K. West¹

¹NeuroRepair Group, Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, Tasmania 7001, Australia,

²Section of Neuroprotection, Faculty of Health Sciences, University of Copenhagen, Copenhagen DK2200, Denmark,

³School of Animal Biology, University of Western Australia, Nedlands, Western Australia 6907, Australia, and

⁴Institute of Neurosciences and Department of Cellular Biology, Physiology and Immunology, Animal Physiology Unit, Faculty of Sciences, Autonomous University of Barcelona, Bellaterra, Barcelona 08193, Spain


Journal of Biological Chemistry, Vol. 283, Issue 22, 15349-15358, May 30, 2008.

Abstract: 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.
 

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New insight into the molecular pathways of metallothionein-mediated neuroprotection and regeneration

R. S. Chung*, J. Hidalgo† and A. K. West*

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Metallothionein and a peptide modeled after metallothionein, EmtinB, induce neuronal differentiation and survival through binding to receptors of the low-density lipoprotein receptor family.

Malene Ambjørn*, Johanne W. Asmussen*,†, Mats Lindstam*, Kamil Gotfryd*, Christian Jacobsen‡, Vladislav V. Kiselyov*, Søren K. Moestrup‡, Milena Penkowa†, Elisabeth Bock* and Vladimir Berezin*

*Protein Laboratory, Institute of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark
†Section of Neuroprotection, Institute of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark
‡Institute of Medical Biochemistry, University of Aarhus, Aarhus, Denmark
Address correspondence and reprint requests to Vladimir Berezin, Protein Laboratory, Institute of Neuroscience and Pharmacology, University of Copenhagen, Panum Institute Building 6.2, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. E-mail: berezin@plab.ku.dk

Abstract: Accumulating evidence suggests that metallothionein (MT)-I and -II promote neuronal survival and regeneration in vivo. The present study investigated the molecular mechanisms underlying the differentiation and survival-promoting effects of MT and a peptide modeled after MT, EmtinB. Both MT and EmtinB directly stimulated neurite outgrowth and promoted survival in vitro using primary cultures of cerebellar granule neurons. In addition, expression and surface localization of megalin, a known MT receptor, and the related lipoprotein receptor-related protein-1 (LRP) are demonstrated in cerebellar granule neurons. By means of surface plasmon resonance MT and EmtinB were found to bind to both megalin and LRP. The bindings were abrogated in the presence of receptor-associated protein-1, an antagonist of the low-density lipoprotein receptor family, which also inhibited MT- and EmtinB-induced neurite outgrowth and survival. MT-mediated neurite outgrowth was furthermore inhibited by an anti-megalin serum. EmtinB-mediated inhibition of apoptosis occurred without a reduction of caspase-3 activity, but was associated with reduced expression of the pro-apoptotic B-cell leukemia/lymphoma-2 interacting member of cell death (BimS). Finally, evidence is provided that MT and EmtinB activate extracellular signal-regulated kinase, protein kinase B, and cAMP response element binding protein. Altogether, these results strongly suggest that MT and EmtinB induce their neuronal effects through direct binding to surface receptors belonging to the low-density lipoprotein receptor family, such as megalin and LRP, thereby activating signal transduction pathways resulting in neurite outgrowth and survival.

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