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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

1)NeuroRepair Group, Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, Tasmania 7001, Australia,
2)Section of Neuroprotection, Faculty of Health Sciences, University of Copenhagen, Copenhagen DK2200, Denmark,
3)School of Animal Biology, University of Western Australia, Nedlands, Western Australia 6907, Australia, and
4)Institute 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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Metallothionein-IIA promotes neurite growth via the megalin receptor

 

Melinda Fitzgerald (1) , Pia Nairn (1), Carole A. Bartlett (1), Roger S. Chung (3), Adrian K. West (3) and Lyn D. Beazley (1, 2)

(1) Experimental and Regenerative Neurosciences, School of Animal Biology, University of Western Australia, Hackett Drive, Crawley, 6009, WA, Australia
(2) Western Australian Institute of Medical Research, University of Western Australia, Hackett Drive, Crawley, 6009, WA, Australia
(3) Neurorepair Group, Menzies Research Institute, University of Tasmania, Private Bag 58, Hobart, TAS, 7001, Australia

 

Journal:    Experimental Brain Research, Volume 183, Number 2 / November, 2007, p. 171-180

 

Open: Entire document  (Subscription to the journal required).


 

Received: 20 February 2007 Accepted: 13 June 2007 Published online: 19 July 2007

Abstract: Metallothionein (MT)-I/II has been shown to be neuroprotective and neuroregenerative in a model of rat cortical brain injury. Here we examine expression patterns of MT-I/II and its putative receptor megalin in rat retina. At neonatal stages, MT-I/II was present in retinal ganglion cells (RGCs) but not glial or amacrine cells; megalin was present throughout the retina. Whilst MT-I/II was absent from adult RGC in normal animals and after optic nerve transection, the constitutive megalin expression in RGCs was lost following optic nerve transection. In vitro MT-IIA treatment stimulated neuritic growth: more RGCs grew neurites longer than 25 μm (P < 0.05) in dissociated retinal cultures and neurite extension increased in retinal explants (P < 0.05). MT-IIA treatment of mixed retinal cultures increased megalin expression in RGCs, and pre-treating cells with anti-megalin antibodies prevented MT-IIA-stimulated neurite extension. Our results indicate that MT-IIA stimulates neurite outgrowth in RGCs and may do so via the megalin receptor; we propose that neurite extension is triggered via signal transduction pathways activated by the NPxY motifs of megalin’s cytoplasmic tail.


Keywords Metallothionein - Retinal ganglion cells - Neuroregeneration

 

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Metal binding of metallothionein-3 versus metallothionein-2: lower affinity and higher plasticity

Peep Palumaa (1), , Indrek Tammiste (2), Keiu Kruusel (1), Liina Kangur (1), Hans Jörnvall (3) and Rannar Sillard (3)


(1) Department of Gene Technology, Tallinn Technical University Akadeemia tee 23, 12618 Tallinn, Estonia
(2) National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia
(3) Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

 

Journal:    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics. Volume 1747, Issue 2, 14 March 2005, Pages 205-211


Received 19 August 2004; revised 16 November 2004; accepted 16 November 2004. Available online 8 December 2004.


Abstract
Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc and copper and in cytoprotection against toxic metals and reactive oxygen species. MT-3 plays a specific role in the brain and is down-regulated in Alzheimer's disease. To evaluate differences in metal binding, we conducted direct metal competition experiments with MT-3 and MT-2 using electrospray ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+ and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. Titration with Cd2+ ions demonstrates that metal-binding affinities of individual clusters of MT-2 and MT-3 are decreasing in the following order: four-metal cluster of MT-2>three-metal cluster of MT-2≈four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3 and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted cellular conditions, we studied the secondary structures of apo-MT-3 and apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have almost equal helical content (approximately 10%) in aqueous buffer, but that MT-3 had slightly higher tendency to form α-helical secondary structure in TFE–water mixtures. Secondary structure predictions also indicated some differences between MT-3 and MT-2, by predicting random coil for common MTs, but 22% α-helical structure for MT-3. Combined, all results highlight further differences between MT-3 and common MTs, which may be related with their functional specificities.

Keywords: MT-3; Alzheimer's disease; ESI-MS; Secondary structure prediction; CD spectroscopy

Abbreviations: MT, metallothionein; DTT, dithiothreitol; TFE, 2,2,2,-trifluoroethanol; CD, circular dichroism; ESI MS, electrospray ionization mass spectroscopy

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Metal binding to brain-specific metallothionein-3 studied by electrospray ionization mass spectrometry.

 

Palumaa P (1), Eriste E (2), Kruusel K (1), Kangur L, (1) Jörnvall H (2), Sillard R (2).


(1) Department of Gene Technology, Tallinn Technical University, Ehitajate tee 5, EE-19086 Tallinn, Estonia.

(2) Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.


Journal:    Cell Mol Biol (Noisy-le-grand). 2003 Jul;49(5):763-768.

 

Abstract
Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.

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Brain-Specific Metallothionein-3 Has Higher Metal-Binding Capacity than Ubiquitous Metallothioneins and Binds Metals Noncooperatively

Peep Palumaa, Elo Eriste, Olga Njunkova, Lesja Pokras, Hans Jörnvall, and Rannar Sillard

Centre for Gene Technology, Tallinn Technical University, Ehitajate tee 5, EE-19086 Tallinn, Estonia,

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden,

National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, EE-12618 Tallinn, Estonia

Journal:     : Biochemistry. 2002 May 14;41(19):6158-6163.

 

Received February 11, 2002, Revised Manuscript Received March 26, 2002

Abstract:
Zinc metabolism in the cells is largely regulated by ubiquitous small proteins, metallothioneins (MT). Metallothionein-3 is specifically expressed in the brain and is down regulated in Alzheimer's disease. We demonstrate by mass spectrometry that MT-3, in contrast to common MTs, binds Zn2+ and Cd2+ in a noncooperative manner and can also bind higher stoichiometries of metals than seven. MT-3 reconstituted with seven metals exists in a dynamic equilibrium of different metalloforms, where the prevalent metalloform is Me7MT-3, but metalloforms with 6, 8, and even 9 metals are also present. The results from pH and stability studies demonstrate that the heterogeneity of metalloforms originates from the N-terminal -cluster, whereas the C-terminal -cluster of MT-3 binds four metal ions such as that of common MTs. Experiments with EDTA demonstrate that the -cluster of ZnMT-3 has a higher metal transfer potential than the -cluster of Zn7MT-2. Moreover, ZnMT-3 loses metals during ultrafiltration. MT-3, reconstituted with an excess of Zn2+ or Cd2+, exists as a dynamic mixture of metalloforms with higher than 7 metal stoichiometries (8-11). Such forms of ZnMT-3 are unstable and decompose partly already during a rapid gel filtration, whereas CdMT-3 forms are more stable. Extra metal ions may bind to the -cluster region as well as to the carboxylates of MT-3. The specific metal-binding properties of MT-3 could be functionally implemented for buffering of fluctuating concentrations of zinc in zincergic neurons and for transfer of zinc to synaptic vesicles.
 

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