Title: Redefining the Role of Metallothionein within the Injured Brain
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. 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. The mechanisms by which certain protective proteins expressed by astrocytes affect neuronal regeneration are not well understood. As an example, we have demonstrated that mice lacking the ability to express a protein produced predominantly by astrocytes in the CNS, 3The abbreviations used are: CNS, central nervous system; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; GAP-43, growth-associated protein-43; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; IL-1α, interleukin-1α; MT, metallothionein; PBS, phosphate-buffered saline; RAP, receptor-associated protein; RGC, retinal ganglion cell; RIA, radioimmunoassay; siRNA, short interfering RNA. metallothionein isoforms I/II (MT-I/-II), exhibit significantly worse outcomes following a range of CNS injuries (1Penkowa M. Carrasco J. Giralt M. Moos T. Hidalgo J. J. Neurosci. 1999; 1: 2535-2545Crossref Google Scholar, 2Giralt M. Penkowa M. Lago N. Molinero A. Hidalgo J. Exp. Neurol. 2002; 173: 114-128Crossref PubMed Scopus (127) Google Scholar). Likewise, MT-I/-II-deficient animals fare worse following stroke, experimental autoimmune encephalomyelitis (an experimental animal model of multiple sclerosis), and motor neuron disease (3Campagne M.L. Thibodeaux H. van Bruggen N. Cairns B. Gerald R. Palmer J.T. Williams S.P. Lowe D.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12870-12875Crossref PubMed Scopus (165) Google Scholar, 4Penkowa M. Hidalgo J. Exp. Neurol. 2001; 170: 1-14Crossref PubMed Scopus (103) Google Scholar, 5Puttaparthi K. Gitomer W.L. Krishnan U. Son M. Rajendran B. Elliott J.L. J. Neurosci. 2002; 22: 8790-8796Crossref PubMed Google Scholar, respectively). Hence, perturbation of an astrocytic protein has major consequences in the injured CNS, including an increase in apoptotic neurons and impaired neuronal regenerative growth. Indeed, genetically modified animals have been produced that exhibit the full range of possible astrocytic MT-I/-II expression, from null to overexpressing strains, and there is a robust correlation between MT-I/-II expression and the ability of the animal to recover from CNS insult or degenerative disease (1Penkowa M. Carrasco J. Giralt M. Moos T. Hidalgo J. J. Neurosci. 1999; 1: 2535-2545Crossref Google Scholar, 6Penkowa M. Espejo C. Martinez-Caceres E.M. Poulsen C.B. Montalban X. Hidalgo J. J. Neuroimmunol. 2001; 119: 248-260Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 7Xie T. Tong L. McCann U.D. Yuan J. Becker K.G. Mechan A.O. Cheadle C. Donovan D.M. Ricaurte G.A. J. Neurosci. 2004; 24: 7043-7050Crossref PubMed Scopus (34) Google Scholar). These studies clearly demonstrate that MT-I/-II represented an important mechanism of protection and regeneration in the injured CNS. There are a number of ways that MT-I/-II might conceivably enhance the ability of astrocytes to promote neuronal regeneration. Metallothioneins, as exemplified by family members MT-I/-II, are zinc-binding proteins that may have roles in metal homeostasis or free radical scavenging (for reviews see Refs. 8Hidalgo J. Aschner M. Zatta P. Vasak M. Brain Res. Bull. 2001; 55: 133-145Crossref PubMed Scopus (366) Google Scholar, 9West A.K. Chuah M.I. Vickers J.C. Chung R.S. Rev. Neurosci. 2004; 15: 157-166Crossref PubMed Scopus (36) Google Scholar), although their role in any tissue remains a matter of debate. Because they lack conventional secretion sequences (10Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8428-8430Crossref PubMed Scopus (605) Google Scholar) and demonstrably accumulate in the astrocytic cytoplasm after neuronal injury (11Chung R.S. Adlard P.A. Dittmann J. Vickers J.C. Chuah M.I. West A.K. J. Neurochem. 2004; 88: 454-461Crossref PubMed Scopus (67) Google Scholar), the general consensus based upon more than 40 years of research is that MTs likely act within the expressing cell itself, and they may, for example, be part of the mechanism by which astrocytes handle toxic intermediates such as reactive oxygen molecules. However, we have shown that the role of MT-I/-II is potentially more complex than simply acting within astrocytes per se. We recently reported that MT-I/-II strongly increases post-injury regenerative sprouting when added directly to injured neurons in culture (12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar). In these experiments, there are no glial or immune system cells present, indicating that extracellular MT-I/-II can act directly on injured neurons, i.e. outside the context of astrocytic cytoplasm. These experiments have since been replicated elsewhere showing that "exogenous" MT-I/-II strongly promotes regenerative neurite growth of cortical (12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar), dopaminergic, and hippocampal neurons (13Kohler L.B. Berezin V. Bock E. Penkowa M. Brain Res. 2003; 992: 128-136Crossref PubMed Scopus (54) Google Scholar) and retinal ganglion cells (14Fitzgerald M. Nairn P. Bartlett C.A. Chung R.S. West A.K. Beazley L.D. Exp. Brain Res. 2007; 183: 171-180Crossref PubMed Scopus (62) Google Scholar) suggesting that there is a robust and generic neuronal response to extracellular MT-I/-II. Based upon these data and the existing literature, we have hypothesized a model to explain the role for extracellular MT-I/-II within the injured brain. We suggested that astrocytes respond to neural trauma by up-regulating MT-I/-II expression with the MT-I/-II being subsequently secreted, allowing direct interaction with neurons which promotes neuronal regeneration and survival following injury (15Chung R.S. West A.K. Neuroscience. 2004; 123: 595-599Crossref PubMed Scopus (87) Google Scholar). Although there are numerous studies investigating MT-I/-II expression following injury, little is known about the mechanism(s) whereby MT-I/-II might interact with neurons to exert their neuroregenerative effect. To examine these mechanisms, we addressed four questions. (a) Is MT-I/-II released by astrocytes in culture and following brain injury? (b) Is the interaction of extracellular MT-I/-II with neurons receptor-mediated? (c) Can MT-I/-II transfer from astrocytes to neurons be observed directly? (d) Is uptake of exogenous MT-I/-II associated with axon regeneration in vivo? We find that astrocytes are capable of releasing MT-I/-II in culture and that these proteins can be detected within the extracellular environment of the injured brain. In tissue culture experiments we identify a receptor whereby extracellular MT-I/-II directly interacts with neurons and demonstrate the direct transfer of MT-I/-II from astrocytes to neurons. Finally, we demonstrate in vivo that exogenous MT-I/-II uptake is associated with robust axon regeneration following optic nerve transection. The results suggest that transfer of MT-I/-II from astrocytes to neurons is an important component of the response of the CNS to injury. Metallothionein Protein—Because these studies have been performed in four different laboratories collaborating together, slightly different forms of MT protein have been used. The particular MT form that is used for each experiment has been specifically stated within the text and includes rabbit Zn-MT-IIA (Bestenbalt LLC), rabbit Zn-MT-II (Sigma catalog number M 9542), and a mixture of rabbit Zn-MT-I and Zn-MT-II (Sigma catalog number M 7641). There are small differences in the amino acid structure of these proteins, although all maintain the 20 conserved cysteine residues that characterize mammalian MTs. We have recently reported that MT proteins from a variety of sources, including mammalian and Drosophila MTs, appear to have a similar ability to promote wound healing following CNS injury (35Penkowa M. Tio L. Giralt M. Quintana A. Molinero A. Atrian S. Vasak M. Hidalgo J. J. Neurosci. Res. 2006; 83: 974-984Crossref PubMed Scopus (38) Google Scholar), so we do not expect any issues in using the slightly different MT forms in this study. Focal Injury to the Adult Rat Neocortex, Collection of Gelfoam, and Western Blotting—Injuries were made to the Par 1 region of the adult rat neocortex as reported previously (12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar). At the appropriate time, rats were re-anesthetized and transcardially perfused, and brains were then removed and post-fixed overnight in 4% paraformaldehyde at 4 °C. They were then sectioned at 50-μm thickness by vibratome (Leica VT1000E). In some experiments, absorbent gelfoam, which had been used to seal the burr hole in the skull during the initial surgery, was recovered at several time points post-injury (three animals per group). Gelfoam pieces were pooled for each time point, and soluble proteins were removed from the gelfoam by the addition of 100 μl of PBS, followed by vortexing. Samples were briefly centrifuged at 15,000 rpm for 2 min, and the supernatant was collected and stored at -20° C for subsequent Western blotting. Western blotting was performed as described previously (36Chung R.S. Holloway A.F. Eckhardt B.L. Harris J.A. Vickers J.C. Chuah M.I. West A.K. Biochem. J. 2002; 365: 323-328Crossref PubMed Google Scholar), under reducing conditions to minimize any potential oxidation of MT proteins. In subsequent Western blots, similar procedures were performed with the same samples, but either mouse anti-GFAP (1:500, Chemicon) or rabbit anti-ferritin (1:1000, ICN) primary antibodies were used. Primary Astrocyte Cultures, Culture Media Collection, and MT-I/-II Radioimmunoassay—Primary astrocyte cultures were prepared as described previously (37Vincent A.J. Taylor J.M. Choi-Lundberg D.L. West A.K. Chuah M.I. Glia. 2005; 51: 132-147Crossref PubMed Scopus (93) Google Scholar). Astrocyte cultures were at least 98% pure (results not shown). Confluent astrocytes cultures were maintained in a total of 500 μl of culture medium and treated with either 10 units/ml IL-1α (Chemicon catalog number IL001), 10 μm ZnSO4, or 10 units/ml IL-1α + 10 μm ZnSO4 combined for the allotted time period, and media were collected, centrifuged at 13,000 × g (5 min), and stored at -20 °C. MT-I/-II was measured by radioimmunoassay as described previously (16Gasull T. Rebollo D.V. Romero B. Hidalgo J. J. Immunoassay. 1993; 14: 209-225Crossref PubMed Scopus (69) Google Scholar). Primary Neuron Cell Cultures—Cortical neuron cultures were prepared as reported previously (12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar) and maintained in a culture medium consisting of Neurobasal™ medium, supplemented with 10% fetal bovine serum, 0.1% (final concentration) B-27 supplement, 0.1 mm (final concentration) l-glutamine, and 200 units/ml gentamicin. At the appropriate time, cells were fixed with 4% paraformaldehyde for 20 min, and a permeabilizing immunocytochemistry method was used to visualize intracellular proteins, with most membrane-bound proteins lost using this protocol. This procedure was performed using an antibody diluent containing 0.03% Triton X detergent. For immunocytochemistry, mouse anti-MT (1:500; Dako), rabbit anti-megalin (1:500; Santa Cruz Biotechnology), and rabbit anti-Tau (1:5000; DAKO) antibodies were used. Secondary labeling was performed using appropriate AlexaFluor-488- or -594-conjugated secondary antibodies (Molecular Probes). Cerebellar granule neurons were prepared from postnatal day 7 Wistar rats (Charles River, Sulzfeld, Germany) as described previously by Schousboe et al. (38Schousboe A. Meier E. Drejer J. Hertz L. Shahar A. de Vellis J. Vernadakis A. Haber B A Dissection and Tissue Culture Manual of the Nervous System. Alan R. Liss, Inc, New York1989: 203-206Google Scholar). For the cerebellar granule neuron neurite outgrowth assay, MT-I/-II was applied to neurons immediately after plating and fixed 24 h after treatment with 4% (v/v) formaldehyde for 20 min and immunostained using primary rabbit antibodies against GAP-43. Alexa-Fluor secondary goat anti-rabbit Ig antibodies were used to visualize the cells. In some experiments, MT-I/-II was immobilized onto coverslips by incubation on Permanox plasticware overnight and washed three times with PBS, and the neurons were then plated onto these coverslips, and neurite outgrowth was assessed 24 h later. For the analysis of neurite outgrowth, we have used a computer-assisted fluorescence microscopy technique described previously (32Rønn L.C. Ralets I. Hartz B.P. Bech M. Berezin A. Berezin V. Moller A. Bock E. J. Neurosci. Methods. 2000; 100: 25-32Crossref PubMed Scopus (145) Google Scholar). In brief, digital images of at least 200 neurons for each group in each individual experiment were obtained systematically; a frame was superimposed on each image, and the number of intersections between neurites and test lines in a given frame was counted and divided by the number of neuron bodies, whereby a relationship between the number of neurite intersections and neurons is obtained. The neurite growth value of control cells was then considered 100%, and all the experimental groups are compared with it. Statistical evaluation was performed by the Student'ns paired t test using Fig-P, 2.98 (Biosoft, Cambridge, UK). Co-immunoprecipitation and Western Blotting Studies—To confirm the interaction between MT and megalin, co-immuno-precipitation experiments were performed whereby an MT antibody was used to pull down MT and MT complexes, which were then probed for the presence of megalin by Western blotting. Briefly, 14-day in vitro cortical neurons plated at a density of 1 × 106 cells/flask were treated with MT, and after 24 h the cells were lysed in 250 μl of ice-cold resuspension buffer (20 mm Tris, pH 7.4, 400 mm NaCl, 7.5 mm MgCl2, 0.2 mm EDTA, 1.0 mm dithiothreitol). The cell lysate was collected into an Eppendorf tube, to which 250 μl of dilution buffer (20 mm Tris, pH 7.4, 100 mm NaCl, 7.5 mm MgCl2, 0.2 mm EDTA, 0.75% Ipegal, 10% glycerol) was added. Lysates were pre-cleared by adding 50 μl of 50% protein-A-Sepharose slurry for 1 h at 4 °C with shaking on a rotational shaker to remove nonspecific proteins that potentially bind to the protein-A-Sepharose beads. The following steps were all performed in a 4 °C cold room. The cell lysate was centrifuged at 3000 rpm for 5 min to pellet the Sepharose beads, and the supernatant was transferred to a fresh Eppendorf tube. The MT antibody (10 μl; Dako) was added to the lysate and incubated at 4 °C with shaking for 1 h. To this, 50 μl of 50% protein-A-Sepharose was added, and the lysate was incubated overnight at 4 °C with shaking. The sample was centrifuged at 3000 rpm for 5 min, and the supernatant was transferred to a new Eppendorf tube. The beads were washed three times with 200 μl of dilution buffer, centrifuging each time at 3000 rpm for 5 min. The final pellet was resuspended in 15 μl of Nu-PAGE lithium dodecyl sulfate sample buffer (Invitrogen) and immediately used for Western blotting. Western blotting was performed as described previously (32Rønn L.C. Ralets I. Hartz B.P. Bech M. Berezin A. Berezin V. Moller A. Bock E. J. Neurosci. Methods. 2000; 100: 25-32Crossref PubMed Scopus (145) Google Scholar), using the Nu-PAGE system (Invitrogen) and 10-20% gradient gels. For all Western blots, the total protein content of samples was determined by the Bradford assay, and equal protein amounts were loaded for all samples. Membranes were probed with a rabbit anti-megalin antibody (1:500; Santa Cruz Biotechnology) and goat anti-rabbit horseradish peroxidase secondary antibody (1:1000; Dako). AlexaFluor-594 Labeling of Rabbit MT-IIA—Rabbit MT-IIA was chemically conjugated to AlexaFluor-594 using an AlexaFluor probe labeling kit (Molecular Probes). Following the conjugation reaction, AlexaFluor-594 bound MT-IIA (MT-594) was separated from excess AlexaFluor-594 dye by standard gel chromatography over a G-75 column. Western blotting analysis confirmed the separation of the fluorescently tagged MT-IIA from excess dye. The excess fluorescent dye, which becomes nonreactive during this process, was used in later studies as an experimental control. Megalin siRNA Transfections into Neurons—In some experiments cortical neurons at a density of 1 × 106 cells were transfected with two pre-designed siRNA molecules targeting different regions of the rat megalin gene (Ambion catalog numbers 199330 and 199331). Transfections were performed by electroporation using a rat hippocampal neuron protocol and the Nucleofector™ (Amaxa) upon neurons at the end of the culturing process and immediately prior to plating out. 6 nm siRNA was used for each transfection. As a control, neurons underwent the entire transfection protocol but in the absence of siRNA. Western blotting 24 h later confirmed that transfection with either siRNA molecule greatly reduced expression of megalin (results not shown). Astrocyte Transfection Studies—The coding sequence for human MT-IIA was removed from a pre-existing bacterial expression vector (12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar) and inserted into the pEGFP vector (BD Biosciences) such that the plasmid expresses an MT-IIA-GFP fusion protein. The pEGFP vector served as the control for all transfection studies. For transfection, a minimum of 1 × 106 astrocytes was transfected using the Amaxa Nucleofector™ and the Nucleofector™ rat astrocyte transfection kit (VPG-1006). In preliminary studies, transfection efficiencies were ∼30%, and immunocytochemistry confirmed co-localization of MT immunolabeling with MT-GFP (results not shown). Following transfection, the astrocytes were seeded into 14-day in vitro cortical neuron cultures (5 × 104 cells) at a seeding density of 1 × 104 cells and maintained for up to 7 days. At the appropriate time, cells were fixed with 4% paraformaldehyde, and immunocytochemistry was performed as discussed previously. For neuronal immunolabeling, a rabbit anti-Tau antibody (DAKO) was used at a concentration of 1:5000. For detection of MT-GFP and GFP in culture medium, samples were collected and concentrated ∼10-fold using Centricon-3 ultrafiltration columns (Millipore). Briefly, 500 μl of media was placed into each spin column and centrifuged at 10,000 × g for 90 min. Concentrated samples were used for Western blotting studies, using the protocol described earlier. A monoclonal anti-GFP antibody (BD Biosciences) was used at a concentration of 1:4000, detected with a secondary goat anti-mouse horseradish peroxidase-conjugated antibody (DAKO) used at a concentration of 1:1000. Complete Optic Nerve Transection Surgeries—Adult male Hooded Wistar rats were deeply anesthetized; the right optic nerve was exposed intraorbitally, and the nerve sheath was cut transversely except for the ventral aspect that was left intact to avoid damage to the ophthalmic blood vessels. The nerve parenchyma was lifted using hooked forceps and completely transected with iridectomy scissors ∼1 mm from the back of the eye. The cut ends of the nerve sheath were then sutured together (10-0 thread). The completeness of transection was confirmed by anterograde labeling. Animals with ischemic retinae (determined ophthalmoscopically) were euthanized. Rabbit MT-I/-II (Sigma) or control solutions (PBS or ZnSO4) were administered via a stereotactically positioned 30-gauge needle attached to a 10-μl Hamilton syringe. A final volume of 2.5 μl was administered via two injection sites (nasal and temporal). Eight animals received MT-I/-II injections, and eight received vehicle (saline). Statistical Analysis—All data are expressed as mean ± S.E. For cell culture experiments, statistical evaluation was performed by the Student'ns paired t test using either SigmaStat (Systat Software) or Fig-P version 2.98 (Biosoft, Cambridge, UK). 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 (16Gasull T. Rebollo D.V. Romero B. Hidalgo J. J. Immunoassay. 1993; 14: 209-225Crossref PubMed Scopus (69) Google Scholar, 17Hidalgo J. Garcia A. Olivia A.M. Giralt M. Gasull T. Gonzalez B. Milnerowicz H. Wood A. Bremner I. Chem. Biol. Interact. 1994; 93: 197-219Crossref PubMed Scopus (62) Google Scholar). 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). In these cultures, intracellular MT-I/-II expression as observed by immunocytochemistry was low in most astrocytes. Astrocytes were then exposed to either zinc (ZnSO4, 10 μm) or interleukin-1 (recombinant human IL-1, 10 units/ml) or both. Co-treatment with zinc and interleukin-1 has been shown previously to lead to accumulation of MT-I/-II in a granular form near the cell membrane (18Kikuchi Y. Irie M. Kasahara T. Sawada J. Terao T. FEBS Lett. 1993; 317: 22-26Crossref PubMed Scopus (40) Google Scholar), feasibly suggesting MT-I/-II localization within secretory vesicles. When either zinc or IL-1 was added, there was no observable increase in extracellular MT-I/-II within the culture media over a 72-h period (Fig. 1A). However, the zinc and IL-1 co-treatment resulted in a statistically significant (p < 0.01; analysis of variance) increase in extracellular MT-I/-II within culture media, with levels increasing 2.5-fold by 72 h (Fig. 1A). The amount of extracellular MT-I/-II was calculated as a percentage of the total MT-I/-II protein in the cultures, and the levels increased from 6% in the control cultures to 13% 72 h after zinc and IL-1 co-treatment. No cell lysis was observed at any time point using lactate dehydrogenase assays (data not shown). To confirm these observations, astrocytes were cultured in flasks (1 × 106 cells/flask) and treated with either zinc or IL-1 alone, or zinc and IL-1 together (at the concentrations used above). After 72 h the culture medium was collected, concentrated using Millipore Ultrafiltration columns, and the presence of MT in culture medium detected by Western blotting (Fig. 1B). Although there was a low level of MT detectable within the culture medium of untreated cells, this was mildly increased following treatment with zinc or IL-1. However, the dual zinc and IL-1 treatment induced much greater levels of MT secretion by astrocytes (Fig. 1B), in accordance with the RIA results above. Note that equal amounts of protein were loaded for each sample. In parallel experiments, from the same media samples we performed Western blots for albumin and noted no change in albumin levels within the culture medium (Fig. 1B). Detection of Extracellular MT-I/-II Following Focal Cortical Injury—To address the issue of whether MT-I/-II is found extracellularly following brain injury, we used a model of focal cortical injury to the adult rat brain as described previously (11Chung R.S. Adlard P.A. Dittmann J. Vickers J.C. Chuah M.I. West A.K. J. Neurochem. 2004; 88: 454-461Crossref PubMed Scopus (67) Google Scholar, 12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar). Absorbent gelfoam was placed over the pial surface above the lesion (Fig. 2A) at the time of injury and left in place for 1-14 days. Proteins were extracted from the gelfoam and analyzed by SDS-PAGE and Western blotting for MT-I/-II, GFAP, and ferritin (Fig. 2B). The latter two proteins were examined as a control for the presence of cells or cell debris (astrocytes and microglia, respectively) that may have invaded the gel foam. An adult rat brain homogenate was used as a positive control for all antisera. As shown in Fig. 2B, MT-I/-II was initially undetectable, but it could be clearly detected in the gelfoam extract at both 7 and 14 days post-injury. The absence of signature intracellular proteins from the two major classes of MT-expressing cells in the CNS, astrocytes and microglia, suggests that the MT-I/-II, which had accumulated in the gelfoam, was derived from an extracellular pool. The adult rat brain homogenate was positive for all three antigens, confirming that Western blotting was successful. Immobilized Extracellular MT-I/-II Is Not Neuritogenic—We have recently demonstrated that extracellular MT-I/-II can promote neurite outgrowth and axon regeneration, both in culture (12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar, 13Kohler L.B. Berezin V. Bock E. Penkowa M. Brain Res. 2003; 992: 128-136Crossref PubMed Scopus (54) Google Scholar, 14Fitzgerald M. Nairn P. Bartlett C.A. Chung R.S. West A.K. Beazley L.D. Exp. Brain Res. 2007; 183: 171-180Crossref PubMed Scopus (62) Google Scholar) and in vivo following an injury to the brain (12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar) or to a peripheral (sciatic) nerve (19Ceballos D. Lago N. Verdu E. Penkowa M. Carrasco J. Navarro X. Palmiter R.D. Hidalgo J. Cell. Mol. Life Sci. 2003; 60: 1209-1216Crossref PubMed Scopus (31) Google Scholar). To investigate whether it is necessary for extracellular MT to be internalized to mediate neurite outgrowth of cultured neurons as we have reported previously (12Chung R.S. Vickers J.C. Chuah M.I. West A.K. J. Neurosci. 2003; 23: 3336-3342Crossref PubMed Google Scholar, 13Kohler L.B. Berezin V. Bock E. Penkowa M. Brain Res. 2003; 992: 128-136Crossref PubMed Scopus (54) Google Scholar, 14Fitzgerald M. Nairn P. Bartlett C.A. Chung R.S. West A.K. Beazley L.D. Exp. Brain Res. 2007; 183: 171-180Crossref PubMed Scopus (62) Google Scholar), rabbit MT-I/-II was bound to Permanox-coated slides. This technique allows the tethered protein to interact with the cell membrane of adhered cells but blocks intracellular import. Compared with the neuritogenic activity of untethered MT-I/II, immobilized MT-I/II was relatively inactive (Fig. 3). Addition of mobile MT-I/-II restored neurite outgrowth in the presence of immobilized MT-I/-II (Fig. 3). As an experimental control, a previously described peptide (P2d) was used (20Pedersen M.V. Kohler L.B. Ditlevsen D.K. Li S. Berezin V. Bock E. J. Neurosci. Res. 2004; 75: 55-65Crossref PubMed Scopus (3