Cell death in the injured brain: Roles of metallothioneins

ARTICLE IN PRESS PROGRESS IN HISTOCHEMISTRY AND CYTOCHEMISTRY Progress in Histochemistry and Cytochemistry 44 (2009) 1–27 www.elsevier.de/proghi

Cell death in the injured brain: Roles of metallothioneins Mie Ø Pedersena,, Agnete Larsenb, Meredin Stoltenbergb, Milena Penkowaa a

Section of Neuroprotection, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark b Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark Received 28 April 2008; accepted 2 October 2008

Abbreviations: ADP, adenosine diphosphate; AIF, apoptosis-inducing factor; AMPA, a-amino-3hydroxy-5-methyl-4-isoxazolyl-propionic acid; Apaf-1, apoptotic protease-activating factor 1; Atg, autophagy gene; Bak, Bcl-2-homologous antagonist/killer; Bax, Bcl-2-associated X protein; Bid, BH3 interacting domain death agonist; BBB, blood–brain barrier; Bcl-2, B-cell lymphoma-2 protein; BDNF, brain-derived neurotrophic factor; CMA, chaperone-mediated autophagy; CNS, central nervous system; DRAM, damage-regulated autophagy modulator; EAE, experimental autoimmune encephalomyelitis; FADD, Fas-associated death domain; FasL, Fas ligand; FGF, fibroblast growth factor; GCEE, gammaglutamylcysteinyl ethyl ester; GDNF, glial cell line-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; Hq, Harlequin mice; HSC, heat shock cognate; ICAM, intercellular adhesion molecule; IL, interleukin; iNOS, inducible nitric oxide synthase; LC-3, microtubule-associated protein light chain 3; LTa, lymphotoxin-a; Mac-1, macrophage activator factor; MDA, malondialdehyde; MMP, matrix metalloproteinases; MOMP, mitochondria outer membrane permeabilization; MT, metallothionein; MTKO, metallothionein knock-out mice; mTOR, mammalian target of rapamycin; NGF, nerve growth factor; NITT, nitrotyrosine; NMDA, N-methyl-D-aspartate; NOXA, phorbol-12-myristate-13-acetateinduced protein 1; NSC, neural stem cell; NT, neurotrophin; PARP, poly(ADP-ribose)polymerase; PBN, alpha-phenyl-N-tertbutyl-nitrone; PCD, programmed cell death; PI3K, phosphoinositide 3 kinase; PIDD, p53-induced protein with a death domain; PKB, protein kinase B; PSA-NCAM, polysialic acid-neural cell adhesion molecule; PUMA, p53-upregulated modulator of apoptosis; RAIDD, RIP-associated protein with a death domain; RIP, ribosome-inactivating protein; ROS, reactive oxygen species; SVZ, subventricular zone; TBI, traumatic brain injury; TGFb, transforming growth factor-beta; TgMT, transgenic metallothionein mice; TNFa, tumor necrosis factor-alpha; TRAMP, TNF-receptor-related apoptosis-mediated protein; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated de-oxyuridine triphosphate (dUTP)-biotin nick-end labeling; TWEAK, tumor necrosis factor-like weak inducer of apoptosis; VEGF, vascular endothelial growth factor; WT, wild-type mice. Corresponding author. Tel.: +45 35 32 72 23. E-mail address: [email protected] (M.Ø. Pedersen). 0079-6336/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.proghi.2008.10.002

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Abstract In traumatic brain injury (TBI), the primary, irreversible damage associated with the moment of impact consists of cells dying from necrosis. This contributes to fuelling a chronic central nervous system (CNS) inflammation with increased formation of proinflammatory cytokines, enzymes and reactive oxygen species (ROS). ROS promote oxidative stress, which leads to neurodegeneration and ultimately results in programmed cell death (secondary injury). Since this delayed, secondary tissue loss occurs days to months following the primary injury it provides a therapeutic window where potential neuroprotective treatment could alleviate ongoing neurodegeneration, cell death and neurological impairment following TBI. Various neuroprotective drug candidates have been described, tested and proven effective in pre-clinical studies, including glutamate receptor antagonists, calcium-channel blockers, and caspase inhibitors. However, most of the scientific efforts have failed in translating the experimental results into clinical trials. Despite intensive research, effective neuroprotective therapies are lacking in the clinic, and TBI continues to be a major cause of morbidity and mortality. This paper provides an overview of the TBI pathophysiology leading to cell death and neurological impairment. We also discuss endogenously expressed neuroprotectants and drug candidates, which at this stage may still hold the potential for treating brain injured patients. r 2008 Elsevier GmbH. All rights reserved.

1. Introduction Due to limited neuroprotective interventions for patients suffering traumatic brain injury (TBI), it has become a leading cause of morbidity and mortality (Erlich et al., 2006; Plesnila et al., 2007; Werner and Engelhard, 2007). Worldwide over 10 million people are injured annually (Hyder et al., 2007), and at least 11.5 million people are currently living with TBI-related disabilities (Bramlett and Dietrich, 2004; Schouten, 2007). The primary, irreversible tissue damage that occurs at the moment of impact results in immediate necrotic cell death (Waldmeier, 2003; Morganti-Kossmann et al., 2007), breakdown of the blood–brain barrier (BBB) (Habgood et al., 2007; Vajtr et al., 2008), impaired regulation of cerebral blood flow (CBF) (Bramlett and Dietrich, 2004; Werner and Engelhard, 2007), edema formation (Vajtr et al., 2008), accumulation of lactic acid and changes in pH (Marino et al., 2007; Werner and Engelhard, 2007), impaired cellular metabolism (e.g. impaired calcium homeostasis) (Marino et al., 2007), increased zinc accumulation (Doering et al., 2007), excitotoxicity (Morganti-Kossmann et al., 2007), mitochondrial dysfunction (Kim et al., 2006), and unfolded protein response (UPR) (Maiuri et al., 2007; Truettner et al., 2007). Moreover, the initial necrosis (cell lysis) contributes to the neuroinflammatory response following TBI (Won et al., 2002; Bramlett and Dietrich, 2004; Morganti-Kossmann et al., 2007; Werner and Engelhard, 2007). This response is characterized by the activation of resident microglia and bloodderived monocytes that transform into round macrophages (Kelley et al., 2007),

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astrocytes showing reactive astrogliosis, and haematogenous leukocytes being recruited to the central nervous system (CNS) (McIntosh et al., 1998; Penkowa, 2006a). The activated macrophages generate proinflammatory mediators such as cytokines (e.g. interleukin (IL)-1a/b, IL-6, IL-12, tumour necrosis factor (TNF)-a), prostaglandins, complement, adhesion molecules (e.g. intercellular adhesion molecule (ICAM-1)), enzymes and proteases (e.g. matrix metalloproteinases (MMP)); and ultimately reactive oxygen species (ROS) are generated (McIntosh et al., 1998; Lenzlinger et al., 2001; Allan and Rothwell, 2003; Lucas et al., 2006; Williams et al., 2007). Even though the proinflammatory mediators contribute to the host defence response, they also have detrimental actions resulting in secondary (delayed) tissue damage. For example, IL-1b and TNF-a have been shown to contribute to demyelination and neuron loss in various autoimmune, demyelinating and neurodegenerative brain diseases (Lee et al., 2000; Allan and Rothwell, 2003; Lu et al., 2005). In addition, ROS have detrimental actions since the brain is highly vulnerable to oxidative damage due to its high oxidative metabolic rate, relatively low antioxidant capacity, low activity of repair mechanisms, high membrane surface to cytoplasmic ratio and high amounts of unsaturated fatty acids that are prone to lipid peroxidation (Leker and Shohami, 2002). The primary injury following TBI provides several initiators that can trigger different forms of programmed cell death (PCD) in an overlapping manner, and these stimuli consist of both extracellular mediators (e.g. excessive calcium, excitotoxicity, proinflammatory cytokines) and intracellular events (e.g. ROS formation, oxidative stress, mitochondrial dysfunction, damaged organelles, misfolded or aggregated proteins, p53 activation and DNA damage) (Bredesen et al., 2006; Maiuri et al., 2007). Together they result in neurodegeneration (neuronal accumulation of abnormal proteins) and delayed death of neurons and glia cells, which result in subsequent secondary brain damage and neurological impairment (Galluzzi et al., 2007; Krantic et al., 2007; Werner and Engelhard, 2007). However, inflammation has a dual role in that it also provides protective mechanisms, including isolation of the injured area, removal of affected cells and regenerative stimuli. Following the initial insult, reactive astrocytes increase their expression and production of neuroprotective and angiogenic factors (e.g. Transforming growth factor-beta (TGFb) (Morganti-Kossmann et al., 2007), fibroblast growth factor (FGF) (Penkowa et al., 2006a), vascular endothelial growth factor (VEGF) (Morgan et al., 2007)), antioxidants (e.g. metallothionein (MT) (Penkowa, 2006a), neurotrophins (e.g. neurotrophin (NT-3, NT-4), and brainderived neurotrophic factor (BDNF)) (Penkowa et al., 2006a) that are involved in synaptic plasticity. Furthermore, some proinflammatory cytokines (e.g. TNFa and IL-6) have been shown to mediate significant delayed neuroprotection (for reviews see Morganti-Kossmann et al., 2002, 2007). For instance, neuroprotection mediated by TNFa in TBI has been described in mice lacking TNF-receptors (p55 and p75), where animals showed enhanced tissue damage and BBB breakdown following injury (Morganti-Kossmann et al., 2002). Moreover, IL-6 has the ability to inhibit TNF synthesis, induce nerve growth factor (NGF) expression, promote neuronal

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differentiation and survival, and counteract N-methyl-D-aspartate (NMDA)mediated toxicity (Morganti-Kossmann et al., 2002). Furthermore, IL-6 knockout mice show reduced astrogliosis, increased tissue damage and reduction of antioxidant enzymes following experimental TBI (Penkowa et al., 2000a). Thus, the host defence reactions also promote neuronal survival and CNS regeneration. While necrosis is a result of the primary injury (Yakovlev and Faden, 2004), it has lately been recognized that PCD (apoptosis and autophagy) likely contributes substantially to the delayed tissue damage associated with TBI (Zweckberger et al., 2003; Raghupathi, 2004; Zhang et al., 2005a; Plesnila et al., 2007). This delayed secondary tissue damage provides a therapeutic window with the opportunity for limiting neurological damage and death in these patients. Over the years, experimental TBI studies have provided numerous promising drug compounds, which mediate neuroprotection and/or repair. These include, among others, glutamate receptor antagonists, calcium-channel blockers, spin trap agents, and caspase inhibitors (Royo et al., 2003; Marklund et al., 2006; Savitz, 2007; Schouten, 2007). However, the research efforts have faced serious problems in translating the experimental results into the clinic. Accordingly, most of the mentioned drug candidates have failed to meet the expectations based upon animal studies. One of recent examples is NXY-059, a compound with free radical trapping properties, which conferred substantial neuroprotection in animal stroke models but in its second clinical trial, NXY-059 failed to improve the outcome of stroke patients (Savitz, 2007). Other drug candidates have not only been ineffective in clinical trials, but have resulted in worsened outcome, even though they had been effective in preclinical testing. As an example of this, D-CPP-ene (SDZ EAA 494; Saphirs), a competitive NMDA receptor antagonist, was evaluated in 51 European Centers, enrolling 920 patients where administration of the drug resulted in a worse outcome at 6 months follow-up (Marklund et al., 2006). Another example is the a-amino-3hydroxy-5-methyl-4-isoxazolyl-propionic acid (AMPA) antagonist ZK200775, which transiently worsened the neurological condition and increased serum markers of glial toxicity in a clinical study of ischemia (Marklund et al., 2006). The investigation of new potential neuroprotective compounds is therefore warranted. However, even though the past two decades have provided a great expansion in our understanding of TBI, the pathophysiology is complex and heterogenous, and it has therefore proved difficult to design pre-clinical studies of neuroprotective drug compounds, that can be translated into human efficacy. In order to achieve this, it is important to evaluate the candidate drug in the relevant animal models, and these studies should be performed in a randomized and blinded manner, as well as inclusion of animals of both sexes and various ages. Furthermore, the candidate drug must be administered in a time-window that is realistic in the clinical setting, and histological and functional outcome measures should be assessed both short-term and long-term, to ensure that the drug leads to clinically meaningful and sustained benefit over time. We here review the current knowledge concerning PCD mechanisms in the development of neurological damage following TBI. We also discuss the possible pharmacological application in the future of MT isoproteins or MT-based

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compounds. MT holds the potential to be more successful in the treatment of TBI than several previously tested drug candidates, since it is an endogenous protein expressed in almost all cell types and tissues, therefore not acting as a potential antigen that could activate the human immune system – as opposed to the synthetic drug compounds. Also, MT is rapidly metabolized and excreted. The MT compounds have been investigated in our laboratory over the last decades and have shown great potential as a neuroprotective agent during various neuropathologies – including TBI – since MT’s mechanisms of action involve both antiinflammatory, antioxidative and antiapoptotic actions, as well as promoting neuroregeneration.

2. Programmed cell death in the injured brain Cell death is broadly classified into three types: necrosis, apoptosis (type 1 programmed cell death (PCD)) and autophagy (type 2 PCD) (for exhaustive reviews see Bredesen et al., 2006; Galluzzi et al., 2007; Werner and Engelhard, 2007) (Fig. 1). In contrast to necrosis, PCD is a highly regulated and energy demanding process (Waldmeier, 2003; Bramlett and Dietrich, 2004; Kajta, 2004), which may present with a delayed time window of days, weeks or months (Shaw et al., 2001; Williams et al., 2001; Raghupathi, 2004), and may be initiated by the primary necrosis and inflammation (Waldmeier, 2003; Bramlett and Dietrich, 2004; Kajta, 2004; Diskin et al., 2005; Erlich et al., 2006). Studies show that this secondary cell death may eventually account for up to 40% of the total tissue loss (Zweckberger et al., 2003;

Fig. 1. Overview over the three different modes of cell death (necrosis, apoptosis and autophagy) that can be morphologically distinguished within the cell. Although debated, this classification is now generally accepted.

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Raghupathi, 2004; Plesnila et al., 2007), thus playing a determining role in the outcome following TBI and hence presenting an important drug target for neuroprotective treatment. It is therefore essential to gain insight into these delayed cell death mechanisms and their involvement in TBI-related pathology. The following sections will therefore provide a general overview of the current knowledge about the role of PCD following TBI. 2.1. Apoptosis (type 1-programmed cell death) Apoptosis involves the activation of catabolic enzymes (in particular proteases), de novo synthesis of apoptosis-related proteins (e.g. capsases), and programmed decomposition of cellular structures and organelles (Maiuri et al., 2007). This leads to characteristic morphological signs of apoptosis: nuclear pyknosis (chromatin condensation), DNA cleavage, karyorrhexis (nuclear fragmentation), budding of the cell membrane, and finally the fragmentation of the dying cell into apoptotic bodies (Krantic et al., 2007; Maiuri et al., 2007). The membrane-bound apoptotic bodies are then removed by macrophages – without inducing manifest inflammatory host responses – in a process signalled by a phospholipid flip-flop process in the cellular membrane where phosphatidylserine is exposed on the cellular surface and annexin is moved to the inner leaflet of the membrane, (Bratton et al., 1999; Fadeel, 2004; Bredesen et al., 2006; Werner and Engelhard, 2007). Apoptosis is usually divided into two major pathways; caspase-dependent and caspase-independent apoptosis (Zhang et al., 2005a; Kim et al., 2006). However, since the pathways have been shown to occur independently or through common molecular mechanisms in vivo (Krantic et al., 2007), the different signalling pathways should not automatically be considered as separate events, even though often being discussed as such. 2.1.1. Caspase-dependent apoptosis Two different pathways of caspase-dependent apoptosis can be identified. In the extrinsic pathway, the caspases are activated by extracellular death inducing ligand–receptor interactions (e.g. TNFa interacting with TNFa-receptor, Fas ligand (FasL) binding to the Fas/CD95-receptor, and tumor necrosis factor-like weak inducer of apoptosis (TWEAK), which binds to TNF-receptor-related apoptosismediated protein (TRAMP) (Kajta, 2004; Yakovlev and Faden, 2004; Zhang et al., 2005a). Ligand binding triggers receptor oligomerization and recruitment of adaptor proteins (e.g. Fas-associated death domain (FADD)) that further propagate death signals in three ways; (i) via proteolysis of the pro-apoptotic BH3 interacting domain death agonist (Bid), with consequent mitochondrial outer membrane permeabilization (MOMP) and subsequent release of cytochrome c (ii) by direct proteolytic activation of procaspase-8 or -10 or (iii) via activation of ribosome-inactivating protein (RIP) kinases (Hutchison et al., 2001; Bredesen et al., 2006; Kim et al., 2006). The final result is activation of caspases-3, -6 and -7 and irreversible cell death (Leker and Shohami, 2002; Kajta, 2004; Zhang et al., 2005a; Bredesen et al., 2006) (Fig. 2). In contrast; the intrinsic pathway is initiated by intracellular organellar stress and

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Fig. 2. Overview of classical apoptosis. Depending on the routes of caspase-3 activation two different pathways can be identified; extrinsic pathway and intrinsic pathway. Both pathways converge on the activation of executioner pro-caspases 3, 6 and 7, which lead to nucleosomal DNA fragmentation. See text for further details. *This pathway can also be activated in the intrinsic pathway. Abbreviations: Apaf-1 – apoptotic protease-activating factor-1; Bak – Bcl-2homologous antagonist/killer; Bax – Bcl-2-associated X protein; Bid – BH3 interacting domain death agonist; FADD – Fas-associated death domain; MOMP – mitochondrial outer membrane permeabilization; NOXA – phorbol-12-myristate-13-acetate-induced protein 1; PIDD – p53-induced protein with a death domain; PUMA – p53-upregulated modulator of apoptosis; RAIDD – RIP-associated protein with a death domain; RIP – ribosomeinactivating protein; TNFa – tumor necrosis factor alpha.

DNA damage, which results in activation of Bcl-2-associated X protein (Bax) and/or Bcl-2-homologous antagonist/killer (Bak) (Kim et al., 2006) or activation of caspase2 by a multinuclear complex involving p53-induced protein with a death domain (PIDD) and RIP-associated protein with a death domain (RAIDD). This leads to increased MOMP and the subsequent release of release of cytochrome c from mitochondria and activation of caspase-9 and apoptotic protease-activating factor 1 (Apaf-1) (Waldmeier, 2003; Kajta, 2004; Bredesen et al., 2006). Both pathways converge on the activation of executioner pro-caspases-3, -6 and -7, and their activation leads to DNA fragmentation that is characteristic for apoptosis (Kajta, 2004; Krantic et al., 2007; Zhang et al., 2005a) (Fig. 2).

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A role of caspase-dependent apoptosis in neuronal death following TBI is supported by in vivo studies showing that pan-caspase inhibitors (such as z-DEVDfmk), which inhibit caspase activation as well as partially inhibiting cytochrome c release from mitochondria (Kim et al., 2006), can reduce brain tissue damage and improve neurological outcome in mice and rats following experimental TBI (Hutchison et al., 2001; Zhang et al., 2005a). However, since caspase-inhibitors do not prevent the loss of mitochondrial membrane potential, and the resulting production of ROS (Kim et al., 2006), they have been shown to shift the mode of cell death to caspase-independent mechanisms (Kim et al., 2006; Vandenabeele et al., 2006). This complicates the use of caspase inhibitors as a neuroprotective treatment following TBI, and hence it would be valuable to find other pharmacological interventions that could inhibit these pro-apoptotic pathways. 2.1.2. Caspase-independent apoptosis A role for caspase-independent apoptosis in TBI is supported by the fact that characteristic morphological features of apoptosis are present in cells cultures with cells devoid of caspase-3 or Apaf-1 (Susin et al., 2000), and following pharmacological caspase-inhibition in vivo (Krantic et al., 2007; Zhang et al., 2005a). The mitochondrial flavoprotein apoptosis-inducing factor (AIF) has recently been acknowledged as one of the main mediators of caspase-independent apoptosis (Bredesen et al., 2006; Culmsee and Landshamer, 2006). The release of AIF from the mitochondria can be induced by poly(ADP-ribose) polymerase (PARP) (ADP – adenosine diphosphate), p53-induced caspase-2 activation, oxidative stress, or excitotoxicity (Culmsee and Landshamer, 2006; Kim et al., 2006; Krantic et al., 2007). Activation of these mediators results in mitochondria outer membrane permeabilization (MOMP) and subsequent activation of calpains and/or cathepsins that promote AIF release from the mitochondria (Fig. 3). AIF is subsequently translocated to the nucleus via its C-terminal nuclear localization sequence, ultimately mediating chromatin condensation and large-scale DNA fragmentation (Zhang et al., 2005a; Culmsee and Landshamer, 2006; Krantic et al., 2007). Studies both in vitro and in vivo suggest that AIF could be involved in cell death associated with TBI, as well as with other neurodegenerative diseases (Zhang et al., 2005b; Culmsee et al., 2005; Culmsee and Landshamer, 2006). In vitro findings show intracellular redistribution of AIF correlating with large-scale DNA fragmentation and chromatin condensation, which occur even in cells lacking Apaf-1 or caspase-3 (Yakovlev and Faden, 2004; Zhang et al., 2005b; Krantic et al., 2007). Furthermore, studies with cultured cortical neurons from mutant Harlequin (Hq) mice (which display a reduction of 480% in AIF expression due to a proviral insertion in the AIF gene (Klein et al., 2002)), have demonstrated that the decreased expression of AIF conferred neuroprotection after exposure to excitotoxicity (Culmsee et al., 2005). Furthermore, in vitro data have shown a significant reduction of cell death by siRNA-mediated AIF downregulation in glutamate- and oxygen–glucose-deprivation neuronal injury models (Culmsee et al., 2005), which suggests that AIF is a mediator of glutamate excitotoxicity.

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Fig. 3. The different pathways responsible for activation of apoptosis-inducing factor (AIF) and caspase-independent apoptosis. AIF is released from the mitochondria in response to poly(ADP-ribose)polymerase (PARP), p53-induced caspase-2 activation, oxidative stress due to reactive oxygen species (ROS), and glutamate-mediated excitotoxicity. AIF is then translocated to the nucleus where it mediates chromatin condensation and large-scale DNA fragmentation.

Moreover, accumulating in vivo data support a role for AIF-mediated caspaseindependent pathways in acute neuronal cell death after cerebral ischemia and brain trauma, as well as in models of neurodegenerative diseases (Ferrer et al., 2003; Zhang et al., 2005b; Culmsee and Landshamer, 2006). For example, reduced AIF levels either in Harlequin mutant mice or following pharmacological AIF siRNA treatment have been found to significantly protect neurons from lethal stress after cerebral ischemia or kainate-induced excitotoxicity in vivo, where a smaller infarct volume and reduced neuronal cell death was seen in the ischemic penumbra following middle cerebral artery occlusion, as compared with controls (Culmsee et al., 2005). Furthermore, AIF has been shown to rapidly translocate to the nucleus of injured neurons and colocalized with DNA damage and apoptotic nuclear condensation following ischemic insult, brain trauma and Parkinson’s disease in vivo (Zhang et al., 2002, 2005b; Plesnila et al., 2004). Importantly, the translocation of AIF following ischemia was not affected by the caspase inhibitor z-VAD-fmk (Zhang et al., 2005b).

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It remains to be determined, however, if this caspase-independent pathway is initiated as a separate event that occurs in parallel with caspase-dependent pathways, or if it represents a default pathway, activated only when caspases are inhibited. Also, as discussed above, it remains to be shown, that the findings from these in vivo animal studies can be successfully translated to human subjects.

2.2. Autophagy (type 2-programmed cell death) Autophagy has recently been acknowledged as an evolutionarily conserved cellular process, in which cellular proteins and organelles are degraded in response to cellular stress and starvation (Diskin et al., 2005; Erlich et al., 2006; Gozuacik and Kimchi, 2007; Klionsky, 2007). In mammals, at least three different modes of autophagy have been identified: macroautophagy, microautophagy, and chaperonemediated autophagy (CMA) (Boland and Nixon, 2006; Bredesen et al., 2006; Huang and Klionsky, 2007). The different types of autophagy share a common organellar endpoint, the lysosome, but they differ with respect to the conditions by which the process is preferentially activated, their regulation, the cellular components that are transported and the pathway by which this material is delivered to the lysosome (Boland and Nixon, 2006; Martinez-Vicente and Cuervo, 2007; Xiao, 2007; Thorburn, 2008) (Fig. 4).

Fig. 4. The three major routes of autophagy (chaperone-mediated autophagy (CMA), microautophagy and macroautophagy) that are currently acknowledged.

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The most commonly employed method for distinguishing between the different forms of autophagy has been by studying the morphological characteristics in the electron microscope. However, several biochemical markers have recently emerged for detecting autophagic activity, and for distinguishing the different types of autophagy from each other (He et al., 2007). For example, since autophagic mechanisms are related to the lysosomes, an increased expression of lysosomal proteins, such as a-hexosaminidase, cathepsin B and cathepsin D can be detected during autophagy (He et al., 2007). Furthermore, heat shock cognate (HSC) protein 73 is a key protein marker for CMA, while Beclin-1, microtubule-associated protein 1 light chain 3 (LC3-I) and the phosphatidylethanolamine conjugated form of LC3-I (LC3-II), are very specific markers of the autophagosome (and thus markers for macroautophagy) (He et al., 2007; Klionsky et al., 2007). 2.2.1. Macroautophagy Macroautophagy is the form of autophagy believed to play the most important role in relation to human pathology (e.g. infections, heart disease, cancer and neurodegenerative diseases) (Huang and Klionsky, 2007; Martinez-Vicente and Cuervo, 2007; Rubinsztein et al., 2007; Levine and Kroemer, 2008; Mizushima et al., 2008). It is regulated by autophagy genes (Atgs) in a process that can be divided into at least four steps: (1) induction, (2) formation of autophagosome, (3) lysosomal fusion and (4) degradation (Huang and Klionsky, 2007; Xiao, 2007). The induction is regulated by classes I and III phosphoinositide 3 kinases (PI3Ks) (Levine and Kroemer, 2008). Activation of class I PI3K inhibits autophagy through activation of protein kinase B (PKB)/Akt and mammalian target of rapamycin (mTOR) phosphorylation (Boland and Nixon, 2006; Erlich et al., 2006; Rubinsztein et al., 2007; Levine and Kroemer, 2008), while activation of the class III PI3K complex containing Beclin-1 (a mammalian homolog of yeast Atg6) promotes autophagy (Boland and Nixon, 2006, Erlich et al., 2006; Lai et al., 2008; Levine and Kroemer, 2008). The second step in the process is initiated by the production of a double membrane, which sequesters the cytosolic target components (e.g. proteins, sugars, lipids, RNA, and organelles (e.g. mitochondria and perixosomes)), thus forming the autophagosome (Boland and Nixon, 2006; Galluzzi et al., 2007; Xiao, 2007). The formation of the autophagosomes double membrane is mediated by two ubiquitinlike conjugation systems: microtubule-associated protein light chain 3 (LC3) (a mammalian homolog of yeast Atg8) and Atg12–Atg5 (Lai et al., 2008; Rubinsztein et al., 2007; Thorburn, 2008). In the third step the outer membrane of the autophagosome fuses with the lysosomal membrane in a process mediated by Rab7, a Rab guanine-triphosphatase (GTPase), hereby generating the autophagolysosome (Klionsky et al., 2007). This leads to the final step where the content of the autophagolysosome is degraded by hydrolytic lysosomal enzymes, such as cathepsin B and D (He et al., 2007) (Fig. 5). Activation of macroautophagy preferentially occurs under stress conditions, such as nutrient and energy starvation, oxidative stress, mitochondrial dysfunction, cytokines, pathogens, misfolded or aggregated proteins, damaged organelles and p53 activation (Bredesen et al., 2006; Klionsky, 2007; Martinez-Vicente and Cuervo,

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Fig. 5. The four major steps in the execution of macroautophagy: (1) induction, (2) formation of autophagosome, (3) lysosomal fusion and (4) degradation – as well as some of the major mediators of this process. Abbrevations: Atg – autophagy gene, LC-3 – microtubule-associated protein light chain 3; mTOR – mammalian target of rapamycin; PI3K – phosphoinositide 3 kinase; PKB – protein kinase B.

2007; Maiuri et al., 2007). It serves to generate essential macromolecules and energy for cell survival under conditions of nutritional scarcity, and as a mechanism for removing damaged intracellular components (Martinez-Vicente and Cuervo, 2007; Maiuri et al., 2007). However, although initially considered a survival process, it is now believed that if cellular damage is too extensive, macroautophagy can also result in cell death (autophagic, apoptotic, or necrotic) (Kim et al., 2006; Klionsky, 2007; Yousefi and Simon, 2007; Thorburn, 2008). Recent research studies also point towards a role for macroautophagy in cell death following TBI (Diskin et al., 2005; Erlich et al., 2006; Lai et al., 2008; Clark et al., 2008), and several studies have revealed a role for macroautophagy in several other human pathologies, such as Alzheimer’s disease (Boland and Nixon, 2006; Martinez-Vicente and Cuervo, 2007; Rubinsztein et al., 2007), Parkinson’s disease (Huang and Klionsky, 2007; Martinez-Vicente and Cuervo, 2007), Huntington’s disease (Boland and Nixon, 2006; Huang and Klionsky, 2007), bacterial infections (Klionsky, 2007; Rubinsztein et al., 2007), cerebral ischemia (He et al., 2007) and cancer (Rubinsztein et al., 2007).

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In relation to TBI, it has been shown that increased expression of Beclin-1 in both neurons and astrocytes can be detected in vivo for at least 3 weeks following head injury (Diskin et al., 2005; Erlich et al., 2006). The study by Diskin et al. (2005) further demonstrated that correlation between dying cells (as indicated by TUNEL staining) and Beclin-1 expression was found only in neurons, which indicates that autophagy may serve to discard injured neurons following injury. In addition, most of the nuclei of the TUNEL-positive neurons with increased Beclin-1 expression were round and not pyknotic or fragmented (which is typically seen in apoptotic cells) (Diskin et al., 2005). This high expression of Beclin-1 and the morphology of the nuclei may suggest that, at least in some injured neurons, autophagic cell death takes place. In line with this, autophagic cell death is usually thought to generate a caspase-independent form for cell death, since some of the best examples of autophagic cell death were demonstrated in cells defect in the apoptotic machinery or in the presence of caspase inhibitors (Kim et al., 2006; Gozuacik and Kimchi, 2007; Yousefi and Simon, 2007; Thorburn, 2008). However, the current understanding of the pathophysiological roles of autophagy in specific brain pathologies remains unclear and further research is needed to elucidate more specifically the role of autophagy following TBI and its potential as drug target in neuroprotective treatment.

3. Regulation of programmed cell death The functional relationships between autophagic and apoptotic cell death is complex in that activation of autophagy can abrogate cell death, result in autophagic cell death, or turn into necrosis/apoptosis (2006; Maiuri et al., 2007). Moreover, autophagy and apoptosis may be initiated and regulated by common upstream signals. As an example, increases in Ca, TNFa, ROS, mitochondrial dysfunction, and protein aggregation have all been shown to initiate both autophagy and apoptosis, while downstream regulators, such as p53 and Bcl-2 family proteins have been implicated in both types of PCD – sometimes resulting in combined autophagy and apoptosis (Kim et al., 2006; Maiuri et al., 2007; Scherz-Shouval et al., 2007; Thorburn, 2008). As an example, ROS enhances lysosomal membrane permeabilization, resulting in the release of lysosomal proteases, which can trigger apoptosis or activate autophagic mechanisms (Kim et al., 2006; Scherz-Shouval et al., 2007). Similarly, TNFa can induce both apoptosis (by activating death receptors), as well as autophagic cell death (through the activation of FADD) (Kim et al., 2006; Thorburn, 2008). Furthermore, the tumor suppressor p53 is a transcription factor activated by many types of stress, including DNA damage, hypoxia, oxidative stress, metabolic compromise and calcium overload (Culmsee and Mattson, 2005; Culmsee and Landshamer, 2006; Plesnila et al., 2007). p53 is a well-known promoter of apoptosis (through enhanced activation of specific target genes) that has been implicated in the activation of both extrinsic and intrinsic apoptotic pathways (Culmsee and Mattson,

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2005). Mice models of experimental TBI have demonstrated a rapid accumulation and nuclear translocation of p53 in the neurons of the injured brain tissue within 12 hours following trauma (Plesnila et al., 2007). Following nuclear translocation p53 has been shown to mediate transcriptional activation of the BH3-only proteins (such as p53-upregulated modulator of apoptosis (PUMA) and phorbol-12-myristate-13acetate-induced protein 1 (NOXA)) that can promote mitochondrial membrane permeabilization via Bax/Bak activation (Maiuri et al., 2007). Alternatively, DNA damage can activate apoptosis through caspase-2 activation in a multinuclear complex that involves the p53-induced protein with a death domain (PIDD) and the RAIDD (Maiuri et al., 2007) (Figs. 2 and 3). However, p53 can also induce autophagy through increased expression of a direct p53 target gene called damageregulated autophagy modulator (DRAM) (Thorburn, 2008). Another example supporting the molecular cross-talk between apoptosis and autophagy is provided by the B-cell lymphoma-2 (Bcl-2) family of proteins. The Bcl2 protein family has been shown to contain both pro- and antiapoptotic members that have been shown to regulate both caspase-dependent and caspase-independent apoptosis (Shacka and Roth, 2005; Zhang et al., 2005a; Schmitt et al., 2007). An important role of Bcl-2 proteins in TBI has been demonstrated with in vivo TBI models, and by the observation that upregulation of Bcl-2 occurs in human brain and cerebrospinal fluid (CSF) following TBI (Clark et al., 2000; Zhang et al., 2005a). For example, in paediatric patients, lower concentrations of Bcl-2 were detected in patients who died than in those who survived (Clark et al., 2000; Hutchison et al., 2001; Zhang et al., 2005a), supporting a pro-survival role for Bcl-2. Similarly, the pro-apoptotic Bax was detectable in samples of brain tissue from TBI patients, and patients with detectable Bax combined with an undetectable level of Bcl-2 were shown to have a less favourable outcome than patients in whom both Bax and Bcl-2 were detectable (Ng et al., 2000; Zhang et al., 2005a), thus supporting its proapoptotic function. In addition to inhibiting apoptosis, Bcl-2 has recently been shown also to inhibit Beclin-1 in vitro, hereby blocking autophagic cell death (Pattingre et al., 2005; Yousefi and Simon, 2007; Thorburn, 2008). Bcl-2 has also been shown to inhibit autophagy by blocking calcium release from the endoplasmic reticulum (Høyer-Hansen et al., 2007). In doing so, the activation of Ca2+/ calmodulin-dependent kinase kinase-b and AMP-activated protein kinases are inhibited, which keeps mTOR activated and hence inhibits autophagy (Thorburn, 2008). Since Bcl-2 has antioxidant functions, it has been speculated that ROSmediated PCD may be partially inhibited by antioxidants (Kim et al., 2006). In line with this in vitro studies by Scherz-Shouval et al. (2007) showed that N-acetyl-Lcysteine (NAC), a general antioxidant, and catalase, which specifically decomposes H2O2, caused a 60% and 25% inhibition in starvation-induced degradation in the cell cultures, respectively. Other studies have also shown that catalase was selectively eliminated during autophagic cell death and that this depletion could be prevented by inhibiting autophagy as well as by antioxidant treatment (Maiuri et al., 2007). Recently there have also been attempts to alleviating neuronal cell death in vivo following TBI by blocking autophagy with antioxidants. By treating mice with the antioxidant gamma-glutamylcysteinyl ethyl ester (GCEE), these studies have showed

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that the treatment with GCEE preserved total antioxidant reserves, reduced LC3-II in injured brains, and improved both behavioural and histological outcome after TBI (Clark et al., 2008; Lai et al., 2008). These research results highlight three important points: (1) that oxidative stress contributes to overall neuropathology following TBI, (2) that the role of oxidative stress could be, at least partially, by influencing autophagy in neurons and (3) that oxidative stress can initiate both apoptosis and autophagy. In relation to this, we have shown that the antioxidant MT plays an important role following experimental TBI (for details refer to Penkowa, 2006a, b; Penkowa et al., 2006a). MT-I+II are multipurpose proteins involved in a broad range of functions, including ROS scavenging, immune defence responses, mitochondrial respiration, angiogenesis, and cell survival and differentiation (Penkowa, 2006b). Even though astrocytes are the main source of MT-I+II in the brain, we have recently shown that astrocytes actively secrete MT in a regulated manner, and that the MT proteins are subsequently taken up by neurons with the aid of the megalin-receptor (Chung et al., 2008). This is the first evidence showing MT transportation from the extracellular to the intracellular space in neurons. In vitro studies have also demonstrated that MT can alleviate lipid peroxidation, Bax up-regulation, and Bcl-2 down-regulation in striatal fetal stem cells in vitro (Sharma and Ebadi, 2003), as well as inactivating p53 – hereby promoting survival through induction of a ‘‘p53-null state’’ (Hainaut and Mann, 2001; Ostrakhovitch et al., 2006). Moreover, cell cycle analyses have revealed that down-regulation of MT-I abrogated proliferation, migration and tube formation of endothelial cells in vitro as well as angiogenesis in vivo (Miyashita and Sato, 2005). Inhibition of MT-I or II by siRNA treatment also increased G0/G1-phase cell population, decreased S-phase population, inhibited cell migration and network formation in both endothelial cells (Miyashita and Sato, 2005) and cancer cells (Yamasaki et al., 2007). These results indicate that MT-I+II are important for cell cycle progression and angiogenesis, which is in accordance with findings showing a transient nuclear localization of MT in cells at the G1-to-S phase transition in rapidly proliferating and injured cells (Cherian and Kang, 2006). By studying the response to experimental TBI in wild-type (WT) mice, we have demonstrated that MT I+II mRNA levels increase in the brain within 24 h after the injury, followed by a significant increase in the protein levels seen typically 1–3 days post-injury, peaking by day 3–10 and returning to basic levels by day 20–30 (Penkowa, 2006a, b). Furthermore, by comparing the response WT mice, MT-I+II knockout mice (MTKO) and transgenic MT-I overexpressing (TgMT) mice following experimental TBI, we have further demonstrated that MT-I+II act as antiinflammatory factors that significantly reduce cerebral activation and recruitment of macrophages and lymphocytes following TBI (Penkowa, 2006a), as well as reducing expression of proinflammatory cytokines IL-1b, IL-6, IL-12 and TNF-a (Penkowa, 2006a; Stankovic et al., 2007). Furthermore, MT-I+II were shown to have antioxidant and antiapoptotic effects, in that they reduce the levels of inducible nitric oxide synthase (iNOS), nitrotyrosine (NITT – a marker of protein nitration), malondialdehyde (MDA – a marker for lipid peroxidation), 8-oxoguanine (a marker

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for oxidative DNA damage), DNA fragmentation (TUNEL), caspase-1 levels, caspase-3 activity and mitochondrial leakage of cytochrome c following TBI (Penkowa, 2006a). In accordance with these results, brain injured MTKO mice show enhanced inflammatory responses including increased recruitment of macrophages and lymphocytes, as well as enhanced expression of proinflammatory factors like IL-1b, IL-3, IL-6, IL-12, TNF-a, lymphotoxin-a (LTa), macrophage activator factor (Mac-1) and ICAM-1 (Penkowa, 2006b). Furthermore, MTKO mice display increased and prolonged ROS formation, oxidative stress, neurodegeneration, and apoptotic cell death, compared with those of WT controls (Penkowa, 2006a, b). In contrast, TgMT mice show significantly reduced numbers of activated microglia/ macrophage and T-lymphocytes, reduced levels of proinflammatory cytokines, ROS, cytochrome c, caspase-3 and caspase-9, as compared with WT controls (Giralt et al., 2002; Penkowa, 2006a, b) (Figs. 6 and 7). This was followed by reduced oxidative stress, neurodegeneration and cell death in the TgMT mice with TBI. We have also repeatedly demonstrated that, in addition to reducing inflammation, oxidative stress and cell death, MT-I+II also play an important role for neuroregeneration following head trauma and other neurodegenerative conditions. These findings are partially explained by the fact that MT-I+II increase the expression of antiinflammatory and/or regenerative cytokines and growth factors; such as IL-10, FGF, FGF-receptor (FGF-R), TGF-b, TGF-b-receptor (TGF-b-R), VEGF, NGF, NT-3–5, BDNF, and glial cell line-derived neurotrophic factor (GDNF) following TBI in vivo (Giralt et al., 2002; Penkowa, 2006b) (Figs. 8 and 9). In line with this, TgMT mice display increased angiogenesis and levels of proangiogenic factors relative to controls following TBI (Penkowa, 2006a), and the TgMT mice show enhanced cortical wound healing and repair; including astrogliotic scar formation, and vascular remodeling relative to wildtype controls (Penkowa et al., 2006a). Furthermore, as judged by GAP-43 and P-40 stainings, TgMT mice show enhanced growth cone formation in surviving neurons situated outside the lesioned Fig. 6. Illustrates lectin histochemistry of wild-type (WT) and transgenic MT (TgMT) mice. (A) Lesioned WT mice at 1 days post-lesion (dpl), showing numerous lectin+macrophages around and inside of the lesion. (B) Lesioned TgMTI mice at 1 dpl, showing a decreased number of lectin+cells around and inside of the lesion. (C) Lesioned WT mice at 3 dpl, showing numerous lectin+macrophages around and inside of the lesion, compared with lesioned TgMT mice at 3 dpl, which showed a decreased number of lectin+macrophages (D). (E) Lesioned WT mice at 6 dpl, still displaying numerous lectin+macrophages. (F) Lesioned TgMT mice at 6 dpl, showing that the lesion has decreased considerably in size as well as in the number of lectin+round macrophages surrounding the lesion has decreased compared with that seen at 3 dpl. (G) Lesioned normal mice at 10 dpl, showing a lesion site comparable to that seen at 3 dpl of normal mice. (H) Lesioned TgMT mice at 10 dpl, showing that the lesioned tissue has regenerated and the lesion is replaced by glial scar tissue. (I) Higher magnification of the square in C, showing ameboid or round macrophages of lesioned WT mice at 3 dpl. (J) Higher magnification of the square in D, showing a decreased number of ameboid or round macrophages in lesioned TgMT mice at 3 dpl relatively to normal mice. Bars: A–D, 457 mm; E–H, 400 mm; I, J, 29 mm (Giralt et al., 2002).

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area (Penkowa, 2006a). Moreover, immunostaining for synaptophysin and syntaxin (presynaptic markers), as well as for spinophilin (a marker of dendritic spines), have shown that exogenous MT treatment increased synaptic plasticity in WT mice, as

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Fig. 7. Immunohistochemical staining for oxidative stress (malondialdehyde (MDA) and nitrotyrosine (NITT)) and apoptosis (as determined by TUNEL) in wild-type (WT) and transgenic MT (TgMT) mice. Unlesioned WT mice show few cells positive for MDA (A), NITT (D), and TUNEL (G), while lesioned WT mice show increased numbers of positive cells for MDA (B), NITT (E), and TUNEL (H) 3 days post-lesion. Lesioned TgMT mice show stainings for MDA (C), NITT (F), and TUNEL (I) at 3 days post-lesion, which are comparable to those of unlesioned mice. Bar: 29 mm (Giralt et al., 2002).

compared with saline-treated controls following experimental TBI (Penkowa et al., 2006a). This is in accordance with in vivo studies on rats with experimental autoimmune encephalomyelitis (EAE), where MT-II treatment significantly increased the number of growth cones and neurites expressing P-40 and GAP-43 in both gray and white matter of the CNS, as compared with untreated controls (Penkowa and Hidalgo, 2003). Moreover, TgMT mice and mice injected with MT-II have shown significant increases in the number of neural stem cells (NSC) in the subventricular zone (SVZ), and inside the brain parenchyma, likely indicating migration in the direction of the lesion cavity (Penkowa et al., 2006b). In line with this, both lesioned and unlesioned MTKO mice display a reduction in NSCs in the SVZ of the lateral ventricles compared with those of WT controls (Penkowa et al., 2006b). This is also in accordance with previous results obtained in the EAE model, where MTKO mice

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Fig. 8. Cellular counts from 0.5 mm2 matched areas at the border of the cortical lesion in brain injured MT knockout (MTKO) mice treated with saline (control), human native MT-IIa (nMT-Iia), or human recombinant MT-III (rMT-III). *Po0.01 vs. saline-injected mice. Abbrevations: BDNF – brain-derived neurotrophic factor; bFGF – basic fibroblast growth factor; FGF-R – fibroblast growth factor receptor; GFAP – glial fibrillary acidic protein; NITT – nitrotyrosine; NT – neurotrophin; PSA-NCAM – polysialic acid-neural cell adhesion molecule; TNFa – tumor necrosis factor-alpha; TrkB – tyrosin kinase receptor-B; TUNEL – terminal deoxynucleotidyl transferase (TdT)-mediated de-oxyuridine triphosphate (dUTP)biotin nick-end labeling (Penkowa et al., 2006a).

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displayed an overall impairment of progenitor cells, as well as impaired functional outcome, compared with WT mice (Penkowa et al., 2003a). Accordingly, MT-I+II are likely activating stem/progenitor cell populations derived from the stem cell niches in the brain, opening exciting perspectives regarding the putative therapeutical use of these proteins. In summary there is considerable evidence for mechanisms of cross-communication and interplay between apoptotic and autophagic cell death signals in response to various stimuli seen following TBI. Moreover, autophagy may play a dual role following trauma, starting as an attempt to save the damaged cells, but potentially contributing to cell death (Boland and Nixon, 2006; Galluzzi et al., 2007; Lai et al., 2008; Maiuri et al., 2007). Since MT-I+II not only act antiinflammatory and antiapoptotic by inhibiting several of the mediators that have been shown to

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contribute both to activation and execution of apoptosis and autophagy, but also increases angiogenesis, neuronal regrowth and brain tissue regeneration following TBI, it holds the potential to limit neurological impairment and improve functional outcome following TBI.

4. Discussion Excitotoxicity, calcium-mediated events, ROS, proinflammatory mediators, mitochondrial damage, apoptosis and lately autophagy are commonly discussed as possible drug targets for neuroprotective therapy following TBI (Bramlett and Dietrich, 2004). Delayed neuronal death following TBI may contribute with as much as 40% of the total neural cell loss (Zweckberger et al., 2003; Raghupathi, 2004; Plesnila et al., 2007), making it a determining role in the outcome following TBI. This delayed cell death is primarily a result of PCD induced by inflammatory mediators (e.g. cytokines, ROS), excitotoxicity and metabolic disturbances (e.g. dysregulation of calcium and zinc homeostasis), and may be detected as TUNELpositive human neurons up to 12 months after TBI (McIntosh et al., 1998; Williams et al., 2001; Waldmeier, 2003; Zhang et al., 2005a). This suggests that a wider therapeutic window may exist for the administration of neuroprotective drugs aiming to reduce neurodegeneration and cell death following brain injury, hereby Fig. 9. (A) Shows the inflammatory responses of F4/80+microglia/macrophages and glial fibrillary acidic protein (GFAP)-positive astrocytes in brain injured MT knockout (MTKO) mice treated with saline (control), human native MT-IIa (nMT-Iia), or human recombinant MT-III (rMT-III). (A–C): F4/80+microglia/macrophages seen at the cortical lesion site in saline (A), nMT-IIa (B), and rMT-III (C) treated mice. (D–F): Higher magnification of framed areas in A–C. (G–I): GFAP-positive reactive astrogliosis seen at the lesion border of saline (G), nMT-IIa (H), and rMT-III (I) treated mice. Scale bars ¼ 200 mm (A–C); 28 mm (D–F); 33 mm (G–I) (Penkowa et al., 2006a). B: shows oxidative stress, neurodegeneration and apoptosis in the parenchyma surrounding the lesion cavity of MTKO mice treated with saline (control), human nMT-IIa or rMT-III. (A–C) nitrotyrosine (NITT) immunoreactivity in saline (A), nMT-IIa (B), and rMT-III (C) treated mice showing that mainly perilesional neurons are suffering oxidative stress. (D–F) Immunoreactivity for neurofibrillary tangles indicating neurodegeneration in saline (D), nMT-IIa (E), and rMT-III (F) treated mice. (G–I) TUNEL in saline (G), nMT-IIa (H), and rMT-III (I) treated mice showing apoptotic cell death inside the lesion (top) and perilesional (bottom). Brain injury-induced oxidative stress, neurodegeneration and apoptosis were reduced by nMT-IIa relative to saline treatment, whereas rMT-III had no significant effects. Scale bars ¼ 52 mm (A–C); 60 mm (D–F); 70 mm (G–I). (Penkowa et al., 2006a). C: depicts the expression of neurotrophins, synaptic plasticity, and repair factors (neurotrophin (NT)-3, brain-derived neurotrophic factor (BDNF), syntaxin, spinophilin, and polysialic acid-neural cell adhesion molecule (PSA-NCAM)) in the tissue surrounding the lesion of MTKO mice treated with saline (control), human nMT-II or human rMT-III. Neurotrophins, synaptic plasticity, and repair factors were enhanced by nMT-IIa relative to saline treatment, whereas rMT-III tended to reduce these. Scale bars ¼ 60 mm (A–F); 58 mm (G–L); 17 mm (M–O). (Penkowa et al., 2006a).

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alleviating permanent neurological damage. However, even though numerous neuroprotective drugs have been developed, tested and proven effective in experimental models and pre-clinical studies, these drugs have so far failed in clinical TBI trials (Royo et al., 2003; Yakovlev and Faden, 2004; Marklund et al., 2006; Schouten, 2007), making the search for new potential drugs imperative. Since it is increasingly being acknowledged that biochemical and morphological features of more than one type of PCD can simultaneously exist within a single cell (Waldmeier, 2003; Kajta, 2004), that inhibition of one form of cell death can lead to domination of another (Waldmeier, 2003; Galluzzi et al., 2007; Maiuri et al., 2007), and that various types of cell death can share common pathways of execution (Yakovlev and Faden, 2004; Maiuri et al., 2007), it can be hypothesized that this complex interplay and molecular cross-talk has substantial implications for the development of more effective neuroprotective strategies, and suggests that treatment regimes should optimally be directed toward alleviation of multiple cell death pathways/mediators. By comparing the response to experimental TBI in WT mice, MTKO and TgMT mice we have repeatedly demonstrated the important roles for MT in the injured brain. These studies have shown that MT-I+II have an antiinflammatory action as they reduce cerebral recruitment of monocytes/macrophages and T- and B-lymphocytes (Penkowa, 2006a; Stankovic et al., 2007), as well as reducing activation of recident microglia (Penkowa, 2006a) (Fig. 7). Hereby MT reduces the expression of proinflammatory cytokines following TBI (Penkowa, 2006a; Stankovic et al., 2007). In addition, MT-I+II have antioxidant effects in that they reduce the levels of iNOS, protein nitration, lipid peroxidation, oxidative DNA damage and DNA fragmentation. Importantly, MT-I+II prevent apoptosis – presumably as a result of their antiinflammatory and antioxidant effects – as shown by the reduction in caspase-1 levels, caspase-3 cleavage and mitochondrial leakage of cytochrome c (Penkowa, 2006a) (Fig. 7), thereby reducing delayed (secondary) tissue damage and neurological damage after experimental TBI. Since it has lately been shown that there is an extensive overlap between the initiating and regulatory factors that play a role in apoptosis and autophagy, and MT-I+II have been shown to influence several of these (e.g. cytokines, ROS, Bcl-2 family proteins) the neuroprotective effects of MT should, hypothetically (even though so far not investigated), be a combined result of reduced apoptotic and autophagic cell death. Thus, by reducing the levels of ROS, proinflammatory cytokines and other factors initiating PCD following TBI, it is clear that MT-I+II inhibit several of the detrimental mechanisms that induce delayed neurodegeneration and neuron loss following TBI. Furthermore, levels of zinc have been shown to be increased in the damaged areas following TBI (Doering et al., 2007) and have previously been implicated in neurotoxicity following TBI (Suh et al., 2006). Since MT is known to regulate zinc homeostasis (Penkowa, 2006b) and protect tissues from heavy metal toxicity (Thirumoorthy et al., 2007), it can be hypothesized that this role of MT could also play a protective role following TBI. However, in addition to their antiinflammatory, antioxidant and antiapoptotic effects, we have also demonstrated that MT-I+II stimulate astroglial expression of

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antiinflammatory and/or regenerative cytokines and growth factors (such as IL-10, FGF, FGF-R, TGF-b, VEGF, NGF, NT-3–5, BDNF, GDNF) following TBI (Giralt et al., 2002; Penkowa, 2006b) (Figs. 8 and 9). MT-I+II hereby not only prevent neurodegeneration and cell death, but also improve brain tissue repair, including neuronal regrowth, scar formation, stem cell proliferation and angiogenesis following injury. This is demonstrated by the fact that TgMT mice show enhanced growth cone formation in surviving neurons situated outside the lesioned area (Penkowa, 2006a), and exogenous MT treatment increases synaptic plasticity, in comparison to saline-treated mice (Penkowa et al., 2006a). It is also supported by the findings that exogenous MT-II significantly increases the number of NSC in the SVZ, and inside the brain parenchyma, while MTKO mice display reduced number of NSCs compared with wild-type controls (Penkowa et al., 2006b). Accordingly, MT-I+II are likely activating stem/progenitor cell populations derived from the stem cell niches in the brain, opening exciting perspectives regarding the putative therapeutical use of these proteins. Thus, not only do endogenous MT-I+II prevent neurodegeneration and cell death, and improve brain tissue repair (including neuronal regrowth, scar formation, NSC proliferation/survival, and angiogenesis), but exogenous therapeutic MT-I+II administration has equivalent effect both in TBI models, as well as in other disease models, such as EAE, and without causing any visible side effects or toxicity (Penkowa, 2006b). In conclusion: in the past decades we have witnessed an enormous expansion in our understanding of events that determine the fate of brain cells following TBI. Cell death following trauma is mediated by a complex interplay of a number of pathophysiologically distinct mechanisms. Inhibiting secondary damage as well as stimulating the induction of endogenous protection and repair mechanisms deserves increased efforts to overcome the difficulties in translating positive pre-clinical results into effective clinical trial-outcomes. To achieve this, it is important to acknowledge the complexity and heterogeneity of TBI pathobiology and the important impact of age and co-morbidity. There will most likely be no ‘one size fits all’ approach for effective TBI treatment. However, some drugs may have a wider implication than other – and we believe that MT could be such a compound. The fact that MT is an endogenous protein, has a multi-target action, and that it can be administered exogenously with the same effect as the endogenous protein sets it apart from most other drugs tested in experimental TBI studies, and we therefore believe that it holds high potential for treatment of brain injuries.

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