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Neurodegeneration and neuroprotective strategies after traumatic brain injury
Vol. 2, No. 4 2005
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Nervous system MECHANISMS
Neurodegeneration and neuroprotective strategies after traumatic brain injury Asla Pitka¨nen1,2,*, Luca Longhi3, Niklas Marklund4, Diego M. Morales5, Tracy K. McIntosh3 1 A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Epilepsy Research Laboratory, Neulaniementie 2, PO Box 1627, FIN-70 211 Kuopio, Finland 2 Department of Neurology, Kuopio University Hospital, PO Box 1777, FIN-70 211 Kuopio, Finland 3 Neurosurgical Intensive Care Unit, Department of Anesthesia and Critical Care Medicine, Fondazione IRCCS, Ospedale Maggiore Policlinico, Regina Elena e Mangiagalli, Milano, Italy 4 Department of Neurosurgery, Uppsala University Hospital, SE 751 85 Uppsala, Sweden 5 Department of Clinical Care Medicine, University of Florence, Florence, Italy
The reorganization of neuronal circuits after traumatic brain injury (TBI) consists of several neurobiological alterations that are orchestrated in parallel and serial fashion. These include cellular death, axonal and den-
Section Editors: Andrey Mazarati – Department of Pediatrics, UCLA, USA Claude Wasterlain – Department of Cardiology, UCLA, USA
dritic plasticity, neurogenesis and gliogenesis, vascular alterations, axonal damage and remodelling of extracellular matrix and cellular membranes. Based on current knowledge, prevention or alleviation of neurodegeneration remains an attractive target for therapeutic attempts to improve the outcome following TBI. Here, we focus on the most recent studies that have advanced our understanding of the molecular mechanisms of TBI-induced neuronal death. These data provide candidate targets for design of novel therapies and for identification of biomarkers or surrogate markers for predicting the outcome or therapy response. Introduction It is estimated that traumatic brain injury (TBI) annually affects about 1.5 million people in the USA [1]. Of these, *Corresponding author: A. Pitka¨nen ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2005.11.011
230,000 people are hospitalized and survive, 80,000 people are estimated to be discharged from the hospital with some TBI-related disability and 50,000 people die. Mortality rates are in the range of 1% for minor injury, 18% for mild injury, and 48% for severe head injury. Approximately 44% of TBI are related to vehicle crashes and 25% to falls. Most studies indicate that males are far more likely to incur a TBI than females. An estimated 5.3 million people in the USA are living today with disability related to TBI, with an annual cost of US$48.3 billion. It is important to realize that TBI is a heterogeneous disorder and the molecular, pathologic and clinical consequences of TBI are multifaceted and complex and depend on the severity and type of injury, patient, treatment and the time point at which the consequences are assessed. Finally, the consequences of TBI can be categorized based on the timing of the appearance of damage (primary or secondary injury), type of impact or damage (contact or acceleration/deceleration), or distribution of damage (focal or diffuse/multifocal). www.drugdiscoverytoday.com
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Glossary DIABLO: direct inhibitor of apoptosis-binding protein with low pI, a murine analog of Smac. EndoG: endonuclease G is an apoptotic nuclease that translocates from mitochondria to nucleus upon apoptotic stimulus and degradates DNA. Granzyme B: serine protease that induces caspase activation. NCX: a Na+/Ca2+ exchanger, a transporter that exchanges three Na+ for one Ca2+. Omi (also known as HtrA2): is a mammalian homology of the procaryotic HtrA proteins. It is a proapoptotic mitochondrial serine protease that is involved in caspase-dependent as well as caspaseindependent cell death. It stimulates apoptosis by its protease activity in addition to its physical interaction with inhibitor of apoptosis protein (IAPs). Smac: second mitochondria-derived activator of caspase. Proapoptotic small protein (239 amino acids) that is located in the intermembranous space in mitochondria. After release it binds to X-linked inhibitor of apoptosis protein (XIAP). TRPM7: transient receptor potential melastatin, a bifunctional protein containing both channel and kinase properties regulating Ca2+ and Mg2+ homeostasis.
Timing and mechanisms of neurodegeneration following TBI Neuronal death in TBI is caused by direct mechanical forces to the brain as well as by consequent vascular, cellular and molecular alterations that result in acute and delayed neuronal death or injury [2–5]. Neurodegeneration can continue for months after TBI both in humans and in experimental models [4]. Importantly, the mechanisms underlying posttraumatic cell death can differ depending on the time delay from TBI (see subsequent sections). There is also evidence that genetic background, gender, age, type and severity of injury, metabolic state of the brain and other conditions (e.g. other diseases, medication) associated with TBI can affect the severity and/or mechanisms of neurodegeneration [6]. Morphologically, neuronal death can be divided into two major types: necrosis and apoptosis. The two categories provide some clues about the underlying mechanisms of neuronal death even though the usefulness of morphologic criteria as a starting point for understanding biochemical cascades has been recently challenged [7]. Electronmicroscopic morphology of necrosis consists of swollen cellular appearance with swollen mitochondria, vacuolated cytoplasm, dilation of endoplasmic reticulum (ER), pycnotic nuclei and plasma membrane rupture. Necrosis can be initiated by mechanical damage that leads to neuronal membrane failure, Ca2+ influx and disruption of ionic homeostatis which triggers the release of glutamate (Fig. 1) ([8]; see subsequent section). Disruptions of membrane potential can also be caused by TBI-associated ischemia or hypoglycemia leading to an inability to sustain plasma membrane potential resulting in membrane depolarization, glutamate release and overstimulation of postsynaptic N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5410
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methyl-4-isoxazolepropionic acid (AMPA) receptors (vide infra). These events result in high intracellular Ca2+ in postsynaptic neurons leading to oxidative stress, mitochondrial failure and activation of Ca2+-dependent proteases (e.g. calpains) that cause cytoskeletal destruction in postsynaptic neurons [9]. Other mechanisms leading to necrosis but not yet investigated in TBI include Ca2+ overload via reactive oxygen/nitrogen activated TRPM7 (see Glossary) channel and activation of acidosis sensitive Ca2+ permeable acid-sensing ion channels (ASICs) [10]. In addition, spillover of lysosomal cathepsins contributes to necrotic death. Inflammatory responses caused by the extracellular release of cytoplasmic components and/or breakdown of the blood–brain barrier is another factor that exposes adjacent neurons to deadly signals and secondary damage (e.g. activation of caspase 1). Necrotic appearance is most common for cells dying within minutes or few days after TBI. Morphologic features of apoptosis include nuclear and cytoplasmic condensation, internucleosomal DNA cleavage and packing of the cell into apoptotic bodies that are engulfed by phagocytes, preventing release of intracellular components. Apoptotic neurodegeneration can occur via caspasedependent or caspase-independent pathways, as summarized in Table 1 and Fig. 1. Evidence from humans and experimental models shows that neurons (as well as glial cells) with apoptotic morphology can be found several months after TBI. A large number of studies have focused on a mitochondriamediated pathway that is triggered by the release of cytochrome c and the formation of an apoptosome, in which procaspase 9 binds to cytochrome c and apoptosis protease activation factor-1 (Apaf-1) leading to formation of active caspase 9 and activation of the executioner caspase 3 in an ATP-dependent manner. Substrates for caspase 3 include cytoskeletal proteins such as spectrin, DNA-repairing enzymes like poly(ADP-ribose)polymerase (PARP), cell-cycle proteins and enzymes involved in signal transduction. Existing data suggest that the mitochondria-mediated pathway is activated both in experimental and human TBI. Expression and activation of various components of the pathway are most prominent during the first 3 days following TBI [5]. Death receptor pathways are activated by the binding of ligands (e.g. TNF-a or FasL) to death receptors (TNFR-1 and Fas, respectively) that are members of the tumor necrosis factor receptor (TNFR) superfamily. This triggers a series of downstream cascades (c-Jun-N-terminal kinase) that lead to activation of caspases [5]. Much less is known concerning the two other pathways leading to caspase activation: the endoplasmatic reticulum (ER)-mediated pathway and the GRANZYME B (see Glossary) mediated pathway, which have been shown to play a role in some other neurodegenerative diseases [10]. Neurons can also show caspase-independent apoptotic features, and in some cases co-activation of several parallel death pathways is required for apoptotic cell death, including
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Figure 1. Summary of crosstalk between various pathways leading to cell death. Intracellular Ca2+ overload is a central mechanism that can initiate both necrotic and apoptotic pathways. Ca2+can enter the postsynaptic cell via several channels (AMPA, NMDA, ASIC, TRPM7). Elevation in intracellular Ca2+results in calpain activation that can cleave NMDA and NCX channels. This results in impairment of Ca2+extrusion via NCX channels and further elevation of intracellular Ca2+. Calpains, cathepsins, and caspases are the major enzymes responsible for degradation of various cellular components. Calpains are activated by Ca2+. Calpains can activate cathepsins that might also become activated as a consequence of lysosomal damage. Interestingly, these two phenomena can occur independently. Elevated intracellular calcium as well as activation of calpains can result in activation of caspase pathways via mitochondria. Calpains can also directly activate caspases. In addition to mitochondrial pathways, caspases can be activated by death receptors (TNFR, Fas) located in cell membranes (data collected from Refs [7,8,42,43]).
activation of calpains and cathepsins [7]. Upregulation or downregulation of proteins that control, either directly or indirectly, the consequences of caspase activity has been studied in TBI (Table 1). For example, inhibitor of apoptosis protein (IAP) or survival-promoting proteins (Bcl-2, Bcl-xL) has been shown to be upregulated in a fluid-percussion injury (FPI) model of TBI (Table 1). Otherwise, other molecules that act at the interface between caspase-mediated and nonmediated programmed cell death (e.g. Bid) are also affected by TBI. Little is known, however, about proteins other than cytochrome c released from mitochondria, including apoptosis inducing factor (AIF), SMAC/DIABLO and Omi/HtrA2 (both act as IAP inhibitors, see Glossary) or EndoG (see Glossary). Future studies will also show whether the other factors listed in Table 1, and known to regulate programmed cell death, form candidate targets for therapeutic attempts in TBI.
Neuroprotection in TBI – pharmacological strategies To date, more than 100 pharmacological studies have been performed in the lateral FPI model alone and a detailed overview of each study and compound evaluated is beyond the scope of the present review. The most important treatment options from a clinical perspective are summarized in the following sections (see also Table 2).
Attenuation of excitotoxicity Numerous reports have evaluated attenuation of glutamatemediated excitotoxicity for the treatment of experimental TBI, including the inhibition of the two major classes of glutamate receptors, ionotropic (NMDA, KA and AMPA) and metabotropic (Groups I–III) receptors, coupled to intracellular second messengers. In experimental TBI, many reports have targeted the NMDA receptor (Table 2), where www.drugdiscoverytoday.com
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Table 1. Summary of cell death pathways in human and experimental traumatic brain injury (TBI) Pathway
Human TBI change
Rat TBI FPIa change
Caspase-dependent cell death Mitochondria-mediated pathway Cytochrome c Apaf-1d Caspase-9 (initiator caspase) Caspase-3 (effector caspase) Caspase-6 (effector caspase) Caspase-7 (effector caspase) Death receptor-mediated pathway TNFg-a TNFRh-1 FasLi Fas Caspase-8 (initiator caspase) Caspase-10 (initiator caspase)
Refs CCIb change
c
nde nd nd f
[44–46] [47] [48–50] [49–52]
nd
nd nd
nd
nd [53,54] [55–57] [58] [59–61] [58,60–63] [49,50,58,60,64]
nd nd 0j
nd
nd
ER -mediated pathway Caspase-12
nd
nd
Granzyme B-mediated pathway Granzyme B Perforin
nd nd
nd nd
nd
k
[65] nd nd
Caspase-independent cell death Calpains Cathepsins Cathepsin B Cathepsin H Cathepsin L Other factors regulating cell death Mitochondrial proteins Smac/Diablol Omi/HtrmA2 EndoGn AIFo
[50,66–68] nd nd nd
nd
nd nd nd nd
0 nd nd nd
Bcl family proteins controlling mitochondria Bcl-2 Bcl-XL nd Bax Bak nd Bid nd Bim nd IAPp
[69]
nd nd nd
[70]
[69]
[71] [45,52,72–74] [74] [52,74]
nd nd nd
nd
PARPq
nd [50,75] nd nd
[49,70]
nd
Caspase 1 a
FPI: fluid percussion injury. b CCI: controlled cortical impact. c : increase. d Apaf-1: apoptotic protease activating factor. e nd: no data available. f : decrease. g TNF: tumor necrosis factor. h TNFR: tumor necrosis factor receptor. i FasL: Fas ligand. j 0: no change. k ER: endoplasmic reticulum. l Smac/Diablo: a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. m Omi/Htr: a proapoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent cell death. n EndoG: endonuclease G. o AIF: apoptosis-inducing factor. p IAP: inhibitor of apoptosis proteins. q PARP: poly(ADP-ribose) polymerase.
412
nd nd nd
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[76,77] q
0
[46,78,79]
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Table 2. Targets of neuroprotection in traumatic brain injury Pharmacological target
Examples of compounds
Refs
EAAa modulation NMDAb AMPAg/KAh Metabotropic EAA release inhibition
Mg2+, CPI 101-606c, MK-801d, HU-211e, Memantinef RPR117824i, YM872j, NBQXk MCPGl, AIDAm, CPCCOetn, LY-367385o, MPEPp, DCG-IVq, LY354740r BW1003C87s, 619C89t, Riluzoleu
[12]
ROSv scavenging
PBNw, S-PBNx, Vitamin E, L-NAMEy, CDPCz, PEG-SODaa
[80]
ab
ac
Calcium-mediated damage
LOE908 , SNX-111
Modulators of inflammation
IA29ad, Ibuprofen, HU-211, CP-0127ae
Miscellaneous
af
[81]
ag
GPI 6150 , CsA , CCPA , Lactate ai
aj
Neurotrophic factors
NGF , GDNF , BDNF
Z-VAD-fmkal, Z-DEVD-fmkam an
TRH
[81]
ak
Inhibitors of apoptosis Endocrinology
[18]
ah
ao
[23] [5] ap
analogs, IGF-1 , DHEAS , progesterone
[81]
a
EAA: excitatory amino acid. b NMDA: N-methyl-D-aspartate. c CPI 101-606: (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol. d MK-801: dizocilpine maleate. e HU-211: (+)-(3S,4S)-7-hydroxy-D-6 tetrahydro-cannabinol 1,1-dimethylheptyl. f Memantine: 3,5-dimethyl-1-adamantanamine. g AMPA: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. h KA: kainate receptors 1 and 2. i RPR117824: 9-carboxymethyl-imidazo-[1-2a]indenol[1-2e]. j YM872: zonampanel monohydrate. k NBQX: 6-nitro-7-sulfamoylbenzo (F) quinoxaline-2,3-dione. l MCPG: (S)-a-4-caboxyphenylglycine. m AIDA: (RS)-1-aminoindan-1,5-dicarboxylic acid. n CPCCOet; 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester. o LY367385: (S)-(+)-a-amino-4-carboxy-2-methylbezeneacetic acid. p MPEP: 2-methyl-6-(phenylethynyl)-pyridine. q DCG-IV: 2, (20 ,30 )-dicarboxycyclopropylglycin. r LY354740: (1S,2S,5R,6S)-(+)-2-aminobicyclo[3.1.0]hexane -2,6-dicarboxylic acid. s BW1003C87: 5-(2,3,5-trichlorophenyl) pyrimidine 2.4-diamine ethane sulfonate. t 619C89: [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-trichlrophenyl)pyrimidine mesylate monohydrate]. u Riluzole: 2-amino-6-trifluro methoxy benzthiazole. v ROS: reactive oxygen species. w PBN: a-phenyl-N-tert-butyl-nitrone. x S-PBN: sodium 2-sulfophenyl-N-tert-butyl nitrone. y L-NAME: nitro-L-arginine methyl ester. z CDPC: cytidine 50 -diphosphocholine. aa PEG-SOD: polyethylene glycol-conjugated superoxide dismutase. ab LOE908: (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide. ac SNX-111: Ziconotide. ad IA29: anti-ICAM-1 monoclonal antibody. ae CP-0127: Bradycor or deltibant, bissuccimidohexane (L-Cys6)-1. af GPI 6150: (1,11b-dihydro-[2H]bezopyrano[4,3,2-de]isoquinolin-3-one. ag CsA: cyclosporin A. ah CCPA: 2-chloro-N(6)-cyclopentyladenosine. ai NGF: nerve growth factor. aj GDNF: glial cell-derived neurotrophic factor. ak BDNF: brain-derived neurotrophic factor. al Z-VAD-fmk: acetyl-Tyr-Val-Ala-Asp-chloromethyl-ketone. am Z-DEVD-fmk: N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone. an TRH: thyrotropin-releasing hormone. ao IGF-1: insulin-like growth factor-1. ap DHEAS: dehydroepiandrosterone sulfate.
earlier compounds such as MK-801, phencyclidine (PCP) and ketamine showed strong psychomimetic effects, these adverse effects were diminished in later generations of NMDA receptor antagonists such as NPS 1506 and NPS 846. To date, the NMDA antagonists magnesium, HU-211 (dexanabinol), memantine and CP-101,606 have generated clinical interest owing to the improved functional and histological outcome
in several clinically relevant TBI models and these compounds are all in the clinical trial phase [11]. In addition to antagonism of excitatory amino acid (EAA) receptor function, modulation of EAA receptor activity might also be accomplished by inhibition of EAA release. Examples of glutamate release inhibitors with neuroprotective action in TBI include the compound riluzole which is in a Phase III www.drugdiscoverytoday.com
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clinical trial for TBI in Europe. Despite promising preclinical documentation, all glutamate blockers evaluated, to date, have shown to be ineffective in Phase III clinical trials [12] (Table 2).
Calcium channel blockage and calpain inhibition The Ca2+ blockers (S)-emopamil, LOE 908, S100B, BMS204352 and SNX-111, have all been shown to attenuate neurological motor and cognitive deficits following experimental TBI [11]. However, a clinical trial using SNX-111 after TBI was suspended owing to higher mortality in SNX-111treated patients than in patients receiving placebo. Much clinical interest, to date, has focused on the Ca2+-channel blocker nimodipine that improved outcome in a randomized study of TBI patients. However, a systemic review of randomized controlled trials of calcium channel blockers in acute TBI patients shows that considerable uncertainty remains over their efficacy [13]. Downstream inhibition of calpains (Ca2+-dependent proteases) might be of therapeutic value; therefore, the calpain inhibitor AK295, infused intra-arterially, was shown to reduce both motor and cognitive deficits one week after lateral FPI in the rat although without effect on cortical lesion size or apoptotic cell death. In a model of traumatic axonal injury in the rat, the calpain inhibitor MDL-28170 reduced the damage of brainstem fiber tracts and further investigation into the efficacy of calpain inhibition following TBI is warranted.
Reactive oxygen and nitrogen scavengers Although tirilazad mesylate (TM) and its related pyrrolopyrimidines, including U-101033E, showed promising effects in a series of TBI studies in mice, rats and cats [11], Phase III clinical trials for ischemia, spinal cord injury, TBI and subarachnoid hemorrhage all failed to show a positive outcome [14]. Inhibition of lipid peroxidation and/or attenuation of hydroxyl radicals is of clinical interest and further testing is necessary to evaluate these compounds using longer and clinically relevant time windows. Cytidine 50 -diphosphocholine (CDPC), or citicoline, compounds that attenuate the activation of phosholipase A2, have been shown to be neuroprotective and possess neurobehavioral efficacy in experimental TBI models [15]. In a randomized study enrolling 216 TBI patients, CDPC improved the motor and cognitive outcome, implying a potential role for CDPC in the treatment of human TBI.
Superoxide scavenging Administration of the antioxidant enzyme superoxide dismutase (SOD) conjugated to enhance blood–brain barrier penetration (PEG-SOD, pegogortein) improved survival and neurological recovery, attenuated cerebral edema and decreased hippocampal cell loss across TBI models in rats 414
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although conflicting results exist (see Table 2). Unfortunately, PEG-SOD failed in the randomized, Phase III multicenter trial, which included 463 patients. The reasons for this failure are probably multifactorial but might be related to the inclusion of many severely brain-injured patients, the extended time window chosen (8 h) and the chosen outcome measures in addition to the possibility of inadequate dosing or lack of sufficient efficacy by the compound [16].
Inflammation and anti-inflammatory compounds The nonselective cyclooxygenase (COX) inhibitors ibuprofen and indomethacin and the selective COX-2 inhibitor nimesulide have all been shown to improve some of the posttraumatic deficits including changes in intracranial pressure and cerebral blood flow and impairment of cognitive and motor function [17]. Cytokines, including tumor necrosis factor (TNF) and interleukins (IL), are increased in human and experimental TBI [18]. Improved outcome following experimental TBI with compounds (e.g. pentoxyfylline, TNF-a binding protein, HU-211 and soluble TNF-a receptor fusion protein) that block the action of TNF-a has been reported [19]. The role of TNF-a (and IL-6) in the treatment of TBI remains elusive because these cytokines have been reported to possess both neuroprotective and neurotoxic properties. Glucocorticoids (GCs) were introduced in the early 1960s because they were found to markedly reduce edema from brain tumors and attenuate ROS-induced lipid peroxidation and reduce inflammation. Although GCs might attenuate delayed edema and inflammation following TBI in the rat, excess GCs might be neurotoxic and the GC receptor inhibitor mifepristone (RU 486) attenuated hippocampal cell death following controlled cortical impact (CCI) injury in the rat. High-dose GC treatment received widespread use in TBI patients, but because of the publication of the large, controlled, randomized, multi-center corticosteroid randomization after significant head injury (CRASH) trial where a worse outcome was observed in patients receiving GCs [20], the use of steroids in the treatment of TBI is generally not recommended.
Pharmacological inhibition of caspases and pro-apoptotic cascades One strategy to prevent acute cell death after TBI might be to inhibit caspases, enzymes involved in the process leading to apoptosis. Several caspase inhibitors of varying specificity have been successfully evaluated in models of experimental TBI including ketones such as the pan-caspase inhibitor zVAD-fmk, the caspase-1 specific inhibitor acetyl-Tyr-Val-AlaAsp-chloromethyl ketone and the caspase-3-specific z-DEVDfmk [5]. However, the role for anti-apoptotic compounds in TBI is controversial and, to date, more preclinical research is necessary to evaluate their clinical potential.
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Several studies have demonstrated that exogenously administered progesterone or its metabolite allepregnanolone reduced post-traumatic increase in cerebral edema, improved cognitive performance and functional deficits, reduced lesion volume and apoptotic cell death following TBI in the rat [21]. Currently, there is an ongoing National Institute of Neurological Disorders and Stroke (NINDS)sponsored single-center, controlled clinical trial of the efficacy of intravenous administration of progesterone in human TBI.
Neurotrophic factors Four major classes of neurotrophins have been characterized in mammals: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4/5) plus additional non-neuronal trophic factors such as basic fibrillary growth factor (bFGF), glial derived neurotrophic factor (GDNF), insulin-like growth factor (IGF), ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) [22,23]. Neurotrophins mediate several types of intercellular communication and have been shown to act as (i) retrogradely transported, target-derived factors that influence afferent neurons; (ii) locally released paracrine factors affecting both neurons and non-neuronal cells; (iii) autocrine factors acting on the same cells that produce and release it and (iv) as endocrine factors that are transported through the blood stream [24]. Administration of trophic factors has been evaluated as a neuroprotective strategy in TBI. Dietrich et al. [25] reported that acute administration of bFGF attenuated cortical cell loss following lateral FPI in rats, whereas McDermott et al. [26], using the same model, demonstrated that delayed intraparenchymal administration of bFGF, beginning 24 h after injury, significantly improved post-traumatic cognitive deficits in the rat. Post-traumatic BDNF infusion [into BDNF (/) mice following CCI brain injured and into rats following lateral FPI] did not attenuate behavioral and histological damage, suggesting that BDNF does not provide a trophic support in the traumatically injured brain [27]. Intracerebroventricular (i.c.v.) administration of GDNF following CCI brain injury in rats decreased the post-traumatic neuronal loss in the hippocampus at 1 week postinjury [28], while i.c.v. administration of NGF attenuates cognitive deficits and cholinergic cell loss in the forebrain following FPI and CCI brain injury in rats [29]. An optimal delivery system for neurotrophins (using viral vectors or genetically modified cells) should be the object of investigation to ensure a constant release of trophic factors to a targeted area without the adverse effects related to systemic and/or intracerebroventricular administration. To this end, Longhi et al. [30] observed that ex vivo gene therapy transplanting the human neuroteratocarcinoma-derived neuronal cells (NT2N) transduced with a lentiviral vector to release NGF into the basal
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forebrain of mice at 24 h postinjury attenuated the cognitive deficits at 1 month postinjury, suggesting that gene therapy (to deliver trophic factor) could be a promising therapeutic strategy following TBI.
Neuroprotection in TBI – cell-based therapy Neuronal replacement might be a viable therapeutic approach to replace the lost tissue/cells following TBI. Several cell lines have been evaluated over the past decade with positive results in terms of survival and integration of the transplant into the injured brain and in terms of attenuation of the post-traumatic behavioral sequelae and histological damage (Table 3) [31]. Early studies reported that fetal cortical tissue that was transplanted 24 h postinjury (alone or coupled with NGF infusion) survived, integrated into the injured brain and attenuated behavioral deficits and histological damage including hippocampal cell loss [32–34]. Human NT2N cells have been observed to (safely) survive and integrate into the injured brain when transplanted up to 1 month postinjury, however, they were not associated with attenuation of post-traumatic behavioral and histological damage [35]. Using a different approach involving bone marrow stromal cells, intraparenchymal transplantation of whole bone marrow into the pericontusional tissue at 24 h after CCI brain injury in rats resulted in improved functional outcome and differentiation of transplanted cells into cells expressing neuronal and glial markers [36]. In addition, bone marrow-derived cells administered at 24 h postinjury were subsequently observed in the pericontusional area of the brain expressing both neuronal and glial markers and were associated with improved functional outcome, increased expression of NGF and BDNF and increased endogenous cellular proliferation [37,38]. Stem cells have the property of unlimited expansion in cultures and differentiation in different cellular lineage. Several lines of stem/progenitor cells have been transplanted in experimental models of TBI and have been associated with reduced behavioral deficits (up to 1 year postinjury) and histological damage [31]. In addition cells transplanted into the hemisphere contralaterally to the injury migrated across the corpus callosum towards the site of injury [39], suggesting that the environment associated with TBI is able to modulate the migratory patterns of engrafted cells. The tropism of stem cells for pathology in combination with the ability to genetically engineer them might provide a platform for delivery of therapeutic genes into target areas of the injured brain. However, even if neural transplantation has been successful in experimental TBI, further work needs to be performed to better understand the mechanisms associated with the behavioral and histological outcome. In addition, the clinical application of cell replacement strategy following TBI, to date, remains speculative. www.drugdiscoverytoday.com
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Table 3. Summary of brain transplantation studies following TBI Source
Model
Main results of the study
Refs
Fetal tissue
FPIa
Graft survival when transplanted days 2–14 PIb c CA3 cell loss at 4 weeks PI
[32]
Fetal tissue + NGFd
FPI
NT2Nf cells
FPI
NT2N cells + NGF
CCIh
Cognitive deficits at 4 weeks
[30]
HiB5 + NGF
FPI
Cognitive deficits Motor recovery CA3 cell loss at 1 week PI
[82]
MHP36j
FPI
Cognitive deficits at 4 months PI
[83]
C17.2
CCI
Motor recovery over 12 weeks PI
[84]
Pre-differentiated stem cells into neuronal and glial precursors
CCI
Motor recovery over 4 weeks PI Lesion volume at 40 days PI
[85]
Progenitor cells
CCI
Cognitive deficits Motor recovery over 1 year PI Migration toward injury at 14 months PI
[86]
C17.2 + EGFRl vIII receptor
FPI
Migration toward injury at 2 weeks PI
[39]
Bone marrow stromal cells
CCI
Umbilical cord blood
CCI
i
k
e
Cognitive deficits Motor recovery over 2 weeks PI
[34]
Graft survival up to 16 weeks PI g Motor recovery over 16 weeks PI
[35]
Motor recovery NGF and BDNFm expression Endogenous cellular proliferation at 2 weeks PI Migration toward injury Motor recovery over 4 weeks PI
[37,38]
[87]
a
FPI: fluid percussion brain injury. PI: postinjury. c : increase. d NGF: nerve growth factor. e : decrease. f NT2N: neuroteratocarcinoma-derived neuronal cells. g : unaffected. h CCI: controlled cortical impact brain injury. i HiB5: conditionally immortalized progenitor cell derived from an embryonic (E16) rat hippocampus. j MHP: fibroblast growth factor 2-responsive Maudsley hippocampal cell line clone 36. k C17.2: clonal multipotent progenitor cell derived from the external germinal layer of the neonatal murine cerebellum. l EGFR: epidermal growth factor receptor. m BDNF: brain derived neurotrophic factor. b
Conclusions and future challenges Understanding the details of molecular cascades underlying neurodegeneration after TBI remains a significant challenge. The complexity of the task is multiplied by the fact that not only neurons but also astrocytes, microglia and oligodendrocytes can undergo degeneration in the traumatized brain. Death of non-neuronal cells can compromise, for example, trophic support for neurons or re-myelinization of damaged axons. Understanding the mechanisms of survival and death of newly born cells after TBI is still in infancy. More data are also needed to understand the mechanisms of degeneration of transplanted cells to optimize the benefits of cell therapies (vide infra and 31). TBI can also cause axonal injury affecting the survival of somata by mechanisms that differ from that of somatic injury [8]. Recent data from single cell profiling studies have added a new aspect to the complexity of factors that eventually determine the fate and death mechanisms of 416
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individual neurons [88]. As shown by Hellmich et al. [40], expression level of survival promoting factors in adjacent neurons, some of which die and some survive, can vary tremendously at the time of TBI. Whether this is related, for example, to the metabolic state of the cell remains to be investigated [41]. Interestingly, recent observations in patients suggest that compromised postinjury metabolic state is associated with poor outcome [8]. To date, in most reports evaluating pharmacological compounds for the treatment of TBI, a single compound has been used with the hope that this compound (the magic bullet) will attenuate all or most of the deficits associated with the injury. However, owing to the heterogeneity and the complex pathophysiology of TBI, it might seem more probable that several different pharmacological strategies (a ‘cocktail’) will be required to ameliorate post-traumatic deficits. However, combination therapy has been inadequately studied in
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TBI and several reports have also shown decreased efficacy of neuroprotective compounds when used in combination with other compounds. More research into the effects of drug interaction and polypharmacy in TBI is needed. Finally, it is apparent that a wide range of candidate mechanism has not yet been tested. Moreover, it is probable that many mechanisms remain to be discovered. The beneficial effects of various treatments in experimental models, even though not yet translated to clinical practice, are encouraging, and certainly maintain the interest in the search of novel, more efficient, less toxic and easier to administer compounds.
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Acknowledgements
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Supported by the Academy of Finland, the Sigrid Juselius Foundation, the Finnish Cultural Foundation and the Paulo Foundation to AP; NIH NS08803 and NS40978 to TKM; Swedish Brain Foundation to NM.
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References 1 Centers for Disease Control and Prevention (2001) Traumatic Brain Injury in the United States: A Report to Congress. http://www.cdc.gov/ncipc/pub-res/ TBI_in_US_04/TBI_ED.htm 2 Pitka¨nen, A. and McIntosh, T.K. Animal models of post-traumatic epilepsy. J. Neurotrauma (in press) 3 Laurer, H.L. and McIntosh, T.K. (2001) Pharmacologic therapy in traumatic brain injury: update on experimental treatment strategies. Curr. Pharm. Des. 7, 1505–1516 4 Royo, N.C. et al. (2003) From cell death to neuronal regeneration: building a new brain after traumatic brain injury. J. Neuropathol. Exp. Neurol. 62, 801–811 5 Raghupathi, R. (2004) Cell death mechanisms following traumatic brain injury. Brain Pathol. 14, 215–222 6 Thompson, H.J. et al. (2005) Lateral fluid percussion brain injury: a 15-year review and evaluation. J. Neurotrauma 22, 42–75 7 Artal-Sanz, M. and Tavernarakis, N. (2005) Proteolytic mechanisms in necrotic cell death and neurodegeneration. FEBS Lett. 579, 3287–3296 8 Povlishock, J.T. and Katz, D.I. (2005) Update of neuropathology and neurological recovery after traumatic brain injury. J. Head Trauma Rehabil. 20, 76–94 9 Saatman, K.E. et al. (2000) Behavioral efficacy of posttraumatic calpain inhibition is not accompanied by reduced spectrin proteolysis, cortical lesion, or apoptosis. J. Cereb. Blood Flow Metab. 20, 66–73 10 Wang, Z-B. et al. (2005) Pathways to caspase activation. Cell Biol. Int. 29, 489–496 11 Morales, D.M. et al. Experimental models of traumatic brain injury: do we really need to build a better mousetrap? Neuroscience 2005 Oct 19 [Epub ahead of print] 12 Marklund, N. et al. (2004) Excitotoxicity and traumatic brain injury. Pathology, treatment approaches and controversies. In Excitotoxicity in Neurological Diseases (Ferrarese, C and Beal, M.F., eds), pp. 254–271, Kluwer Academic Publishers 13 Langham, J. et al. (2003) Calcium channel blockers for acute traumatic brain injury. Cochrane Database Syst. Rev. 4, CD000565 14 Roberts, I. et al. (2000) Aminosteroids for acute traumatic brain injury. Cochrane Database Syst. Rev. 4, CD001527 15 Dempsey, R.J. and Raghavendra, R.V. (2003) Cytidinediphosphocholine treatment to decrease traumatic brain injury-induced hippocampal neuronal death, cortical contusion volume, and neurological dysfunction in rats. J. Neurosurg. 98, 867–873 16 Young, B. et al. (1996) Effects of pegorgotein on neurologic outcome of patients with severe head injury. A multicenter, randomized controlled trial. JAMA 276, 538–543
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Larner, S.F. et al. (2004) increased expression and processing of caspase-12 after traumatic brain injury in rats. J. Neurochem. 88, 78–90 McCracken, E. et al. (1999) Calpain activation and cytoskeletal protein breakdown in the corpus callosum of head-injured patients. J. Neurotrauma 16, 749–761 Farkas, O. et al. (2005) Spectrin breakdown products in the cerebrospinal fluid in severe head injury – preliminary observations. Acta Neurochir. 147, 855–861 Saatman, K.E. et al. (1996) Prolonged calpain-mediated spectrin breakdown occurs regionally following experimental brain injury in the rat. J. Neuropathol. Exp. Neurol. 55, 850–860 Natale, J.E. et al. (2003) Gene expression profile changes are commonly modulated across models and species after traumatic brain injury. J. Neurotrauma 20, 907–927 Lotocki, G. et al. (2003) Monoubiquitination and cellular distribution of XIAP in neurons after traumatic brain injury. J. Cereb. Blood Flow Metab. 23, 1129–1136 Zhang, X. et al. (2002) Intranuclear localization of apoptosis-inducing factor (AIF) and large scale DNA fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite. J. Neurochem. 82, 181–191 Nathoo, N. (2004) Influence of apoptosis on neurological outcome following traumatic cerebral contusion. J. Neurosurg. 101, 233–240 Shimamura, M. et al. (2005) Analysis of long-term gene expression in neurons of the hippocampal subfields following traumatic brain injury in rats. Neuroscience 131, 87–97 Strauss, K. (2004) Common patterns of Bcl-2 family gene expression in two traumatic brain injury models. Neurotoxicity Res. 6, 333–342 Franz, G. et al. (2002) Temporal and spatial profile of Bid cleavage after experimental traumatic brain injury. J. Cereb. Blood Flow Metab. 22, 951– 958 Ang, B.T. et al. (2003) Poly(adenosine diphosphate-ribose) polymerase expression in human traumatic brain injury. J. Neurosurg. 99, 125–130 Besson, V.C. et al. (2005) Beneficial effect of PJ34 and INO-1001, two novel water-soluble poly(ADP-ribose) polymerase inhibitors, on the consequences of traumatic brain injury in rat. Brain Res. 1041, 149–156 Clark, R.S.B. et al. (1999) Increases in Bcl-2 and cleavage of caspase-1 and caspase-3 in human brain after head injury. FASEB J. 13, 813–821 Yakovlev, A.G. et al. (1997) Activation of CPP32-like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury. J. Neurosci. 17, 7415–7424 Lewen, A. et al. (2000) Free radical pathways in CNS injury. J. Neurotrauma 17, 871–890 Marklund, N. et al. Evaluation of pharmacological treatment strategies in traumatic brain injury. Curr Pharm Design (in press) Philips, M.F. et al. (2001) Neuroprotective and behavioral efficacy of nerve growth factor-transfected hippocampal progenitor cell transplants after experimental traumatic brain injury. J. Neurosurg. 94, 765–774 Lenzlinger, P.M. et al. (2002) Early transplantation of immortalized hippocampal progenitorcells (MHP36) following experimental traumatic brain injury in rats improves motor and cognitive function at 4 months postinjury. Exp. Neurol. 175, 421–422 Riess, P. et al. (2002) Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury. Neurosurgery 51, 1043–1052 Hoane, M.R. et al. (2004) Transplantation of neuronal and glial precursors dramatically improves sensorimotor function but not cognitive function in the traumatically injured brain. J. Neurotrauma 21, 163–174 Shear, D.A. et al. (2004) Neural progenitor cell transplants promote longterm functional recovery after traumatic brain injury. Brain Res. 1026, 1– 22 Lu, D. et al. (2002) Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant. 11, 275–281 Davis, J.E. et al. (2004) Methodological considerations regarding singlecell gene expression profiling for brain injury. Neurochem. Res. 29, 1113– 1121
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Nervous system MECHANISMS
Neurodegeneration and neuroprotective strategies after traumatic brain injury Asla Pitka¨nen1,2,*, Luca Longhi3, Niklas Marklund4, Diego M. Morales5, Tracy K. McIntosh3 1 A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Epilepsy Research Laboratory, Neulaniementie 2, PO Box 1627, FIN-70 211 Kuopio, Finland 2 Department of Neurology, Kuopio University Hospital, PO Box 1777, FIN-70 211 Kuopio, Finland 3 Neurosurgical Intensive Care Unit, Department of Anesthesia and Critical Care Medicine, Fondazione IRCCS, Ospedale Maggiore Policlinico, Regina Elena e Mangiagalli, Milano, Italy 4 Department of Neurosurgery, Uppsala University Hospital, SE 751 85 Uppsala, Sweden 5 Department of Clinical Care Medicine, University of Florence, Florence, Italy
The reorganization of neuronal circuits after traumatic brain injury (TBI) consists of several neurobiological alterations that are orchestrated in parallel and serial fashion. These include cellular death, axonal and den-
Section Editors: Andrey Mazarati – Department of Pediatrics, UCLA, USA Claude Wasterlain – Department of Cardiology, UCLA, USA
dritic plasticity, neurogenesis and gliogenesis, vascular alterations, axonal damage and remodelling of extracellular matrix and cellular membranes. Based on current knowledge, prevention or alleviation of neurodegeneration remains an attractive target for therapeutic attempts to improve the outcome following TBI. Here, we focus on the most recent studies that have advanced our understanding of the molecular mechanisms of TBI-induced neuronal death. These data provide candidate targets for design of novel therapies and for identification of biomarkers or surrogate markers for predicting the outcome or therapy response. Introduction It is estimated that traumatic brain injury (TBI) annually affects about 1.5 million people in the USA [1]. Of these, *Corresponding author: A. Pitka¨nen ([email protected]) 1740-6765/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2005.11.011
230,000 people are hospitalized and survive, 80,000 people are estimated to be discharged from the hospital with some TBI-related disability and 50,000 people die. Mortality rates are in the range of 1% for minor injury, 18% for mild injury, and 48% for severe head injury. Approximately 44% of TBI are related to vehicle crashes and 25% to falls. Most studies indicate that males are far more likely to incur a TBI than females. An estimated 5.3 million people in the USA are living today with disability related to TBI, with an annual cost of US$48.3 billion. It is important to realize that TBI is a heterogeneous disorder and the molecular, pathologic and clinical consequences of TBI are multifaceted and complex and depend on the severity and type of injury, patient, treatment and the time point at which the consequences are assessed. Finally, the consequences of TBI can be categorized based on the timing of the appearance of damage (primary or secondary injury), type of impact or damage (contact or acceleration/deceleration), or distribution of damage (focal or diffuse/multifocal). www.drugdiscoverytoday.com
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Glossary DIABLO: direct inhibitor of apoptosis-binding protein with low pI, a murine analog of Smac. EndoG: endonuclease G is an apoptotic nuclease that translocates from mitochondria to nucleus upon apoptotic stimulus and degradates DNA. Granzyme B: serine protease that induces caspase activation. NCX: a Na+/Ca2+ exchanger, a transporter that exchanges three Na+ for one Ca2+. Omi (also known as HtrA2): is a mammalian homology of the procaryotic HtrA proteins. It is a proapoptotic mitochondrial serine protease that is involved in caspase-dependent as well as caspaseindependent cell death. It stimulates apoptosis by its protease activity in addition to its physical interaction with inhibitor of apoptosis protein (IAPs). Smac: second mitochondria-derived activator of caspase. Proapoptotic small protein (239 amino acids) that is located in the intermembranous space in mitochondria. After release it binds to X-linked inhibitor of apoptosis protein (XIAP). TRPM7: transient receptor potential melastatin, a bifunctional protein containing both channel and kinase properties regulating Ca2+ and Mg2+ homeostasis.
Timing and mechanisms of neurodegeneration following TBI Neuronal death in TBI is caused by direct mechanical forces to the brain as well as by consequent vascular, cellular and molecular alterations that result in acute and delayed neuronal death or injury [2–5]. Neurodegeneration can continue for months after TBI both in humans and in experimental models [4]. Importantly, the mechanisms underlying posttraumatic cell death can differ depending on the time delay from TBI (see subsequent sections). There is also evidence that genetic background, gender, age, type and severity of injury, metabolic state of the brain and other conditions (e.g. other diseases, medication) associated with TBI can affect the severity and/or mechanisms of neurodegeneration [6]. Morphologically, neuronal death can be divided into two major types: necrosis and apoptosis. The two categories provide some clues about the underlying mechanisms of neuronal death even though the usefulness of morphologic criteria as a starting point for understanding biochemical cascades has been recently challenged [7]. Electronmicroscopic morphology of necrosis consists of swollen cellular appearance with swollen mitochondria, vacuolated cytoplasm, dilation of endoplasmic reticulum (ER), pycnotic nuclei and plasma membrane rupture. Necrosis can be initiated by mechanical damage that leads to neuronal membrane failure, Ca2+ influx and disruption of ionic homeostatis which triggers the release of glutamate (Fig. 1) ([8]; see subsequent section). Disruptions of membrane potential can also be caused by TBI-associated ischemia or hypoglycemia leading to an inability to sustain plasma membrane potential resulting in membrane depolarization, glutamate release and overstimulation of postsynaptic N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5410
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methyl-4-isoxazolepropionic acid (AMPA) receptors (vide infra). These events result in high intracellular Ca2+ in postsynaptic neurons leading to oxidative stress, mitochondrial failure and activation of Ca2+-dependent proteases (e.g. calpains) that cause cytoskeletal destruction in postsynaptic neurons [9]. Other mechanisms leading to necrosis but not yet investigated in TBI include Ca2+ overload via reactive oxygen/nitrogen activated TRPM7 (see Glossary) channel and activation of acidosis sensitive Ca2+ permeable acid-sensing ion channels (ASICs) [10]. In addition, spillover of lysosomal cathepsins contributes to necrotic death. Inflammatory responses caused by the extracellular release of cytoplasmic components and/or breakdown of the blood–brain barrier is another factor that exposes adjacent neurons to deadly signals and secondary damage (e.g. activation of caspase 1). Necrotic appearance is most common for cells dying within minutes or few days after TBI. Morphologic features of apoptosis include nuclear and cytoplasmic condensation, internucleosomal DNA cleavage and packing of the cell into apoptotic bodies that are engulfed by phagocytes, preventing release of intracellular components. Apoptotic neurodegeneration can occur via caspasedependent or caspase-independent pathways, as summarized in Table 1 and Fig. 1. Evidence from humans and experimental models shows that neurons (as well as glial cells) with apoptotic morphology can be found several months after TBI. A large number of studies have focused on a mitochondriamediated pathway that is triggered by the release of cytochrome c and the formation of an apoptosome, in which procaspase 9 binds to cytochrome c and apoptosis protease activation factor-1 (Apaf-1) leading to formation of active caspase 9 and activation of the executioner caspase 3 in an ATP-dependent manner. Substrates for caspase 3 include cytoskeletal proteins such as spectrin, DNA-repairing enzymes like poly(ADP-ribose)polymerase (PARP), cell-cycle proteins and enzymes involved in signal transduction. Existing data suggest that the mitochondria-mediated pathway is activated both in experimental and human TBI. Expression and activation of various components of the pathway are most prominent during the first 3 days following TBI [5]. Death receptor pathways are activated by the binding of ligands (e.g. TNF-a or FasL) to death receptors (TNFR-1 and Fas, respectively) that are members of the tumor necrosis factor receptor (TNFR) superfamily. This triggers a series of downstream cascades (c-Jun-N-terminal kinase) that lead to activation of caspases [5]. Much less is known concerning the two other pathways leading to caspase activation: the endoplasmatic reticulum (ER)-mediated pathway and the GRANZYME B (see Glossary) mediated pathway, which have been shown to play a role in some other neurodegenerative diseases [10]. Neurons can also show caspase-independent apoptotic features, and in some cases co-activation of several parallel death pathways is required for apoptotic cell death, including
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Figure 1. Summary of crosstalk between various pathways leading to cell death. Intracellular Ca2+ overload is a central mechanism that can initiate both necrotic and apoptotic pathways. Ca2+can enter the postsynaptic cell via several channels (AMPA, NMDA, ASIC, TRPM7). Elevation in intracellular Ca2+results in calpain activation that can cleave NMDA and NCX channels. This results in impairment of Ca2+extrusion via NCX channels and further elevation of intracellular Ca2+. Calpains, cathepsins, and caspases are the major enzymes responsible for degradation of various cellular components. Calpains are activated by Ca2+. Calpains can activate cathepsins that might also become activated as a consequence of lysosomal damage. Interestingly, these two phenomena can occur independently. Elevated intracellular calcium as well as activation of calpains can result in activation of caspase pathways via mitochondria. Calpains can also directly activate caspases. In addition to mitochondrial pathways, caspases can be activated by death receptors (TNFR, Fas) located in cell membranes (data collected from Refs [7,8,42,43]).
activation of calpains and cathepsins [7]. Upregulation or downregulation of proteins that control, either directly or indirectly, the consequences of caspase activity has been studied in TBI (Table 1). For example, inhibitor of apoptosis protein (IAP) or survival-promoting proteins (Bcl-2, Bcl-xL) has been shown to be upregulated in a fluid-percussion injury (FPI) model of TBI (Table 1). Otherwise, other molecules that act at the interface between caspase-mediated and nonmediated programmed cell death (e.g. Bid) are also affected by TBI. Little is known, however, about proteins other than cytochrome c released from mitochondria, including apoptosis inducing factor (AIF), SMAC/DIABLO and Omi/HtrA2 (both act as IAP inhibitors, see Glossary) or EndoG (see Glossary). Future studies will also show whether the other factors listed in Table 1, and known to regulate programmed cell death, form candidate targets for therapeutic attempts in TBI.
Neuroprotection in TBI – pharmacological strategies To date, more than 100 pharmacological studies have been performed in the lateral FPI model alone and a detailed overview of each study and compound evaluated is beyond the scope of the present review. The most important treatment options from a clinical perspective are summarized in the following sections (see also Table 2).
Attenuation of excitotoxicity Numerous reports have evaluated attenuation of glutamatemediated excitotoxicity for the treatment of experimental TBI, including the inhibition of the two major classes of glutamate receptors, ionotropic (NMDA, KA and AMPA) and metabotropic (Groups I–III) receptors, coupled to intracellular second messengers. In experimental TBI, many reports have targeted the NMDA receptor (Table 2), where www.drugdiscoverytoday.com
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Table 1. Summary of cell death pathways in human and experimental traumatic brain injury (TBI) Pathway
Human TBI change
Rat TBI FPIa change
Caspase-dependent cell death Mitochondria-mediated pathway Cytochrome c Apaf-1d Caspase-9 (initiator caspase) Caspase-3 (effector caspase) Caspase-6 (effector caspase) Caspase-7 (effector caspase) Death receptor-mediated pathway TNFg-a TNFRh-1 FasLi Fas Caspase-8 (initiator caspase) Caspase-10 (initiator caspase)
Refs CCIb change
c
nde nd nd f
[44–46] [47] [48–50] [49–52]
nd
nd nd
nd
nd [53,54] [55–57] [58] [59–61] [58,60–63] [49,50,58,60,64]
nd nd 0j
nd
nd
ER -mediated pathway Caspase-12
nd
nd
Granzyme B-mediated pathway Granzyme B Perforin
nd nd
nd nd
nd
k
[65] nd nd
Caspase-independent cell death Calpains Cathepsins Cathepsin B Cathepsin H Cathepsin L Other factors regulating cell death Mitochondrial proteins Smac/Diablol Omi/HtrmA2 EndoGn AIFo
[50,66–68] nd nd nd
nd
nd nd nd nd
0 nd nd nd
Bcl family proteins controlling mitochondria Bcl-2 Bcl-XL nd Bax Bak nd Bid nd Bim nd IAPp
[69]
nd nd nd
[70]
[69]
[71] [45,52,72–74] [74] [52,74]
nd nd nd
nd
PARPq
nd [50,75] nd nd
[49,70]
nd
Caspase 1 a
FPI: fluid percussion injury. b CCI: controlled cortical impact. c : increase. d Apaf-1: apoptotic protease activating factor. e nd: no data available. f : decrease. g TNF: tumor necrosis factor. h TNFR: tumor necrosis factor receptor. i FasL: Fas ligand. j 0: no change. k ER: endoplasmic reticulum. l Smac/Diablo: a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. m Omi/Htr: a proapoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent cell death. n EndoG: endonuclease G. o AIF: apoptosis-inducing factor. p IAP: inhibitor of apoptosis proteins. q PARP: poly(ADP-ribose) polymerase.
412
nd nd nd
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0
[46,78,79]
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Table 2. Targets of neuroprotection in traumatic brain injury Pharmacological target
Examples of compounds
Refs
EAAa modulation NMDAb AMPAg/KAh Metabotropic EAA release inhibition
Mg2+, CPI 101-606c, MK-801d, HU-211e, Memantinef RPR117824i, YM872j, NBQXk MCPGl, AIDAm, CPCCOetn, LY-367385o, MPEPp, DCG-IVq, LY354740r BW1003C87s, 619C89t, Riluzoleu
[12]
ROSv scavenging
PBNw, S-PBNx, Vitamin E, L-NAMEy, CDPCz, PEG-SODaa
[80]
ab
ac
Calcium-mediated damage
LOE908 , SNX-111
Modulators of inflammation
IA29ad, Ibuprofen, HU-211, CP-0127ae
Miscellaneous
af
[81]
ag
GPI 6150 , CsA , CCPA , Lactate ai
aj
Neurotrophic factors
NGF , GDNF , BDNF
Z-VAD-fmkal, Z-DEVD-fmkam an
TRH
[81]
ak
Inhibitors of apoptosis Endocrinology
[18]
ah
ao
[23] [5] ap
analogs, IGF-1 , DHEAS , progesterone
[81]
a
EAA: excitatory amino acid. b NMDA: N-methyl-D-aspartate. c CPI 101-606: (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol. d MK-801: dizocilpine maleate. e HU-211: (+)-(3S,4S)-7-hydroxy-D-6 tetrahydro-cannabinol 1,1-dimethylheptyl. f Memantine: 3,5-dimethyl-1-adamantanamine. g AMPA: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. h KA: kainate receptors 1 and 2. i RPR117824: 9-carboxymethyl-imidazo-[1-2a]indenol[1-2e]. j YM872: zonampanel monohydrate. k NBQX: 6-nitro-7-sulfamoylbenzo (F) quinoxaline-2,3-dione. l MCPG: (S)-a-4-caboxyphenylglycine. m AIDA: (RS)-1-aminoindan-1,5-dicarboxylic acid. n CPCCOet; 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester. o LY367385: (S)-(+)-a-amino-4-carboxy-2-methylbezeneacetic acid. p MPEP: 2-methyl-6-(phenylethynyl)-pyridine. q DCG-IV: 2, (20 ,30 )-dicarboxycyclopropylglycin. r LY354740: (1S,2S,5R,6S)-(+)-2-aminobicyclo[3.1.0]hexane -2,6-dicarboxylic acid. s BW1003C87: 5-(2,3,5-trichlorophenyl) pyrimidine 2.4-diamine ethane sulfonate. t 619C89: [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-trichlrophenyl)pyrimidine mesylate monohydrate]. u Riluzole: 2-amino-6-trifluro methoxy benzthiazole. v ROS: reactive oxygen species. w PBN: a-phenyl-N-tert-butyl-nitrone. x S-PBN: sodium 2-sulfophenyl-N-tert-butyl nitrone. y L-NAME: nitro-L-arginine methyl ester. z CDPC: cytidine 50 -diphosphocholine. aa PEG-SOD: polyethylene glycol-conjugated superoxide dismutase. ab LOE908: (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide. ac SNX-111: Ziconotide. ad IA29: anti-ICAM-1 monoclonal antibody. ae CP-0127: Bradycor or deltibant, bissuccimidohexane (L-Cys6)-1. af GPI 6150: (1,11b-dihydro-[2H]bezopyrano[4,3,2-de]isoquinolin-3-one. ag CsA: cyclosporin A. ah CCPA: 2-chloro-N(6)-cyclopentyladenosine. ai NGF: nerve growth factor. aj GDNF: glial cell-derived neurotrophic factor. ak BDNF: brain-derived neurotrophic factor. al Z-VAD-fmk: acetyl-Tyr-Val-Ala-Asp-chloromethyl-ketone. am Z-DEVD-fmk: N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone. an TRH: thyrotropin-releasing hormone. ao IGF-1: insulin-like growth factor-1. ap DHEAS: dehydroepiandrosterone sulfate.
earlier compounds such as MK-801, phencyclidine (PCP) and ketamine showed strong psychomimetic effects, these adverse effects were diminished in later generations of NMDA receptor antagonists such as NPS 1506 and NPS 846. To date, the NMDA antagonists magnesium, HU-211 (dexanabinol), memantine and CP-101,606 have generated clinical interest owing to the improved functional and histological outcome
in several clinically relevant TBI models and these compounds are all in the clinical trial phase [11]. In addition to antagonism of excitatory amino acid (EAA) receptor function, modulation of EAA receptor activity might also be accomplished by inhibition of EAA release. Examples of glutamate release inhibitors with neuroprotective action in TBI include the compound riluzole which is in a Phase III www.drugdiscoverytoday.com
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clinical trial for TBI in Europe. Despite promising preclinical documentation, all glutamate blockers evaluated, to date, have shown to be ineffective in Phase III clinical trials [12] (Table 2).
Calcium channel blockage and calpain inhibition The Ca2+ blockers (S)-emopamil, LOE 908, S100B, BMS204352 and SNX-111, have all been shown to attenuate neurological motor and cognitive deficits following experimental TBI [11]. However, a clinical trial using SNX-111 after TBI was suspended owing to higher mortality in SNX-111treated patients than in patients receiving placebo. Much clinical interest, to date, has focused on the Ca2+-channel blocker nimodipine that improved outcome in a randomized study of TBI patients. However, a systemic review of randomized controlled trials of calcium channel blockers in acute TBI patients shows that considerable uncertainty remains over their efficacy [13]. Downstream inhibition of calpains (Ca2+-dependent proteases) might be of therapeutic value; therefore, the calpain inhibitor AK295, infused intra-arterially, was shown to reduce both motor and cognitive deficits one week after lateral FPI in the rat although without effect on cortical lesion size or apoptotic cell death. In a model of traumatic axonal injury in the rat, the calpain inhibitor MDL-28170 reduced the damage of brainstem fiber tracts and further investigation into the efficacy of calpain inhibition following TBI is warranted.
Reactive oxygen and nitrogen scavengers Although tirilazad mesylate (TM) and its related pyrrolopyrimidines, including U-101033E, showed promising effects in a series of TBI studies in mice, rats and cats [11], Phase III clinical trials for ischemia, spinal cord injury, TBI and subarachnoid hemorrhage all failed to show a positive outcome [14]. Inhibition of lipid peroxidation and/or attenuation of hydroxyl radicals is of clinical interest and further testing is necessary to evaluate these compounds using longer and clinically relevant time windows. Cytidine 50 -diphosphocholine (CDPC), or citicoline, compounds that attenuate the activation of phosholipase A2, have been shown to be neuroprotective and possess neurobehavioral efficacy in experimental TBI models [15]. In a randomized study enrolling 216 TBI patients, CDPC improved the motor and cognitive outcome, implying a potential role for CDPC in the treatment of human TBI.
Superoxide scavenging Administration of the antioxidant enzyme superoxide dismutase (SOD) conjugated to enhance blood–brain barrier penetration (PEG-SOD, pegogortein) improved survival and neurological recovery, attenuated cerebral edema and decreased hippocampal cell loss across TBI models in rats 414
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although conflicting results exist (see Table 2). Unfortunately, PEG-SOD failed in the randomized, Phase III multicenter trial, which included 463 patients. The reasons for this failure are probably multifactorial but might be related to the inclusion of many severely brain-injured patients, the extended time window chosen (8 h) and the chosen outcome measures in addition to the possibility of inadequate dosing or lack of sufficient efficacy by the compound [16].
Inflammation and anti-inflammatory compounds The nonselective cyclooxygenase (COX) inhibitors ibuprofen and indomethacin and the selective COX-2 inhibitor nimesulide have all been shown to improve some of the posttraumatic deficits including changes in intracranial pressure and cerebral blood flow and impairment of cognitive and motor function [17]. Cytokines, including tumor necrosis factor (TNF) and interleukins (IL), are increased in human and experimental TBI [18]. Improved outcome following experimental TBI with compounds (e.g. pentoxyfylline, TNF-a binding protein, HU-211 and soluble TNF-a receptor fusion protein) that block the action of TNF-a has been reported [19]. The role of TNF-a (and IL-6) in the treatment of TBI remains elusive because these cytokines have been reported to possess both neuroprotective and neurotoxic properties. Glucocorticoids (GCs) were introduced in the early 1960s because they were found to markedly reduce edema from brain tumors and attenuate ROS-induced lipid peroxidation and reduce inflammation. Although GCs might attenuate delayed edema and inflammation following TBI in the rat, excess GCs might be neurotoxic and the GC receptor inhibitor mifepristone (RU 486) attenuated hippocampal cell death following controlled cortical impact (CCI) injury in the rat. High-dose GC treatment received widespread use in TBI patients, but because of the publication of the large, controlled, randomized, multi-center corticosteroid randomization after significant head injury (CRASH) trial where a worse outcome was observed in patients receiving GCs [20], the use of steroids in the treatment of TBI is generally not recommended.
Pharmacological inhibition of caspases and pro-apoptotic cascades One strategy to prevent acute cell death after TBI might be to inhibit caspases, enzymes involved in the process leading to apoptosis. Several caspase inhibitors of varying specificity have been successfully evaluated in models of experimental TBI including ketones such as the pan-caspase inhibitor zVAD-fmk, the caspase-1 specific inhibitor acetyl-Tyr-Val-AlaAsp-chloromethyl ketone and the caspase-3-specific z-DEVDfmk [5]. However, the role for anti-apoptotic compounds in TBI is controversial and, to date, more preclinical research is necessary to evaluate their clinical potential.
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Several studies have demonstrated that exogenously administered progesterone or its metabolite allepregnanolone reduced post-traumatic increase in cerebral edema, improved cognitive performance and functional deficits, reduced lesion volume and apoptotic cell death following TBI in the rat [21]. Currently, there is an ongoing National Institute of Neurological Disorders and Stroke (NINDS)sponsored single-center, controlled clinical trial of the efficacy of intravenous administration of progesterone in human TBI.
Neurotrophic factors Four major classes of neurotrophins have been characterized in mammals: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4/5) plus additional non-neuronal trophic factors such as basic fibrillary growth factor (bFGF), glial derived neurotrophic factor (GDNF), insulin-like growth factor (IGF), ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) [22,23]. Neurotrophins mediate several types of intercellular communication and have been shown to act as (i) retrogradely transported, target-derived factors that influence afferent neurons; (ii) locally released paracrine factors affecting both neurons and non-neuronal cells; (iii) autocrine factors acting on the same cells that produce and release it and (iv) as endocrine factors that are transported through the blood stream [24]. Administration of trophic factors has been evaluated as a neuroprotective strategy in TBI. Dietrich et al. [25] reported that acute administration of bFGF attenuated cortical cell loss following lateral FPI in rats, whereas McDermott et al. [26], using the same model, demonstrated that delayed intraparenchymal administration of bFGF, beginning 24 h after injury, significantly improved post-traumatic cognitive deficits in the rat. Post-traumatic BDNF infusion [into BDNF (/) mice following CCI brain injured and into rats following lateral FPI] did not attenuate behavioral and histological damage, suggesting that BDNF does not provide a trophic support in the traumatically injured brain [27]. Intracerebroventricular (i.c.v.) administration of GDNF following CCI brain injury in rats decreased the post-traumatic neuronal loss in the hippocampus at 1 week postinjury [28], while i.c.v. administration of NGF attenuates cognitive deficits and cholinergic cell loss in the forebrain following FPI and CCI brain injury in rats [29]. An optimal delivery system for neurotrophins (using viral vectors or genetically modified cells) should be the object of investigation to ensure a constant release of trophic factors to a targeted area without the adverse effects related to systemic and/or intracerebroventricular administration. To this end, Longhi et al. [30] observed that ex vivo gene therapy transplanting the human neuroteratocarcinoma-derived neuronal cells (NT2N) transduced with a lentiviral vector to release NGF into the basal
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forebrain of mice at 24 h postinjury attenuated the cognitive deficits at 1 month postinjury, suggesting that gene therapy (to deliver trophic factor) could be a promising therapeutic strategy following TBI.
Neuroprotection in TBI – cell-based therapy Neuronal replacement might be a viable therapeutic approach to replace the lost tissue/cells following TBI. Several cell lines have been evaluated over the past decade with positive results in terms of survival and integration of the transplant into the injured brain and in terms of attenuation of the post-traumatic behavioral sequelae and histological damage (Table 3) [31]. Early studies reported that fetal cortical tissue that was transplanted 24 h postinjury (alone or coupled with NGF infusion) survived, integrated into the injured brain and attenuated behavioral deficits and histological damage including hippocampal cell loss [32–34]. Human NT2N cells have been observed to (safely) survive and integrate into the injured brain when transplanted up to 1 month postinjury, however, they were not associated with attenuation of post-traumatic behavioral and histological damage [35]. Using a different approach involving bone marrow stromal cells, intraparenchymal transplantation of whole bone marrow into the pericontusional tissue at 24 h after CCI brain injury in rats resulted in improved functional outcome and differentiation of transplanted cells into cells expressing neuronal and glial markers [36]. In addition, bone marrow-derived cells administered at 24 h postinjury were subsequently observed in the pericontusional area of the brain expressing both neuronal and glial markers and were associated with improved functional outcome, increased expression of NGF and BDNF and increased endogenous cellular proliferation [37,38]. Stem cells have the property of unlimited expansion in cultures and differentiation in different cellular lineage. Several lines of stem/progenitor cells have been transplanted in experimental models of TBI and have been associated with reduced behavioral deficits (up to 1 year postinjury) and histological damage [31]. In addition cells transplanted into the hemisphere contralaterally to the injury migrated across the corpus callosum towards the site of injury [39], suggesting that the environment associated with TBI is able to modulate the migratory patterns of engrafted cells. The tropism of stem cells for pathology in combination with the ability to genetically engineer them might provide a platform for delivery of therapeutic genes into target areas of the injured brain. However, even if neural transplantation has been successful in experimental TBI, further work needs to be performed to better understand the mechanisms associated with the behavioral and histological outcome. In addition, the clinical application of cell replacement strategy following TBI, to date, remains speculative. www.drugdiscoverytoday.com
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Table 3. Summary of brain transplantation studies following TBI Source
Model
Main results of the study
Refs
Fetal tissue
FPIa
Graft survival when transplanted days 2–14 PIb c CA3 cell loss at 4 weeks PI
[32]
Fetal tissue + NGFd
FPI
NT2Nf cells
FPI
NT2N cells + NGF
CCIh
Cognitive deficits at 4 weeks
[30]
HiB5 + NGF
FPI
Cognitive deficits Motor recovery CA3 cell loss at 1 week PI
[82]
MHP36j
FPI
Cognitive deficits at 4 months PI
[83]
C17.2
CCI
Motor recovery over 12 weeks PI
[84]
Pre-differentiated stem cells into neuronal and glial precursors
CCI
Motor recovery over 4 weeks PI Lesion volume at 40 days PI
[85]
Progenitor cells
CCI
Cognitive deficits Motor recovery over 1 year PI Migration toward injury at 14 months PI
[86]
C17.2 + EGFRl vIII receptor
FPI
Migration toward injury at 2 weeks PI
[39]
Bone marrow stromal cells
CCI
Umbilical cord blood
CCI
i
k
e
Cognitive deficits Motor recovery over 2 weeks PI
[34]
Graft survival up to 16 weeks PI g Motor recovery over 16 weeks PI
[35]
Motor recovery NGF and BDNFm expression Endogenous cellular proliferation at 2 weeks PI Migration toward injury Motor recovery over 4 weeks PI
[37,38]
[87]
a
FPI: fluid percussion brain injury. PI: postinjury. c : increase. d NGF: nerve growth factor. e : decrease. f NT2N: neuroteratocarcinoma-derived neuronal cells. g : unaffected. h CCI: controlled cortical impact brain injury. i HiB5: conditionally immortalized progenitor cell derived from an embryonic (E16) rat hippocampus. j MHP: fibroblast growth factor 2-responsive Maudsley hippocampal cell line clone 36. k C17.2: clonal multipotent progenitor cell derived from the external germinal layer of the neonatal murine cerebellum. l EGFR: epidermal growth factor receptor. m BDNF: brain derived neurotrophic factor. b
Conclusions and future challenges Understanding the details of molecular cascades underlying neurodegeneration after TBI remains a significant challenge. The complexity of the task is multiplied by the fact that not only neurons but also astrocytes, microglia and oligodendrocytes can undergo degeneration in the traumatized brain. Death of non-neuronal cells can compromise, for example, trophic support for neurons or re-myelinization of damaged axons. Understanding the mechanisms of survival and death of newly born cells after TBI is still in infancy. More data are also needed to understand the mechanisms of degeneration of transplanted cells to optimize the benefits of cell therapies (vide infra and 31). TBI can also cause axonal injury affecting the survival of somata by mechanisms that differ from that of somatic injury [8]. Recent data from single cell profiling studies have added a new aspect to the complexity of factors that eventually determine the fate and death mechanisms of 416
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individual neurons [88]. As shown by Hellmich et al. [40], expression level of survival promoting factors in adjacent neurons, some of which die and some survive, can vary tremendously at the time of TBI. Whether this is related, for example, to the metabolic state of the cell remains to be investigated [41]. Interestingly, recent observations in patients suggest that compromised postinjury metabolic state is associated with poor outcome [8]. To date, in most reports evaluating pharmacological compounds for the treatment of TBI, a single compound has been used with the hope that this compound (the magic bullet) will attenuate all or most of the deficits associated with the injury. However, owing to the heterogeneity and the complex pathophysiology of TBI, it might seem more probable that several different pharmacological strategies (a ‘cocktail’) will be required to ameliorate post-traumatic deficits. However, combination therapy has been inadequately studied in
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TBI and several reports have also shown decreased efficacy of neuroprotective compounds when used in combination with other compounds. More research into the effects of drug interaction and polypharmacy in TBI is needed. Finally, it is apparent that a wide range of candidate mechanism has not yet been tested. Moreover, it is probable that many mechanisms remain to be discovered. The beneficial effects of various treatments in experimental models, even though not yet translated to clinical practice, are encouraging, and certainly maintain the interest in the search of novel, more efficient, less toxic and easier to administer compounds.
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22
Acknowledgements
23
Supported by the Academy of Finland, the Sigrid Juselius Foundation, the Finnish Cultural Foundation and the Paulo Foundation to AP; NIH NS08803 and NS40978 to TKM; Swedish Brain Foundation to NM.
24
25
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