Zinc in traumatic brain injury: from neuroprotection to neurotoxicity Deborah R. Morris a and Cathy W. Levenson a,b

Purpose of review In light of the recent recognition that even mild forms of traumatic brain injury (TBI) can lead to long-term cognitive and behavioral deficits, this review examines recent data on the neuroprotective and neurotoxic roles of zinc after brain injury. Recent findings Data show that treatment using dietary and parenteral zinc supplementation can reduce TBI-associated depression and improve cognitive function, specifically spatial learning and memory. However, excessive release of free zinc, particularly in the hippocampus associated with acute injury, can lead to increases in protein ubiquitination and neuronal death. Summary This work shows the need for future research to clarify the potentially contradictory roles of zinc in the hippocampus and define the clinical use of zinc as a treatment following brain injury in humans. This is particularly important given the finding that zinc may reduce TBI-associated depression, a common and difficult outcome to treat in all forms of TBI. Keywords behavior, supplementation, traumatic brain injury, ubiquitin, zinc

INTRODUCTION Traumatic brain injury (TBI) affects approximately 1.7 million Americans every year [1] with common causes including falls, motor vehicle crashes, sportsrelated injuries or other recreational activities, as well as military service. Many individuals who sustain a TBI suffer a variety of short-term and longterm impairments in cognition, communication, sensory processing, and other behavioral problems including depression and anxiety. Treatment interventions are limited. The essential trace element zinc has been implicated in many disorders of the central nervous system such as TBI, ischemia, stroke, and mood disorders including depression [2].

TRAUMATIC BRAIN INJURY AND THE DEVELOPMENT OF ZINC DEFICIENCY In patients with a brain injury, there are clear acute increases in urinary zinc excretion and significant decreases in serum zinc levels that have been shown to be proportional to the severity of injury [3]. A recently published article provides insight into the mechanisms responsible for this observation. In mice subjected to a cortical injury, there was an

initial release of zinc from the liver. This resulted in an upregulation of the expression of the hepatic zinc-binding proteins metallothionein-I and II. These proteins resulted in the hepatic sequestration of zinc such that liver zinc levels returned to preinjury levels [4 ]. Although these data suggest that zinc released from the liver is then shuttled back to the liver, virtually all cells synthesize metallothionein-I/II which serves as an efficient storage form of zinc. Thus, metallothionein could potentially sequester zinc from other sources, contributing to reductions in serum zinc. &

USE OF ZINC AS A TREATMENT IN TRAUMATIC BRAIN INJURY Given that serum levels of zinc have been shown to decrease after TBI, both preclinical and clinical work a Department of Biomedical Sciences and bProgram in Neuroscience, Florida State University College of Medicine, Tallahassee, Florida, USA

Correspondence to Cathy W. Levenson, PhD, Florida State University, College of Medicine, Tallahassee, FL 32306-4300, USA. Tel: +1 850 644 4122; fax: +1 850 644 5781; e-mail: [email protected] Curr Opin Clin Nutr Metab Care 2013, 16:708–711 DOI:10.1097/MCO.0b013e328364f39c Volume 16  Number 6  November 2013

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Zinc in traumatic brain injury Morris and Levenson

zinc deficiency and TBI have been associated with oxidative stress [9,10]

KEY POINTS  This review examines the neuroprotective and neurotoxic roles of zinc after brain injury.  The use of dietary and parenteral zinc supplementation as a treatment following brain injury improves neuropsychological function.  Zinc alterations after brain injury can lead to increases in protein ubiquitination and neuronal death.

have been conducted to test the efficacy of zinc supplementation in patients suffering from a moderate to severe TBI. In the single human study to date, patients were assigned to either a zincadequate or a zinc-supplemented group. Zinc supplementation was associated with increased visceral proteins such as prealbumin and retinal-binding protein and improvements in Glasgow Coma Scale (GCS) scores by 2 weeks after the start of treatment [5]. Preclinical work has also suggested that 4 weeks of zinc supplementation (180 ppm) prior to injury can be used to provide resiliency to the behavioral effects of TBI including depression, stress-related anxiety, and cognition [6,7]. Recent work has examined the potential of zinc supplementation as a treatment for its ability to improve behavioral outcomes. After TBI, rats were put on a zinc-adequate (30 ppm) or zincsupplemented (180 ppm) diet. Additional rats were given either a zinc-adequate or a zinc-supplemented diet and a single intraperitoneal injection of zinc (30 mg/kg) 1 h after injury to account for reduced food consumption on the day following a brain injury. Unlike the findings of the earlier resiliency study, in which zinc was provided before the injury [6], supplementation with zinc after the injury via either dietary or parenteral routes did not reduce anxiety-like behaviors. However, zinc supplementation and the early zinc injection significantly improved depression-like behaviors following TBI. Interestingly, dietary supplementation or the zinc injection alone was not effective [8 ]. The work also showed that zinc supplementation can be used to improve cognitive outcomes after TBI. Although there were improvements in time spent finding a hidden platform in the Morris Water Maze (MWM), a test that examines spatial learning and memory, in rats given the zinc-supplemented diet alone after injury, an early zinc injection alone did not enhance MWM performance [8 ]. Together these data strongly suggest that zinc status should be monitored and maintained after a brain injury, not only because of the reported neurocognitive outcomes, but also because both &


POTENTIAL NEUROTOXIC EFFECTS OF ZINC IN TRAUMATIC BRAIN INJURY Although there are both preclinical and clinical evidences for zinc as a neuroprotective agent following TBI, excessive zinc, released from neurons acutely after TBI, has been associated with neuronal death and damage. An abundance of previous work has shown that free zinc, sequestered in synaptic vesicles, is released from neurons into the synaptic cleft where it results in postsynaptic neuronal death [2]. Although the release of synaptic zinc after injury is well accepted in the field, there have been indicators that other pools of free zinc may contribute to neuronal damage after injury. For example, using an in-vitro model of ischemic brain injury in which cells were exposed to 3 h of oxygen–glucose deprivation (OGD) followed by incubation under normal conditions, free zinc levels were quantified with ratiometric mitochondrial and cytoplasmic zinc sensors. Mitochondrial free zinc levels 1 h after OGD increased to 10 pmol/l from 0.2 pmol/l at control levels, whereas at the same time cytosolic free zinc concentrations were decreased significantly. Mitochondrial free zinc eventually tapered off to normal physiological levels by 2 h after OGD, whereas cytosolic free zinc gradually increased by about 10-fold, 24 h after OGD [11]. This work suggests that changes in the subcellular localization of free zinc after neuronal injury are complex and are likely to have a significant impact on neuronal survival. Although these data provide further evidence that disruption of zinc homeostasis during a brain insult can be detrimental to the survival and viability of neurons, caution should be used when interpreting data, such as those above, utilizing fluorescein or rhodamine-containing free zinc indicators after TBI in fresh frozen or fixed tissue sections. A recent report has shown that fluorescein, the fluorescein-containing zinc probe Newport Green (Invitrogen/Molecular Probes, Eugene, Oregon, USA), and the rhodamine-containing calcium probe Rhod-5N (Invitrogen/Molecular Probes, Eugene, Oregon, USA) stained brain sections after injury, but did not stain zinc in sham (uninjured) rat brains. Furthermore, the fluorescence of these probes was not diminished after zinc chelation in TBI-injured brain sections suggesting that these zinc dyes may be binding to injured neurons rather than zinc. Further research will need to clarify the usefulness of these and other fluorescent zinc dyes in live brain slices, organotypic cultures, and cell culture [12].

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The mechanisms of free zinc accumulation and neurodegeneration following TBI were further examined in recent in-vitro and in-vivo studies. In cultured rat, hippocampal neurons zinc-induced protein ubiquitination was mediated by the p38 mitogen-activated protein kinase signaling pathway [13 ]. Application of zinc chloride in the culture media resulted in the rapid phosphorylation of p38. This was followed by ubiquitination of proteins at zinc treatment levels of 100–600 mmol/l, and the subsequent loss of mitochondrial activity and neuronal death beginning 6 h after zinc application. The ubiquitination was prevented by pretreatment of cells with the inhibitor of p38, SB239063, suggesting that zinc-induced ubiquitination of neuronal proteins is dependent on p38 [13 ]. These data were subsequently confirmed in rat hippocampal slices and homogenates [14 ]. TBI increased hippocampal ubiquitin protein conjugates (and decreased free ubiquitin) beginning at 6 h postTBI. This was followed by neuronal death in the cornu ammonis 1 and cornu ammonis 3 regions of the hippocampus around 48 h post-TBI. Zinc depletion with the chelator Calcium EDTA blocked this ubiquitin conjugation and prevented neurodegeneration. Samples collected from human TBI patients and uninjured controls also revealed increases in protein ubiquitination in the cortex [14 ]. Taken together, the timing of these results suggests that p38 activation and neuronal protein ubiquitination are not simply the result of zincmediated neuronal damage and death, but may be part of the mechanisms responsible for cell death after TBI. The authors of these articles conclude that p38 phosphorylation and activation lead to a disruption of proteolysis via the proteasome, leaving increased ubiquitinated proteins in the cell that eventually cause neuronal damage and death. However, proteosomal activity was not measured in these experiments. Thus, an alternate explanation for the data could simply be that p38 activation leads to increased ubiquitination and targeting of vital proteins to the proteasome and subsequent neuronal death. Finally, it is worth noting that investigations are ongoing to develop clinically relevant treatments to prevent zinc-mediated neuronal death. A 3 h pretreatment of mouse hippocampal primary neurons with urokinase plasminogen activator (uPA), a serine protease that converts plasminogen to active plasmin, inhibited zinc-induced cell death. Thus, future work should be conducted to determine the extent to which uPA can be utilized as a potential treatment and neuroprotective agent in zincinduced cellular and neuronal damage [15]. &&





CONCLUSION As little can be done to improve the primary damage that results from TBI, it is important to intervene as quickly as possible to minimize secondary cell injury and death in the cortex and other regions of the brain, such as the hippocampus. Although zinc supplementation has been shown in both humans and animal models of TBI to be an effective treatment for a number of TBI-related behavioral deficits, the timing of zinc intervention appears to be important. There has been only one published clinical study using zinc as a treatment for TBI, and none that used zinc to improve resilience. Animal studies used the standard supplementation levels typically used in the literature (180 ppm). Thus, although zinc supplementation prior to an injury may be especially important for populations that are at risk of brain injury, such as athletes and military personnel, future research will be needed to establish optimal zinc doses needed to provide behavioral resilience or function as a treatment in human populations. Furthermore, reports that zinc causes hippocampal death via a variety of mechanisms including p38 and protein ubiquitination illustrate the importance of future work to understand the cellular and molecular actions of supplemental zinc as well as neurotoxic zinc in the hippocampus. Acknowledgements The authors wish to acknowledge funding from National Institute for General Medicine (GM081382) and the Department of Defense, US Army Medical Research Material Command (MRMC). Conflicts of interest There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Faul M, Xu L, Wald MM, Coronado VG. Traumatic brain injury in the United States: emergency department visits, hospitalizations, and deaths. Atlanta (GA): Centers for Disease Control and Prevention, National Center for Injury Prevention and Control; 2010. 2. Gower-Winter SD, Levenson CW. Zinc in the central nervous system: From molecules to behavior. Biofactors 2012; 38:186–193. 3. McClain CJ, Twyman DL, Ott LG, et al. Serum and urine zinc response in head-injured patients. J Neurosurg 1986; 64:224–230. 4. Pankhurst MW, Gell DA, Butler CW, et al. Metallothionein (MT) -I and MT-II & expression are induced and cause zinc sequestration in the liver after brain injury. PLoS One 2012; 7:e31185. After brain injury in mice, zinc that is initially released from the liver is sequestered back to the liver by the upregulation of hepatic zinc-binding proteins metallothionein I/II. Because metallothionein I/II is found in many organs and could potentially sequester zinc from other sources, this observation provides a possible explanation for why patients become zinc deficient after a TBI.

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Zinc in traumatic brain injury Morris and Levenson 5. Young B, Ott L, Kasarskis E, et al. Zinc supplementation is associated with improved neurologic recovery rate and visceral protein levels of patients with severe closed head injury. J Neurotrauma 1996; 13:25–34. 6. Cope EC, Morris DR, Scrimgeour AG, et al. Zinc supplementation provides behavioral resiliency in a rat model of traumatic brain injury. Physiol Behav 2011; 104:942–947. 7. Cope EC, Morris DR, Levenson CW. Improving treatments and outcomes: an emerging role for zinc in traumatic brain injury. Nutr Rev 2012; 70:410– 413. 8. Cope EC, Morris DR, Scrimgeour AG, Levenson CW. Use of zinc as a & treatment for traumatic brain injury in the rat: effects on cognitive and behavioral outcomes. Neurorehabil Neural Repair 2012; 26:907–913. In a rat model of TBI, dietary zinc supplementation (180 ppm) and an early zinc injection (30 mg/kg) after injury improved depression-like behaviors. Dietary zinc supplementation after injury also improved spatial learning and memory. This is the first animal study to examine the effect of supplemental zinc after TBI on behavioral deficits such as depression and cognitive impairments. 9. Wessels I, Haase H, Engelhardt G, et al. Zinc deficiency induces production of the proinflammatory cytokines IL-1b and TNFa in promyeloid cells via epigenetic and redox-dependent mechanisms. J Nutr Biochem 2013; 24: 289–297.

10. Clausen F, Marklund N, Lewe´n A, et al. Interstitial F2-Isoprostane 8-IsoPGF2a As a biomarker of oxidative stress after severe human traumatic brain injury. J Neurotrauma 2012; 29:766–775. 11. McCranor BJ, Bozym RA, Vitolo MI, et al. Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion. J Bioenerg Biomembr 2012; 44:253–263. 12. Hawkins BE, Frederickson CJ, Dewitt DS, Prough DS. Fluorophilia: fluorophore-containing compounds adhere nonspecifically to injured neurons. Brain Res 2012; 1432:28–35. 13. Zhu L, Ji XJ, Wang HD, et al. Zinc neurotoxicity to hippocampal neurons in vitro && induces ubiquitin conjugation that requires p38 activation. Brain Res 2012; 1438:1–7. This article elucidates mechanisms of zinc neurotoxicity and neurodegeneration. Application of zinc to cultured hippocampal neurons induced phosphorylation of p38 and induction of ubiquitinated protein conjugates, followed by neuronal death. 14. Sun KJ, Zhu L, Wang HD, et al. Zinc as mediator of ubiquitin conjugation & following traumatic brain injury. Brain Res 2013; 1506:132–141. This article shows that zinc accumulation facilitated TBI-induced neuronal death by increased ubiquitin conjugation in both rat hippocampal and human cortical neurons. 15. Cho E, Lee KJ, Seo JW, et al. Neuroprotection by urokinase plasminogen activator in the hippocampus. Neurobiol Dis 2012; 46:215–224.

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Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Zinc in traumatic brain injury: from neuroprotection to neurotoxicity.

In light of the recent recognition that even mild forms of traumatic brain injury (TBI) can lead to long-term cognitive and behavioral deficits, this ...
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