Zinc homeostasis and zinc signaling in white matter development and injury

Neuroscience Letters 707 (2019) 134247

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Review article

Zinc homeostasis and zinc signaling in white matter development and injury a,b,⁎

Christopher M. Elitt a b c

c

a,b

, Christoph J. Fahrni , Paul A. Rosenberg

T

Boston Children’s Hospital, Department of Neurology and the F.M. Kirby Neurobiology Center, 300 Longwood Avenue, Boston, MA, United States Program in Neuroscience, Harvard Medical School, Boston, MA, 02115, USA School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, United States

ARTICLE INFO

ABSTRACT

Keywords: Oligodendrocyte White matter Myelin Zinc Zinc transporters White matter injury of prematurity Periventricular leukomalacia Multiple sclerosis

Zinc is an essential dietary micronutrient that is abundant in the brain with diverse roles in development, injury, and neurological diseases. With new imaging tools and chelators selectively targeting zinc, the field of zinc biology is rapidly expanding. The importance of zinc homeostasis is now well recognized in neurodegeneration, but there is emerging data that zinc may be equally important in white matter disorders. This review provides an overview of zinc biology, including a discussion of clinical disorders of zinc deficiency, different zinc pools, zinc biomarkers, and methods for measuring zinc. It emphasizes our limited understanding of how zinc is regulated in oligodendrocytes and white matter. Gaps in knowledge about zinc transporters and zinc signaling are discussed. Zinc-induced oligodendrocyte injury pathways relevant to white matter stroke, multiple sclerosis, and white matter injury of prematurity are reviewed and examples of zinc-dependent proteins relevant to myelination highlighted. Finally, a novel ratiometric zinc sensor is reviewed, revealing new information about mobile zinc during oligodendrocyte differentiation. With a better understanding of zinc biology in oligodendrocytes, new therapeutic targets for white matter disorders may be possible and the necessary tools to appropriately study zinc are finally available.

Zinc signaling in the brain is increasingly recognized to play essential roles in synaptic transmission, neurodegeneration, stroke, differentiation, and proliferation [1–3]. The study of zinc biology has been hampered by a lack of appropriate tools to detect relatively small free zinc fluctuations with specificity and sensitivity, but in the last decade appropriate chelators and zinc sensors have become available [4–8]. Similar to developments in the calcium field in the 1980’s, these tools are beginning to shed light on multiple important roles for zinc signals in the brain. Here we provide an overview of zinc biology and discuss examples of zinc signals in the brain relevant to white matter development and injury, followed by a review of a powerful new ratiometric zinc sensor to visualize zinc fluxes the use of which led to important new information about the role of zinc in oligodendrocyte differentiation. 1. Zinc: an essential dietary micronutrient Zinc is the second most abundant transition metal in the brain, second only to iron, and is obtained exclusively through dietary intake [3,9,10]. The importance of dietary zinc was recognized in animals models where zinc deficiency leads to profound brain malformations [11] and in humans from developing countries where zinc deficiency is ⁎

common and leads to cognitive deficits and linear growth stunting in children [10,12,13]. Animal models have demonstrated delayed myelination when employing a zinc-deficient diet [14,15], although direct versus indirect effects of zinc deficiency on myelination remain to be elucidated. In human infants, there are two recognized clinical disorders associated with severe zinc deficiency in newborns, acrodermatitis enteropathica and transient neonatal zinc deficiency [16]. Acrodermatitis enteropathica is caused by an autosomal recessive mutation in a zinc transporter in the intestine (ZIP4) preventing absorption of zinc. The result is a severe form of zinc deficiency leading to a characteristic dermatitis, irritability, failure to thrive, infections and death if not recognized. With zinc supplementation, the deficits in absorption can be overcome with complete recovery (reviewed in [17]). While typically recognized and treated, a case report from an untreated 6-month-old with acrodermatitis enteropathica showed diffuse cerebral atrophy by head computed tomography (CT) which was reversed upon zinc supplementation for two months. The authors raise the intriguing possibility that the recovery could potentially be explained by improved myelination [18]. A second recognized clinical disorder associated with severe zinc deficiency is transient neonatal zinc deficiency, resulting from a mutation in a zinc transporter (ZnT2) in the mammillary gland

Corresponding author. E-mail address: [email protected] (C.M. Elitt).

https://doi.org/10.1016/j.neulet.2019.05.001 Received 1 February 2019; Received in revised form 29 April 2019; Accepted 1 May 2019 Available online 04 May 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.

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of lactating mothers preventing zinc secretion into breast milk [19]. There are increasing polymorphisms identified in zinc transporters likely leading to heterogeneous availability of zinc to newborns. While acrodermatitis enteropathica and transient neonatal zinc deficiency are well recognized, there are likely other mild or moderate forms of zinc deficiencies, particularly in preterm infants that have impaired zinc stores and zinc absorption [20].

antibodies conjugated to gold nanoparticles to allow ICP-MS quantification on single cells [31,32]. However, single-cell analysis has not yet been performed in developing oligodendrocytes or injured oligodendrocytes. The ICP-MS technique has the limitation of metal leaching during tissue harvesting and fixation and provides only information on the total zinc content. Synthetic fluorescent sensors such as chromis-1 (vide infra) as well as zinc-responsive MRI contrast agents represent promising new tools to visualize dynamic changes in the cellular zinc status within live cells and tissues, and thus are areas of active investigation [33,34].

2. Zinc pools: total, bound, and mobile zinc The physiological function of zinc may greatly vary depending both on its location within the diverse set of cellular compartments in the brain as well as its local coordination environment within protein hosts and low-molecular weight bioligands. Many intracellular organelles, such as mitochondria, lysosomes, the nucleus, or synaptic vesicles sequester significant levels of zinc, where it is likely bound to proteins, such as enzymes, zinc finger transcription factors, and metallothioneins [4,21]. While the total zinc concentration in the brain is around 100–300 μM, zinc levels within some organelles and compartments such as glutamatergic synaptic vesicles may approach low millimolar concentrations [22]. Despite strong association of zinc with bioligands, increasing evidence suggests that a significant portion of the total cellular zinc is kinetically labile and can readily exchange with exogenous ligands. Concluding from in situ measurements with fluorescent probes, the cytosolic labile pool is buffered in the low nanomolar to high picomolar concentration range [22]. At the same time, the total concentration of labile cellular zinc, which is best described as the buffer depth, remains unclear but might well approach tens to hundreds of micromolar. The labile nature of this pool implies a complex dynamic equilibrium that is in constant flux, modulated by varying protein affinities for zinc, changes in membrane permeability pathways, and release from intracellular storage forms. Injuries such as hypoxia-ischemia, inflammation, and oxidative/nitrative stress can promote zinc dyshomeostasis [23–26], for example through oxidation of sulfhydryl groups involved in zinc binding or the alterations of zinc binding affinities due to pH changes. Altogether, the physiologic concentration range that allows for zinc signaling appears to be tightly regulated whereas excess or inadequate zinc rapidly leads to toxicity. The distinction between zinc that is exchangeable or labile and the static fraction of zinc that is buried within the active sites of proteins becomes especially important when considering the effect of chelators and zinc ionophores, as well as zinc deprivation, or zinc supplementation, on altering zinc signaling. The non-exchangeable, static fraction of cellular zinc is likely to be unaffected, whereas the labile pool may be vulnerable in zinc-deficient states. Such distinctions are particularly important when considering developmental effects of zinc signaling where newly synthesized transcription factors may require zinc for initial activation.

4. Control of zinc homeostasis Intracellular free zinc concentrations are regulated by Zrt-and-Irt-like Proteins (ZIPs), zinc transporters (ZnTs), and zinc storage proteins, primarily metallothioneins (MTs). There are 14 members of the ZIP family and 10 members of the ZnT family [35–37]. The expression of members of this family has been confirmed in the brain and other organs (reviewed in [38]), thus highlighting the importance of zinc homeostasis in the central nervous system. RNAseq studies indicate expression of many of these in differentiating oligodendrocytes [39] (see Table 1), but scarce data exist regarding the protein expression or function of specific zinc transporters in oligodendrocytes. A single study demonstrated expression of ZnT1 in O4-positive oligodendrocytes in culture, with expression more robust than in astrocytes or microglia [40]. There is also a single study assessing Zn65 uptake in oligodendrocyte progenitors providing evidence for a saturable influx pathway mediated by one or more specific zinc transporters as the application of glutamate receptor or calcium receptor antagonists did not block the uptake [41]. A much more comprehensive characterization of transporter expression and function is required in differentiating oligodendrocytes and how transporters might be modulated during white matter injuries. 5. Zinc in neuronal and oligodendrocyte injury Because of the tight regulation of zinc homeostasis in the brain, both zinc deficiency and excess can rapidly lead to cell death via necrotic, apoptotic, or autophagic pathways (reviewed in [1,4]). Release of zinc from intracellular stores such as mitochondria or metallothioneins, as well as extracellular release primarily from zinc-rich glutamatergic synaptic vesicles, can be toxic. Important studies from Dennis Choi’s laboratory suggested a critical role for intracellular zinc accumulation as an early step leading to neuronal death following global ischemia by showing that injection of a chelating agent could block neurodegeneration but not when that agent was saturated with zinc [23]. Specific zinc entry pathways have been defined, including through opening of calcium-permeable glutamate receptors, voltage-gated calcium channels, TRP channels, and via specific zinc transporters [42–45]. More recent studies using oxygen-glucose deprivation have also demonstrated regional differences in the source of intracellular zinc with CA1 neuronal death dependent on zinc released from metallothionein-3 (MT-3), but CA3 neuronal death dependent on presynaptic zinc release and permeation through Ca2+ and Zn2+ permeable AMPA receptors [46]. Many important downstream signaling pathways activated by intracellular zinc accumulation in neurons have been identified (reviewed in [4,47]). As one example, zinc entry and accumulation in mitochondria is a key early step in ischemia-induced death cascades (reviewed in [48]). Specifically, excess zinc leads to loss of the mitochondrial membrane potential due to opening of the mitochondrial permeability transition pore, leading to release of pro-apoptotic peptides, generation of reactive oxygen species, and ultimately cell death [42,46,49]. Less is known about zinc-induced injury in oligodendrocytes. Prior work from our group has shown that liberation of zinc from intracellular stores contributes to mature oligodendrocyte toxicity during nitrative and oxidative injuries [25]. Zinc triggers downstream

3. Zinc biomarkers and zinc measurements A central problem in zinc biology has been the lack of appropriate biomarkers to measure the zinc status in humans. This is a general challenge with micronutrients. In some cases, such as iron, there are ways to assess sufficiency, for example by using serum concentrations, total iron binding capacity, and ferritin levels. The current consensus to assess zinc status is to use dietary zinc intake, plasma zinc concentrations, and height-for-age of growing infants and children [10]. In adults, there has been some suggestion that the measurement of metallothioneins in leukocytes may be useful in determining zinc status [27]. However, there are likely to be regional differences in zinc sufficiency or zinc depletion and thus brain-specific biomarkers for zinc (and other trace metals) are critically needed. Measurement of total zinc content in myelin from autopsy specimens is possible using inductively coupled plasma mass spectrometry (ICP-MS) [28–30]. This technique can be further combined with cell-specific 2

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Table 1 Expression of Zinc Transporters in Oligodendrocytes. RNAseq data for Zinc Transporters (ZnTs) and Zrt-and-Irtlike Proteins (ZIPs) from 3 stages in the oligodendrocyte lineage: oligodendrocyte progenitors, newly formed oligodendrocytes and myelinating oligodendrocytes. Expressed as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Genes highlighted in red show developmental downregulation and genes highlighted in green show developmental upregulation. Data from [39]. https://web.stanford.edu/group/barres_lab/brain_rnaseq. html. (For interpretation of the references to color in this table legend, the reader is referred to the web version of this article.)

phosphorylation of ERK, activation of 12-lipoxygenase, and generation of reactive oxygen species (ROS). Chelation of mobile zinc with TPEN could prevent the toxicity, as could inhibitors of the downstream targets (reviewed in [50]). Other groups have similarly demonstrated intracellular zinc release as an important step in oligodendrocyte death following anoxia. In the oxygen-glucose deprivation model, a death pathway involves an increase in intracellular mobile zinc, followed by sustained activation of ERK, increase in poly-[ADP]-ribosylation proteins and generation of ROS [51]. Death could be blocked using TPEN, an ERK inhibitor, a poly-[ADP]-ribose polymerase 1 inhibitor, and antioxidants. A third group has demonstrated a role for zinc in excitotoxic injury to oligodendrocytes [52]. Activation of AMPA receptors led to a calcium-dependent mobilization of intracellular zinc from mitochondria and protein-bound pools, but, interestingly, without generation of ROS. Toxicity could be blocked both in vitro and in situ in isolated optic nerves with TPEN. Thus, in response to oxidative, nitrative, ischemic, and excitotoxic stimuli, release of zinc from intracellular stores is a key step leading to oligodendrocyte death. Since many of these stimuli are implicated in diseases such as white matter stroke, multiple sclerosis, and premature brain injury, it is likely that zinc dyshomeostasis is important in all of these disorders.

studies have shown a critical dependence for zinc in mitosis as well [57,58]. Furthermore, in response to growth factors that stimulate proliferation in cell lines, the intracellular labile zinc pool transiently expands, mediated by alterations in expression of zinc transporters to allow adequate zinc supplies for proliferation [59]. Within the brain, prior studies have focused on neurons and identified important roles in proliferation [60]. Pregnant rats that were fed a marginally zinc-deficient diet throughout gestation resulted in E19 pups with decreased neural progenitor cell proliferation in the ventricular zone [61]. In this study, effects were attributed to decreased ERK phosphorylation secondary to reduced zinc inhibition of an ERK targeting phosphatase, protein phosphatase 2A. Follow up work has shown that reductions in neuronal number persist into adulthood and, in addition to alterations in ERK signaling, critical transcription factors for neuronal development such as Sox2, Pax6, Tbr1, and Tbr2 are also modulated by zinc deficiency [62]. Neurogenesis in the adult hippocampus is reduced with dietary zinc deficiency [60,63–65], whereas dietary zinc supplementation promotes neurogenesis [66,67]. It is reasonable to hypothesize that zinc may play similar roles in dividing oligodendrocyte precursors (OPCs), although such experiments have not been performed.

6. Zinc and proliferation

7. Zinc and signaling

Zinc plays a role in proliferation in many cells types [53,54]. For example, during the final steps of oocyte maturation, intracellular zinc concentrations increase by more than 50%, followed by ejection of “zinc sparks” after fertilization to lower the concentration of intracellular zinc and allow cell cycle progression [55,56]. Multiple

Nearly 10% of the proteome binds zinc [68], including many enzymes and transcription factors critical in myelination. The ERK pathway has been well studied in the setting of zinc dyshomeostasis. ERK activity is altered by zinc-mediated regulation of protein phosphatases, as discussed above. ERK is critical for oligodendrocyte 3

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proliferation and differentiation [69,70], so alterations in zinc homeostasis would be predicted to change zinc-mediated ERK function. This has already been shown in states of injury (see oligodendrocyte injury section), but may also be important during normal oligodendrocyte development and myelination. HDACs also play essential roles in oligodendrocyte differentiation [71,72] and bind zinc [73,74]. Many HDAC inhibitors function as zinc chelators [75], illustrating the necessity of zinc for proper enzyme function. The regulation of these proteins might be accomplished by alterations in zinc affinity from changes in the local environment or protein modifications, or from increased zinc binding if the proteins are not fully saturated at baseline. There are numerous examples of zinc-finger transcription factors such as Zfp488, Znf16 l, Znf24 and CXXC5 [76–80], highlighting another avenue for zinc signaling in the brain, particularly during development.

pathogenesis of MS, similar to the function described earlier during global ischemia, and, importantly, that restoration of zinc homeostasis is protective in this disorder. It may seem paradoxical that both zinc deficiency and excess play a role in MS. It is important to recognize that MS is a complex disorder with both demyelination and remyelination phases. Similar to other disorders where zinc has been implicated, the critical distinction is likely the specific local zinc requirements. The two EAE model studies discussed above target synaptically released zinc, preventing neuronal toxicity and immune activation. The authors focused on prevention of demyelination and showed that excess zinc is clearly toxic. However, the remyelination phase and effects on oligodendrocyte survival and differentiation were not addressed in these studies. While excess zinc is likely to be toxic to oligodendrocytes, during the subsequent remyelination phase there may be a necessity for influx of zinc for remyelination to proceed. Based on the limited human studies in brains from MS patients showing reductions in total zinc in MS and no evidence of accumulation of zinc in plaques to support a zinc-overload hypothesis [85], it is reasonable to think that local zinc deficiency may play a role in remyelination failure. Using zinc modulating agents and genetic manipulation of zinc homeostasis during remyelination are important future studies. Thus, relative zinc levels will likely depend on location, timing, and cell type.

8. Zinc in multiple sclerosis There is increasing evidence that zinc dyshomeostasis occurs in patients with multiple sclerosis (MS) (reviewed in [81]). Some studies have suggested a lower serum concentration of zinc in patients with MS [82–84]. Synchrotron X-ray fluorescence imaging and quantification of the total zinc content in autopsy specimens collected from MS patients demonstrated that zinc was decreased in most white matter lesions and paralleled myelin loss [85]. These results are intriguing given that prior work has suggested an important structural role for zinc ions in maintaining myelin integrity and compaction [86–88]. Exogenous zinc inhibited dissociation of MBP (myelin basic protein) from myelin membranes as well as accumulation of intact MBP in incubation media [89]. Furthermore, gel filtration studies revealed that endogenous zinc co-eluted with both MBP and proteolipid protein (PLP), thus implicating zinc in stabilizing MBP in myelin membranes by promoting binding to PLP. White matter contains substantial zinc, estimated to be in the 0.05 mM range [89,90] and retained with extensive washing or application of EDTA, suggesting at least a portion is tightly bound [91]. More recently, Tsang and colleagues identified 3 major zinc-binding proteins in porcine brain homogenates, one of which was identified to be MBP [88]. They also showed that MBP-induced aggregation of phospholipid vesicles was enhanced in the presence of zinc, suggesting stabilizing effects on MBP–membrane interactions. In addition to interactions with MBP, zinc also binds to the cytoplasmic domain of myelin-associated glycoprotein (MAG), inducing a conformational change that leads to increased surface hydrophobicity [92], which in turn was predicted to alter protein-protein interactions and secondary structure. Together, these studies implicate zinc as a component of several major myelin proteins, likely with important functional roles in myelin homeostasis. MS animal models also provide evidence that restoration of zinc homeostasis may promote myelin repair. Using clioquinol, which serves as both a chelator capable of binding zinc (and copper) as well as an ionophore capable of delivering zinc (and copper) to local areas with relative zinc deficiency, another group showed reduced spinal cord white matter injury and improved motor behavior scores in the experimental autoimmune encephalomyelitis model [93]. In this study, the effects were attributable primarily to clioquinol chelation of zinc released at synapses, which could be visualized by a fluorescent zinc sensor TSQ. There was also reduced activation of metal metalloprotease-9 (MMP9, a zinc-dependent protease), reduced activation of microglia, T-cells and B-cells, and increased autophagy. In this case, increased autophagy is thought to be beneficial by quickly removing damaged cells, thus preventing more extensive injury. The same researchers subsequently demonstrated that mice lacking ZnT3, the primary zinc transporter within synaptic vesicles in a subset of glutamatergic neurons [94], also had reduced demyelination, immune cell activation, MMP9 activation, blood-brain barrier disruption, and improved behavioral scores [95]. The protective effect of ZnT3 knockout suggests that vesicular zinc from neurons may play a role in the

9. New tools to probe zinc signals in the brain As discussed above, a central challenge in studying zinc signals in the brain has been the absence of sensitive and specific tools to visualize dynamic changes in vivo or in vitro. In the last decade, new zinc-selective fluorescent probes and chelators have been developed, finally allowing a window into this important biology. From a historical perspective, parallels with calcium biology are inevitable. Calcium concentrations are orders of magnitude lower than magnesium concentrations and tools to differentiate the two ions were not available until Roger Tsien developed the ratiometric calcium probe fura-2 and the calcium selective chelator BAPTA in the 1980’s [96], ultimately unraveling the essential role of intracellular calcium signals modulating neuronal activity [97]. Similar work to differentiate zinc from calcium has been undertaken by multiple groups leading to the synthesis of fluorescent probes such as Newport green, FluoZin-3, zinpyr-1, chromis-1, and genetically encoded zinc sensors [8,98–104]. The novel extracellular zinc chelator ZX1 [6], which does not bind to calcium or magnesium, has become an indispensable tool for manipulating zinc-dependent neuronal processes in situ. 10. Non-ratiometric flurorescent zinc probes in neurons and oligodendrocytes The initial development of sensitive and zinc-selective sensors permitted many important discoveries in zinc biology in the brain. For example, Newport green allowed visualization of synaptically released zinc and discovery of the importance of zinc in mitochondrial ROS production leading to neuronal injury [42]. It similarly allowed differentiation of calcium and zinc responses to thiol oxidation, highlighting the importance of zinc in induction of neuronal apoptosis [24]. FluoZin-3 used in combination with fura-2, allowed separation of calcium and zinc signals in the same neurons [105], ultimately allowing recognition that increase in mobile zinc may be the trigger for neurodegeneration rather than calcium [106]. In oligodendrocytes, FluoZin-3 was essential to demonstrate that activation of AMPA receptors triggered calcium-dependent release of intracellular zinc from mitochondria [52]. Similarly, our prior studies also used FluoZin-3 to demonstrate intracellular zinc release in oligodendrocytes in response to peroxynitrite, allowing characterization of a zinc-dependent toxicity pathway in response to oxidative/nitrative stress [25]. While invaluable in identifying qualitative changes in zinc and differentiating them 4

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from calcium signals, they do not allow for quantifying zinc concentrations.

and MAG, highlighting the likely importance of zinc signaling in oligodendrocytes and white matter. Ratiometric zinc probes such as chromis-1 significantly expand the toolbox necessary for unraveling the role of zinc signals in myelination and oligodendrocyte development in the future.

11. Chromis-1, a zinc-responsive emission-ratiometric fluorescent probe Developed for visualizing labile zinc pools by two-photon excitation microscopy (TPEM), the fluorescent probe chromis-1 revealed dynamic changes of zinc concentrations in differentiating oligodendrocytes [8]. A central problem in white matter injury of prematurity, remyelination failure in multiple sclerosis, and failure of repair following white matter stroke is a block in oligodendrocyte differentiation such that developing oligodendrocytes do not mature into myelin basic protein expressing cells [107–110]. Because of the importance of zinc in proliferation and differentiation, changes in intracellular zinc levels might be important drivers of oligodendrocyte differentiation programs. With a Kd of 2.4 nM, chromis-1 covers a dynamic range from high pico- to low nanomolar buffered zinc concentrations. In differentiating oligodendrocytes, chromis-1 revealed a 2-fold reduction in buffered zinc levels of mature oligodendrocytes compared to developing oligodendrocytes. While traditional turn-on fluorescence probes produce a signal that depends on both the probe and analyte concentration, ratiometric probes respond with a spectral shift upon analyte binding such that the ratio of fluorescence intensities, simultaneously acquired through two bandpass filters, is not affected by uneven cellular loading or non-uniform subcellular probe distributions. Upon in vitro calibration of the ratiometric response using solutions with well-defined buffered zinc concentrations, cellular zinc levels can then be estimated based on the intensity ratios derived from imaging data. Using this approach, chromis-1 revealed buffered zinc levels in the subnanomolar range in oligodendrocytes, which is in agreement with prior studies of other cell types using genetically encoded probes [98,103]. There are multiple possibilities for the drop in buffered zinc concentrations observed in more mature oligodendrocytes. As discussed earlier, zinc binds to multiple proteins in the myelin sheath, likely contributing to important structural characteristics of myelin. Chromis-1 may not equilibrate with myelin-bound zinc due to either insufficient binding affinity or lack of access to the myelin-containing cellular compartment. A second possibility is that chromatin binding proteins necessary for myelination programs might competitively bind zinc. This would likely produce an increase in nuclear zinc and also require translocation of zinc from the cytoplasm into the nucleus. This scenario could be further explored with a nuclear-targeted zinc probe of appropriate binding affinity, combined with synchrotron x-ray fluorescence imaging studies or ICPMS to assess total zinc levels. A third possibility is that zinc associates with cytoplasmic proteins important in signaling initiation of myelination. Thus, ratiometric probes such as chromis-1 represent powerful new tools to image changes in buffered zinc levels in a variety of cellular contexts, including development, injury, and physiological activation. Specifically developed for two-photon microscopy, chromis-1 is particularly well suited for deep-tissue imaging applications and in vivo imaging studies requiring continuous observation over extended time periods with minimal phototoxicity or photobleaching.

Acknowledgements This work was supported in part by the National Institute of Neurological Disorders and Stroke Grant K12NS079414 (CME), the Baby Alex Foundation Grant (CME), the Philip R. Dodge Young Investigator Award from the Child Neurology Society (CME), internal funding from the Department of Neurology at Boston Children’s Hospital (CME), the National Institute of General Medical Sciences Grant R01GM067169 (CJF), the National Eye Institute Grants R01EY024481 (PAR) and R01EY027881 (PAR), and the Intellectual and Developmental Disabilities Research Center at Boston Children’s Hospital (IDDRC) HD018655. References [1] S.L. Sensi, P. Paoletti, A.I. Bush, I. Sekler, Zinc in the physiology and pathology of the CNS, Nat. Rev. Neurosci. 10 (2009) 780–791. [2] N. Levaot, M. Hershfinkel, How cellular Zn(2+) signaling drives physiological functions, Cell Calcium 75 (2018) 53–63. [3] C.J. Frederickson, J.Y. Koh, A.I. Bush, The neurobiology of zinc in health and disease, Nat. Rev. Neurosci. 6 (2005) 449–462. [4] S.L. Sensi, P. Paoletti, J.Y. Koh, E. Aizenman, A.I. Bush, M. Hershfinkel, The neurophysiology and pathology of brain zinc, J. Neurosci. 31 (2011) 16076–16085. [5] J.M. Goldberg, S.J. Lippard, New tools uncover new functions for mobile zinc in the brain, Biochemistry 57 (2018) 3991–3992. [6] E. Pan, X.A. Zhang, Z. Huang, A. Krezel, M. Zhao, C.E. Tinberg, S.J. Lippard, J.O. McNamara, Vesicular zinc promotes presynaptic and inhibits postsynaptic long-term potentiation of mossy fiber-CA3 synapse, Neuron 71 (2011) 1116–1126. [7] C.E.R. Richardson, L.S. Cunden, V.L. Butty, E.M. Nolan, S.J. Lippard, M.D. Shoulders, A method for selective depletion of Zn(II) ions from complex biological media and evaluation of cellular consequences of Zn(II) deficiency, J. Am. Chem. Soc. 140 (2018) 2413–2416. [8] D. Bourassa, C.M. Elitt, A.M. McCallum, S. Sumalekshmy, R.L. McRae, M.T. Morgan, N. Siegel, J.W. Perry, P.A. Rosenberg, C.J. Fahrni, Chromis-1, a ratiometric fluorescent probe optimized for two-photon microscopy reveals dynamic changes in labile Zn(II) in differentiating oligodendrocytes, ACS Sens. 3 (2018) 458–467. [9] J.C. King, Zinc: an essential but elusive nutrient, Am. J. Clin. Nutr. 94 (2011) 679S–684S. [10] J.C. King, K.H. Brown, R.S. Gibson, N.F. Krebs, N.M. Lowe, J.H. Siekmann, D.J. Raiten, Biomarkers of nutrition for development (BOND)-zinc review, J. Nutr. 146 (Suppl) (2016) 858S–885S. [11] L.S. Hurley, H. Swenerton, Congenital malformations resulting from zinc deficiency in rats, Proc. Soc. Exp. Biol. Med. 123 (1966) 692–696. [12] A.S. Prasad, A.R. Schulert, A. Miale Jr, Z. Farid, H.H. Sandstead, Zinc and iron deficiencies in male subjects with dwarfism and hypogonadism but without ancylostomiasis, schistosomiasis or severe anemia, Am. J. Clin. Nutr. 12 (1963) 437–444. [13] A.S. Prasad, A. Miale Jr, Z. Farid, H.H. Sandstead, A.R. Schulert, Zinc metabolism in patients with the syndrome of iron deficiency anemia, hepatosplenomegaly, dwarfism, and hypognadism, J. Lab. Clin. Med. 61 (1963) 537–549. [14] H. Liu, P.I. Oteiza, M.E. Gershwin, M.S. Golub, C.L. Keen, Effects of maternal marginal zinc deficiency on myelin protein profiles in the suckling rat and infant rhesus monkey, Biol. Trace Elem. Res. 34 (1992) 55–66. [15] I.E. Dreosti, S.J. Manuel, R.A. Buckley, F.J. Fraser, I.R. Record, The effect of late prenatal and/or early postnatal zinc deficiency on the development and some biochemical aspects of the cerebellum and hippocampus in rats, Life Sci. 28 (1981) 2133–2141. [16] N. Danbolt, Acrodermatitis enteropathica, Br. J. Dermatol. 100 (1979) 37–40. [17] A.S. Prasad, Discovery of human zinc deficiency: its impact on human health and disease, Adv. Nutr. 4 (2013) 176–190. [18] A. Ohlsson, Acrodermatitis enteropathica reversibility of cerebral atrophy with zinc therapy, Acta Paediatr, Scand. 70 (1981) 269–273. [19] Y. Golan, T. Kambe, Y.G. Assaraf, The role of the zinc transporter SLC30A2/ZnT2 in transient neonatal zinc deficiency, Metallomics 9 (2017) 1352–1366. [20] G. Terrin, R. Berni Canani, M. Di Chiara, A. Pietravalle, V. Aleandri, F. Conte, M. De Curtis, Zinc in early life: a key element in the fetus and preterm neonate, Nutrients 7 (2015) 10427–10446. [21] W. Maret, Zinc biochemistry: from a single zinc enzyme to a key element of life, Adv. Nutr. 4 (2013) 82–91. [22] W. Maret, Zinc in cellular regulation: the nature and significance of "zinc signals", Int. J. Mol. Sci. 18 (2017).

12. Conclusions In summary, zinc is an essential micronutrient that is enriched in the brain and tightly regulated. There are multiple risk factors for zinc deficiency with deleterious effects on brain development and myelination in particular. Control of zinc homeostasis in the brain is critical, and primarily accomplished by zinc transporters and zinc storage proteins. The function of specific zinc transporters in oligodendrocytes remains to be determined. There is convincing data that an increase in intracellular zinc leads to oligodendrocyte death in multiple injury paradigms, but less is known about zinc signaling in development. Many enzymes and transcription factors bind to zinc, including MBP 5

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