Neurulation and neurite extension require the zinc transporter ZIP12 (slc39a12) Winyoo Chowanadisaia,b,1, David M. Grahamc, Carl L. Keena, Robert B. Ruckera, and Mark A. Messerlib,c aDepartment of Nutrition, University of California, Davis, CA 95616; bCellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA 02543; and cEugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543 Edited by Yuh Nung Jan, Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, and approved May 1, 2013 (receivedfor review December 19, 2012) Zn2+ is required for many aspects of neuronal structure and func- We observed that ZIP12 is important for multiple aspects of + tion. However, the regulation of Zn2 in the nervous system remains neuronal differentiation, including activation of cAMP response poorly understood. Systematic analysis of tissue-profiling microar- element-binding protein (CREB), tubulin polymerization, and ray data showed that the zinc transporter ZIP12 (slc39a12)ishighly neurite extension, in vitro. We show that ZIP12 is required for expressed in the human brain. In the work reported here, we con- neurulation and embryonic viability during Xenopus tropicalis + + firmed that ZIP12 is a Zn2 uptake transporter with a conserved development. These findings show that the Zn2 transporter + patternofhighexpressioninthemouseandXenopus nervous sys- ZIP12 represents a point of regulation that links Zn2 directly to tem. Mouse neurons and Neuro-2a cells produce fewer and shorter nervous system development. + neurites after ZIP12 knockdown without affecting cell viability. Zn2 + chelation or loading in cells to alter Zn2 availability respectively Results mimicked or reduced the effects of ZIP12 knockdown on neurite ZIP12 Is Highly Expressed in the Brain. We analyzed a previously outgrowth. ZIP12 knockdown reduces cAMP response element-bind- published microarray dataset (14) for human genes in the SLC30 ing protein activation and phosphorylation at serine 133, which is and SLC39 families with brain-specific patterns of expression a critical pathway for neuronal differentiation. Constitutive cAMP that are likely to be important for nervous system development response element-binding protein activation restores impairments and function (Fig. S1A). Of 32,878 probe sets, 1,130 genes + in neurite outgrowth caused by Zn2 chelation or ZIP12 knockdown. (3.44%) had a brain expression ratio greater than 5; of these, ZIP12 knockdown also reduces tubulin polymerization and increases 1,118 genes (3.40%) were annotated with a gene symbol, Na- MEDICAL SCIENCES sensitivity to nocodazole following neurite outgrowth. We find that tional Center for Biotechnology Information accession number, ZIP12 is expressed during neurulation and early nervous system de- or Celera transcript number (Dataset S1). The gene expression velopment in Xenopus tropicalis, where ZIP12 antisense morpholino of slc39a12 is 47-fold higher in the human brain than in other knockdown impairs neural tube closure and arrests development tissues (Fig. S1A). The top five genes with the highest brain- during neurulation with concomitant reduction in tubulin polymer- expression ratios (Dataset S1) have documented nervous system- + ization in the neural plate. This study identifies a Zn2 transporter specific expression patterns and functions. The high expression that is specifically required for nervous system development and of ZIP12 in the human brain identified in our analysis is con- + provides tangible links between Zn2 , neurulation, and neuronal sistent with a previous transcriptome analysis using expressed differentiation. sequence tag data (8). Next, we examined slc39a12 expression in mice to gain insight brain development | CREB | neural tube defect | zinc deficiency | into the possible physiological roles of ZIP12. Our gene- birth defects expression analysis revealed high levels of mZIP12 mRNA in brain (Fig. 1A). We created an mZIP12-specific antibody that pproximately 12% of Americans fail to consume the Esti- binds to an N-terminal epitope before the first transmembrane + Amated Average Requirement for Zn2 and could be at risk domain (Fig. 1B). mZIP12 protein expression was primarily in + for zinc deficiency (1). Zn2 is a nutrient that plays critical roles in the mouse brain (Fig. 1C) and was detected in the hippocampus, more than 1,000 proteins, including enzyme catalysis, cell signaling, frontal cortex, striatum, hypothalamus, and cerebellum (Fig. 1D). and DNA repair (2), and as a result is essential for neural de- Primary mouse neurons have endogenous ZIP12 expression at the E velopment, learning, and memory (3). Gene–nutrient interactions plasma membrane (Fig. 1 ). mZIP12 localization to the plasma fi influence brain development, structure, and function throughout membrane also was con rmed via epitope labeling in unpermea- 2+ bilized CHO cells transfected with mZIP12-HA (Fig. S1 B–E). all stages of life (4). Zn supplementation can reduce the risk 2+ 2+ – Sequestration of extracellular Zn with the Zn chelator N,N,N′, for certain pregnancy complications (5 7), including congenital ′ defects, by preventing primary deficiencies caused by diet or by N -tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) resulted in treating secondary deficiencies, such as acrodermatitis enter- a redistribution of mZIP12 from the perinuclear space to the cy- 2+ toplasm and plasma membrane, as detected by indirect immuno- opathica, a genetic disorder caused by a defect in intestinal Zn 2+ fluorescence (Fig. S1D) and cell-surface protein biotinylation (Fig. transport secondary to a mutation in the Zn IRT-like protein + S1E). TPEN also increased Zn2 uptake in both control and ZIP4 (5). Members of the solute carrier 39 (SLC39) gene family + mZIP12-transfected cells (Fig. S1F), indicating that mZIP12 is encoding the Zn2 IRT-like proteins (ZIPs) are important com- + present at the plasma membrane of transfected CHO cells. ponents of cellular Zn2 homeostasis and encode proteins that + promote cellular Zn2 uptake in a wide range of species (8). In vertebrates, mutations in some SLC39 members have been linked – Author contributions: W.C., D.M.G., R.B.R., and M.A.M. designed research; W.C., D.M.G., to developmental and metabolic disorders (5, 9 13), generally and M.A.M. performed research; C.L.K. contributed new reagents/analytic tools; W.C., D.M.G., leading to pleiotropic phenotypes. and M.A.M. analyzed data; and W.C., D.M.G., C.L.K., R.B.R., and M.A.M. wrote the paper. Here, we analyzed published genome-wide microarray data The authors declare no conflict of interest. (14) to determine that slc39a12 (ZIP12) is highly expressed in the This article is a PNAS Direct Submission. human brain relative to other SLC30 and SLC39 transporters. We 1To whom correspondence should be addressed. E-mail: [email protected]. used a reverse genetics approach to demonstrate that ZIP12 is an This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2+ essential Zn transporter predominantly expressed in the brain. 1073/pnas.1222142110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1222142110 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 + ZIP12 Expression and Intracellular Zn2 Increase During Stimulated Neurite Outgrowth. We conducted studies to determine if neuro- + nal differentiation is accompanied by changes in cellular Zn2 homeostasis and mZIP12 expression. We used the mouse neuro- blastoma cell line, Neuro2a (N2a), a well-characterized model for retinoic acid (RA)-induced differentiation (16). Quantitative trans- cript analysis of endogenous mZIP12 in N2a cells revealed an approximately threefold increase in mZIP12 gene expression in differentiated cells versus control and reduced serum conditions (Fig. S2A). A similar increase in mZIP12 mRNA expression was found in neuronal precursor cells (NPCs) (Fig. S2B) following differentiation. Endogenous mZIP12 protein expression increased in N2a cells after differentiation (Fig. S2C). After 48 h differenti- + ated N2a cells displayed significantly increased Zn2 uptake (Fig. S2D), as measured by 65Zn, and increased fluorescence of the zinc fluorophore Zinpyr-1 in perinuclear compartments, as measured Fig. 1. ZIP12 is primarily expressed in the human and mouse brain. (A)ZIP12 by microscopy (Fig. S2E)andflow cytometry (Fig. S2 F and G), mRNA expression detected in various mouse tissues by RT-PCR. (B) Detection compared with undifferentiated cells. Time-dependent increases in of mouse ZIP12 by immunoblotting in mZIP12-transfected CHO cells. (C and D) neurite extension, an early morphological sign of neuronal differ- ZIP12 protein expression detected in various mouse tissue lysates (C) and H brain region lysates (D) by immunoblotting. (C) Tissues include (from left to entiation, were observed in N2a cells treated with RA (Fig. S2 ). 2+ right): brain, lung, skeletal muscle, liver, small intestine, heart, kidney, and These changes in Zn metabolism induced by differentiation oc- pancreas. (D) Brain regions include (from left to right): hippocampus (Hipp), curred without affecting cell viability (Fig. S2I). frontal cortex (FC), striatum (Str), hypothalamus (Hyp), and cerebellum (Cerb). + (E) ZIP12 is present at the plasma membrane in primary mouse neurons. (Scale ZIP12 Knockdown Reduces Zn2 Uptake and Neurite Extension During bar: 10 μm.) (F–I) ZIP12 is present in coronal sections of various regions of the Differentiation. We used a ZIP12-specific