Neurulation and neurite extension require the zinc transporter ZIP12 (slc39a12)

Abstract

Zn(2+) is required for many aspects of neuronal structure and function. However, the regulation of Zn(2+) in the nervous system remains poorly understood. Systematic analysis of tissue-profiling microarray data showed that the zinc transporter ZIP12 (slc39a12) is highly expressed in the human brain. In the work reported here, we confirmed that ZIP12 is a Zn(2+) uptake transporter with a conserved pattern of high expression in the mouse and Xenopus nervous system. Mouse neurons and Neuro-2a cells produce fewer and shorter neurites after ZIP12 knockdown without affecting cell viability. Zn(2+) chelation or loading in cells to alter Zn(2+) availability respectively mimicked or reduced the effects of ZIP12 knockdown on neurite outgrowth. ZIP12 knockdown reduces cAMP response element-binding protein activation and phosphorylation at serine 133, which is a critical pathway for neuronal differentiation. Constitutive cAMP response element-binding protein activation restores impairments in neurite outgrowth caused by Zn(2+) chelation or ZIP12 knockdown. ZIP12 knockdown also reduces tubulin polymerization and increases sensitivity to nocodazole following neurite outgrowth. We find that ZIP12 is expressed during neurulation and early nervous system development in Xenopus tropicalis, where ZIP12 antisense morpholino knockdown impairs neural tube closure and arrests development during neurulation with concomitant reduction in tubulin polymerization in the neural plate. This study identifies a Zn(2+) transporter that is specifically required for nervous system development and provides tangible links between Zn(2+), neurulation, and neuronal differentiation.

Keywords: CREB; birth defects; brain development; neural tube defect; zinc deficiency.

Fig. 1.

ZIP12 is primarily expressed in the human and mouse brain. (A) ZIP12 mRNA expression detected in various mouse tissues by RT-PCR. (B) Detection of mouse ZIP12 by immunoblotting in mZIP12-transfected CHO cells. (C and D) ZIP12 protein expression detected in various mouse tissue lysates (C) and brain region lysates (D) by immunoblotting. (C) Tissues include (from left to right): brain, lung, skeletal muscle, liver, small intestine, heart, kidney, and pancreas. (D) Brain regions include (from left to right): hippocampus (Hipp), frontal cortex (FC), striatum (Str), hypothalamus (Hyp), and cerebellum (Cerb). (E) ZIP12 is present at the plasma membrane in primary mouse neurons. (Scale bar: 10 μm.) (F–I) ZIP12 is present in coronal sections of various regions of the mouse brain. Brain regions include (F) cerebellum (including Purkinje cell layer), (G) medulla oblongata, (H) frontal cortex, and (I) corpus callosum (and cortex). Arrows in F and G indicate neuronal staining. [Scale bar: 50 μm (60× magnification) in F and G; 200 μm (20× magnification) in H and I.]

Fig. 2.

ZIP12 is a high-affinity, Zn²⁺- specific transporter. (A) Zn²⁺ uptake at different concentrations of free external Zn²⁺ by ZIP12 in CHO cells was measured using ⁶⁵Zn (n = 3, ± SE). The curve marked ZIP12 – Control represents net Zn²⁺ uptake in ZIP12-transfected cells minus uptake in control-transfected cells. (B) Zn²⁺ uptake specificity of ZIP12 relative to other metals determined using ⁶⁵Zn (n = 3, ± SE). Excess Zn²⁺ indicates cold Zn²⁺ added to uptake buffer during assay. (C) Increased Zn²⁺ content relative to cell number by ZIP12 was measured by ICP-MS (n = 6, ± SE). (D) ZIP12 increases MRE activation, measured by reporter assay (n = 6, ± SE). (E) ZIP12 overexpression does not affect cell viability at 48 h posttransfection, measured by Trypan blue exclusion (n = 6, ± SE). **P < 0.01; ***P < 0.001 versus control cells.

Fig. 3.

Neurite extension is dependent on Zn²⁺ transport by ZIP12 and is affected by Zn²⁺ availability. (A and B) ZIP12 shRNA reduces neurite length in N2a cells (n = 50, ± SE). (C) Chelation of extracellular Zn²⁺ with DTPA mimics the impairment of ZIP12 knockdown on neurite length in N2a cells (n = 50, ± SE). (D) Zn²⁺ carrier pyrithione (ZP) restores neurite outgrowth impaired by ZIP12 shRNA knockdown in N2a cells (n = 50, ± SE). (Scale bars: 100 μm.) **P < 0.0, ***P < 0.001 versus control cells without DTPA or ZP; ^##P < 0.01, ^###P < 0.001 versus control cells with DTPA or ZIP12-shRNA cells without ZP.

Fig. 4.

ZIP12 is required for CREB signaling and tubulin polymerization. (A and B) Differentiation of N2a cells increases cAMP response element (CRE) activation (n = 6, ± SE) and CREB phosphorylation, both of which are blunted by ZIP12 shRNA-mediated knockdown. ***P < 0.001 versus control undifferentiated cells; ^##P < 0.01 versus control differentiated cells. (C) Extracellular Zn²⁺ chelation by DTPA mimics the blunting of CREB activation in differentiated cells induced by ZIP12 shRNA knockdown (n = 6, ± SE). ***P < 0.001 versus control undifferentiated cells; ^##P < 0.01 versus control differentiated cells; ⁺⁺⁺P < 0.001 versus cells treated with DTPA. (D) Intracellular Zn²⁺ chelation by TPEN reduces phosphorylation of CREB regardless of ZIP12 shRNA knockdown. (E) Constitutive activation of CREB by VP16-CREB1 transfection increases neurite extension in the absence of RA. ***P < 0.001 versus cells transfected with control plasmid. (F and G) Neurite outgrowth induced by constitutive activation of CREB is not affected by (F) extracellular Zn²⁺ chelation or (G) ZIP12 shRNA-mediated knockdown. ***P < 0.001 versus cells transfected with control plasmid in reduced (Red) serum medium. ^###P < 0.001 versus cells transfected with control plasmid and differentiating medium. (H) ZIP12 knockdown alters soluble and polymerized tubulin fractions in N2a cells without affecting total tubulin protein expression. (I) ZIP12 knockdown in differentiated N2a cells increases sensitivity to neurite retraction following microtubule destabilization by nocodazole (n = 50, ± SE). ***P < 0.001 versus cells before nocodazole.

Fig. 5.

ZIP12 is expressed primarily in the neural tube and brain of X. tropicalis and is required for neural tube closure and embryonic viability. (A and B) ZIP12 and β-actin or GAPDH expression was determined in various adult tissues and developmental stages by RT-PCR. (C) ZIP12 mRNA expression is elevated in the neural plate, as determined by quantitative RT-PCR (n = 6, ± SE). ***P < 0.001 versus whole embryo; ^###P < 0.001 versus rest of embryo. (D) ZIP12 mRNA (slc39a12) is expressed during neurulation and early nervous system development, analyzed by in situ hybridization. (E–M) ZIP12 knockdown by antisense morpholino microinjection impairs X. tropicalis development during neurulation. (N) Microinjection of slc39a12MO1 does not affect tubulin protein content, analyzed by immunoblotting. (O) Microinjection of slc39a12MO1 affects the ratio of polymerized to soluble β2-tubulin, as analyzed by polymerized tubulin fractionation and immunoblotting. (Scale bars: 250 μm in all images.)