Zinc Deficiency Decreases Neurite Extension via CRMP2 Signal Pathway

Abstract

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3β (pGSK3β) relative to GSK3β in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3β inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3β. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

INTRODUCTION

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children under 5 years of age.^1,2) In experiments using rats, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood.³⁾ Zinc supplementation of pregnant women and children with zinc deficiency has been reported to reduce premature births and low birth weight, and to reduce death and stunted growth in children due to infectious diarrhea.^4,5) Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail.

Collapsin response mediator protein 2 (CRMP2) forms dimers with Tubulin to promote neuroaxonal elongation.⁶⁾ Phosphorylation of Thr-514 in CRMP2 by glycogen synthase kinase 3β (GSK3β) inhibits neuroaxonal elongation.⁷⁾ It has been reported that CRMP2 could be related to various neural functions such as axonogenesis, neuronal migration, synaptogenesis, and synaptic plasticity, as well as in neurodegenerative diseases.⁸⁾ However, there are no reports of relationship between the CRMP pathway and neurodevelopmental depression due to disruption of zinc homeostasis. This study was performed to determine the effects of low zinc status on neurite outgrowth and CRMP2 signal pathway.

MATERIALS AND METHODS

Cell Culture

Human neuroblastoma cell line (SH-SY5Y) was cultured in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich, St. Louis, MO, U.S.A.) supplemented with 10% fetal bovine serum (FBS) under 5% CO₂ at 37 °C.

Preparation of Zinc-Free DMEM

Zinc-free DMEM was prepared based on previous studies.^9,10) Briefly, 100 g of Chelex^®100 (BioRad, Hercules, CA, U.S.A.) was added in 500 mL of H₂O, adjusted with HCl to pH 7.4, and filtered. The adjusted Chelex^®100 resin was then added in 500 mL of FBS and stirred at room temperature for 4 h, followed by stirring at 4 °C overnight. The Chelex-treated FBS was filtered and sterilized, and the removal of zinc was confirmed by atomic absorption spectrophotometry (SHIMADZU, Kyoto, Japan).

Differentiation of SH-SY5Y Cells

SH-SY5Y cells were seed at 2.0 × 10⁵ cells/mL in 6-well plate for Western blot, and at 1.5 × 10⁵ cells/mL in 24-well plate for immunofluorescence stain. At 24 h after seeding, cells were differentiated in DMEM supplemented with 2% of zinc-free FBS and 10 µM of retinoic acid). Four micromolar of ZnSO₄ was treated to cells at the beginning of differentiation as control group.

Immunofluorescence Stain and Determination of Neurite Extension

Cells were fixed in PHEM buffer (60 mM PIPES, 25 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 10 mM ethylenediaminetetraacetic acid (EDTA), 2 mM MgCl₂) containing 4% of paraformaldehyde, 0.25% of glutaraldehyde. Immunofluorescence stain was performed according to the method a slight modified from the previous study.¹¹⁾ Primary antibody of mouse monoclonal anti-TUJ1 antibody (1 : 2000 dilution) (Sigma-Aldrich) (RRID:AB_1841228) and second antibody (Alexa Fluor^®488 goat anti-mouse immunoglobulin G (IgG) H&L) (1 : 200 dilution) (Thermo Fisher Scientific, Waltham, MA, U.S.A.) were used. Nuclei was stained using 4′,6-diamidino-2-phenylindole (DAPI) (1 : 2000 dilution) (Thermo Fisher Scientific) (RRID:AB_2534088). Fluorescence images were obtained by In Cell Analyzer 2200 (GE Healthcare Life Sciences, Chicago, IL, U.S.A.) or Evos FL Auto2 imaging system (Thermo Fisher Scientific) and were analyzed for neurite extension by In Cell Investigator software (GE Healthcare Life Sciences) or Celleste Image Analysis Software (Thermo Fisher Scientific). Data of neurite length was calculated as sum of neurite length in total cells divided by number of nuclear, and this data was calculated per picture. Measurement of neurite length was performed in 34–36 or 100 pictures per experimental group.

Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting

Cells were lysed with radio immunoprecipitation assay (RIPA) buffer and protein sample was prepared. SDS-PAGE and Western blotting (WB) were performed according to the previous studies.^12,13) Primary antibodies used for WB in this study were following; mouse anti-β-actin antibody (Sigma-Aldrich) (1 : 1000 dilution) (RRID:AB_476692), rabbit anti-CRMP2 antibody (Abcam, Cambridge, U.K.) (1 : 1000 dilution) (RRID:AB_1078573), rabbit anti-pCRMP2 (T-514) antibody (Abcam) (1 : 1000 dilution) (RRID:AB_942229), rabbit anti-GSK3β antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) (1 : 1000 dilution) (RRID:AB_490890), rabbit anti-pGSK3β antibody (Ser-9) (Cell Signaling Technology) (1 : 1000 dilution) (RRID:AB_2115201). Second antibodies used for WB in this study were following; goat anti-mouse IgG antibody, peroxidase conjugated, H + L (1 : 5000 dilution) (Merck KGaA, Darmstadt, Germany) (RRID:AB_90456), goat anti-rabbit IgG antibody, peroxidase conjugated, H + L (1 : 5000 dilution) (Merck KGaA) (RRID:AB_90264). The band image was obtained by Fusion Solo S (Vilber Lourmat, Eberhardzell, Germany). Intensity of band was quantified by ImageJ software (NIH, Bethesda, MD, U.S.A.).

Statistical Analysis

Data was presented as means ± standard error of the mean (S.E.M.) or boxplot. All statistical tests were performed by StatView (Abacus, Baltimore, MD, U.S.A.) and IBM SPSS Statistics ver.19.0 (IBM, Armonk, NY, U.S.A.). Statistical significance was set at p < 0.05.

RESULTS AND DISCUSSION

This study was performed to determine the effects of low zinc on neurite outgrowth during neuronal differentiation and its mechanisms, with a focus on the CRMP2 signal related to neurite extension. It has been reported that zinc deficiency not only inhibits neuronal differentiation but also causes impaired brain development and apoptosis.^14,15) In this study, low zinc suppressed neurite outgrowth in SH-SY5Y cells on days 1, 2, and 3 of neuronal differentiation induction (Fig. 1). Low-zinc-induced inhibitory changes in neurite outgrowth were greater at 3 d post-neuronal differentiation (Fig. 1D). Neurite outgrowth measurements under normal neuronal differentiation conditions (intact) versus 4 µM ZnSO₄ control (control) showed no difference between the two groups (Supplementary Fig. 1). We then examined the signaling pathway involving CRMP2 in the suppression of neurite outgrowth by low zinc.

Fig. 1. Effects of Low Zinc on Neurite Extension

(A–C) Neurite length was determined in SH-SY5Y cells differentiated in low zinc condition. Immunostaining for detection of neurite was used anti-β-tubulin (TUJ1) antibody. Scale bar is 100 µm. (D) Quantitative data of neurite length was presented. Data of neurite length was calculated as sum of neurite length in total cells divided by number of nuclear, and these data were calculated per picture. Data were expressed as boxplot (n = 36 pictures/group). Significance was determined by Student’s t-test at each time point (* p < 0.05).

CRMP2 is a protein that forms dimers with Tublin and is involved in the formation of nerve axons.⁶⁾ CRMP2 is phosphorylated by GSK3β and inhibits neurite outgrowth⁷⁾ (Fig. 2A). Therefore, we analyzed changes in phosphorylation of CRMP2 and GSK3β proteins. On days 1, 2, and 3 of neuronal differentiation induction, low zinc caused increased expression of pCRMP2 relative to CRMP2 (Fig. 2B) and decreased expression of pGSK3β relative to GSK3β (Fig. 2C). These results suggest that GSK3β–CRMP2 signaling is altered by low zinc status in the direction of neurite outgrowth inhibition. Next, we examined the causal relationship between GSK3β–CRMP2 signaling and low zinc-induced neurite inhibition using the GSK3β inhibitor CHIR99021. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3β inhibitor CHIR99021 (Fig. 3). Thus, low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3β. Although it is expected that phosphorylation of CRMP2 in Low-Zn group would be inhibited by treatment with CHIR99021, we could not analyze that far in this study. This will be considered as a future study.

Fig. 2. Changes in Phosphorylated CRMP2 (pCRMP2) and Phosphorylated GSK3β (pGSK3β) Level

(A) Relationship between GSK3β-CRMP2 signaling and neurite extension. (B, C) The level of pCRMP2, CRMP2 (B), pGSK3β and GSK3β (C) were measured in SH-SY5Y cells differentiated in low zinc condition. Data was expressed as means ±S.E.M. (n = 3/group). Significance was determined by Student’s t-test (* p < 0.05).

Fig. 3. Effects of CHIR99021, GSK3β Inhibitor, on Low-Zinc-Induced Inhibition of Neurite Extension

(A) Neurite length was determined in SH-SY5Y cells differentiated in low zinc condition and under CHIR99021 treatment. Scale bar is 100 µm. (B) Quantitative data of neurite length were presented. Data of neurite length were calculated as sum of neurite length in total cells divided by number of nuclear, and this data was calculated per picture. Data were expressed as boxplot (n = 34–36 pictures/group). Significance was determined by a two-way ANOVA followed by Bonferroni’s post hoc test (* p < 0.05).

The previous report has shown that zinc increases tyrosine phosphorylation in skeletal muscle cells by inhibiting tyrosine phosphatase.¹⁶⁾ On the other hand, tyrosine kinases have been reported to cause activation of protein kinase B (Akt), which is responsible for phosphorylation of GSK3β.^17,18) Therefore, changes in Akt phosphorylation upstream of GSK3β may also occur in this study.

Other than the present study, it was reported that knockdown of Zip12, a zinc transporter, in N2a cells, a mouse neuroblastoma cell type, reduced cellular zinc uptake and neurite outgrowth, and that phosphorylation of cAMP response element binding protein (CREB) was involved in this mechanism.¹⁹⁾ The interaction between various zinc transporters and the inhibition of neurite outgrowth by low zinc via CRMP2 signaling should also be analyzed in the future. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

Acknowledgments

This research was supported by a Grant-in-Aid for Scientific Research on Innovative Areas JSPS KAKENHI (JP19H05767A02) to I.H., JSPS KAKENHI (17K18001) and Grant of The Uehara Memorial Foundation (2018) to H.K.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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