Establishing an in vivo p48ZnF bioluminescence mouse brain imaging model

Neuroscience Letters 542 (2013) 97–101

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Establishing an in vivo p48ZnF bioluminescence mouse brain imaging model Klaus Heese ∗ Department of Biomedical Engineering, College of Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 133-791, Republic of Korea

h i g h l i g h t s  In vivo p48ZnF mouse brain bioluminescence imaging.  p48ZnF interacts with Drg1 and Pcbp1.  p48ZnF regulates gene transcription and translation.

a r t i c l e

i n f o

Article history: Received 12 January 2013 Received in revised form 20 February 2013 Accepted 23 February 2013 Keywords: p48ZnF Dfrp1 Drg1 Zinc finger Gene transcription

a b s t r a c t p48ZnF is a C3H1 zinc finger domain-containing protein that is involved in the control of gene transcription and translation. In the present study a novel transgenic p48ZnF mouse model is described that is useful for in vivo brain imaging using luciferase as bioluminescence-mediating reporter gene. Yeast two-hybrid screening and western blot analyses revealed Drg1 (developmentally regulated GTP binding protein 1) and Pcbp1 (poly (rC)-binding protein 1) as p48ZnF-associated proteins. Interestingly, p48ZnF’ cellular location of action depends on the cell’s differentiation status: nuclear in proliferating cells and cytoplasmic in differentiated neurons. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

2. Materials and methods

Many transcription factors or DNA regulatory proteins contain zinc-finger domains which enable these proteins to bind to DNA and regulate DNA expression [11]. The recently identified novel protein p48ZnF is glutamate/lysine-rich and contains two characteristic C-x8-C-x5-C-x3-H (CCCH) zinc finger domains [3] (Fig. 1). C3H1-type domains are relatively rare – only 58 discrete C3H1type domains can be found in the human and murine genomes and most of these domains have yet to be characterized [10]. Thus, to get further insights into the neuronal function of p48ZnF, in the present study a transgenic mouse model has been established where brain p48ZnF protein levels could be observed in vivo in a living mouse via a bioluminescence-mediated luciferase reporter gene expression. Alongside, p48ZnF’s signaling pathways have been explored by in vitro molecular and cellular methods as well as by biocomputational analyses.

2.1. Reagents All reagents used for experiments were purchased from Sigma–Aldrich (Milwaukee, WI, USA) unless otherwise stated. 2.2. Antibodies Anti-␣-tubulin: 1:1000, mouse monoclonal (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-beta-actin (Actb, 1:3000, goat polyclonal; Santa Cruz), anti-p48ZnF (rabbit polyclonal, 1:3000; raised against peptide aa cSGGRAENGERSDLEE), anti-Drg1 (developmentally regulated GTP binding protein 1, rabbit polyclonal, 1:400; kindly provided by Inuoe et al. [8]), anti-Gapdh (glyceraldehyde 3-phosphate dehydrogenase, goat polyclonal, 1:5000; Santa Cruz), anti-Pcbp1 (poly (rC)-binding protein 1, mouse polyclonal, 1:1000; Abnova, Taiwan), and anti luciferase (rabbit polyclonal, 1:1000; Abcam, Cambridge, UK). 2.3. Animal experiments

∗ Tel.: +82 2 2220 0398; fax: +82 2 2296 5943. E-mail addresses: [email protected], [email protected] 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.02.044

Experimental methods, including the killing of animals, were performed in accordance with the International Guiding Principles

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K. Heese / Neuroscience Letters 542 (2013) 97–101

vector was constructed as indicated (Fig. 1B). The transgene vector was injected in different sessions into zygotes of C57BL/6N female mice obtained from Charles River Wiga GmbH (Sulzfeld, Germany), superovulated and mated with C57BL/6N males. Injected zygotes were cultivated overnight and transferred into pseudo-pregnant B6CBAF1 females (Charles River) and the animals were kept in individually ventilated cages (IVG). Transgenic founder mice resulting from second-round pronucleus injections were identified by polymerase chain reaction (PCR). From the founders the F1 generations were produced and bred to maintain as heterozygous lines (PolyGene AG, Switzerland). Expression of p48ZnF/luciferase in transgenic mice was confirmed by western blot (data not shown) and bioluminescence luciferase activity-based imaging via the NightOWL LB983 Imager (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Brain tissue luciferase assay was performed as described previously. Adult mice were anesthetized via intraperitoneal injection of ketamine/xylazine (RBI, Natick, MA, USA) and brain tissue was taken and subjected to luciferase assay using reagents obtained from Promega (Madison, WI, USA) [14]. Representative pictures are shown. Mouse brain immunohistochemistry (IHC) was performed as described previously [13,14,20]. 2.5. Cell culture of PC12 cells

Fig. 1. Overexpression of p48ZnF in transgenic mice brain using a bioluminescence reporter gene. (A) Schematic diagram of the p48ZnF (also known as Zc3h15, Dfrp1, Lerepo4 or Tma46; locus: AAR24540 and NP 001010963) amino acid sequence. The two zinc finger domains (aa100–aa125, aa175–aa211) depicted are of the C3H1-type with a DNA-binding domain at aa147–aa188. In addition to the lysinerich region (aa4–aa175), two bipartite nuclear localization signals (NLS, aa4–aa36, aa262–aa279), one nuclear export domain (NES, aa415–aa424) and a DFRP domain (aa234–aa295) have been identified [3,8]. (B) Schematic diagram of the transgene vector insertion construct used to establish the transgenic p48ZnF mouse line. The mouse synapsin-I promotor (Syn1P) with its natural intron was introduced behind the rabbit ␤-globulin 5 UTR (untranslated region) followed by the mouse p48ZnF gene, an internal ribosome entry site (IRES), the luciferase gene, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and the rabbit ␤globulin 3 UTR containing its own polyadenylation signal (pA). (C) In vivo mouse brain bioluminescence luciferase activity imaging in alive mice showing p48ZnF expression in the brain of a transgenic (tg) mouse compared with a corresponding wild-type littermate (top: female, 1 week old, wild type; bottom: female, 1 week old, tg p48ZnF strain 37.1013). Small inserted panel: ex vivo mouse brain bioluminescence luciferase activity imaging (tg p48ZnF strain 37.1013 (female, 1 week old)). (D) IHC analysis of transgenic (tg) mouse brain using anti-luciferase (tg-luc) and antip48ZnF antibodies confirmed neuronal expression of p48ZnF in the hippocampus and dentate gyrus (same mouse brain but different slices). Wild-type (wt) mice also expressed endogenous neuronal p48ZnF in the hippocampal area. Tg- and wt-mice: both female, 3 months old. Scale bar represents 100 ␮m.

for Animal Research (WHO) and were approved by the local Institutional Animal Care and Use Committee. Mouse tissues were isolated from C57BL/6J mice after the humane killing of the animals using approved anesthetic methods to isolate brain tissue and to analyze p48ZnF expression. All efforts were made to minimize animal suffering and to reduce the number of animals used [1,14,15,17].

Rat pheochromocytoma PC12 cells (American Type Culture Collection, ATCC, Manassas, VA, USA) were propagated at 37 ◦ C in humidified 5% CO2 /95% air, in Dulbecco’s Modified Eagle’s Medium (DMEM, GlutaMaxTM ; Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS, Invitrogen), non-essential amino acids (Invitrogen), and antibiotic–antimycotic (Invitrogen). All media used for cell culture were from Gibco unless otherwise stated. Cells were cultured at 37 ◦ C in humidified 5% CO2 /95% air. For neuronal differentiation PC12 cells were seeded in poly-d-lysine (PDL)-coated six-well plates (Becton Dickinson, San Jose, CA, USA) at 1.5 × 104 cells per well. The cells were allowed to attach and divide for 20 h, after which 100 ng/ml NGF (nerve growth factor) was added for 1 week. Images were captured via an inverted microscope (Nikon Eclipse TE2000, Tokyo, Japan). Vehicle controls were treated with the solvent only. Experiments were performed twice, with each set repeated in triplicates and representative data are shown [3,15]. 2.6. Cell lysis and protein extraction Adherent cells were washed in the dish using ice-cold Ca2+ /Mg2+ -free saline phosphate buffer (PBS), lysed using a disposable cell scraper (Greiner Bio-One GmbH, Frickenhausen, Germany). Lysed cells were centrifuged at 10,000 × g at 4 ◦ C for 10 min. Lysis buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 40 mM NaF, 5 mM EDTA, 0.5 mM dithiothreitol (DTT), 1% Triton-X100, 1 mM sodium orthovanadate, 1% (vol/vol) Nonidet P-40, 0.1% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 ng/ml of aprotinin and 10 ng/ml leupeptin plus a EDTA-containing (serine- and cysteine-) protease inhibitor (Roche) as well as serine/threonine/tyrosine phosphatase inhibitor cocktails 1 and 2 (Sigma)) was added and incubated on ice for 5–10 min before applying for SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis [13].

2.4. Establishment of the transgene and the transgenic p48ZnF mouse lines

2.7. Co-immunoprecipitation

The p48ZnF mouse lines were generated analogously to a previously described transgenic ‘synapsin-I-p60TRP’ mouse [14]. Fig. 1 shows a schematic diagram of the insertion construct used to establish the transgenic p48ZnF mouse line. The transgene

For immunoprecipitation [13], PC12 cells were lysed in lysis buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 40 mM NaF, 1 mM sodium orthovanadate, 1% (vol/vol) Nonidet P-40, 0.1% (wt/vol) sodium deoxycholate, 1% (vol/vol) Triton-X-100, 1 mM

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PMSF, 20 ng/ml of aprotinin and 20 ng/ml leupeptin), followed by centrifugation at 14,000 rpm at 4 ◦ C for 15 min. After protein quantification, equal amounts of the protein supernatants were incubated with polyclonal rabbit anti-p48ZnF antibodyloaded protein A/G-sepharose at 4 ◦ C overnight. As a control, the lysates were incubated with a non-specific rabbit polyclonal antibody-loaded protein A/G-sepharose. The immunoprecipitates were then washed five times in lysis buffer, and the bound proteins were recovered by boiling the beads in 2× SDS sample buffer and separated by SDS–PAGE, followed by western blot with a specific antibody (anti-Pcbp1 or anti-Drg1) as indicated. Experiments were performed three times and representative data are shown.

2.8. SDS–PAGE and western blot analysis Twenty micrograms of cell lysates were resolved by 8–12% SDS–PAGE at 0.02 A of constant current and transferred to a polyvinylidine fluoride (PVDF) membrane (0.22 ␮m; Amersham, Piscataway, NJ, USA) using the ‘semi-dry’-transfer method (BioRad, Singapore) for 60 min at 0.12 A in buffer containing 25 mM Tris, 192 mM glycine, 20% methanol, and 0.01% (wt/vol) SDS. The membrane was blocked with 5% bovine serum albumin (BSA) (BioRad) in Tris-buffered saline (TBS) solution plus 0.1% Tween-20 (TBS-T) or PBS-T for 2 h at room temperature (RT), washed three times in PBST for 10 min each, and incubated with primary antibody (diluted in 2% BSA in PBS-T) for overnight at 4 ◦ C. The membranes were washed as described above, incubated with a horseradish peroxidase (HRP)conjugated secondary antibody for 1 h at RT, and developed using the ECL plus western-blot detection reagent (Amersham). X-ray films (Konica Minolta Inc., Tokyo, Japan) were exposed to the membranes before film development in a Kodak X-OMAT 2000 processor (Kodak, Ontario, Canada). For equal sample loading, protein quantification was performed using a ‘2D Quant’ kit (Amersham) with two independent replicates. BSA was used as a standard for protein quantification. To re-probe the same membrane with another primary antibody, Pierce’s (Pierce Biotechnology, Inc., Rockford, IL, USA) ‘stripping solution’ was used to strip the membranes. In addition, equal sample loading was confirmed using Gapdh or Actb as a reference protein (data not shown). Western blot experiments were performed three times and representative blots are shown [13].

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2.9. Immunocytochemistry (ICC) Cells seeded on poly-l-lysine (PLL; Sigma)-coated coverslips were fixed with 4% paraformaldehyde in PBS for 10 min at RT, washed twice with PBS and then permeabilized with 0.5% TritonX100 (Sigma) in PBS for 5 min at RT. The cells were washed thrice with PBS and incubated with primary antibody diluted in TBST with 4% BSA (AppliChem GmbH, Denmark) for 1 h at RT. Following incubation, cells were washed thrice with TBST and incubated for 30 min with a secondary antibody (labeled with FITC (AlexaFluor 568, goat polyclonal anti-rabbit, 1:400, or AlexaFluor 488, goat monoclonal anti-mouse, 1:400; Molecular Probes, Eugene, OR, USA)) in TBST with 4% BSA for 1 h in the dark at RT. After three final washes with TBST, the coverslips were mounted onto microscope glass slides using anti-fade solution containing DAPI (4 ,6-diamidino-2-phenylindole; Chemicon, Temecula, CA, USA). Slides were visualized using an Axiovert 200 M inverted florescence compound microscope (Carl Zeiss, Germany) and images were analyzed with Axiovision software. Experiments were performed three times and representative figures are shown [16,20]. 2.10. ProQuestTM two-hybrid-system with GatewayTM technology The two-hybrid system is an in vivo yeast-based system that identifies the interaction between two proteins (for instance X = p48ZnF and Y = cDNA library, Pcbp1 or Drg1) by reconstituting an active transcription factor. Analyses were performed according to the manufacturer’s protocol (Invitrogen’s brain ProQuestTM twohybrid-system, Singapore) using p48ZnF as bait. In the ProQuestTM two-hybrid-system, in comparison to standard two-hybrid systems, false positives are reduced because three independent transcription events (from distinct promoters) must occur at independent chromosomal loci. Positive clones were confirmed by retransformation assays and co-immuneprecipitation analyses [4,13]. 3. Results 3.1. In vivo mouse brain imaging of neuronal p48ZnF expression A novel transgenic mouse model has been developed in which the expression of p48ZnF was driven by the synapsin-1 promoter

Fig. 2. p48ZnF interacts with Pcbp1 and Drg1. Co-immune precipitation (Co-IP) performed with the anti-p48ZnF antibody. (A) Western blot membrane probed with the anti-Drg1 antibody: lane 1: Co-IP product obtained with the anti-p48ZnF antibody; lane 2: total cell lysate. (B) Western blot membrane probed with the anti-Pcbp1 antibody: lane 1: Co-IP product obtained with the anti-p48ZnF antibody; lane 2: total cell lysate. (C) Western blot membrane probed with the anti-p48ZnF antibody: lane 1: Co-IP product obtained with the anti-p48ZnF antibody; lane 2: total cell lysate.

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Fig. 3. Localization of p48ZnF in non-differentiated and NGF-mediated (100 ng/ml) differentiated neuronal PC12 cells. (A) p48ZnF co-localizes with nuclear DAPI-stained DNA in non-differentiated cells. (B) In the differentiated cells, p48ZnF was distributed evenly in both cytoplasm and nucleus. Scale bar represents 10 ␮m.

in neurons to investigate the significance of p48ZnF in neuronal function in the brain. Luciferase, a bioluminescence reporter protein, was used to visualize p48ZnF in vivo in the transgenic p48ZnF mouse model (Fig. 1). 3.2. p48ZnF interacts with Pcbp1 and Drg1 Yeast two-hybrid screening analyses on potential interacting partners of protein p48ZnF provided data of Drg1 and Pcbp1 as binding partners of p48ZnF. To confirm the physical association of p48ZnF with Pcbp1 and Drg1, PC12 cell lysates were immunoprecipitated with an anti-p48ZnF antibody, and co-precipitated Drg1 and Pcbp1 were detected by immunoblotting (Fig. 2). These data confirmed the association of p48ZnF with these two proteins Drg1 and Pcbp1. 3.3. Immunocytochemistry reveals intracellular localization of p48ZnF PC12 cells were immunostained to determine the intracellular sub-localization of p48ZnF. DNA and ␣-tubulin staining was

done to indicate cytoplasmic and nuclear localizations, respectively. In non-differentiated PC12 cells, p48ZnF was predominantly nuclear. However, when neuronal differentiation of PC12 cells was induced by NGF, p48ZnF changed its localization from predominantly nuclear to a combination of nuclear and cytoplasmic distribution (Fig. 3).

4. Discussion In the present study an in vivo imaging mouse model has been developed for the detection of brain neuronal p48ZnF expression via a bioluminescence reporter gene. p48ZnF belongs to the C3H1type zinc finger proteins that interacts with Drg1 and Pcbp1. The interaction forms a polysomal Drg1–p48ZnF complex which participates in eukaryotic translation by providing protein stability to Drg1 possibly by blocking poly-ubiquitination [2,7]. Though physical association of Drg1 with p48ZnF has been established [7,8], the neuro-functional significance of p48ZnF–Pcbp1 interaction remains to be studied.

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Acknowledgements This study was supported by the Hanyang University. I would like to thank Ms. B. Jiao, Ms. Y.P. Lee, Ms. M. Mishra, Dr. O. Islam and Mr. B. Cher for technical assistance. References

Fig. 4. STRING-9.0 analysis (at http://string-db.org/; default mode) of human p48ZnF’s potential interactive signaling pathways. The neuro-functional significance of p48ZnF within the PCBP1 and DRG1 networks remains to be elucidated, in particular in view of its potential function in terms of neuronal development, axon guidance and synaptic plasticity. HNRNPA2B1, heterogeneous nuclear ribonucleoprotein A2/B1; HNRNPD, HNRNP D (AU-rich element RNA binding protein 1); LMNA, lamin A/C; PTBP1, polypyrimidine tract binding protein 1; SF3A2, splicing factor 3a, subunit 2; SRSF2, serine/arginine-rich splicing factor 2; SRSF9, serine/arginine-rich splicing factor 9; YBX1, Y box binding protein 1.

Pcbp1 belongs to the heterogeneous nuclear ribonucleoprotein family (hnRNP) and participates in transcriptional and translational regulation. Pcbps are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). Although Pcbp1 is known to affect the stability of gene expression through post-translational regulation, its exact function in neuronal axonal navigation and connectivity is still unclear [9]. The presence of Pcbp1 and p48ZnF interaction suggests a close transcriptional–translational pathway with p48ZnF regulating processes on the DNA and protein levels while Pcbp1 is involved in a diverse set of post-translational control pathways such as mRNA stabilization, translational activation and translational silencing [12]. Meanwhile, coupled with Drg1, which is a cytoplasmic developmentally regulated GTP-binding protein that regulates the transcriptional–translational process by modulating GTP availability, the Drg1–p48ZnF–PcbpP1 complex may govern cytoplasmic and nuclear transcriptional/translational processes potentially involved in neuronal differentiation, axon guidance and synaptic plasticity (Fig. 4). Besides, Pcbp1 is an iron chaperone that binds iron and delivers it to ferritin, a cytosolic iron storage protein. Pcbp1 is also required for the iron incorporation into iron-dependent prolyl hydroxylases (PHDs). Such data suggests a broad role for Pcbp1 in delivering iron to cytosolic non-heme iron enzymes [19,21]. Since Pcbp1 may control the expression or activity of Wnt-, TGF-␤ and muopioid receptor (MOR) signaling pathway genes too [5,6,18], the p48ZnF mouse model could be useful to study these pivotal neurodevelopmental signaling cascades as it still remains a challenging research area.

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