Low-Dose Ionizing Radiation Rapidly Affects Mitochondrial and

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Low-Dose Ionizing Radiation Rapidly Affects Mitochondrial and Synaptic Signaling Pathways in Murine Hippocampus and Cortex Stefan J. Kempf,† Simone Moertl,† Sara Sepe,‡ Christine von Toerne,§ Stefanie M. Hauck,§ Michael J. Atkinson,†,∥ Pier G. Mastroberardino,‡ and Soile Tapio*,† †

Institute of Radiation Biology, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany ‡ Department of Genetics, Erasmus Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands § Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany ∥ Chair of Radiation Biology, Technical University Munich, Arcisstrasse 21, 80333 Munich, Germany S Supporting Information *

ABSTRACT: The increased use of radiation-based medical imaging methods such as computer tomography is a matter of concern due to potential radiation-induced adverse effects. Efficient protection against such detrimental effects has not been possible due to inadequate understanding of radiation-induced alterations in signaling pathways. The aim of this study was to elucidate the molecular mechanisms behind learning and memory deficits after acute low and moderate doses of ionizing radiation. Female C57BL/6J mice were irradiated on postnatal day 10 (PND10) with gamma doses of 0.1 or 0.5 Gy. This was followed by evaluation of the cellular proteome, pathwayfocused transcriptome, and neurological development/disease-focused miRNAome of hippocampus and cortex 24 h postirradiation. Our analysis showed that signaling pathways related to mitochondrial and synaptic functions were changed by acute irradiation. This may lead to reduced mitochondrial function paralleled by enhanced number of dendritic spines and neurite outgrowth due to elevated long-term potentiation, triggered by increased phosphorylated CREB. This was predominately observed in the cortex at 0.1 and 0.5 Gy and in the hippocampus only at 0.5 Gy. Moreover, a radiation-induced increase in the expression of several neural miRNAs associated with synaptic plasticity was found. The early changes in signaling pathways related to memory formation may be associated with the acute neurocognitive side effects in patients after brain radiotherapy but might also contribute to late radiation-induced cognitive injury. KEYWORDS: synaptic plasticity, microRNA, proteomics, LTP, dendritic spine, learning, memory, CREB, ionizing radiation

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pediatric diagnostics may be higher than 1.5 million per year.9 The presumptive threshold of the brain dose still causing delayed damage may be as low as 0.1 Gy.10 The infant brain may be especially sensitive to ionizing radiation. This was highlighted by a recent follow-up study showing that particularly the hippocampus and cortex are persistently affected in children treated with high doses of cranial irradiation and associated with neurocognitive decline.11 Hall et al. showed that irradiation of the infant brain with dose levels overlapping those imparted by CT may adversely affect intellectual development.1 During early phases of childhood, the volume of the gray matter within the cortex consisting of mainly neuronal cell bodies increases rapidly but shrinks later in life.12 This specific phase where brain maturation takes place is called the brain growth spurt.13 It involves phases of axonal and dendritic growth

INTRODUCTION Central nervous system (CNS) malignancies are effectively treated by ionizing radiation, yet the benefits are overshadowed by acute side effects such as learning and memory deficits originating from high radiation doses during treatment (fractions of ∼2.0 Gy up to a cumulative dose of 40.0 Gy). In comparison, epidemiological studies indicate that even low radiation doses may lead to acute and permanent deficits in learning and memory,1−3 particularly if the exposure occurred during childhood.4,5 Therefore, it is of concern that the number of computer tomography (CT) scans is rapidly increasing.6 The radiation doses per single head scan range between 30 and 70 mGy.7 Importantly, a French large-scale multicenter study with 27 362 children showed that the mean number of CT examinations per child was 1.6 (range 1−43); 63% of all CT scans were applied to the head region with the brain receiving the highest cumulative doses with a mean dose of 22 mGy.8 Thus, the cumulative dose of CT scans may by far exceed the low-dose range (≤100 mGy). Overall, the number of CT scans used for © XXXX American Chemical Society

Received: November 6, 2014

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(Erasmus University Medical Center EDC). The radiation field was homogeneous within ±3%, as verified by TLD-100 dosimetry. Animals from three litters were used within each irradiation group to minimize litter effects; each group was irradiated together.

including establishment and breakup of neuronal circuits.14,15 The time of the brain growth spurt is species-specific and lasts in humans until the third to fourth postnatal year. In rodents, the comparable time window is restricted to the second and fourth postnatal week.16 It has been shown that toxic agents administered to neonatal mice within this susceptibility window can lead to disruption of adult brain function17 and impair cognitive behavior when combined with low-dose ionizing radiation on postnatal day ten (PND10).18 It has been shown that the networks responsible for effective neurotransmission in mature neurons may function as radiation targets, as recently demonstrated in the murine hippocampus at both moderate (1.0 Gy) and high therapeutic (10 Gy) doses of irradiation 30 days postirradiation.19 Similar observations were made by others using hippocampal primary neuronal cultures exposed to an acute dose of 30 Gy following an immediate in situ morphological analysis.20 In this context, synapses are a key element of neurotransmission modulating the information flow by constant dynamic alteration called synaptic plasticity.21 This process depends on rapid conversion of the cytoskeleton supporting synaptic morphology, stabilizing the postsynaptic density, and anchoring neuronal receptors of glutamate.22,23 Importantly, this is an energy-demanding process highly dependent on efficient mitochondrial function, as up to 50% of neuronal energy is used to keep up actin dynamics.24 In general, neurotransmission follows the activation of neuronal receptors on the postsynapse coupled to phosphorylation and dephosphorylation events. While long-term potentiation (LTP) is associated with the activation of protein kinases phosphorylating target proteins, long-term depression (LTD) originates from activation of calcium-dependent phosphatases.25 LTP and LTD modulate nuclear transcription factors regulating the transcription of synaptic proteins and shifting the excitability of synapses. One of the best described transcription factors involved in learning and memory is the cAMP-responsive element binding (CREB) protein.26,27 We have previously shown that acute ionizing radiation (1.0 Gy, 24 h postirradiation) affects synaptic signaling pathways in vitro (HT22 cells) and in vivo (murine NMRI hippocampus and cortex).28 The aim of this study was to determine signaling pathways induced by acute ionizing radiation using doses in the range of those applied in a single (0.1 Gy) or several head CT scans (0.5 Gy). Female C57BL/6J mice were irradiated on PND10 that corresponds to the period of brain growth spurt and is the point where the highest brain sensitivity to exogenous stressors is seen.17,18 We evaluated the changes in the hippocampal and cortical proteome and targeted pathwayfocused gene expression as well as neurological disorder-focused miRNAome 24 h postirradiation. By these means, we gained knowledge about radiation-affected signaling pathways related to learning and memory. We show here that signaling pathways associated with mitochondrial function and synaptic plasticity were rapidly altered by acute low-dose radiation exposure.

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Tissue Collection

Animals were killed 24 h postirradiation via cervical dislocation. Brains were excised and transferred to ice-cold phosphatebuffered saline (PBS), rinsed carefully, and dissected under stereomicroscopic inspection under cold conditions (ice-cold PBS). Hippocampi and whole cortices from each hemisphere were separately sampled, gently rinsed in ice-cold PBS, and snapfrozen in liquid nitrogen. Samples were shipped on dry ice to Helmholtz Zentrum München, Munich, Germany, and were stored at −80 °C until isolation of protein and RNA. Isolation of Total Protein and RNA

Tissue samples were homogenized in 6 M guanidine hydrochloride (SERVA Electrophoresis) on ice. Homogenates were briefly vortexed, sonicated, and cleared by centrifugation (20 000g, 1 h, 4 °C). Protein concentration of the supernatants was determined using the Bradford assay kit (Thermo Fisher) following the manufacturer’s instructions. Total RNA from individual frozen hippocampi and cortices was isolated and purified by mirVana Isolation Kit (Ambion) according to the manufacturer’s instructions. The optical density (OD) ratio of 260/280 was measured using a Nanodrop spectrophotometer (PeqLab Biotechnology); it ranged between 1.9 and 2.1. RIN values were determined (TapeStation, Lab901− genomax Technologies); they ranged between 8.6 and 9.0. Mass-Spectrometry-Based Proteome Analysis

The isotope-coded protein label (ICPL) approach was used to quantify proteome alterations as recently described.28 In brief, the protein lysates (20 μg in 20 μL of 6 M guanidine hydrochloride from each biological replicate) were reduced, alkylated, and labeled with the three different ICPL reagents as follows: control with ICPL-0, 0.1 Gy sample with ICPL-4, and 0.5 Gy sample with ICPL-6. The three labeled samples representing each radiation dose were combined and overnight precipitated with 80% acetone at −20 °C to purify the labeled protein content. Four biological replicates in each dose group and brain region were used. Protein precipitates were separated by 12% SDS−polyacrylamide gel electrophoresis, followed by Coomassie Blue staining. Gel lanes were cut into at least four equal slices, destained, and trypsinised overnight as previously described.28 Peptides were extracted and acidified with 1% formic acid, followed by analysis via mass spectrometry. LC−MS/MS analysis was performed as previously described on a LTQ-Orbitrap XL (Thermo Fisher).29 In brief, the prefractionated samples were automatically injected and loaded onto the trap column (Acclaim PepMap100, C18, 5 μm, 100 Å pore size, 300 μm ID × 5 mm μ-Precolumn, no. 160454; Thermo Scientific). After 5 min, the peptides were eluted and passed to the analytical column (Acclaim PepMap100, C18, 3 μm, 100 Å pore size, 75 μm ID × 15 cm, nanoViper - no. 164568; Thermo Scientific) by reversed-phase chromatography, which was operated on a nano-HPLC (Ultimate 3000, Dionex). A nonlinear 170 min gradient was used for elution with a mobile phase of 35% acetonitrile in 0.1% formic acid in water (A) and 0.1% formic acid in 98% acetonitrile (B) at a flow rate of 300 nL/min. The gradient settings were: 5−140 min: 14.5−90% A, 140−145 min: 90% A−

MATERIALS AND METHODS

Ethics Statement and Irradiation of Animals

Experiments were carried out according to protocol number 13912-30 approved by the animal experiments committee (EMC no. 3018). Female C57BL/6J (Charles River) mice were total-bodyirradiated on PND10 with a single exposure of gamma irradiation (137Cs, 0.082 Gy/min) at doses of 0 (sham), 0.1, and 0.5 Gy B

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Figure 1. Venn diagram of all deregulated proteins and corresponding pathways. Venn diagram showing the number of all and shared deregulated proteins found in hippocampus [H] and cortex [C] of female C57BL/6 mice irradiated at doses of 0.1 and 0.5 Gy at PND10 24 h postirradiation using global proteomics approach (A). The number above each dose shows the total number of deregulated proteins at this dose. Altered signaling pathways at all doses using the Ingenuity Pathway Analysis (IPA) software are shown in panel B. High color intensity represents high significance (p value) of the pathway. All colored boxes have a p value of ≤0.05; white boxes have a p value of ≥0.05 and are not significantly altered; n = 4 in each dose group. H: hippocampus, C: cortex.

comparison analysis takes into account the signaling pathway rank according to the calculated p value and reports it hierarchically. The software generates significance values (p values) between each biological or molecular event and the imported proteins based on the Fischer’s exact test (p ≤ 0.05). We used only the database information on experimental and predictive origin regarding CNS to be confident about the potential affected signaling pathways.

95% B, and 145−150 min: 95% B, followed by equilibration for 15 min to starting conditions. The 10 most abundant peptide ions were selected from the MS prescan for fragmentation in the linear ion trap if they exceeded an intensity of at least 200 counts and were at least doubly charged. During fragment analysis via collision-induced fragmentation (collision energy: 35 V), a highresolution (60.000 full width at half-maximum) MS spectrum was acquired in the Orbitrap with a mass range of 300 to 1500 Da. Target peptides were dynamically excluded for 60 s if already selected for MS/MS. MS/MS spectra were searched against the ENSEMBL mouse database (version: 2.4, 56 416 sequences) via MASCOT search engine (version 2.3.02; Matrix Science). A mass tolerance of 10 ppm for peptide precursors and 0.6 Da for MS/MS peptide fragments was applied, allowing not more than one missed cleavage. Fixed modifications included carbamidomethylation of cysteine and ICPL-0, ICPL-4, and ICPL-6 for lysine. Proteins were identified and quantified based on the ICPL pairs using the Proteome Discoverer software (version 1.3, Thermo Fisher). To ensure that only high-confident identified peptides were used for protein quantification, we applied the MASCOT percolator algorithm (q value filter of 0.01). Subsequently, these peptides were filtered against a Decoy database, resulting in a false discovery rate (FDR) of each LC−MS run; the significance threshold was set to 0.01 to ensure that only highly confident peptide identifications were used for protein quantification. Proteins from each LC−MS run were normalized against the median of all quantifiable proteins. Proteins were considered to be significantly deregulated if they fulfilled the following criteria: (i) identification by at least two unique peptides in n − 1 massspectrometry runs (n: number of biological replicates), (ii) quantification with an ICPL-variability of ≤30%, and (iii) a foldchange of ≥1.3 or ≤ −1.3. A threshold for the fold change of ±1.3 was used based on our average experimental technical variance of the multiple analyses of hippocampal and cortical technical replicates.30 The raw-files of the obtained MS/MS spectra are deposited at http://storedb.org/project_details.php?projectid=43 with the ProjectID 43.

Immunoblotting

Immunoblotting was performed as recently described28 using 10 μg of hippocampal and cortical protein extracts on 12% SDS polyacrylamide gels. The following antibodies were used: GAPDH (dilution 1:200) [sc-47724, murine monoclonal IgG1 raised against recombinant GAPDH of human origin; Santa Cruz], CREB (dilution 1:500) [no. 4820, rabbit monoclonal antibody raised against full length GST-CREB fusion protein; Cell signaling], phospho-CREB (dilution 1:200) [no. 9191, rabbit polyclonal antibody raised against a synthetic phosphopeptide corresponding to residues surrounding Ser133 of human CREB - Cell signaling], and Stat1α/β (dilution 1:1000) [no. 9172, rabbit polyclonal antibody raised against a synthetic peptide corresponding to the sequence of human Stat1 - Cell signaling]. An antibody cocktail comprising Atp5a (Complex V), Uqcrc2 (Complex III), and Ndufb8 (Complex I) (1:1000) [ab110413, mouse monoclonal antibody cocktail (MitoProfile Total OXPHOS rodent WB antibody cocktail)] was used to detect mitochondrial complex proteins. The expression of GAPDH was used for normalization of the levels of CREB, phospho-CREB, and Stat1α/β data because it was not changed in any sample during the proteomic and transcriptomic investigation. The levels of mitochondrial complexes were normalized against total protein content of the relevant immunoblots visualized by Ponceau-S staining. Immunoblots were quantified with TotalLab TL100 software (www.totallab.com) using software-suggested background correction. Three biological replicates were used for statistical analysis (unpaired Student’s t test, two-sided) with a significance threshold of 0.05.

Bioinformatics Analysis

Quantification of mRNA and miRNA via Quantitative PCR

The analyses of affected signaling pathways from all deregulated proteins were performed with the INGENUITY Pathway Analysis (IPA) (http://www.ingenuity.com) software. Deregulated proteins with their protein accession number and foldchanges were imported into the IPA core analysis, followed by a hierarchical comparison analysis by the IPA software. The IPA

100 ng of RNA isolates were used to quantify miRNA expression (miScript miRNA PCR Array “Neurological Development & Disease” [MIMM-107Z, Qiagen]) and gene expression (RT2 Profiler PCR Array “Synaptic Plasticity” [PAMM-126Z, Qiagen]) levels according to the manufacturer’s protocol on a C

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Figure 2. Protein expression of mitochondrial subunits, stathmin1, and CREB following ionizing radiation exposure. Data from immunoblotting of mitochondrial subunits (Complex V: Atp5a, Complex III: Uqcrc2, and Complex I: Ndufb8), stathmin1α and -β, and both total CREB and phosphorylated CREB are depicted in panels A−D. The columns represent the fold changes with standard errors of the mean (SEM); n = 3. The visualization of protein bands shows the representative change from the biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student’s t test). Normalization was performed against endogenous GAPDH for Stat1α, Stat1β, CREB, and p-CREB and Ponceau intensity from total protein content of each lane for mitochondrial proteins. C: cortex, H: hippocampus.

StepOnePlus device. Expression levels of miRNAs and mRNAs were calculated based on the 2−ΔΔCt method with normalization against the median of all target miRNAs/mRNAs, respectively. Changes were considered significant if they reached a p value of ≤0.05 (unpaired Student’s t test, two-sided). Three biological replicates per dose group and brain region were used for each assay. Importantly, the total variance calculated as the standard error of the mean (SEM) of all target mRNAs was comparable in both hippocampus and cortex (hippocampus: 0 Gy: 0.09, 0.1 Gy: 0.09, 0.5 Gy: 0.08; cortex: 0 Gy: 0.08, 0.1 Gy: 0.07:0.5 Gy: 0.05) (SI Tables S2 and S3). This indicates that the high number of altered mRNAs in the irradiated cortex compared with the hippocampus did not arise from technical limitations such as sample generation, processing, or measurement.

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was 35 and 39 at 0.1 and 0.5 Gy, respectively. In the cortex, 39 and 51 proteins were deregulated at 0.1 and 0.5 Gy, respectively (Figure 1A). The proteome analysis showed that very few of these proteins were shared in both hippocampus and cortex. In addition, the proteome alterations induced by the low and moderate dose exposures were very distinct. Only protein kinase C gamma (Prkcc) was found to be always down-regulated, independent of dose or brain region (fold-changes in hippocampus: 0.1 Gy: −1.43, 0.5 Gy: −1.54; fold-changes in cortex: 0.1 Gy: −1.44, 0.5 Gy: −1.47) (SI Table S1). To obtain information about the signaling pathways affected by these changes, we performed a bioinformatics analysis of all deregulated proteins using IPA software. The analysis was done by a hierarchical comparison against the p values within each radiation condition. Despite the relatively small number of shared deregulated proteins between the brain regions and doses, the pathways altered by ionizing radiation showed a considerable overlap (Figure 1B). The signaling pathways showing the highest significance scores were oxidative phosphorylation, mitochondrial dysfunction, synaptic LTP, and stathmin1 signaling, followed by cell-adhesion-associated signaling pathways (remodelling of epithelial adherens junctions, cell junction signaling, and gap junction signaling) (Figure 1B).

RESULTS

Acute Irradiation Alters Synaptic Remodelling and Mitochondrial Signaling Pathways

The mice were irradiated with doses of 0.1 and 0.5 Gy, followed by a global quantitative proteome analysis 24 h postirradiation. SI Table S1 shows the complete list of deregulated proteins. The number of significantly deregulated proteins in the hippocampus D

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Journal of Proteome Research Low-Dose Radiation Reduces Mitochondrial Complex I Levels

lated CREB showed a strong increase in the cortex at both doses, whereas it was not altered in the hippocampus (Figure 2C,D). A high phosphorylation status of CREB may lead to a pronounced transcription of CREB target genes.31 Accordingly, gene expression analysis demonstrated an increase in the CREB regulated gene Crem at both doses in the cortex (0.1 Gy: 1.52, 0.5 Gy: 1.61) but not in the hippocampus (SI Table S2 and S3). Moreover, we found an increased gene expression associated with the development and maturation of the CNS via neuronal survival, proliferation, and synaptic plasticity only in the cortex at 0.1 Gy (Ntf 3, Ntf5, Ntrk2, Rela [Nf kb3], Nf kb1, and Homer1) and 0.5 Gy (Ntf 3, Ntrk2, Rela [Nf kb3], Nf kb1, and Homer1) (SI Table S3).

Immunoblotting using antibodies against core subunit proteins of the mitochondrial respiratory complexes I, III, and V showed a significant reduction of Ndufb8 (Complex I) protein levels at 0.1 and 0.5 Gy in the cortex (Figure 2A,B). This was in agreement with the mass spectrometry data demonstrating reductions in Ndufs7 (0.1 Gy) and Ndufs1 (0.5 Gy) subunits of mitochondrial Complex I (SI Table S1). Atp5a (Complex V) expression investigated by immunoblotting demonstrated no change, whereas mass spectrometry data indicated reduced levels of a number of other Complex V components (Atp6v1a, Atp1a3, Atp5c1) (SI Table S1). In the hippocampus, similar changes were observed as in the cortex; however, we noted a reduction of Ndufb8 (Complex I) protein only at 0.5 Gy (Figure 2A,B). This correlated with reduced Ndufa9 and Ndufs3 levels obtained by mass spectrometry at this dose (SI Table S1). Atp5a (Complex V) was unchanged in immunoblot analysis (Figure 2A,B), although mass spectrometry data indicated reduced levels of Atp6v1c1, Atp1b2, Atp1a2 (0.1 Gy), and Atp6v1c1 (0.5 Gy) (Complex V) (SI Table S1). Proteins of Complex III were unchanged both in massspectrometry-based approach (SI Table S1) and by immunoblotting of Uqcrc2 levels (Complex III) in cortex and hippocampus (Figure 2A,B). Taken together, the changes seen in signaling pathways associated with oxidative phosphorylation and mitochondrial dysfunction were due to reduced expression of Complex I subunits but may as well involve Complex V impairment.

Irradiation Reduces Stathmin1 Levels

To verify the changes seen in Stathmin1 signaling according to the proteomics data and bioinformatics analysis, we evaluated both stathmin1α and stathmin1β protein expressions by immunoblotting. Data showed that both were significantly reduced in the cortex at both doses, whereas only the α-chain was reduced in the hippocampus at 0.5 Gy (Figure 2C,D). In contrast, proteomics data showed an increase in stathmin1 (foldchange: 1.324) only in the hippocampus at 0.5 Gy (SI Table S1). Moreover, gene expression analysis demonstrated that also mRNA transcripts involved in cytoskeletal reorganization were altered in the cortex at both doses (increase in Pick and Plcg1) but were not changed in the hippocampus (SI Table S2 and S3). Additionally, our proteomics data indicated an elevated expression of microtubule/microtubule-associated proteins in the cortex (0.1 Gy: Tubb5, Tuba1a, Mtap1b; 0.5 Gy: Tubb5, Mapt, Tubb3, Mtap1b) and hippocampus (0.1 Gy: Tubb3; 0.5 Gy: Tuba1a, Tuba4a) (SI Table S1).

Signaling Pathway of LTP is Enhanced via Increased Phosphorylation of CREB

Cell-Adhesion-Associated Signaling Pathways Are Influenced by Irradiation Especially in the Cortex

To validate the observed deregulation of the synaptic signaling pathways, we quantified the expression of 84 mRNA transcripts of genes that are associated with synaptic plasticity (SI Table S2 and S3). We also performed immunoblotting of key proteins involved in these pathways to confirm the status of the pathway. The pathway-focused gene expression analysis as well as immunoblotting of the CREB protein demonstrated several alterations associated with LTP signaling. We noted an increase in the level of adenylate cyclases in both hippocampus (0.1 Gy: Adcy1 and Adyc8; 0.5 Gy: Adcy1) and cortex (0.5 Gy: Adcy1) (SI Table S2 and S3) accompanied by a number of molecular alterations in hippocampal neuronal receptors (0.1 Gy: reduced Gria2, increased Grm2; 0.5 Gy: increased Ephb2 and Grm2) (SI Table S2). Compared with hippocampus, we observed increased expression in a much larger number of neuronal receptors, particularly for glutamate at 0.1 Gy (Gabra5, Gria4, Grin2a, Grm3, and Grm7) and 0.5 Gy (Gabra5, Gria1, Gria2, Gria3, Gria4, Grin2a, Grin2b, Grm1, Grm3, Grm5, Grm7, Grm8, and Ngf r) in the cortex (SI Table S3). We also noted an elevation in the gene expression of protein kinases and phosphatases associated with LTP signaling only in the cortex at both 0.1 Gy (Ppp1ca, Ppp2ca, Ppp3ca, Prkca, and Prkg1) and at 0.5 Gy (Ppp1ca, Ppp1cc, Ppp2ca, Ppp3ca, Prkca, and Prkg1) (SI Table S3). Quantification of CREB and phosphorylated CREB levels as a downstream transcription factor of LTP signaling cascades showed a reduced total CREB level in the hippocampus at 0.5 Gy, whereas it was unaffected in cortex or in hippocampus at 0.1 Gy (Figure 2C,D); however, quantification of total phosphory-

We did not note any changes in the gene expression associated with cell adhesion and cell-adhesion-associated proteolytic processing in the hippocampus (SI Table S2). In contrast, we observed a number of alterations in the expression of cortical genes involved in cell adhesion (0.1 and 0.5 Gy: increase in Cdh2, Ncam1, Pcdh8) and cell-adhesion associated proteolytic processing (0.1 and 0.5 Gy: increase in Adam10, Reln, and Timp1) (SI Table S3). Similarly, the proteomics data showed an increased expression of several cell adhesion proteins in cortex (0.1 Gy: Cdh13, Chl1 and L1cam; 0.5 Gy: Chl1) but also in the hippocampus (0.1 Gy: L1cam; 0.5 Gy: Ctnna2 and Cntn1) (SI Table S1). miRNA Expression Associated with Cognition Is Increased by Ionizing Radiation

Recently, we demonstrated that miRNAs play an important role in the acute radiation response in vitro and in vivo.28,32,33 Furthermore, miRNAs are rapidly emerging as central regulators of gene expression in the postnatal mammalian brain, and their role in synaptic plasticity processes involving neuronal circuits, higher cognitive abilities, and neuropsychiatric diseases has been reported.34 Thus, we studied the profile of 84 miRNAs associated with neurological development and neurological cognitive diseases such as autistic disorders, schizophrenia, Alzheimer’s disease, and Huntington’s disease (SI Table S4). In hippocampus, miR-125b-5p and miR-128-3p were significantly increased only at 0.5 Gy. In cortex, we found enhanced levels of miR-106b-5p, miR-181a-5p, and miR-203-3p at both doses, whereas the expression of let-7e-5p, miR-15a-5p, E

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Figure 3. Radiation-induced deregulation of miRNAs in hippocampus and cortex. Data from miRNA quantification in hippocampus [H] and cortex [C] from C57BL/6 mice exposed on PND10 with doses of 0.1 and 0.5 Gy. The measurement was performed 24 h postirradiation. The columns represent the fold changes with standard errors of the mean (SEM); n = 3. Data were normalized against the median of all quantified 84 miRNAs on the assay plate. *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student’s t test).

and miR-376b-3p was increased only at 0.1 Gy and the expression of miR-146b-5p only at 0.5 Gy (Figure 3A−C).

Changes in Synaptic Plasticity and Outgrowth Due to Altered Cytoskeletal and Cell Adhesion Dynamics

DISCUSSION The aim of this study was to elucidate the effect of low-dose radiation on learning- and memory-related signaling pathways using omics approaches. Perinatal mice were exposed to doses similar to those received in single and repeated CT scanning of the head. Our proteomics and pathway-focused transcriptomics data suggest that signaling pathways associated with learning and memory formation such as LTP, mitochondrial function, and cytoskeletal and cell adhesion dynamics were the main radiationinduced changes. These are all involved in synaptic plasticity processes of neuronal networks. The rapid response after acute irradiation in vivo suggests that even low radiation doses are able to elicit alterations in signaling pathways related to cognition. Additionally, a regulatory role of miRNAs seems to be profound in this immediate response.

Our immunoblotting data showed decreased levels of stathmin1, especially in the cortex, where the effect was seen already at 0.1 Gy. Interestingly, a reduced stathmin1 expression has been noted in the frontal and temporal cortex of Alzheimer’s disease and Down syndrome patients.35 Similar observations were made by Jin et al. using primary neuronal cultures.36 Because stathmin1 functions as an important regulator of microtubule dynamics in neuronal dendrites,37,38 we suggest that low-dose radiation induces alterations in cortical dendritic microtubule cytoskeletal dynamics and thus dendrite arborisation. This is also highlighted by the increased expression of Pick1 only in the cortex. Pick1 functions as an adaptor that organizes the subcellular localization of membrane proteins such as neuronal AMPA receptors. It also negatively regulates Arp2/3-mediated actin cytoskeleton polymerization that is critical for the development of neuronal architecture.39 This is in good agreement with the downregulation of Arpc4 (actin related protein 2/3 complex, subunit

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Journal of Proteome Research 4) and Actr3b (Arp3 actin-related protein 3) that we find dysregulated in the mass-spectrometry-based proteome analysis in the cortex at 0.5 Gy. The discrepancy between proteomics-based and immunoblotting analysis in stathmin1 quantification may be due to the high sequence homology between Stat1α and Stat1αβ subunits, hindering their efficient discrimination in the mass spectrometry analysis. Interestingly, Buratovic et al. demonstrated that NMRI mice that had received a single total body dose of 0.5 Gy on PND10 showed a reduction of the microtubule-associated Tau-protein (Mapt) in the hippocampus 1 day postirradiation. This was accompanied by cognitive dysfunction in a novel environment behavioral test 2 and 4 months later.40 Our proteomics analysis demonstrated an increase in Mapt (fold-change: 1.35) in the cortex at 0.5 Gy but not in the hippocampus. We also noted an increased expression of miR-125b in the hippocampus at 0.5 Gy in this study. Injection of miR-125b into the hippocampus of mice has been shown to impair associative learning and increase tau phosphorylation, a marker of Alzheimer’s.41 Moreover, miR-125b was found to be differentially expressed in fetal hippocampus, aged brain, and Alzheimer’s hippocampus.42 We additionally noted an increase in mir-128-3p in the irradiated hippocampus (0.5 Gy). The neuronal miR-128 has been reported to regulate motor behavior by modulating neuronal signaling networks and excitability via alteration in ion channels, whereas an overexpression of miR-128 attenuated neuronal responsiveness and motor activity.43 Additional supporting evidence that low-dose ionizing radiation rapidly affects especially cortical synaptic plasticity and neuronal outgrowth comes from our observation of the increased gene expression in both Adam10 and Ncam1 levels in the cortex. The neural cell adhesion molecule (Ncam) plays an important role in directing and regulating the efficiency of neurite outgrowth in the brain.44 Ncam functions by interacting with Pak1 in growth cones of neurons.45 Recently, we showed that acute irradiation of NMRI mice with a gamma dose of 1.0 Gy affected the expression of Rac1, which is the upstream regulator of Pak1, in both hippocampus and cortex.28 Protease-directed cleavage and endocytosis of adhesion molecules might modulate cell responses on neurite outgrowth; Ncam-dependent shedding by Adam, a disintegrin metalloprotease, down-regulates neurite branching and outgrowth in cortical neurons.46 Furthermore, we found an increase in cortical Ntf 3, Ntf5, and neurotrophin receptor Ntrk2 in this study. Neurotrophic factors such as neurotrophin-3 (Ntf3) interacting with neurotrophin receptors can mediate neuronal survival and also neurite outgrowth, as reviewed elsewhere.47 Taken together, the process of axonal outgrowth and guidance requires the coordination of both actin and microtubule dynamics to promote synaptic shape and change of locomotion in neurites.48 This is in accordance with the cortical alterations of stathmin1 levels seen in this study, probably influencing microtubule formation and actin cytoskeleton signaling; however, this has to be elucidated by in situ neuromorphometric analysis in further experiments.

Furthermore, the transcriptional regulation changes noted by Lowe et al. resembled those found in the aged human brain and in the brain of Alzheimer’s patients;49 however, this study was performed using total coronal sections that included different brain regions such as motor cortex, portions of the frontal and parietal lobes, hippocampus, and diencephalons. In contrast, we separately analyzed the molecular biosignatures in both hippocampus and cortex. Also, Raber et al.50 observed similar findings in whole-body high-LET 28Si-irradiated 11-week old C57BL/6J mice after a dose of 0.25 Gy; the hippocampi were sliced and analyzed 3 weeks postirradiation, and the results showed that the magnitude of LTP was enhanced.50 We also noted an effect in LTP transmission in both brain regions, whereby the cortex seemed to be more affected by acute low-dose irradiation than the hippocampus in regard to number and intensity of deregulation of the molecular targets. In our study, an increased gene expression of several neuronal receptors, mainly metabotropic glutamate receptors (Grm’s) but also AMPA receptors (Grias) and NMDA receptors (Grins), was found in the cortex but not in the hippocampus. Interestingly, our miRNA analysis showed that several miRNAs were increased only in the cortex at both doses (miR-181-5p, miR-203-3p, and miR-106b-5p). They have been shown to be involved in neurotransmitter targeting (miR-181a: AMPA2, mGlu5, GABAA receptor α-1 subunit, and dopamine transporter DAT; miR-203: GABAA receptor α-1 subunit; miR-106b: NMDAR) in vivo and in vitro,51 affecting synaptic plasticity. Noteworthy, miR-181a has been shown to be enriched in the axons and growth cones of cortical neurons compared with the neuronal soma, suggesting involvement in local protein regulation during the formation of synaptic plasticity.52 It has also been shown that miR-181a inhibited dendritic growth in cultured neurons.53 A radiation-induced (1.0 Gy, 55 day old male and female C57BL/6 mice, 6 and 96 h postirradiation) deregulation of miR-203 and miR-106 in the cortex has also been observed by others.54 Thus, the data presented here demonstrate an altered function in intracellular signaling by neuronal receptors and synaptic plasticity within the synaptic compartment on the molecular level. These rearrangements include changes in the expression of several miRNAs. Furthermore, this was accompanied by increased gene expression of the effector kinases (adenylate cyclases, Adcys) as well as the downstream protein kinases (Prks) and phosphatases (Ppps) that are involved in the LTP formation, yet the increased phosphorylation status of CREB protein and the increased transcription of the CREB-mediated gene cAMP-responsive element modulator Crem55 are characteristic for LTP activation.56 The differences in the phosphorylation status of CREB between hippocampus and cortex have also been observed by others in male but not in female mice after acute radiation exposure (0.5 Gy, X-rays, 45 day old C57BL/6 mice, 2 h postirradiation).57 Because we found differences in the phosphorylation status of CREB between hippocampus and cortex using perinatal female mice, the gender-specific responses may be characteristic only to mice irradiated at a mature age. Given the essential role of CREB in neuroprotection and neuronal survival, for example, via upregulation of neurothropins (Ntfs)58 that was also seen in our study, enhanced cortical CREB activation may act as an adaptive response toward acute radiation exposure. The increased CREB activation in the cortex but not in the hippocampus may be reasoned with the different cellular structures as, for example, the hippocampal dentate granule cells exhibit marked neuronal survival.59,60

Activation of LTP Signaling

It was previously shown that whole-body irradiation using a gamma dose of 0.1 Gy altered the gene expression in the brain tissue of 8 to 10 week old B6C3F1/HSD male mice 4 h postirradiation.49 These alterations were associated with changes in the expression of ion channels and LTP formation.49 G

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exposure. It remains to be seen whether this dose that corresponds to the cumulative dose received after a few CT scans is able to cause long-term impairment of memory and cognition.

Nevertheless, alterations in CREB signaling can contribute to cognitive impairment61 and may lead to increased incidence and progression of neurodegenerative diseases such as Huntington’s, Alzheimer’s, and Parkinson’s disease.62,63

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Defects in Synaptic Plasticity May Reflect Altered Mitochondrial Function

S Supporting Information *

We have shown previously that ionizing radiation (2.0 Gy) leads to persistent biological alterations in the murine heart mitochondria due to inactivation of Complex I and III and an accompanying reduced respiratory capacity several weeks postirradiation.64,65 Similar data were obtained in vitro, demonstrating that 5.0 Gy gamma-ray irradiation can lead to decreased NADH dehydrogenase activity (Complex I) 12 h after radiation exposure.66 These data are in good agreement with our results, indicating acute mitochondrial dysfunction due to a presumptive reduction in the expression of mitochondrial Complex I subunits in the hippocampus and cortex immediately after acute low-dose radiation. Because the mitochondrial antibody cocktail used for the immunoblotting consists only of the core proteins important for the assembly of the mitochondrial complex, the discrepancy between proteomics and immunoblotting data as particularly in the case of Complex V is not unexpected, and has been observed before.64 Thus, a dysfunction of Complex III and presumably Complex V, as indicated in the proteomic analysis, cannot be ruled out in the low-dose irradiated brain. In neurodegenerative diseases such as Alzheimer’s, a functional decline of mitochondria has been observed.67 Because mitochondria are present in synapses,68 changes in synaptic morphology and signaling pathways as suggested in our study may be coupled to alterations in synaptic mitochondria.69,70 The damage in synapses includes defects on NMDA and AMPA glutamate receptor signaling71,72 and also metabotropic Gprotein coupled glutamate receptors73 as early molecular events. We observed alterations on the gene expression level of these receptor components. Moreover, synaptic signaling depends on intact synaptic mitochondria to sustain the high energy and calcium buffer levels that are required for this process.74 Nevertheless, we cannot distinguish whether the alterations originated from synaptic mitochondria or neuronal soma mitochondria in this study. On the basis of our molecular data suggesting alterations in synapses, it is highly likely that it is the synaptic mitochondria that are mainly affected in the acute radiation response, yet it is necessary to follow the presumptive mitochondrial damage over a longer period of time, analyzing mitochondrial complex activity and respiration capacity separately from isolated neuronal soma and synaptosomes.

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ASSOCIATED CONTENT

SI Table S1: Deregulated proteins found in mass spectrometrybased proteomics of hippocampus and cortex. SI Table S2: Analysis of the pathway-focused transcriptome of the hippocampus. SI Table S3: Analysis of the pathway-focused transcriptome of the cortex. SI Table S4: Quantified target miRNAs. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 89 3187 3445. Fax: +49 89 3187 3378. E-mail: soile. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research leading to these results was supported by a grant from the European Community’s Seventh Framework Programme (EURATOM) contract no. 29552 (CEREBRAD). The funders had no role in study design, data collection, analysis and interpretation, decision to publish, or preparation of the manuscript. We thank Stefanie Winkler, Klaudia Winkler and Sandra Helm for their excellent technical assistance.

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ABBREVIATIONS: Gy, gray; IPA, ingenuity pathway analysis; H/L, heavy-to-light ratio; ICPL, isotope-coded protein label; LC−MS, liquid chromatography mass spectrometry; OD, optical density; PBS, phosphate-buffered saline; PND, postnatal day; CNS, central nervous system; LTP, long-term potentiation; LTD, long-term depression; CREB, cAMP responsive element binding protein; CT, computer tomography

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REFERENCES

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CONCLUSIONS

Overall, our study demonstrates the importance of immediate radiation-induced mitochondrial dysfunction in the synaptic process affecting signaling pathways of LTP and neurotransmission, especially in the murine cortex but also in the hippocampus. Several signaling pathways found altered in this study are involved in neurocognitive disorders such as Alzheimer’s. In particular, our data highlight the role of synapses as a putative target of ionizing radiation in the hippocampus and cortex. This is congruent with our initial hypothesis that PND10 mice are highly sensitive to ionizing radiation due to the high state of synaptic remodelling at this time. Importantly, our data emphasize a potential harmful effect of doses as low as 0.1 Gy seen immediately after the radiation H

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