Presymptomatic Alterations in Energy Metabolism and Oxidative

Article pubs.acs.org/jpr

Presymptomatic Alterations in Energy Metabolism and Oxidative Stress in the APP23 Mouse Model of Alzheimer Disease Daniela Hartl,*,† Victoria Schuldt,† Stephanie Forler,† Claus Zabel,† Joachim Klose,† and Michael Rohe*,‡ †

Institute for Medical Genetics and Human Genetics, Charité-University Medicine, Berlin, Germany Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany

‡

S Supporting Information *

ABSTRACT: Glucose hypometabolism is the earliest symptom observed in the brains of Alzheimer disease (AD) patients. In a former study, we analyzed the cortical proteome of the APP23 mouse model of AD at presymptomatic age (1 month) using a 2-D electrophoresis-based approach. Interestingly, long before amyloidosis can be observed in APP23 mice, proteins associated with energy metabolism were predominantly altered in transgenic as compared to wild-type mice indicating presymptomatic changes in energy metabolism. In the study presented here, we analyzed whether the observed changes were associated with oxidative stress and confirmed our previous findings in primary cortical neurons, which exhibited altered ADP/ATP levels if transgenic APP was expressed. Reactive oxygen species produced during energy metabolism have important roles in cell signaling and homeostasis as they modify proteins. We observed an overall up-regulation of protein oxidation status as shown by increased protein carbonylation in the cortex of presymptomatic APP23 mice. Interestingly, many carbonylated proteins, such as Vilip1 and Syntaxin were associated to synaptic plasticity. This demonstrates an important link between energy metabolism and synaptic function, which is altered in AD. In summary, we demonstrate that changes in cortical energy metabolism and increased protein oxidation precede the amyloidogenic phenotype in a mouse model for AD. These changes might contribute to synaptic failure observed in later disease stages, as synaptic transmission is particularly dependent on energy metabolism. KEYWORDS: Alzheimer disease, APP23, mouse model, energy metabolism, Abeta, proteome

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INTRODUCTION Alzheimer disease (AD) is the most common neurodegenerative disease among the elderly. Although most cases of AD are sporadic, there are also rare and highly aggressive familial, early onset cases of AD (FAD). FAD mutations occur in three genes, the amyloid precursor protein (APP) and its processing secretases, presenilin 1 (PS1) and presenilin 2 (PS2).1 Introduced into animal models, FAD mutations provided useful insight into the pathomechanisms of AD. The APP23 mouse model overexpresses human APP carrying the Swedish (KM670/671NL, APPswe) mutation. This mutation results in increased Abeta production and causes cerebral amyloidosis.2−4 Plaque pathology as one important symptom of the human disease was observed in APP23 mice beginning at 6 months of age.2,5 Cognitive impairment was also observed. This symptom progressively worsens with increasing amyloid load.5−7 In an earlier study we have reported that a high number of proteome alterations were found at very early age in presymptomatic stages in two brain regions, cortex and hippocampus, of APP23 mice.8 We also observed a peak in the number of protein alterations during adolescence (around 2 months of age), a developmental stage associated with major structural changes in synaptic connections in the brain. Importantly, many proteome alterations, in particular of proteins involved in synaptic plasticity, observed in wild-type mice during adolescence were absent in APP23 mice. © 2012 American Chemical Society

Therefore we concluded that adolescent synaptic plasticity was disturbed in APP23 mice. In AD patients a decrease in synaptic density in affected brain regions is strongly correlated to cognitive impairment.9 The detailed molecular mechanism causing synaptic failure remains elusive, but APP is located at synapses where its processing and Abeta release are activity-dependent.9,10 In the study presented here we analyzed very young, one month old APP23 mice, representing a presymptomatic disease stage to determine possible causes of pathology. When analyzing the cortical proteome of one month old APP23 and wild-type mice, we observed that glycolysis-associated proteins were predominantly altered at this early age. In addition we observed increased carbonylation of proteins in transgenic mice and cultured transgenic cortical neurons. These changes might induce synaptic failure observed later in disease, as synaptic transmission is particularly dependent on energy metabolism.

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MATERIALS AND METHODS

Mouse Models and Analyzed Tissues

APP23 mice express human APP751 with the Swedish double mutation (KM670/671NL) and were backcrossed to C57Bl/6 Received: January 9, 2012 Published: May 8, 2012 3295

dx.doi.org/10.1021/pr300021e | J. Proteome Res. 2012, 11, 3295−3304

Journal of Proteome Research

Article

mice for over 20 generations.2 Proteome analysis of different age stages has been published previously.8 For enrichment analysis, results from cortex of 1 month old mice (presymptomatic stage) were considered. Sample size was n = 6 (biological replicates). All experiments performed with mice were conducted according to the guidelines of the German Animal Welfare Law. The study was approved by the State Office of Health and Social Affairs Berlin (approval number T0297/01).

for individual peptides using MS identification was equivalent to p < 0.05 for each peptide as calculated by MASCOT. Analysis of Functional Categories

Gene symbols were used to annotate identified proteins to gene ontology (GO, only biological functions were considered) categories. This was done using the open-access tool Webgestalt (Gene set analysis toolkit V2, http://bioinfo.vanderbilt.edu/ webgestalt/index.php). Enrichment analysis was conducted by applying the following settings provided by Webgestalt: Hypergeometric test, adjustment for multiple testing (BH; Benjamini and Hochberg), p ≤ 0.05, more than 3 proteins per category.

Protein Extraction

Protein extracts were prepared from cortex tissue according to our updated protein extraction protocol.11,12 Briefly, frozen tissue samples and sample buffer (4% CHAPS, 50 mM TRIZMA Base (Sigma-Aldrich), 50 mM KCl and 20% w/v glycerol at pH 7.5) as well as a proteinase and phosphatase inhibitor cocktail (Complete; PhosStop, Roche Diagnostics) were ground to fine powder in liquid nitrogen. Samples were then thawed and sonicated on ice (6 times for 10 s). Afterward, DNase, urea (6.5M) and thiourea (2 M) were added. The protein extracts were then supplied with 70 mM dithiothreitol and 2% v/w of ampholyte mixture (Servalyte pH 2−4, Serva) and stored at −80 °C until separation by 2-D electrophoresis.

Immunoblotting and OxyBlot Experiments

Protein samples were separated by the large-gel 2-DE technique developed in our laboratory as described previously.11,12 The original gel format for proteome analysis was 40 cm (isoelectric focusing) × 30 cm (SDS-PAGE) × 1.0 mm (gel width).8 For OxyBlot experiments, protein samples were separated on smallsize 2-D gels (5 ×10 cm).

Protein concentration was determined using the Roti-Nanoquant assay (Carl Roth). Protein extracts or immunoprecipitates were separated using 12% SDS-PAGE gels and blotted to PVDF membranes according to standard immunoblotting procedures. For OxyBlot experiments, 200 μg of protein were separated by small 2-DE. After IEF, gels were incubated in 2,4-dinitrophenylhydrazine (DNPH)-containing solution for 15 min following manufacturer’s instructions (OxyBlot Protein Oxidation Detection Kit, Millipore). After running the second dimension, gels were blotted to PVDF membranes, and DNP-lated proteins were detected using the DNP-specific antibody. Negative control samples were treated with DNPH-free control solution and positive controls of pre DNP-lated proteins (provided by Millipore) were analyzed to verify results. Four biological replicates were made for 1 month old APP23 and wild-type mice, respectively. Significant spot alterations were determined by quantification of spots using Delta2D (t test, p ≤ 0.05).

Spot Evaluation Procedure

Immunoprecipitation

Protein spot patterns from 2-D OxyBlots were evaluated by Delta2D imaging software (version 4.0, Decodon).13 Briefly, protein patterns were matched to each other using “exact” mode of Delta2D. Subsequently, a fusion image was generated employing “union” mode, creating a protein pattern containing all spots from all 2-D blots within the project. Digital spot detection was carried out on the fusion image, followed by manual control if the spots were detected correctly. Percent volume of spot pixel intensities was used for quantitative analysis of protein expression. Normalized values (after background subtraction) were used for statistical analysis. All significantly altered protein spots were assigned to their respective positions in silver-stained gels.

In order to purify carbonylated proteins from cortical or neuronal protein extracts, we immunoprecipitated DNP-lated proteins using DNP-specific antibody. 1200 μg (10 μg/μL) of protein of each protein lysate were DNP-lated as described before and incubated with DNP-specific antibody at 4 °C overnight. In addition, one (transgenic) sample was treated with DNPH-free control solution and served as negative control. The samples were then incubated with Protein-G agarose beads (Protein G Agarose, Pierce) for 2 h at room temperature. After binding of antibody to the beads, samples were repeatedly (6 times) pelleted and washed to remove unbound protein. Samples were then eluted from beads by incubation with SDS-PAGE sample buffer for 10 min at 90 °C and separated by SDS-PAGE. For MS analysis of proteins precipitated from cortex of one month old mice, gels were stained with Coomassie Brilliant Bue and each lane was cut to 18 pieces. Gel pieces were digested, and samples were analyzed by MS as described previously. All samples (wild-type, transgenic and negative control) were analyzed in one run to avoid technical variation and to allow relative quantification (emPAI). Validation of results by Western blot analysis was carried out for six proteins in DNP-immunoprecipitates (n = 3−6, biological replicates) using specific antibodies for the respective proteins.

Two-Dimensional Gel Electrophoresis (2-DE)

Protein Identification

For protein identification by mass spectrometry, 640 μg of protein extract were separated on 2-DE gels and stained with a MScompatible silver staining protocol.11 Protein spots of interest were excised from 2-DE gels and subjected to in-gel tryptic digestion. Peptides were analyzed by an ESI-tandem-MS/MS on a LCQ Deca XP ion trap instrument (Thermo Finnigan, Waltham, MA).8,14,15 Mass spectra were analyzed using our in-house MASCOT software package (version 2.1) automatically searching the SwissProt database (version 51.8/513877 sequences). MS/MS ion search was performed with the following set of parameters: (i) taxonomy: Mus musculus, (ii) proteolytic enzyme: trypsin, (iii) maximum of accepted missed cleavages: 1, (iv) mass value: monoisotopic, (v) peptide mass tolerance 0.8 Da, (vi) fragment mass tolerance: 0.8 Da, and (vii) variable modifications: oxidation of methionine and acrylamide adducts (propionamide) on cysteine. No fixed modifications were considered. Only proteins with scores corresponding to p < 0.05, with at least two independent peptides identified were considered. The cutoff score

Preparation and Treatment of Primary Cortical Neurons

Primary cortical neurons of wild-type and APP23 mice were prepared from newborn mice at postnatal day 1. Cortical cells were dissociated using papain (1 h at 37 °C) and cultured on poly-Dlysine/collagen coated culture dishes. Neurons were cultured for 14 days in Neurobasal-A medium (Gibco) containing B27 supplement (Sigma), and GlutaMAX (Invitrogen). Expression of human transgenic APP was tested with antibodies (WO2) specific for human 3296



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Figure 1. Carbonylated proteins on 2-D blots of presymptomatic APP23 and wild-type mice. Representative OxyBlots with spot signals detected in cortex of one month old wild-type and APP23 mice are shown. Protein signals altered in expression between APP23 and wild-type mice (n = 4) are indicated on selected regions of the 2-D blots (left part of the figure). Five spots were up- (1−5) and one spot was down-regulated (6) in transgenic mice.

proteins of a reference brain proteome data set of 1022 nonredundant proteins including all proteins identified so far in mouse brain on our 2-D gels.17 Proteins associated with Glycolysis or OXPHOS accounted for 2 or 3% of all proteins in our reference brain proteome data set, respectively. All proteins associated to these categories, the direction of expression change in APP23 mice and the numbers of changed spots are shown in Supporting Information Table S1. Because of the observed enrichment of glycolysis in young AD mice, we assume that alterations of proteins from these categories are specific for the introduced mutation and not caused by mere overrepresentation on 2-D gels. In order to investigate whether the observed enrichment of functional categories reached statistical significance, we conducted an enrichment analysis using Webgestalt.16 Enrichment analysis of GO-terms revealed a significant overrepresentation (enrichment, p = 0.02) of Glycolysis in presymptomatic APP23 mice. No other category was enriched. In summary, categorization of altered proteins revealed that Glycolysis was the most abundant functional group in presymptomatic APP23 mice. Enrichment analysis confirmed these results.

APP, and this analysis revealed that transgenic APP was already expressed in neurons from APP23 mice. Cells were harvested in ice cold PBS, and cell pellets were frozen immediately in liquid nitrogen. Twelve individual samples of APP23 and wild-type neurons were collected, respectively (n = 12). ADP/ATP Measurement

ATP and ADP levels were measured using the bioluminescent ADP/ATP ratio assay kit (Abcam). Primary cortical neurons (cell pellets) were incubated with 50 μL of sample buffer, and ATP/ADP levels were measured according to the manufacturer’s protocol.

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RESULTS

Enrichment of “Glycolysis” among Proteins Altered in the Cortex of Presymptomatic APP23 Mice

In a previous study of the APP23 mouse model, we analyzed the cortical proteome of transgenic mice at different age stages using large-gel 2-DE in combination with 2-D fluorescence difference gel electrophoresis (DIGE) and mass spectrometry.8 Numerous protein expression changes were observed even at an early, presymptomatic age of mice. Those proteins are of special interest since they might give insights into disease development rather than showing secondary protein changes unrelated to primary pathology. On the basis of the results of our previous study, we now investigated the enrichment of functional protein groups altered in cortex of one month old presymptomatic APP23 mice as compared to wild-type mice. Proteins were annotated with gene ontology (GO) terms using the open-access tool Webgestalt.16 We then determined the shares of distinct functional categories within our set of altered, nonredundant proteins.17 The two categories with the highest shares among altered proteins were Glycolysis (7 proteins, 13% of altered proteins) and Oxidative Phosphorylation (OXPHOS, 4 proteins, 7% of altered proteins). Since proteins associated with energy metabolism are highly abundant in cells and might therefore be over-represented on 2D-gels, we next determined the share of these two categories (Glycolysis and OXPHOS) among

Quantitative Changes of Protein Carbonylation in APP23 Mice Revealed by 2-D OxyBlots

Since proteins associated with energy metabolism were predominantly altered in presymptomatic mice, we next asked whether there might also be changes of oxidative protein modifications in presymptomatic APP23 mice. Those changes have already been observed in other mouse models for AD at an older age and in the brains of AD patients.18 Reactive oxygen species (ROS), produced mainly in mitochondria through OXPHOS, either directly oxidate proteins or induce oxidative modifications of proteins via secondary byproduct of oxidative stress. Protein carbonylation is one modification induced by ROS, and it can be used as a global indicator of oxidative stress. Moreover, protein carbonylation can be assessed relatively easy by derivatization of carbonyl groups with dinitrophenylhydrazone (DNPH). Regarding the functional relevance, carbonylation 3297



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Table 1. Proteins Altered in Expression between Wild-Type and Transgenic Mice As Analyzed by 2-D OxyBlots spot ID

gene name

protein name

1

Ube2v1

2 3 4 5 6

Cfl1 Pgam1 Lasp1 not identified Aco2 Aconitate hydratase, mitochondrial

regulation (trans/wild-type)

Ubiquitin-conjugating enzyme E2 variant 1 Cofilin-1 Phosphoglycerate mutase 1 LIM and SH3 domain protein 1

MOWSE score

sequence coverage (%)

number of unique peptides

up

111

25

2

up up up up down

293 427 438

33 35 32

4 6 7

177

6

3

Figure 2. Elevated carbonylation of proteins in APP23 mice. (A) Numbers of proteins identified in immunoprecipitates of carbonylated proteins prepared from wild-type (WT) or transgenic (APP23) mice and from a negative control sample (neg., transgenic cortex treated with DNPH-free solution). The Venn diagram shows an overlap of 55 proteins identified in both APP23 and WT mice. 28 and 9 proteins were only identified in APP23 or WT immunoprecipitates, respectively. (B) Western blot (anti-DNP) of DNP-immunoprecipitates from one positive control (pos., DNP-lated protein standard), one negative control, and two DNP-lated brain lysates (WT and APP23). (C) Representative Western blots of six proteins detected in immunoprecipitates of carbonylated proteins (DNP-IP, n = 3−6). Signal intensities of each protein were unaltered between APP23 and WT in protein lysates (Input). Levels of carbonylated protein in DNP-IP for all six proteins were significantly induced in APP23 mice. (D) Densitometric quantification of Western blot signals. All differences observed in DNP-IP’s reached statistical significance (*). WT signals were set to 100%.

In order to identify underlying proteins, we matched protein spots with altered expression between wild-type and transgenic samples to their corresponding positions in silver-stained gels. Spots were then excised from gels, and underlying proteins were identified by mass spectrometry (Table 1). One spot yielded no significant identification. The other spots were identified. Except for one protein (LIM and SH3 domain protein 1, Lasp1), all proteins altered in transgenic cortex were also identified in immunoprecipitates of carbonylated proteins (see following paragraph; Ubiquitin-conjugating enzyme E2 variant 1 was identified in immunoprecipitates but only with one significant peptide, which did not meet our significance criteria; the identification was therefore excluded from the list).

of proteins is thought to result in a loss of protein functions, and some carbonylated proteins have been shown to undergo degradation. However, protein carbonylation and decarbonylation was also recently proposed to serve as a mechanism of signal transduction (see review19). We separated DNPderivatized proteins by 2-DE followed by Western blotting and probed blots with DNP-specific antibodies (OxyBlot).20,21 We analyzed protein carbonylation in cortices of presymptomatic (1 month old) APP23 mice using the OxyBlot method. After separation of proteins by IEF (first dimension of 2-DE), carbonyls were derivatized by DNPH. DNP-lated proteins were then separated by SDS-PAGE (second dimension of 2-DE) and transferred to blot-membranes allowing analysis of carbonylated proteins using DNP-specific antibody. Comparison of wild-type and transgenic samples revealed significant up-regulation of five protein spots in APP23 mice. In addition, one spot group (corresponding to one protein) was down-regulated in APP23 mice (Figure 1, n = 4, biological replicates).

Changes of Protein Carbonylation in APP23 Mice Revealed by Immunoprecipitation

In order to identify more carbonylated proteins and to verify our previous results, we immunoisolated carbonylated proteins (derivatized by DNPH) from the cortex of two presymptomatic wild-type and two APP23 mice, using a DNP-specific antibody. 3298



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Table 2. Proteins Identified in Immunoprecipitates of Carbonylated Proteins from Two Wild-Type (Wildtype) and Two APP23 (APP23) Samplesa functional category

gene name

functional category

protein name

gene name

protein name

Proteins with higher abundance in transgenic immunoprecipitates

Proteins with higher abundance in transgenic immunoprecipitates

Atp6v1e1 Vcp

V-type proton ATPase subunit E 1 Transitional endoplasmic reticulum ATPase Slc25a4 ADP/ATP translocase 1 Transport Slc1a2 Excitatory amino acid transporter 2 (EAAT2) Vdac1 Voltage-dependent anion-selective channel protein 1 Calcium signaling Hpcal4 Hippocalcin-like protein 4 Vsnl1 Visinin-like protein 1 Endo-/Exocytosis Rab1A Ras-related protein Rab-1A Rab3b Ras-related protein Rab-3B Signaling Ywhae 14-3-3 protein epsilon Ywhah 14-3-3 protein eta Ywhaq 14-3-3 protein theta Cytoskeleton Arpc3 Actin-related protein 2/3 complex subunit 3 Tubb4 Tubulin beta-4 chain Myh10 Myosin-10 Myh9 Myosin-9 Dync1h1 Cytoplasmic dynein 1 heavy chain 1 Actbl2 Beta-actin-like protein 2 Proteins with higher abundance in wild-type immunoprecipitates

Proteins identified in wild-type and transgenic immunoprecipitates Energy Metabolism Mdh2 Malate dehydrogenase, (Glycolysis, OXPHOS, mitochondrial TCA) Atp5a1 ATP synthase subunit alpha, mitochondrial Atp5h ATP synthase subunit d, mitochondrial Ndufa4 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4 Aco2 Aconitate hydratase, mitochondrial Pgam1 Phosphoglycerate mutase 1 ATPase Atp1a1 Sodium/potassium-transporting ATPase subunit alpha-1 Atp1a3 Sodium/potassium-transporting ATPase subunit alpha-3 Ckb Creatine kinase B-type Atp6v1a V-type proton ATPase catalytic subunit A Calcium signaling Camk2a Calcium/calmodulin-dependent protein kinase type II alpha chain Endo-/Exocytosis Snap25 Synaptosomal-associated protein 25 Stx1a Syntaxin-1A Stxbp1 Syntaxin-binding protein 1 Signaling Ywhab 14-3-3 protein beta/alpha Ywhag 14-3-3 protein gamma Cytoskeleton associated Spna2 Spectrin alpha chain, brain Protein folding Hsp90aa1 Heat shock protein HSP 90-alpha Proteins identified in transgenic immunoprecipitates only Energy metabolism Atp5f1 ATP synthase subunit b, mitochondrial Atp5o ATP synthase subunit O, mitochondrial Uqcrc2 Cytochrome b-c1 complex subunit 2, mitochondrial Got2 Aspartate aminotransferase, mitochondrial Pgk1 Phosphoglycerate kinase 1 Pkm2 Pyruvate kinase isozymes M1/M2 Nme1 Nucleoside diphosphate kinase A ATPase/ATP transport Atp1a2 Sodium/potassium-transporting ATPase subunit alpha-2 Atp2b2 Plasma membrane calciumtransporting ATPase 2

Proteins identified in wild-type and transgenic immunoprecipitates Calcium signaling Calm1 Calmodulin Signaling Pebp1 Phosphatidylethanolamine-binding protein 1 (RKIP) Protein folding Hspa5 78 kDa glucose-regulated protein Proteins identified in wild-type immunoprecipitates only Energy metabolism Aldoc Fructose-bisphosphate aldolase C Calcium signaling Camkv CaM kinase-like vesicle-associated protein Endo-/Exocytosis Sptbn1 Spectrin beta chain, brain 1 Syt1 Synaptotagmin-1 Arf1 ADP-ribosylation factor 1 Cytoskeleton Tubb3 Tubulin beta-3 chain Protein folding/protein Hsp90ab1 Heat shock protein HSP 90-beta degradation Uba1 Ubiquitin-like modifier-activating enzyme 1 Other Hist1h4a Histone H4

a Biological replicates; only proteins not identified in the negative control sample were considered. Identified proteins were grouped into functional categories and according to abundance in APP23 and wild-type samples (in case proteins were identified in both wild-type and transgenic samples, emPAI values were considered to assess relative protein abundance).

To check for unspecific binding of proteins to beads or DNPantibody, we also treated one cortex sample of a transgenic mouse with DNPH-free control solution (negative control, Figure 2B). Immunoisolates were then separated by SDS-PAGE, and proteins were identified by mass spectrometry. All samples were identified in the same run to allow relative quantification of protein abundance in each of the samples. 62 and 82 proteins were identified in the wild-type and transgenic samples, respectively (Figure 2A). Of these proteins, an overlap of 33 proteins was identified in wild-type and transgenic samples. Thus, 9 and 28 proteins were uniquely identified in wild-type and transgenic cortex samples, respectively. In addition, 23 proteins were identified in the negative control, and 22 of these proteins were also

identified in wild-type and transgenic samples. Functional categorization of identified proteins revealed their association with different functional categories including energy metabolism, calcium signaling, endo/exocytosis, signaling, cytoskeleton, protein folding and others. All identified proteins and functional categories are shown in Supporting Information Table S2. Although mass spectrometry is not intrinsically quantitative, the number of identified peptides can be converted into “emPAI” values (exponentially modified protein abundance index), an approximate measure of absolute protein abundance.22 We used emPAI values calculated by Mascot to estimate the relative amount of the proteins present in wild-type and transgenic immunoprecipitation samples. We grouped proteins according to their relative abundance into 3299



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Figure 3. Energetic status and protein carbonylation in APP23 neurons and Abeta treated neurons. (A) ADP/ATP ratios as measured in primary cortical neurons of APP23 and wild-type (CTRL) mice (n = 12). Primary cortical neurons were differentiated for 14 days. ADP/ATP ratios were significantly down-regulated in APP23 as compared to CTRL neurons. (B) Western blot analysis of Vilip1 as observed in input or DNPImmunoprecipitates of primary cortical neurons. Carbonylation of Vilip1 was up-regulated in APP23 neurons (n = 4).

proteins with higher abundance in APP23 or wild-type immunoprecipitates (Table 2; only proteins with reproducible expression changes are shown). In addition, proteins only identified in either APP23 or wild-type immunoprecipitates were also considered to be more abundant in the respective samples (Table 2, all proteins identified along with emPAI values are shown in Suppporting Information Table S2) In order to confirm our results, we conducted Western blot analysis of selected proteins in DNP-immunoprecipitates of wild-type and transgenic mice (n = 3−6, biological replicates). Analysis of Visinin-like protein 1 (Vilip1), Sodium/potassiumtransporting ATPase (Atp1a2), Excitatory amino acid transporter 2 (EAAT2), Syntaxin-binding protein 1 (Stxbp1), Syntaxin-1 (Stx1) and Phosphoglycerate kinase 1 (Pgk1) confirmed that carbonylated forms of the respective proteins were significantly up-regulated in transgenic mice (Figure 2B and C). Quantification of Western blot signals revealed that carbonylated Vilip1, Atp1a2, EAAT2, Stxbp1, Stx1 and Pgk1 were up-regulated by 193, 25, 167, 250, 52 and 10%, respectively (mean values). All changes reached the criteria of significance (p ≤ 0.05; Mann−Whitney U test). We next asked if the total amounts of the respective proteins were altered in APP23 mice and which fractions of the proteins were modified by carbonylation. We therefore analyzed cortical lysates of 1 month old APP23 and wild-type mice by Western blot analysis (n = 3−6, biological replicates). Quantification of protein signals revealed no significant changes in the total amounts of Vilip1, Atp1a2, EAAT2, Stxbp1, Stx1 and Pgk1 when transgenic were compared to wild-type samples (Mann−Whitney U test, detection of tubulin serving as loading control). In a next step, we quantified the signals of carbonylated protein fractions (as observed in DNP-immunoprecipitates from cortical lysates) in relation to their total protein signals (as observed in cortical lysates of APP23 mice). Our analysis revealed that 6% of Vilip1, 5% of Atp1a2, 4% of EAAT2, 5% of Stxbp1, 2% of Stx1 and 3% of Pgk1 were carbonylated (mean values). These values might represent an underestimation since we cannot exclude that not the entire fraction of carbonylated proteins was immunoprecipitated from protein lysates. We conclude that an increased fraction of proteins was carbonylated in APP23 as compared to wild-type mice. Western blot analysis of six selected proteins confirmed our results and revealed that a minimum fraction of 2−6% of each of the proteins analyzed was carbonylated in APP23 mice. No significant differences in total protein expression of the analyzed proteins in APP23 as compared to wild-type mice were observed. This indicates that total protein expression was not altered in APP23 mice to compensate for protein carbonylation.

Together, our results indicate enhanced oxidative stress in the cortex of presymptomatic APP23 mice as protein carbonylation can be used as a global indicator for oxidative stress. Down-Reglation of ADP/ATP Ratios and Increased Protein Carbonylation in APP23 and Abeta-Treated Neurons

Our previous results indicated changes in cortical energy metabolism, which is linked to oxidative stress. Next, we asked whether changes in energy metabolism substrates (such as ATP) can be observed in transgenic mice and whether these changes take place in neurons, the cell type mainly affected in AD. We therefore analyzed the ADP/ATP ratio in primary cortical neurons of APP23 and wild-type mice. Statistical analysis revealed a significant downregulation of the ADP/ATP ratio in APP23 as compared to wild-type neurons (Mann−Whitney U test, p = 0.034, n = 12, biological replicates, Figure 3A). Since increased protein carbonylation was observed in the cortex of presymptomatic APP23 mice, we analyzed if this was also the case in primary cortical neurons. Analysis of Visinin-like protein 1 (Vilip1) in DNP-immunoprecipitates of primary cortical neurons (n = 4) revealed that levels of carbonylated Vilip1 were significantly increased in transgenic neurons as compared to controls (Mann− Whitney U test, p = 0.007, Figure 3B). Together our results indicate that changes in neuronal energy metabolism are triggered by overexpression of mutated human APP.

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DISCUSSION We analyzed proteome changes in the cortex of the APP23 mouse model of AD at one month of age. In an earlier study, we have compared APP23 and wild-type mice at five different age stages and found many proteome alterations even in young, presymptomatic APP23 mice.8 Functional categorization of proteins altered in one month old APP23 mice now revealed that the categories with the highest shares were Glycolysis and OXPHOS. One of the first observable symptoms in the brain of patients with AD are abnormalities identified in positron emission tomography (PET) scans that indicate significantly decreased glucose utilization.23 Moreover these changes also occur in patients with mild cognitive impairment (MCI), and some studies even described glucose hypometabolism as best predictor for AD progression in cognitively normal persons, appearing more than four decades before onset of dementia and years before occurrence of AD histopathology (as neurofibrillary tangles and amyloid plaques).24−29 Thus, impairment of glucose metabolism is believed to be rather a cause than a consequence of neurodegeneration due to its early 3300



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appearance.30−32 Importantly, glycolysis rates show regional variation in the human brain with especially high rates found in the default mode network, a group of brain regions that participate in internal modes of cognition and that are also highly vulnerable to AD symptoms such as Abeta plaque deposition.33−37 It was therefore hypothesized that an elevated glycolysis rate is the underlying cause that predisposes the DMN above all other brain regions for AD pathology.33 In rodent models for AD, presymptomatic energy metabolism was only analyzed in two mouse models so far. In the 3xTg mouse model bearing mutations in three genes (human APPSwe,TauP301L, and PS1M146 V), presymptomatic decrease in Pyruvate dehydrogenase expression levels and activity, enhanced production of reactive oxygen species (ROS), and a decrease in mitochondrial respiration combined with up-regulation of the glycolysis rate were described.38 In the Tg mAPP mouse model overexpressing human APP, presymptomatic deficits in synaptic mitochondria were described.21 However, our findings that proteins associated to glycolysis and OXPHOS were predominantly altered in presymptomatic APP23 mice provide further evidence linking dysregulation of energy metabolism to AD. Moreover, we observed downregulation of ADP/ATP levels in primary cortical neurons of APP23 mice as compared to wild-type neurons. This demonstrates that dysregulation of energy metabolism is localized in neurons of APP23 mice. Similar findings have been described in neurons and SH-SY5Y cells treated with Abeta, in APP-overexpressing SH-SY5Y cells and in the forebrain of aged 3xtg mice.39−42 Together, these results suggest that neuronal energy metabolism might be altered by Abeta. ROS production is an inevitable consequence of energy metabolism, and ROS are believed to play an important regulatory role in cellular metabolism and signaling. Importantly, production of ROS after exposure to Abeta has previously been described38,41−46 (see also reviews46,47). ROS directly impact on protein function through oxidative modification. Thus, a disequilibrium in the production and detoxification of ROS can have farreaching consequences.45,48 Glycolytic enzymes, subunits of the mitochondrial ATPase complex and other energy metabolism associated proteins were previously identified as targets of increased oxidative modifications (rendering them dysfunctional) in the brains of AD and MCI patients as well as in mouse models for AD.13,49−55 Accordingly, our analysis of protein carbonylation via 2-D gels (OxyBlots) and immunoprecipitation of carbonylated proteins revealed enhanced carbonylation of proteins in APP23 as compared to wild-type mice. Moreover, the majority of carbonylated proteins were associated to energy metabolism. Interestingly, this was observed at presymptomatic age of mice, which has not been shown before and might point toward a direct connection between APP overexpression or mutation (leading to enhanced Abeta production in APP23 mice) and energy metabolism dysregulation. This is also in line with energy metabolism associated proteins being predominantly altered in presymptomatic APP23 mice. Carbonylation and decarbonylation of proteins downstream of ROS might have a role in cell signaling (see review19). Isolation of carbonylated proteins by immunoprecipitation and subsequent identification by mass spectrometry revealed many proteins that were only identified in the APP23 cortex, which might be due to their higher abundance in APP23 as compared to wild-type cortex. In addition, many proteins were identified in both tissues, but had higher relative abundance (according to emPAI values) in transgenic as compared to wild-type immunoprecipitates. Those proteins can be categorized to proteins

associated to energy metabolism, ATPase subunits, cytoskeletal components, 14-3-3 proteins and proteins associated to endo/ exocytosis and calcium signaling. Interestingly, the latter four groups contained many proteins important for synaptic functions, and all groups contained proteins that were previously described to be altered in AD. Three ATPases identified in immunoprecipitates of APP23 mice have previously been related to AD: (i) Cerebral activity of the Na+/K+-transporting ATPase (Atp1a2), which is of fundamental importance for maintenance of neuronal excitability, and calcium homeostasis is inhibited by Abeta.56,57 Given that more Atp1a2 was carbonylated in transgenic mice, one might speculate that Abeta (indirectly) inhibits the activity of this enzyme via carbonylation. (ii) Vesicular V-ATPase (V-type H+-ATPase), which is essential for the cycling of neurotransmitters at the synapse, is targeted to lysosomes by the APP-cleaving enzyme subunit PS1.58 (iii) The activity of plasma membrane calciumtransporting ATPase has specifically been shown to be decreased in the brain upon Abeta administration.59 The brain consumes the most energy of any tissue in the human body. The high energy demand results from excitatory synaptic transmission where the restoration of ion gradients after depolarization and removal of glutamate from the synaptic cleft into astrocytes require large amounts of energy in form of ATP.60−62 Thus, synaptic transmission is tightly coupled to energy metabolism. Accordingly, among proteins with up-regulated carbonylation in transgenic mice, many were associated to synaptic transmission. Excitatory amino acid transporter 2 (EAAT2), for example, is the dominant glutamate transporter in cerebral cortex and hippocampus. Importantly increased oxidative modification of EAAT2 in AD has been demonstrated before.63 However, any dysfunction of EAAT2 results in excess glutamate leading to excitotoxicity and finally the death of synapses and neurons, a mechanism that is well-described in brains of AD patients.46,47 Other carbonylated proteins related to synaptic transmission were 14-3-3 proteins, protein members of the N-ethylmaleimide-sensitive-factor attachment receptor (SNARE; Syntaxin and Snap-25), Syntaxin-binding protein 1, Vesicle-associated membrane protein 2 (Vamp2) and Clathrin. Interestingly, 14-3-3 gamma has also previously been shown to be oxidatively modified in brains of MCI patients.18 Concerning proteins associated to calcium signaling, it was striking that two proteins of the calmodulin family of calcium sensor proteins, Visinin-like protein 1 (Vilip-1) and Hippocalcinlike protein 4 (Hpcal4) were identified in immunoprecipitates of transgenic mice, and increased carbonylation of Vilip-1 in transgenic mouse brains and primary cortical neurons was confirmed by Western blot analysis. Increased carbonylation of Vilip-1 has previously been demonstrated in response to induced ROS-production in the mouse brain.64 Vilip-1 may play a role in synaptic plasticity by influencing neuronal excitability. It interacts with alpha4beta2 nicotinic acetylcholine receptors (α4β2 receptors) enhancing acetylcholine responsiveness of the receptors and has been identified in a functional protein complex with glutamate receptors.65,66 Vilip-1 also forms a signaling complex with P2X2 receptors, in which it regulates surface expression of the receptor and its sensitivity for ATP.67 Importantly, Vilip-1 has recently been identified as potential biomarker for AD, as Vilip-1 concentrations were increased in the CSF and decreased in the cortex of AD patients.68−70 Increased Vilip-1 immunoreactivity is localized in close vicinity to Abeta-plaques and fibrillar tau-tangles in the brain, and it was shown that Vilip-1 promotes tau hyperphosphorylation.71 Although it is 3301



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not clear how carbonylation influences the function of Vilip-1, our results indicate an early involvement of Vilip-1 in AD pathology.

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CONCLUSION We observed predominant alterations in proteins associated to energy metabolism in cortex of APP23 mice at presymptomatic age. Moreover, oxidative modification (i.e., carbonylation) of proteins was increased at this age stage, and we could show that energy metabolism was altered in cultured neurons of APP23 mice. Together, our results indicate that altered energy metabolism might be the earliest observable consequence of APP mutation/overexpression in mice. Our study also indicates that impairment of synaptic transmission, a hallmark of AD, could be the consequence of energy metabolism changes as oxidative protein modification provides a link between energy metabolism, synaptic transmission and calcium homeostasis.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1: Glycolysis and OXPHOS associated proteins altered in cortex of 1 month old APP23 as compared to wild-type mice. Table S2: Proteins identified in immunoprecipitates of carbonylated proteins from two wild-type, two APP23, and one negative control sample. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 30 450566258. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Matthias Staufenbiel for providing the mouse model analyzed in this study. We thank Marion Herrmann, Yvonne Kläre, and Angelika Krajewski for excellent technical assistance and Grit Nebrich and Oliver Klein for mass spectrometry analysis. This study was financially supported by the “Deutsche Forschungsgemeinschaft” (DFG), Project HA6155/1-1. Acknowledgement is also made to the donors of ADR, a program of the American Health Assistance Foundation (AHAF), for support of this research.

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REFERENCES

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