Expression Profile of Rat Hippocampal Neurons Treatedwith the Neuroprotective Compound 2,4-Dinitrophenol:Up-Regulation of cAMP Signaling Genes

Adriano Sebollela • Leo Freitas-Correa • Fabio F. Oliveira • Camila T. Mendes •

Ana Paula Wasilewska-Sampaio • Juliana Camacho-Pereira • Antonio Galina • Helena Brentani •

Fabio Passetti • Fernanda G. De Felice • Emmanuel Dias-Neto • Sergio T. Ferreira

Received: 11 June 2009 / Revised: 22 October 2009 / Accepted: 3 November 2009

� Springer Science+Business Media, LLC 2009

Abstract 2,4-Dinitrophenol (DNP) is classically known

as a mitochondrial uncoupler and, at high concentrations, is

toxic to a variety of cells. However, it has recently been

shown that, at subtoxic concentrations, DNP protects

neurons against a variety of insults and promotes neuronal

differentiation and neuritogenesis. The molecular and cel-

lular mechanisms underlying the beneficial neuroactive

properties of DNP are still largely unknown. We have now

used DNA microarray analysis to investigate changes in

gene expression in rat hippocampal neurons in culture

treated with low micromolar concentrations of DNP. Under

conditions that did not affect neuronal viability, high-

energy phosphate levels or mitochondrial oxygen con-

sumption, DNP induced up-regulation of 275 genes and

down-regulation of 231 genes. Significantly, several up-

regulated genes were linked to intracellular cAMP signal-

ing, known to be involved in neurite outgrowth, synaptic

plasticity, and neuronal survival. Differential expression of

specific genes was validated by quantitative RT-PCR using

independent samples. Results shed light on molecular

mechanisms underlying neuroprotection by DNP and point

to possible targets for development of novel therapeutics

for neurodegenerative disorders.

Keywords Neuronal cultures � Hippocampus �Neuroprotection � DNP � Gene expression � Cyclic AMP

Introduction

2,4-Dinitrophenol (DNP) is classically known as a mito-

chondrial uncoupler. At high concentrations, DNP disrupts

the proton gradient across the mitochondrial membrane and

inhibits oxidative phosphorylation (Parascandola 1974;

Hanstein 1976). Surprisingly, however, recent in vitro and

in vivo studies have shown that at low subtoxic concen-

trations, DNP protects neurons against a variety of insults

and promotes neuronal differentiation and neurite out-

growth (reviewed in De Felice and Ferreira 2006; De

Felice et al. 2007a). For example, DNP reduces brain

damage caused by striatal injection of the NMDA receptor

agonist quinolinic acid (Maragos et al. 2003; Korde et al.

2005a), focal ischemia-reperfusion (Korde et al. 2005b),

and traumatic brain injury (Pandya et al. 2007), all of

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12640-009-9133-y) contains supplementarymaterial, which is available to authorized users.

A. Sebollela � L. Freitas-Correa � F. F. Oliveira �A. P. Wasilewska-Sampaio � J. Camacho-Pereira � A. Galina �F. G. De Felice � S. T. Ferreira (&)

Instituto de Bioquımica Medica, Programa de Bioquımica e

Biofısica Celular, Universidade Federal do Rio de Janeiro,

Rio de Janeiro, RJ 21944-590, Brazil

e-mail: ferreira@bioqmed.ufrj.br

C. T. Mendes � E. Dias-Neto

Laboratorio de Neurociencias (LIM27), Instituto de Psiquiatria,

Faculdade de Medicina da Universidade de Sao Paulo,

Sao Paulo, SP 05403-010, Brazil

H. Brentani

Laboratorio de Bioinformatica, Hospital do Cancer AC

Camargo, Sao Paulo, SP, Brazil

F. Passetti

Laboratorio de Bioinformatica e Biologia Computacional,

Servico de Pesquisa Clınica, Coordenacao de Pesquisa (CPQ),

Instituto Nacional do Cancer (INCA), Rio de Janeiro,

RJ 20231-050, Brazil

E. Dias-Neto

Centro de Pesquisas do Hospital do Cancer, Sao Paulo,

SP 01509-900, Brazil

123

Neurotox Res

DOI 10.1007/s12640-009-9133-y

which are associated with increased generation of reactive

oxygen species (ROS) (Dugan et al. 1995; Mattson 2003;

Korde et al. 2005a; Sullivan et al. 2005). DNP treatment

also improves mitochondrial function and attenuates oxi-

dative damage in a spinal cord contusion model in rats (Jin

et al. 2004). These observations led to the proposal that the

neuroprotective actions of DNP are due to mild mito-

chondrial uncoupling causing a reduction in formation of

toxic ROS (Papa and Skulachev 1997; Brand 2000).

On the other hand, we have previously shown that, at

low micromolar concentrations, DNP blocks the neuro-

toxicity instigated by both fibrils and soluble oligomers of

the amyloid-b peptide (De Felice et al. 2001, 2004), and

induces neurite outgrowth and neuronal differentiation

under conditions that do not cause an increase in O2 con-

sumption (Wasilewska-Sampaio et al. 2005). These results

indicate that, at least in part, the neuroprotective actions of

DNP do not involve mitochondrial uncoupling. Interest-

ingly, DNP induces an increase in intraneuronal levels of

the second messenger cyclic AMP (cAMP) in primary

cultures of both cortical and hippocampal neurons as well

as in a neuroblastoma cell line (Wasilewska-Sampaio et al.

2005). cAMP is a key messenger in a number of important

neuronal processes, including control of neurite outgrowth,

neuronal differentiation, and regeneration (De Felice et al.

2007a), and is also known to regulate mRNA expression of

several memory-related genes (Kandel 2001).

Despite the potential applications of DNP in the devel-

opment of novel approaches to treat neurodegenerative

disorders, the mechanisms underlying its beneficial neu-

ronal actions remain to be fully elucidated. In order to gain

insight into such mechanisms, we have now performed a

DNA microarray analysis to investigate changes in gene

expression in rat hippocampal neurons in culture treated

with DNP. This revealed a set of 275 up-regulated genes

and 231 down-regulated genes. Interestingly, several up-

regulated genes are linked to cAMP signaling pathways,

substantiating the involvement of cAMP signaling in the

neuroactive properties of DNP.

Materials and Methods

Primary Neuronal Cultures

Hippocampal neuronal cultures were prepared from

18-day-old rat embryos as previously described (Paula-

Lima et al. 2005, 2009). Briefly, hippocampi were dis-

sected in PBS-glucose, mechanically dissociated, and cells

were plated onto poly-L-lysine-coated wells at densities of

1.5 9 106 or 5 9 104 cells/well (for 35 mm wells or 96

well plates, respectively) in Neurobasal/B27 medium

(Invitrogen, Carlsbad, CA, USA) with antibiotics. After

3 days at 37�C under a 5% CO2 atmosphere, cultures were

treated with vehicle (Milli-Q purified H2O) or DNP (Sigma

Chem. Co., St. Louis, MO, USA; from a freshly prepared

2 mM stock solution in H2O) and were further incubated at

37�C for the indicated times.

Cell Viability

The viabilities of neuronal cultures were assessed using the

Live/Dead kit (Molecular Probes, Eugene, OR, USA).

Culture medium was removed and cells were gently

washed three times with PBS-glucose. Cells were then

incubated at room temperature for 40 min in the presence

of 2 lM calcein AM ester and 1 lM ethidium homodimer

in PBS-glucose. Images were acquired on a Nikon Eclipse

TE300 microscope. Live cells were identified by green

calcein fluorescence, and dead cells were identified by red

DNA-bound ethidium fluorescence. Percentages of live

neurons (means ± SEM) were calculated relative to the

total number of neurons in each field.

MTT reduction was assayed as described in Vieira et al.

(2007). After treatment with DNP or vehicle, cultures (in

96-well plates) were incubated for 4 h with 100 lg/ml

MTT (Sigma). Cells were disrupted and formazan blue

crystals were dissolved by addition of 100 ll of a 10%

solution of sodium dodecyl sulfate in 10 mM HCl.

Absorption was measured at 540 nm in a plate reader after

incubation at 25�C for 16 h.

Measurement of Reactive Oxygen Species

Reactive oxygen species formation was measured in live

cultured neurons using the fluorescent probe CM-

H2DCFDA (Molecular Probes) as previously described

(De Felice et al. 2007b). Neurons were loaded for 45 min

with 10 lM probe, rinsed with PBS, and immediately

visualized on the Nikon microscope. Quantitative analysis

of DCF fluorescence was carried out using Image J

(Abramoff et al. 2004). Appropriate thresholding was

employed to eliminate background signal in the images

before histogram analysis. Experiments were carried out in

triplicate wells per experimental condition, and at least

three fields per well were imaged and quantified. In all

experiments, fluorescence levels were normalized by the

number of cells.

Determination of ADP and ATP Levels

Intracellular ADP and ATP levels were determined using a

modification of the protocol described by de Souza Leite

et al. (2007). Cells were lysed in the presence of 6% tri-

chloroacetic acid, followed by immediate neutralization

with a small volume of 1 M Tris solution. Lysates were

Neurotox Res

123

centrifuged at 14,000 rpm for 5 min at 4�C, and superna-

tants were collected. Separation of the nucleotides was

achieved by ion-pair reversed-phase chromatography on an

analytical Supelcosil LC-18 column (Supelco, St. Louis,

MO, USA) equipped with a Supelguard guard column.

Runs were performed on a Shimadzu HPLC system

(Tokyo, Japan) at a flow rate of 1 ml/min. Sample size was

200 ll and the running buffer contained 50 mM KH2PO4,

50 mM K2HPO4, 4 mM TBAB, and 10% methanol (all

from Merck Co., Darmstadt, Germany), pH 6.0. Nucleotide

elution was monitored by absorption at 254 nm, and ADP

and ATP peaks were identified by co-injection of standards

(Sigma). Relative amounts of ADP and ATP were calcu-

lated by the ratio of their respective peak areas normalized

by protein concentration in each sample. Protein concen-

trations were determined using the BCA kit (Pierce,

Rockford, IL, USA).

Oxygen Consumption Measurements in Rat

Hippocampal Slices

Oxygen consumption rates were measured polarographi-

cally using high-resolution respirometry as described in

Kudin et al. (1999) with modifications using an Oroboros

Oxygraph O2K respirometer (Insbruck, Austria). Mea-

surements were performed in an electronically controlled

thermal environment with high temperature stability

(0.001�C). The electrode was calibrated between 0 and

100% (200 lM O2) saturation at atmospheric pressure

(101.3 kPa) at 37�C. Briefly, hippocampal slices (400 lm)

from 2-month-old rats were maintained in Krebs–

Ringer solution and increasing concentrations of DNP

(from 5 to 500 lM) or FCCP (carbonyl cyanide p-tri-

fluoromethoxyphenylhydrazone) (from 0.1 to 0.5 lM)

were sequentially added. A total of four slices were used in

each analysis, and results were normalized by the total

mass of the slices. Results represent means ± SD from

three independent experiments with slices from different

animals. Under our experimental conditions, greater than

95% in oxygen consumption rate [O2 flux per mass: pmol/

(s.mg)] could be blocked by antimycin A (data not shown).

RNA Extraction and Labeled cDNA Synthesis

Total RNA was extracted with Trizol (Invitrogen) follow-

ing manufacturer’s instructions. One millilitre Trizol was

used to extract RNA from 1.5 9 106 cells. Purity and

integrity of RNA preparations were checked by the 260/

280 nm absorbance ratio and by agarose gel electropho-

resis. Only preparations with 260/280 nm ratios C 1.8 and

no signs of rRNA degradation were used. RNA concen-

trations were determined by absorption at 260 nm.

For probe preparation, 10 lg of total RNA was reverse-

transcribed into cDNA incorporating Cy3-dUTP or Cy5-

dUTP using the CyScribe First-Strand cDNA labeling kit

(Amersham Biosciences, Little Chalfond, England) fol-

lowing manufacturer’s instructions. Incorporation of fluo-

rophore was determined by measuring absorption at

555 nm for Cy3 and 655 nm for Cy5.

Microarray Analysis

Equal amounts of labeled cDNA from vehicle- or DNP-

treated cultures were hybridized to 5 K oligo Rat arrays

(DNA Microarray Unit, National Autonomous University,

Mexico) as described in Luna-Moreno et al. (2007). Two

independent hybridizations (both with dye swapping) were

performed, corresponding to a total of four hybridizations

per experimental condition. Array images were acquired

and quantified on a ScanArray 4000 scanner with original

software from Packard BioChips (Billerica, MA, USA).

Images were acquired using 65% photomultiplier gain,

70–75% laser power, and 10 lm resolution at 50% scan

rate. For each spot, Cy3 and Cy5 mean density values and

corresponding background values were determined using

ArrayPro Analyzer software (Media Cybernetics; Silver

Spring, MD). Differentially expressed genes were identi-

fied using the genArise software (Luna-Moreno et al.

2007).

Functional Annotation

Over-represented gene ontology (GO) terms (biological

processes) were detected in the lists of differentially

expressed genes using the functional annotation tool of the

DAVID bioinformatics database (http://david.abcc.ncifcrf.

gov; Dennis et al. 2003). Independent analyses were per-

formed for up- or down-regulated gene using Rattus

novergicus as the background list. Only GO terms with C2

genes represented and EASE scores B0.05 were considered

in the output of the analysis.

Biological pathways affected by DNP treatment were

also identified using the Kegg Automatic Annotation Ser-

ver (http://www.genome.jp/kaas-bin/kaas_main) selecting

the ‘‘rno’’ (R. norvergicus) GENES dataset and bi-direc-

tional best hit options. Input lists consisted of differentially

expressed genes with z-score C 2 (z-score is an index that

measures the deviation, in standard deviation units, from a

data point to the local mean; for more details, see

http://www.ifc.unam.mx/genarise) obtained from two

independent samples. For each selected gene, the nucleo-

tide sequence of the largest available RefSeq mRNA was

retrieved from NCBI (http://www.ncbi.nlm.nih.gov).

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123

Quantitative RT-PCR Assays

One microgram of total RNA was used for cDNA synthesis

using 50 pmol of oligo dT20 and the Superscript III First

Strand cDNA kit (Invitrogen). Quantitative expression

analysis of genes of interest was performed by qRT-PCR

on a 7500 Applied Biosystems Real-Time PCR system

with the Power Sybr kit (Applied Biosystems, Foster City,

USA). b-actin (actb) was routinely used as an endogenous

control for data normalization. qRT-PCR was performed in

20 ll reaction volumes according to manufacturer’s pro-

tocols. Cycle threshold (Ct) values were used to calculate

fold changes in gene expression using the 2-DDCt method

(Livak and Schmittgen 2001). Statistical significance of

changes in expression was evaluated using Student’s t test.

Results

Low Doses of DNP Do Not Affect Cell Viability

or Mitochondrial Oxygen Consumption in Rat

Hippocampal Neurons

We initially asked whether treatment with low concentra-

tions of DNP affected cell viability or metabolic redox

activity in hippocampal neuronal cultures. Treatment with

20 lM DNP for 24 h had no effect on cell viability

(measured using the Live/Dead assay) compared to control,

vehicle-treated cultures (Fig. 1a). Similarly, no effect of

DNP on metabolic redox activity was detected using the

MTT assay (Fig. 1b). In addition, we investigated whether

DNP treatment interfered with high-energy phosphate

levels by directly measuring intraneuronal ADP and ATP

levels. Compared to control cultures, ADP and ATP levels

were unaffected in DNP-treated cultures (Fig. 1c).

Although the results described above indicated that

20 lM DNP had no effect on neuronal viability and mito-

chondrial function, the possibility remained that mild

mitochondrial uncoupling (not sufficient to significantly

affect MTT reduction or overall cellular ATP/ADP levels)

might take place under our experimental conditions. In

order to further investigate this possibility, we directly

measured mitochondrial O2 consumption in rat hippocam-

pal slices using high resolution respirometry. Titration of

DNP concentrations (ranging from 5 to 500 lM) indicated

that there was no significant alteration in O2 flux (mito-

chondrial O2 consumption) up to 50 lM DNP (Fig. 1d). A

tendency (which, however, did not reach statistical signifi-

cance) to increase O2 flux was observed at 100 lM DNP,

and a significant increase in oxygen consumption was only

detected at 200 and 500 lM DNP (Fig. 1d).

As mitochondrial production of ROS is quite sensitive to

mitochondrial uncoupling (Papa and Skulachev 1997), we

also measured ROS levels in control and DNP-treated

neuronal cultures using a ROS-sensitive fluorescent probe.

Treatment with 20 lM DNP had no effect on neuronal

ROS generation (P = 0.12; Suppl. Fig. 1). On the other

hand, a higher DNP dose (500 lM) markedly reduced ROS

levels (P \ 0.01; Supp Fig. 1).

Altogether, our data on cell viability, ATP/ADP levels,

O2 consumption and ROS production in hippocampal

neurons treated with 20 lM DNP show that DNP is not

neurotoxic and does not alter mitochondrial respiratory

activity at this low concentration. This conclusion is in line

with our previous finding that DNP concentrations higher

than 20 lM are necessary to induce mitochondrial uncou-

pling in a neuroblastoma cell line (Wasilewska-Sampaio

et al. 2005).

DNP Induces Changes in Neuronal Gene Expression

Changes in neuronal gene expression induced by DNP

were investigated using a rat 5K DNA microarray chip.

Using a z-score C 2 cutoff, we identified a total of 506

differentially expressed genes (DEGs), with 275 up-regu-

lated and 231 down-regulated genes. A full list of DEGs

can be found in Supplemental Tables I (up-regulated

genes) and II (down-regulated genes).

Functional (GO) classification of up-regulated genes

revealed significant over-representation of five biological

processes related to cAMP signaling pathways among the

top 20 biological processes presenting EASE scor-

es B 0.05 (Table 1). Moreover, among the five biological

processes presenting the highest fold enrichment scores

(which measures the increase in representation of a given

GO term; for details, see http://david.abcc.ncifcrf.gov/

home.jsp) were ‘‘Dopamine receptor signaling pathway’’

and ‘‘G-protein signaling, adenylate cyclase activating

pathway,’’ two cAMP-dependent memory-related path-

ways in neurons (Abel and Kandel 1998; Jay 2003; Bour-

tchouladze et al. 2006). Collectively, the five over-

represented processes related to cAMP signaling comprise

11 genes (Table 2), representing *4.0% of the total

number of up-regulated genes. Significantly, a similar

analysis using the set of genes that are down-regulated by

DNP revealed a lack of GO terms directly related to cAMP

signaling among the top 20 over-represented biological

processes (Table 3).

Another useful approach to extract biological meaning

from lists of DEGs consists of mapping those genes to

pathways to infer effects at the cellular or systemic levels

(Kanehisa et al. 2008). Moreover, identification of multiple

DEGs in a common pathway adds confidence to the results

found for each individual gene (Blalock et al. 2005). Using

the Kegg pathways tool (see ‘‘Materials and Methods’’

section), we identified ‘‘neuroactive ligand-receptor

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123

interaction’’ (Kegg rno04080) as the most represented

pathway in the sets of both up- and down-regulated genes

(Fig. 2). A total of 19 DEGs, representing 8.7% of the total

DEGs with a Kegg assignment, mapped to this pathway,

suggesting that DNP modulates gene expression of

neuronal receptors. Significantly, ‘‘MAPK signaling’’

(Kegg rno04010), a pathway known to involve signal

transduction driven by cAMP, was highly represented in

the classification of up-regulated genes (eight genes, cor-

responding to 6.8% of the total; Fig. 2a) but not in the set

Fig. 1 DNP does not affect cell viability or mitochondrial oxygen

consumption in rat hippocampal neurons. (Panels a–c) After 3 days in

vitro, dissociated neuronal cultures were treated with vehicle (water)

or 20 lM DNP for 24 h prior to Live/Dead and MTT assays or

intracellular ATP/ADP determination. a Representative Live/Dead

fluorescence images (409 magnification) from control (top) and

DNP-treated (center) cultures. Scale bar: 20 lm. The graph (bottom)

shows quantification of cell viability results (triplicate cultures, 5

fields per well). Bars correspond to means ± SD. b MTT reduction

assay. Results from a representative experiment (performed in

triplicate) from a total of three experiments yielding similar results.

c Neuronal levels of ADP and ATP were determined as described in

‘‘Materials and Methods’’ section. Results are means ± SD from

three independent experiments. The inset shows representative HPLC

chromatograms for control (green) or DNP-treated (red, shifted for

visualization) samples. A representative trace from a DNP-treated

sample to which ADP and ATP standards were added is also shown

(dotted line). d Oxygen consumption in rat hippocampal slices in the

absence (c, white bar) or in the presence of increasing concentrations

of DNP (black bars). Oxygen flow rates were measured using high

resolution respirometry, as described in ‘‘Materials and Methods’’

section. O2 flux values are normalized by control levels in each

experiment. Control O2 flux values ranged from 116 to 211 pmol

O2/mg tissue in experiments with hippocampal slice preparations

from three different animals. * P \ 0.05 (ANOVA followed by

Dunnet’s test)

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of down-regulated genes. Together with the GO functional

classification results, these observations suggest that cAMP

signaling is a major target of gene expression induced by

DNP. Kegg pathways with low representation (grouped as

‘‘other’’ in Fig. 2) obtained in both up- and down-regulated

datasets are listed in Supplemental Tables III and IV,

respectively.

Validation of Microarray Results by Quantitative

RT-PCR

Selected DEGs identified by microarray analysis were

validated using quantitative real-time PCR (qRT-PCR).

Selection of candidate genes for qRT-PCR analysis was

based both on their known participation in neuritogenesis,

Table 2 cAMP-related genes up-regulated by DNP treatment

Accession no. Gene symbol Gene name z-score Terma

NM_138915 Caly Calcyon neuron-specific vesicular protein 2.44 1

NM_031034 Gna12 Guanine nucleotide binding protein, alpha 12 2.24 1

NM_024365 Htr6 5-hydroxytryptamine (serotonin) receptor 6 2.51 3, 4

NM_012852 Htr1d 5-hydroxytryptamine (serotonin) receptor 1D 2.26 3, 4

X55812 Cnr1 Cannabinoid receptor 1 (brain) 3.70 3, 4

AF178674 Oprl1 Opioid receptor-like 1 2.87 3, 4, 5

NM_012728 Glp1r Glucagon-like peptide 1 receptor 3.29 2, 3, 4, 5

NM_030999 Crhr1 Corticotropin releasing hormone receptor 1 2.00 2, 3, 4, 5

NM_019132 Gnas Guanine nucleotide binding protein, alpha stimulating complex locus 3.25 2, 3, 4, 5

NM_022600 Adcy5 Adenylate cyclase 5 2.24 1, 2, 3, 4, 5

NM_013071 Oprm1 Opioid receptor, mu 1 2.33 1, 2, 3, 4, 5

a GO terms comprising each individual gene are coded as follows: 1 (Dopamine receptor signaling pathway); 2 (G-protein signaling, adenylate

cyclase activating pathway); 3 (G-protein signaling, coupled to cyclic nucleotide second messenger); 4 (Cyclic-nucleotide-mediated signaling); 5

(G-protein signaling, coupled to cAMP nucleotide second messenger)

Table 1 Functional

classification of up-regulated

genes in DNP-treated neurons

Over-represented GO terms

(biological processes) were

identified using DAVID (see

‘‘Materials and Methods’’

section). Terms presenting

EASE scores B 0.05 were

selected and ranked according

to their fold enrichment scores

(see text). Only top 20 terms are

listed. Terms related to cAMP

signaling processes are

highlighted (bold, italics)

Term Count % EASE

score

Fold

enrichment

Organic acid catabolic process 3 1.11 0.013 16.8

Carboxylic acid catabolic process 3 1.11 0.013 16.8

Dopamine receptor signaling pathway 4 1.48 0.002 14.9

Cofactor catabolism 4 1.48 0.022 6.6

G-protein signaling, adenylate cyclaseactivating pathway

5 1.85 0.015 5.2

Negative regulation of growth 5 1.85 0.018 4.9

Lung development 5 1.85 0.021 4.7

Respiratory tube development 5 1.85 0.023 4.6

Negative regulation of progression through cell cycle 8 2.95 0.003 4.2

Positive regulation of cell differentiation 5 1.85 0.033 4.1

Regulation of growth 12 4.43 0.000 4.0

Fatty acid metabolic process 12 4.43 0.000 3.9

G-protein signaling, coupled to cyclic nucleotidesecond messenger

9 3.32 0.002 3.9

Regulation of cell motility 5 1.85 0.041 3.8

Regulation of cell growth 8 2.95 0.005 3.8

Axon guidance 5 1.85 0.043 3.8

Response to oxidative stress 7 2.58 0.013 3.6

Cyclic-nucleotide-mediated signaling 9 3.32 0.004 3.5

Monocarboxylic acid metabolic process 15 5.54 0.000 3.4

G-protein signaling, coupled to cAMP nucleotidesecond messenger

6 2.21 0.035 3.3

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neuronal survival/differentiation, or synaptic plasticity

(biological processes that have been shown to be modu-

lated by DNP; reviewed in De Felice et al. 2007a) and on

the results from functional classification analysis described

above. Based on these criteria, six up-regulated and five

down-regulated genes were selected for qRT-PCR

(Table 4). mRNA levels of those genes were normalized by

beta-actin (atcb) expression. For some genes (calm3, gnas,

slc8a3), gapdh expression was also used for normalization,

yielding similar results (data not shown). Primer sequences

for all genes are described in Supplemental Table V. Dif-

ferential expression was confirmed by qRT-PCR for 7 out

of 11 genes tested (Table 4). Very low neuronal expression

precluded precise quantification of mRNA levels for htr6

(data not shown), one of the four genes for which differ-

ential expression could not be confirmed.

Discussion

Despite its known toxicity at high concentrations (Para-

scandola 1974), DNP is not cytotoxic at low micromolar

doses and has recently emerged as a lead compound for the

development of novel neuroprotective approaches (De

Felice and Ferreira 2006; De Felice et al. 2007a). DNP

affords efficient protection against neuronal damage

induced by ROS (Korde et al. 2005a, b), oxygen-glucose

deprivation (Mattiasson et al. 2003) and by aggregates of

the b-amyloid peptide (De Felice et al. 2001, 2004). Fur-

thermore, DNP inhibits the formation of amyloid fibrils and

oligomers from various proteins both in vitro (De Felice

et al. 2001, 2004; Raghu et al. 2002; Cardoso et al. 2003;

Vieira et al. 2006) and in vivo (De Felice et al. 2001), a

finding that holds promise for the development of thera-

peutic strategies against different types of amyloidoses.

Importantly, Takahashi et al. (2008) reported good toler-

ance to administration of low DNP doses in mammals. In

that study, no toxicity of DNP was detected upon admin-

istration of up to 10 mg/kg in rats, a dose considerably

higher than those used in in vivo studies reporting neuro-

protection by DNP (Maragos et al. 2003; Korde et al.

2005a, b). Along this line, it is interesting to note that

Caldeira da Silva et al. (2008) recently showed that chronic

oral administration of low DNP doses (1 mg/l in aqueous

solution, equivalent to approximately 100 lg/kg/day) was

not only non-toxic but also increased the lifespan of mice.

Mild mitochondrial uncoupling has been implicated as

the underlying mechanism of neuroprotection by DNP

(Mattiasson et al. 2003; Pandya et al. 2007). However,

recent studies have demonstrated that DNP may also act by

regulating intracellular levels of key proteins. For example,

DNP modulates protein levels of microtubule-associated

protein Tau in neurons (Wasilewska-Sampaio et al. 2005)

and induces a reduction in cell cycle-related protein levels

Table 3 Functional

classification of down-regulated

genes in DNP-treated neurons

Over-represented GO terms

(biological process) were

identified using DAVID (see

‘‘Materials and Methods’’

section). Terms presenting

EASE scores B 0.05 were

selected and ranked according

to their fold enrichment scores

(see text). Only top 20 terms are

listed

Term Count % EASE

score

Fold

enrichment*

Establishment and/or maintenance of

apical/basal cell polarity

3 1.4 0.009 19.8

Glutamate signaling pathway 4 1.8 0.008 9.8

Establishment and/or maintenance of cell polarity 4 1.8 0.011 8.5

Membrane lipid biosynthetic process 5 2.3 0.028 4.4

Neuropeptide signaling pathway 6 2.7 0.013 4.3

Response to protein stimulus 5 2.3 0.031 4.2

Response to unfolded protein 5 2.3 0.031 4.2

Microtubule-based movement 6 2.7 0.020 3.9

Angiogenesis 7 3.2 0.012 3.7

Cytoskeleton-dependent intracellular transport 6 2.7 0.033 3.4

Anatomical structure formation 8 3.6 0.010 3.3

RNA splicing 6 2.7 0.047 3.1

Regulation of transport 7 3.2 0.026 3.1

mRNA processing 7 3.2 0.027 3

Blood vessel morphogenesis 7 3.2 0.031 2.9

Microtubule-based process 9 4.1 0.011 2.9

Cell migration 11 5 0.012 2.5

Cell motility 15 6.8 0.002 2.5

Localization of cell 15 6.8 0.002 2.5

Lipid biosynthetic process 9 4.1 0.034 2.4

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in lung cancer cells (Han et al. 2008). In addition, DNP

treatment in vivo modulates neuronal levels of the amyloid

precursor protein (APP) (Madeiro da Costa, Martinez &

Ferreira, submitted), which may have important implica-

tions in neuronal processes such as neuroregeneration fol-

lowing nerve injury and Alzheimer’s disease.

Here, we show that DNP at a low concentration (20 lM)

causes widespread changes in neuronal gene expression in

the absence of alterations in cell viability, high-energy

phosphate levels, mitochondrial O2 consumption, and ROS

production. These results are in agreement with previous

data showing that low micromolar concentrations of DNP

do not affect oxygen consumption or mitochondrial mem-

brane potential in neuronal cell lines or primary cultures

(Wasilewska-Sampaio et al. 2005).

Interestingly, biological processes related to cAMP

signaling were significantly over-represented (as indicated

by EASE scores B 0.05) among the genes up-regulated by

DNP treatment. This finding is in harmony with our pre-

vious report that DNP stimulates an increase in cAMP

Fig. 2 Functional distribution

of up- and down-regulated

genes in DNP-treated neurons.

Pathways were identified using

the Kegg pathways database.

Percentages refer to the number

of differentially expressed genes

in each pathway relative to the

total number of genes

possessing a Kegg assignment.

Charts in panels a (up) and b(down) were based on 117 out

of 275 up-regulated genes and

100 out of 231 down-regulated

genes, respectively. Pathways

comprising less than four

differentially expressed genes

were collectively grouped as

‘‘other’’

Neurotox Res

123

levels in primary neurons and in a neuroblastoma cell line

(Wasilewska-Sampaio et al. 2005). cAMP is a central

component of intracellular signaling pathways that regulate

a variety of important biological processes, including

synaptic plasticity (Kandel 2001; Ji et al. 2005), neurite

outgrowth (Hernandez et al. 1995), neuronal differentiation

(Sanchez et al. 2004), and neuroregeneration (Teng and

Tang 2006). Thus, it is likely that up-regulation of genes

playing major roles in cAMP signaling is directly related to

the neuroprotective actions of DNP. These observations

give support to the proposal that DNP at low concentra-

tions could be used as a cAMP enhancing compound

against neuronal dysfunction and degeneration in neuro-

logical disorders such as Alzheimer’s disease (De Felice

et al. 2007a).

Functional analysis of the main biological pathways

represented in the lists of up- and down-regulated genes

revealed a network of processes related to cell growth,

learning, and memory (Fig. 3). Interestingly, DNP prefer-

entially activated cAMP-mediated signal transduction

rather than calcium-induced signaling (Fig. 3). This finding

is in agreement with previous results showing that DNP

causes only slight changes in intracellular calcium levels in

a cortical neuronal cell line (Paula Lima et al. 2008) and

that neuronal differentiation promoted by DNP is depen-

dent on activation of the extracellular signal-regulated

kinase, ERK, a downstream target of cAMP signaling

Table 4 Validation of differentially expressed genes by qRT-PCR

Accession number Gene symbol RQ z-score

NM_022606 Pp2c 1.40 ± 0.22* 3.00

NM_022600 Adcy5 1.44 ± 0.06* 2.24

NM_019132 Gnas 1.66 ± 0.45* 3.25

U31554 Lsamp 1.33 ± 0.13* 3.08

NM_012560 Fkhr 1.57 ± 0.16* 2.30

U53420 Slc8a3 0.73 ± 0.09* -2.60

NM_012920 Camk2a 0.75 ± 0.14* -2.65

NM_017237 Uchl1 1.13 ± 0.15 -2.66

NM_012518 Calm3 0.92 ± 0.14 -2.07

NM_022542 Arhb 1.06 ± 0.10 -2.38

Expression levels were determined by relative quantification (RQ),

calculated by the 2-DDCt method, using b-actin for normalization.

Results are means ± standard deviations of at least three independent

experiments. Statistically significant gene expression changes

(P \ 0.05; Student’s t test) are denoted by an asterisk in the RQ

column. Z-score values obtained in the microarray analysis are shown

for comparison

Fig. 3 DNP modulates signaling pathways related to learning/

memory and cell proliferation/differentiation. Gene products are

represented by their Kegg symbols and color—colored according to

microarray data (green for up- and red for down-regulation).

Pathways modulated by DNP treatment were identified using the

Kegg Pathways database. The scheme was created based on

‘‘Neuroactive ligand-receptor interaction’’ (Kegg rno04080), ‘‘MAPK

signaling’’ (Kegg rno04010), ‘‘Calcium signaling’’ (Kegg rno04020),

‘‘GnRH signaling’’ (Kegg rno04912) and ‘‘Long-term potentiation’’

(Kegg rno04720) pathways. Solid lines represent direct interactions,

whereas dashed and doted lines denote indirect interactions and

links to other cellular events, respectively. Symbols are: HTR6

(5-hydroxytryptamine (serotonin) receptor 6); GNAS (Guanine

nucleotide binding protein, alpha stimulating); ADCY5 (Adenylate

cyclase 5); P2RX7 (Purinergic receptor P2X, ligand-gated ion

channel 7); SLC8A3 (Solute carrier family 8, member 3); LHCGH

(Luteinizing hormone/Choriogonadotropin receptor); GNAQ (Guan-

ine nucleotide binding protein Q); CALM3 (Calmodulin 3);

CAMK2A (Calcium/Calmodulin-dependent protein kinase 2); CaV

(calcium channel, voltage-dependent); PLCB (Phospholipase C, beta

1); PKA (cAMP-dependent protein kinase); PLN (phospholamban);

RYR (ryanodine receptor); IP3R (inositol 1,4,5-triphosphate receptor

3); Raf (v-raf-1 murine leukemia viral oncogene homolog 1); MEK1/

2 (mitogen activated protein kinase kinase 1/2); ERK1/2 (extra-

cellular-signal-regulated kinase 1/2); Rsk (ribosomal protein S6

kinase); CREB (cAMP responsive element binding protein)

Neurotox Res

123

(Wasilewska-Sampaio et al. 2005). Activated ERK phos-

phorylates the cAMP-responsive element binding protein

(CREB), a transcription factor that plays a major role in the

regulation of expression of memory-related genes in neu-

rons (reviewed in Carlezon et al. 2005). Significantly, we

recently found that aged rats systemically treated with DNP

exhibited increased brain levels of phosphorylated CREB

and showed improved performance in memory tasks

(Wasilewska-Sampaio, De Felice and Ferreira, unpublished

results). Although CREB activation may also be triggered

by calcium (Lonze and Ginty 2002), signaling cascades

triggered by cAMP and calcium differ in terms of the

downstream targets and functional effects. For instance, it

has been shown that cAMP and calcium stimuli have

opposite effects in the control of MEF2-mediated gene

expression, which in turns participates in neuronal differ-

entiation and plasticity (Belfield et al. 2006). Therefore, it

is conceivable that the neuronal effects instigated by DNP

are specifically driven by cAMP signaling, rather than

calcium signaling.

Based on microarray results and subsequent bioinfor-

matics analysis, we selected a subset of genes for direct

validation by qRT-PCR. Alterations in mRNA levels

induced by DNP were confirmed by qRT-PCR for 70% of

those genes, a proportion that is in good agreement with

recently reported studies and reinforces the reliability of

the microarray findings. Among the differentially expres-

sed genes confirmed by qPCR, five are present in the

pathways represented in Fig. 3, thus substantiating our

conclusions based on functional analysis of microarray

data. In particular, we confirmed the up-regulation of three

genes, Fkhr, Lsamp, and Pp2c, which are directly impli-

cated in synaptic plasticity. Alterations in expression of the

transcription factor FoxG1, product of Fkhr gene, have

been associated with mental retardation (Shoichet et al.

2005) and impaired neurogenesis (Shen et al. 2006), while

protein phosphatase 2C (PP2C, product of the Pp2c gene)

is involved in the regulation of synaptic transmission by

interaction with both neuronal metabotropic glutamate

receptors (Flajolet et al. 2003) and voltage-gated calcium

channels (Li et al. 2005). In addition, the limbic system-

associated membrane protein LAMP (product of the Lsamp

gene) participates in mechanisms of induction of neurite

outgrowth and synaptogenesis (Pimenta et al. 1995).

In addition to cAMP signaling, our results also indicated

other biological processes and pathways affected by DNP.

For example, down-regulation of processes related to

neuronal signaling (such as ‘‘Glutamate signaling path-

way’’ (GO:0007215) and ‘‘Neuropeptide signaling path-

way’’ (GO:0007218) (Table 3) may reflect a

neuroprotective response against toxic excitatory stimuli.

Furthermore, presence of ‘‘Cell adhesion molecules’’ and

‘‘Regulation of actin cytoskeleton’’ pathways in both up-

and down-regulated gene sets (Fig. 2) may represent global

changes in expression of genes required for neurite

outgrowth.

It is also interesting to note that two uncoupling proteins

(ucp3 and ucp4) are present in the list of up-regulated

genes. However, to date these two gene products have not

been categorized into any Kegg pathway in the rat

(R. novergicus) database (http://www.genome.jp/keggbin/

show_organism?menu_type=gene_catalogs&org=rno). As

a result, despite the presence of these genes in the list we

obtained, no multi-gene pathway related to mitochondrial

uncoupling activity could be retrieved from Kegg analysis,

even when the group of underrepresented pathways (Sup-

plemental Table III) was taken into account. Similarly, we

did not detect any GO terms (biological processes) in

which either ucp3 or ucp4 were present in the list of

overrepresented processes (Tables 1, 3, using up- and

down-regulated genes lists, respectively). Therefore, based

on the criteria we have used to extract biological meaning

from our list of DNP-induced DEGs, we conclude that

uncoupling proteins are not a preferential target of DNP-

induced changes in neuronal gene expression. Nonetheless,

the possibility remains that up-regulation of uncoupling

proteins, in particular ucp3 and ucp4, as well as of other

genes found in this study, may play a role in neuropro-

tection instigated by DNP and that this should be further

investigated in future experiments.

In addition to the pathways revealed by the functional

analyses described above, DNP treatment affected the

expression of 96 genes (52 up- and 44 down-regulated)

with no characterized biological functions in the GO

database at the time of our analysis (March, 2009).

Ongoing efforts to improve annotation in the GO database

may result in future functional annotation of additional

DNP-targets.

In conclusion, current results show that DNP affects

gene expression in rat hippocampal neurons in culture.

Accumulating evidence indicates that, at low concentra-

tions, DNP is not neurotoxic and can be considered a

small-molecule neuroprotective compound. Interestingly,

transcriptional up-regulation by DNP included a number

of genes related to cAMP signaling, which may be

involved in the molecular mechanisms of neuroprotection

by DNP.

Acknowledgments This article was supported by grants from

Howard Hughes Medical Institute, Conselho Nacional de Desen-

volvimento Cientıfico e Tecnologico (CNPq/Brazil), Fundacao de

Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ/Brazil) and

Instituto Nacional de Neurociencia Translacional (INNT/Brazil) (to

STF). We thank Lorena Chavez Gonzalez, Simon Guzman Leon, Jose

Luis Santillan Torres and Jorge Ramırez for expert assistance with

microarray analysis, and Gerardo Coello, Gustavo Corral and Ana

Patricia Gomez for genArise software assistance. CTM and EDN

acknowledge the support of Associacao Beneficente Alzira Denise

Neurotox Res

123

Hertzog Silva (ABADHS) and Fundacao de Amparo a Pesquisa do

Estado de Sao Paulo (FAPESP).

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