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Tf-lipoplex-mediated c-Jun silencing improves neuronal survival following

excitotoxic damage in vivo

A.L.C. Cardoso a,b, P. Costa a,b, L.P. de Almeida a,c, S. Simes a,c, N. Plesnila d, C. Culmsee e,1,E. Wagner e, M.C. Pedroso de Lima a,b,a Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugalb Department of Biochemistry, Faculty of Science and Technology, University of Coimbra, Apartado 3126, 3001-401 Coimbra, Portugalc Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, 3000-295 Coimbra, Portugald Department of Neurosurgery & Institute for Surgical Research, University of Munich Medical Center Grohadern, Ludwig-Maximilians University, Germanye Department of Pharmacy, Ludwig-Maximilians University, Munich, Germany

a b s t r a c ta r t i c l e i n f o

Article history:

Received 24 July 2009

Accepted 3 November 2009

Available online xxxx

Keywords:

Tf-lipoplexes

c-Jun

Neuroprotection

Excitotoxicity

Kainate

siRNA

Excitotoxicity is one of the main features responsible for neuronal cell death after acute brain injury and in

several neurodegenerative disorders, for which only few therapeutic options are currently available. In this

work, RNA interference was employed to identify and validate a potential target for successful treatment of

excitotoxic brain injury, the transcription factor c-Jun. The nuclear translocation of c-Jun and its upregulation

are early events following glutamate-induced excitotoxic damage in primary neuronal cultures. We present

evidence for the efficient knockdown of this transcription factor using a non-viral vector consisting of

cationic liposomes associated to transferrin (Tf-lipoplexes). Tf-lipoplexes were able to deliver anti-c-Jun

siRNAs to neuronal cells in culture, resulting in efficient silencing of c-Jun mRNA and protein and in a

significant decrease of cell death following glutamate-induced damage or oxygenglucose deprivation. This

formulation also leads to a significant c-Jun knockdown in the mouse hippocampus in vivo, resulting in the

attenuation of both neuronal death and inflammation following kainic acid-mediated lesion of this region.

Furthermore, a strong reduction of seizure activity and cytokine production was observed in animals treated

with anti-c-Jun siRNAs. These findings demonstrate the efficient delivery of therapeutic siRNAs to the brainby Tf-lipoplexesand validate c-Jun as a promisingtherapeutic target in neurodegenerativedisordersinvolving

excitotoxic lesions.

2009 Elsevier B.V. All rights reserved.

1. Introduction

Hypoxic/ischemic events contribute to neuronal degeneration in

many acute central nervous system disorders, including stroke,

traumaticbraininjury andepilepsyand mayalso play a role in chronic

diseases, such as amyotrophic lateral sclerosis and Alzheimer's disease

[1]. The impact of all these disorders in developed countries is

considerable, with stroke, forexample, being oneof theleading causes

of death and disability worldwide. The fundamental process respon-

sible for triggering neuronal cell death after an ischemic event is

known as excitotoxicity [2] and refers to the excessive stimulation of

excitatory amino acid receptors, with consequent increase in intra-

cellular Ca2+ levels and activation of pro-death signalling pathways.

Calcium-dependent activation of proteases, lipases and nucleases

leads to cytoskeleton breakdown, oxidative stress and nuclear DNA

degradation, resulting in impaired neuronal function and, ultimately,

neuronal death[3]. Cell death mechanisms involve a series of proteins,

ranging from ion-channels and membrane receptors to mitochondrial

proteins and transcription factors. Revealing the role of these proteins

under physiological conditions and after excitotoxic damage is crucial

for the design of new and improved strategies aiming at neuroprotec-

tion and neuronal recovery after brain injury.

Stress-activated pathways, such as the mitogen-activated protein

kinase (MAPK) cascade, were found to be strongly activated following

cerebral ischemia [1,4], brain trauma and seizure [5] and, depending

on the duration of the hypoxic/ischemic insult and extent of energy

depletion, the activation of these processes may be neuroprotective or

detrimental to the cells. For example, cerebral ischemia enhanced the

activity of several members of the MAPK family, extracellular signal-

regulated kinases (ERK1 and ERK2), p38 and c-Jun N-terminal kinases

Journal of Controlled Release xxx (2009) xxxxxx

Corresponding author. Department of Biochemistry, Faculty of Sciences and Technol-

ogy, University of Coimbra, Apartado 3126, 3001-401 Coimbra, Portugal. Tel.: +351 239

820 190; fax: +351 239 853 607.

E-mail addresses: [email protected](C. Culmsee), [email protected]

(M.C. Pedroso de Lima).1 Current address: Clinical Pharmacy Pharmacology and Toxicology, Faculty of

Pharmacy, Philipps-University of Marburg, Karl v. Frisch Strasse 1, 35043 Marburg,

Germany. Tel.: +49 6421 25780; fax: +49 6421 25720.

COREL-05279; No of Pages 12

0168-3659/$ see front matter 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.jconrel.2009.11.004

Contents lists available at ScienceDirect

Journal of Controlled Release

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Please cite this article as: A.L.C. Cardoso, et al., Tf-lipoplex-mediated c-Jun silencing improves neuronal survival following excitotoxic damagein vivo, J. Control. Release (2009), doi:10.1016/j.jconrel.2009.11.004
mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.jconrel.2009.11.004http://www.sciencedirect.com/science/journal/01683659http://dx.doi.org/10.1016/j.jconrel.2009.11.004http://dx.doi.org/10.1016/j.jconrel.2009.11.004http://www.sciencedirect.com/science/journal/01683659http://dx.doi.org/10.1016/j.jconrel.2009.11.004mailto:[email protected]:[email protected]

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(JNK1, JNK2 and JNK3) were shown to be active after ischemia [6]. In

contrast to ERK1 and ERK2, which mediate neuroprotection when

activated by neurotrophins [7], p38 and JNKs are thought to be mostly

involved in inflammatory cytokine production and apoptosis [8,9].

Several studies have shown that JNK inhibition or knockout in the

brain not only improves the functional outcome followingkainic acid-

induced excitotoxic lesion [10,11], but also reduces lesion size after

cerebral ischemia [12]. The downstream events of JNK activation

involve c-Jun phosphorylation and formation of the activated protein-1 (AP-1) transcription factor, which leads to the expression of imme-

diate early genes (IEGs) and production of several proteins important

for processes of neuronal injury and inflammation, such as FasL, Bim,

COX-2 and TNF- [13,14]. Most of these IEGs were found to be

upregulated following hypoxic/ischemic injury, which suggests a

major role for c-Jun and AP-1 in the activation of some of the most

important executing pathways of delayed neuronal cell death.

The aim of the current study was to follow upour previousfindings

on the neuroprotective role of c-Jun silencing [15] in vitro, and

investigate the potential of siRNA-mediated downregulation of c-Jun

as a therapeutic approach in an excitotoxic lesion model in vivo, using

a lipid-based siRNA delivery system [16].

2. Materials and methods

2.1. Materials

The cationic lipid 1,2 dioleoyl-3(trimethylammonium)propane

(DOTAP) and cholesterol (Chol) were purchased from Avanti Polar

Lipids (Alabaster, AL, USA). Iron-saturated human transferrin (Tf) was

obtained from Sigma (Sigma, St.Louis, MO, USA). The anti-c-Jun siRNA

(5-AGTCATGAACCACGTTAAC-3 ) was obtained from Ambion (Ambion,

Austin, Texas, USA). The control non-silencing siRNA as well as all the

QRT-PCR reagentswas obtainedfromQiagen (Qiagen,Hilden,Germany).

The c-Jun antibody was purchased from Cell Signalling (Cell Signalling,

Danvers,USA), the-tubulin antibody was obtained from Sigma (Sigma,

Saint Louis, USA), the CD11b antibody was purchased from Serotec

(Serotec, Oxford, United Kingdom) and the GFAP and NeuN antibodies

were purchased from Millipore (Millipore, Billerica, USA). All otherchemicals were obtained from Sigma unless stated otherwise.

2.2. Animals

All efforts were made to minimize the number of animals and

suffering according to the guidelines of the German animal protection

law and derived guidelines on the ethical use of animals and the

relevant international laws and policies (Directive 86/609/EEC and

Guidefor theCareand useof Laboratory Animals,US NationalResearch

Council, 1996). C57BL/6 mice were obtained from Charles River,

Sulzfeld, Germany. All animals were kept under controlled light and

environmental conditions (12 h dark/light cycle, 231 C, 555%

relative humidity), having free access to food and water.

2.3. Liposome and lipoplex preparation

Cationic liposomes composed of DOTAP:cholesterol (1:1 molar

ratio) were prepared as previously described by Campbell [17] for in

vitro application. Briefly, a mixture of 1 ml of DOTAP and 1.5 ml of

cholesterol in chloroform (from stock solutions of 25 mg/ml DOTAP

and 37.8 mg/ml cholesterol), was dried under nitrogen in order to

obtain a thin lipid film. The film was dissolved in 100 l of ultrapure

ethanol and the resulting ethanol solution was injected into 900 l of

HGB buffer (Hepesglucose buffer: 5% glucose, 20 mM Hepes, pH 7.4)

maintained under continuous vortex, employing a 250 l Hamilton

syringe. The resulting MLV (multilamellar vesicles) were sonicated

briefly to obtainSUV (smallunilamellarvesicles) anddiluted in HBSto

afinal lipidconcentration of 1.43 mM DOTAP (1 mg/ml). Alternatively,

for in vivo application, the dried lipidfilm was hydrated in1.6 ml of 5%

HBG buffer (Hepesglucose buffer: 5% glucose, 20 mM Hepes, pH 7.4)

and sonicated for 5 min. The resulting liposomes were then extruded

21 times through two stacked polycarbonate membranes (50 nm pore

diameter) and diluted in HBG buffer to a final DOTAP concentration of

22.5 mM. The liposomes were stored at 4 C until use.

For the in vitro studies, Tf-lipoplexes were prepared by pre-

incubating a given volume of the liposome suspension with iron-

saturated human transferrin (32 g/g of siRNA) for 15 min, followedby addition of the necessaryvolume of siRNA stock solution to achieve

a final siRNA concentration of 50 or 100 nM in each well and a 2/1

lipid/siRNA charge ratio. The mixture was further incubated for

30 min at room temperature before delivery to the cortical neurons in

culture. Alternatively, for in vivo administration, Tf-lipoplexes

prepared at a 6/1 lipid/siRNA (+/) charge ratio were obtained by

mixing 0.8 l/animal of the liposome stock solution (22.5 mM DOTAP)

with 0.5 l/animal of human Tf solution (192 mg/ml in HBG),

followed by 15 min incubation prior to the addition of siRNAs (2 g/

animal). The resulting mixture was further incubated for 30 min. All

formulations were used immediately after being prepared.

2.4. Primary neuronal cultures

Primary mouse embryonic cortical neurons were obtained from

C57/BL6 mice, at day 16 of gestation, as described previously [18].

After dissociation and centrifugation of the dissected cortices, the tissue

was ressuspended in Neurobasal medium (Invitrogen, San Diego, CA,

USA), enriched with 2% (v/v) B27 supplement (Invitrogen), 2 mM

glutamine and 100 U/ml penicillin/streptomycin (Invitrogen). For

survival experiments, QRT-PCR and Western blot analysis, cells were

plated at a density of 0.5106 cells/well onto 12-well plates previously

coated with poly-L-lysine. For fluorescence microscopy studies, cells

were plated at a density of 0.12106 cells/well onto 12-well plates

containing glass coverslips previously coated with poly-L-lysine.

Characterization of the embryonic neuronal cultures confirmed the

presence of 95% neurons in these cultures, as determined by GFAP and

NeuN-immunostaining. Primary cultures were kept at 37 C in a

humidified atmosphere containing 5% CO2. All experimental treatmentswere performed in 1314 day old cultures.

2.5. In vitro Tf-lipoplex-mediated siRNA delivery and induction of

neuronal cell death

After 13 days in culture, 50 l of Tf-lipoplexes containing anti-c-Jun

or non-silencing (Mut) siRNAs was added to the cells to a final siRNA

concentration of 50 nM per well. After a 4 h incubation period (in 5%

CO2, at 37 C), the Neurobasal medium was replaced with a fresh

medium and the cells were further incubated for different periods of

time (24 h for QRT-PCR analysis and 48 h for Western blot or cell vi-

ability analysis). In order to induce oxygenglucose deprivation (OGD),

glucose-free EBSSmedium(6.8 g/l NaCl,0.4 g/l KCl,0.264 g/l CaCl22H2O,

0.2 g/l MgCl22H2O, 2.2 g/l NaHCO3, 0.14 g/l NaH2PO42H2O, pH 7.2)supplemented with gentamicin (5 mg/l) was purged with 95% N2 /5%

CO2 for 30 min, resulting in an oxygen content of 23%. After 14 days

in culture, neurons were washed 3 times with this medium and incu-

bated for 4 h in an oxygen-free 95% N2/5% CO2 atmosphere (OGD).

Control cultures were incubated in EBSS with 10 mM glucose. In order

to induce glutamate excitotoxicity, neurons (14 days in culture) were

exposed to 125 M glutamate in EBSS with 10 mM glucose, for 20 min.

Following the indicated incubation periods (4 h for OGD and 20 min

for glutamate excitotoxicity), the medium was replaced by standard

Neurobasal medium. Eighteen hours after theonsetof OGDor glutamate

challenge, cells were harvested for cell viability analysis or protein and

mRNA extraction. In parallel experiments, neurons were washed two

times in phosphate-buffered saline (PBS) andfixedin PBS containing 4%

paraformaldehyde for immunocytochemistry studies.

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2.6. Evaluation of cell viability

Cell viability of primary neuronal cultures was determined,

following Tf-lipoplex delivery, by 3-(4,5 dimethylthiazol-2-y1)-2,5-

diphenyltetrazolium bromide (MTT) reduction. Forty-eight hours

after transfection and 18 h after glutamate or OGD exposure, cells

were washed with fresh culture medium and incubated with MTT

(0.25 mg/ml) for 2 h at 37 C. The reaction was terminated by adding

dimethylsulfoxide solution and the absorbance was determined at590 nm and 630 nm in an ELISA microplate reader (Spectra FluorPlus;

Tecan, Durham, NC, USA). Cell viability was calculated as percentage

of control cells (non-treated cells) using the formula: (A590-A630) of

treated cells 100/(A590-A630) of control cells.

2.7. Stereotactic injection of Tf-lipoplexes and kainic acid

For hippocampal injections of Tf-lipoplexes, C57/BL6 mice were

anaesthetized with 10 l/g of avertin (1.3% tribromoethanol and 0.8%

amylalcohol in MiliQ water) and placed in a stereotactic apparatus. A

midline incision was made, the soft tissues were reflected and a burr hole

was made in the skull with the aid of a surgery micro-drill, at a point

2.00 mm (posterior) from bregma and 2.25 mm lateral from the

midline, according to Paxinos and Franklin [19]. A volume of 2 l of Tf-

lipoplexes in HGB, containing 2 g of anti-c-Jun or non-silencing (Mut)

siRNAs was injected at a rate of 0.2 l/30 s in the right hemisphere

(ipsilateral hemisphere) of each animal, via a stainless steel needle

connected to a Hamilton syringe (Hamilton Bonaduz, Bonaduz, GR,

Switzerland), 2.00 mm ventral from dura. For kainic acid (KA) adminis-

tration, each mouse received a single injection of 3 l PBS containing

1 nmol of KA in the lateral ventricle of theleft brain hemisphere, at a point

1.00 mm posterior from bregma, 1.75 mm lateral from themidline and

1.75 mm ventral from dura. Control animals received a single injection of

PBS (sham operated). Five minutes after the injections were completed,

theneedle waswithdrawnslowlyand theskinwassutured.No symptoms

of toxicity or loss of basic activity were observed in any of the mice

following Tf-lipoplex injection or vehicle application.

All animals were sacrificed 1, 3 or 5 days following Tf-lipoplexinjection and 1 or 3 days following KA administration. For histological

evaluation of cell viability and immunohistochemistry analysis, the

animals were transcardially perfused with 20 ml of an ice-cold 0.9%

NaCl solution, followed by further perfusion with 20 ml of ice-cold 4%

paraformaldehyde in 0.9% NaClsolution. The brains wereremoved and

postfixed (12 h) in the samefixative solution, followed by 23 days in

a cryoprotective solution containing 25% sucrose. Afterthis period, the

brains were rapidly frozen in dry ice, dipped in OCT embedding

medium (Sakura Finetek, Mijdrecht, The Netherlands), and 30 m

sections were cut at 20 C in a cryostat (Leica CM 3050 S, Leica,

Wetzlar, Germany) 1000 m anterior and 1000 m posterior from the

injection site and placed in PBS. For protein and mRNA extraction, the

brains were removed and placed on an acrylic matrix. A 2 mm coronal

section containing theinjection site wascut with a stainlesssteel razorand the hippocampal region from both hemispheres of each animal

was dissected and placed in the appropriate ice-cold lysis buffer.

2.8. Monitoring of seizure activity

Following KA administration, the micewere monitored continuously

for 4 h for the onset and extent of seizure activity. Seizures were rated

accordingto a previously definedscale[20]; stage 1: immobility, stage 2:

forelimb and/or tail extension, stage 3: repetitive movements, head

bobbing, stage 4: rearing and falling, stage 5: continuous rearing and

falling, blackouts and stage 6: severe tonic-clonic seizures and death. In

order to be included in this study, mice needed to demonstrate at least

stage 3 seizures.

2.9. Histological evaluation and immunostaining

For histological evaluation using cresyl violet staining, brain sections

containing the hippocampus region were mounted and dried in

gelatine-coated glass slides, and each slide was stained for 5 min in

0.5% cresylvioletsolution in acetatebuffer, rinsedtwice in water, briefly

dehydrated in ethanol, cleared in xylene solution and mounted with

Entellan (Merck, Darmstadt, Germany). The striatal slices were

examined under a Zeiss Axiovert microscope (Zeiss, Thornwood, NewYork, USA) equipped with 5 and 20 objectives. In order to quantify

neuronal loss, neuron counts were made in the CA3 region of every six

section (180 m separation distance), using an unbiased counting

frame. Only neurons with a visible nucleus and in which the entire

outline of the cell was apparent were considered intact. Intensively

stained, condensed and fragmented nuclei were considered damaged

and counted as pycnotic nuclei. For each section, the number of intact

and pycnotic neurons was counted in the CA3 region of both

hemispheres. Cell counts were averaged and mean numbers were

used for statisticalanalysis. Resultswere expressed as the percentage of

intact neurons of the total CA3 neurons per hemisphere.

Immunocytochemistry and immunohistochemistry were per-

formed in cultured cells or in 30 m brain slices, respectively,

according to established protocols. Briefly, the cells or tissue were

permeabilized for 30 min in PBS/0.5% Triton X-100, and non-specific

binding was blocked with PBS/4% goat serum (Invitrogen, Karlsruhe,

Germany) for an additional 30 min period. The cells or tissue were

incubated overnight at 4 C with the primary antibodies anti-c-Jun

(dilution 1:100), anti-NeuN (specific neuronal marker, dilution

1:500), anti-GFAP (specific marker of astrocytes, dilution 1:1000),

anti-CD11b (specific marker of microglia, dilution 1:500) or anti--

tubulin (dilution 1:1000), diluted in PBS containing 0.25% goat serum,

followed by two washing steps in PBS. Alexa Fluor 488 or Alexa

Fluor 594-conjugated secondary antibodies (Molecular Probes,

Leiden, The Netherlands) in PBS/0.25% goat serum were applied at a

1:500 dilution for 2 h at room temperature. After further rinsing twice

in PBS, the coverslips were mounted in glass slidesusing theProlong

Anti-fade kit (Molecular Probes) and the brain slices were mounted

on gelatine-coated glass slides using the FluorSave reagent(Calbiochem, Darmstadt, Germany). All coverslips or glass slides

sections were observed under a Zeiss Axiovert epifluorescence micro-

scope, equipped with the 20 or 40 objectives and the rhodamine,

FITC and DAPI filters. In order to evaluate c-Jun translocation to the

nucleus following KA-induced excitotoxicity, cell counts were

performed following exposure of neuronal primary cultures to KA.

The number of blue nuclei and red nuclei was counted in at least six

separate coverslips per condition and the results were expressed as

the percentage of red nuclei (cells presenting nuclear c-Jun) of the

total cell number per field.

Loss of the neuronal marker NeuN was also evaluated following KA-

induced lesion in vivo, by immunohistochemistry. Neuron counts were

made in the CA3 region of at least four sections per animal. For each

section, the number of red cells (cells positive for NeuN) was counted inthe CA3 region of both hemispheres and results were expressed as a

percentage of NeuNpositive neurons in control animals (sham-operated

animals).

2.10. Extraction of RNA and cDNA synthesis

Total RNA was extracted from 1 106 neuronal cells or from

hippocampal samples using the RNeasy Mini Kit (Qiagen, Hilden,

Germany), according to the manufacturer's recommendations for

cultured cells or brain tissue, respectively. Briefly, after cell lysis, the

total RNA was adsorbed to a silica matrix, washed with the recom-

mended buffers and eluted with 40 l of RNase-free water by centri-

fugation. After RNA quantification, cDNA conversion was performed

using the Superscript III First Strand Synthesis Kit (Invitrogen,

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Karlsruhe, Germany), according to the manufacturer's instructions.

For eachsample, cDNAwas produced from0.5 g of total RNA in a iQ 5

thermocycler (Bio-Rad, Munich, Germany), by applying the following

protocol: 10 min at 25 C, 30 min at 55 C and 5 min at 85 C. After

cDNA synthesis, a 30 min incubation period with RNase H at 37 C was

performed in order to remove any remaining RNA contamination.

Finally, the cDNA was diluted 1:3 with RNase-free water prior to

quantification by QRT-PCR.

2.11. Quantitative real time polymerase chain reaction (QRT-PCR)

Quantitative PCR was performed as described previously [16] inan

iQ5 thermocycler (Bio-Rad) using 96-well microtitre plates and the iQ

SYBR Green Supermix Kit (Bio-Rad). The primers for the target genes

(c-Jun, IL-1, IL-6 and TNF-) and the two tested housekeeping genes

(GAPDH and HPRT) were pre-designed by Qiagen (QuantiTect Primer,

Qiagen). A master mix was prepared for each primer set, containing a

fixed volume of SYBR Green Supermix and the appropriate amount of

each primer to yield a final concentration of 150 nM. For each

reaction, 20 l of master mix were added to 5 l of template cDNA. All

reactions were performed in duplicate (two cDNA reactions per RNA

sample) at a final volume of 25 l per well, using the iQ5 Optical

System Software (Bio-Rad). The reaction conditions consisted of

enzyme activation and well-factor determination at 95 C for 1 min

and 30 s followed by 40 cycles at 95 C for 10 s (denaturation), 30 s at

55 C (annealing) and 30 s at 72 C (elongation). The melting curve

protocol started immediately after amplification and consisted of

1 min heating at 55 C followed by eighty 10 s steps, with 0.5 C

increases in temperature at each step. Threshold values for threshold

cycle determination (Ct) were generated automatically by the iQ5

Optical System Software. The percentage of c-Jun knockdown or

interleukin decrease was determined following the guidelines for

relative mRNA quantification in the presence of target and reference

genes with different amplification efficiencies. The amplification

efficiency for each target or housekeeping gene was determined

according to the formula: E=10(1/S)1, where S is the slope of the

standard curve obtained for each gene.

2.12. Western blot analysis

Protein extracts were obtained fromneuronal primary cultures or

from hippocampal tissue samples homogeneized at 4 C in lysis

buffer (50 mM NaCl, 50 mM EDTA, 1% Triton X-100) containing a

protease inhibitor cocktail (Sigma), 10 g/ml DTT and 1 mM PMSF.

Protein content was determined using the Bio-Rad Dc protein assay

(Bio-Rad) and 20 g of total protein was resuspended in a loading

buffer (20%glycerol, 10% SDS, 0.1%bromophenol blue), incubated for

2 min at 95 C and loaded onto a 10% polyacrylamide gel. After

electrophoresis the proteins were blotted onto a PVDF membrane

according to standard protocols. After blocking in 5% non-fat milk,the membrane was incubated withthe appropriate primary antibody

(anti-c-Jun 1:500 and anti-GFAP 1:1000) overnight at 4 C, and with

the appropriate secondary antibody(1:20000) (Amersham, Uppsala,

Sweden) for 2 h at room temperature. Equal protein loading was

shown by reprobing the membrane with an anti--tubulin antibody

(1:10000) (Sigma) and with the same secondary antibody. After this

incubation period, the blots were washed several times with saline

buffer (TBS/T 25 mM TrisHCl, 150 mM NaCl, 0.1% Tween and

5 mg/ml non-fat powder milk) and incubated with ECF (alkaline

phosphatase substrate; 20 l of ECF/cm2 of membrane) for 5 min at

room temperature and then submitted to fluorescence detection at

570 nm using a Storm-860 (Molecular Dynamics, CA, USA). For each

membrane, the analysis of band intensity was performed using the

Quantity One software (Bio-Rad).

2.13. Statistical analysis

All data are presented as meanstandard deviation (SD). In vitro

data result from three independent experiments, each performed at

least in triplicate. One way ANOVA analysis of variance combined with

Tukey posthoc test was used for multiple comparisons in cell culture

experiments.

In vivo datawere analysed by the KruskalWallisone wayanalysis of

variance, followed by the Dunnett's posthoc test for multiple compar-isons between groups (n =6 for each group). Statistical differences are

presented at probability levels ofpb0.05, pb0.01 and pb0.001. Calcu-

lations were performed with standard statistical software (GraphPad

Prism 4).

3. Results

3.1. c-Jun expression and nuclear translocation following glutamate

toxicity in vitro

c-Jun activation and mRNA increase in neuronal primary cultures

were evaluated at different time points (15 min, 30 min, 1 h, 3 h and

6 h), by immunocytochemistry and QRT-PCR, following excitotoxic

damage mediated by acute exposure to glutamate. c-Jun mRNA levelsincreased significantly within thefirst 30 min after glutamate exposure,

reaching a peak at 30 min1 h after the insult and then decreasing to

basal levels (Fig. 1A). In the same time window, a similar time-

dependent increase in the number of cells showing c-Jun nuclear

translocation was observed (Fig. 1B and C). At 30 min following

glutamate exposure, 70% of neurons showed c-Jun nuclear labelling

(Fig. 1B and C, panels c, f and i) which strongly correlates with the

increase in c-Jun mRNA observed at this time point. No significant

modifications in nuclear morphology and cytoskeleton were observedat

early time points (Fig. 1C, panels a, b, c, j and k), but a degeneration of

neuronal terminals and tubulin filaments was evident, starting at 6 h

after glutamate insult (Fig. 1C, panel l). These results suggest that c-Jun

nuclear translocation is an early event of neuronal degeneration

following excitotoxic damage and precedes nuclear shrinkage andcytoskeleton fragmentation.

3.2. Neuroprotection following in vitro Tf-lipoplex-mediated c-Jun silencing

In a previous study [15], c-Jun silencing mediated by Tf-lipoplexes

containing anti-c-Jun siRNAs was found to result in a neuroprotective

effect in HT-22 cells, a neuronal cell line sensitive to glutamate damage.

Aiming at examining the possible neuroprotective role of c-Jun knock-

down in primary neurons, cortical cultures (day 13 after isolation) were

transfected with Tf-lipoplexes containing anti-c-Jun or Mut (non-

silencing) siRNAs and exposed to glutamate or OGD 24 h later. Results

in Fig. 2 illustrate the c-Jun mRNA and protein knockdown 24 h

following glutamate or OGD insults, as well as the neuronal cell viability

at this time point. A significant decrease in c-Jun mRNA (70% reduction)(Fig. 2A) and protein levels (50% reduction) (Fig. 2B and C) was

observed 48 h after transfection.Gene silencingwas found to be specific,

since no significant alteration of c-Jun levelswas observedafter delivery

of Mut siRNAs. These results demonstrate that efficient c-Jun silencing

by Tf-lipoplexes persists also in neurons exposed to the excitotoxic

damage.

In accordance with ourprevious results in a neuronal cell line, c-Jun

knockdown significantly increased neuronal viability after glutamate

(Fig.2D)orOGD(Fig.2E) exposure,by 25%and 30%respectively,when

compared to untreatedneuronalculturesexposedto these two insults.

No positive effect on cell viability was observed following delivery of

Mut siRNAs, which indicates that the observed neuroprotective effect

is directly related to c-Jun silencing andis not an indirect consequence

of the transfection process.


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3.3. Efficient and non-toxic Tf-lipoplex-mediated c-Jun knockdown in vivo

We have previously demonstrated that association of Tf to lipop-

lexes promotes siRNA delivery and efficient knockdown of reporter

genes in vivo [16]. Therefore, in this work we evaluated whether this

strategy would also result in efficient c-Jun silencing in the mouse

brain. For this purpose, C57/BL6 mice were injected with Tf-lipoplexes

prepared at 6/1 lipid/siRNA charge ratio and containing 2 g of anti-c-

Jun siRNAs or Mut siRNAs. The injections were performed near the

CA3region of thehippocampusof therighthemisphere(ipsilateral) of

each animal and c-Jun knockdown was evaluated 1, 3 or 5 days after

Tf-lipoplex delivery, by QRT-PCR, immunohistochemistry and Western

blot.Fig. 3 provides evidence that stereotactic injection of Tf-lipoplexes

leads to efficient c-Jun silencing in vivo, as observed by the significant

decrease of c-Jun mRNA (Fig. 3A) and protein levels (Fig. 3C and D)

following siRNA delivery. Around 60% decrease in c-Jun mRNA was

observed in the ipsilateral hemisphere of animals treated with anti-c-

Jun siRNAs, when compared to animals treated with Mut siRNAs

(Fig. 3A), 1 day after Tf-lipoplex injection. As expected from a non-viral

delivery system,c-JunmRNAsilencing mediated by siRNAswas found to

be transient and c-Jun mRNA returned to basal levels 5 days after Tf-

lipoplex injection (data not shown). Although no differences were

found in c-Jun protein levels at this same time point, a significant

reduction (45%) was observed starting at day 3 (Fig. 3B, C and D) and

silencing of the c-Jun gene lasted at least 5 days (Fig. 3A and D).

Immunohistochemistry images indicate that proteinknockdownoccurs

mainly in CA3 region (Fig. 3B, panel b), where loss of nuclear and

cytoplasmic c-Jun labelling can be observed in the pyramidal neurons.

In order to evaluate Tf-lipoplex biocompatibility, counterstaining

of brain slices with cresyl violet was performed in animals injected

with Tf-lipoplexes containing Mut or anti-c-Jun siRNAs (Fig. 4,

panels ad). In parallel experiments, immunohistochemical label-

ling of the specific cell markers GFAP (Fig. 4, panels e and f) and

CD11b (Fig. 4, panels g and h) was performed in order to investigate

a possible inflammatory reaction, which could lead to gliosis and

microglia activation.

No relevant signs of toxicity were found throughout the ipsilateral

hippocampus where, similarly to observations in the contralateral

hemisphere, neurons presented a light violet colour and large cell

body characteristic of healthy cells (Fig. 4, panel d). The presence of

vacuolization or large number of apoptotic cells was not detected inany of the injected animals and only a small number of pycnotic cells

were observed in the tissue surrounding the injection site. Moreover,

no differences in the number and morphology of astrocytes and

microglia cells were observed between the contralateral and

ipsilateral hemispheres (Fig. 4, panels eh). Results were similar in

animals receiving anti-c-Jun or Mut siRNAs. These results indicate

that Tf-lipoplexes are well tolerated and do not seem to induce a

significant inflammatory reaction following local application.

3.4. Contribution of c-Jun silencing to neuroprotection following in vivo

KA-induced excitotoxic lesion

Neurotoxicity mediated by overstimulation of glutamate receptors

and massive calcium influx is known to play an important role in

Fig. 1. c-Jun mRNA upregulation and nuclear translocation following glutamate-mediated

excitotoxicity. C-Jun mRNA quantification by QRT-PCR or immunocytochemistry

experiments took place 0 min, 15 min, 30 min, 1 h , 3 h or 6 h after the onset of lesion

(20 min with 125 M glutamate). (A) c-Jun mRNA levels are expressed as fold increase

above c-Jun mRNA levels in control cells. In order to study c-Jun nuclear translocation,

neurons were labelled with Hoechst 33342 (blue), anti-c-Jun antibody (red) and anti-

tubulin antibody (green). (B) The number of c-Jun positive nuclei was determined for

each time point and is expressed as the percentage of total number of nuclei. (C)

Fluorescence microscopy images were acquired at 200 magnification. Representative

images foreach timepoint arepresented separately forthe redand bluechannels andas

a merged image. Results in (A) and (B) are presented as mean valuesSD and are

representative of three independent experiments, each performed in triplicate. pb0.5,

pb

0.01 and

pb

0.001 compared to control cells.


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several pathologies such as stroke, epilepsy, trauma and some neuro-

degenerative diseases. In order to evaluate the potential therapeutic

effect of c-Jun silencingin an excitotoxic context, a selective lesionwas

induced in the mouse brain by administering kainicacid, an agonist of

both kainate and AMPA subtypes of glutamate receptors. Intracer-

ebroventricular injection of 0.1 g of this excitotoxic substance

(Fig. 5A) in the left brain hemisphere was applied to establish a well

defined lesion in the hippocampus according to a previously devel-

oped epilepsy model [21,22]. Tf-lipoplexes containing anti-c-Jun orMut siRNAs were injected near the CA3 region of the right hemisphere

(Fig. 5A) 3 days before or immediately after KA injection, aiming at

evaluating the neuroprotective role of c-Jun silencing in botha pre- and

post-lesion situation. The loss of neuronal integrity was investigated

3 days after KA injection, by both cresyl violet staining (Fig. 5B and

panels aj) and NeuN immunohistochemistry (Fig. 5B and panels kt).

Fig. 5 shows clear signs of neuronal loss after KA injection, in both

hemispheres of Mut siRNA-treated animals and in contralateral hemi-

spheres of animals receiving c-Jun siRNAs. Between 6070% of neuronal

death was observed, following cresyl violet counterstaining (Fig. 5C), as

well as significant loss (75%) of the neuronal marker NeuN (Fig. 5D),

indicating the presence of neurodegeneration and apoptosis and

providing evidence of KA-induced lesion in these brain regions. On

the contrary, no lesion could be observed in the hemispheres (right) of

animals injected with Tf-lipoplexes containing anti-c-Jun siRNAs, both

pre- and post-lesion (Fig. 5, panels f, j, p and t), where few pycnotic cells

were detected and NeuN loss was not significant (1020%), similarly to

what was observed in all sham-operated animals (Fig. 5, panels a, b, k

and l). These results indicate a clear neuroprotective effect of c-Jun

silencing in this excitotoxic model of brain injury.

Concerning seizure activity, all animals were observed continuously

for 4 h following KA injection for the onset and extent of seizures.

Seizures were rated accordingto a previously defined scale [20]. Almost

all control animals (injected with KA only) and animals treated with

Mut siRNAs, pre- (Mut+KA) or post-lesion (Mut/KA) achieved the

expected stage 5 in the Racine's scale (80%), presenting spontaneous

blackouts and continuous rearing and falling (Fig. 6). Moreover, a small

percentage of death animals (1030%) were also observed in these

groups (Fig. 6).In contrast, no deaths were registered in the animals treated with

anti-c-Jun siRNAs pre- (c-Jun+KA) or post-lesion (c-Jun/KA), and in

the group that received pre-treatment most animals only achieved

stages 3 and 4 of status epilepticus, with only 15% of the animals

reaching stage 5. Overall, these data imply that c-Jun silencing, when

performed previously to the onset of seizure activity, can effectively

moderate the amount and severity of seizures.

3.5. Reduction of KA-induced inflammation following Tf-lipoplex-mediated

c-Jun silencing

KA administration is known to induce dramatic changes in the

number and morphology of both astrocytes and microglia, leading to

the production and release of inflammatory cytokines. In order toevaluate the effect of c-Jun knockdown in the modulation of the

inflammatory reaction induced by KA injection, the protein levels of

GFAP, a specific marker of astrocytes, were analysed by Western blot

(Fig. 7A and B) and immunohistochemistry (Fig. 7C, panels kt),

Fig. 2. Recovery of neuronal viability following Tf-lipoplex-mediated c-Jun silencing in

vitro. Twenty-four hours following transfection with Tf-lipoplexes containing c-Jun

siRNA or Mut siRNA, neurons were incubated with glutamate (125 M20 min) or

exposed to oxygenglucose deprivation (OGD) for 4 h. (A) Quantification of c-Jun

mRNA by QRT-PCR, (B) and (C) Western blot analysis of c-Jun protein levels or (D) and

(E) cell viability analysis using the MTT assay were performed 18 h after lesion. Results

in (A), (B), (D) and (E) are expressed as a percentage of control and are presented as

mean valuesSD. All results are representative of three independent experiments,

each performed in triplicate. pb0.01 and pb0.001 compared to cells exposed to

glutamate or OGD.


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3 days after KA administration. In parallel immunohistochemistry

experiments, microglia activation was followed using the specific cell

marker CD11b (Fig. 7C, panels aj).A significant increase in GFAP levels was observed in animals

lesioned with KA and treated with Mut siRNAs (right hemisphere) and

in those lesioned with KA (lateral ventricle) without further treatment,

when compared to control animals, which received a single intra-

cerebroventricular injection of PBS. Similar results were obtained

when Tf-lipoplexes were administered both pre- (c-Jun+KA and

Mut+ KA) and post-lesion (c-Jun/KA and Mut/KA). In contrast, GFAP

levels in the right hemisphere of animals injected with anti-c-Jun

siRNAs were equal to those of the control group, independently of

whether Tf-lipoplexes had been administered 3 days before or

immediately after KA injection (Fig. 7A).

Significant changes in the number of astrocytes could also be

observed in the hemispheres that did not receive anti-c-Jun siRNAs

(Fig. 7C, panelsm, n,o, q,r and s)as wellas a notorious alteration inthe

Fig. 3. Tf-lipoplex-mediated c-Jun silencing in the mouse hippocampus. Mice wereinjected in the CA3 region of the right hippocampus (ipsilateral), with Tf-lipoplexes

(2 gc-JunsiRNAs or Mut siRNAs); noinjection wasperformed in thelefthippocampus

(contralateral). Animals were sacrificed 1, 3 or 5 days postinjection (D1, D3 and D5).

(A) c-Jun mRNA levels were evaluated by QRT-PCR. Results are presented as a

percentage of mRNA levels in sham-operated animals. (B) c-Jun protein knockdown

was investigated by immunohistochemistry (green anti-c-Jun antibody). (ad)

Representative fluorescence microscopy images of the contralateral hemispheres and

ipsilateral hemispheres injected with anti-c-Jun or Mut siRNAs (day 3) are presented at

200 magnification. Protein knockdown was assessed by Western blot. (C) Represen-

tative gel showing c-Jun protein levels at day 3 following Tf-lipoplex delivery. Two

bands, corresponding to the ipsilareral (ips) and contralateral (ct) hemispheres are

presented for each animal (#). (D) Quantification of c-Jun protein knockdown,

corrected for individual -tubulin levels. Results are expressed as a percentage of c-Jun

levels in the contralateral hemispheres. Results in (A) and (D) are presented as mean

valuesSD (n=6). pb0.01 and pb0.05 compared to animals injected with Mut

siRNAs. (For interpretation of the references to colour in this figure legend, the reader is

referred to the web version of this article.)

Fig. 4. Tf-lipoplex biocompatibility in vivo, following injection in the mouse hippocampus.Mice (n=6) were injected, in the CA3 region of the right hippocampus (ipsilateral)

with Tf-lipoplexes (Mut siRNAs); no injection was performed in the left hippocampus

(contralateral). Animals were sacrificed 3 days postinjection. (ad) Biocompatibility of

Tf-lipoplexes was evaluated in brain sections surrounding the injection site by cresyl

violet staining. Representative light microscopy images are presented at (a,b) 50 and

(c,d) 200 magnifications. A possible inflammatory response to the delivery of Tf-

lipoplexes was evaluated by immunohistochemistry. Sections were labelled with (e,f)

an anti-GFAP antibody (green) which specifically labels astrocytes and with (g,h) an

anti-CD11b antibody (red) which labels microglia cells. Representative fluorescence

microscopy images are presented at 200 magnification. (For interpretation of the

references to colour in this figure legend, the reader is referred to the web version of

this article.)


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morphology and activation state of microglia (Fig. 7C, panels c, d, e, g,

h and i). An increase in GFAP and CD11b labelling was observed in the

CA3 region of these hemispheres, specially surrounding the area where

neuronal loss wasmost evident. On theotherhand,GFAP(Fig.7C, panels

p and t)and CD11b(Fig. 7C, panels f and j) labelling in the hemispheres

treated with anti-c-Jun siRNAs remained similar to that of the sham-

operated group (Fig. 7C, panels a, b, k and l), with very few CD11b

positive cells and a small number of astrocytes.

Regarding cytokine production, the mRNA levels of some relevant

inflammatory cytokines (IL-1, IL-6 and TNF-) were analysed by

QRT-PCR, following Tf-mediated treatment and KA administration

(Fig. 8).

Fig.5. Neuroprotection mediatedby c-Junsilencing in vivo, followingexcitotoxic lesion.(A)Mice wereinjected in theCA3 regionof theright hippocampus (ipsilateral), withTf-lipoplexes

(c-Jun siRNAs or Mut siRNAs); 3 days after (c-Jun+KA; Mut+KA) or immediately before (c-Jun/KA; Mut/KA) Tf-lipoplex injection, KA (0.1 nmol3 l) was injected in the lateral

ventricle. Sham-operated animals (PBS) received a single injection of PBS (3 l). Animals were sacrificed 3 days postinjection. (B) Neuronal death was evaluated by cresyl violet

staining and immunohistochemistry. (aj) Representative light microscopy images of both hemispheres stained with cresyl violet are shown at 200 magnification. (kt) Sections

were labelled with anti-NeuN antibody (red), a specific marker of neurons. Representative fluorescence microscopy images are shown at 200 magnification. (C) Quantification of

neuronaldeath.Resultsare expressedas thepercentageof intact neuronswith respectto thetotal CA3neuronsper hemisphere. (D)Quantification of NeuNloss. Results areexpressed

as a percentage ofNeuN positiveneurons in sham-operated animals.Resultsin (C) and(D) arepresented as meanvalues SD obtainedfrom cellcountsperformed inevery sixsection

(n =6). pb0.001 compared to animals injected with KA in the absence of Tf-lipoplex treatment.


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Cytokine mRNA levels were measured in the right hemisphere of

animals treated with Tf-lipoplexes carrying anti-c-Jun or Mut siRNAs,

1 day after KA injection andthe results were compared to the cytokine

mRNA levels of animals lesioned with KA in the absence of Tf-

lipoplex-mediated treatment (control group). In parallel experiments,

the mRNA levels of IL-1, IL-6 and TNF- were also evaluated in

sham-operated animals, in order to determine cytokine levels in

undamaged tissue. While cytokine mRNA levels of animals treated

with Mut siRNAs, pre- or post-lesion, remained similar to those of the

control group, a significant reduction of the tested cytokines was

found in the animals treated with anti-c-Jun siRNAs, which presented

IL-1 and TNF- mRNA levels similar to those observed in the sham-

operated group. It is interesting to note that, while IL-6 levels found in

PBS injected animals are similar to those of the control group,

indicating that IL-6 is not upregulated 1 day after KA-induced lesion,

c-Jun silencing was able to induce a reduction of IL-6 transcription.Overall, these data suggest that the neuroprotective effect of Tf-

lipoplex-mediated c-Jun silencing is related not only to a decrease in

neuronal cell death, but also to the modulation of the inflammatory

reaction that follows an excitotoxic lesion.

4. Discussion

Gene therapy based on RNA interference has been considered a

promising therapeutic approach to a variety of neurological disorders

for which current treatments are largely unsatisfactory [23]. Recent

reports support the use of RNAi both as a research tool, to uncover the

complexity of intracellular signalling pathways involved in cell death,

and as a new therapeutic strategy for genetic and sporadic neurode-

generative diseases [2427]. Nevertheless, few studies have beenperformed using RNAi applications in acute brain disorders, such as

stroke, trauma and epilepsy. In this work, using a lipid-based delivery

vector in a model of excitotoxic brain lesion we were able to demon-

strate the potential of RNA interference to identify and validate a new

promising target for the treatment of acute brain injury in vivo.

Several studies have implicated the JNK protein family and its

downstream target, the transcription factor c-Jun, in the neurode-

generative events resulting from overstimulation of glutamate

receptors [11,14,28]. c-Jun is known to activate the transcription of

several immediate early genes encoding proteins [29], which include

pro-apoptotic mithochondrial proteins and inflammatory cytokines

[30]. Moreover, evidence suggests that the AP-1 transcription factor,

composed of c-Jun and c-Fos dimers, is able to induce the trans-

cription of c-Jun itself, leading to a perpetuation of its activity. In the

present work, we investigated the time-course of c-Jun activation in

the context of excitotoxic injury in neurons. In this model system,

early translocation of c-Jun to the nucleus (Fig. 1B and C) and a

significant increase in c-Jun mRNA were observed, as soon as 15 min

after glutamate exposure (Fig. 1A). These data strongly correlate with

previous reports on JNK and c-Jun activation after excitotoxic lesion

and suggest that c-Jun nuclear translocation and upregulation are

early eventsof thesignalling cascades that occur withina fewminutes

after overstimulation of glutamate receptors.Similarly to other MAPK family members, the role of JNKs and c-Jun

can be neuroprotective or neurotoxic, depending on the cell type and

pathological context of activation. The direct inhibition of JNKs in

neurons has provided substantial protection against various neurode-

generative stimulus including ischemia and excitotoxicity [28,31,32],

suggesting a pro-apoptotic role for these enzymes after acute brain

damage. In order to clarify the relevance of c-Jun activation in this

context, we evaluated the effect of RNAi-mediated c-Jun silencing in

neuronal viability following glutamate insult or oxygenglucose depri-

vation, two well established models of excitotoxic damage. Aiming at

achievingefficient c-Junknockdownin neuronalprimarycultures, a non-

viral strategy previously developed in our laboratory was employed to

mediate efficient delivery of siRNA molecules. Tf-lipoplexes, generated

from complexation of Tf-associated cationic liposomes with anti-c-Jun

siRNAs, where shown to promote efficient c-Jun knockdown in primary

neuronalcultures, similarly to thatdemonstrated for reportergenes,such

as luciferase [16]. Following demonstration of both mRNA and protein

knockdown in vitro mediated by Tf-lipoplexes (Fig. 2A, B and C),

neuronal viability was determined 18 h after both excitotoxic insults

(Fig. 2D and E) and neuronal cell death was found to be significantly

decreased when cells were pre-treated with anti-c-Jun siRNAs, but not

when cells were treated with Mut siRNAs. These results correlate with

indirect observation made in previous studies [14] and strongly suggest

thatc-Jun is directlyinvolvedin neuronaldeath after excitotoxic damage.

Kainic acid is known to cause epileptic seizures and to increase the

levels of extracellular glutamate, thereby mimicking some of the

events that occur after excitotoxic lesion in several neurodegenerative

diseases and acute brain disorders. Here, we used the kainate lesion

model to evaluate the efficiency of c-Jun siRNA Tf-lipoplexes toprovide neuroprotection and attenuate inflammatory responses in

vivo. Considering the difficulties associated with nucleic acid delivery

in vivo, we first evaluated whether Tf-lipoplexes could lead to

successful c-Jun knockdown after local brain injection. Our results

clearly confirm the potential of this strategy to promote in vivo siRNA

delivery and c-Jun knockdown in the mouse brain (Fig. 3). In fact, we

observed an impressive 70% reduction of c-Jun mRNA levels 1 day

after Tf-lipoplex injection, accompanied by a 45% decrease in protein

levels 3 days after siRNA administration mediated by Tf-lipoplexes,

which could be sustained for at least 5 days. This knockdown was

found to be specific, since no decrease in c-Jun levels was observed

upon treatment with Mut siRNAs. Moreover, Tf-lipoplexes did not

induce any kind of toxicity or inflammatory response in the mouse

brain (Fig. 4), showing their high biocompatibility.In the presently applied kainate lesion model, a clear and

reproducible neurodegeneration of CA3 pyramidal neurons could be

observed in the hippocampus of both hemispheres as soon as 3 days

followingKA injection.In order to evaluate the neuroprotective effect of

anti-c-JunsiRNAs, whendeliveredby Tf-lipoplexes, Tf-lipoplexinjection

was performed near the CA3 region, 3 days before or immediately after

KA injection (Fig. 5). An impressive reduction of lesion size could be

observed in all hemispheres treated with anti-c-Jun siRNAs (Fig. 5),

independently of whether the siRNA was delivered 3 days before or

immediately after the lesion. In addition, a strong decrease in seizure

activity was observed in the animals pre-treated with anti-c-Jun siRNAs

(Fig. 6), which translated into a significant decrease in the number of

death animals due to KA-induced convulsions. These findings indicate

that c-Jun silencing results in a neuroprotective and repairing effect in

Fig. 6. Decrease in seizure activity mediated by c-Jun silencing, following kainic acid

injection. Immediately before(c-Jun/KA; Mut/KA)or 3 daysafter(c-Jun+KA; Mut+KA)

Tf-lipoplex injection in the CA3 region of the right hippocampus, mice were injected

with KA (0.1 nmol3 l) in the lateral ventricle. All mice were monitored for 4 h for the

onset and extent of seizure activity, 1 day after KA injection. Seizures were rated

according to the previously described Racine's scale. The number of animals reaching

each stage of status epilepticus was determined for all experimental conditions. Results

areexpressed as a percentage of thetotal numberof animals perexperimental condition

(n=12).


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this model of excitotoxic injury, which seems to be specific for this

target, since it could not be achieved with a Mut siRNA sequence.

Kainic acid-induced lesion was also found to be accompanied by a

strong inflammatory reaction, characterized by astrogliosis and

activation of microglia. While in a healthy brain, resting microglia

andastrocytes contact neuronalcells in order to surveyany changes in

the neuronal environment, in damaged brains astrocytes proliferate at

a high rate and microglia cells are activated and maydamage neurons.

Within 1 to 2 days after KA injection, resident microglia cells change

their morphology and migrate into injured regions. After KA injection,

the number of activated microglia cells and astrocytes increases

during 3 and 4 days due to proliferation, and a high number of these

cells can still be observed one month after the onset of injury [33].

Moreover, production of several cytokines can also be detected in the

Fig. 7. Anti-inflammatory potential of c-Jun silencing mediated by Tf-lipoplexes. Immediately before (c-Jun/KA; Mut/KA) or 3 days after (c-Jun+KA; Mut+KA) Tf-lipoplex delivery in

theright hippocampus, micewere injectedwith KA (0.1 nmol3 l) in thelateral ventricle. In parallel experiments, animals receiveda single injection of KA (0.1 nmol3 l)or PBS in

the lateral ventricle. Animals were sacrificed 3 days postinjection. The presence of an inflammatory reaction was investigated in all experimental groups. (A, B) GFAP levels were

analysed by Western blot. Results from the quantification of GFAPlevels corrected for individual -tubulin levels are presented in (A) andare expressed as foldincrease above GFAP

levels in sham-operated animals. Results correspond to mean valuesSD ( n =6). pb0.01, pb0.001 and n.s. (without statistical significance) compared to the sham-operated

group. A representative gel showing GFAP levels for the most relevant experimental conditions is shown in (B). (C) Microglia activation and gliosis were assessed by

immunohistochemistry. Sections were labelled with (a j) anti-CD11b antibody (red) or (kt) anti-GFAP antibody (green). Representative fluorescence microscopy images are

presented at 200 magnification.


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lesioned tissue, such as TNF-, IL-6and IL-1, which are key players in

cerebral inflammation and neurodegeneration [34]. Upon Tf-lipoplex-

mediated c-Jun silencing, an attenuation of microglia activation and

astrocyte proliferation could be observed in the treated hemispheres,

when compared with the untreated or Mut siRNA-treated hemi-

spheres (Fig. 7). Moreover, a significant decrease in TNF-, IL-6 and

IL-1 was also observed in the hemispheres showing a decrease in

microglia activation (Fig. 8).

Our findings indicate that c-Junsilencing not only protects neurons

from cell death but may also attenuate microglia activation and

astrogliosis, thereby extending the therapeutic relevance of c-Jun

silencing to anti-inflammatory activity. Although this activity could be

attributed simply to the decrease in neuronal cell death due to c-Jun

silencing in neurons, which would be sufficient per se to reduce

microgliaactivation, the observed effect can also be partially related to

c-Jun knockdownin microglial cells. It has beenrecently demonstrated

that JNK inhibition in microglia significantly reduces TNF- and IL-6

production [30] and that c-Jun/AP-1 consensus sequences can be

found in the promoters of the genes coding for these interleukins. It istherefore possible that c-Jun silencing in microglia cells will directly

lead to a reduction in c-Jun/AP-1-mediated expression of these pro-

inflammatory molecules, contributing to the anti-inflammatory effect

observed after Tf-lipoplex delivery of anti-c-Jun siRNAs.

Overall, our work illustrates how RNA interference technology can

help to identify the most relevant proteins responsible for the pro-

gression of cell death following excitotoxic injury, and to silence these

same proteins, achieving a therapeutic effect. In this study we show

that Tf-lipoplexes promote siRNA delivery and siRNA-mediated

protein silencing in the brain, following stereotactic injection, with

high efficiency and minimum toxicity. Although this kind of

administration is not as easy to perform as oral or systemic delivery,

stereotactic injection of a non-viral siRNA delivery system in a specific

brain region is feasible in humans and can be accomplished usingcommon neurosurgical procedures. Moreover, Tf-lipoplexes can be

further optimized for systemic delivery, since Tf receptors are present

not only in neuronal cells, but also in the endothelial cells that

constitute the BBB. This opens the possibility of developing new

therapeutic strategies to neurological disorders based on the RNA

interference technology. Once the major barrier of brain delivery is

surpassed, the chances of success of this kind of therapy increase,

especially if long-term silencing is not required, such as in the case of

acute brain disorders where neuronal loss occurs in a short period of

time following injury. The results presented in this work also provide

evidence that the transcription factor c-Jun is a promising therapeutic

target, whose silencing leads to significant neuroprotection after

excitotoxic injury, mediated by both anti-apoptotic and anti-inflam-

matory effects. This suggests that c-Jun silencing by siRNAs, which in

this study was shown to last at least five days, could reduce neuronal

apoptosis in the crucial period that follows stroke or an epileptic

seizure. This approach prevents loss of neuronal function in the

affected areas and, therefore, represents a therapeutic alternative to

the use of JNK and AP-1 systemic inhibitors, which although

somewhat effective may lead to serious side-effects in other organs.

Acknowledgements

A.L.C. Cardoso is the recipient of a fellowship from the Portuguese

Foundation for Science and Technology (SFRH/BD/17216/2004). This

work was partially financed by a grant from the Portuguese

Foundation for Science and Technology (PTDC/BIO/65627/2006).

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7200.

Fig. 8. Reduction of inflammatory cytokine expression following Tf-lipoplex-mediated c-Jun

silencing. Immediately before (c-Jun/KA; Mut/KA) or 3 days after (c-Jun+KA; Mut+KA)

Tf-lipoplex injection in the right hippocampus (ispilateral), mice were injected with KA

(0.1nmol3 l) in the lateral ventricle. In parallel experiments, animals received a single

injectionof KA or PBSin the lateral ventricle.Animals were sacrificed 1 day postinjection.

The mRNA levels of IL-1, IL-6 and TNF- in the right (ipsilateral) hemispheres were

assessed byQRT-PCR andare expressedas foldreduction with respect tocytokinelevels in

the KA group. Results are presented as mean values SD (n =6), representative of three

independent experiments. pb0.001, pb0.01 and pb0.05 compared to animals

injected with KA in the absence of Tf-lipoplex treatment.


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Please cite this article as: A.L.C. Cardoso, et al., Tf-lipoplex-mediated c-Jun silencing improves neuronal survival following excitotoxic damage

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