Neurobiology of Disease 32 (2008) 528–534

Contents lists available at ScienceDirect

Neurobiology of Disease

j ourna l homepage: www.e lsev ie r.com/ locate /ynbd i

Astrocytic proliferation and mitochondrial dysfunction induced by accumulatedglutaric acidemia I (GAI) metabolites: Possible implications for GAI pathogenesis

Silvia Olivera a, Anabel Fernandez b, Alexandra Latini c, Juan Carlos Rosillo b, Gabriela Casanova d,Moacir Wajner c, Patricia Cassina e, Luis Barbeito a,⁎a Cellular and Molecular Neurobiology Department, Instituto Clemente Estable (IIBCE), Montevideo, Uruguayb Comparative Neuroanatomy (IIBCE)-Associated Unit to the School of Sciences, Universidad de la Republica (UdelaR), Montevideo, Uruguayc Biochemistry Department, Instituto de Ciencias Basicas da Saude (ICBS), Universidad Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazild Cellular Biology Section, School of Sciences, UdelaR, Montevideo, Uruguaye Histology Department, School of Medicine, UdelaR, Montevideo, Uruguay

⁎ Corresponding author. Cellular and Molecular NeurInstituto de Investigaciones Biológicas Clemente EsMontevideo, Av. Italia 3318, CP 11600, 2nd Floor Mon487 5461.

E-mail addresses: lbarb@iibce.edu.uy, barbeito@pastAvailable online on ScienceDirect (www.scienced

0969-9961/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.nbd.2008.09.011

a b s t r a c t

a r t i c l e i n f o

Article history:

Glutaric (GA) and 3-hydrox Received 11 June 2008Revised 2 August 2008Accepted 9 September 2008Available online 30 September 2008

Keywords:Astrocyte proliferationGlutaric acidemia IMitochondria dysfunction

yglutaric (OHGA) acids accumulate in glutaric acidemia I (GAI), a neurometa-bolic disease characterized by acute striatal degeneration and chronic progressive cortical atrophy. Toexplore the hypothesis that astrocytes are involved in GAI pathogenesis and targets of accumulatingmetabolites, we determined the effects of GA and OHGA on cultured rat cortical astrocytes. Remarkably,both acids induced mitochondria depolarization and stimulated proliferation in confluent cultures withoutapparent cell toxicity. Newborn rats injected with GA systemically also showed increased cell proliferationin different brain regions. Most of the proliferating cells displayed markers of immature astrocytes.Antioxidant iron porphyrins prevented both mitochondria dysfunction and increased in vitro and in vivoproliferation, suggesting a role of oxidative stress in inducing astrocytosis. Taken together, the data suggestthat mitochondrial dysfunction induced by GA metabolites causes astrocytes to adopt a proliferativephenotype, which may underlie neuronal loss, white matter abnormalities and macrocephalia character-istics of GAI.

© 2008 Elsevier Inc. All rights reserved.

Introduction

Glutaric acidemia type I (GAI) is an inherited neurodegenerativedisease caused by deficient activity of glutarylCoA dehydrogenase (EC1.3.99.7, GCDH), a mitochondrial enzyme involved in L-tryptophan, L-lysine and L-hydroxylysine catabolism (Goodman and Frerman, 1995;Kolker et al., 2000; Strauss et al., 2003; Funk et al., 2005, Zinnanti et al.,2007). Critical accumulation of glutaric (GA) and 3-hydroxyglutaric(OHGA) acids resulting from GCDH deficiency together with complexcell interplays trigger acute encephalopathic crisis in affected childrenwho suffer permanent neurological deficits including recurrentseizures, dystonia, dyskinesia, growth and cognition impairments(for a review see Hoffmann et al., 1996; Funk et al., 2005). Among thepathological hallmarks of GAI are cortical and striatal degeneration,gliosis and white-matter abnormalities (Goodman and Frerman,1995;Zinnanti et al., 2007). Macrocephalia and increased brain size have

obiology Department (NBCM),table (IIBCE)-Institut Pasteurtevideo, Uruguay. Fax: +598 2

eur.edu.uy (L. Barbeito).irect.com).

l rights reserved.

been also frequently reported (Hoffmann et al., 1996; Funk et al.,2005). Excitotoxicity (Kolker et al., 2000), disruption of mitochondrialenergy metabolism and oxidative stress (Das et al., 2003; Das, 2003;Ferreira et al., 2005; Sauer et al., 2005; Latini et al., 2007; Sauer, 2007)have been proposed as pathogenic mechanisms. So far, the pathogen-esis of GAI is still unknown.

Astrocytes playpivotal rolesprovidingmetabolic and trophic supportto neurons (Chiu and Kriegler, 1994; Aschner et al., 1999; Dienel andHertz, 2001), regulating neuronal activity and synaptic neurotransmis-sion (Chiu and Kriegler, 1994; Araque, 2006) and collaborating tomaintain blood-brain barrier integrity (Simpson et al., 2007). On theother hand, in response to injury, astrocytes proliferate and adopt areactive phenotype expressing and secreting several cell markers andsoluble factors that interact with other cells amplifying injuring loops(Ridet et al., 1997; Araque, 2006). Finally, astrocytes together withmicroglia, underlie pathological states of reactive gliosis and their rolesin neurodegenerative processes are increasingly recognized (Barbeitoet al., 2004; Araque, 2006; Cassina et al., 2008). Thus, we hypothesizedthat astrocytes may be a primary target of GAI accumulating acids andtheir dysfunction elicits pathogenic cascades that may explain some ofthe clinical neuropathological features of GAI.

We have recently shown that altered astrocytic mitochondriaactivity, including depolarization and increased superoxide pro-

529S. Olivera et al. / Neurobiology of Disease 32 (2008) 528–534

duction, cause astrocytes to adopt a neurotoxic phenotype (Cassina etal., 2008). Other authors have shown that mitochondria activateseveral signal transduction pathways that modulate cell proliferationor oppositely cell arrest and apoptosis (Alonso et al., 2004; Carrerasand Poderoso, 2007; Valero et al., 2008). In this context, wehypothesized that brain accumulation of GA and OHGA, both havingtoxicity on mitochondria (Ferreira et al., 2005; Fighera et al., 2006;Latini et al., 2007; Magni et al., 2007), induces major effects onastrocytic phenotype that could underly neurodegeneration in GAI.Previous reports have shown that GAI metabolites interfere withastrocytic glutamate transporters (Porciuncula et al., 2004; Rosa et al.,2007) suggesting a pathogenic role mediated by dysfunctionalastrocytes. Thus, we determined in vitro and in vivo GA and OHGAeffects on astrocytes.We found that both acids impairedmitochondrialactivity and, instead of inducing cell death, they potently stimulatedastrocyte proliferation. Our results suggest that astrocytes are earlytargets of GA and OHGA acids, providing new avenues to theunderstanding of GAI pathogenesis and the development of ther-apeutic approaches.

Materials and methods

Chemicals

Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum(FBS), penicillin/streptomycin, trypsin, CyQuant™ Cell ProliferationAssay kit for cells in culture, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC1), MitoFluor Green™ (MF),MitoTrackerRed™ (MT), propidium iodide (PI) and anthra[1-9-cd]pyrazol-6(2H)-one (SP600125) were purchased from Invitrogen(Carlsbad, CA, USA). Lactate dehydrogenase (LDH) detection kit wasfrom Roche (Mannheim, Germany). 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) was purchased from CellSignaling Technology (Danvers, MA, USA). The iron porphyrins, iron(III)tetrakis(carboxyphenyl)porphyrin (FeTCPP) and iron(III) tetrakis(N-methyl-4′pyridyl)porphyrin pentachloride (FeTMPyP), came fromAlexis Biochemical (San Diego, CA, USA). Commercial antibodies wereobtained from Dako (Carpinteria, CA, USA), Sigma (St Louis, MO, USA)and Invitrogen. All other chemicals of analytical grade were obtainedfrom Sigma.

Animals

Sprague Dawley rats were maintained with food and water adlibitum at the IIBCE animal house. Institutional guidelines andnational laws for the protection of vertebrate animals for experimentaland other scientific purposes, both in accordance with internationalstandards of animal welfare, were followed. Every effort was made tominimize number, pain and animal discomfort.

Astrocyte cultures and treatments

Primary astrocyte cultures were prepared from cortices of 1–2 dayrats according to Cassina et al. (2005; 2008). Astrocytes were plated at2×104 cells/cm2 and maintained in supplemented DMEM (10% FBS,3.6 g/l HEPES, 100 IU/ml penicillin, 100 μg/ml streptomycin). Afterenrichment, astrocyte monolayers were at least 98% pure asdetermined by glial acidic fibrillar protein (GFAP) immunoreactivityand devoid of OX42-positive microglial cells. Experiments were donein confluency (1 week after plating), unless otherwise stated. Beforeeach treatment, cells were incubated with DMEM-2% FBS during 24 hand then submitted to GA or OHGA at concentrations ranging from0.01 to 10 mM (Latini et al., 2007). Appropriate aliquots of a 500 mMacid stock solution, were prepared in 5 N NaOH immediately prior touse to assure pH close to 7.4. In other experiments, under lightprotection, cells were pretreated with 10–50 μM FeTCCP, FeTMPyP,

U0126 or SP600125 24 h previous to acid addition. Concentrations aswell as pre-treatment and incubation periods, were based on previousreports (Pehar et al., 2004; 2007; Cassina et al., 2008). Once incubationwas ended, cell-free medium was collected for determination of LDHactivity while attached cells were used for biochemical analysis andimmunocytochemistry.

Proliferation assays

Proliferation rate was assessed quantifying incorporation of 5-bromo-3′-deoxyuridine (BrdU, Sigma) (Zaidi et al., 2004) andconfirmed with CyQuant™ Cell Proliferation Assay (Invitrogen).Briefly, cultured astrocytes were fixed on ice-cold paraformaldehyde[PFA, 4% in 10 mM phosphate buffer saline (PBS, pH 7.4), 15 min] andthen permeabilized (4 °C, 30 min) with 0.1% X-100 Triton:PBS. A 2 NHCl denaturation was done (37 °C, 30 min) and after washing withborate buffer (10 mM, pH 8) until neutralization, blockade of nonspecific binding was done incubating with blocking solution (5%bovine serum albumin:PBS) during 1 h at room temperature. Cellswere incubated with primary antibodies against BrdU (monoclonal,1:800 in blocking solution, Dako) and glial markers (GFAP, S100,S100β, 1:1000, Sigma) (4 °C, overnight) in a wet closed chamber. After3 washes, cells were incubated with correspondent secondaryantibodies (1:1000, Invitrogen, 25 °C, 2:30 h), mounted and imagedin an Olympus FV300 laser scanning confocal microscope. PositiveBrdU cells were related to total nuclei labelled with diaminopheny-lindole (DAPI) or PI. To perform the CyQuant assay according tofabricant instructions, culture media was aspirated, cells frozen(−80 °C, overnight), slowly thawed and incubated with the commer-cial dye binding solution (37 °C, 40 min). Fluorescent emission wasdetected at 530 nm and related to values obtained in a calibrationcurve done with 500 to 50,000 cells.

Cell viability assessment

Cell viability was measured by LDH release (Porciuncula et al.,2004) and PI labeling of unfixed non permeabilized cells (Kelly et al.,2003). LDH activity was determined in the incubation mediummeasuring the 490 nm absorbance related to a 620 nm referencefilter. Results were expressed as percent of the total LDH contained in2% Triton X-100 lysated cells. To determine PI labeling, living cellswere incubated with 0.1 μg/ml PI plus 1 μg/ml DAPI in complete PBS(PBS+1 g/l glucose, 0.183 g/l CaCl2 and 0.183 g/l MgCl2) during 4 h at37 °C. Cells were washed, fixed with 4% PFA and imaged. Positive rednuclei indicating dying cells were related to total nuclei stained withDAPI. 10–50 μM sodium azide was used as positive control for bothLDH determination and PI incorporation.

Evaluation of mitochondrial activity

Mitochondrial status was evaluated using fluorescent probes suchas MF, MT, JC1 and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-trazolium bromide (MTT) assay, according to fabricant instructions.Briefly, control and treated attached cells were washed, changed tocomplete PBS and then incubated in controlled atmosphere (37 °C,95%O2/5%CO2) with 100 nM MF plus 100 nM MT or with 3 μM JC1during 15 min (Cassina et al., 2008). Fluorescent dyes were washedand living cells were immediately imaged with a FV300 Olympus laserscanning confocal microscope using 488 nm and 546 nm lasers. JC1red to green ratio was determined fluorimetrically measuring at590 nm and 520 nm, respectively. To perform the MTT assay, attachedcontrol and treated cells were incubated with 10 μM MTT (37 °C,45 min); culture media was removed and dimethyl sulfoxide added tosolubilize the purple formazan crystals formed by active mitochondria(Porciuncula et al., 2004). Samples were read at 570 nm (Elysa ReaderDynex Technology).

Fig. 1. GAI acids enhanced proliferation of astrocytes in culture. (A) Proliferation wasestimatedbyBrdUpositive and total nuclei counts 24h after additionofGAI accumulatingacids. Total nuclei are shown in red propidium iodide (PI)fluorescencewhereas those thathave incorporated BrdU are seen in yellow that resulted from merging red and greenfluorescence. Note the increased density of BrdUpositive nuclei in astrocytes treatedwithGAI accumulating acids. (B) Astrocyte proliferation was stimulated by GAI accumulatingacids in non-confluent and confluent astrocytemonolayers submitted toOHGA andGA atconcentrations ranging from 0.05 to 20mM. Results are expressed as the fold-increase ofnormalized BrdU incorporation in controls. Proliferation was higher in non-confluentcultures andmaximal responsewas found at lowmMacid concentration. (C–D) GAI acidsdid not affect astrocyte viability as determined by LDH release to the culture media (C)and microscopic identification of non-viable cells with PI (D). Confluent astrocytes wereexposed to vehicle (DMEM),1 mMOHGA or 5mMGA during 24 h previous estimation ofcell viability. 10 μM sodium azide was used as positive control. Calibration bars: 100 μm.

530 S. Olivera et al. / Neurobiology of Disease 32 (2008) 528–534

In vivo treatment

Each littermate of 1 postnatal day were injected in the Magnacisterna 4th ventricule with a final dose of 2.5 μmol/g GA (treated) orPBS (control) together with a single 60 μg/g BrdU intraperitonealinjection. 5 days later, animals were anesthetized (25 μg/g pentobar-bital) until complete unresponsiveness to nociceptive stimuli and thentranscardially perfused with 0.9% saline and 10% PFA (in 0.1 Mphosphate buffer (PB), pH 7.2–7.4, 1 ml/min). Fixed brains wereremoved, post-fixed by immersion during 24 h and then seriallytransverse sectioned on a vibrating microtome or processed to obtainsemi-thin sections.

Immunohistochemistry

Serial vibratome coronal sections (60–80 μm) throughout thestriatal extension [from P0–8 to −P0–18 according to the Atlas of theDeveloping Rat Brain (Paxinos et al., 1991)] were collected in PBS andhydrolyzed (2 N HCl, 1 h, room temperature). Then passed throughthree washing buffered solutions and incubated with anti-BrdU(1:500 in pH 7.4 0.3% Triton X-100 buffered solution, DAKO, 4 °C,overnight) and one of the following antibodies: anti-GFAP, S100,S100β (1:200 in pH 7.4 0.3% Triton X-100 buffered solution, Sigma), orNeu-N (1:200 in pH 7.4 0.3% Triton X-100 buffered solution,Chemicon). Detection of nuclei was achieved using different specificmarkers according to the secondary antibodies used.

Semithin sections

After 10% PFA perfusion, 3 brains of each experimental conditionwere post-fixed by sequential immersion in 10% PFA (4 °C, 2 h, pH 7.4),2.5 % glutaraldehyde (4 °C, overnight, pH 7.4) and 1% osmium tetroxide(4 °C, 40 min, pH 7.4). After several washes, dehydration wasaccomplished by passing the samples through an ethanol series ofincreasing concentrations and finished with acetone. Pieces wereincluded in araldite (Durcupan ACM, Fluka), sectioned at 750 nmwitha RMC MT-X ultramicrotome, and stained with toluidine blue atstandard conditions. Pictures of all brain sections were obtained witha DP70 OLYMPUS camera adapted to a BX61 Olympus microscope.

Counting of cells

All morphological and quantitative analyses were performed onblind-coded slides. Cells were characterized as BrdU positivewhen thestaining throughout nuclei was more than 50% and intensity at leastdoubled the background. To count in cell monolayers, 5 to 7 randomlyfields that cover at least 80% of cultured cells were chosen previouslyto start the counting. All positive cells were counted in each field,related to the total nuclei and referred to corresponding controls. Forsemithin and vibratome sections, all stained profiles at the striatalregion were counted following systematic random sampling. 5 to 7representative high-power non-overlapping fields were imaged andindividual immunopositive cells were marked by the operator andcounted by Image J cell counter (NIH, USA). Data obtained in each fieldper slice were added together providing a single value for each slice.Values of 3–5 slices per ratwere averaged and the final result was usedto calculate the relationship between GA treated and control slices. Allsamples underwent same parallel procedures.

Statistic analysis

All values shown are the mean±SEM of at least 3 to 5 independentexperiments performed in triplicate for immunostaining or quintu-plicate for biochemical assays. Data analysis was performed usingstandard statistical packages (SigmaStat, Jandel and Origin). Todetermine statistical difference among groups we use a two-factor

analysis of variance (ANOVA). Pb0.05 was considered statisticallysignificant.

Results

GA and OHGA induce astrocyte proliferation without affectingcell viability

To evaluate the astrocyte response to GAI accumulating acids, wefirst studied their effects on the astrocytic proliferation ratedetermined as the percentage of cells that incorporated BrdU (Zaidiet al., 2004). Both GA and OHGA applied to the culture medium for24 h significantly stimulated astrocyte proliferation in a dose-dependent manner (Figs. 1A and B). Maximal values were obtainedwith concentrations ranging from 0.5 to 1 mM for OHGA and 1 to5 mM GA, respectively. Increased proliferation was observed in bothnon-confluent and confluent cultures, with respective peak values of270% and 200% (for astrocytes treated with 5 mM GA). The GAI acidenhanced proliferation rate was confirmed using the CyQuantproliferation assay based on the measurement of total DNA content.After treating confluent cultures with 1 mM OHGA or 5 mM GA, theamount of DNA increased 150% and 170% of the control, respectively.

531S. Olivera et al. / Neurobiology of Disease 32 (2008) 528–534

GAI acid induced no apparent cell death as evaluated by morpholo-gical observation and absence of LDH release (Fig. 1C) and PI live cellstaining (Fig. 1D).

GAI accumulating acids induce astrocytic mitochondrial depolarization

Because both GA and OHGA exert toxic influence on mitochondria(Ferreira et al., 2005; Fighera et al., 2006; Latini et al., 2007; Magni etal., 2007), we determined the effect of GAI acids on the functionalstatus of astrocytic mitochondria. In cultured cortical astrocytes, GAIaccumulating acids significantly reduced mitochondrial activity in aconcentration dependent manner as estimated by the MTT assay,which measures the activity of mitochondrial dehydrogenases (Fig.2A). Interestingly all cells including those bearing decreased mito-chondrial function displayed apparent normal morphology, with nosigns of astrocyte reactivity. Both GA and OHGA also causedmitochondrial depolarization as shown by the predominant greenfluorescence exhibited by MF/MT double staining displayed in livingastrocytes treated with GAI accumulating acids (Fig. 2B). Mitochon-drial dysfunction evoked by GAI accumulating acids was similar tothat evoked by carbonyl cyanide 4-(trifluoromethoxy)phenylhydra-

Fig. 2. GA and OHGA caused astrocytic mitochondrial depolarization. (A) In vivo MTTrepresentative images showing the effect of a 24 h treatment with 1mMOHGA or 5mMGA on mitochondrial enzymatic dehydrogenase activity. Note the inability of astrocyticmitochondria to reduce MTT to formazan (blue dots in microphotographs) and theconcentration dependence of the effect (graph). All values were expressed as a controlpercentage. Readings were performed at 570 nmwith 630 nm as the reference filter. (B)Mitochondrial potential determined by MF/MT staining after exposure to 1 mM OHGA.Red MF staining indicates polarized mitochondria whereas green MF fluorescenceindicated total mitochondrial mass. Note the lack of red polarized mitochondria inOHGA but preservation of mitochondrial number. Images were taken with anepifluorescence microscope (Olympus BX61) setted for green (525±25 nm) and red(590±30 nm) detection.

Fig. 3. Iron porphyrins and MAPK inhibitors abrogated the effects of GAI accumulatingacids on astrocytes. (A) FeTCPP maintains mitochondrial potential. JC1 imaging ofmitochondria and fluorimetric ratio obtained in confluent astrocytes treated with 1mMOHGA, 5 mM GA or preincubated 24 h with 50 μM FeTCPP (FeP) before acid addition.After 24 h of acid treatment, cells were washed, incubated with 3 μM JC1 (15 min, 37 °C)and immediately imaged or the fluorescence measured at 520 nm (green) and 590 nm(red), respectively. Note that FeTCPP (FeP) abrogated the depolarizing effects evoked byGAI accumulating acids, preserving mitochondrial potential at control values (picturesand chart). (B) Quantitation of iron porphyrin and MAPK inhibitor effects on astrocyteproliferation. Proliferation rate was determined after pretreating confluent astrocyteswith 50 μM FeTCPP, 10 μM FeTMPYP (FeM), 20 μM U0126 (U) or 5 μM SP600125 (SP)during 24 h. Then, cells were treated with 1 mM OHGA or 5 mM GA. BrdU was addedsimultaneously to acids. In all conditions, positive BrdU nuclei were counted 24 h laterand related to total nuclei number. Datawere expressed as percent of controls (vehicle)±SEM. Not only FeTCPP but also FeTMPYP and MAPK inhibitors blocked the proliferationpromoting effects of GAI accumulating acids.

zone (FCCP), a well known mitochondrial uncoupler (Cassina et al.,2008).

Iron porphyrins and MAPK inhibitors abrogate GAI acid-inducedastrocyte proliferation

After treatment with GA and OHGA, JC1 red fluorescence decreasedup to 30% indicating a reduction in the mitochondrial potential (Fig. 3A,chart). The depolarizing effect of GAI accumulating acids was preventedby pretreating astrocytes with the antioxidants FeTCPP (Fig. 3A, picturesand chart) or FeTMPyP. Both iron porphyrins also completely abrogatedthe GA and OHGA-induced enhanced astrocyte proliferation (Fig. 3B),suggesting that maintainment of mitochondrial potential is clue tocontrol astrocyte cell cycle. We used the pharmacological inhibitors ofMAPK, UO126 and SP600125, to determinewhether enhanced astrocyteproliferation was linked to activation of this pathway. Incubation ofastrocytes with either inhibitor prevented astrocyte proliferationinduced by GAI accumulating acids (Fig. 3B).

532 S. Olivera et al. / Neurobiology of Disease 32 (2008) 528–534

Systemic GA induces astrocyte proliferation with no immediateneuronal loss

To determine the in vivo effects of GA on cell proliferation, weinjected intraventricularly newborn rat pups with 2.5 μmol/g GA andthen quantified cell and astrocyte proliferation in the striatum. 5 daysafter injection, GA caused a 3-fold increased cell proliferation withrespect to control basal proliferation, both measured as BrdUincorporation rate (Fig. 4A, a,b). Double immunostaining againstBrdU and the early astrocyte marker S100β protein allowed the

Fig. 4. Systemic administration of GA enhanced astrocyte proliferation in newborn ratbrain. (A) Immunohistochemistry against BrdU showing increased striatal proliferationin 2.5 μmol/g GA injected animals (b) as compared to controls injected with vehicle(PBS) (a). Proliferation was evidenced by dark brown nuclei immunopositive to BrdU.Inset in (a) schematizes the striatal area studied. Note that proliferation in controlanimals (a) was restricted to subventricular areas, whereas increased striatalproliferation was seen in pups injected with GA (b). Images (c) and (d) show thedouble immunostaining BrdU (red)/S100β (green) in a striatum of control (c) and GAinjected animal (d). Compare the increased colocalization of both markers after GAinjection (d, d inset) and the spare double positives in the control (c, c inset) indicatingthat proliferating cells in controls are not astrocyte like-cells. (B) Quantitation of striatalcell proliferation. BrdU and BrdU/S100β positive cells were counted in the striatum ofanimals injected with vehicle (control, white column), GA (black column), 50 μMFeTCPP/GA (third column) or 50 μM FeTCPP alone (last column). Note the significantincreases in total cell and astrocyte-cell like proliferation evoked by GA and theblockade of augments by FeTCPP (FeP). (C) The number of cells bearing neuronalphenotype remained unchanged 5 days after systemic administration of GA. Cells withglial and neuronal phenotype were counted in semithin sections of the striatum of5 days after systemic injection of vehicle, GA, FeTCPP/GA or FeTCPP alone, respectively.There were significant changes only in astrocyte like cells whereas the number ofneurons was preserved, regardless of the treatment employed. Images and data arerepresentative of those obtained in 3 separate experiments with 3–5 animals in eachone.

identification of these proliferating cells as glial cells with apparentmorphology of immature astrocytes (Fig. 4A, c, d). GFAP did not labelmost of the newborn immature astrocytes as S100β did.When FeTCPPwas injected 24 h before GA, a very significant blockade of cell andastrocyte proliferation was found (110% and 105%, respectively,Fig. 4B). FeTCPP also slightly decreased the basal proliferation (88%,Fig. 4B). Interestingly, the intraventricular GA administration causedno apparent cell or neuronal toxicity apart from the increased numberof astrocytes as indicated by microscopic observation. This wasconfirmed by a careful analysis of semithin sections. 5 days afterinjection, the number of cells with neuronal morphology remainedunchanged in treated and control pups striata (Fig. 4C) and themorphology of neuronal population seemed preserved. Neuronal losswas observed 2 weeks after GA injection (not shown).

Discussion

Herein, we provide evidence that astrocytes are early target cellsfor the major glutaric acidemia-accumulating metabolites and thus,possible contributors to GAI pathogenesis. Rather than being vulner-able to these acids, astrocytes proliferate before any apparent sign ofneuronal death, the newborn cells displaying a S100β-positive/GFAP-negative phenotype, characteristic of immature astrocytes. In addi-tion, we provide evidence that astrocyte proliferation is associatedwithmitochondrial dysfunction and oxidative stress caused by GA andOHGA, suggesting a yet unknown mechanism regulating astrocytosis.

Evidence indicates that GA and OHGA disrupt the activity of themitochondrial respiratory chain and subsequently induce the genera-tion of reactive oxygen species (ROS) and oxidative stress (Ferreira etal., 2005; Fighera et al., 2006; Latini et al., 2007; Magni et al., 2007).Mitochondria dysfunction has also been reported in GAI patients, asshown by increased excretion of lactate, 3-hydroxybutyrate anddicarboxylic acids (Gregersen and Brandt, 1979; Das, 2003; Sauer etal., 2005; Sauer, 2007). Here, we show that GAI accumulating acidsinduce mitochondria dysfunction in astrocytes at concentrationsbelieved to occur in patients. This was shown by means of differentprobes sensitive to mitochondrial potential (JC1, Mitotracker), andother based upon dehydrogenase activity (MTT assay). Remarkably,astrocytes were not vulnerable to a wide range of GAI accumulatingacid concentrations, as suggested by a lack of cell death induced by theacids both in vivo and in vitro. This featuremay play a role in protectingmore vulnerable neurons and oligodendrocytes against acute raise inacid concentration in GAI patients. The astrocyte mitochondrialdepolarization caused by GAI accumulating acids was reverted by theantioxidant iron porphyrins, suggesting a close association betweenlocal production of ROS and mitochondria dysfunction. In agreement,previous studies have found a protective effect of antioxidants andmetabolic substrates (such as 3-hydroxykynurenin and creatine)against GA effects and evoked electrographic convulsions (Das et al.,2003; Fighera et al., 2006; Magni et al., 2007).

Together with mitochondrial depolarization, a population ofastrocytes proliferated in response to GAI accumulating acids. Thenewborn cells identified by intense labeling with BrdU, displayed acharacteristic S100β-positive/GFAP-negative phenotype. The appear-ance of such newborn astrocytes may underlay the subsequentdevelopment of gliosis reported as one of the GAI hallmarks (Good-man and Frerman, 1995; Hoffmann et al., 1996; Zinnanti et al., 2007).In addition, the appearance of these aberrant immature astrocytesmay greatly disrupt brain development, perhaps contributing to theestablishment of delayed neurological deficits. Remarkably, GAIaccumulating acids trigger astrocyte proliferation in vitro, even inconfluent culture conditions and upon in vivo GA systemic adminis-tration. Increased glial proliferation was dependent on GAI accumu-lating acids ability to induce mitochondrial depolarization andoxidative stress, since antioxidants abrogated the effect. Intracellularand mitochondria-derived ROS levels regulate cellular proliferation in

533S. Olivera et al. / Neurobiology of Disease 32 (2008) 528–534

a complex manner, being the mitochondrial potential one of the waysinwhichmitochondria regulate cell cycle (Alonso et al., 2004; Carrerasand Poderoso, 2007, Valero et al., 2008). In some cell types,mitochondrial depolarization leads to cell cycle arrest and subsequentapoptosis (Knudson and Brown, 2008; Valero et al., 2008). In others,mitochondrial depolarization elevates intracellular calcium therebystimulating cell proliferation and abrogating apoptosis, a mechanismmodulated by potassium channels (Moudgil et al., 2006). Thus, wehypothesize that the mitochondria depolarizing effect caused by GAand OHGA is the main signal triggering astrocyte proliferation.Importantly, astrocytes compensate such mitochondrial dysfunctionby switching their metabolism to the glycolytic pathway in order tomaintain the energetic levels (Moncada and Bolanos, 2006, Hertz etal., 2007; Rouleau et al., 2007). This unique metabolic feature mayexplain why astrocytes are resistant to GAI accumulating acids,escaping necrosis or apoptosis.

Remarkably, systemic administration of GA for 2 dayswas sufficientto induced astrocyte proliferation in the forebrain, including thestriatum. We found that the in vivo GA effects can be abrogated by awell-known neuroprotective antioxidant FeTCPP, which also pre-vented mitochondrial dysfunction. FeTCPP and other iron porphyrinshave been shown to exert neuroprotection in a number of injuringconditions such as increased oxidative stress, ischemia-reperfusioninjury and spinal cord trauma (Choi et al., 2001; Wu et al., 2003;Cassina et al., 2005; 2008;Genovese et al., 2007; Stefanutti et al., 2007).In addition to prevent proliferation in pups treated with GA, FeTCPPalso decreased cell proliferation in control animals, suggesting it mayexert more complex effects in brain development. Moreover, the factthat inhibitors of the MAPK cascade prevented the effects of GAIaccumulating acids, suggest a link between mitochondria dysfunctionand MAPK-dependent astrocyte proliferation. This is in agreementwith reports showing that extracellular signal-regulated proteinkinases 1 and 2 (ERK1/2) and their upstream phospho-MAPK (MEK1/2) are located and active in brainmitochondria during rat development(Alonso et al., 2004), possibly anticipating a putative signal transduc-tion mechanism specifically activated by GAI accumulating acids.

What is the impact of astrocyte proliferation on GAI pathogenesis?The role of proliferating astrocytes in the neurodegenerative process isstill a matter of debate. In themature CNS, glial cells mainly proliferatein response to damage. The effect is mediated by complex mechan-isms including cytokines and fibroblast growth factors (Giulian et al.,1988; Ridet et al., 1997). Interestingly, suchmediators can also activateastrocytes and caused them to become neurotoxic to surroundingneurons (Pehar et al., 2004; 2007; Cassina et al., 2005). Thus, GA-induced proliferating astrocytes may also become neurotoxic andpromote elimination of a subset of damaged neurons and oligoden-drocytes, as suggested by reports showing that GAI acids display lowdirect toxicity in neuronal cultures (Freudenberg et al., 2004; Lundet al., 2004). Upon GA administration, most of the newly dividing cellswere not GFAP-positive but astrocyte-like cells labelled with the S100antibodies, suggesting that most of the newborn astrocytes are notderived from GFAP-positive differentiated astrocytes. In addition, GAalso stimulated the proliferation of neuroglican 2 (NG2)-positive glialprogenitor cells (not shown), which represent the main reservoir ofastrocytes and oligodendrocytes in the developing CNS and appears tobe greatly activated after brain injury (Lepore et al., 2008).Alternatively, GA may induce de-differentiation of GFAP-positiveastrocytes, allowing them to activate their retained capacity to resumeproliferation (Buffo et al., 2008).

GA stimulated astrocyte-like cell proliferation is not likely to besecondary to neuron death because it occurredwithout apparent signsof neuronal loss. Our results suggest that astrocytes but not neuronsare the first cells targeted in GAI. The fact that mice lackingglutarylCoA dehydrogenase activity and exhibiting increased GAIaccumulating acids show extensive white matter alterations but notapparent neuronal loss (Koeller et al., 2002), suggest a specific

vulnerability of glial cells to GAI acids during development or repairprocesses. As the primary target of GAI accumulating acids, astrocytescould adopt reactive phenotypes with yet unknown consequences onneuronal and oligodendrocyte survival, establishment of synapticnetworks and myelinization. It is also worthwhile to note thatsustained astrocyte proliferation could participate in the macroce-phalia described in a number of GAI patients as an early sign precedingneurodegeneration (Hoffmann et al., 1996).

In conclusion, the present data suggest that mitochondrialdysfunction and oxidative stress induced by GA and OHGA acidscause astrocytes to adopt a proliferative phenotype both in vivo and invitro. Through inducing major changes in astrocyte phenotype GAIacid accumulation may lead to neuronal loss, white matter abnorm-alities and macrocephaly characteristics of GAI. Antioxidant com-pounds such as FeTCPP that abrogated the early astrocyte reactionmay become useful as adjuvant therapeutics against GAI acidneurotoxicity. Further experiments addressed to determine theneurotoxic potential of astrocytes submitted to GAI accumulatingacids are necessary to shed light on the underlying mechanismsinvolved in neuron death elicited by astrocyte dysfunction.

Acknowledgments

This study was supported by PDT Salud 76/23 and PEDECIBABiologia, Uruguay.

References

Alonso, M., Melani, M., Converso, D., Jaitovich, A., Paz, C., Carreras, M.C., Medina, J.H.,Poderoso, J.J., 2004. Mitochondrial extracellular signal-regulated kinases 1/2 (ERK1/2)are modulated during brain development. J. Neurochem. 89, 248–256.

Araque, A., 2006. Astrocyte-neuron signaling in the brain—implications for disease.Curr. Opin. Investig. Drugs 7, 619–624.

Aschner, M., Allen, J.W., Kimelberg, H.K., LoPachin, R.M., Streit, W.J., 1999. Glial cells inneurotoxicity development. Annu. Rev. Pharmacol. Toxicol. 39, 151–173.

Barbeito, L.H., Pehar, M., Cassina, P., Vargas, M.R., Peluffo, H., Viera, L., Estevez, A.G.,Beckman, J.S., 2004. A role for astrocytes in motor neuron loss in amyotrophiclateral sclerosis. Brain Res. Brain Res. Rev. 47, 263–274.

Buffo, A., Rite, I., Tripathi, P., Lepier, A., Colak, D., Horn, A.P., Mori, T., Gotz, M., 2008.Origin and progeny of reactive gliosis: a source of multipotent cells in the injuredbrain. Proc. Natl. Acad. Sci. U.S.A. 105, 3581–3586.

Carreras, M.C., Poderoso, J.J., 2007. Mitochondrial nitric oxide in the signaling of cellintegrated responses. Am. J. Physiol. Cell Physiol. 292, C1569–C1580.

Cassina, P., Pehar, M., Vargas, M.R., Castellanos, R., Barbeito, A.G., Estevez, A.G.,Thompson, J.A., Beckman, J.S., Barbeito, L., 2005. Astrocyte activation by fibroblastgrowth factor-1 and motor neuron apoptosis: implications for amyotrophic lateralsclerosis. J. Neurochem. 93, 38–46.

Cassina, P., Cassina, A., Pehar,M., Castellanos, R., Gandelman,M., de León, A., Robinson, K.M., Mason, R.P., Beckman, J.S., Barbeito, L., Radi, R., 2008. Mitochondrial dysfunctionin SOD1G93A-bearing astrocytes promotesmotor neuron degeneration: preventionby mitochondrial-targeted antioxidants. J. Neurosci. 28, 4115–4122.

Chiu, S.Y., Kriegler, S., 1994. Neurotransmitter-mediated signaling between axons andglial cells. Glia 11, 191–200.

Choi, I.Y., Lee, S.J., Nam, W., Park, J.S., Ko, K.H., Kim, H.C., Shin, C.Y., Chung, J.H., Noh, S.K.,Choi, C.R., Shin, D.H., Kim, W.K., 2001. Augmented death in immunostimulatedastrocytes deprived of glucose: inhibition by an iron porphyrin FeTMPyP.J. Neuroimmunol. 112, 55–62.

Das, A.M., 2003. Regulation of the mitochondrial ATP-synthase in health and disease.Mol. Genet. Metab. 79, 71–82.

Das, A.M., Lucke, T., Ullrich, K., 2003. Glutaric aciduria I: creatine supplementationrestores creatinephosphate levels in mixed cortex cells from rat incubated with3-hydroxyglutarate. Mol. Genet. Metab. 78, 108–111.

Dienel, G.A., Hertz, L., 2001. Glucose and lactate metabolism during brain activation.J. Neurosci. Res. 66, 824–838.

Ferreira Gda, C., Viegas, C.M., Schuck, P.F., Tonin, A., Ribeiro, C.A., Coelho Dde, M.,Dalla-Costa, T., Latini, A., Wyse, A.T., Wannmacher, C.M., Vargas, C.R., Wajner, M.,2005. Glutaric acid administration impairs energy metabolism in midbrain andskeletal muscle of young rats. Neurochem. Res. 30, 1123–1231.

Fighera, M.R., Royes, L.F., Furian, A.F., Oliveira, M.S., Fiorenza, N.G., Frussa-Filho, R.,Petry, J.C., Coelho, R.C., Mello, C.F., 2006. GM1 ganglioside prevents seizures, Na+,K+-ATPase activity inhibition and oxidative stress induced by glutaric acid andpentylenetetrazole. Neurobiol. Dis. 22, 611–623.

Freudenberg, F., Lukacs, Z., Ullrich, K., 2004. 3-Hydroxyglutaric acid fails to affect theviability of primary neuronal rat cells. Neurobiol. Dis. 16, 581–584.

Funk, C.B., Prasad, A.N., Frosk, P., Sauer, S., Kolker, S., Greenberg, C.R., Del Bigio, M.R.,2005. Neuropathological, biochemical andmolecular findings in a glutaric acidemiatype 1 cohort. Brain 128, 711–722.

534 S. Olivera et al. / Neurobiology of Disease 32 (2008) 528–534

Giulian, D., Young, D.G., Woodward, J., Brown, D.C., Lachman, L.B., 1988. Interleukin-1 isan astroglial growth factor in the developing brain. J. Neurosci. 8, 709–714.

Genovese, T., Mazzon, E., Esposito, E., Muia, C., Di Paola, R., Bramanti, P., Cuzzocrea, S.,2007. Beneficial effects of FeTSPP, a peroxynitrite decomposition catalyst, in amouse model of spinal cord injury. Free Radic. Biol. Med. 43, 763–780.

Goodman, S.I., Frerman, F.E., 1995. Organic acidemias due to defects in lysine oxidation:2-ketoadipic acidemia and glutaric acidemia. In: Scriver, C., Beudet, A., Sly, W., Valle,D. (Eds.), The Metabolic and Molecular Bases of Inherited Disease. McGraw Hill,New York, pp. 2195–2204.

Gregersen, N., Brandt, N.J., 1979. Ketotic episodes in glutaryl-CoA dehydrogenasedeficiency (glutaric aciduria). Pediatr. Res. 13, 977–981.

Hertz, L., Peng, L., Dienel, G.A., 2007. Energy metabolism in astrocytes: high rate ofoxidative metabolism and spatiotemporal dependence on glycolysis/glycogenoly-sis. J. Cereb. Blood Flow Metab. 27, 219–249.

Hoffmann, G.F., Athanassopoulos, S., Burlina, A.B., Duran, M., de Klerk, J.B., Lehnert, W.,Leonar, J.V., Monavari, A.A., Müller, E., Muntau, A.C., Naughten, E.R., Plecko-Starting,B., Superti-Furga, A., Zschocke, J., Christensen, E., 1996. Clinical course, earlydiagnosis, treatment, and prevention of disease in glutaryl-CoA dehydrogenasedeficiency. Neuropediatrics 27, 115–123.

Kelly, K.J., Sandoval, R.M., Dunn, K.W., Molitoris, B.A., Dager, P.C., 2003. A novel methodto determine specificity and sensitivity of TUNEL reaction in the quantitation ofapoptosis. Am. J. Physiol. Cell Physiol. 284, 1309–1318.

Knudson, C.M., Brown, N.M., 2008. Mitochondrial potential, bax “activation”, andprogrammed cell death. Methods Mol. Biol. 414, 95–108.

Koeller, D.M., Woontner, M., Crnic, L.S., Kleinschmidt-DeMasters, B., Stephens, J., Hunt,E.L., Goodman, S.I., 2002. Biochemical, pathologic and behavioral analysis of amouse model of glutaric acidemia type I. Hum. Mol. Genet. 11, 347–357.

Kolker, S., Ahlemeyer, B., Krieglstein, J., Hoffmann, G.F., 2000. Cerebral organic aciddisorders induce neuronal damage via excitotoxic organic acids in vitro. AminoAcids 18, 31–40.

Latini, A., Ferreira, G.C., Scussiato, K., Schuck, P.F., Solano, A.F., Dutra-Filho, C.S., Vargas,C.R., Wajner, M., 2007. Induction of oxidative stress by chronic and acute glutaricacid administration to rats. Cell. Mol. Neurobiol. 27, 423–438.

Lepore, A.C., Dejea, C., Carmen, J., Rauck, B., Kerr, D.A., Sofroniew, M.V., Maragakis, N.J.,2008. Selective ablation of proliferating astrocytes does not affect disease outcomein either acute or chronic models of motor neuron degeneration. Exp. Neurol. [Epubahead of print].

Lund, T.M., Christensen, E., Kristensen, A.S., Schousboe, A., Lund, A.M., 2004. On theneurotoxicity of glutaric, 3-hydroxyglutaric, and trans-glutaconic acids in glutaricacidemia type. J. Neurosci. Res. 77, 143–147.

Magni, D.V., Oliveira,M.S., Furian, A.F., Fiorenza, N.G., Fighera,M.R., Ferreira, J.,Mello, C.F.,Royes, L.F., 2007. Creatine decreases convulsions and neurochemical alterationsinduced by glutaric acid in rats. Brain Res. 1185, 336–345.

Moncada, S., Bolanos, J.P., 2006. Nitric oxide, cell bioenergetics and neurodegeneration.J. Neurochem. 97, 1676–1689.

Moudgil, R., Michelakis, E.D., Archer, S.L., 2006. The role of k+ channels in determiningpulmonary vascular tone, oxygen sensing, cell proliferation, and apoptosis:implications in hypoxic pulmonary vasoconstriction and pulmonary arterialhypertension. Microcirculation 13, 615–632.

Paxinos, G., Törk, I., Tecott, L.H., Valentino, K.L., 1991. Atlas of the Developing Rat Brain.Academic Press, San Diego.

Pehar, M., Cassina, P., Vargas, M.R., Castellanos, R., Viera, L., Beckman, J.S., Estevez, A.G.,Barbeito, L., 2004. Astrocytic production of nerve growth factor in motor neuronapoptosis: implications for amyotrophic lateral sclerosis. J. Neurochem. 89, 464–473.

Pehar, M., Vargas, M.R., Robinson, K.M., Cassina, P., Diaz-Amarilla, P., Hagen, T.M., Radi,R., Barbeito, L., Beckman, J.S., 2007. Mitochondrial superoxide production andnuclear factor erythroid 2-related factor 2 activation in p75 neurotrophin receptor-induced neuron apoptosis. J. Neurosci. 27, 7777–7785.

Porciuncula, L.O., Emanuelli, T., Tavares, R.G., Schwarzbold, C., Frizzo, M.E., Souza, D.O.,Wajner, M., 2004. Glutaric acid stimulates glutamate binding and astrocytic uptakeand inhibits vesicular glutamate uptake in forebrain from young rats. Neurochem.Int. 45, 1075–1086.

Ridet, J.L., Malhotra, S.K., Privat, A., Gage, F.H., 1997. Reactive astrocytes: cellular andmolecular clues to biological function. Trends Neurosci. 20, 570–577.

Rouleau, C., Rakotoarivelo, C., Petite, D., Lambert, K., Fabre, C., Bonadet, A., Mercier, J.,Baldet, P., Privat, A., Langley, K., Mersel, M., 2007. Pyruvate modifies glycolytic andoxidative metabolism of rat embryonic spinal cord astrocyte cell lines and preventstheir spontaneous transformation. J. Neurochem. 100, 1589–1598.

Rosa, R.B., Dalcin, K., Schmidt, A.L., Gerhardt, D., Ribeiro, C.A.J., Ferreira, G.C., Schuck, P.F.,Wyse, A.T.S., Porciúncula, L.O., Wofchuk, S., Salbego, C.G., Souza, D.O., Wajner, M.,2007. Evidence that glutaric acid reduces glutamate uptake by cerebral cortex ofinfant rats. Life Sci. 81, 1668–1676.

Sauer, S.W., Okun, J.G., Schwab, M.A., Crnic, L.R., Hoffmann, G.F., Goodman, S.I., Koeller,D.M., Kölker, S., 2005. Bioenergetics in glutaryl-coenzyme A dehydrogenasedeficiency: a role for glutaryl-coenzyme A. J. Biol. Chem. 280, 21830–21836.

Sauer, S.W., 2007. Biochemistry and bioenergetics of glutaryl-CoA dehydrogenasedeficiency. J. Inherit. Metab. Dis. 30, 673–680.

Simpson, I.A., Carruthers, A., Vannucci, S.J., 2007. Supply and demand in cerebral energymetabolism: the role ofnutrient transporters. J. Cereb.BloodFlow.Metab. 27,1766–1791.

Stefanutti, G., Pierro, A., Smith, V.V., Klein, N.J., Eaton, S., 2007. Peroxynitritedecomposition catalyst FeTMPyP provides partial protection against intestinalischemia and reperfusion injury in infant rats. Pediatr. Res. 62, 43–48.

Strauss, K.A., Puffenberger, E.G., Robinson, D.L., Morton, D.H., 2003. Type I glutaricaciduria, part 1: natural history of 77 patients. Am. J. Med. Genet. C. Semin. Med.Genet. 121, 38–52.

Valero, R.A., Senovilla, L., Nuñez, L., Villalobos, C., 2008. The role of mitochondrialpotential in control of calcium signals involved in cell proliferation. Cell Calcium[Epub ahead of print].

Wu, A.S., Kiaei, M., Aguirre, N., Crow, J.P., Calingasan, N.Y., Browne, S.E., Beal, M.F., 2003.Iron porphyrin treatment extends survival in a transgenic animal model ofamyotrophic lateral sclerosis. J. Neurochem. 85, 142–150.

Zaidi, A.U., Bessert, D.A., Ong, J.E., Xu, H., Barks, J.D., Silverstein, F.S., Skoff, R.P., 2004.New oligodendrocytes are generated after neonatal hypoxic-ischemic brain injuryin rodents. Glia 46, 380–390.

Zinnanti, W.J., Lazovic, J., Housman, C., LaNoue, K., O'Callaghan, J.P., Simpson, I.,Woontner, M., Goodman, S.I., Connor, J.R., Jacobs, R.E., Cheng, K.C., 2007. Mechanismof age-dependent susceptibility and novel treatment strategy in glutaric acidemiatype I. J. Clin. Invest. 117, 3258–3270.