copyright © 2011 cognizant communication corporation

Copyright © 2011 Cognizant Communication Corporation

CT‐0511 Cell Transplantation Epub; submitted 04/11/2011, provisional acceptance 08/15/2011

DOI: 10.3727/096368911X600957

CCTT--00551111 AAcccceepptteedd 88//2299//22001111 ffoorr ppuubblliiccaattiioonn iinn ““CCeellll TTrraannssppllaannttaattiioonn””

Altered oxygen metabolism associated to neurogenesis of induced pluripotent stem

cells derived from a schizophrenic patient

Authors and Affiliations

Bruna da Silveira Paulsen1*, Renata de Moraes Maciel1*, Antonio Galina2, Mariana

Souza da Silveira3, Cleide dos Santos Souza1, Hannah Drummond1, Ernesto Nascimento

Pozzatto1, Hamilton Silva Junior1, Leonardo Chicaybam4, Raffael Massuda5, Pedro

Setti-Perdigão9, Martin Bonamino4, Paulo Silva Belmonte-de-Abreu5, Newton

Gonçalves Castro9, Helena Brentani6,7,8, Stevens Kastrup Rehen1#

1Laboratório Nacional de Células–Tronco Embrionárias, Instituto de Ciências

Biomédicas, UFRJ, Rio de Janeiro, RJ, 21941-913, Brazil; 2Laboratório de

Bioenergética e Fisiologia Mitocondrial, Instituto de Bioquímica Médica, UFRJ, Rio de

Janeiro, RJ, 21941-590, Brazil; 3Laboratório de Neurogênese, Instituto de Biofisica

Carlos Chagas Filho, UFRJ, Rio de Janeiro, RJ, 21941-902, Brazil; 4Divisão de

Medicina Experimental, Instituto Nacional de Câncer, Rio de Janeiro, RJ, 20231-050,

Brazil; 5Departamento de Psiquiatria, Faculdade de Medicina, Universidade Federal do

Rio Grande do Sul, Porto Alegre, RS, 90035-003, Brazil; 6Departamento de Psiquiatria,

Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, 05403-010, Brazil;

7Instituto Nacional de Psiquiatria do Desenvolvimento, Faculdade de Medicina,

Universidade de São Paulo, São Paulo, SP, 05403-010, Brazil; 8Laboratório de

Investigação Médica, Faculdade de Medicina, Universidade de São Paulo, São Paulo,

SP, 05403-010, Brazil; 9Laboratório de Farmacologia Molecular, Instituto de Ciências

Biomédicas, UFRJ, Rio de Janeiro, RJ, 21941-902, Brazil.

*These authors provided equal contribution to this work.



#Address correspondence to Stevens K. Rehen, National Laboratory for Embryonic

Stem Cell Research, UFRJ, Hospital Universitário Clementino Fraga Filho – HUCFF,

Rua Rodolpho Paulo Rocco, 255 - 4º andar – CEPEDIP - Cidade Universitária Rio de

Janeiro – RJ, Brasil. Phone: + 55 21 2562 2928; E-mail: [email protected]

ABSTRACT

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in

the process of thoughts, perceptions and emotions, usually leading to a mental

deterioration and affective blunting. Studies have shown altered cell respiration and

oxidative stress response in schizophrenia; however, most of the knowledge has been

acquired from post-mortem brains analyses or from non-neural cells. Here we describe

that neural cells, derived from induced pluripotent stem cells generated from skin

fibroblasts of a schizophrenic patient, presented a 2-fold increase in extra-mitochondrial

oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when

compared to controls. This difference in ROS levels was reverted by the mood stabilizer

valproic acid. Our model shows evidence that metabolic changes occurring during

neurogenesis are associated with schizophrenia, contributing to a better understanding

of the development of the disease and highlighting potential targets for treatment and

drug screening.

Key words: Schizophrenia; Oxygen Metabolism; Induced Pluripotent Stem Cells; ROS

production

Running head: ROS associated to neurogenesis in schizophrenia



INTRODUCTION

Schizophrenia is characterized by both positive and negative symptoms, such as

auditory hallucinations, paranoid delusions and social impairment. The disability of

schizophrenia is largely due to cognitive deficits, such as lack of attention and working

memory. Usually, first symptoms are identified in the adolescence or in early adulthood

and after that, the disease enters into a chronic evolution, regardless of culture, social

class, racial group and gender (31).

Brain mitochondria play a key role in neuroenergetics, as they are essentials regulators

of intracellular Ca2+ and reactive oxygen species (ROS) homeostasis, as well as of the

glutamate-lactate metabolism. Therefore, an imbalance between mitochondrial and non-

mitochondrial ROS production (12,25,37), in addition to impaired levels of antioxidant

defenses, may lead to altered intracellular signaling radicals or oxidative stress,

conditions that have been associated with neurodysfunction (20,38,40,47). In fact, a

large number of studies have shown affected efficiency of oxidative phosphorylation

and ROS levels in some psychiatric diseases such as schizophrenia (11). On the other

hand, even though it is not known whether schizophrenia involves ROS overproduction

by non-mitochondrial sources, an impaired oxidative stress response has been



associated with the disorder for a long time (21), in which changes in glutathione (GSH)

synthesis were reported, as well as polymorphisms in key enzyme genes (22,44,49).

Much of the knowledge about schizophrenia biology is the result of analyses in post-

mortem brains, which are not always preserved under ideal conditions (35) and are

usually a representation of the final stage of the disease, not providing proper clues

about its development. In order to overcome these limitations, others have used non-

neural cells as a model for studying schizophrenia; however, it is not clear how

informative these cells are for understanding the onset of the disease (36).

Since schizophrenia has been proposed as a neurodevelopmental disorder (27), the

generation of a new model by reprogramming somatic cells into induced pluripotent

stem (iPS) cells has the advantage of enabling the study of neurogenesis and early

phenotypic changes associated to the mental illness, avoiding interferences such as

therapeutic treatment.

Understanding the role of oxygen metabolism, both in diseased brain and throughout

development, is of critical importance to elucidate pathophysiological mechanisms in

psychiatric disorders. Taking all this into account, we decided to explore oxygen

consumption and ROS levels changes in neural cells derived from reprogrammed

fibroblasts obtained from a schizophrenic patient.

In the current study, we successfully generated two clones of iPS cells from skin

fibroblasts of one patient with schizophrenia. When compared to controls, neural

precursor cells derived from these two independent schizophrenic iPS clones showed

altered extra-mitochondrial oxygen consumption associated with an increase in the

levels of ROS, which was reverted by the use of valproic acid (VPA). Using the iPS



model, our results provide the first evidence of a developmental metabolic alteration in

schizophrenia, which can be reverted by pharmacological intervention.



MATERIAL AND METHODS

Schizophrenic patient

The patient with DSM-IV-TR (Diagnostic and Statistical Manual of Mental Disorders)

diagnosis of schizophrenia was recruited from the outpatient clinic (Schizophrenia

Program) at Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Brazil.

Psychiatric diagnosis was defined after a comprehensive assessment, including clinical

interview, application of the Structured Clinical Interview for DSM-IV-Axis I Disorders

(SCID-I), and Operational Criteria (OPCRIT), based on medical history, interview with

caregivers and clinical examination. The patient is a 48 year-old woman considered as

Clozapine-resistant. Skin tissue for use in reprogramming experiments was obtained

after patient and caregivers signed an informed consent approved by the HCPA

Institutional Review Board (IRB00000921). Data was analyzed anonymously and all

clinical investigations were conducted according to the principles expressed in the

Declaration of Helsinki.

Derivation of primary human fibroblast from schizophrenic patient

Skin fibroblasts from schizophrenic patient were obtained after patient and caregivers

signed an informed consent approved by the Ethics Committee of the Institution

(Institutional Review Board). Using sterile technique, a 10-mm full-thickness skin cut

biopsy was taken from the neck surface of the patient by a plastic surgeon. The biopsy

was cut into 2x2 mm pieces. The pieces were plated in a T75 bottle and maintained in 2

mL of DMEM/F12 supplemented with 10 % fetal bovine serum (FBS) and 20 µg/mL



ciprofloxacin. After fibroblast migration (10–14 days), cells were detached using

TrypLETM Express (Invitrogen) and maintained in DMEM/F12 medium supplemented

with 10 % FBS without antibiotics. Age-matched male control fibroblasts were obtained

from a commercial source (Excellion, Brazil).

Cell culture H9 female human embryonic stem (ES) cells and iPS cells were cultured on Matrigel

(BD Biosciences) -coated plates in mTeSR™1 (StemCell Technologies). All splits were

performed mechanically as colonies clumps. To form embryoid bodies (EBs), confluent

undifferentiated iPS cells were treated with accutase (Millipore), scrapped out and

transferred to 60 mm, non-adherent dishes in DMEM/F12 medium supplemented by

15% of knockout serum replacement (KSR; Invitrogen), 1% nonessential amino acid

solution (Invitrogen), 2mM L-glutamine (Invitrogen), 0.1mM β-mercaptoethanol

(Invitrogen), 40μg/mL gentamicin sulfate (Schering-Plough).

iPS cell generation

The pMX-based retroviral vectors encoding human Octamer binding transcription factor

4 [Oct4], (sex determining region Y) box 2 [Sox2], Krüppel-like factor 4 [Klf4] and c-

Myc cDNAs (48) were prepared as described previously by Paulsen and coworkers

(41). For generation of iPS cells, human primary fibroblasts were transduced overnight

with equal amounts of concentrated supernatants containing each of the four

retroviruses, supplemented with 8 g/mL polybrene. Twenty-four hours after

transduction, virus-containing medium was replaced with the second round of

supernatant. Two days after transduction, 50 g/mL of L-Ascorbic Acid (USB

Corporation) were added to cultures, as described by Esteban and coworkers (17). Six



days after transduction cells were replated (5x104 cells per 6-well plate) on mitotically

inactivated mouse embryonic fibroblasts (MEFs). In the following day, medium was

replaced by mTeSR™1 and changed every day. Eight days after transduction, 1 mM of

valproic acid (VPA, Sigma) was added to the medium for another seven days. About

twenty days after transduction, colonies were picked up and transferred to 6-well plates

coated with Matrigel in mTeSRTM1. Pluripotent-like colonies were selected based on

morphological criteria, and pluripotency was verified by RT-PCR analyses and

immunostaining assays using OCT3/4 (Santa Cruz Biotechnology; mouse, 1:100),

stage-specific embryonic antigen 4 (SSEA4; Chemicon; mouse, 1:100), TRA-1-60

(Chemicon; mouse, 1:100) and TRA-1-81 (Chemicon; mouse, 1:100) primary

antibodies.

The cell lines derived in this work, as well as controls, are available in the cell bank of

the National Laboratory for Embryonic Stem Cell Research at Federal University of Rio

de Janeiro, LaNCE-RJ (Brazil).

RNA isolation and PCR analysis

Total RNA was isolated using the Trizol reagent (Invitrogen) and digested with DNase I

using DNA-freeTM Kit (Applied Biosystems), following manufacturer’s instructions.

Complementary DNA was generated from 1 g total RNA using High Capacity RNA-

to-cDNA Kit (Applied Biosystems), according to manufacturer’s recommendations.

Reverse transcription (RT)–PCR was performed using specific primer sequences (Table

S1). Each RT-PCR reaction was carried out for 35 cycles in a reaction mixture

containing 1U Taq DNA Polymerase (NB Science), 1 X Taq DNA Polymerase Buffer

containing 25 mM MgCl2 (NB Science), 500 nM of each primer (Forward and Reverse),



200 M dNTP mixture containing the four deoxyribonucleotides (dATP, dCTP, DTTP,

dGTP) and 1 L of cDNA.

Neural differentiation of iPS cells

Generation of neural precursor cells (NPCs) was performed using retinoic acid (RA) as

previously described (3), and immunological analyses were done using primary

antibodies for Nestin (Chemicon; mouse, 1:100), III-tubulin (Sigma; mouse, 1:100)

and NeuN (Chemicon; mouse, 1:100).

High-resolution respirometry

Oxygen consumption was measured by high-resolution respirometry using the

OROBOROS Oxygraph 2k at 37 °C with chamber volumes set at 2 mL. DatLab

software (Oroboros Instruments, Innsbruck, Austria) was used for data acquisition and

analysis. Human fibroblasts, iPS cells and NPCs were enzymatically harvested,

centrifuged and resuspended in serum free DMEM. Cell routine respiration, which is

defined as respiration in cell-culture medium without additional substrates or effectors,

was determined under baseline conditions for 10-15 minutes. After observing routine

respiration, ATP synthase was inhibited by oligomycin (2 g/mL). Oxygen

consumption coupled to ATP synthesis by oxidative phosphorylation was derived from

the difference between routine respiration and oligomycin-resistant respiration. To

uncouple oxidative phosphorylation, stepwise titration of FCCP (carbonyl cyanide p-

trifluoromethoxyphenylhydrazone) was used in the range of 100-700 nM. After FCCP

titration, the electron transport chain of oxidative phosphorylation was stopped/blocked

by Antimycin A (1 g/mL) and/or KCN (0.5 mM) and the oxygen flow rate detected in

this condition was defined as extra-mitochondrial oxygen consumption.



Reactive oxygen species (ROS) production

Formation of intracellular ROS was measured by incubating NPC-Control (NPCs from

a control patient) and NPC-SZP (NPCs from a schizophrenic patient) in the presence of

5-(and-6)-carboxy-2`,7`-dichlorodihydrofluorescein diacetate (CM-H2-DCFDA,

Molecular Probes). Control groups included cells without VPA treatment. VPA

treatment was performed by adding 1 mM of valproic acid to medium for 24 h. After

that, medium was replaced by fresh DMEM/F12 supplemented with 2 mM CM-H2-

DCFDA. Cells were incubated for 40 min at 37 °C and 5% CO2, and randomly chosen

fields were examined under a Zeiss Axio Observer.Z1 inverted microscope, equipped

with a 20X objective lens and an AxioCam MRm camera at a fixed exposure time. Five

independent fields were counted for each experimental condition, which was carried out

in triplicate.

Electrophysiology

Voltage-activated currents and synaptic activity were measured after further culturing

NPC-Control and NPC-SZP neural tube-like structures onto 5 μg/mL laminin and 15

μg/mL poly L-ornithine-coated tissue culture dishes in Neurobasal medium (Invitrogen)

supplemented with 1X B27 (Invitrogen) and 1X N2 (Invitrogen) for 20 days. Whole-

cell membrane currents were recorded at a membrane holding potential of –70 mV with

an EPC-7 (List, Germany) patch-clamp system. Currents were low-pass filtered at 3

kHz and digitized with a LabMaster interface under the control of pClamp software

(Axon Instruments, USA). The standard extracellular solution was 165 mM NaCl, 5

mM KCl, 2 mM CaCl2, 10 mM D-glucose, 5 mM HEPES, and ~2 mM NaOH, pH 7.34.

This solution replaced the culture medium approximately 20 minutes before the



recordings and was continuously perfused at a rate of ~1 mL/min throughout the

experiments. Patch micropipettes were made from borosilicate glass capillaries (WPI,

USA) in a P-97 horizontal puller (Sutter Instruments, USA). The intracellular solution

was 80 mM CsCl, 80 mM CsF, 10 mM EGTA, 10 mM HEPES, and ~26 mM CsOH, pH

7.3. The filled patch microelectrodes had resistances of 2 to 5 MΩ in the bath; the

access resistance was left uncompensated. Approximately 2 minutes after achieving the

whole-cell configuration, voltage sensitive sodium currents were evoked by 50 ms-

depolarizing square pulses ranging from –40 mV to +10 mV from a holding potential of

–70 mV. Leak currents were subtracted by a fractional method (P/N) using four scaled

hyperpolarizing subpulses. Immediately after this pulse, 3-minute recordings of the

spontaneous activity were made for each cell. Recordings were made at room

temperature (~23°C).

Statistical Analyses

Statistical analyses were performed with GraphPad Prism 5 and validated by applying

Student’s t tests to compare two groups individually, or One-Way ANOVA followed by

Newman-Keuls post-test to compare more than two groups. Confidence intervals were

defined at 95% confidence level (p<0.05 was considered to be statistically

significant).RESULTS

Characterization of iPS cells generated from fibroblasts of a patient with schizophrenia

Skin fibroblasts obtained from one patient with schizophrenia and one control with

similar age (Fig. 1A, B) were reprogrammed through retroviral transduction. Two SZP

iPS clones (SZP-C11 and SZP-C2) (Fig. 1B; Fig. S1A, B, respectively) and one control

iPS clone (Fig. 1A) were manually selected based on their human ES cells-like

morphology. Both clones from the schizophrenic patient presented similar results



regarding all the analyses. Data presented here refers to SZP-C11, while data from SZP-

C2 can be found at the supplemental material. All iPS clones used had similar rates of

growth and stable karyotypes with 46 chromosomes (data not shown). Analysis by RT-

PCR demonstrated that both iPS-Control and iPS-SZPs cells present similar expression

of pluripotency markers compared to one of the most widely used human embryonic

stem (hES) cells lines, H9 (Fig. 1C) (19). Pluripotency markers were also verified by

immunostaining for OCT3/4, SSEA4, Tra-1-60 and Tra-1-81 (Fig. 1D, E).

Since teratoma formation is not required for iPS cells in vitro applications (16), and in

this work we intended to use those cells as a tool for studying schizophrenia-associated

events during neurogenesis, we adopted differentiation of the selected iPS cells into

embryoid bodies (EBs), which were similar to those generated from H9 cells (data not

shown), and also toward early neuroectoderm, mesoderm, and endoderm, to allow

assessment of their differentiation capacity (Fig. 1F-H). Moreover, iPS cells were able

to differentiate into spontaneously beating cells in vitro (data not shown) or, when co-

cultured with the mouse bone marrow derived stromal PA6 cell line, into neural cells

expressing the dopaminergic neuron marker tyrosine hydroxylase (TH) as well as other

neuronal markers (Fig. S2).

Direct differentiation of iPS cells into neural precursor cells

To induce iPS cells to direct neural differentiation, we performed an adaptation of

Baharvand and coworkers protocol (3) (Fig. 2A). During the first stage of the neural

induction, the cells underwent drastic morphological changes. During the second stage,

rosettes and neural tube-like structures (Fig. S3, left panels) were noticed amongst

differentiating cells. The emergence of neural processes could be observed at the final



stage of differentiation, in both iPS-Control and iPS-SZP cells (Fig. S3, right panels).

By the end of 18 days of differentiation, we obtained a population of neural precursor

cells (NPCs) that similarly expressed the neuronal marker gene nestin in both NPC-

Control (51.9 ± 4.3%) and NPC-SZP (53.7 ± 2.8%). Other neuronal marker genes were

expressed by these cells such as βIII-Tubulin and NeuN [neuronal nuclei] (Fig. 2B; Fig.

S1E), as well as aromatic amino acid decarboxylase [AADC] and dopamine transporter

[DAT] (dopaminergic neurons), choline acetyltransferase [ChAT] (cholinergic neurons)

and LIM homeobox transcription factor 1, beta [LMX1B] (serotonergic neurons) were

evidenced by RT-PCR analysis (Fig. 2C; Fig. S1H). Furthermore, after more 20 days in

culture, both NPC-Control and NPC-SZP expressed functional voltage activated

channels and formed functional synapses, as indicated by the presence of spontaneous

postsynaptic currents (Fig. 3). No apparent changes in neuronal differentiation or

expression of neuronal marker genes were observed amongst the experimental groups.

Changes in endogenous oxygen consumption are only observed in SZP neural precursor

cells but not in fibroblasts or iPS cells

Endogenous oxygen consumption is a result of two main components of O2 sink

systems: mitochondria and extra-mitochondrial oxidases (28). Total cellular oxygen

flow parameters were assessed in fibroblasts, iPS cells and NPCs from control and

schizophrenic individual by using high-resolution respirometry (Fig. 4; Fig. S4).

Respiratory control ratios were determined from titrations with the FoF1-ATP synthase

specific inhibitor, oligomycin, and the oxidative phosphorylation uncoupler, FCCP (Fig.

4A-C). These ratios yield important information regarding control of electron flux

through respiratory chain by the FoF1-ATP synthase, and also about maximal respiratory

capacity of mitochondrial electron transfer system (ETS) per cell. Oligomycin-inhibited



respiration represents O2 consumption coupled to ATP synthesis (Fig. 4D). When the

proton ionophore FCCP is added to cells, it dissipates the proton gradient, and

mitochondrial oxygen flow is settled to a maximal rate (Fig. 4). It was observed that

routine, oligomycin-insensitive and maximal ETS respiration (FCCP-treated) were

much higher in fibroblasts than in iPS cells and NPCs (Fig. 4A-C). In addition, the

portion of respiration coupled to ATP synthesis was five to eight times higher in

fibroblasts than in iPS cells and NPCs, but no significant difference was observed

amongst all cell types when Control and SZP were compared (Fig. 4D). Analysis of the

same parameters in embryonic stem cells, H9, place them on a similar zone to iPS cells

and NPCs, displaying much lower rates than those detected in fibroblasts (Fig. 4B). The

slight difference observed between respiration parameters level of H9 and iPS cells lines

is consistent with other recently described metabolic differences between these two

types of pluripotent stem cells (50). Remarkably, after differentiation, all respiration

parameters (routine, oligomycin- and FCCP-treated rates) were down-regulated

exclusively in NPC-Control cells (Fig. 4C, black bars), while in NPC-SZP they

remained similar to those observed for iPS cells (Fig. 4C, grey bars).

In accordance with previous observations (26), fibroblasts derived from both control

and schizophrenic patient present over 80% of oxygen consumption attributed to

mitochondrial respiration, as shown when antimycin A or KCN-insensitive values are

compared to routine O2 flow in these cells (Fig. 4A, E). On the other hand, our results

show that, in iPS cells and NPCs, the main O2 consumption fraction is extra-

mitochondrial (Fig. 4B, C, E; Fig. S4A, C). The new finding consists in the fact that

fibroblasts from Control and SZP, as well as iPS cells from theses two groups, present



no difference in extra-mitochondrial O2 consumption, while NPC-SZP shows 2-fold

higher levels than NPC-Control (Fig. 4E).

NPCs from schizophrenic patient present higher ROS levels, which is reverted by

valproic acid

Then, we analyzed whether the mood stabilizer VPA could interfere in two oxidative

activities: (i) extra-mitochondrial O2 consumption (Fig. 5A) and (ii) ROS levels (Fig.

5B, C). After VPA treatment, both NPC-Control and NPC-SZP presented a 2 to 3-fold

increase in extra-mitochondrial O2 flow. This result raised the possibility that there

could be an increase in ROS accumulation in cells mediated by oxidases activity

(1,23,42,52). In order to evaluate ROS levels, the ROS-sensitive fluorescent probe CM-

H2-DCFDA was used (Fig. 5B, C). It was observed that ROS detection was more than

2-fold higher in NPC-SZP than NPC-Control, in accordance to the extra-mitochondrial

O2 consumption pattern. However, although VPA treatment enhanced extra-

mitochondrial O2 consumption, this was not followed by ROS increase (Fig. 5),

suggesting that ROS levels detected in response to VPA might be determined by a

balance between ROS generation and antioxidant defenses. In fact, after VPA treatment,

we observed that NPC-SZP ROS levels were reduced to NPC-Control levels (Fig 5C).

DISCUSSION

Due to difficulties in obtaining biopsies of the central nervous system, the study of

neuropathological abnormalities in patients, especially during the early stages of the

disease, has long been halted. Moreover, there is speculation regarding the credibility of



data obtained from adult patients, that could reflect effects of secondary cause (i.e.

constant use of a drug) rather than a disease phenotype (35).

Because iPS cells have a genetic background similar to that of the original donor cell

and are capable of generating all cell types, including neural cells, they appear as an

attractive model for the study of neuropathologies (4,32). It also facilitates

characterization of specific changes that might occur during nervous system

development (33). The potential of iPS cells to generate models of neurodegenerative

diseases has been successfully demonstrated by reprogramming of somatic cells from a

single patient with amyotrophic lateral sclerosis (13), one patient with spinal muscular

atrophy (15), one patient with Leucine-rich repeat kinase 2 (LRRK2) mutation of

Parkinson’s disease (39), one patient with X-linked chronic granulomatous disease (X-

CGD) (54), and four patients with Rett Syndrome (34).

However, modeling a multifactorial disorder, such as schizophrenia, is an enormous

challenge to the iPS cells field. Schizophrenia, for example, is a consequence of many

different combinations of gene malfunction and also the influence of environmental

factors (27). Because cellular and molecular mechanisms underlying the disease state

remain unclear, schizophrenia innovative models (i.e. iPS cells) are required as a

complementation to current models, such as post-mortem brains and animals (7).

Recently, fibroblasts from two schizophrenic patients with a mutation in DISC1

(Disrupted in schizophrenia 1) were reprogrammed into iPS cells without any specific

phenotype being described in vitro (10). In spite of being considered a risk factor for

developing schizophrenia, little is known about how DISC1 contributes to a spectrum of

the mental disorder (10). Furthermore, since schizophrenia is now considered a



neurodevelopmental disorder, specific phenotypes associated with the disease in vitro

may only be revealed after the onset of neurogenesis, as described here.

Moreover, after reprogramming iPS cell from fibroblasts of four schizophrenic patients,

Brennand et al reported changes in gene expression of many components of the cyclic

AMP and WNT signaling pathways and also identified neuronal phenotypes associated

with the mental disorder, such as reduced neuronal connectivity, decreased neurite

number, post synaptic density protein 95 (PSD95)-protein levels and glutamate receptor

expression (7).

Here, we described a novel and different approach to the elucidation of changes in

respiration and ROS production in a patient with schizophrenia. We further show that

neurons derived from a schizophrenic patient developed electrophysiological properties

in vitro similar to those of mature neurons, as recently reported (7). In addition, we also

show that synaptic connections are formed amongst neurons from SZP without the

support of exogenous glial cells or co-cultured neurons. Therefore, one can envisage the

development of networked derived cell systems, which might better model the

complexities of schizophrenia. Since we could recapitulate at least some cellular aspects

of the disorder, our methodology may lead to the development of screening assays of

new compounds capable of modulating these aspects of schizophrenia pathophysiology.

However, as discussed by Brennand et al., due to sample size, our findings are

necessarily preliminary.

Increasing evidence indicates that schizophrenia may encompass a combination of many

different disorders with different outcomes, caused by distinct lesions that contribute to

the final clinical scenario (27,31). Therefore, it is worth mentioning that the expectation



for the next decade is turned towards development of personalized approach to

treatment and prevention, aiming more accurate diagnosis and pathophysiologic

understanding based on individual risk detection (27). In this context, iPS cells seem to

be a valuable tool.

The use of oxygen consumption as an index of oxidative metabolism has been applied

for decades and revealed important mechanisms and enzyme activities related either to

redox or bioenergetic metabolisms (24,28,43).

Although there are consistent reports in which specific alterations in mitochondrial

physiology are described as a factor that contributes for the onset of psychiatric

disorders, including schizophrenia (5,11), the original causal relationship between the

disease and mitochondrial dysfunction has not been clearly identified.

Here the analysis of oxygen flow, which includes both mitochondrial and extra-

mitochondrial consumption rates, showed there was no detectable distinction in all

respiratory coupling states analyzed using oligomycin and FCCP, either in fibroblasts or

iPS cells from both Control and SZP. This observation supports the notion that

phenotypic differences between Control and SZP are not seen in non-disease-specific

cell types. The fact that routine, ATP-coupled or maximal ETS respirations rates are all

lower in iPS cells than in fibroblasts is in accordance with previous observations

demonstrating that mitochondria energy supply is lower in stem cells, in which

glycolysis assumes the spare energetic role (8,9,18). However, the novelty is that all the

above-mentioned respiration parameters are decreased in NPC-Control without loss of

the amount of ATP-coupled respiration. We described that these differences were

related to extra-mitochondrial oxygen consumption.



Alterations observed in oxygen consumption of schizophrenic patient-derived cells

cannot be attributed to improper integration of the vectors in the genome since both SZP

clones (C2 and C11) presented similar results (see supplemental data).

One possibility for the total extra-mitochondrial O2 flow decrease observed in NPC-

Control is that cells undergoing differentiation have to reset their ROS levels for novel

signaling programs, as suggested for other cell types (30,43).

In contrast, oxygen flow in NPC-SZP remains similar to iPS-SZP levels (about 2-fold

higher than NPC-Control), which correlates to higher ROS levels. Some reports have

attributed to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) a

relevant role in oxygen consumption and ROS generation in various cell types,

including hematopoietic and neural stem cells (30,43). Signaling roles for NOX-

produced ROS were also recently proposed (30), but other oxidases cannot be excluded.

The fact that NPC-SZP present higher levels of ROS in comparison with NPC-Control

could be related not only to an aberrant production of ROS, but also, to an impaired

regulation of antioxidant responses. In fact, it has been previously suggested that ROS

imbalance might be relevant to schizophrenia pathophysiology (6,14).

More interesting is the new finding that this neuroenergetic failure in NPC-SZP

(increased ROS production) might have emerged during neural differentiation and can

be attenuated pharmacologically.

Comparing oxygen consumption in response to VPA treatment to ROS levels, it

becomes clear that antioxidant responses are critically affected under these conditions.



In fact, VPA and other mood stabilizers were previously shown to interfere in pathways

able to revert oxidative stress (2,51).

The way in which VPA interacts with its cellular targets is still not fully understood.

However, evidences suggest that the mechanism of mood-stabilizing action of VPA

comprises acute effects due to the enhancement of gamma-aminobutyric acidergic

(GABAergic) transmission, followed by a variety of longer-term effects caused by

changes in gene expression, mediated in one hand by the glycogen synthase kinase

(GSK)-3 and/or inositol monophosphatase (IMPase) cascades (46), and in the other by

histone deacetylases inhibition. These changes in the expression of multiple genes are

involved in transcription regulation, cell survival, ion homeostasis, cytoskeletal

modifications and signal transduction (45). Besides that, it has been long established

that VPA protects against oxidative stress (29).

The effect of VPA in our model, reverting the schizophrenic-specific neuronal

phenotype in culture, may serve as a proof of principle for future high-throughput

screening of different drugs, including other epigenetic modifiers with similar effects as

VPA.

High-throughput screening technology using iPS cells is a novel alternative to current

drug discovery and toxicological tests, as it promotes the improvement of drug selection

and decreases costs and time of drug development. However, to validate this model,

further understanding and technology development are required to confirm that iPS cells

would be able to recapitulate such a complicated system, as the human organism.

Furthermore, quality criteria have to be monitored during reprogramming process in



order to certify the biologic relevance of iPS cell system for solving specific

toxicological questions (53).

In conclusion, the application of this approach of generating neural cells from iPS of a

schizophrenic patient revealed metabolic changes during neurogenesis, which might not

only contribute to the development of the disease but also represent an important target

for treatment.

SUPPLEMENTAL DATA can be downloaded from the link http://www.lance‐

ufrj.org/supplemental‐data‐schizophrenia‐2011‐cell‐transplantation‐9182736455.html.

ACKNOWLEDGEMENTS: We thank Dr. Aline Marie Fernandes for assistance with the

biopsies, Paulo André Nóbrega Marinho for assistance with cell cultures, Sylvie

Devalle for manuscript editing, Ismael Gomes and Sandra Pellegrini for technical

assistance. This work was supported by Fundação Carlos Chagas Filho de Amparo a

Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e

Tecnologia (INCTC), Instituto do Milênio – Terapia Gênica (CNPq), Instituto Nacional

do Câncer (INCA) and INCT for Excitotoxicity and Neuroprotection/CNPq.

Authors declare no conflicts of interest.

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FIGURE LEGENDS

Figure 1. Reprogrammed induced pluripotent stem (iPS) cells are generated from

fibroblasts after biopsy. (A, B) Representative images of selected colonies with

embryonic stem (ES)-cell-like morphology derived from control and schizophrenic

(SZP) fibroblasts, respectively. (C) Expression of ES cell-marker transcripts in the iPS

clones in comparison to H9, a human embryonic stem cell line. (D) RT-PCR analysis

shows that iPS from both control and SZP patient express markers from the three germ

layer. (E, F) As shown by immunocytochemistry, control (E) and SZP patient iPS (F)

express pluripotency markers. Nuclei were stained with DAPI (blue). (G, H) Embryoid

bodies (EBs) from control and SZP iPS cells, respectively. Scale bars, 50 m.

GAPDH = Glyceraldehyde 3-phosphate dehydrogenase; OCT3/4 = octamer-binding

transcription factor 3/4; SOX2 = (sex determining region Y)-box 2; KLF4 = Krüppel-

like factor 4; DPPA2 = developmental pluripotency associated 2; ESG1 = Embryonic

stem cell-specific gene; FGF4 = fibroblast growth factor 4; GDF3 = Growth

differentiation factor-3; TERT = telomerase reverse transcriptase; REX1 = RNA

exonuclease 1; -RT = no reverse transcriptase; AFP = alpha fetoprotein; FOXA2 =

forkhead box A2; CK8 = cytokeratin 8; MSX1 = muscle segment homeobox 1; GFAP =



glial fibrillary acidic protein; MAP2 = microtubule associated protein 2; PAX6a =

paired box gene 6a.

Figure 2. iPS cells differentiate into neural precursor cells (NPCs). (A) Schematic view

of the protocol used for differentiation. (B) Control and SZP iPS cells express the

neuroectodermal marker Nestin, and generate III-Tubulin and NeuN positive neural

cells after eighteen days of differentiation. Nuclei were stained with DAPI (blue). Scale

bars, 50 m. (C) RT-PCR showing that NPCs express neurochemical neuron markers.

RA= retinoic acid; AADC= aromatic amino acid decarboxylase; DAT = dopamine

transporter; ChAT = choline acetyltransferase; LMX1b = LIM homeobox transcription

factor 1, beta.

Figure 3. NPC-Control and NPC-SZP develop mature neuronal electrophysiological

properties after 20 days in culture. (A) Sample traces showing spontaneous postsynaptic

currents recorded at –70 mV in each group. (B) Averaged traces from five

representative synaptic currents from one cell in each case. (C) Voltage-activated

currents elicited by depolarizing pulses ranging from –40 mV to +10 mV in 10 mV

steps from a holding potential of –70 mV. Similar results were obtained from four

Control and four SZP neurons.

Figure 4. Neural precursor cells (NPCs) from the schizophrenic patient consume more

oxygen than control. (A, B, C) Different respiration conditions in fibroblasts (A), iPS /

H9 (B) and NPC (C) cells from Control (black bars) and SZP patient (gray bars). (D)

Respiration coupled to ATP synthesis in fibroblasts (Fib), iPS, NPCs and H9. (E) Extra-



mitochondrial oxygen consumption. Bars represent mean ± s.e.m. (n=3 * p<0.01, **

p<0.001, with respect to NPC-Control). FCCP = carbonyl cyanide p-

trifluoromethoxyphenylhydrazone).

Figure 5. NPC-SZP generates more reactive oxygen species (ROS) than NPC-Control

and valproic acid (VPA) treatment reverts this condition. (A) Extra-mitochondrial

respiration on VPA treated and non-treated NPCs. (B) Fluorescence microscopy of

ROS derived from NPC-Control and NPC-SZP cells probed with 5-(and-6)-carboxy-

2`,7`-dichlorodihydrofluorescein diacetate (CM-H2-DCFDA), Scale bar, 100 m. (C)

Reactive oxygen species generation of VPA-treated and non-treated NPC-Control and

NPC-SZP. Bars represent mean ± s.e.m. (n=3,* p<0.05, ** p<0.001, with respect to

NPC-Control or as indicated by black lines).



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