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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 postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial 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.

Keywords

Schizophrenia Oxygen metabolism Induced pluripotent stem cells Reactive oxygen species (ROS) production

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 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 nonmitochondrial 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 nonmitochondrial 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 postmortem 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 nonneural 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 level 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 extramitochondrial 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.

Materials 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 were 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 the 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 2 × 2-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 TrypLE™ 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 colony clumps. To form embryoid bodies (EBs), confluent undifferentiated iPS cells were treated with accutase (Millipore), scrapped out, and transferred to 60-mm nonadherent dishes in DMEM/F12 medium supplemented by 15% of knockout serum replacement (KSR; Invitrogen), 1% nonessential amino acid solution (Invitrogen), 2 mM l-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 40 μg/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 μg/ml polybrene. Twenty-four hours after transduction, virus-containing medium was replaced by the second round of supernatant. Two days after transduction, 50 μg/ml of l-ascorbic acid (USB Corporation) was added to cultures, as described by Esteban and coworkers (17). Six days after transduction cells were replated (5 × 10⁴ cells per six-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 7 days. About 20 days after transduction, colonies were picked up and transferred to six-well plates coated with Matrigel in mTeSR™1. 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-free™ Kit (Applied Biosystems), following the manufacturer's instructions. Complementary DNA was generated from 1 μg total RNA using High Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer's recommendations. Reverse transcription (RT)-PCR was performed using specific primer sequences (Table S1; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html). Each RT-PCR reaction was carried out for 35 cycles in a reaction mixture containing 1 U Taq DNA Polymerase (NB Science), 1x Taq DNA Polymerase Buffer containing 25 mM MgCl₂ (NB Science), 500 nM of each primer (forward and reverse), 200 μM dNTP mixture containing the four deoxyribonucleotides (dATP, dCTP, DTTP, dGTP), and 1 μl 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), βIII-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 min. After observing routine respiration, ATP synthase was inhibited by oligomycin (2 μg/ 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 μg/ml) and/or KCN (0.5 mM) and the oxygen flow rate detected in this condition was defined as extramitochondrial 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 min 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 2–5 MΩ in the bath; the access resistance was left uncompensated. Approximately 2 min after achieving the whole-cell configuration, voltage-sensitive sodium currents were evoked by 50-ms depolarizing square pulses ranging from −40 to +10 mV from a holding potential of −70 mV. Leak currents were subtracted by a fractional method (P/N) using four scaled hyperpolarizing sub-pulses. Immediately after this pulse, 3-min 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; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html) 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 refer to SZP-C11, while data from SZP-C2 can be found in 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. 1E, F).

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 layers. (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 μm. 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, α-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.

Since teratoma formation is not required for iPS cells in 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. 1D, G, H). Moreover, iPS cells were able to differentiate into spontaneously beating cells in vitro (data not shown) or, when cocultured 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; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html).

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; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html) were noticed among 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; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html). 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; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html), 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, β (LMX1B) (serotonergic neurons) were evidenced by RT-PCR analysis (Fig. 2C; Fig. S1H; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html). Furthermore, after more than 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 among the experimental groups.

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 βIII-tubulin and NeuN-positive neural cells after 18 days of differentiation. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (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, β.

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 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.

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 extramitochondrial 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; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html). 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. 4A–C). 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 among 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 downregulated exclusively in NPC-control cells (Fig. 4C, black bars), while in NPC-SZP they remained similar to those observed for iPS cells (Fig. 4C, gray bars).

Figure 4.

Neural precursor cells (NPCs) from the schizophrenic patient consume more oxygen than control. (A–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) Extramitochondrial oxygen consumption. Bars represent mean ± SEM (n = 3, *p < 0.01, **p < 0.001, with respect to NPC-control). FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

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 extramitochondrial (Fig. 4B, C, E; Fig. S4A, C; supplemental material available from http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html). 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 extramitochondrial O₂ consumption, while NPC-SZP shows twofold 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) extramitochondrial O₂ consumption (Fig. 5A) and (ii) ROS levels (Fig. 5B, C). After VPA treatment, both NPC-control and NPC-SZP presented a two- to threefold increase in extramitochondrial 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 twofold higher in NPC-SZP than NPC-control, in accordance to the extramitochondrial O₂ consumption pattern. However, although VPA treatment enhanced extramitochondrial 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).

Figure 5.

NPC-SZP generates more reactive oxygen species (ROS) than NPC-control and valproic acid (VPA) treatment reverts this condition. (A) Extramitochondrial respiration on VPA-treated and nontreated 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 μm. (C) Reactive oxygen species generation of VPA-treated and nontreated NPC-control and NPC-SZP. Bars represent mean ± SEM (n = 3, *p < 0.05, **p < 0.001, with respect to NPC-control or as indicated by black lines).

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, which 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 postmortem 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, postsynaptic 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 among neurons from SZP without the support of exogenous glial cells or cocultured 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 extramitochondrial 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 extramitochondrial 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 available at http://www.lance-ufrj.org/supplemental-data-schizophrenia-2011-cell-transplantation-9182736455.html).

One possibility for the total extramitochondrial 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 twofold 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 γ-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.

Footnotes

Acknowledgments

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, and 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. The authors declare no conflicts of interest.

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Altered Oxygen Metabolism Associated to Neurogenesis of Induced Pluripotent Stem Cells Derived from a Schizophrenic Patient

Abstract

Keywords

Introduction

Materials and Methods

Schizophrenic Patient

Derivation of Primary Human Fibroblast from Schizophrenic Patient

Cell Culture

iPS Cell Generation

RNA Isolation and PCR Analysis

Neural Differentiation of iPS Cells

High-Resolution Respirometry

Reactive Oxygen Species (ROS) Production

Electrophysiology

Statistical Analyses

Results

Characterization of iPS Cells Generated from Fibroblasts of a Patient with Schizophrenia

Direct Differentiation of iPS Cells Into Neural Precursor Cells

Changes in Endogenous Oxygen Consumption Are Only Observed in SZP Neural Precursor Cells But Not in Fibroblasts or iPS Cells

NPCs from Schizophrenic Patient Present Higher ROS Levels, Which Is Reverted by Valproic Acid

Discussion

Footnotes

Acknowledgments

References