Valproate reverts zinc and potassium imbalance in schizophrenia-derived reprogrammed cells

https://doi.org/10.1016/j.schres.2014.02.007Get rights and content

Abstract

Schizophrenia has been considered a devastating clinical syndrome rather than a single disease. Nevertheless, the mechanisms behind the onset of schizophrenia have been only partially elucidated. Several studies propose that levels of trace elements are abnormal in schizophrenia; however, conflicting data generated from different biological sources prevent conclusions being drawn. In this work, we used synchrotron radiation X-ray microfluorescence spectroscopy to compare trace element levels in neural progenitor cells (NPCs) derived from two clones of induced pluripotent stem cell lines of a clozapine-resistant schizophrenic patient and two controls. Our data reveal the presence of elevated levels of potassium and zinc in schizophrenic NPCs. Neural cells treated with valproate, an adjunctive medication for schizophrenia, brought potassium and zinc content back to control levels. These results expand the understanding of atomic element imbalance related to schizophrenia and may provide novel insights for the screening of drugs to treat mental disorders.

Introduction

Schizophrenia is a devastating mental disorder characterized by symptoms such as auditory hallucinations, paranoid delusions, and social impairment (Lewis and Lieberman, 2000, Krishnan et al., 2009). Schizophrenia is a very heterogeneous syndrome whose etiology is not yet fully understood. For this reason, it has been considered a clinical syndrome rather than a single disease (Kirkpatrick et al., 2001). This explains why patients have different symptoms and why resistance to certain drugs varies among individuals.

Essential trace elements are chemical elements that play fundamental roles in cellular processes, including gene expression, but which are present in just small amounts in the organism. They include such elements as copper (Cu), manganese (Mn), selenium (Se), zinc (Zn), and iron (Fe). The idea that abnormal levels of these elements are related to schizophrenia is not recent (Blaustein and Ickowicz, 1983, Gould et al., 1983, Yanik et al., 2003, Grabrucker et al., 2011). However, this hypothesis has been raised based on studies done in non-neural tissues (blood, hair, and liquor) or postmortem neural tissues, which may not be a bona fide representation of what occurs in the brain of schizophrenic patients. Additionally, the results reported to date are inconsistent, with studies showing opposite data (Arinola and Idonije, 2009, Rahman et al., 2009).

Induced pluripotent stem cells (iPSCs) represent a novel strategy to study psychiatry disorders (Brennand et al., 2011, Chiang et al., 2011, Pedrosa et al., 2011, Paulsen et al., 2012, Robicsek et al., 2013), as they can be differentiated into all cell types, including neurons. Previously, our group showed that neural progenitor cells (NPCs) derived from schizophrenic iPSCs presented increased both extramitochondrial oxygen consumption and reactive oxygen species (ROS), which correlated with the first events associated with the onset of the disease (Paulsen et al., 2012). Treating schizophrenic NPCs with the adjunctive therapy for schizophrenia valproate (VPA) brought ROS levels back to levels similar to controls.

In this study, we investigated essential trace element composition during the early neurodevelopmental stages of iPSCs. We also asked whether VPA could affect the levels of trace elements. To this end, NPCs were examined with synchrotron radiation micro X-ray microfluorescence spectroscopy (SR-μXRF), a non-destructive technique used to investigate atomic elements on a micrometric scale (Miller et al., 2006, Cardoso et al., 2011). We described that abnormal levels of zinc and potassium in schizophrenic neural cells reverted to control levels after treatment with VPA. These findings may provide further insights on mechanisms leading to schizophrenia especially regarding unrevealed effects of adjunctive medications for this syndrome.

Section snippets

Cell reprogramming

Fibroblasts derived from biopsies were expanded and reprogrammed according to Paulsen et al. (2012). Briefly, fibroblasts were transduced using pMX-based retroviral vectors encoding Oct-4, Sox-2, Klf-4, and c-Myc each. Six days after transduction, cells were transferred to mitotically inactivated mouse embryonic fibroblasts (MEFs) and cultured in mTeSR™1 (Stem Cell Technologies, Tukwila, WA, USA). Approximately 20 days after transduction, pluripotent-like colonies were collected and inoculated

Schizophrenic neural cells have higher levels of potassium and zinc

To compare the elemental composition of iPSCs reprogrammed from a clozapine-resistant schizophrenic patient to controls (healthy donor iPSCs, referred to as iPSC-control, and a human embryonic stem cell line, referred to as ES-control), we used cells as pluripotent colonies and as neurospheres (Fig. 1A), which present NPCs expressing nestin, a neural progenitor marker (Fig. 1B). Schizophrenia-related differences were considered as remarkable when significant changes were detected in the

Discussion

In this study, we examined the levels of trace elements in neural progenitor cells derived from human embryonic stem cells, control, and schizophrenic iPSCs. To this end, we adopted a technique able to detect trace elements on a micrometric scale. Analysis of the atomic content of NPCs revealed that potassium and zinc were increased in neural progenitor cells of the schizophrenic clones (Fig. 2).

To our knowledge, no studies have analyzed the levels of potassium in samples of schizophrenic

Role of funding source

This work was supported by Laboratório Nacional de Luz Síncrotron (LNLS), Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Banco Nacional do Desenvolvimento (BNDES), and Agência Brasileira da Inovação (Financiadora de Estudos e Projetos—FINEP).

Contributors

BSP, MPS, and DVC wrote the main manuscript text. BSP and MPS prepared the figures. BSP and MPS were responsible for cell culture and differentiation assays. MPS and SCC were responsible for XRF analyses. SCC and SKR conceived the study and supervised the experiment design. All authors reviewed the manuscript. Co-authors BSP, SCC, and MPS contributed equally to this work.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by Laboratório Nacional de Luz Síncrotron (LNLS), Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Banco Nacional do Desenvolvimento (BNDES), and Agência Brasileira da Inovação (Financiadora de Estudos e Projetos—FINEP). This manuscript was reviewed by a professional science editor and by a

References (60)

  • U. Meyer

    Developmental neuroinflammation and schizophrenia

    Prog. Neuropsychopharmacol. Biol. Psychiatry

    (2013)
  • L.M. Miller et al.

    Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease

    J. Struct. Biol.

    (2006)
  • C.J. Phiel et al.

    Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen

    J. Biol. Chem.

    (2001)
  • M. Raffa et al.

    Decreased glutathione levels and antioxidant enzyme activities in untreated and treated schizophrenic patients

    Prog. Neuropsychopharmacol. Biol. Psychiatry

    (2009)
  • M. Raffa et al.

    Reduced antioxidant defense systems in schizophrenia and bipolar I disorder

    Prog. Neuropsychopharmacol. Biol. Psychiatry

    (2012)
  • M. Raffa et al.

    The reduction of superoxide dismutase activity is associated with the severity of neurological soft signs in patients with schizophrenia

    Prog. Neuropsychopharmacol. Biol. Psychiatry

    (2012)
  • S. Suda et al.

    Valproic acid attenuates ischemia-reperfusion injury in the rat brain through inhibition of oxidative stress and inflammation

    Eur. J. Pharmacol.

    (2013)
  • T. Suzuki et al.

    Effects of long-term anticonvulsant therapy on copper, zinc, and magnesium in hair and serum of epileptics

    Biol. Psychiatry

    (1992)
  • K. Tada et al.

    Trace elements in the hair of schizophrenics

    Biol. Psychiatry

    (1986)
  • Z. Vukadinovic et al.

    Abnormalities in thalamic neurophysiology in schizophrenia: could psychosis be a result of potassium channel dysfunction?

    Neurosci. Biobehav. Rev.

    (2012)
  • Y. Yilmaz et al.

    The influence of valproic acid treatment on hair and serum zinc levels and serum biotinidase activity

    Eur. J. Paediatr. Neurol. EJPN Off. J. Eur. Paediatr. Neurol. Soc.

    (2009)
  • X.Y. Zhang et al.

    Effects of risperidone and haloperidol on superoxide dismutase and nitric oxide in schizophrenia

    Neuropharmacology

    (2012)
  • O. Arinola et al.

    Status of plasma nitric oxide and non-enzymatic antioxidants before and after antipsychotic treatment in Nigerian patients with schizophrenia

    J. Res. Med. Sci. Off. J. Isfahan Univ. Med. Sci.

    (2009)
  • H. Baharvand et al.

    Neural differentiation from human embryonic stem cells in a defined adherent culture condition

    Int. J. Dev. Biol.

    (2007)
  • M.P. Blaustein et al.

    Phencyclidine in nanomolar concentrations binds to synaptosomes and blocks certain potassium channels

    Proc. Natl. Acad. Sci. U. S. A.

    (1983)
  • K.J. Brennand et al.

    Modelling schizophrenia using human induced pluripotent stem cells

    Nature

    (2011)
  • F. Calabrese et al.

    Modulation of neuronal plasticity following chronic concomitant administration of the novel antipsychotic lurasidone with the mood stabilizer valproic acid

    Psychopharmacology (Berl)

    (2013)
  • S.C. Cardoso et al.

    Synchrotron radiation X-ray microfluorescence reveals polarized distribution of atomic elements during differentiation of pluripotent stem cells

    PLoS ONE

    (2011)
  • G. Chen et al.

    The mood-stabilizing agent valproate inhibits the activity of glycogen synthase kinase-3

    J. Neurochem.

    (1999)
  • C.-H. Chiang et al.

    Integration-free induced pluripotent stem cells derived from schizophrenia patients with a DISC1 mutation

    Mol. Psychiatry

    (2011)
  • Cited by (38)

    • Non-enzymatic antioxidants, macro-minerals and monocyte/high-density lipoprotein cholesterol ratio among patients with bipolar disorder

      2023, Journal of Affective Disorders
      Citation Excerpt :

      However, other studies have shown that no differences were found between clinical groups during different phases of BD and healthy controls concerning albumin and bilirubin (De Berardis et al., 2008). Macro-minerals, such as Sodium (Na), Potassium (K), Calcium (Ca), levels of which can be obtained quickly in routine blood tests, are required to maintain a proper healthy life and play a vital role in the human immune system and antioxidant mechanisms (Paulsen Bda et al., 2014). Chowdhury's study (Chowdhury et al., 2017) showed significantly lower levels of Na, K and Ca in the BD group when compared with the control group.

    • Applying stem cells and CRISPR engineering to uncover the etiology of schizophrenia

      2021, Current Opinion in Neurobiology
      Citation Excerpt :

      That being said, decreased levels of the synaptic protein PSD95 found in the first hiPSC-based study of schizophrenia were subsequently replicated using hiPSC neurons from independent cases and controls [12,28]; moreover, decreased synaptic puncta density has been reported in idiopathic schizophrenia hiPSC lines and those from patients with mutations in the schizophrenia-linked gene DISC1 [28,36,38,39]. Increased oxidative stress was also broadly indicated across multiple studies [26,28,37]. Nonetheless, the large genetic variation between individuals means that case and control cohort designs are underpowered to detect subtle phenotypes such as genome-wide differential gene expression and the molecular mechanisms underpinning cellular phenotypes [25].

    • Contribution of induced pluripotent stem cell technologies to the understanding of cellular phenotypes in schizophrenia

      2019, Neurobiology of Disease
      Citation Excerpt :

      Paulsen et al. observed a two-fold increase in extramitochondrial oxygen consumption and elevated levels of reactive oxygen species (ROS) in NPCs differentiated from hiPSCs established from a clozapine resistant schizophrenia patient (Paulsen Bda et al., 2012). This was more pronounced in NPCs than in iPSCs or fibroblasts, and was rescued by treatment with valproic acid, suggestive of mitochondrial deficits associated with aberrant neurogenesis (Paulsen Bda et al., 2014). Another group used hair follicle keratinocytes to establish iPSCs from patients with schizophrenia, and further differentiated the cells into dopaminergic and glutamatergic neuronal cells (Robicsek et al., 2013).

    • Stem cell models of schizophrenia, what have we learned and what is the potential?

      2019, Schizophrenia Research
      Citation Excerpt :

      Development of new treatments for SCZ would be a large step forward considering the limited options there are today. iPSC models of SCZ have successfully been used to evaluate the effect of previously known treatments like valproate that bring potassium and zinc content back to normal in patient NPC (Paulsen Bda et al., 2014), but since one third of the SCZ patients do not respond to available antipsychotic treatments (Hasan et al., 2012), justifies adding resources into screening for novel drug candidates. Recently, an expression-based study investigated SCZ-linked transcriptional responses to 135 small molecules when introduced to iPSC-derived NPCs from several individuals with SCZ and healthy controls (Readhead et al., 2018).

    View all citing articles on Scopus
    1

    Authors with equal contributions.

    2

    Present address: Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Avenida Carlos Chagas, 373, bloco H, Rio de Janeiro 21941, Brazil.

    View full text