Valproate reverts zinc and potassium imbalance in schizophrenia-derived reprogrammed cells
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
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Authors with equal contributions.
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Present address: Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Avenida Carlos Chagas, 373, bloco H, Rio de Janeiro 21941, Brazil.