ICOSR 2009—Studying Cultured Cells From Patients With Schizophrenia
As part of our ongoing coverage of the 2009 International Congress on Schizophrenia Research (ICOSR), 28 March to 1 April 2009, in San Diego, California, we bring you session summaries from some of the Young Investigator travel award winners. On the morning of March 30, the congress featured a symposium showcase of molecular and functional studies of cultured cells from patients with schizophrenia, including peripheral lymphocytes, fibroblasts, olfactory epithelial neurons, and pluripotent stem cells. We are grateful for this summary by Vibeke S. Catts of the University of Queensland, Australia, who in fact was a travel awardee at the 2007 meeting and was generous enough to file a report this year as well!
14 April 2009. In opening the symposium, Akira Sawa of Johns Hopkins University proposed an ideal research paradigm in which multiple cell types are collected from all patients for molecular profiles and drug screening experiments. The field of schizophrenia research has mainly utilized lymphoblasts and fibroblasts, which are easily obtained but may not reflect the neuronal disease phenotype. Olfactory epithelial (OE)-derived neurons address this limitation to some extent; however, not all neuronal cell types are represented in this culture system. More recently, induced pluripotent stem (iPS) cells have been established from which any cell type can be obtained, though at present it remains an expensive and time-consuming process to establish each cell line. Sawa stated that while autopsied brain tissue is useful for schizophrenia research, it cannot be used for functional assays and is limited by the confounding effects of postmortem delay, medication, substance use, and smoking. End-stage disease tissue may not necessarily reflect neurodevelopmental pathological processes.
Sawa presented a study on susceptibility to metabolic and oxidative stress in lymphoblasts from patients with schizophrenia and matched controls. Small increases in NAD+/NADH (an index of an oxidative environment in cells) and reactive oxygen species were observed in lymphoblasts from patients with schizophrenia under normal culture conditions. These differences were much greater when the glucose concentration of the culture medium was increased fivefold, highlighting the value of functional assays. Linking these results with findings of interneuron deficits in postmortem tissue from patients with schizophrenia, Sawa and colleagues developed the hypothesis that perturbation of the oxidative stress cascade may interfere with interneuron maturation, contributing to the neuropathology of schizophrenia. This hypothesis is consistent with the therapeutic effects of NADPH oxidase inhibitors and N-acetyl-L-cysteine (NAC) in the treatment of patients.
Informed by the observations in patient-derived cells and tissue, Sawa asked whether oxidative stress conditions play a role in animal models of schizophrenia. Modelling an interaction between genetic and environmental risk factors, Sawa and colleagues investigated the dominant-negative DISC1 (disrupted in schizophrenia-1) transgenic mouse model (Hikida et al., 2007) in the absence and presence of neonatal poly I:C treatment (a proxy for neonatal infection). They found that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Siah signalling cascade, an oxidative stress-elicited signalling pathway (Hara et al., 2005; Sen et al., 2008), was perturbed in the transgenic and poly I:C-treated mice. Sawa reasoned that if the GAPDH-Siah cascade is abnormal in patients with schizophrenia, then deprenyl derivatives that block the GAPDH-Siah cascade but lack monoamine oxidase inhibition might be an effective therapeutic for schizophrenia.
The next talk, by Nicola Cascella of Johns Hopkins University, focused on the utility of OE neurons for schizophrenia research. Cascella referred to findings on smell identification deficits in patients with schizophrenia, which studies have been found to be associated with negative symptoms (especially anhedonia, affective flattening, and apathy) and lower IQ. As olfactory deficits are reportedly present in the earliest stages of the disease, they may be related to a genetic vulnerability evident in OE tissue. Molecular profiling of OE samples in fixed and non-fixed biopsies was compared. It was noted that RNA extraction of fixed tissue regularly resulted in loss of samples due to quality assurance criteria, and that non-fixed tissue was better suited for microarray analysis. To enrich the samples for cell types of interest, Cascella and colleagues microdissected out the neuronal layer of the nasal biopsy using laser capture techniques prior to RNA extraction. Using these techniques, the researchers observed negative correlations between expression of RNA binding and ribosomal proteins, and olfactory function. Cascella said that one limitation of OE neuron biopsies is that dopamine D2 receptors appear not to be expressed in these tissues, though dopamine D4, AMPA, NMDA, and insulin receptors are.
Cascella presented a cluster analysis of expression profiles in biopsy samples of OE, cultured OE, and lymphoblasts from the same individuals. The analysis clustered the cultured OE cell expression profiles as intermediate between those of the OE biopsies and lymphoblasts.
The third talk of the symposium, given by Guo-li Ming of Johns Hopkins University, focused on the potential of pluripotent human stem cells in understanding the pathogenic mechanisms of schizophrenia. It is now well known that there is continuous generation and integration of new neurons in discrete regions of adult mammalian brain (Ge et al., 2008). Using a retroviral method to label and genetically modify dividing progenitor cells in adult mice, Ming and colleagues tracked the formation and integration of newly generated neurons with DISC1 knockdown in adult mice. Their experiments showed that DISC1 regulates morphogenesis and positioning of adult-born neurons. A knockdown of DISC1 in these cells also results in migration of newly divided neurons beyond the granule cell layer into the molecular layer of the hippocampal formation (Duan et al., 2007). The DISC1 knockdown also results in an initial increase in the formation of synaptic outputs of newly differentiated neurons; however, these synapses fail to mature and are lost eight weeks following cell division (Faulkner et al., 2008).
Ming and colleagues described exciting new methodologies they have developed to culture human embryonic stem cells (hESCs) and examine their neuronal development both in vitro and in vivo after in utero transplantation into E13.5 mice. Using recently introduced technology to reprogram human skin fibroblasts into induced pluripotent stem cells (iPSCs) comparable to embryonic stem cells, it is now possible to study the effects of human-specific genes that relate to schizophrenia. Ming described how their group had investigated axonal and dendritic differentiation and synapse formation in neurosphere-derived neurons as well as the development of human neural cells in animals after in utero transplantation of GFP-labelled hESC into embryonic mouse brain. The group is currently extending their research to patient samples.
Completing the symposium, Chang-Gyu Hahn of University of Pennsylvania described functional studies using both ex vivo sections and in vitro cultures of OE cells (Hahn et al., 2005). Hahn illustrated a few ways to utilize OE tissues and cultures for pathophysiologic study of schizophrenia. First, human OE cultures, particularly organotypic cultures, can be utilized to recapitulate some neurodevelopmental dysregulations as postulated in schizophrenia. Second, Hahn showed robust presence of neurotransmitter receptors and their functionality in human OE cultures (Borgmann-Winter et al., 2009), which can be used to study receptor signalling associated with schizophrenia. Indeed, Hahn showed data indicating altered NMDA-induced calcium accumulation in human OE cultures from patients with schizophrenia, as well as altered tyrosine phosphorylation of NR2A and NR2B subunits.
Hahn alerted the audience to a number of potential confounds affecting OE studies. For example with ex vivo sections, the consistency of biopsy location is an important variable to control for, and with in vitro cultures the passage number may confound, as the proportion of glial cells increases with culture age. Such variability should be taken into consideration for study designs to capture disease-specific molecular or cellular characteristics.
In summary, the symposium demonstrated the utility of OE biopsies and peripheral cell cultures in the study of schizophrenia, with a particular focus on functional assays not possible in postmortem brain tissue (Sawa and Cascella, 2009). The talk by Guo-li Ming highlighted the exciting future possibilities of using human OE-derived cells or iPSCs in in vivo murine brain assays. This area of research promises to be an interesting and fruitful field to follow.—Vibeke S. Catts.