April 14, 2014. Neural progenitor cells (NPCs) derived from the skin cells of patients with schizophrenia show abnormal neuronal migration and elevated levels of oxidative stress—alterations that are suspected in the illness—reports a new study published online April 1 in Molecular Psychiatry. Led by Kristen Brennand and Fred Gage of the Salk Institute in La Jolla, California, the report also demonstrates that using NPCs to model aspects of schizophrenia is a viable alternative to their more high-maintenance neuronal descendants that have garnered so much attention.
The ground-breaking 2006 report from Shinya Yamanaka demonstrating that human and mouse fibroblasts could be reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated into any desired cell type, including neurons, marked a new way to study the neurobiology of brain diseases and test potential therapeutics. However, despite substantial efforts, differentiating iPSCs into neurons has proved to be more challenging than expected (see SRF related conference report).
The technique first came to schizophrenia in 2011, when Gage and Brennand, then a postdoc in Gage’s lab, reported that iPSC-derived neurons from four schizophrenia patients exhibited reduced connectivity and neurite number, as well as many gene expression changes (see SRF related news report; SRF related conference report). In addition, treatment with the antipsychotic loxapine reversed many of these deficits.
The use of iPSC-derived neurons as a model of schizophrenia has several caveats. Within a single culture, not all neurons have the same fate or age, and consequently they form artificial networks that are both immature and lack myelination. In addition, coaxing iPSCs into pyramidal cells and interneurons is a very labor-intensive process that takes several months. But the biggest caveat is that just how these in vitro phenotypes will relate to the human disease process is currently unknown.
In the current study, Brennand (now at Mt. Sinai School of Medicine, New York City) and Gage focused on the same patients’ neural precursor cells (NPCs)—the intermediaries between iPSCs and neurons. NPCs aren’t neurons, of course, and therefore don’t form synapses, but they can be used to assay a cellular phenotype distinct from that of iPSCs. They are much easier to grow than neurons—they don’t require months in culture to mature—and in the new report the researchers show that NPCs derived from schizophrenia patients have a gene expression signature that overlaps significantly with that of the iPSC-derived neurons from the earlier study.
By comparing the microarray gene expression profiles of the NPCs and neurons to that of human tissue from the Allen BrainSpan Atlas, the researchers found that the gene expression profiles of both the iPSC-derived NPCs and neurons most closely matched tissue from a third trimester fetus. “That’s why we’re careful to say we’re modeling the predisposition to [the illness], and not schizophrenia per se,” said Brennand at an April 8 presentation of the new data at the 4th Schizophrenia International Research Society Conference in Florence, Italy.
A total of 481 genes had aberrant expression levels in the NPCs from patients compared to controls. Using weighted gene co-expression network analysis, the researchers found five modules of co-expressed genes from within the 481: neuron differentiation and synaptic transmission (the one with the most differentially expressed genes), glutamate receptor signaling, insulin signaling, neuronal migration, and synaptic vesicle function. Gene ontology analyses suggested that the abnormally expressed genes were involved in synapse formation, synaptic transmission, and cell adhesion. A quantitative protein mass spectroscopy analysis was not able to replicate the synaptic gene expression changes; however, it did find altered levels of many cytoplasmic cytoskeleton proteins (such as profilins and cofilins) as well as those involved in oxidative stress (such as thioredoxin).
These data prompted Brennand and colleagues to look for perturbed neuronal migration as well as evidence of elevated oxidative stress in the patient-derived NPCs. Using three different assays—neurosphere migration, microfluidic device migration, and laminin spot chaining—the researchers observed reduced migration in the patient-derived NPCs. In her talk, Brennand emphasized that the results suggest aberrant migration rather than decreased migration, because a fourth assay actually revealed increased migration in the patient-derived NPCs. The findings also point to cell adhesion differences rather than motility deficits in the cells.
To assay oxidative stress, Brennand and colleagues used a dye called JC1 that changes from red to green fluorescence as the potential across the inner mitochondrial membrane decreases, an indicator of oxidative stress. The schizophrenia NPCs showed significantly more oxidative stress than control-derived NPCs. Two other groups have also reported evidence of increased oxidative stress in schizophrenia patient-derived NPCs (Paulsen et al., 2012; Robicsek et al., 2013).
“While iPS is still a very new field for studying schizophrenia,” said Brennand, “three groups in three different countries have all now shown one common phenotype: oxidative stress,” a process that is gaining increasing attention in schizophrenia (see SRF related news report; SRF related conference report).
Fellow symposium speaker Akira Sawa of Johns Hopkins University in Baltimore, Maryland, commented that the oxidative stress findings are a “really important confirmation” of the multitude of animal studies implicating the process in the illness that have not yet been extended to humans (Emiliani et al., 2014).
Although NPCs are certainly not a perfect model of the intact schizophrenia brain, they still have a lot to offer. “The goal is not to replace clinical- or animal-based models,” remarked Brennand. “It’s to see if we can potentially add on [to other research] with this new model.”—Allison A. Curley.
Brennand K, Savas JN, Kim Y, Tran N, Simone A, Hashimoto-Torii K, Beaumont KG, Kim HJ, Topol A, Ladran I, Abdelrahim M, Matikainen-Ankney B, Chao SH, Mrksich M, Rakic P, Fang G, Zhang B, Yates JR, Gage FH. Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia. Mol Psychiatry . 2014 Apr 1. Abstract