An important obstacle in understanding the molecular and physiological basis of neuropsychiatric disorders is the inaccessibility of the human brain. Investigators have had to resort to autopsy specimens and peripheral blood leukocytes to carry out such studies. Studying blood leukocytes is extremely limited in terms of extrapolating findings to abnormalities in the human brain. And, although a number of interesting findings have emerged in studying gene expression profiling in autopsy samples obtained from subjects with schizophrenia (SZ), there are numerous confounding factors that could affect data interpretation including co- morbid nicotine, alcohol and drug abuse, and chronic use of psychotropic medications. In addition, studying postmortem samples in neuropsychiatric disorders, such as SZ and autism spectrum disorders (ASD) that are both viewed as neurodevelopmental in origin, is limiting. One exciting technological advance we and others are using that could address these limitations is induced pluripotent stem cells (iPSCs). In an ongoing NIHM funded study, we have been able to cultivate human glutamatergic neurons from iPSCs using fibroblasts derived from patients with SZ and controls. That study focuses on analyzing mRNA expression profiles and carrying out electrophysiolgical studies. This current proposal is designed to capitalize on this unique biological resource for microRNA (miRNA) profiling using next generation sequencing technology (miRNA-Seq). MicroRNAs play a key role in brain development and synaptogenesis, and have been postulated to be involved in SZ pathophysiology. One of the most compelling arguments favoring a role for miRNAs in SZ comes from an analysis of velocardiofacial syndrome (VCFS), which is caused by a 22q11 microdeletion. Approximately 1/3 of patients with VCFS suffer from SZ. Although the genes responsible for the psychiatric manifestations have not been unequivocally identified, a promising candidate is DGCR8, which codes for a nuclear protein involved in miRNA biogenesis. DGCR8 combines with Drosha to form the so-called "microprocessor," which produces miRNAs from long primary miRNAs. An analysis of knockout mice by another investigator shows that hemizygosity for Dgcr8 affects behavior, alters dendritic complexity, and influences the expression of several miRNAs in the hippocampus and cortex. Since our original iPSC study includes the cultivation of iPSCs from patients with SZ with and without 22q11 deletions, as well as controls, we have a unique biological resource to expand on the mouse knockout studies. An expected finding is that RNA-Seq analysis in the neurons cultivated from iPSCs with the 22q11 deletion will show altered expression of a number of miRNAs, similar to mouse knockout model. Indeed, considering the increased complexity of gene expression in the human brain compared with lower animals, we expect that the number of miRNAs (and their targeted mRNAs) will be higher in human neurons than in the mouse model. We are especially interested in determining whether their expression is also altered in patients with SZ who do not have 22q11.2 deletions. This would point to common molecular pathways underlying disease pathophysiology, an important consideration for a genetically heterogeneous disorder like SZ. Such common pathways would be ideal targets for medication development that could be effective in a large subgroup of patients.
This proposal describes experiments that will help understand how a class of gene regulators called microRNAs could be involved in the development of schizophrenia. MicroRNAs are involved in regulating genes responsible for the growth and function of neurons (brain cells). Experiments will be carried out using a novel experimental cell system known as induced pluripotent stem cells (iPSCs), which have the capacity to be turned into (differentiate) into any other cell type, including neurons. We have generated several iPSC lines from patients with schizophrenia, as well as control subjects, and are now able to grow their neurons in our lab. These will be used for the microRNA analysis. The goal is to find miRNAs that are abnormally regulated in schizophrenia, which will present investigators with targets for the development of novel treatment strategies.