RNA-Seq of Human Neurons Derived from iPS Cells Reveals Candidate Long Non-Coding RNAs Involved in Neurogenesis and Neuropsychiatric Disorders

et al. (2011) RNA-Seq of Human Neurons Derived from iPS Cells Reveals Candidate Long Non-Coding RNAs Involved in Neurogenesis and Neuropsychiatric Disorders. PLoS ONE 6(9): e23356. doi:10.1371/journal.pone.0023356 RNA-Seq of Human Neurons Derived from iPS Cells Reveals Candidate Long Non-Coding RNAs Involved in Neurogenesis and Neuropsychiatric Disorders Mingyan Lin 0 Erika Pedrosa 0 Abhishek Shah 0 Anastasia Hrabovsky 0 Shahina Maqbool 0 Deyou 0 Zheng 0 Herbert M. Lachman 0 Takeo Yoshikawa, Rikagaku Kenkyu sho Brain Science Institute, Japan 0 1 Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Dominick Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, United States of America, 4 Department of Neurology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 5 Department of Medicine, Albert Einstein College of Medicine , Bronx, New York , United States of America Genome-wide expression analysis using next generation sequencing (RNA-Seq) provides an opportunity for in-depth molecular profiling of fundamental biological processes, such as cellular differentiation and malignant transformation. Differentiating human neurons derived from induced pluripotent stem cells (iPSCs) provide an ideal system for RNA-Seq since defective neurogenesis caused by abnormalities in transcription factors, DNA methylation, and chromatin modifiers lie at the heart of some neuropsychiatric disorders. As a preliminary step towards applying next generation sequencing using neurons derived from patient-specific iPSCs, we have carried out an RNA-Seq analysis on control human neurons. Dramatic changes in the expression of coding genes, long non-coding RNAs (lncRNAs), pseudogenes, and splice isoforms were seen during the transition from pluripotent stem cells to early differentiating neurons. A number of genes that undergo radical changes in expression during this transition include candidates for schizophrenia (SZ), bipolar disorder (BD) and autism spectrum disorders (ASD) that function as transcription factors and chromatin modifiers, such as POU3F2 and ZNF804A, and genes coding for cell adhesion proteins implicated in these conditions including NRXN1 and NLGN1. In addition, a number of novel lncRNAs were found to undergo dramatic changes in expression, one of which is HOTAIRM1, a regulator of several HOXA genes during myelopoiesis. The increase we observed in differentiating neurons suggests a role in neurogenesis as well. Finally, several lncRNAs that map near SNPs associated with SZ in genome wide association studies also increase during neuronal differentiation, suggesting that these novel transcripts may be abnormally regulated in a subgroup of patients. - Funding: Dr. Lachman is supported by grants from the National Institute of Mental Health (NIMH) (MH087840 and MH073164). Dr. Zheng is supported by a grant from NIMH(MH087840) and the National Heart, Lung and Blood Institute (NHLBI) (HL104444). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Research on the biological basis of SZ and other neuropsychiatric disorders has been hampered by the inaccessibility of the human brain. However, the discovery of iPSC technology has the potential to address this problem by providing investigators with patient-specific neurons that can be used for disease modeling. In the past few years, investigators have taken advantage of this opportunity to establish iPSC lines in a variety of neuropsychiatric disorders including Rett Syndrome, Parkinson Disease, Amyotrophic Lateral Sclerosis, Familial Dysautonomia, and most recently, SZ [112]. In the study reported by Brennand et al. neurons derived from SZ-specific iPSCs showed diminished neuronal connectivity, reduced PSD95-protein levels and altered expression of WNT signaling pathways [1]. Similarly, we have also been developing iPSC lines from patients with SZ, a data set that includes patients with 22q11.2 deletions (in press). In addition to their utility for disease modeling in neuropsychiatric problems, iPSCs can also be used to study early differentiating human neurons in vitro to gain insight into neurogenesis, which is particularly relevant to both SZ and ASD considering that both appear to have a neurodevelopmental basis [1316]. With these aspects of disease pathogenesis in mind, we have analyzed the transcriptome of human neurons derived from iPSCs using RNA-Seq, a method that provides increased sensitivity with the capacity to detect low-copy transcripts, novel transcripts, lncRNAs, and splice isoforms [1720]. The key role played by cell type-specific splicing in neuronal differentiation, particularly in genes coding for cell adhesion proteins, and the growing recognition that lncRNAs play a role neurogenesis lend further support for the value of deep sequencing transcriptome analysis [2123]. Finally, a global, unbiased transcriptome analysis could help determine the biological significance of SNP markers associated with neuropsychiatric disorders identified in GWAS carried out in SZ, ASD, BD, many of which map to intergenic regions or deep within large introns where important regulatory lncRNAs may be found [2426]. Our findings show that early differentiating neurons undergo an extraordinary array of quantitative changes in gene expression and in splice isoform generation, similar to that reported in differentiating human neurons derived from human embryonic stem cells (hESCs) [27]. In addition, we describe dramatic changes in expression of lncRNA genes during differentiation, one of which is HOTAIRM1, a cis-acting regulator of the HOXA cluster during myelopoiesis [28]. Contrary to previous reports suggesting that HOTAIRM1 is not expressed in brain, a 54.6-fold increase in transcripts was detected in differentiating neurons, suggesting a novel role in neural differentiation. In addition, some GWAS SNPs found in SZ, BD and ASD mapped to lncRNA genes that are expressed in differentiating neurons, suggesting a role for these novel regulators in a subgroup of patients. This study was approved by the Internal Review Board of the Albert Einstein College of Medicine (protocol number 1996-013). Informed consent was obtained and the data were analyzed anonymously in accordance with the Declaration of Helsinki. iPSCs and neural differentiation Details regarding the generation of iPSCs and the neuronal differentiation protocol used are described in Text S1. RNA-Seq Total RNA was isolated from cells using the miRNeasy Kit (Qiagen) according to the manufacturers protocol. An additional DNase1 digestion step was performed to ensure that the (...truncated)