Abstract
Direct conversion technique to produce induced-neuronal (iN) cells from human fibroblasts within 2 weeks is expected to discover unknown neuronal phenotypes of neuropsychiatric disorders. Here, we present unique gene expression profiles in iN cells from patients with neurofibromatosis type 1 (NF1), a single-gene multifaceted disorder with comparatively high co-occurrence of autism spectrum disorder (ASD). Microarray-based transcriptomic analysis on iN cells from male healthy controls and male NF1 patients (NF1-iN cells) revealed that 149 genes expressions were significantly different (110 upregulated and 39 downregulated). We validated that mRNA of MEX3D (mex-3 RNA binding family member D) was lower in NF1-iN cells by real-time PCR with 12 sex-mixed samples. In NF1-iN cells on day 14, higher expression of FOS mRNA was observed with lower expression of MEX3D mRNA. Interestingly, BCL2 mRNA was higher in NF1-iN cells on day 5 (early-period) but not on day 14. Our data suggest that aberrant molecular signals due to NF1 mutations may disturb gene expressions, a subset of which defines continuum of the neuronal phenotypes of NF1 with ASD. Further translational studies using induced pluripotent stem (iPS) cell-derived neuronal cells are needed to validate our preliminary findings especially confirming meanings of analysis using early-period iN cells.
Highlights
Neurofibromatosis type 1 (NF1: known as von Recklinghausen disease) is a multifaceted disease, which shows a variety of physical symptoms including multiple café-au-lait spots, Lisch nodules, neurofibromas, scoliosis, and vision disorder[1,2,3]
Total RNA was extracted from mixed culture dishes including these neuronal cells, and gene expression was quantified by real-time PCR (LightCycler 480 real-time PCR system: Roche Diagnostics, Mannheim, Germany)
Knockdown of Mex3d did not change the mRNA expression level of Nf1 in Neuro2A cells (Supplementary Fig. 4D), and knockdown of Nf1 did not change the mRNA expression level of Mex3d in Neuro2A cells (Supplementary Fig. 4E,F). These results suggest that the lower expression level of MEX3D mRNA found in NF1-iN cells is not validated in a mouse neuronal cell line, Neuro2A, suggesting the importance of analyzing human cells in disease models
Summary
Neurofibromatosis type 1 (NF1: known as von Recklinghausen disease) is a multifaceted disease, which shows a variety of physical symptoms including multiple café-au-lait spots, Lisch nodules, neurofibromas, scoliosis, and vision disorder[1,2,3]. Twenty to thirty percent of NF1 patients are known to have autism spectrum disorder (ASD)[9,10,11]. These clinical reports have suggested some neurodevelopmental pathophysiology in the brains of NF1 patients. Some studies have shown that neurofibromin 1 regulates Ras-GTPase and adenylyl cyclases (ACs) in various cell types[14]. No study has shown whether such dysfunctions exist in human living neuronal cells of NF1 patients, due to the difficulty of directly analyzing human brain cells, including neuronal cells. The main purpose of the present pilot study is to clarify dysregulated gene expressions using comprehensive microarray-based transcriptomic analysis of iN cells from NF1 patients, especially via ACs in the presence or absence of forskolin, a typical ACs activator
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