Direct conversion techniques, which generate induced-neuronal (iN) cells from human fibroblasts in less than two weeks, are expected to discover unknown neuronal phenotypes of neuropsychiatric disorders. Here, we present unique gene expression and cell morphology profiles in iN cells derived from neurofibromatosis type 1 (NF1) patients. NF1 is a single-gene multifaceted disorder with relatively high co-occurrence of autism spectrum disorder (ASD). Adenylyl cyclase (AC) dysfunction is one of the candidate pathways in abnormal neuronal development in the brains of NF1 patients. In our study, microarray-based transcriptomic analysis of iN cells from healthy controls (males) and NF1 patients (males) revealed significantly different gene expression of 149 (110 were upregulated and 39 were downregulated). In iN cells derived from NF1 patients (NF1-iN cells), there was a change in the expression level of 90 genes with the addition of forskolin, an AC activator. Furthermore, treatment with forskolin dramatically changed the cell morphology, especially that of NF1-iN cells, from flat-form to spherical-form. Current pilot data indicate the potential therapeutic effect of forskolin or AC activators on neuronal growth in NF1 patients. Further translational research is needed to validate the pilot findings for future drug development of ASD.
- adenylyl cyclases (ACs)
- autism spectrum disorder (ASD)
- induced-neuronal (iN) cells
- neurofibromatosis type 1 (NF1)
Neurofibromatosis type 1 (NF1: also called von Recklinghausen disease) is a multifaceted disease with a variety of physical manifestations, including multiple café-au-lait spots, neurofibromas, Lisch nodules, scoliosis, and visual impairment [1, 2, 3]. NF1 patients also exhibit a variety of psychiatric symptoms such as mental retardation, epilepsy, and cognitive dysfunction/learning disabilities [4, 5]. Approximately half of NF1 patients exhibit impaired social information processing and disturbed social behavior [6, 7, 8]. In addition, about 30% of NF1 patients have comorbid autism spectrum disorder (ASD) [9, 10, 11, 12, 13]. These clinical reports suggest some kind of neurodevelopmental pathophysiology in the brains of NF1 patients.
NF1 is a monogenic disease, and the causative gene is the
Few studies have shown whether such dysfunction is present in human living neuronal cells of NF1 patients. This is because it is difficult to directly analyze human brain cells containing neuronal cells. Very recently, a study has been published that reveals patient-common and mutation-dependent abnormalities using neural progenitor cells and cerebral organoids derived from induced pluripotent stem (iPS) cells in NF1 patients . Regenerative medicine technologies using iPS cells from human tissues have been attracting attention to clarify the pathophysiology of brain disorders, including psychiatric disorders, at the cellular level [19, 20]. Direct conversion technologies without using iPS cells have also attracted attention as a useful translational research tool [21, 22]. The cells directly converted to neuronal cells are called “induced-neuronal (iN) cells” and were first developed from mouse fibroblasts transfected with the three transcriptional factors
In this chapter, we present the results obtained to date on abnormalities in gene expression and cell morphology in iN cells derived from NF1 patients, and describe future prospects.
2. Dysregulated gene expression in the neuronal cells of NF1 patients
Direct conversion methods that generate human induced-neuronal (iN) cells from fibroblasts within two weeks are expected to discover unknown neuronal phenotypes in neuropsychiatric disorders. Here, we introduce unique gene expression profiles in iN cells of neurofibromatosis type 1 (NF1) patients, a single-gene multifaceted disorder with relatively high co-occurrence with autism spectrum disorder (ASD). The association between NF1 and adenylyl cyclases (ACs) activity has been reported in animal model studies, [16, 17] as far as we know, there are no experimental studies using human neuronal cells. To clarify how abnormalities in the ACs pathway affect the gene expression pattern of iN cells derived from NF1 patients (NF1-iN cells), a group treated with forskolin, an ACs activator, was included in the microarray analysis. First, an unbiased microarray analysis was performed to investigate aberrant gene expression in NF1-iN cells (6 male samples including 3 healthy controls (HC) and 3 NF1). Interestingly, in the iN cells, the expression of 149 genes was significantly different in NF1-iN cells compared to HC-iN cells (Figure 2). It is strongly suggested that these aberrant gene expressions in NF1 patients are shown only in iN cells and not in fibroblasts. In NF1-iN cells, the expression level of 90 genes was changed by the addition of the AC activator forskolin. Among the above149 genes (HC-iN cells vs. NF1-iN cells) and 90 genes (NF1-iN cells without forskolin vs. NF-iN cells with forskolin), 31 genes were overlapped (Figure 2). Interestingly, all of their expression levels in NF1-iN cells were rescued to HC level by the application of forskolin (Figure 3). These 31 genes may be strongly dysregulated via the AC pathway in neurons of NF1 patients (especially males).
To confirm the validity of the differences in expression of the 31 gene mentioned above, all samples on hand (3 male HC samples and 3 female NF1 samples) were added and reassessed by real-time PCR analysis. Unfortunately, when we validated these results with real-time PCR analysis using a total of six HC and six NF1 samples, including female samples, we could not reproduce most of the differences in the expression of the 31 genes. Recent epidemiological studies have shown that NF1 patients have a high comorbidity with ASD, and prevalence of ASD is about twice as high in males than females [12, 33]. Further investigation with larger samples may clarify our novel hypothesis about the tendency for neuronal pathologies to develop especially in male NF1 patients, and may lead to a better understanding of gender differences in ASD and other neuropsychiatric disorders.
MEX3Dgene expression in the neuronal cells of NF1 patients
Interestingly, in the real-time PCR analysis described above, only the gene expression of the
To evaluate whether the reduction of
BCL2gene expression in the neuronal cells of NF1 patients
A previous study has shown that BCL2, an anti-apoptotic protein, is elevated in neuronal stem cells (NSCs) from
The morphology of Day-5 iN cells (early-stage iN cells) was not significantly different from that of fibroblasts. Surprisingly, forskolin transformed iN cells from a fibroblast-like shape to a long-branched neuron-like morphology even at Day 5. These Day-5 iN cells showed higher levels of
MAGEL2gene expression in the neuronal cells of NF1 patients
Aberrant gene expression in NF1-iN cells has also been discovered from a completely different approach. Akamine
3. Aberrant cell morphology of the neuronal cells of NF1 patients
Adenylyl cyclase (AC) dysfunction is one of the candidate pathways in abnormal neuronal development in the brain of NF1 patients, but its dynamic abnormalities have not been observed. Therefore, we observed the dynamic effects of forskolin on iN cells. In HC-iN cells, most of cells were neuron-like spherical-form. On the other hand, in NF1-iN cells, most of the cells were thin and flat. Interestingly, after only 20 minutes of AC activation by forskolin treatment, most NF1-iN cells had a dense cell contour and their cell morphology changed dramatically to neuron-like spherical-form. This result suggests that most NF1-iN cells were unable to form neuron-like spherical-form cell morphology due to lack of AC ability. Counting the number of cells, NF1-iN cells had a significantly higher number of flat-form cells than HC-iN cells (Figure 4, p = 0.0164), and their cell morphology was significantly restored by forskolin treatment (Figure 4, p = 0.0059) . In addition, forskolin appeared to promote neurite outgrowth in iN cells, so quantitative experiments and analysis with more samples should be conducted in the near future.
Forskolin activates intracellular ACs and increases intracellular cyclic adenosine monophosphate (cAMP) levels, and it has previously reported that forskolin regulates cytoskeletal formation in Y1 cells, a cell line derived from mouse adrenocortical tumors . When intracellular cAMP levels increase, dephosphorylation of paxillin occurs at the cell edge, and paxillin moves from the focal adhesion to the cytoplasm . Patients with NF1 have aberrant gene expression of neurofibromin that is known to regulate the activity of ACs and the intracellular cAMP levels . Recently, we have shown that neurofibromin gene expression is also low in NF1-iN cells,  suggesting that intracellular cAMP levels are low in NF1-iN cells. As mentioned above, NF1-iN cells tend to have flat-form cell morphology compared to HC-iN cells, and these cell morphologies are restored by application of forskolin . Thus, such morphological abnormalities may be attributed by abnormal cytoskeleton development due to decreased dephosphorylation levels of paxillin due to decreased activation of ACs and decreased intracellular cAMP levels in NF1-iN cells. Paxillin has been shown to be involved in neurite outgrowth in PC12 cells, a cell line derived from rat adrenal medulla pheochromocytoma . Similarly, our findings suggest that forskolin alter the phosphorylation level of paxillin and activated neurite outgrowth.
Our pilot experiment showed that activation of ACs may normalize the development of neuronal cells in the brain of NF1 patients. We propose that administration of forskolin or forskolin-like AC activators into the brain during neurodevelopmental periods of NF1 patients may contribute to the prevention of neurodevelopmental disorders such as ASD and neuropsychiatric disorders in subsequent life.
In this chapter, we have presented unique gene expressions and cell morphology profiles in induced-neuronal (iN) cells of patients with neurofibromatosis type 1 (NF1), a single-gene multifaceted disorder with relatively high co-occurrence of autism spectrum disorder (ASD). Microarray analysis revealed that the expression of 149 genes was abnormal in the neuronal cells of NF1 male patients, and that the expression of 90 genes was altered in the presence of forskolin. Of these, 31 genes in particular were suggested to be normalized by improvement of the AC pathway. These abnormalities of gene expressions may be male-specific and may be related to gender differences in the development of ASD. Further cellular analysis, especially considering gender-specific neuronal dysregulation, should be performed to reveal unknown neurobiological roles of gender underlying the pathophysiology of ASD.
We also introduced that the effects of forskolin shows dramatic changes not only in gene expression but also in the cell morphology of neuronal cells in NF1 patients. We propose that research is needed to prevent the development of ASD and neuropsychiatric disorder later in life by administering forskolin and other AC activators, which are easily introduced in to the brain, to NF1 patients early in their developmental period.
Furthermore, we found that the expression of
Moreover, the findings presented here should be validated by additional analyses such as apoptosis analysis, protein level analysis and functional analysis of neurons. On the other hand, more detailed molecular mechanisms, especially the interactions between NF1, MEX3D, FOS, JUN, BCL2, and MAGEL2, will be the subject of future work. In addition,
Our studies shown in this paper were partially supported by Grant-in-Aid for Scientific Research on (1) Innovative Areas “Will-Dynamics” of The Ministry of Education, Culture, Sports, Science, and Technology, Japan (JP16H06403 to T.A.K.), (2) The Japan Agency for Medical Research and Development (AMED) (“Syogaisya-Taisaku-Sogo-Kenkyu-Kaihatsu-Jigyo” JP19dk0307047 & JP19dk0307075, and “Yugo-no” JP19dm0107095 to T.A.K.), (3) KAKENHI - the Japan Society for the Promotion of Science (“Wakate A” JP26713039 and “Kiban A” JP18H04042 to T.A.K., and “Wakate B” JP26860932 & JP17K16386 to N.S.), (4) SENSHIN Medical Research Foundation (to T.A.K.), (5) Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.A.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interest
The authors declare no conflict of interest.
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