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, 9 (12), e01473

Nystagmus-related FRMD7 Gene Influences the Maturation and Complexities of Neuronal Processes in Human Neurons

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Nystagmus-related FRMD7 Gene Influences the Maturation and Complexities of Neuronal Processes in Human Neurons

Jiali Pu et al. Brain Behav.

Abstract

Aims: Idiopathic congenital nystagmus (ICN) is an oculomotor disorder caused by the defects in the ocular motor control regions of the brain. Mutations in FRMD7, a member of the FERM family of proteins, associated with cytoskeletal dynamics, are the most frequent causes of X-linked ICN. Previous studies illustrated that FRMD7 is involved in the elongation of neurites during neuronal development; however, almost all the studies were performed on mice cell models. The complexity in the human neuronal network might suggest a unique vulnerability of human neurons to FRMD7 mutations.

Methods: Herein, we successfully established human neuronal cell models with FRMD7 mutations, from fibroblasts-reprogrammed neurons (iNs). In these neurons, the complexity of the neuronal processes was measured by the induced ratio, total neurite length, the number of terminals, and the number of maturation neurons.

Results: The complexity of the neuronal processes was greatly reduced during various reprogramming stages in the presence of FRMD7 mutations. Consistently, the expression of the three main Rho GTPases was significantly increased by FRMD7 mutations. Interestingly, a slightly diverse phenotype is observed in different derived neurons.

Conclusion: We established ideal human neuron models and confirmed that the mutation in FRMD7 influences the maturation and complexities of neuronal processes, which might be involved with the Rho GTPase signaling.

Keywords: CRISPR/Cas9; FRMD7; Rho GTPases; idiopathic congenital nystagmus; induced neurons; neuronal outgrowth.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1
CRISPR/Cas9‐mediated mutagenesis of FRMD7 gene in human fibroblast cell line. (a, b) Schematic representation of the targeting strategy for the FRMD7 locus using CRISPR/Cas9. The PCR primer sites for homologous recombination test are indicted as arrows (P1 For and P1 Rev). The exons of the FRMD7 gene locus are indicated by blue boxes, and arrows indicate the genomic site cut by FRMD7‐CRISPR/Cas9. PX458: CRISPR/Cas9 template plasmid; sgRNA: guided RNA. (c) FRMD7‐CRISPR/Cas9 activity assay by site‐directed mutagenesis assay. FRMD7‐CRISPR/Cas9 was transiently transfected in human embryonic kidney 293FT cells. Prior to PCR amplification, genomic DNA was digested with DdeI, which was present at the FRMD7‐CRISPR/Cas9 target site. (d) The sequencing results of the clones. Out of 10, seven of the randomly sequenced clones harbored the mutations. (e, f) Examples of the mutated clones showing the mutant sites
Figure 2
Figure 2
Direct conversion of human fibroblasts into neurons. (a) Conversion of fibroblast cells into neurons. Cells were reprogrammed to induced neurons (iNs) in neural induction medium with lentiviruses expressing Ascl1, p53 shRNA, plus miR124. FM: Full medium. (b) Cell morphology during HFF fibroblast reprogramming. Day 0: fibroblast cell morphology; day 4: the cells gradually changed their morphological characteristics; days 9 and 20: The sharp of the cells exhibited neuron‐like morphology. F7, FRMD7 mutant; N, control; scale bars: 100 μm
Figure 3
Figure 3
FRMD7 attenuation modifies the complexity of neurons from directly converted human fibroblasts. (a) The induced cells were stained at day 9 with antibodies against the neuronal marker b3‐tubulin (Tuj1). (b) Percentage of Tuj1+ cells in all cells (DAPI+) at day 9. *p < .05, unpaired, two‐tailed Student's t tests versus Tuj1+ in control, respectively. n = 6 wells from three independent experiments for each condition. Average numbers of Tuj1+ or DAPI+ cells per field under × 10 lenses were plotted. Total neurite length (c) and number of neurites (d) per Tuj‐1+ neurons. *p < .05, unpaired, two‐tailed Student's t tests versus control; n = 30–50 Tuj‐1+ neurons from three independent experiments for each condition
Figure 4
Figure 4
FRMD7 mutants influence the maturation of human fibroblasts‐reprogrammed neurons. (a) The induced cells were stained at day 15 with antibodies against the neuronal marker microtubule‐associated protein 2 (MAP2) in two different fibroblast cell lines. (b) Percentage of MAP2+ and Tuj1+ cells in all cells (DAPI+) at day 15. The ratio MAP2+/Tuj‐1+ was calculated. *p < .05, unpaired, two‐tailed Student's t tests versus MAP2+/Tuj‐1+ in control, respectively. The experiments were repeated three times, and the graphs represent the average of three independent experiments (columns, mean; bars, SEM.; *p < .05, **p < .01)
Figure 5
Figure 5
Characterization of the fibroblast‐induced neurons. Costaining of induced neurons with antibodies against Tuj‐1 and other neuronal markers, such as postsynaptic density‐95 (PSD95), synapsin, and neural cell adhesion molecule (NCAM). Scale bar, 10 mm
Figure 6
Figure 6
Mutations in FRMD7 increase the expression levels of neuron‐specific Rho GTPases during induced time lapse. The mRNA expression levels of three main Rho GTPases (Rac1, Cdc42, and RhoA) were measured by real‐time PCR during different time points. The expression of all of the three Rho GTPases was increased in the mutant FRMD7‐induced neurons and significantly for Rac1. With prolonged induction time, the increase in Rho GTPases expression was observed markedly. *p < .05, unpaired, two‐tailed Student's t tests versus control, respectively. Data are presented as the fold change in expression as compared to the controls. All experiments were performed in triplicate, and the graph shows the average for the three measurements (columns = means; bars = SEM; *p < .05, **p < .01 vs. controls)

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