Neurofibromin 1 mutations impair the function of human induced pluripotent stem cell-derived microglia

ABSTRACT Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by germline mutations in the neurofibromin 1 (NF1) gene. Children with NF1 are prone to the development of multiple nervous system abnormalities, including autism and brain tumors, which could reflect the effect of NF1 mutation on microglia function. Using heterozygous Nf1-mutant mice, we previously demonstrated that impaired purinergic signaling underlies deficits in microglia process extension and phagocytosis in situ. To determine whether these abnormalities are also observed in human microglia in the setting of NF1, we leveraged an engineered isogenic series of human induced pluripotent stem cells to generate human microglia-like (hiMGL) cells heterozygous for three different NF1 gene mutations found in patients with NF1. Whereas all NF1-mutant and isogenic control hiMGL cells expressed classical microglia markers and exhibited similar transcriptomes and cytokine/chemokine release profiles, only NF1-mutant hiMGL cells had defects in P2X receptor activation, phagocytosis and motility. Taken together, these findings indicate that heterozygous NF1 mutations impair a subset of the functional properties of human microglia, which could contribute to the neurological abnormalities seen in children with NF1.


Fig. S2 .
Fig. S2.Immunohistochemical staining and TREM2 expression in CTL and NF1-mutant hiMGL cells A CTL and NF1-mutant hiMGL cells labelled with DAPI are immunopositive for IBA1 and TMEM119 expression.Merged images show the combined signal for DAPI, IBA1 and TMEM119.Scale bars, 50 μm.B Relative mRNA expression levels of the TREM2 microglia marker were assessed in CTL and NF1-mutant hiMGL cells by quantitative RT-qPCR.Relative expression (R.E.) was normalized relative to the TATA box binding protein (TBP) housekeeping gene (n=3).Results are represented as the mean ± SEM.Data were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test.

Fig. S6 .C
Fig. S6.Basal membrane properties of CTL and NF1-mutant hiMGL cells A Sample patch clamp recordings of CTL and NF1-mutant hiMGL cells.Membrane currents were obtained during a series of voltage steps for 50 ms ranging from -170 mV to +60 mV from a holding potential of -70 mV.B Average current density-voltage relationships of hiMGL cells obtained from the recordings shown in A. C Distribution of the reversal potentials (indicative of the membrane potentials).N: number of patched cells.D: Summary of the membrane capacities of CTL and NF1-mutant hiMGL cells.

Fig. S7 .
Fig. S7.Expression of P2RX4 and P2RX7 in CTL and NF1-mutant hiMGL cells A: Relative P2RX4 (left) and P2RX7 mRNA expression (right) levels in CTL and NF1-mutant hiMGL cells by quantitative RT-PCR.TATA box binding protein (TBP) was used as a housekeeping gene for normalization (n = 3).Data were then normalized to mRNA expression levels in CTL hiMGL cells.Results are represented as the mean ± SD.Data were analyzed by one-way ANOVA.B: P2 receptor expression from the RNA sequencing data revealed that only P2RY4 expression was different in NF1-mutant hiMGL cells relative to CTL hiMGL cells.P values are included in the graph.All other genes were not statistically different between NF1-mutant and CTL hiMGL cell groups.

Fig. S8 .
Fig. S8.Motility of CTL and NF1-mutant hiMGL cells A Relative mRNA expression levels of Toll-like receptor 2 (TLR2) in CTL and NF1-mutant hiMGL cells by quantitative RT-PCR.Relative expression (R.E.) is shown relative to CTL hiMGL cells.TATA box binding protein (TBP) mRNA expression was used as a housekeeping gene for normalization (n = 3).Data were then normalized to TLR2 expression of CTL hiMGL cells.Results are represented as the mean ± SEM.Data were analyzed by one-way ANOVA.B Motility was assessed using a standardized wound scratch assay (RWD) using an Incucyte Zoom System.hiMGL cells were incubated with or without Pam2CSK4 (100 ng/ml).Relative Wound Density (RWD) was assessed over the course of two days comparing CTL and NF1mutant hiMGL cells.Results are represented as the mean ± SEM.N = 5 for CTL and N = 3-4 for NF1-mutant hiMGL cells.Data indicated by the asterisks were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test.*P<0.05;**P< 0.01, ***P<0.001.Comparisons between basal and Pam2CSK4 conditions were performed using a Student's t-test: #P<0.05,###P<0.001.

Fig. S9 .
Fig. S9.Phagocytic activity of CTL and NF1-mutant hiMGL cells Phagocytic activity was assessed by microscopy using fluorescent microbeads.CTL and NF1mutant hiMGL cells were incubated for 1h with beads with or without addition of 1 µg/ml LPS.A: Representative images of CTL and NF1-mutant hiMGL cells at the end of the assay period under LPS stimulation conditions.IBA1 staining is indicated in blue and beads in white.Scale bars, 20 µm.B: Phagocytic activity presented as the phagocytic index, which is a measure of the percentage of cells harboring 0, 1, 2 or >3 engulfed beads.Data indicated by asterisks were analyzed by oneway ANOVA followed by Tukey's multiple comparisons test.*P<0.05;**P< 0.01, ***P<0.001.Comparisons between basal and UDP conditions were performed using a Student's t-test: ###P<0.001.Results are represented as the mean ± SEM.N = 5 for CTL and n = 3 for NF1mutant hiMGL cells.