Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation

Abstract

Recent studies suggest that induced neuronal (iN) cells that are directly transdifferentiated from nonneuronal cells provide a powerful opportunity to examine neuropsychiatric diseases. However, the validity of using this approach to examine disease-specific changes has not been demonstrated. Here, we analyze the phenotypes of iN cells that were derived from murine embryonic fibroblasts cultured from littermate wild-type and mutant mice carrying the autism-associated R704C substitution in neuroligin-3. We show that neuroligin-3 R704C-mutant iN cells exhibit a large and selective decrease in AMPA-type glutamate receptor-mediated synaptic transmission without changes in NMDA-type glutamate receptor- or in GABAA receptor-mediated synaptic transmission. Thus, the synaptic phenotype observed in R704C-mutant iN cells replicates the previously observed phenotype of R704C-mutant neurons. Our data show that the effect of the R704C mutation is applicable even to neurons transdifferentiated from fibroblasts and constitute a proof-of-concept demonstration that iN cells can be used for cellular disease modeling.

Keywords: cellular reprogramming; neurexin; postsynaptic density; stem cells; synapse.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Generation of iN cells from MEFs isolated from WT and Nlgn3 R704C-mutant mice. (A) Schematic representation of the experimental design. MEFs were seeded on day 0 and transduced with the BAM transcription factors on day 1, and transgene expression was induced by the addition of doxycycline to the medium on day 2. Olfactory bulb neurons were cultured from newborn WT mice on day 5, tau-EGFP–positive iN cells were isolated by FACS sorting and placed on top of the primary neurons on day 6, and electrophysiological and imaging experiments were performed on days 22–24. (B) Representative image of R704C-mutant iN cells stained 14 d after transduction of MEFs for the pan-neuronal marker Tuj1. (C) Representative quantitative RT-PCR analysis of Nlgn3 expression in MEFs, mouse brain, and WT and R704C-mutant iN cells that were analyzed without FACS sorting 20 d posttransduction. (D) (Left) FACS analysis illustrating the efficiency of the conversion of MEFs into iN cells (defined as tau-EGFP–positive cells) in R704C-mutant and WT cells. (Right) The bar graph shows the average abundance of tau-EGFP–positive cells (n = 5 experiments). (E and F) Immunofluorescence analysis of iN cells (cultured on glia only). At day 20, iN cells were stained for DAPI (blue) and MAP2 (red) (E) and for tau-GFP (green), synapsin (red), and DAPI (blue) (F).

Fig. 2.

iN cells derived from WT and Nlgn3 R704C-mutant mice have similar intrinsic electrical properties. (A and B) Analysis of AP firing properties in WT (blue) and R704C-mutant iN cells (red). iN cells were patched in current-clamp mode and injected with current pulses increasing in 10-pA increments. (A) Experimental protocol (Top) and representative traces of AP firing patterns consisting of multiple (Middle) or single (Bottom) APs. (B) Quantitative analyses of (i) the number of APs induced by a current pulse, (ii) the resting membrane potential, (iii) the AP firing threshold, and (iv) the AP amplitude. No statistically significant difference (P > 0.5) between WT and R704C-mutant iN cells was detected in any parameter. In all subpanels, the number of cells/cultures analyzed is indicated in the corresponding bar graphs. (C) Analysis of Na+ and K+ currents in WT (blue) and R704C-mutant (red) iN cells. (Left) The experimental voltage pulse step protocol (black, Top), and examples voltage-clamp recording traces (Vhold = −70 mV). Expanded views of Na+ currents are shown in the dashed boxes below the traces. The black line below the protocol diagram depicts the time period used for calculating average K+ currents. (Right) The average Na+ currents (INa, filled squares) and K+ currents (IK, filled circles) recorded from WT and R704C-mutant iN cells and plotted as a function of the step-voltage amplitudes. (Inset) The reversal potential (Vrev) for K+ currents. No significant difference was found between WT and R70C-mutant iN cells at any step size tested (P > 0.2, unpaired, one-tailed t test). (D and E) Analysis of the capacitance (D) and input resistance (E) of WT (blue) and R704C-mutant iN cells (red). No significant difference was found for either parameter (P > 0.8, t test).

Fig. 3.

iN cells derived from Nlgn3 R704C-mutant mice show significant decreases in AMPAR-mediated synaptic transmission. (A) Analysis of spontaneous mEPSCs. (Left) Sample traces.The dashed box depicts an expanded trace to illustrate the EPSC kinetics. (Right) Cumulative probability plots and bar graphs of the mEPSC amplitude (Upper) and frequency (Lower). mEPSCs were recorded from WT (blue) and R704C-mutant iN (red) cells in 50 μM picrotoxin and 1 μM TTX. (B) Spontaneous EPSCs measured in WT (blue) and R704C-mutant iN (red) cells in 50 μM picrotoxin and 50 μM AP5. Data are presented as in A. (C) As in B, except that spontaneous IPSCs were examined in the presence of 50 μM CNQX. Data shown in the bar diagrams are means ± SEM; number of cells/independent cultures analyzed are indicated in the bars. Statistical significance levels were assessed by unpaired, one-tailed Student t test (*P < 0.05, **P < 0.01, and ***P < 0.001, all versus control).

Fig. 4.

R704C-mutant iN cells exhibit reduced evoked AMPAR- but not GABAR- mediated synaptic responses. (A) Analysis of evoked AMPAR-mediated EPSCs in WT (blue) and R704C-mutant iN (red) cells. EPSCs were evoked by extracellular stimulation in the presence of 50 μM picrotoxin and 50 μM AP5. (Left) Example traces show three consecutive trials for each example (shades) that are overlaid with a small lateral shift for better visibility. (Center) Cumulative probability plots depict the EPSC amplitude (Upper Center) and coefficient of variation as a measure of release probability (Lower Center). (Right) Bar graphs display the means ± SEM of the respective parameters. (B) As in A, but for NMDAR-mediated EPSCs without analysis of the coefficient of variation. Traces depict only a single example. (C) Analysis of PPRs (interstimulus interval, 50 ms) to assess the release probability. (Left) Scaled sample traces. (Center) Cumulative probability plots of PPRs. (Right) Mean PPR. (D) As in B, but for IPSCs. (E) As in C, but for IPSCs. (F) Analysis of EPSCs induced by direct application of AMPA (50 μM) in WT (blue) and R704C-mutant iN (red).cells. (Left) Representative traces. (Center) Cumulative probability plots of the EPSC amplitude (Upper) and charge transfer (Lower). (Right) Bar graphs of the mean values for the same parameters. Data shown in the bar graphs are means ± SEM; numbers of cells/independent cultures analyzed are stated in the bars. *P < 0.05; **P < 0.01, Student t test.