Figure 2

Evaluation of synapses, neurite length, and neuronal firing in Control and MJD neurons. Control, MJD CLA, MJD CLB, and MJD CLC iPSC-derived NESC (CNT, MJD CLA, MJD CLB, and MJD CLC, respectively) were differentiated into neural cultures for 3 weeks. Representative immunofluorescence images showing (A) inhibitory postsynaptic terminals, defined as gephyrin-positive (green) puncta on (B, merge) β3 Tubulin-positive (red) neurite. (C) Number of inhibitory postsynaptic terminals per neurite length normalized for CNT NESC (n = 3–4 independent experiments, analyzed neurons: CNT n = 40, MJD CLA n = 22, MJD CLB n = 41, MJD CLC n = 22). Representative immunofluorescence images of excitatory synapses, defined as instances of (D) VGluT1 (green) and (E) PSD95 (red) puncta (F) colocalization on MAP2-positive neurite. (G) Number of excitatory synapses per neurite length normalized for CNT NESC (n = 3–4 independent experiments, analyzed neurons: CNT n = 23, NESC CLA n = 21, MJD CLB n = 33, MJD CLC n = 29). (A, B and D-F) scale bars: 10 μm. (H) Representative immunofluorescence image of MAP2-positive (red) neurite of CNT NESC. (I) Total neurites length per neuron normalized for CNT NESC (n = 2–5 independent experiments, analyzed neurites: CNT n = 128, MJD CLA n = 197, MJD CLB n = 107, MJD CLC n = 101). Scale bar: 20 μm, DAPI: blue. (J-L) Neuronal firing evaluation through the variation of intracellular calcium concentration in neurons by single-cell calcium imaging. Representative images of the calcium fluorescence indicator (Fluo-4, green) in neurons (J) before and (K) after potassium (K⁺) stimulus. (L) Percentage of cells responding to potassium (neurons) and histamine (neural progenitors) stimulus (n = 4 independent experiments, analyzed neurons: CNT n = 271, MJD CLA n = 271, MJD CLB n = 59, MJD CLC n = 138); (J, K) scale bars: 50 μm. Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, and ****p < 0.0001, One-way ANOVA followed by Tukey’s post-test.