Human iPSC-derived microglia sense and dampen hyperexcitability of cortical neurons carrying the epilepsy-associated SCN2A-L1342P mutation

Abstract

Neuronal hyperexcitability is a hallmark of seizures. It has been recently shown in rodent models of seizures that microglia, the brain's resident immune cells, can respond to and modulate neuronal excitability. However, how human microglia interacts with human neurons to regulate hyperexcitability mediated by epilepsy-causing genetic mutation found in human patients remains unknown. The SCN2A genetic locus is responsible for encoding the voltage-gated sodium channel Nav1.2, recognized as one of the leading contributors to monogenic epilepsies. Previously, we demonstrated that the recurring Nav1.2-L1342P mutation identified in patients with epilepsy leads to hyperexcitability in a hiPSC-derived cortical neuron model from a male donor. While microglia play an important role in the brain, these cells originate from a different lineage (yolk sac) and thus are not naturally present in hiPSCs-derived neuronal culture. To study how microglia respond to diseased neurons and influence neuronal excitability, we established a co-culture model comprising hiPSC-derived neurons and microglia. We found that microglia display altered morphology with increased branch length and enhanced calcium signal when co-cultured with neurons carrying the Nav1.2-L1342P mutation. Moreover, the presence of microglia significantly lowers the action potential firing of neurons carrying the mutation. Interestingly, we further demonstrated that the current density of sodium channels in neurons carrying the epilepsy-associated mutation was reduced in the presence of microglia. Taken together, our work reveals a critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing mutation present in human neurons, highlighting the importance of neuron-microglia interactions in human pathophysiology.

Keywords: channelopathy; electrophysiology; human induced pluripotent stem cells (hiPSCs); hyperexcitability; monogenic diseases; seizures; voltage-gated sodium channel Nav1.2.

Figure 1.. Characterization of hiPSC-derived cortical neurons and microglia.

(A) Schematic illustrating the protocol for generating hiPSC-derived cortical neurons. (B) Representative Fluorescent image of hiPSC-derived neurons stained for somatodendritic marker MAP2 (magenta), synaptic vesicle proteins SYN1/2 (green), and DAPI (blue). (C) Schematic illustrating the protocol for generating hiPSC-derived microglia. Human iPSCs are differentiated into hematopoietic progenitor cells for 12 days and cultured in microglia differentiation media for 24 days. The microglia maturation process is then carried out for up to 12 days. (D) Representative images of hiPSC-differentiated microglia expressing microglial-specific markers: IBA1 (D, top panel, green, n=13 fields of view, two differentiations), TMEM119 (D, middle panel, yellow, n=18 fields of view, two differentiations), P2RY12 (D, lower panel red, n=19 fields of view, two differentiations). DAPI was used to stain nuclei. Data is presented as mean ± s.e.m. Scale bar=100 μm. (E) Phagocytosis of pHrodo-myelin by wild-type (control) hiPSC-derived microglia. Data was obtained from one differentiation of three wells (48 images per well). Representative images at 0 hours and 24 hours after the addition of pHrodo-myelin. Human iPSC-derived microglia phagocytosed the pHrodo-labeled bioparticles, showing a gradually increasing red fluorescent signal over time. Scale bar=25 μm. hiPSC, human induced pluripotent stem cells; EB, Embryoid body; NP, Neural Progenitors; MAP2, microtubule-associated protein-2; SYN1/2, Synapsin1/2; IBA1, Ionized calcium-binding adaptor molecule 1; TMEM119, transmembrane protein 119; P2RY12, Purinergic Receptor P2Y12.

Figure 2.. Human microglia in co-culture with L1342P neurons display morphological changes.

(A) The hiPSC-derived neurons and microglia were matured separately, and then microglia were seeded on top of neurons for seven days before imaging. (B) Representative images of co-cultured neurons stained for neuron-specific marker MAP2 (green), microglia stained for IBA1 (red), and DAPI (blue) as a nuclear stain. (C-D) Human iPSC-derived microglia in co-culture with control (WT) neurons (WT+M, C) and Nav1.2-L1342P neurons (L1342P+M, D). IBA1+ microglia co-cultured with the hyperexcitable Nav1.2-L1342P neurons displayed an extended ramified process compared to co-culture with control (WT) neurons. Images are pseudo-colored in a rainbow gradient to facilitate identification, and the skeletonized view was included to detail branches. (E) The microglial average branch length increases in co-culture with Nav1.2-L1342P cortical neurons (WT+M: n=43 fields of view and L1342+M: n = 49 fields of view, three differentiations, two clones per condition). (F) The total microglial area did not change in co-culture with Nav1.2-L1342P neurons (WT+M: n=43 fields of view and L1342+M: n=49 fields of view, three differentiations, two clones per condition). (G) Microglial perimeter is enhanced in co-culture with Nav1.2-L1234P neurons, indicating extended processes (WT+M: n=43 fields of view and L1342+M: n=49 fields of view, three differentiations, two clones per condition). (H) Microglial circularity is decreased in co-culture with Nav1.2-L1342P neurons, indicating that they are less ameboid-like (WT+M: n=43 fields of view, and L1342+M: n=49 fields of view, three differentiations, two clones per condition). Each dot represents the mean value of a parameter per field of view. Data are presented as mean ± s.e.m. Scale bar=50 μm. Data was pooled from three differentiations. Data was analyzed by nested t-test; **p < 0.01 and ****p < 0.0001.

Figure 3.. GCaMP6f calcium signal is enhanced in human microglia processes when co-culturedwith hyperexcitable hiPSC-derived L1342P neurons.

(A) Fluorescent images of hiPSC-derived microglia expressing GCaMP6f co-cultured with WT (top) and mutant Nav1.2-L1342P (bottom) hiPSC-derived neurons. The GCaMP6f calcium signal is pseudocolored, with dark green indicating low signal and yellow hues depicting high signal. (B) Representative ∆F/F traces of GCaMP6f global calcium activity of microglia in co-culture with control (WT) neurons (top, black) or Nav1.2-L1342P neurons (bottom, red). Three representative cells per condition are shown. (C) The global average microglia calcium signal area increases in co-culture with Nav1.2-L1342P neurons. (D) The global average microglial calcium spike amplitude increases in co-culture with Nav1.2-L1342P neurons. (E) The average microglial signal area in the soma microdomain is not statistically different in the two co-culture conditions. (F) The average microglial spike amplitude in the soma microdomain is not statistically different in the two co-culture conditions. (G) The average microglial calcium signal area of the processes microdomain is increased in co-culture with Nav1.2-L1342P neurons. (H) The processes’ average microglial calcium spike amplitude increases in co-culture with L1342P neurons. Data was collected from two independent differentiations per genotype. Data in C, D, E, F, G, and H were analyzed with Mann-Whitney’s U test. ****p < 0.0001, and n.s (not significant).

Figure 4.. The repetitive firing of hiPSC-derived neurons carrying the Nav1.2-L1342P mutation is reduced in co-culture with microglia.

(A) hiPSC-derived cortical neurons were transduced with an AAV-CaMKIIa-EGFP to allow the detection of excitatory neuronal populations for patch-clamp electrophysiology. Neurons and microglia were co-cultured for seven days before patch clamp measurements. (B) Representative action potential (AP) firings from hiPSC-derived control (WT) cortical neurons alone (left) and with microglia (right). (C) Representative AP firings from hiPSC-derived Nav1.2-L1342P cortical neurons alone (left) and with microglia (right). (D) The number of action potentials presented no statistical difference between WT neurons alone and with microglia co-culture (WT: n=12 neurons, two differentiations; WT+M: n = 18 neurons, two differentiations). (E) The maximum number of APs triggered from each neuron under the 0 to 125-pA current injections range presented no statistical difference between WT neurons with and without microglia co-culture (WT: n=12 neurons, two differentiations; WT+M: n = 18 neurons, two differentiations). (F) The action potential number of Nav1.2-L1342P neurons in co-culture with microglia consistently fired fewer APs than Nav1.2-L1342P neurons alone (L1342P: n = 14 neurons, two differentiations; L1342P+M: n = 25 neurons, two differentiations). (G) The maximum number of APs triggered from each neuron between the 0 to 125 pA current injections range was reduced for Nav1.2-L1342P neurons in co-culture with microglia compared to Nav1.2-L1342P neurons alone (L1342P: n=14 neurons, two differentiations; L1342P+M: n=25 neurons, two differentiations). Nav1.2-L1342P neurons co-cultured with microglia display a reduction in the maximum number of action potentials compared to Nav1.2-L1342P neurons alone. Data are presented as mean ± s.e.m. Unpaired Student’s t-test analyzed data in E and G. Each dot corresponds to one neuron. Data in D and F were analyzed by Repeated Measures of Two-Way ANOVA, with data pooled from at least two differentiations per condition. *p < 0.05, and n.s. (not significant).

Figure 5.. hiPSC-derived Nav1.2-L1342P neurons display reduced sodium current when co-cultured with human microglia.

(A) Representative sodium current trace of Nav1.2-L1342P cortical neurons. (B) Representative sodium current trace of Nav1.2-L1342P cortical neurons in co-culture with hiPSC-derived microglia. In both A and B, the outward current was blocked using the tetraethylammonium chloride in the bath solution. (C) Sodium channel activation curve over varying voltage for Nav1.2-L1342P neurons in isolation or co-culture with microglia was plotted (left). The average maximum current density was significantly decreased in L1342P+M neurons compared with L1342P neuron alone (right) (L1342P: n=31 neurons, three differentiations: L1342P+M: n=28 neurons from two differentiations). Data are presented as mean ± s.e.m. Data in C right were analyzed by Student’s t-test. Each dot represents one neuron (right panel). Data were collected from at least two independent differentiations per genotype. *p < 0.05.