Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.

Sleep bruxism (SB) involves involuntary jaw movements during sleep and is potentially caused by motor neuronal hyperexcitability and GABAergic system dysfunction. However, the molecular basis remains unclear. In this study, we aimed to investigate changes in the expression of several genes associated with the pathophysiology of SB. Bulk RNA sequencing (bulk RNA-seq) and single-nucleus RNA sequencing (snRNA-seq) of neurons derived from patient and control human induced pluripotent stem cells (hiPSCs) were performed to comprehensively assess gene expression and cell type-specific alterations, respectively. Bulk RNA-seq revealed significant upregulation of calcium signaling-related genes in SB neurons, including those encoding G protein-coupled receptors and receptor-operated calcium channels. snRNA-seq confirmed the increased expression of GRIN2B (an N-methyl-D-aspartate receptor subunit) and CHRM3 (an M3 muscarinic acetylcholine receptor), particularly in glutamatergic and GABAergic neurons. These alterations were linked to hyperexcitability, with GRIN2B contributing to glutamatergic signaling and CHRM3 contributing to cholinergic signaling. These findings suggest that disrupted calcium signaling and overexpression of GRIN2B and CHRM3 drive neuronal hyperexcitability, providing insight into the pathophysiology of SB. Targeting these pathways may inform therapeutic strategies for SB treatment.

Sleep bruxism (SB) involves involuntary jaw movements during sleep and has been linked to impaired inhibitory regulation of brainstem circuits (particularly those involving GABAergic neurons). While previous studies using patch-clamp electrophysiology have demonstrated intrinsic hyperexcitability in neurons differentiated from SB patient-derived human induced pluripotent stem cells (hiPSCs), the low-throughput nature of this technique limits large-scale phenotypic screening. We aimed to establish a robust, high-throughput, multielectrode array (MEA)-based platform capable of quantitatively assessing electrophysiological phenotypes of SB-derived neurons. hiPSCs from three patients with SB and three healthy controls were differentiated to become ventral brainstem-like neurons. Their neuronal composition was evaluated using immunocytochemistry for total neurons (TUBB3⁺) and GABAergic neurons (GAD1/2⁺). Spontaneous neuronal firing was assessed using MEA under steady-state conditions and immediately after medium change. Extracellular GABA levels were quantified via ELISA to indirectly assess GABAergic hyperexcitability. Differentiation efficiency to TUBB3⁺ or GAD1/2⁺ neurons did not differ among cell lines. Under steady-state conditions, the weighted mean firing rate (wMFR) did not differ between groups. The SB group showed significantly elevated extracellular GABA concentrations and a significant increase in wMFR immediately after medium change (GABA removal), suggesting that GABA hypersecretion concealed the underlying hyperexcitability of SB-derived neurons. We developed a robust, high-throughput MEA platform that reliably quantifies SB-related electrophysiological phenotypes. Our findings indicate that SB-derived GABAergic neurons exhibit constitutive hyperexcitability and excessive GABA release, providing mechanistic insights and a scalable framework for therapeutic discovery.

Parkinson’ disease is increasingly recognized as a systemic disorder involving pathological communication along the brain–gut axis, yet the cellular mechanisms underlying inter-organ α-synuclein propagation remain poorly defined. Here, we establish a human induced pluripotent stem cell–derived brain–intestinal assembloid that integrates a developmentally specified medullary hindbrain organoid with an enteric neuron–enriched intestinal organoid. Using this model, we show the propagation of α-synuclein from brainstem neurons to enteric neurons. Ligand–receptor analysis combined with genetic perturbation identifies NRXN1–NLGN2 signaling as a key mediator of brain-to-gut α-synuclein propagation, with NRXN1 being upregulated in Parkinson’s disease patient datasets in vivo . Together, these findings establish a human stem cell–based platform for investigating brain–gut communication and uncover molecular mechanisms underlying α-synuclein propagation in Parkinson’s disease.