Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and the subsequent loss of the RNA-binding protein FMRP. N6-methyladenosine (m6A) is a prevalent mRNA modification essential for post-transcriptional regulation. FMRP binds and regulates the stability of m6A-containing transcripts. However, how FMRP deficiency impacts transcriptome-wide m6A modifications in FXS remains unknown. To address this, we generated cortical neurons from induced pluripotent stem cells (iPSCs) derived from healthy individuals and FXS patients. Electrophysiology recordings revealed synaptic and neuronal network defects in FXS iPSC-derived neurons. Transcriptome-wide analysis revealed striking m6A hypermethylation predominantly affecting synapse-associated transcripts. Mechanistically, we demonstrated that FMRP deficiency drives the aberrant translational upregulation of core m6A writers, a causal relationship definitively validated using CGG-corrected isogenic control lines. Targeted genetic knockdown of the m6A writer METTL3 successfully rescued synaptic phenotypes in FXS neurons, whereas its overexpression in control neurons phenocopied these synaptic defects, confirming the causal role of m6A dysregulation in FXS pathology. Notably, pharmacological intervention with the METTL3 inhibitor STM-2457 normalized methylation on synapse-associated transcripts and restored synaptic transmission in FXS neurons. Together, our findings uncover an FMRP-dependent epitranscriptomic mechanism contributing to FXS pathogenesis and suggest a promising avenue for m6A-targeted therapies.

Cells derived from rejuvenated human induced pluripotent stem cells (hiPSCs) require extended culture periods to achieve functional maturation, and it remains difficult to recapitulate cellular senescence in these cells in vitro. This limitation hinders the accurate and efficient modeling of age-related neurodegenerative diseases. Here, we aimed to establish a simple approach to promote neuronal maturation and improve the efficiency of hiPSC-based disease modeling. Using a small-molecule inhibitor library, we identified an ATM kinase inhibitor, KU60019, that promotes both maturation-associated features and senescence-associated phenotypes in hiPSC-derived neurons and fibroblasts. KU60019 treatment promoted the manifestation of disease-relevant phenotypes in hiPSC models of age-related neurodegenerative diseases. Furthermore, senolytic analyses suggested that KU60019-induced senescent cells depend on pro-survival pathways, including HSP90-associated signaling. These findings suggest that KU60019 provides a simple and useful tool for accelerating phenotypic recapitulation in hiPSC models of age-related neurodegenerative diseases.

Fragile X syndrome (FXS), the leading genetic cause of intellectual disability, arises from FMR1 gene silencing and loss of the FMRP protein. N6-methyladenosine (m 6 A) is a prevalent mRNA modification essential for post-transcriptional regulation. FMRP is known to bind to and regulate the stability of m 6 A-containing transcripts. However, how loss of FMRP impacts on transcriptome-wide m 6 A modifications in FXS patients remains unknown. To answer this question, we generated cortical neurons differentiated from induced pluripotent stem cells (iPSC) derived from healthy subjects and FXS patients. In electrophysiology recordings, we validated that synaptic and neuronal network defects in iPSC-derived FXS neurons corresponded to the clinical EEG data of the patients from which the corresponding iPSC line was derived. In analysis of transcriptome-wide methylation, we show that FMRP deficiency led to increased translation of m 6 A writers, resulting in hypermethylation that primarily affecting synapse-associated transcripts and increased mRNA decay. Conversely, in the presence of an m 6 A writer inhibitor, synaptic defects in FXS neurons were rescued. Taken together, our findings uncover that an FMRP-dependent epi-transcriptomic mechanism contributes to FXS pathogenesis by disrupting m 6 A modifications in FXS, suggesting a promising avenue for m 6 A- targeted therapies.

The human blood-brain barrier (hBBB) is a highly specialized structure that regulates passage across blood and central nervous system (CNS) compartments. Despite its critical physiological role, there are no reliable in vitro models that can mimic hBBB development and function. Here, we constructed hBBB assembloids from brain and blood vessel organoids derived from human pluripotent stem cells. We validated the acquisition of blood-brain barrier (BBB)-specific molecular, cellular, transcriptomic, and functional characteristics and uncovered an extensive neuro-vascular crosstalk with a spatial pattern within hBBB assembloids. When we used patient-derived hBBB assembloids to model cerebral cavernous malformations (CCMs), we found that these assembloids recapitulated the cavernoma anatomy and BBB breakdown observed in patients. Upon comparison of phenotypes and transcriptome between patient-derived hBBB assembloids and primary human cavernoma tissues, we uncovered CCM-related molecular and cellular alterations. Taken together, we report hBBB assembloids that mimic the core properties of the hBBB and identify a potentially underlying cause of CCMs.

The neuron-glia cross-talk is critical to brain homeostasis and is particularly affected by neurodegenerative diseases. How neurons manipulate the neuron-astrocyte interaction under pathological conditions, such as hyperphosphorylated tau, a pathological hallmark in Alzheimer’s disease (AD), remains elusive. In this study, we identified excessively elevated neuronal expression of adenosine receptor 1 (Adora1 or A1R) in 3×Tg mice, MAPT P301L (rTg4510) mice, patients with AD, and patient-derived neurons. The up-regulation of A1R was found to be tau pathology dependent and posttranscriptionally regulated by Mef2c via miR-133a-3p. Rebuilding the miR-133a-3p/A1R signal effectively rescued synaptic and memory impairments in AD mice. Furthermore, neuronal A1R promoted the release of lipocalin 2 (Lcn2) and resulted in astrocyte activation. Last, silencing neuronal Lcn2 in AD mice ameliorated astrocyte activation and restored synaptic plasticity and learning/memory. Our findings reveal that the tau pathology remodels neuron-glial cross-talk and promotes neurodegenerative progression. Approaches targeting A1R and modulating this signaling pathway might be a potential therapeutic strategy for AD.

α-Synuclein (αS), the causative protein of Parkinson’s disease and other α-synucleinopathies, aggregates from a low molecular weight form (LMW-αS) to a high molecular weight αS oligomer (HMW-αSo). Aggregated αS accumulates intracellularly, induces intrinsic apoptosis, is released extracellularly, and appears to propagate disease through prion-like spreading. Whether extracellular αS aggregates are cytotoxic, damage cell wall, or induce cell death is unclear. We investigated cytotoxicity and cell death caused by HMW-αSo or LMW-αS. Extracellular HMW-αSo was more cytotoxic than LMW-αS and was a crucial factor for inducing plasma membrane damage and cell death. HMW-αSo induced reactive oxygen species production and phospholipid peroxidation in the membrane, thereby impairing calcium homeostasis and disrupting plasma membrane integrity. HMW-αSo also induced extrinsic apoptosis and cell death by activating acidic sphingomyelinase. Thus, as extracellular HMW-αSo causes neuronal injury and death via cellular transmission and direct plasma membrane damage, we propose an additional disease progression pathway for α-synucleinopathies.

Sleep bruxism (SB) is a sleep-related movement disorder characterized by grinding and clenching of the teeth during sleep. We previously found a significant association between SB and a single nucleotide polymorphism (SNP), rs6313, in the neuronal serotonin 2A receptor gene (HTR2A), and established human induced pluripotent stem cell (iPSC)-derived neurons from SB patients with a genetic variant. To elucidate the electrophysiological characteristics of SB iPSC-derived neural cells bearing an SB-related genetic variant, we generated ventral hindbrain neurons from SB patients and unaffected controls, and explored the intrinsic membrane properties of these neurons using the patch-clamp technique. We found that the electrophysiological properties of iPSC-derived neurons mature in a time-dependent manner in long-term control cultures. SB neurons exhibited higher action potential firing frequency, higher gain, and shorter action potential half duration. This is the first in vitro modeling of SB using patient-specific iPSCs. The revealed electrophysiological characteristics may serve as a benchmark for further investigation of pathogenic mechanisms underlying SB. Moreover, our results on long-term cultures provide a strategy to define the functional maturity of human neurons in vitro, which can be implemented for stem cell research of neurogenesis, and neurodevelopmental disorders.

The hiPSCs of two sleep bruxism (SB) patients with the risk allele (C) of HTR2A SNP rs6313 (SB1 and SB2) and two unaffected controls without the risk allele (C1 and C2) were established and differentiated to ventral hindbrain neurons. qRT-PCR analyses were performed to exhibit neuronal characteristics. Patch-clamp recordings were conducted to explore the membrane properties of hiPSC-derived neurons.

Motoneurons that innervate the jaw-closing and jaw-opening muscles play a critical role in oro-facial behaviors, including mastication, suckling, and swallowing. These motoneurons can alter their physiological properties through the postnatal period during which feeding behavior shifts from suckling to mastication; however, the functional synaptic properties of developmental changes in these neurons remain unknown. Thus, we explored the postnatal changes in glutamatergic synaptic transmission onto the motoneurons that innervate the jaw-closing and jaw-opening musculatures during early postnatal development in rats. We measured miniature excitatory postsynaptic currents (mEPSCs) mediated by non-NMDA receptors (non-NMDA mEPSCs) and NMDA receptors in the masseter and digastric motoneurons. The amplitude, frequency, and rise time of non-NMDA mEPSCs remained unchanged among postnatal day (P)2-5, P9-12, and P14-17 age groups in masseter motoneurons, whereas the decay time dramatically decreased with age. The properties of the NMDA mEPSCs were more predominant at P2-5 masseter motoneurons, followed by reduction as neurons matured. The decay time of NMDA mEPSCs of masseter motoneurons also shortened remarkably across development. Furthermore, the proportion of NMDA/non-NMDA EPSCs induced in response to the electrical stimulation of the supratrigeminal region was quite high in P2-5 masseter motoneurons, and then decreased toward P14-17. In contrast to masseter motoneurons, digastric motoneurons showed unchanged properties in non-NMDA and NMDA EPSCs throughout postnatal development. Our results suggest that the developmental patterns of non-NMDA and NMDA receptor-mediated inputs vary among jaw-closing and jaw-opening motoneurons, possibly related to distinct roles of respective motoneurons in postnatal development of feeding behavior.

The serotonin 5-HT 2A receptor (5-HT 2A R) has been receiving increasing attention because its genetic variants have been associated with a variety of neurological diseases. To elucidate the pathogenesis of the neurological diseases associated with 5-HT 2A R gene ( HTR2A ) variants, we have previously established a protocol to induce HTR2A -expressing neurons from human-induced pluripotent stem cells (hiPSCs). Here, we investigated the maturation stages and electrophysiological properties of HTR2A -positive neurons induced from hiPSCs and constructed an HTR2A promoter-specific reporter lentivirus to label the neurons. We found that neuronal maturity increased over time and that HTR2A expression was induced at the late stage of neuronal maturation. Furthermore, we demonstrated successful labelling of the HTR2A -positive neurons, which had fluorescence and generated repetitive action potentials in response to depolarizing currents and an inward current during the application of TCB-2, a selective agonist of 5-HT 2A Rs, respectively. These results indicated that our in vitro model mimicked the in vivo dynamics of 5-HT 2A R. Therefore, in vitro monitoring of the function of HTR2A -positive neurons induced from hiPSCs could help elucidate the pathophysiological mechanisms of neurological diseases associated with genetic variations of the HTR2A gene.