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Biology subjects

Lee, S.-K.

Publications and source records attributed to Lee, S.-K..

13 recordsLinked to original sources

AAV-mediated neuronal expression of FOXG1 restores oligodendrocyte maturation, myelination, and hippocampal structure in mouse models of FOXG1 Syndrome

FOXG1 syndrome is a devastating neurodevelopmental disorder caused by haploinsufficiency of the transcription factor FOXG1, leading to intellectual disability, epilepsy, and white-matter deficits. Although FOXG1 is well known for its neuronal functions, its role in glial pathology remains poorly understood. Here, we show that reducing FOXG1 selectively in neurons impairs oligodendrocyte lineage progression and myelination, establishing a critical non-cell-autonomous role for neuronal FOXG1 in glial maturation. To restore FOXG1 in neurons, we developed AAV vectors expressing human FOXG1 under neuron-specific promoters. Neonatal administration of these vectors normalized oligodendrocyte precursor cell (OPC) accumulation, enhanced myelination, and corrected hippocampal structural abnormalities in Foxg1 conditional heterozygous mice. To test therapeutic robustness under stringent conditions, we used the patient-specific W300X heterozygous model, which combines FOXG1 loss-of-function with a toxic truncated protein and represents one of the most severe FOXG1 syndrome genotypes. Remarkably, neuron-restricted AAV-FOXG1 delivery produced substantial rescue even in this high-bar model, suppressing OPC overaccumulation, restoring myelination, and progressively improving dentate gyrus morphology, with benefits persisting into adulthood. Moreover, adolescent administration remained highly effective, rescuing myelination, axonal bundle thickness, and microglial activation. These findings identify neuronal FOXG1 as a master regulator of neuron-glia interactions and establish neuron-targeted AAV-FOXG1 as a potent and clinically translatable therapeutic strategy across diverse severities of FOXG1 syndrome.

developmental biology↗

Human patient-specific FOXG1 syndrome mouse model revealed FOXG1-MYCN-mediated regulation of protein homeostasis in neurodevelopmental disorder

Neurodevelopmental disorders are characterized by disruptions in brain development, resulting in cognitive, behavioral, and neurological impairments. FOXG1 syndrome (FS), caused by heterozygous mutations in the FOXG1 gene, exemplifies a severe monogenic neurodevelopmental disorder. To investigate its pathogenesis, we generated a patient-specific W300X mouse model carrying a truncation variant of FOXG1. We found that the truncated FOXG1 protein in W300X-heterozygous (W300X-Het) mice is more abundant and more nuclear-localized than the full-length FOXG1 protein, implicating a pathogenic mechanism involving the truncated protein. Interestingly, W300X-Het mice exhibited profound abnormalities in the dentate gyrus, including disrupted neurogenesis, impaired granule cell migration, and altered dendritic morphology. Transcriptomic profiling identified broad dysregulation in protein homeostasis pathways, particularly ribosomal biogenesis, translation, and proteostasis. Disruption of the FOXG1-MYCN pathway, critical for robust protein synthesis during neural stem cell division, synaptogenesis, and synaptic plasticity, emerged as a key mechanism underlying these defects. In parallel, microglial activation and inflammation were markedly increased in the dentate gyrus, contributing to a pro-inflammatory environment that exacerbates neurogenic and structural deficits. Consistent with hippocampal dysfunction in FS patients, W300X-Het mice exhibited significant spatial learning and memory impairments. Together, our study highlights disrupted protein homeostasis and neuroinflammation as key drivers of FS pathogenesis, providing a framework for developing therapeutic strategies targeting these pathways.

neuroscience↗

Searching for influencers among placental immune cells in preeclampsia.

Cells in maternal and fetal immune systems may communicate, leading to immune tolerance during pregnancy; however, this hypothesis remains controversial. Here, we profiled single-cell transcriptional signatures in placental layers comprising the maternal-fetal interface and deep placenta, then searched for genes associated with preeclampsia. To investigate the underlying principle of the failure of immune tolerance, we started by clarifying the systemic framework, comprising models of immune interaction frequency (IIF) and specific triggers (i.e., influencers) of tolerance (IT). We generated single-cell transcriptional profiles of normal term (Norms) and preeclampsia preterm (PePT) parturitions. Fetal and maternal cells are admixed across the placenta, for both Norms and PePTs, rejecting the IIF model of immune failure during pregnancy posed by excessive interactions between fetomaternal cells. Whereas placental layers are well mixed with maternal cells, we identified a conserved gradual immune transition of fetal T-cells in both PePT and Norm, disproving the IIF model. To search for influencers of PePT in the IT model, we established and validated a classification model for PePT and Norm immune cells, including T-cells, and then prioritized major contributors to the classifier model, which are highly enriched in ligands and receptors (p = 5.98e-5). Among the prioritized ligand receptors, SPP1 and CD44 are suggested as influencers of inflammation signatures and were experimentally validated by the exclusive colocalization of SPP1- and CD44-expressing cells in the PePT placentas. Different interleukin-4 and interferon-{psi} levels in the serum and urine of PePTs further support the contribution of SPP1 to associated pathways, including allograft rejection. Our findings provide insight into the influence of specific immune interactions between cells in the human placenta and their influencer-derived impact on PePT.

bioinformatics↗

The patient-specific mouse model with Foxg1 frameshift mutation provides insights into the pathophysiology of FOXG1 syndrome

AbstractsSingle allelic mutations in the forebrain-specific transcription factor gene FOXG1 lead to FOXG1 syndrome (FS). To decipher the disease mechanisms of FS, which vary depending on FOXG1 mutation types, patient-specific animal models are critical. Here, we report the first patient-specific FS mouse model, Q84Pfs heterozygous (Q84Pfs-Het) mice, which emulates one of the most predominant FS variants. Remarkably, Q84Pfs-Het mice recapitulate various human FS phenotypes across cellular, brain structural, and behavioral levels, such as microcephaly, corpus callosum agenesis, movement disorders, repetitive behaviors, and anxiety. Q84Pfs-Het cortex showed dysregulations of genes controlling cell proliferation, neuronal projection and migration, synaptic assembly, and synaptic vesicle transport. Interestingly, the FS-causing Q84Pfs allele produced the N-terminal fragment of FOXG1, denoted as Q84Pfs protein, in Q84Pfs-Het mouse brains. Q84Pfs fragment forms intracellular speckles, interacts with FOXG1 full-length protein, and triggers the sequestration of FOXG1 to distinct subcellular domains. Q84Pfs protein also promotes the radial glial cell identity and suppresses neuronal migration in the cortex. Together, our study uncovered the role of the FOXG1 fragment derived from FS-causing FOXG1 variants and identified the genes involved in FS-like cellular and behavioral phenotypes, providing essential insights into the pathophysiology of FS.

neuroscience↗

Mll4 regulates tooth enamel development

Amelogenesis, the process of enamel formation, is tightly regulated and essential for producing the tooth enamel that protects teeth from decay and wear. Disruptions in amelogenesis can result in amelogenesis imperfecta, a group of genetic conditions characterized by defective enamel, including enamel hypoplasia, marked by thin or underdeveloped enamel. Mutations in the KMT2D (MLL4) gene, which encodes a histone H3-lysine 4-methyltransferase, are associated with Kabuki syndrome, a developmental disorder that can involve dental anomalies such as enamel hypoplasia. However, the specific role of KMT2D in amelogenesis remains poorly understood. To address this gap, we generated a conditional knockout mouse model with ectoderm-specific deletion of Kmt2d (Krt14-Cre;Kmt2dfl/fl, or Kmt2d-cKO) and characterized the resulting enamel defects using gross, radiographic, histological, cellular, and molecular analyses. Micro-computed tomography and scanning electron microscopy revealed that adult Kmt2d-cKO mice exhibited 100% penetrant amelogenesis imperfecta, characterized by hypoplastic and hypomineralized enamel, partially phenocopying human Kabuki syndrome. Additionally, Kmt2d-cKO neonates developed molar tooth germs with subtle cusp shape alterations and mild delays in ameloblast differentiation at birth. RNA-seq analysis of the first molar tooth germ at birth revealed that 33.7% of known amelogenesis-related genes were significantly downregulated in the Kmt2d-cKO teeth. Integration with KMT2D CUT&RUN-seq results identified 8 overlapping genes directly targeted by KMT2D. Re-analysis of a single-cell RNA-seq dataset in the developing mouse incisors revealed distinct roles for these genes in KMT2D-regulated differentiation across various cell subtypes within the dental epithelium. Among these genes, Satb1 and Sp6 are likely direct targets involved in the differentiation of pre-ameloblasts into ameloblasts. Taken together, we propose that KMT2D plays a crucial role in amelogenesis by directly activating key genes involved in ameloblast differentiation, offering insights into the molecular basis of enamel development and related dental pathologies.

developmental biology↗

Mll4 regulates postnatal palate growth and midpalatal suture development

MLL4, also known as KMT2D, is a histone methyltransferase that acts as an important epigenetic regulator in various organogenesis programs. Mutations in the MLL4 gene are the major cause of Kabuki syndrome, a human developmental disorder that involves craniofacial birth defects, including anomalies in the palate. This study aimed to investigate the role of MLL4 and the underlying mechanisms in the development and growth of the palate. We generated a novel conditional knockout (cKO) mouse model with tissue-specific deletion of Mll4 in the palatal mesenchyme. Using micro-computed tomography (CT), histological analysis, cell mechanism assays, and gene expression profiling, we examined palate development and growth in the Mll4-cKO mice. Gross craniofacial examination at adult stages revealed mild midfacial hypoplasia and midline defects of the palate in Mll4-cKO mice, including a widened midpalatal suture and disrupted midline rugae pattern. Micro-CT-based time-course skeletal analysis during postnatal palatogenesis through adulthood demonstrated a transverse growth deficit in overall palate width in Mll4-cKO mice. Whole-mount and histological staining at perinatal stages identified that the midline defects in the Mll4-cKO mice emerged as early as one day prior to birth, presenting as a widened midpalatal suture, accompanied by increased cell apoptosis in the suture mesenchyme. Genome-wide mRNA expression analysis of the midpalatal suture tissue revealed that MLL4 is essential for the timely expression of major cartilage development genes, such as Col2a1 and Acan, at birth. Immunofluorescence staining for osteochondral differentiation markers demonstrated a marked decrease in the chondrogenic marker COL2A1, while the expression of the osteogenic marker RUNX2 remained unchanged, in the Mll4-cKO midpalatal suture. Additionally, SOX9, a master regulator of chondrogenesis, exhibited a significant decrease in protein expression. Indeed, time-course histological analysis during postnatal palate growth revealed retardation in the development of the suture cartilage in Mll4-cKO mice. Taken together, our results demonstrate that MLL4 is essential for orchestrating key cellular and molecular events that ensure proper midpalatal suture development and palate growth.

developmental biology↗

Detection of changes in membrane potential by magnetic resonance imaging

Membrane potential plays a crucial role in various cellular functions. However, existing techniques for measuring membrane potential are often invasive or have limited recording depth. In contrast, magnetic resonance imaging (MRI) offers noninvasive imaging with desirable spatial resolution over large areas. This study investigates the feasibility of utilizing MRI to detect responses of cultured cells and in vivo rat cortex to membrane potential-modulating ionic solutions by measuring magnetic resonance parameters. Our findings reveal that depolarizing (or hyperpolarizing) ionic solutions increase (or decrease) the T2 relaxation time, while the ratio of bound to free water proton shows the opposite trend. These findings also suggest a potential approach to noninvasively detect changes in membrane potential using MRI.

biophysics↗

Antibody-conjugating nanogel (Conjugel) with two immune checkpoint inhibitors for enhanced cancer immunotherapy

Cancer immunotherapy by immune checkpoint inhibitors (ICI) acts on antitumor responses by stimulating the immune system to attack cancer cells. However, this powerful therapy is hampered by its high treatment cost and limited efficacy. Here, we show the development of an antibody-conjugating system (Conjugel) that potentiates the efficacy of bispecific immunotherapy that simultaneously targets CTLA-4 and PD-L1. The Conjugel, consisting of highly deformable nanogels and antibody-binding protein, was loaded with two ICI monoclonal antibodies (mAb). Compared with mAb treatment alone, treatment with a bispecific Conjugel loaded with the both ICIs significantly decreased both the survival of MCF-7 and MDA-MB-231 breast cancer cells in vitro and the size of 4T1-Luc2-derived orthotopic syngeneic tumors in vivo. Furthermore, the ICI-loaded Conjugel was less toxic in vivo than the combination treatment delivered as a bolus. Our findings have important implications for Conjugel-based immunotherapy, developing the safer and higher efficacy of ICIs to treat breast cancers.

bioengineering↗

In vivo direct imaging of neuronal activity at high temporo-spatial resolution

There has been a longstanding demand for noninvasive neuroimaging methods capable of detecting neuronal activity at both high temporal and spatial resolution. Here, we propose a novel method that enables Direct Imaging of Neuronal Activity for functional MRI (termed DIANA-fMRI) that can dynamically image spiking activity in milliseconds precision, while retaining the original benefit of high spatial resolution of MRI. DIANA-fMRI was demonstrated through in vivo mice brain imaging at 9.4 T applying electrical whisker-pad stimulation, directly imaging the spiking activity as well as capturing its sequential propagation along the thalamocortical pathway, as further confirmed through in vivo spike recording and optogenetics. DIANA-fMRI will open up new avenues in brain science by providing a deeper understanding of the brains functional organization including neural networks.

neuroscience↗

Neural correlates of episodic memory modulated by temporally delayed rewards

When a stimulus is associated with an external reward, its chance of being consolidated into long-term memory is boosted via dopaminergic facilitation of long-term potentiation in the hippocampus. Given that higher temporal distance (TD) has been found to discount the subjective value of a reward, we hypothesized that memory performance associated with a more immediate reward will result in better memory performance. We tested this hypothesis by measuring both behavioral memory performance and brain activation using functional magnetic resonance imaging (fMRI) during memory encoding and retrieval tasks. Contrary to our hypothesis, both behavioral and fMRI results suggest that the TD of rewards might enhance the chance of the associated stimulus being remembered. The fMRI data demonstrate that the lateral prefrontal cortex, which shows encoding-related activation proportional to the TD, is reactivated when searching for regions that show activation proportional to the TD during retrieval. This is not surprising given that this region is not only activated to discriminate between future vs. immediate rewards, it is also a part of the retrieval-success network. These results provide support for the conclusion that the encoding-retrieval overlap provoked as the rewards are more delayed lead to better memory performance of the items associated with the rewards.

neuroscience↗

Selection of HIV-1 for Resistance to Fourth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in the high drug concentrations that are achived in vivo. Little is known about the de novo resistance pathway for DRV. We have selected for resistance to high drug concentrations against ten PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small chemical changes in the inhibitor P1 side chain led to preferential use of one pathway over the other. Changes in the inhbitor P2 side chain determined differences in potency that were retained in the resistant viruses. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fourth generation PIs, and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.

microbiology↗

HIV-1 Protease Uses Bi-Specific S2/S2’ Subsites To Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PR) have a unique specificity that allows cleavage of sites with or without a P1 proline. A P1 proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 iterations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1-P2), with two complementary sets of rules. When P1 is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2 amino acid interacts with a hydrophobic region of the S2 subsite. When P1 is not proline, the orientations of the P2 and P2 side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite while the P2 amino acid usually engages hydrophilic amino acids in the S2 subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2 subsites to accommodate the steric effects imposed by a P1 proline on the orientation of P2 and P2 substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.

biochemistry↗

Therapeutic effects of Hypoxia-Inducible Factor-1α (HIF-1α) on bone formation around implants in diabetic mice

Patients with uncontrolled diabetes are susceptible to implant failure due to impaired bone metabolism. Hypoxia-Inducible Factor 1 (HIF-1), a transcription factor that is up-regulated in response to reduced oxygen condition during the bone repair process after fracture or osteotomy, is known to mediate angiogenesis and osteogenesis. However, its function is inhibited under hyperglycemic conditions in diabetic patients. The aim of this study is to evaluate the effects of exogenous HIF-1 on bone formation around implants by applying HIF-1 to diabetic mice via a novel PTD-mediated DNA delivery system. Smooth surface implants (1mm in diameter; 2mm in length) were placed in the both femurs of diabetic and normal mice. HIF-1 and placebo gels were injected to implant sites of the right and left femurs, respectively: Normal mouse with HIF-1 gel (NH), Normal mouse with placebo gel (NP), Diabetic mouse with HIF-1 gel (DH), and Diabetic mouse with placebo gel (DP). RNA sequencing was performed 4 days after surgery. Based on RNA sequencing, Differentially Expressed Genes (DEGs) were identified and HIF-1 target genes were selected. Histologic and histomorphometric results were evaluated 2 weeks after the surgery. The results showed that bone-to-implant contact (BIC) and bone volume (BV) were significantly greater in the DH group than the DP group (p < 0.05). A total of 216 genes were differentially expressed in DH group compared to DP group. On the other hand, there were 95 DEGs in the case of normal mice. Twenty-one target genes of HIF-1 were identified in diabetic mice through bioinformatic analysis of DEGs. Among the target genes, NOS2, GPNMB, CCL2, CCL5, CXCL16 and TRIM63 were manually found to be associated with wound healing-related genes. In conclusion, local administration of HIF-1 via PTD may help bone formation around the implant and induce gene expression more favorable to bone formation in diabetic mice.

molecular biology↗