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Cancer stem cells (CSCs) are a group of specially programmed tumor cells that possess the characteristics of perpetual cell renewal, increased invasiveness, and often, drug resistance. Hence, eliminating CSCs is a major challenge for cancer treatment. Understanding the cellular programs that maintain CSCs, and identifying the critical regulators for such programs, are major undertakings in both basic and translational cancer research. Recently, we have reported that RAB4A is a major regulator of epithelial-to-mesenchymal transition (EMT) and it does so mainly through regulating the activation of RAC1 GTPase. In the current study, we have delineated a new signaling circuitry through which RAB4A transmits its control of cancer stemness. Using in vitro and in vivo studies, we show that RAB4A, as the upstream regulator, relays signal stepwise to NUMB, NOTCH1, RAC1, and then SOX2 to control the self-renewal property of multiple cancer cells of diverse tissue origins. Knockdown of NUMB, or overexpression of NICD (the active fragment NOTCH1) or SOX2, rescued the in vitro sphere-forming and in vivo tumor-forming abilities that were lost upon RAB4A knockdown. Furthermore, we discovered that the chain of control is mostly through transcriptional regulation at every step of the pathway. The discovery of the novel signaling axis of RAB4A-NUMB-NOTCH-SOX2 opens the path for further expansion of the signaling chain and for the identification of new regulators and interacting proteins important for CSC functions, which can be explored to develop new and effective therapies., (© 2024. The Author(s).)

Background: CHRDL1 belongs to a novel class of mRNA molecules. Nonetheless, the specific biological functions and underlying mechanisms of CHRDL1 in oral squamous cell carcinoma (OSCC) remain largely unexplored., Methods: RT-qPCR and immunohistochemical staining were employed to assess the mRNA and protein expression levels of the MED29 gene in clinical samples of OSCC. Additionally, RT-qPCR and Western Blot analyses were conducted to investigate the mRNA and protein expression levels of the MED29 gene specifically in OSCC. The impact of MED29 on epithelial-mesenchymal transition (EMT), invasion, and migration of OSCC was evaluated through scratch assay, transwell assay, and immunofluorescence staining. Furthermore, wound healing assay and Transwell assay were utilized to examine whether CHRDL1 influences the malignant behavior of OSCC by modulating MED29 in vitro. The regulatory role of CHRDL1 on MED29 was further elucidated in vivo through a tail vein lung metastasis model in nude mice., Results: MED29 expression was elevated in tumor tissues of OSCC patients compared with adjacent cancer tissues. Moreover, in CAL27 and SCC25 cell lines, MED29 was upregulated and associated with increased cell migration and invasion abilities. Overexpression of MED29 facilitated EMT in OSCC cell lines, whereas knockdown of MED29 impeded EMT, resulting in diminished cell migration and invasion capacities. CHRDL1 exerted inhibitory effects on the expression of MED29, thereby suppressing EMT progression and consequently restraining the invasion and migration of OSCC cells. Furthermore, CHRDL1 mediated the inhibition of migration of OSCC cell lines to the OSCC through its regulation of MED29., Conclusions: MED29 facilitated the epithelial-mesenchymal transition process in OSCC, thereby promoting migration and invasion. On the other hand, CHRDL1 exerted inhibitory effects on the invasion and metastasis of OSCC by suppressing MED29 through the inhibition of the MAPK signaling pathway., (© 2024. The Author(s).)

Background: Renal cell carcinoma (RCC), especially clear cell RCC (ccRCC), significantly impacts health, and results in particularly poor outcomes in patients at the advanced stage. Resistance to vascular endothelial growth factor (VEGF) pathway-targeting tyrosine kinase inhibitors (TKIs) is a major barrier in effective ccRCC treatment. Herein, we aim to explore how decitabine mediates bridging integrator 1 (BIN1) and spectrin repeat containing nuclear envelope protein 1 (SYNE1) to impact resistance of ccRCC to sorafenib., Methods: Employing bioinformatics on datasets GSE64052 and CancerSea, we identified genes linked to TKI resistance, ultimately focusing on SYNE1. We assessed influences of SYNE1 overexpression and BIN1 knockdown via quantitative real-time PCR (qRT-PCR) and Western blot. Assessment of cell viability and apoptosis was accomplished using cell counting kit-8 (CCK-8) assays and flow cytometry. The investigation into the potential interactions between SYNE1 and BIN1, as well as their impacts on sorafenib sensitivity was accomplished by Co-Immunoprecipitation (Co-IP) and Glutathione-S-transferase (GST) Pull-down., Results: SYNE1 was substantially down-regulated in sorafenib-resistant ccRCC cells, and its overexpression increased sorafenib sensitivity, decreased viability and enhanced apoptosis. Interaction between BIN1 and SYNE1 was confirmed, with BIN1 level lower in resistant cells. BIN1 knockdown reduced the beneficial effects of SYNE1 overexpression on sorafenib sensitivity. Decitabine treatment elevated both SYNE1 and BIN1, while boosting apoptosis and reducing sorafenib resistance., Conclusions: SYNE1 contributes to the modulation of sorafenib resistance in ccRCC cells through interacting with BIN1. Decitabine treatment enhances expressions of these two proteins to improve TKI response, suggesting a potential strategy for counteracting resistance and bettering patient outcomes., (© 2024 The Author(s). Published by IMR Press.)

Background: Nesfatin-1 is a neuropeptide that regulates the hypothalamic-pituitary-gonadal axis and may play a role in uterus function. It is co-expressed with other peptides, such as phoenixin, which can influence sex hormone secretion. Our previous research has confirmed that phoenixin-14 is involved in the development of cystic endometrial hyperplasia (CEH) and pyometra in dogs. Therefore, based on the similarities and interactions between these neuropeptides, we hypothesized that nesfatin-1 might also regulate the reproductive system in dogs. This study aimed to determine the expression of nesfatin-1 and its interaction with phoenixin-14 in dogs with CEH or pyometra compared to healthy females, and concerning animals' body condition score (BCS 4-5/9 vs. BCS > 5/9)., Results: The analysis of nesfatin-1 in the uterus of bitches consisted of qPCR, western blot and immunofluorescence assays, and ELISAs. The results showed significantly higher nesfatin-1 encoding gene, nucleobindin-2 mRNA (Nucb2) and nesfatin-1 protein expression in overweight females and those suffering from CEH or pyometra compared to healthy animals. The immunoreactivity of nesfatin-1 was elevated in the uteri of bitches with higher BCS > 5/9. Moreover, nesfatin-1 blood concentrations increased in all examined overweight bitches. In the case of phoenixin signals, we found opposite results, regardless of the female body condition score., Conclusion: The etiology of CEH and pyometra are not fully known, although we have expanded the level of knowledge with respect to the possible interaction of nesfatin-1 and phoenixin in female dogs' uteri. They interact oppositely. With increasing female body weight, the expression of nesfatin-1 in the uterus and its peripheral blood concentration increased. However, for female dogs affected by CEH and pyometra, a decreased level of phoenixin-14, irrespective of their body condition score is characteristic. This knowledge could be crucial in the development of biomarkers for these conditions, which may lead to earlier recognition., (© 2024. The Author(s).)

How tissue-specific progenitor cells generate adult tissues is a puzzle in organogenesis. Using single-cell RNA sequencing of control and Six3 and Six6 compound-mutant mouse embryonic eyecups, we demonstrated that these two closely related transcription factors jointly control diverse target genes in multiple cell populations over the developmental trajectories of mouse embryonic retinal progenitor cells. In the Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) graph of control retinas, naïve retinal progenitor cells had two major trajectories leading to ciliary margin cells and retinal neurons, respectively. The ciliary margin trajectory was from naïve retinal progenitor cells in the G1 phase directly to ciliary margin cells, whereas the neuronal trajectory went through an intermediate neurogenic state marked by Atoh7 expression. Neurogenic retinal progenitor cells (Atoh7+) were still proliferative; early retinal neurons branched out from Atoh7+ retina progenitor cells in the G1 phase. Upon Six3 and Six6 dual deficiency, both naïve and neurogenic retinal progenitor cells were defective, ciliary margin differentiation was enhanced, and multi-lineage neuronal differentiation was disrupted. An ectopic neuronal trajectory lacking the Atoh7+ state led to ectopic neurons. Additionally, Wnt signaling was upregulated, whereas FGF signaling was downregulated. Notably, Six3 and Six6 proteins occupied the loci of diverse genes that were differentially expressed in distinct cell populations, and expression of these genes was significantly altered upon Six3 and Six6 dual deficiency. Our findings provide deeper insight into the molecular mechanisms underlying early retinal differentiation in mammals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ferrena et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Post-cardiac arrest brain (PCABI) is a severe medical condition characterized by a significant risk of neurological impairment and death. Nevertheless, the specific process and essential molecules responsible for its development are not fully understood. Profiling based on competing endogenous RNAs (ceRNA) has been implicated in the onset and progression of neurological disorders, yet its role in PCABI remains unclear. In this study, we performed RNA transcriptome sequencing analysis to identify differentially expressed genes in the rat model for cardiac arrest and cardiopulmonary resuscitation (CA/CPR). A hub ceRNA regulatory network was constructed using miRWalk 2.0 and Cytohubba plug-in in Cytoscape. Subsequently, real-time quantitative reverse transcription-polymerase chain reaction and dual-luciferase activity assays validated MSTRG.13,871, miR-155-5p, and Grip1 as differentially expressed in CA/CPR group, with MSTRG.13,871 capable of targeting both miR-155-5p and Grip1. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed the ceRNA network enrichment in immunoregulation mechanisms such as mitochondrion, apoptotic process, and negative regulation cell death. Our research highlights the mechanism of PCABI by revealing a critical regulatory axis involving MSTRG.13,871-miR-155-5p-Grip1 in the hippocampus CA1 region after CA/CPR in rats, proposing a feasible controlled mechanism, which may serve as a theoretical basis for designing innovative therapies., (© 2024. The Author(s).)

Autism Spectrum Disorder (ASD) is a developmental disorder characterized by impaired social communication and repetitive behaviors. In recent years, a pharmacological mouse model of ASD involving maternal administration of valproic acid (VPA) has become widely used. Newborn pups in this model show an abnormal balance between excitatory and inhibitory (E/I) signaling in neurons and exhibit ASD-like behavior. However, the molecular basis of this model and its implications for the pathogenesis of ASD in humans remain unknown. Using quantitative secretome analysis, we found that the level of leucine-rich repeat and immunoglobulin domain-containing protein 2 (Lingo2) was upregulated in the conditioned medium of VPA model neurons. This upregulation was associated with excitatory synaptic organizer activity. The secreted form of the extracellular domain of Lingo2 (sLingo2) is produced by the transmembrane metalloprotease ADAM10 through proteolytic processing. sLingo2 was found to induce the formation of excitatory synapses in both mouse and human neurons, and treatment with sLingo2 resulted in an increased frequency of miniature excitatory postsynaptic currents in human neurons. These findings suggest that sLingo2 is an excitatory synapse organizer involved in ASD, and further understanding of the mechanisms by which sLingo2 induces excitatory synaptogenesis is expected to advance our understanding of the pathogenesis of ASD., (© 2024. The Author(s).)

Astrocytes are morphologically complex cells that serve essential roles. They are widely implicated in central nervous system (CNS) disorders, with changes in astrocyte morphology and gene expression accompanying disease. In the Sapap3 knockout (KO) mouse model of compulsive and anxiety-related behaviors related to obsessive-compulsive disorder (OCD), striatal astrocytes display reduced morphology and altered actin cytoskeleton and Gi-G-protein-coupled receptor (Gi-GPCR) signaling proteins. Here, we show that normalizing striatal astrocyte morphology, actin cytoskeleton, and essential homeostatic support functions by targeting the astrocyte Gi-GPCR pathway using chemogenetics corrected phenotypes in Sapap3 KO mice, including anxiety-related and compulsive behaviors. Our data portend an astrocytic pharmacological strategy for rescuing phenotypes in brain disorders that include compromised astrocyte morphology and tissue support., Competing Interests: Declaration of interests UCLA filed a US provisional patent (no. 63/658,760) based on this work. B.S.K. is on the editorial advisory board of Neuron., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The WW and C2 domain-containing protein (WWC2) is implicated in several neurological disorders. Here, we demonstrate that WWC2 interacts with inhibitory, but not excitatory, postsynaptic scaffolds, consistent with prior proteomic identification of WWC2 as a putative component of the inhibitory postsynaptic density. Using mice lacking WWC2 expression in excitatory forebrain neurons, we show that WWC2 suppresses γ-aminobutyric acid type-A receptor (GABA A R) incorporation into the plasma membrane and regulates HAP1 and GRIP1, which form a complex promoting GABA A R recycling to the membrane. Inhibitory synaptic transmission is increased in CA1 pyramidal cells lacking WWC2. Furthermore, unlike the WWC2 homolog KIBRA (kidney/brain protein; WWC1), a key regulator of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking at excitatory synapses, the deletion of WWC2 does not affect synaptic AMPAR expression. In contrast, loss of KIBRA does not affect GABA A R membrane expression. These data reveal synapse class-selective functions for WWC proteins as regulators of ionotropic neurotransmitter receptors and provide insight into mechanisms regulating GABA A R membrane expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fate determination of neural stem cells (NSCs) is crucial for cortex development and is closely linked to neurodevelopmental disorders when gene expression networks are disrupted. The transcriptional corepressor chromodomain Y-like (CDYL) is widely expressed across diverse cell populations within the human embryonic cortex. However, its precise role in cortical development remains unclear. Here, we show that CDYL is critical for human cortical neurogenesis and that its deficiency leads to a substantial increase in gamma-aminobutyric acid (GABA)-ergic neurons in cortical organoids. Subsequently, neuronatin (NNAT) is identified as a significant target of CDYL, and its abnormal expression obviously influences the fate commitment of cortical NSCs. Cross-species comparisons of CDYL targets unravel a distinct developmental trajectory between human cortical organoids and the mouse cortex at an analogous stage. Collectively, our data provide insight into the evolutionary roles of CDYL in human cortex development, emphasizing its critical function in maintaining the fate of human cortical NSCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Double C2-like domain beta (DOC2B) is a vesicle priming protein critical for glucose-stimulated insulin secretion in β-cells. Individuals with type 1 diabetes (T1D) have lower levels of DOC2B in their residual functional β-cell mass and platelets, a phenotype also observed in a mouse model of T1D. Thus, DOC2B levels could provide important information on β-cell dys(function)., Objective: Our objective was to evaluate the DOC2B secretome of β-cells. In addition to soluble extracellular protein, we assessed DOC2B localized within membrane-delimited nanoparticles - extracellular vesicles (EVs). Moreover, in rat clonal β-cells, we probed domains required for DOC2B sorting into EVs., Method: Using Single Extracellular VEsicle Nanoscopy, we quantified EVs derived from clonal β-cells (human EndoC-βH1, rat INS-1 832/13, and mouse MIN6); two other cell types known to regulate glucose homeostasis and functionally utilize DOC2B (skeletal muscle rat myotube L6-GLUT4myc and human neuronal-like SH-SY5Y cells); and human islets sourced from individuals with no diabetes (ND). EVs derived from ND human plasma, ND human islets, and cell lines were isolated with either size exclusion chromatography or differential centrifugation. Isolated EVs were comprehensively characterized using dotblots, transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting., Results: DOC2B was present within EVs derived from ND human plasma, ND human islets, and INS-1 832/13 β-cells. Compared to neuronal-like SH-SY5Y cells and L6-GLUT4myc myotubes, clonal β-cells (EndoC-βH1, INS-1 832/13, and MIN6) produced significantly more EVs. DOC2B levels in EVs (over whole cell lysates) were higher in INS-1 832/13 β-cells compared to L6-GLUT4myc myotubes; SH-SY5Y neuronal-like cells did not release appreciable DOC2B. Mechanistically, we show that DOC2B was localized to the EV lumen; the tandem C2 domains were sufficient to confer sorting to INS-1 832/13 β-cell EVs., Discussion: Clonal β-cells and ND human islets produce abundant EVs. In cell culture, appreciable DOC2B can be packaged into EVs, and a small fraction is excreted as a soluble protein. While DOC2B-laden EVs and soluble protein are present in ND plasma, further studies will be necessary to determine if DOC2B originating from β-cells significantly contributes to the plasma secretome., Competing Interests: Part of the methodology used in this work is the subject of a provisional patent application. Patent holders may be entitled to certain compensation through their institutions’ respective intellectual property policies in the event such intellectual property is licensed. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Esparza, Lima, Abuelreich, Ghaeli, Hwang, Oh, Lenz, Gu, Jiang, Kandeel, Thurmond and Jovanovic-Talisman.)

Introduction: Because MRGPRX2 is now recognized as the mast cell receptor for basic secretagogues, there is currently a tremendous interest in whether MRGRPX2 could play an important role in various pruritic dermatoses such as chronic spontaneous urticaria. Therefore, we sought to identify new potent and selective antagonists to pharmacologically characterize the biological role of MRGPRX2., Methods: Various relevant in vitro , ex vivo , and in vivo model systems were used to investigate the role of MRGPRX2. This included the study of freshly isolated human skin mast cells and human basophils as well as an ex vivo human skin microdialysis preparation. The additivity of MRGPRX2 and FcεR1-mediated degranulation was also investigated. Human MRGPRX2 knock-in mice were generated to interrogate pharmacokinetic/pharmacodynamic relationships because both antagonists studied were shown to be human specific., Results: Two novel and structurally distinct MRGPRX2 antagonists were identified with one, Compound B, being orally active and demonstrating high potency in blocking Substance P-mediated degranulation using freshly isolated human skin mast cells with half maximal inhibitory concentration (IC 50 ) at 0.42 nM. Compound B also potently blocked Substance P-stimulated histamine release from resident mast cells in a human skin explant setup as well as blocking itch in an established behavioral scratching model using MRGPRX2 knock-in mice. Unlike human mast cells, Substance P failed to elicit a functional response in human basophils., Conclusion: These data fully support the investigation of MRGPRX2 receptor antagonists in mast cell-driven allergic skin disorders such as chronic spontaneous urticaria., Competing Interests: CHM, DVP, TR, DAG, SKP, and ZW are all employees of GSK. PSS is the Head of R&D at RefLab, and KB is an employee of RefLab. XD is the scientific co-founder and a consultant of Escient Pharmaceuticals and collaborator with GSK on MRGPRX projects. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Macphee, Dong, Peng, Paone, Skov, Baumann, Roethke, Goldspink, Pearson and Wu.)

Macrophages adopt distinct phenotypes in response to environmental cues, with type-2 cytokine interleukin-4 promoting a tissue-repair homeostatic state (M2 IL4 ). Glucocorticoids (GC), widely used anti-inflammatory therapeutics, reportedly impart a similar phenotype (M2 GC ), but how such disparate pathways may functionally converge is unknown. We show using integrative functional genomics that M2 IL4 and M2 GC transcriptomes share a striking overlap mirrored by a shift in chromatin landscape in both common and signal-specific gene subsets. This core homeostatic program is enacted by transcriptional effectors KLF4 and the glucocorticoid receptor, whose genome-wide occupancy and actions are integrated in a stimulus-specific manner by the nuclear receptor cofactor GRIP1. Indeed, many of the M2 IL4 :M2 GC -shared transcriptomic changes were GRIP1-dependent. Consistently, GRIP1 loss attenuated phagocytic activity of both populations in vitro and macrophage tissue-repair properties in the murine colitis model in vivo. These findings provide a mechanistic framework for homeostatic macrophage programming by distinct signals, to better inform anti-inflammatory drug design., (© 2024. The Author(s).)

Microglial abnormality and heterogeneity are observed in autism spectrum disorder (ASD) patients and animal models of ASD. Microglial depletion by colony stimulating factor 1-receptor (CSF1R) inhibition has been proved to improve autism-like behaviors in maternal immune activation mouse offspring. However, it is unclear whether CSF1R inhibition has extensive effectiveness and pharmacological heterogeneity in treating autism models caused by genetic and environmental risk factors. Here, we report pharmacological functions and cellular mechanisms of PLX5622, a small-molecule CSF1R inhibitor, in treating Cntnap2 knockout and valproic acid (VPA)-exposed autism model mice. For the Cntnap2 knockout mice, PLX5622 can improve their social ability and reciprocal social behavior, slow down their hyperactivity in open field and repetitive grooming behavior, and enhance their nesting ability. For the VPA model mice, PLX5622 can enhance their social ability and social novelty, and alleviate their anxiety behavior, repetitive and stereotyped autism-like behaviors such as grooming and marble burying. At the cellular level, PLX5622 restores the morphology and/or number of microglia in the somatosensory cortex, striatum, and hippocampal CA1 regions of the two models. Specially, PLX5622 corrects neurophysiological abnormalities in the striatum of the Cntnap2 knockout mice, and in the somatosensory cortex, striatum, and hippocampal CA1 regions of the VPA model mice. Incidentally, microglial dynamic changes in the VPA model mice are also reported. Our study demonstrates that microglial depletion and repopulation by transient CSF1R inhibition is effective, and however, has differential pharmacological functions and cellular mechanisms in rescuing behavioral deficits in the two autism models., (© 2024. The Author(s).)

While many mRNAs contain more than one translation initiation site (TIS), the functions of most alternative TISs and their corresponding protein isoforms (proteoforms) remain undetermined. Here, we showed that alternative usage of CUG and AUG TISs in neuronal pentraxin receptor (NPR) mRNA produced two proteoforms, of which the ratio was regulated by RNA secondary structure and neuronal activity. Downstream AUG initiation truncated the N-terminal transmembrane domain and produced a secreted NPR proteoform sufficient in promoting synaptic clustering of AMPA-type glutamate receptors. Mutations that altered the ratio of NPR proteoforms reduced AMPA receptors in parvalbumin-positive interneurons and affected learning behaviors in mice. In addition to NPR, upstream AUU-initiated N-terminal extension of C1q-like synaptic organizers anchored these otherwise secreted factors to the membrane. Together, these results uncovered the plasticity of N-terminal signal sequences regulated by alternative TIS usage as a potentially widespread mechanism in diversifying protein localization and functions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Reelin, a secreted glycoprotein, plays a crucial role in guiding neocortical neuronal migration, dendritic outgrowth and arborization, and synaptic plasticity in the adult brain. Reelin primarily operates through the canonical lipoprotein receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr). Reelin also engages with noncanonical receptors and unidentified coreceptors; however, the effects of which are less understood. Using high-throughput tandem mass tag (TMT) liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics and gene set enrichment analysis (GSEA), we identified both shared and unique intracellular pathways activated by Reelin through its canonical and noncanonical signaling in primary murine neurons of either sex during dendritic growth and arborization. We observed pathway cross talk related to regulation of cytoskeleton, neuron projection development, protein transport, and actin filament-based process. We also found enriched gene sets exclusively by the noncanonical Reelin pathway including protein translation, mRNA metabolic process, and ribonucleoprotein complex biogenesis suggesting Reelin fine-tunes neuronal structure through distinct signaling pathways. A key discovery is the identification of aldolase A, a glycolytic enzyme and actin-binding protein, as a novel effector of Reelin signaling. Reelin induced de novo translation and mobilization of aldolase A from the actin cytoskeleton. We demonstrated that aldolase A is necessary for Reelin-mediated dendrite growth and arborization in primary murine neurons and mouse brain cortical neurons. Interestingly, the function of aldolase A in dendrite development is independent of its known role in glycolysis. Altogether, our findings provide new insights into the Reelin-dependent signaling pathways and effector proteins that are crucial for dendritic development., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Dysregulated appetite is common in autism spectrum disorder (ASD) and it includes excessive interest in tasty foods. Overconsumption of palatable fluids has been found in the valproic acid-induced ASD rat. Though ASD has a strong genetic component, the link between ASD-related genes and appetite for palatable foods remains elusive. We focused on the CNTNAP2 gene whose deletion in mice recapitulates human ASD symptoms. We investigated whether Cntnap2-/- male mice consume greater amounts of palatable 10% sucrose, 0.1% saccharin, and 4.1% intralipid solutions offered in episodic meals either in a no-choice paradigm or a two-bottle choice test. We examined how sucrose intake affects c-Fos immunoreactivity in feeding-related brain areas. Finally, we determined doses at which intraperitoneal oxytocin decreases sucrose intake in mutants. In the single-bottle tests, Cntnap2-/- mice drank more sucrose, saccharin, and intralipid compared to WTs. Given a choice between two tastants, Cntnap2-/- mice had a higher preference for sucrose than intralipid. While the standard 1 mg/kg oxytocin dose reduced sucrose intake in WTs, a low oxytocin dose (0.1 mg/kg) decreased sucrose intake in Cntnap2-/- mice. Sucrose intake induced a more robust c-Fos response in wild-type (WT) than Cntnap2-/- mice in the reward and hypothalamic sites and it increased the percentage of Fos-immunoreactivity oxytocin neurons in WTs, but not in mutants. We conclude that Cntnap2-/- mice overconsume palatable solutions, especially sucrose, beyond levels seen in WTs. This excessive consumption is associated with blunted c-Fos immunoreactivity in feeding-related brain sites, and it can be reversed by low-dose oxytocin., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Three-needle electroacupuncture (TNEA) has shown promise as a non-pharmacological treatment for post-stroke depression (PSD). However, the underlying mechanisms of its therapeutic effects remain unclear. In this study, we investigated the potential molecular and synaptic mechanisms by which TNEA ameliorates depressive-like behaviors in a mouse model of PSD. Male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) to induce PSD and subsequently treated with TNEA for three weeks at specific acupoints (GV24 and bilateral GB13). Through a combination of behavioral tests, neuronal activation assessment, synaptic function examination, transcriptomic analysis, and various molecular techniques, we found that TNEA treatment significantly improved anxiety and depressive-like behaviors in PSD mice. These improvements were accompanied by enhanced neuronal activation in the medial prefrontal cortex (mPFC) and primary somatosensory cortex (PSC), as well as the promotion of excitatory synapse formation and transmission function in the mPFC. Transcriptomic analysis revealed that TNEA upregulated the expression of Netrin-G Ligand-3 (NGL-3), a postsynaptic cell adhesion molecule, in the mPFC. Further investigation showed that the extracellular domain of NGL-3 binds to the presynaptic protein L1cam, promoting the formation of Vesicular Glutamate Transporter 1 (vGluT1) puncta on neuronal dendrites. Notably, cortical neuron-specific knockout of NGL-3 abolished the antidepressant-like effects of TNEA in PSD mice, confirming the crucial role of the NGL-3/L1cam pathway in mediating the therapeutic effects of TNEA. These findings provide novel insights into the molecular and synaptic mechanisms underlying the therapeutic effects of acupuncture in the treatment of PSD and highlight the potential of targeting the NGL-3/L1cam pathway for the development of alternative interventions for PSD and other depressive disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Neuroinflammation has been gaining attention as one of the potential causes of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis in recent years. The suppression of excessive proinflammatory responses is expected to be a target for the treatment and prevention of neurodegenerative diseases. Collapsin response mediator protein 4 (CRMP4) is involved in cytoskeleton-associated axonal guidance in the developing brain. Recently, the involvement of CRMP4 in several pathological conditions, including inflammation induced by lipopolysaccharide (LPS), a widely used inflammatory molecule, has been reported. However, the role of CRMP4 in LPS-induced inflammation in vivo remains largely unknown. In this study, we generated microglia-specific CRMP4 knockout mice for the first time and examined the role of CRMP4 in an LPS-induced brain inflammation model. We found that microglia after LPS injection in substantia nigra was significantly reduced in Crmp4 -/- mice compared to Crmp4 +/+ mice. The increased expression of IL-10 in striatum samples was downregulated in Crmp4 -/- mice. A significant reduction in Iba1 expression was also observed in microglia-specific Crmp4 knockout mice compared with that in control mice. In contrast, the expression of IL-10 did not change in these mice, whereas arginase 1 (Arg1) expression was significantly suppressed. These results demonstrate the involvement of CRMP4 in LPS-induced inflammation in vivo, that CRMP4 suppresses microglial proliferation in a cell-autonomous manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ursolic acid (UA), a pentacyclic triterpene, exhibits diverse pharmacological effects, including potential treatment for allergic diseases. It downregulates thymic stromal lymphopoietin (TSLP) and disrupts mast cell signaling pathways. However, the exact molecular mechanism by which UA interferes with mast cell action remains unclear. Therefore, the current study aimed to uncover molecular entities underlying the effect of UA on mast cells and its potential antipruritic effect, specifically investigating its modulation of key molecules such as TRPV4, PAR2, and MRGPRX2, which are involved in TSLP regulation and sensation. Calcium imaging experiments revealed that UA pretreatment significantly suppressed MRGPRX2 activation (and its mouse orthologue MrgprB2), a G protein-coupled receptor predominantly expressed in mast cells. Molecular docking predictions suggested potential interactions between UA and MRGPRX2/MrgprB2. UA pretreatment also reduced mast cell degranulation through MRGPRX2 and MrgprB2-dependent mechanisms. In a dry skin mouse model, UA administration decreased tryptase and TSLP production in the skin, and diminished TSLP response in the sensory neurons. While PAR2 and TRPV4 activation enhances TSLP production, UA did not inhibit their activity. Notably, UA attenuated compound 48/80-induced scratching behaviors in mice and suppressed spontaneous scratching in a dry skin model. The present study confirms the effective inhibition of UA on MRGPRX2/MrgprB2, leading to reduced mast cell degranulation and suppressed scratching behaviors. These findings highlight the potential of UA as an antipruritic agent for managing various allergy- or itch-related conditions., Competing Interests: Declaration of competing interest The authors state no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

UNC13 family (also known as Munc13) proteins are evolutionarily conserved proteins involved in the rapid and regulated secretion of vesicles, including synaptic vesicles and cytotoxic granules. Fast and regulated secretion at the neuronal and immunological synapses requires multiple steps, from the biogenesis of vesicles to membrane fusion, and a complex array of proteins for each step. Defects at these steps can lead to various genetic disorders. Recent studies have shown multiple roles of UNC13D in the secretion of cytotoxic granules by immune cells. Here, the molecular structure and detailed roles of UNC13D in the biogenesis, tethering, and priming of cytotoxic vesicles and in endoplasmic reticulum are summarized. Moreover, its association with immune diseases, including familial hemophagocytic lymphohistiocytosis type 3, macrophage activation syndrome, juvenile idiopathic arthritis, and autoimmune lymphoproliferative syndrome, is reviewed. Finally, the therapeutic application of CRISPR/Cas9-based gene therapy for genetic diseases is introduced., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Duong, Lee, Kim and Oh.)

Previous in vitro studies have suggested that SLIT ligands could play roles in regulating ovarian granulosa cell proliferation and gene expression, as well as luteolysis. However, no in vivo study of Slit gene function has been conducted to date. Here, we investigated the potential role of Slit1 in ovarian biology using a Slit1-null mouse model. Female Slit1-null mice were found to produce larger litters than their wild-type counterparts due to increased ovulation rates. Increased ovarian weights in Slit1-null animals were found to be due to the presence of greater numbers of healthy antral follicles with similar numbers of atretic ones, suggesting both an increased rate of follicle recruitment and a decreased rate of atresia. Consistent with this, treatment of cultured granulosa cells with exogenous SLIT1 induced apoptosis in presence or absence of follicle-stimulating hormone, but had no effect on cell proliferation. Although few alterations in the messenger RNA levels of follicle-stimulating hormone-responsive genes were noted in granulosa cells of Slit1-null mice, luteinizing hormone target gene mRNA levels were greatly increased. Finally, increased phospho-AKT levels were found in granulosa cells isolated from Slit1-null mice, and SLIT1 pretreatment of cultured granulosa cells inhibited the ability of both follicle-stimulating hormone and luteinizing hormone to increase AKT phosphorylation, suggesting a mechanism whereby SLIT1 could antagonize gonadotropin signaling. These findings therefore represent the first evidence for a physiological role of a SLIT ligand in the ovary, and define Slit1 as a novel autocrine/paracrine regulator of follicle development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Skin mast cells (MCs) mediate acute allergic reactions in the cutaneous environment and contribute to chronic dermatoses, including urticaria, and atopic or contact dermatitis. The cAMP response element binding protein (CREB), an evolutionarily well conserved transcription factor (TF) with over 4,000 binding sites in the genome, was recently found to form a feedforward loop with KIT, maintaining MC survival. The most selective MC function is degranulation with its acute release of prestored mediators. Herein, we asked whether CREB contributes to the expression and function of the degranulation-competent receptors FcεRI and MRGPRX2. Interference with CREB by pharmacological inhibition (CREBi, 666-15) or RNA interference only slightly affected the expression of these receptors, while KIT was strongly attenuated. Interestingly, MRGPRX2 surface expression moderately increased following CREB-knockdown, whereas MRGPRX2-dependent exocytosis simultaneously decreased. FcεRI expression and function were regulated consistently, although the effect was stronger at the functional level. Preformed MC mediators (tryptase, histamine, β -hexosaminidase) remained comparable following CREB attenuation, suggesting that granule synthesis did not rely on CREB function. Collectively, in contrast to KIT, FcεRI and MRGPRX2 moderately depend on unperturbed CREB function. Nevertheless, CREB is required to maintain MC releasability irrespective of stimulus, insinuating that CREB may operate by safeguarding the degranulation machinery. To our knowledge, CREB is the first factor identified to regulate MRGPRX2 expression and function in opposite direction. Overall, the ancient TF is an indispensable component of skin MCs, orchestrating not only survival and proliferation but also their secretory competence.

Synapses are organized into nanocolumns that control synaptic transmission efficacy through precise alignment of postsynaptic neurotransmitter receptors and presynaptic release sites. Recent evidence show that Leucine-Rich Repeat Transmembrane protein LRRTM2, highly enriched and confined at synapses, interacts with Neurexins through its C-terminal cap, but the role of this binding interface has not been explored in synapse formation and function. Here, we develop a conditional knock-out mouse model (cKO) to address the molecular mechanisms of LRRTM2 regulation, and its role in synapse organization and function. We show that LRRTM2 cKO specifically impairs excitatory synapse formation and function in mice. Surface expression, synaptic clustering, and membrane dynamics of LRRTM2 are tightly controlled by selective motifs in the C-terminal domain. Conversely, the N-terminal domain controls presynapse nano-organization and postsynapse AMPAR sub-positioning and stabilization through the recently identified Neurexin-binding interface. Thus, we identify LRRTM2 as a central organizer of pre- and post- excitatory synapse nanostructure through interaction with presynaptic Neurexins., (© 2024. The Author(s).)

Autism spectrum disorder (ASD) is characterized by social and neurocognitive impairments, with mutations of the SHANK3 gene being prominent in patients with monogenic ASD. Using the InsG3680 mouse model with a Shank3 mutation seen in humans, we revealed an unknown role for Shank3 in postsynaptic oligodendrocyte (OL) features, similar to its role in neurons. This was shown by impaired molecular and physiological glutamatergic traits of InsG3680-derived primary OL cultures. In vivo, InsG3680 mice exhibit significant reductions in the expression of key myelination-related transcripts and proteins, along with deficits in myelin ultrastructure, white matter, axonal conductivity, and motor skills. Last, we observed significant impairments, with clinical relevance, in induced pluripotent stem cell-derived OLs from a patient with the InsG3680 mutation. Together, our study provides insight into Shank3's role in OLs and reveals a mechanism of the crucial connection of myelination to ASD pathology.

Odontoblasts are primarily responsible for synthesizing and secreting extracellular matrix proteins, which are crucial for dentinogenesis. Our previous single-cell profile and RNAscope for odontoblast lineage revealed that cyclic adenosine monophosphate responsive element-binding protein 3 like 1 (Creb3l1) was specifically enriched in the terminal differentiated odontoblasts. In this study, deletion of Creb3l1 in the Wnt1+ lineage led to insufficient root elongation and dentin deposition. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing were performed to revealed that in CREB3L1-deficient mouse dental papilla cells (mDPCs), the genes near the closed chromatin regions were mainly associated with mesenchymal development and the downregulated genes were primarily related to biological processes including cell differentiation, protein biosynthesis and transport, all of which were evidenced by a diminished ability of odontoblastic differentiation, a significant reduction in intracellular proteins, and an even greater decline in extracellular supernatant proteins. Dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp), and transmembrane protein 30B (Tmem30b) were identified as direct transcriptional regulatory targets. TMEM30B was intensively expressed in the differentiated odontoblasts, and exhibited a significant decline in both CREB3L1-deficient odontoblasts in vivo and in vitro. Deletion of Tmem30b impaired the ability of odontoblastic differentiation, protein synthesis, and protein secretion in mDPCs. Moreover, overexpressing TMEM30B in CREB3L1-deficient mDPCs partially rescued the extracellular proteins secretion. Collectively, our findings suggest that CREB3L1 participates in dentinogenesis and facilitates odontoblastic differentiation by directly enhancing the transcription of Dmp1, Dspp, and other differentiation-related genes and indirectly promoting protein secretion partially via TMEM30B., (© 2024. The Author(s).)

FOXG1 syndrome is a rare neurodevelopmental disorder of the telencephalon, for which there is no cure. Underlying heterozygous pathogenic variants in the Forkhead Box G1 ( FOXG1 ) gene with resulting impaired or loss of FOXG1 function lead to severe neurological impairments. Here, we report a patient with a de novo pathogenic single nucleotide deletion c.946del (p.Leu316Cysfs*10) of the FOXG1 gene that causes a premature protein truncation. To study this variant in vivo, we generated and characterized Foxg1 c946del mice that recapitulate hallmarks of the human disorder. Accordingly, heterozygous Foxg1 c946del mice display neurological symptoms with aberrant neuronal networks and increased seizure susceptibility. Gene expression profiling identified increased oligodendrocyte- and myelination-related gene clusters. Specifically, we showed that expression of the c946del mutant and of other pathogenic FOXG1 variants correlated with overexpression of proteolipid protein 1 ( Plp1 ), a gene linked to white matter disorders. Postnatal administration of Plp1 -targeting antisense oligonucleotides (ASOs) in Foxg1 c946del mice improved neurological deficits. Our data suggest Plp1 as a new target for therapeutic strategies mitigating disease phenotypes in FOXG1 syndrome patients.

α-Neurexins are essential and highly expressed presynaptic cell-adhesion molecules that are frequently linked to neuropsychiatric and neurodevelopmental disorders. Despite their importance, how the elaborate extracellular sequences of α-neurexins contribute to synapse function is poorly understood. We recently characterized the presynaptic gain-of-function phenotype caused by a missense mutation in an evolutionarily conserved extracellular sequence of neurexin-3α (A687T) that we identified in a patient diagnosed with profound intellectual disability and epilepsy. The striking A687T gain-of-function mutation on neurexin-3α prompted us to systematically test using mutants whether the presynaptic gain-of-function phenotype is a consequence of the addition of side-chain bulk (i.e., A687V) or polar/hydrophilic properties (i.e., A687S). We used multidisciplinary approaches in mixed-sex primary hippocampal cultures to assess the impact of the neurexin-3α A687 residue on synapse morphology, function and ligand binding. Unexpectedly, neither A687V nor A687S recapitulated the neurexin-3α A687T phenotype. Instead, distinct from A687T, molecular replacement with A687S significantly enhanced postsynaptic properties exclusively at excitatory synapses and selectively increased binding to neuroligin-1 and neuroligin-3 without changing binding to neuroligin-2 or LRRTM2. Importantly, we provide the first experimental evidence supporting the notion that the position A687 of neurexin-3α and the N-terminal sequences of neuroligins may contribute to the stability of α-neurexin-neuroligin-1 trans-synaptic interactions and that these interactions may specifically regulate the postsynaptic strength of excitatory synapses., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Mediator, a co-regulator complex required for RNA Polymerase II activity, interacts with tissue-specific transcription factors to regulate development and maintain homeostasis. We observe reduced Mediator subunit MED15 expression in endocrine hormone-producing pancreatic islets isolated from people living with type 2 diabetes and sought to understand how MED15 and Mediator control gene expression programs important for the function of insulin-producing β-cells. Here we show that Med15 is expressed during mouse β-cell development and maturation. Knockout of Med15 in mouse β-cells causes defects in β-cell maturation without affecting β-cell mass or insulin expression. ChIP-seq and co-immunoprecipitation analyses found that Med15 binds β-cell transcription factors Nkx6-1 and NeuroD1 to regulate key β-cell maturation genes. In support of a conserved role during human development, human embryonic stem cell-derived β-like cells, genetically engineered to express high levels of MED15, express increased levels of maturation markers. We provide evidence of a conserved role for Mediator in β-cell maturation and demonstrate an additional layer of control that tunes β-cell transcription factor function., (© 2024. The Author(s).)

Repulsive guidance molecule b (RGMb), a glycosylphosphatidylinositol-anchored member of the RGM family, is initially identified as a co-receptor of bone morphogenetic protein (BMP) in the nervous system. The expression of RGMb is transcriptionally regulated by dorsal root ganglion 11 (DRG11), which is a transcription factor expressed in embryonic DRG and dorsal horn neurons and plays an important role in the development of sensory circuits. RGMb is involved in important physiological processes such as embryonic development, immune response, intercellular adhesion and tumorigenesis. Furthermore, RGMb is mainly involved in the regulation of RGMb-neogenin-Rho and BMP signalling pathways. The recent discovery of programmed death-ligand 2 (PD-L2)-RGMb binding reveals that the cell signalling network and functional regulation centred on RGMb are extremely complex. The latest report suggests that down-regulation of the PD-L2-RGMb pathway in the gut microbiota promotes an anti-tumour immune response, which defines a potentially effective immune strategy. However, the biological function of RGMb in a variety of human diseases has not been fully determined, and will remain an active research field. This article reviews the properties and functions of RGMb, focusing on its role under various physiological and pathological conditions.

Calorie restriction increases lifespan. Among the tissue-specific protective effects of calorie restriction, the impact on the gastrointestinal tract remains unclear. We report increased numbers of chromogranin A-positive (+), including orexigenic ghrelin+ cells, in the stomach of calorie-restricted mice. This effect was accompanied by increased Notch target Hes1 and Notch ligand Jag1 and was reversed by blocking Notch with DAPT, a gamma-secretase inhibitor. Primary cultures and genetically modified reporter mice show that increased endocrine cell abundance is due to altered Lgr5+ stem and Neurog3+ endocrine progenitor cell proliferation. Different from the intestine, calorie restriction decreased gastric Lgr5+ stem cells, while increasing a FOXO1/Neurog3+ subpopulation of endocrine progenitors in a Notch-dependent manner. Further, activation of FOXO1 was sufficient to promote endocrine cell differentiation independent of Notch. The Notch inhibitor PF-03084014 or ghrelin receptor antagonist GHRP-6 reversed the phenotypic effects of calorie restriction in mice. Tirzepatide additionally expanded ghrelin+ cells in mice. In summary, calorie restriction promotes Notch-dependent, FOXO1-regulated gastric endocrine cell differentiation., (© 2024 McKimpson et al.)

Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy., (© 2024. The Author(s).)

Social memory impairment is a key symptom of many brain disorders, but its underlying mechanisms remain unclear. Neuroligins (NLGs) are a family of cell adhesion molecules essential for synapse development and function and their dysfunctions are linked to neurodevelopmental and neuropsychiatric disorders, including autism and schizophrenia. Although NLGs are extensively studied in neurons, their role in glial cells is poorly understood. Here we show that astrocytic deletion of NLG3 in the ventral hippocampus of adult male mice impairs social memory, attenuates astrocytic Ca 2+ signals, enhances the expression of EAAT2 and prevents long-term potentiation, and these impairments are rescued by increasing astrocyte activity, reducing EAAT2 function or enhancing adenosine/A2a receptor signaling. This study has revealed an important role of NLG3 in astrocyte function, glutamate homeostasis and social memory and identified the glutamate transporter and adenosine signaling pathway as potential therapeutic strategies to treat brain disorders., (© 2024. The Author(s).)

Aims: This study investigates the neuroprotective effects of lipidized analogues of 2-SS-CART(61-102) derived from anorexigenic neuropeptide cocaine- and amphetamine-regulated transcript peptide (CARTp) in light of the link between obesity, its comorbidities, and the development of Alzheimer's disease., Methods: We introduce novel lipidized analogues derived from 2-SS-CART(61-102), a specific analogue of natural CART(61-102), with two disulfide bridges. Using hypothermic PC12 cells, we tested the effect of the most potent analogues on Tau phosphorylation. We further described the anorexigenic and neuroprotective potential of subcutaneously (SC) injected lipidized CARTp analogue in a mouse model with prediabetes and obesity induced by neonatal monosodium glutamate (MSG) administration., Results: Compared to the non-lipidized 2-SS-CART(61-102), all lipidized analogues exhibited a potent binding affinity to PC12 cells and enhanced in vitro stability in rat plasma. Two most potent lipidized analogues attenuated hypothermia-induced Tau hyperphosphorylation at multiple epitopes. Subsequently, chronic SC treatment with palm-2-SS-CART(61-102) significantly decreased body weight and food intake, improved metabolic parameters, decreased level of pTau and increased neurogenesis in hippocampi of obese MSG mice., Conclusion: Our unique CARTp analogue palm-2-SS-CART(61-102) shows promise as a potent anti-obesity and neuroprotective agent., Competing Interests: Declaration of competing interest The authors declare no competiting interests., (Copyright © 2024. Published by Elsevier B.V.)

It has been proposed that the phenotypic differences in cognitive abilities between humans and our closest living relatives, chimpanzees, are largely due to changes in the regulation of neurodevelopmental genes. We have previously found that the neurodevelopmental transcription factor gene NPAS3 accumulates the largest number of human accelerated regions (HARs), suggesting it may play some role in the phenotypic evolution of the human nervous system. In this work, we performed a comparative functional analysis of NPAS3-HAR202 using enhancer reporter assays in transgenic zebrafish and mice. We found that the Homo sapiens HAR202 ortholog failed to drive reporter expression to the zebrafish nervous system, in high contrast to the strong expression displayed by the rest of the vertebrate ortholog sequences tested. Remarkably, the HAR202 ortholog from archaic humans (Neanderthals/Denisovans) also displayed a pan-vertebrate expression pattern, despite the fact that archaic and modern humans have only one nucleotide substitution. Moreover, similar results were found when comparing enhancer activity in transgenic mice, where we observed a loss of activity of the modern human version in the mouse developing brain. To investigate the functional importance of HAR202, we generated mice lacking HAR202 and found a remarkable decrease of Npas3 expression in the forebrain during development. Our results place HAR202 as one of the very few examples of a neurodevelopmental transcriptional enhancer displaying functional evolution in the brain as a result of a fast molecular evolutionary process that specifically occurred in the human lineage., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

This study focuses on exploring the role of Six2 in the progression of hepatocellular carcinoma (HCC) and its resistance to the chemotherapy drug 5-fluorouracil (5-FU). Using Hep3B and Huh7 cell lines, we analyzed how Six2 affects various cellular functions, including viability, proliferation, apoptosis, and invasion. Our research also delved into Six2's regulatory impact on DNMT1 levels, E-cadherin expression, and the methylation of the E-cadherin promoter, all of which are crucial for 5-FU resistance in HCC cells. Additionally, we examined the effects of Six2 knockdown on the PI3K/AKT/mTOR signaling pathway. Our findings indicate that overexpression of Six2 enhances cell viability and proliferation, encourages invasive behavior, increases methylation at the E-cadherin promoter, and reduces apoptosis. These changes correspond with increased levels of DNMT1 and decreased levels of E-cadherin, culminating in heightened resistance to 5-FU. Conversely, knocking down Six2 increases the sensitivity of HCC cells to 5-FU and reduces activation of the PI3K/AKT/mTOR pathway. These results suggest that Six2 plays a significant role in promoting HCC proliferation, invasion, and chemotherapy resistance, particularly through mechanisms involving DNMT1 and the PI3K/AKT/mTOR pathway, highlighting its potential as a target for HCC treatment.

Distal metastases result from metastatic microenvironment and tumour epithelial cell interactions, the cellular heterogeneity of primary colorectal cancer (CRC) and liver metastases (LM) was evaluated by integrating single-cell sequencing data, and the collected gene expression data from metastatic epithelial cell subsets was used to construct a prognostic model and to identify intercellular receptor-ligand interactions between epithelial and immune cells in CRC and LM. Multiplex immunofluorescence staining, and in vitro wound healing, cell migration and cell apoptosis assays were performed to further explore the biological relevance of identified potential regulatory molecules. In this study, approximately 17 epithelial cell subtypes were detected, with Epi-11 cells being highly expressed in LM tissues compared with CRC samples. Furthermore, patients with high expression of the metastasis-related genetic profile of Epi-11 had a poorer prognosis. By predicting receptor-ligand interactions, Epi-11 cells were found to interact more with myeloid and T/natural killer cells in LM tissues when compared to primary CRC samples, which was mediated by the PLXNB1/SEMA4D axis. In addition, high SEMA4D expression was correlated with decreased overall survival of patients with CRC, whereas PLXNB1 was not. SEMA4D knockdown prevented the migration and promoted the apoptosis of HCT116 cells in vitro. In summary, Epi-11 cells, an important subset of epithelial cells, may drive the LM of CRC and act by crosstalk with immune cells through the PLXNB1/SEMA4D signalling axis., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Glioblastoma represents the most frequently diagnosed malignant neoplasm within the central nervous system. Human glioblastoma cells can be phenotypically reprogrammed into neuron-like cells through the forced expression of NEUROG2 and SOXC factors. NEUROG2 serves as a pioneer factor, establishing an initial framework for this transformation. However, the specific role of SOXC factors has not been fully elucidated., Methods: In this study, we used ChIP-seq to determine the potential target gene of NGN2. RNA-seq has been used to evaluate the transcriptional change during NGN2-SOX11-mediated neuron reprogramming. Immunofluorescence was used to determine the neuron reprogramming efficacy and cell proliferation ability. ChIP-qPCR, Co-IP, and Western Blot were performed to investigate the mechanism., Results: Our findings reveal that SOXC factors, in contrast to their previously identified function as transcriptional activators, act as transcriptional repressors. They achieve this by recruiting TRIM28 to suppress the expression of ECT2, a RhoGEF. This suppression results in the differential regulation of RhoA, RAC1, and CDC42 activities throughout the reprogramming process. We further establish that small molecules targeting RhoA and its effectors can substitute for SOXC factors in facilitating the neuronal reprogramming of glioblastoma cells., Conclusion: These results underscore the pivotal role of SOXC factors' transcriptional repression and illuminate one of their specific downstream targets., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Neuropeptide cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the brains of teleosts, amphibians, birds, and mammals and has emerged as a conserved regulator of energy balance across these vertebrate phyla. However, as yet, there is no information on CART in the reptilian brain. We characterized the cDNA encoding CART and mapped CART-containing elements in the brain of gecko, Hemidactylus leschenaultii (hl) using a specific anti-CART antiserum. We report a 683-bp hlcart transcript containing a 336-bp open reading frame, which encodes a putative 111-amino acid hl-preproCART. The 89-amino acid hl-proCART generated from hl-preproCART produced two putative bioactive hl-CART-peptides. These bioactive CART-peptides were > 93% similar with those in rats/humans. Although reverse transcription-polymerase chain reaction (RT-PCR) detected hlcart-transcript in the brain, CART-containing neurons/fibers were widely distributed in the telencephalon, diencephalon, mesencephalon, rhombencephalon, spinal cord, and retina. The mitral cells in olfactory bulb, neurons in the paraventricular, periventricular, arcuate (Arc), Edinger-Westphal, and brainstem nuclei were intensely CART-positive. In view of antagonistic roles of neuropeptide Y (NPY) and CART in energy balance in the framework of mammalian hypothalamus, we probed CART-NPY interaction in the hypothalamus of H. leschenaultii. Double immunofluorescence showed a dense NPY-innervation of Arc CART neurons. Ex vivo hypothalamic slices treated with NPY/NPY-Y 1 -receptor agonist significantly reduced hlcart-mRNA levels in the Arc-containing tissues and CART-ir in the dorsal-Arc. However, CART-ir in ventral-Arc was unaffected. NPY via Y 1 -receptors may regulate energy balance by inhibiting dArc CART neurons. This study on CART in a reptilian brain fills the current void in literature and underscores the conserved feature of the neuropeptide across the entire vertebrate phyla., (© 2024 The Author(s). The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Background: Atrial fibrillation GWAS (genome-wide association studies) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding Nesprin-2, which connects the nuclear membrane with the cytoskeleton., Methods: Reporter gene vector transfection and CRISPR-Cas9 editing were used to identify the causal variant regulating the expression of SYNE2α1 . After SYNE2 knockdown or SYNE2α1 overexpression in human stem cell-derived cardiomyocytes, nuclear phenotypes were assessed by imaging and atomic force microscopy. Gene expression was assessed by RNAseq and gene set enrichment analysis. Fura-2 AM staining assessed calcium transients. Optical mapping assessed action potential duration and conduction velocity., Results: The risk allele of rs1152591 had lower promoter and enhancer activity and was significantly associated with lower expression of the short SYNE2α1 isoform in human stem cell-derived cardiomyocytes, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 overexpression had dominant negative effects on the nucleus with its overexpression or SYNE2 knockdown leading to increased nuclear area and decreased nuclear stiffness. Gene expression results from SYNE2α1 overexpression demonstrated both concordant and nonconcordant effects with SYNE2 knockdown. SYNE2α1 overexpression had a gain of function on electrophysiology, leading to significantly faster calcium reuptake and decreased assessed action potential duration, while SYNE2 knockdown showed both shortened assessed action potential duration and decreased conduction velocity., Conclusions: rs1152591 was identified as a causal atrial fibrillation variant, with the risk allele decreasing SYNE2α1 expression. Downstream effects of SYNE2α1 overexpression include changes in nuclear stiffness and electrophysiology, which may contribute to the mechanism for the risk allele's association with AF., Competing Interests: None.

Social deficits represent a core symptom domain of autism spectrum disorder (ASD), which is often comorbid with sleep disturbances. In this issue of the JCI, Sun et al. explored a medial septum (MS) circuit linking these behaviors in a neuroligin 3 conditional knockout model of autism. They identified GABAergic neuron hyperactivity following neuroligin 3 deletion in the MS. This hyperactivity resulted in the inhibition of the downstream preoptic area (POA) and hippocampal CA2 region, resulting in sleep loss and social memory deficits, respectively. Inactivating the hyperactive MS GABA neurons or activating the POA or CA2 rescued the behavioral deficits. Together, these findings deepen our understanding of neural circuits underlying social and sleep deficits in ASD.

A synaptic protein, Neuronal Pentraxin 2 (NPTX2), has emerged as a pivotal biomarker for Alzheimer's dementia (AD). We identified candidate miRNAs targeting NPTX2 and performed association and mediation analyses using multi-omics data (N = 702). Among 44 candidate miRNAs, miR-133b was significantly associated with AD and Braak positivity. Higher miR-133b expression was also associated with higher NPTX2 gene expression and better cognition. Mediation analysis showed that miR-133b partially influences AD and cognition through the NPTX2 protein. Our integrated approach suggests a potential role of miR-133b in synaptic integrity and offers new insights into AD pathogenesis., (© 2024 The Author(s). Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Hair cells of the inner ear and lateral-line system rely on specialized ribbon synapses to transmit sensory information to the central nervous system. The molecules required to assemble these synapses are not fully understood. We show that Nrxn3, a presynaptic adhesion molecule, is crucial for ribbon-synapse maturation in hair cells. In both mouse and zebrafish models, the loss of Nrxn3 results in significantly fewer intact ribbon synapses. We show in zebrafish that, initially, Nrxn3 loss does not alter pre- and postsynapse numbers but, later, synapses fail to pair, leading to postsynapse loss. We also demonstrate that Nrxn3 subtly influences synapse selectivity in zebrafish lateral-line hair cells that detect anterior flow. Loss of Nrxn3 leads to a 60% loss of synapses in zebrafish, which dramatically reduces pre- and postsynaptic responses. Despite fewer synapses, auditory responses in zebrafish and mice are unaffected. This work demonstrates that Nrxn3 is a crucial and conserved molecule required for the maturation of ribbon synapses. Understanding how ribbon synapses mature is essential to generating new therapies to treat synaptopathies linked to auditory or vestibular dysfunction., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Previous studies have suggested that N6-methyladenosine (mA) modification of RNA affects fundamental aspects of RNA metabolism, and mA dysregulation is implicated in various human diseases, including Alzheimer's disease (AD). This study is designed to explore the role and mechanism of methyltransferase-like 14 (METTL14) in the pathogenesis of AD. SK-N-SH cells were treated with Aβ1-42 to establish an in vitro model of AD. Cerebellin 4 (CBLN4) and METTL14 expression levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability and apoptosis were analyzed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry assay. B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), C-caspase-3, total-caspase-3, C/EBP homologous protein (CHOP), and glucose-related protein 78 (GRP78) protein levels were determined using Western blot. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) levels were analyzed using ELISA. Reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) products were examined using special assay kits. Interaction between CBLN4 and METTL14 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assays. CBLN4 and METTL14 expression was decreased in Aβ1-42-treated SK-N-SH cells. Upregulation of CBLN4 relieved Aβ1-42-induced SK-N-SH cell apoptosis, inflammation, oxidative stress, and endoplasmic reticulum (ER) stress in vitro. At the molecular level, METTL14 could improve the stability and expression of CBLN4 mRNA via m6A methylation. Our findings indicated that m6A methylase METTL14-mediated upregulation of CBLN4 mRNA stability could repress Aβ1-42-triggered SK-N-SH cell injury, providing a promising therapeutic target for AD treatment., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In recent years, there has been a surge in interest in investigating the mechanisms underlying memory consolidation. However, our understanding of the behavioural tagging (BT) model and its establishment in diverse brain regions remains limited. This study elucidates the contributions of the anterior cingulate cortex (ACC) and hippocampus in the formation of long-term memory (LTM) employing behaviour tagging as a model for studying the underlying mechanism of LTM formation in rats. Existing knowledge highlights a protein synthesis-dependent phase as imperative for LTM. Brain-derived neurotrophic factor (BDNF) stands as a pivotal plasticity-related protein (PRP) in mediating molecular alterations crucial for long-term synaptic plasticity and memory consolidation. Our study offers evidence suggesting that tropomyosin receptor kinase B (TrkB), the receptor of BDNF, may act as a combined "behavioural tag/PRP". Interfering with the expression of these molecules resulted in impaired LTM after 24 h. Furthermore, augmenting BDNF expression led to an elevation in Arc protein levels in both the ACC and hippocampus regions. Introducing novelty around weak inhibitory avoidance (IA) training resulted in heightened step-down latencies and expression of these molecules, respectively. We also demonstrate that the increase in Arc expression relies on BDNF synthesis, which is vital for the memory consolidation process. Additionally, inhibiting BDNF using an anti-BDNF function-blocking antibody impacted Arc expression in both the ACC and hippocampus regions, disrupting the transformations from labile to robust memory. These findings mark the initial identification of a "behavioural tag/PRP" combination and underscore the involvement of the TrkB-BDNF-Arc cascade in the behavioural tagging model of learning and memory., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Autologous adipose tissue is an ideal material for soft tissue filling and transplantation; however, high volumes of fat absorption over time lead to a relatively low overall survival percentage. The survival and differentiation of adipose-derived stem cells (ADSCs) in the transplanted microenvironment might improve adipose graft survival. Adipocytes have been reported to affect ADSC activation. However, its underlying mechanisms remain unclear., Methods: Human ADSCs were incubated in a culture medium supplemented with hypoxic or normoxic conditioned culture medium (CM) derived from human adipocytes. Neuronal Pentraxin 1 (NPTX1) was overexpressed or knocked down in human adipocytes using an overexpression vector (NPTX1 OE) or small interfering RNA (siRNA) transfection, respectively. ADSC differentiation and paracrine secretion were assessed. Nude mice were implanted with human adipocytes and ADSCs. The adipose tissue was subsequently evaluated by histological analysis., Results: CM from hypoxic-stimulated human adipocytes significantly facilitated the differentiation ability and paracrine levels of ADSCs. NPTX1 was significantly up-regulated in human adipocytes exposed to hypoxic conditions. In vitro, CM derived from hypoxia-stimulated human adipocytes or NPTX1-overexpressing human adipocytes exposed to normoxia promoted ADSC differentiation and paracrine; after silencing NPTX1, the facilitating effects of hypoxia-treated human adipocytes on ADSC activation were eliminated. Similarly, in vivo, the NPTX1 OE + normoxia-CM group saw improved histological morphology and fat integrity, less fibrosis and inflammation, and increased vessel numbers compared with the OE NC + normoxia-CM group; the adipocyte grafts of the si-NC + hypoxia-CM group yielded the most improved histological morphology, fat integrity, and the most vessel numbers. However, these enhancements of ADSC activation and adipose graft survival were partially abolished by NPTX1 knockdown in human adipocytes., Conclusion: NPTX1 might mediate the facilitating effects of hypoxia-stimulated human adipocytes on ADSC activation, thereby improving adipose tissue survival rate after autologous fat transplantation and the effectiveness of autologous fat transplantation through promoting ADSC activation., Level of Evidence Iii: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 ., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature and International Society of Aesthetic Plastic Surgery.)

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor. Our observations suggest a multi-layered genetic response to drive tumor recurrence and implicate PTP4A2 (protein tyrosine phosphatase 4A2) as a modulator of self-renewal, proliferation and tumorigenicity in recurrent GBM. Genetic perturbation or small-molecule inhibition of PTP4A2 acts through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and its downstream molecular players, exploiting a functional dependency on ROBO signaling. Because a pan-PTP4A inhibitor was limited by poor penetrance across the blood-brain barrier in vivo, we engineered a second-generation chimeric antigen receptor (CAR) T cell therapy against ROBO1, a cell surface receptor enriched across recurrent GBM specimens. A single dose of ROBO1-targeted CAR T cells doubled median survival in cell-line-derived xenograft (CDX) models of recurrent GBM. Moreover, in CDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma, ROBO1 CAR T cells eradicated tumors in 50-100% of mice. Our study identifies a promising multi-targetable PTP4A-ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Autism spectrum disorder (ASD) encompasses a range of neurodevelopmental conditions. Different mutations on a single ASD gene contribute to heterogeneity of disease phenotypes, possibly due to functional diversity of generated isoforms. SHANK2, a causative gene in ASD, demonstrates this phenomenon, but there is a scarcity of tools for studying endogenous SHANK2 proteins in an isoform-specific manner. Here, we report a point mutation on SHANK2, which is found in a patient with autism, located on exon of the SHANK2B transcript variant (NM_133266.5), hereby SHANK2B Y29X . This mutation results in an early stop codon and an aberrant splicing event that impacts SHANK2 transcript variants distinctly. Induced pluripotent stem cells (iPSCs) carrying this mutation, from the patient or isogenic editing, fail to differentiate into functional dopamine (DA) neurons, which can be rescued by genetic correction. Available SMART-Seq single-cell data from human midbrain reveals the abundance of SHANK2B transcript in the ALDH1A1 negative DA neurons. We then show that SHANK2B Y29X mutation primarily affects SHANK2B expression and ALDH1A1 negative DA neurons in vitro during early neuronal developmental stage. Mice knocked in with the identical mutation exhibit autistic-like behavior, decreased occupancy of ALDH1A1 negative DA neurons and decreased dopamine release in ventral tegmental area (VTA). Our study provides novel insights on a SHANK2 mutation derived from autism patient and highlights SHANK2B significance in ALDH1A1 negative DA neuron., (© 2024. The Author(s).)

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.