Autophagy controls neuronal differentiation by regulating the WNT-DVL signaling pathway.

Macroautophagy/autophagy dysregulation is associated with various neurological diseases, including Vici syndrome. We aimed to determine the role of autophagy in early brain development. We generated neurons from human embryonic stem cells and developed a Vici syndrome model by introducing a loss-of-function mutation in the EPG5 gene. Autophagy-related genes were upregulated at the neuronal progenitor cell stage. Inhibition of autolysosome formation with bafilomycin A1 treatment at the neuronal progenitor cell stage delayed neuronal differentiation. Notably, WNT (Wnt family member) signaling may be part of the underlying mechanism, which is negatively regulated by autophagy-mediated DVL2 (disheveled segment polarity protein 2) degradation. Disruption of autolysosome formation may lead to failure in the downregulation of WNT signaling, delaying neuronal differentiation. EPG5 mutations disturbed autolysosome formation, subsequently inducing defects in progenitor cell differentiation and cortical layer generation in organoids. Disrupted autophagy leads to smaller organoids, recapitulating Vici syndrome-associated microcephaly, and validating the disease relevance of our study.Abbreviations: BafA1: bafilomycin A1; co-IP: co-immunoprecipitation; DVL2: dishevelled segment polarity protein 2; EPG5: ectopic P-granules 5 autophagy tethering factor; gRNA, guide RNA; hESC: human embryonic stem cells; KO: knockout; mDA, midbrain dopamine; NIM: neural induction media; NPC: neuronal progenitor cell; qPCR: quantitative polymerase chain reaction; UPS: ubiquitin-proteasome system; WNT: Wnt family member; WT: wild type.

A peptide against the N-terminus of myristoylated alanine-rich C kinase substrate promotes neuronal differentiation in SH-SY5Y human neuroblastoma cells.

Myristoylated alanine-rich protein kinase C substrate (MARCKS) plays crucial roles in neuronal functions and differentiation. However, specific effects of the myristoylated N-terminal sequence (MANS) peptide, a widely used MARCKS modulator comprising the initial 24 amino acids of MARCKS, on neuronal cells remain unclear. Therefore, in this study, we aimed to examine the effects and action mechanisms of the MANS peptide on SH-SY5Y human neuroblastoma cells, which served as the in vitro neuronal cell models. MANS treatment of SH-SY5Y cells resulted in significant neurite outgrowth within 24 hr, which was as prominent as that induced by seven days of treatment with all-trans retinoic acid, the most common agent used to induce SH-SY5Y cell differentiation. Levels of synaptophysin, a neuronal marker protein, were significantly increased in the MANS peptide-treated cells. Additionally, increased MARCKS levels and decreased MARCKS phosphorylation were observed in MANS peptide-treated cells. Notably, neurite outgrowth induced by the MANS peptide was significantly reduced in MARCKS-knocked-down cells. Overall, these results suggest the MANS peptide as a novel agent for SH-SY5Y cell differentiation, particularly for the analysis of MARCKS functions.

Real-Time, Non-Invasive Monitoring of Neuronal Differentiation Using Intein-Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell-Based Biosensors.

Real-time and non-invasive monitoring of neuronal differentiation will help increase our understanding of neuronal development and help develop regenerative stem cell therapies for neurodegenerative diseases. Traditionally, reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence (IF) staining have been widely used to investigate stem cell differentiation; however, their limitations include endpoint analysis, invasive nature of monitoring, and lack of single-cell-level resolution. Several limitations hamper current approaches to studying neural stem cell (NSC) differentiation. In particular, fixation and staining procedures can introduce artificial changes in cellular morphology, hindering our ability to accurately monitor the progression of the process and fully understand its functional aspects, particularly those related to cellular connectivity and neural network formation. Herein, we report a novel approach to monitor neuronal differentiation of NSCs non-invasively in real-time using cell-based biosensors (CBBs). Our research efforts focused on utilizing intein-mediated protein engineering to design and construct a highly sensitive biosensor capable of detecting a biomarker of neuronal differentiation, hippocalcin. Hippocalcin is a critical protein involved in neurogenesis, and the CBB functions by translocating a fluorescence signal to report the presence of hippocalcin externally. To construct the hippocalcin sensor proteins, hippocalcin bioreceptors, AP2 and glutamate ionotropic receptor AMPA-type subunit 2 (GRIA2), were fused to each split-intein carrying split-nuclear localization signal (NLS) peptides, respectively, and a fluorescent protein was introduced as a reporter. Protein splicing (PS) was triggered in the presence of hippocalcin to generate functional signal peptides, which promptly translocated the fluorescence signal to the nucleus. The stem cell-based biosensor showed fluorescence signal translocation only upon neuronal differentiation. Undifferentiated stem cells or cells that had differentiated into astrocytes or oligodendrocytes did not show fluorescence signal translocation. The number of differentiated neurons was consistent with that measured by conventional IF staining. Furthermore, this approach allowed for the monitoring of neuronal differentiation at an earlier stage than that detected using conventional approaches, and the translocation of fluorescence signal was monitored before the noticeable expression of class III ß-tubulin (TuJ1), an early neuronal differentiation marker. We believe that these novel CBBs offer an alternative to current techniques by capturing the dynamics of differentiation progress at the single-cell level and by providing a tool to evaluate how NSCs efficiently differentiate into specific cell types, particularly neurons.

DVL/GSK3/ISL1 pathway signaling: unraveling the mechanism of SIRT3 in neurogenesis and AD therapy.

BACKGROUND: The established association between Alzheimer's disease (AD) and compromised neural regeneration is well-documented. In addition to the mitigation of apoptosis in neural stem cells (NSCs), the induction of neurogenesis has been proposed as a promising therapeutic strategy for AD. Our previous research has demonstrated the effective inhibition of NSC injury induced by microglial activation through the repression of oxidative stress and mitochondrial dysfunction by Sirtuin 3 (SIRT3). Nonetheless, the precise role of SIRT3 in neurogenesis remains incompletely understood. METHODS: In vivo, SIRT3 overexpression adenovirus was firstly injected by brain stereotaxic localization to affect the hippocampal SIRT3 expression in APP/PS1 mice, and then behavioral experiments were performed to investigate the cognitive improvement of SIRT3 in APP/PS1 mice, as well as neurogenic changes in hippocampal region by immunohistochemistry and immunofluorescence. In vitro, under the transwell co-culture condition of microglia and neural stem cells, the mechanism of SIRT3 improving neurogenesis of neural stem cells through DVL/GSK3/ISL1 axis was investigated by immunoblotting, immunofluorescence and other experimental methods. RESULTS: Our findings indicate that the overexpression of SIRT3 in APP/PS1 mice led to enhanced cognitive function and increased neurogenesis. Additionally, SIRT3 was observed to promote the differentiation of NSCs into neurons during retinoic acid (RA)-induced NSC differentiation in vitro, suggesting a potential role in neurogenesis. Furthermore, we observed the activation of the Wnt/ß-catenin signaling pathway during this process, with Glycogen Synthase Kinase-3a (GSK3a) primarily governing NSC proliferation and GSK3ß predominantly regulating NSC differentiation. Moreover, the outcomes of our study demonstrate that SIRT3 exerts a protective effect against microglia-induced apoptosis in neural stem cells through its interaction with DVLs. CONCLUSIONS: Our results show that SIRT3 overexpressing APP/PS1 mice have improved cognition and neurogenesis, as well as improved neurogenesis of NSC in microglia and NSC transwell co-culture conditions through the DVL/GSK3/ISL1 axis.

Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation.

NGN2-driven induced pluripotent stem cell (iPSC)-to-neuron conversion is a popular method for human neurological disease modeling. In this study, we present a standardized approach for generating neurons utilizing clonal, targeted-engineered iPSC lines with defined reagents. We demonstrate consistent production of excitatory neurons at scale and long-term maintenance for at least 150 days. Temporal omics, electrophysiological, and morphological profiling indicate continued maturation to postnatal-like neurons. Quantitative characterizations through transcriptomic, imaging, and functional assays reveal coordinated actions of multiple pathways that drive neuronal maturation. We also show the expression of disease-related genes in these neurons to demonstrate the relevance of our protocol for modeling neurological disorders. Finally, we demonstrate efficient generation of NGN2-integrated iPSC lines. These workflows, profiling data, and functional characterizations enable the development of reproducible human in vitro models of neurological disorders.

Magnetic Induction Heating in a Conducting Polymer for Biomedical Applications.

In this study, we investigate the magnetic induction heating induced in a conducting polymer (CP) under alternative magnetic fields (AMFs). Experimental results and numerical simulations have proved that the magneto-thermal conversion of the CP is caused by the induced eddy current, which is related to the shape and intensity of the applied external AMF, and the intrinsic electrical conductivity, macrostructure and microstructure of the CP. By employing various fabrication methods, specific temperature distribution and control of thermal field within conducting polymer films and aerogels could be achieved. To exploit the potential of magnetic induction heating in CP for biomedical applications, we designed a conducting polymer aerogel-based self-adaptive heat patch and demonstrated its AMF-enabled localized heating of skin. In addition to the thermal ablation of tumor cells via magneto-thermal conversion of the CP, the promotion of neuronal differentiation at mild temperature by noninvasive magneto-electrical stimulation has also been demonstrated to be an effective strategy for tissue engineering.

Neurodevelopmental disorders: 2024 update.

Neurodevelopmental disorders encompass a range of conditions such as intellectual disability, autism spectrum disorder, rare genetic disorders and developmental and epileptic encephalopathies, all manifesting during childhood. Over 1,500 genes involved in various signaling pathways, including numerous transcriptional regulators, spliceosome elements, chromatin-modifying complexes and de novo variants have been recognized for their substantial role in these disorders. Along with new machine learning tools applied to neuroimaging, these discoveries facilitate genetic diagnoses, providing critical insights into neuropathological mechanisms and aiding in prognosis, and precision medicine. Also, new findings underscore the importance of understanding genetic contributions beyond protein-coding genes and emphasize the role of RNA and non-coding DNA molecules but also new players, such as transposable elements, whose dysregulation generates gene function disruption, epigenetic alteration, and genomic instability. Finally, recent developments in analyzing neuroimaging now offer the possibility of characterizing neuronal cytoarchitecture in vivo, presenting a viable alternative to traditional post-mortem studies. With a recently launched digital atlas of human fetal brain development, these new approaches will allow answering complex biological questions about fetal origins of cognitive function in childhood. In this review, we present ten fascinating topics where major progress has been made in the last year.

Transcriptomic Analysis Reveals the Heterogeneous Role of Conducting Films Upon Electrical Stimulation.

Central nervous system (CNS) injuries and neurodegenerative diseases have markedly poor prognoses and can result in permanent dysfunction due to the general inability of CNS neurons to regenerate. Differentiation of transplanted stem cells has emerged as a therapeutic avenue to regenerate tissue architecture in damaged areas. Electrical stimulation is a promising approach for directing the differentiation outcomes and pattern of outgrowth of transplanted stem cells, however traditional inorganic bio-electrodes can induce adverse effects such as inflammation. This study demonstrates the implementation of two organic thin films, a polymer/reduced graphene oxide nanocomposite (P(rGO)) and PEDOT:PSS, that have favorable properties for implementation as conductive materials for electrical stimulation, as well as an inorganic indium tin oxide (ITO) conductive film. Transcriptomic analysis reveals that electrical stimulation improves neuronal differentiation of SH-SY5Y cells on all three films, with the greatest effect for P(rGO). Unique material- and electrical stimuli-mediated effects are observed, associated with differentiation, cell-substrate adhesion, and translation. The work demonstrates that P(rGO) and PEDOT:PSS are highly promising organic materials for the development of biocompatible, conductive scaffolds that will enhance electrically-aided stem cell therapeutics for CNS injuries and neurodegenerative diseases.

In Ovo Electroporation of Embryonic Chicken Spinal Cord in Combination with Ex Vivo Slice Culture for Live Imaging of Vertebrate Neuronal Differentiation.

To investigate the cell behavior underlying neuronal differentiation in a physiologically relevant context, differentiating neurons must be studied in their native tissue environment. Here, we describe an accessible protocol for fluorescent live imaging of differentiating neurons within ex vivo embryonic chicken spinal cord slice cultures, which facilitates long-term observation of individual cells within developing tissue.

Stanniocalcin 2 Promotes Neuronal Differentiation in Neural Stem/Progenitor Cells of the Mouse Subventricular Zone Through Activation of AKT Pathway.

Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone (SVZ) throughout life, responding to various pathophysiological stimuli and playing a crucial role in central nervous system repair. Although numerous studies have elucidated the role of stanniocalcin 2 (STC2) in regulating cell differentiation processes, its specific function in NSPCs differentiation remains poorly understood. Clarifying the role of STC2 in NSPCs is essential for devising novel strategies to enhance the intrinsic potential for brain regeneration postinjury. Our study revealed the expression of STC2 in NSPCs derived from the SVZ of the C57BL/6N mouse. In cultured SVZ-derived NSPCs, STC2 treatment significantly increased the number of Tuj1 and DCX-positive cells. Furthermore, STC2 injection into the lateral ventricle promoted the neuronal differentiation of NSPCs and migration to the olfactory bulb. Conversely, the STC2 knockdown produced the opposite effect. Further investigation showed that STC2 treatment enhanced AKT phosphorylation in cultured NSPCs, whereas STC2 inhibition hindered AKT activation. Notably, the neuronal differentiation induced by STC2 was blocked by the AKT inhibitor GSK690693, whereas the AKT activator SC79 reversed the impact of STC2 knockdown on neuronal differentiation. Our findings indicate that enhancing STC2 expression in SVZ-derived NSPCs facilitates neuronal differentiation, with AKT regulation potentially serving as a key intracellular target of STC2 signaling.

A comprehensive protocol for efficient differentiation of human NPCs into electrically competent neurons.

BACKGROUND: The study of neurons is fundamental to unraveling the complexities of the nervous system. Primary neuronal cultures from rodents have long been a cornerstone of experimental studies, yet limitations related to their non-human nature and ethical concerns have prompted the development of alternatives. In recent years, the derivation of neurons from human-induced pluripotent stem cells (hiPSCs) has emerged as a powerful option, offering a scalable source of cells for diverse applications. Neural progenitor cells (NPCs) derived from hiPSCs can be efficiently differentiated into functional neurons, providing a platform to study human neural physiology and pathology in vitro. However, challenges persist in achieving consistent and reproducible outcomes across experimental settings. COMPARISON WITH EXISTING METHODS: Our aim is to provide a step-by-step methodological protocol, augmenting existing procedures with additional instructions and parameters, to guide researchers in achieving reproducible results. METHODS AND RESULTS: We outline procedures for the differentiation of hiPSC-derived NPCs into electrically competent neurons, encompassing initial cell density, morphology, maintenance, and differentiation. We also describe the analysis of specific markers for assessing neuronal phenotype, along with electrophysiological analysis to evaluate biophysical properties of neuronal excitability. Additionally, we conduct a comparative analysis of three different chemical methods-KCl, N-methyl-D-aspartate (NMDA), and bicuculline-to induce neuronal depolarization and assess their effects on the induction of both fast and slow post-translational, transcriptional, and post-transcriptional responses. CONCLUSION: Our protocol provides clear instructions for generating reliable human neuronal cultures with defined electrophysiological properties to investigate neuronal differentiation and model diseases in vitro.

Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-ß), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

Integrative analysis of long isoform sequencing and functional data identifies distinct cortical layer neuronal subtypes derived from human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated nonneuronal cells in the culture as compared with those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, ß tubulin, and Synapsin I) and whole cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.NEW & NOTEWORTHY Low differentiation efficiency is one of the major drawbacks of the existing protocols to differentiate iPSCs into neuronal lineages. Here, we present time-efficient and robust approach of neuronal differentiation leading to the generation of functional brain units, cortical layer neurons. We found iPSCs cultured in Essential 8 media (E8M) resulted in neuronal differentiation without the signs of growth of spontaneously differentiated cells in culture at any point in 35 days compared with Stemflex media (SFM).

Reduction of Glyoxalase 1 Expression Links Fetal Methylmercury Exposure to Autism Spectrum Disorder Pathogenesis.

Glyoxalase 1 (Glo1) is an essential enzyme to detoxify methylglyoxal (MGO), a cytotoxic byproduct of glycolysis. Accumulating studies have shown an important role of Glo1 in regulating cortical development and neurogenesis, potentially contributing to the pathogenesis of autism spectrum disorder (ASD) when impaired. We have previously shown that prenatal exposure to non-apoptotic low-dose methylmercury (MeHg), an environmental pollutant, induces premature cortical neurogenesis and ASD-like behaviors in a rodent model. In this study, we aimed to determine the underlying molecular mechanisms that mediate prenatal MeHg-induced premature neuronal differentiation and abnormal neurodevelopment. Using single-cell RNA sequencing (scRNA-seq) and real-time quantitative PCR (RT-qPCR), we found that prenatal MeHg exposure at a non-apoptotic dose significantly reduced Glo1 gene expression in embryonic cultured radial glia precursors (RGPs). In cultured RGPs, the knockdown of Glo1 expression increased neuronal production at the expense of the cultured RGPs population, while overexpression of Glo1 restored MeHg-induced neuronal differentiation back to normal levels. Furthermore, we found that co-treatment with both MeHg and multiple MGO scavengers or a CREB inhibitor (iCREB) mitigated MeHg-induced premature neuronal differentiation, reinforcing the role of Glo1 and CREB in mediating MeHg-induced neuronal differentiation. Our findings demonstrate a direct link between MeHg exposure and expression of an ASD risk gene Glo1 in cortical development, supporting the important role of gene-environment interaction in contributing to the etiology of neural developmental disorders, such as ASD.

The Placenta as a Source of Human Material for Neuronal Repair.

Stem cell therapy has the potential to meet unsolved problems in tissue repair and regeneration, particularly in the neural tissues. However, an optimal source has not yet been found. Growing evidence indicates that positive effects produced in vivo by mesenchymal stem cells (MSCs) can be due not only to their plasticity but also to secreted molecules including extracellular vesicles (EVs) and the extracellular matrix (ECM). Trophic effects produced by MSCs may reveal the key to developing effective tissue-repair strategies, including approaches based on brain implants or other implantable neural electrodes. In this sense, MSCs will become increasingly valuable and needed in the future. The placenta is a temporary organ devoted to protecting and supporting the fetus. At the same time, the placenta represents an abundant and extremely convenient source of MSCs. Nonetheless, placenta-derived MSCs (P-MSCs) remain understudied as compared to MSCs isolated from other sources. This review outlines the limited literature describing the neuroregenerative effects of P-MSC-derived biomaterials and advocates for exploiting the potential of this untapped source for human regenerative therapies.

Altered nucleocytoplasmic export of adenosine-rich circRNAs by PABPC1 contributes to neuronal function.

Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive. Comparing the nuclear and cytoplasmic circRNAs between H9 cells and H9-derived forebrain (FB) neurons, we identify that a subset of adenosine (A)-rich circRNAs are restricted in H9 nuclei but exported to cytosols upon differentiation. Such a subcellular relocation of circRNAs is modulated by the poly(A)-binding protein PABPC1. In the H9 nucleus, newly produced (A)-rich circRNAs are bound by PABPC1 and trapped by the nuclear basket protein TPR to prevent their export. Modulating (A)-rich motifs in circRNAs alters their subcellular localization, and introducing (A)-rich circRNAs in H9 cytosols results in mRNA translation suppression. Moreover, decreased nuclear PABPC1 upon neuronal differentiation enables the export of (A)-rich circRNAs, including circRTN4(2,3), which is required for neurite outgrowth. These findings uncover subcellular localization features of circRNAs, linking their processing and function during neurogenesis.

Dopamine-Functionalized, Red Carbon Quantum Dots for In Vivo Bioimaging, Cancer Therapeutics, and Neuronal Differentiation.

One of the crucial requirements of quantum dots for biological applications is their surface modification for very specific and enhanced biological recognition and uptake. Toward this end, we present the green synthesis of bright, red-emitting carbon quantum dots derived from mango leaf extract (mQDs). These mQDs are conjugated electrostatically with dopamine to form mQDs-dopamine (mQDs:DOPA) bioconjugates. Bright-red fluorescence of mQDs was used for bioimaging and uptake in cancerous and noncancerous cell lines, tissues, and in vivo models like zebrafish. mQDs exhibited the highest uptake in brain tissue compared to the heart, kidney, and liver. mQD:DOPA conjugates killed breast cancer cells and increased uptake in epithelial RPE-1 cells and zebrafish. Additionally, mQDs:DOPA promoted neuronal differentiation of SH-SY5Y cells to differentiated neurons. Both mQDs and mQDs:DOPA exhibited the potential for higher collective cell migrations, implicating their future potential as next-generation tools for advanced biological and biomedical applications.

Comparing the biological activity and composition of Cerebrolysin with other peptide preparations.

Neurological disorders, ranging from acute forms such as stroke and traumatic brain injury to neurodegenerative diseases like dementia, are the leading cause of disability-adjusted life years (DALYs) worldwide. A promising approach to address these conditions and promote nervous system regeneration is the use of the neuropeptide preparation Cerebrolysin, which has been shown to be effective in both clinical and preclinical studies. Despite claims of similar clinical efficacy and safety by several peptide preparations, concerns regarding their generic composition and efficacy have been previously raised. Based on these reports, we analyzed the peptide composition and neurotrophic activity of several peptide preparations allegedly similar to Cerebrolysin and approved in some countries for treating neurological diseases. Our results demonstrate that these preparations lack relevant biological activity and that the peptide composition is significantly different from Cerebrolysin. peptide.

Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of stem cell maintenance and neuronal differentiation.

Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.

Transplantation of Wnt5a-modified Bone Marrow Mesenchymal Stem Cells Promotes Recovery After Spinal Cord Injury via the PI3K/AKT Pathway.

Spinal cord injury (SCI) is a severe neurological condition that can lead to paralysis or even death. This study explored the potential benefits of bone marrow mesenchymal stem cell (BMSC) transplantation for repairing SCI. BMSCs also differentiate into astrocytes within damaged spinal cord tissues hindering the cell transplantation efficacy, therefore it is crucial to enhance their neuronal differentiation rate to facilitate spinal cord repair. Wnt5a, an upstream protein in the non-classical Wnt signaling pathway, has been implicated in stem cell migration, differentiation, and neurite formation but its role in the neuronal differentiation of BMSCs remains unclear. Thus, this study investigated the role and underlying mechanisms of Wnt5a in promoting neuronal differentiation of BMSCs both in vivo and in vitro. Wnt5a enhanced neuronal differentiation of BMSCs in vitro while reducing astrocyte differentiation. Additionally, high-throughput RNA sequencing revealed a correlation between Wnt5a and phosphoinositide 3-kinase (PI3K)/protein kinase B(AKT) signaling, which was confirmed by the use of the PI3K inhibitor LY294002 to reverse the effects of Wnt5a on BMSC neuronal differentiation. Furthermore, transplantation of Wnt5a-modified BMSCs into SCI rats effectively improved the histomorphology (Hematoxylin and eosin [H&E], Nissl and Luxol Fast Blue [LFB] staining), motor function scores (Footprint test and Basso-Beattie-Bresnahan [BBB]scores)and promoted neuron production, axonal formation, and remodeling of myelin sheaths (microtubule associated protein-2 [MAP-2], growth-associated protein 43 [GAP43], myelin basic protein [MBP]), while reducing astrocyte production (glial fibrillary acidic protein [GFAP]). Therefore, targeting the Wnt5a/PI3K/AKT pathway could enhance BMSC transplantation for SCI treatment.