Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities.2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation.Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation , we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that CaDetailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.The online version contains supplementary material available at 10.1186/s12860-021-00343-z. Human neuronal tissue lacks regenerative capacity, leaving few treatments available following neuronal injury or neurodegeneration. In the past decade, an interest in direct neuronal reprogramming of stem cells into motor neurons (MNs) has emerged as a solution to generate human neuronal tissue for therapeutic applications. MNs form synapses to potentiate electrical signals from the CNS into peripheral tissues. They play a critical role in the formation of neuromuscular junctions (NMJs), where MN axons terminate on muscle fibers and neurotransmitters are released to trigger muscle contractions. NMJs are cholinergic synapses, where the neurotransmitter acetylcholine (ACh) is released from the presynaptic MN terminal for uptake by postsynaptic ACh receptors on the target muscle cell . This crSeveral in vitro protocols have been developed to convert progenitor cells, such as human inducible pluripotent stem cells (iPSCs), into MNs –4. ThereHere, we followed an established protocol  to diffeGenome-wide transcriptome studies provide in-depth knowledge of regulatory pathways that shape cellular morphology and function. Such information is crucial for the design of novel neuron regenerative therapies and cell-based drug discovery platforms. Previous studies based on single cell transcriptomics of Amyotrophic Lateral Sclerosis (ALS) patient-derived iPSCs discovered the underlying mechanisms of disease pathology  and the Here we performed a comprehensive transcriptomic analysis of in vitro iPSCs-derived MNs to characterize the key principles of MN development by analyzing bulk transcriptome data from five crucial time points of MN neurogenesis: iPSCs (D0), NSCs (D7), post-mitotic MNP cells D13), eMNs (D18), and mature MNs (D28). We interrogated the transcriptomic signatures using next-generation RNA-sequencing (RNA-Seq) technology and performed in situ validation of key pluripotency and MN specific biomarker expression. Additionally, we characterized the functionality of iPSC-derived MNs via electrophysiological analysis of neuronal network connectivity. Our results corroborate transcriptomic profiles previously identified in the astrocyte-to-neuron transformation process [, eMNs (D2+) signaling through cyclic adenosine monophosphate (cAMP) and LPC is downregulated during the conversion of the iPSC to NSC and remains silenced until the final stage of MN maturation when the regulatory pathway becomes a driving force for the neuronal synaptic activity. Another key finding is that during the in vitro development of MN in 2D monocultures, ionotropic nAChR are expressed in the early MN stage and are subsequently replaced by the G protein coupled receptors (GPCR) -type muscarinic AChR in mature MN, probably due to lack of metabolic stimuli that are naturally released by the muscle tissue in vivo. While our findings provide unique insights into the temporal mechanism of iPSC-derived MNs they also indicate the advantages of using a co-culture system, of MN and muscle, to enable the development of physiologically relevant MNs types akin to that obtained in vivo.By applying a LANL-developed Ontology Pathway Analysis software (OPaver) we found that calcium  is driven by inhibition of TGFβ, activin, nodal, and BMP signaling pathways while simultaneously activating the Wnt signaling pathway to sustain cell proliferation. Therefore, to convert undifferentiated iPSCs into a NSC lineage the culture media was supplemented with SB431542 (SB) and DMH-1, inhibitors of activin receptor-like kinases , and CHIR99021 (CHIR), a Wnt pathway activator. MNPs and early motor neurons (eMNs) were patterned through activation of the Shh pathway by Purmorphamine (Pur). The final step of MN maturation and specification was aided by a Notch signaling pathway inhibitor, Compound E (CpdE), to synchronize the maturation process through the elimination of dividing cells. Neurotrophic and growth factors were supplemented to facilitate neuronal growth and maturation  recordings of cellular electrophysiological responses. This non-invasive method allows for repetitive recordings of spontaneous electrical firings at various times during neuronal differentiation. Spontaneous oscillations in extracellular action potential (AP) were recorded after day 20 of neuronal differentiation, and the AP frequency increased the longer the cells differentiated on the MEA  of global transcriptome data revealed tight clustering of all replicates, indicating high reproducibility of gene expression profiles for each stage of development. Differentiation timepoints showed distinct genetic programs, as evidenced by the PCA variances Fig. a. ApplyiWe analyzed RNA-Seq data with the NCBI Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8  which cap < 0.05) altered during iPSC conversion to MNs , indicating that this gene may act as a positive regulator of cell division in human neuronal cells. In contrast, ARHGAP36 and CRMP1 genes were downregulated upon transition of iPSCs to NSCs (D7) and responded to Pur with gradual stimulation of gene expression in MNs at D18–28  increased throughout the course of neuronal maturation  were dramatically downregulated , along with MYC, Ras guanyl-releasing protein 2 (RASGRP2), and GNG12. Gene groups encoding the auxiliary subunits of high-voltage activated (HVA) Cavs were upregulated during the early stages of neuronal development [CACNB3 (induced 4-fold at D7) and CACNA2D3 (16-fold activation at D7-D13). The late stage of MN formation (D18-D28) was marked by upregulation of CACNA1E and CACNG2. Several gene groups encoding auxiliary subunits of low-voltage activated (LVA) Cavs (CACNG5 and CACNG8), were initially downregulated in NSC and later upregulated during the MN maturation stage (D18-D28). Accumulation of Cavs gene transcripts throughout the MN differentiation process is consistent with the increased excitability of neuronal cells  and downregulated during the transition from MNP to eMN (D13-D18). KITLG transcription was again upregulated in MNs (D18–28). KIT signaling promotes cell survival through the activation of PIK3, PLC, and AKT1 pathways and we detected upregulation of KITLG gene expression in MNs which may be a response to the addition of IGF-1 in the culture media. Upregulation of various genes regulating cell survival and proliferation in the post-mitotic MN, such as KDR, PTPRR, PTPN5, and PRKCB, further exemplifies the MN response to IGF-1 pro-survival stimulus.Other key components of MAPK-signaling showed oscillatory patterns of gene expression throughout MN formation and maturation. Gene transcripts of 2+-signaling (Table HTR7). In addition, adenylate cyclase 2 (ADCY2) was also downregulated at this time and is known to act immediately downstream of HTRs. Together, the downregulation of HTR7 and ADCY2 indicates a functional silencing of the cAMP-dependent signaling pathway. Furthermore, multiple genes involved in the activation of Ca2+ signaling were downregulated: GPCR subunit alpha-14 (GNA14), an activator of phospholipase C (LPC); two gene groups encoding inositol 1,4,5-triphosphate receptors (ITPR2 and ITPR3); and ryanodine receptor 2 (RYR2).  during the transition into NSCs.CaERBB4) transcripts. ERBB4 is activated by epidermal growth factor proteins, neuregulins2/3, to shape the development of neuronal cells through activation of MAPK3/ERK [ERBB4 expression was upregulated by 18-fold in D7-D13, followed by upregulation of CACNA1B, CACNA1E, GRIN2A and P2RX3 (D13-D18). Formation of eMNs (D13-D18) coincided with upregulated gene expression of HVA N- and R-type (CACNA1B and CACNA1E) voltage-dependent Ca2+ channels, glutamate ionotropic receptor (GRIN2A), and the ligand-gated ion channel responsible for peripheral pain responses, P2RX3. These gene expression profiles indicate the selective pressure of chemical stimuli lead to the formation of eMNs with characteristics of the sympathetic nerve system [A hallmark of the NSC to MNP transition was the accumulation of receptor tyrosine-protein kinase erB-4 , signifying cell readiness to release Ca2+ from the sarcoplasmic reticulum in response to adrenergic (sympathetic) stimulation  and metabotropic (GRM1) glutamate receptors, together with the upregulation of neuromedin-K receptor (TACR3), accentuated the role of the IP3-Ca2+ second messenger system to activate ERK1/2 as well as classical PKC signaling pathways [PRKCB gene transcripts in MNs compared to eMNs , we observed upregulation of gene groups activating Caion Fig. d. This fpathways –31. We a8, we obsCHRM3) and inhibitory (CHRM2) mAChRs were specifically activated in late stage MN maturation  were upregulated in mature MNs D28). Genes encoding for excitatory  were upregulated in NSCs, as well as GPCR response to sphingosine-1-phosphate signaling receptor (S1PR1), suggesting changes in cytoskeleton dynamics and mitosis [CNR1and CHRNB4) were upregulated during the conversion of NSCs into MNPs and remained activated until eMNs were formed.During the first step of iPSC commitment to a neuronal lineage, multiple glutamate receptors were altered, notably, gene expression levels of two ionotropic glutamate receptors were upregulated in the NSCs,  mitosis . Genes etachykinin, and its receptor (TACR3), were upregulated in MNs. In spinal neurons, tachykinins evoke synthesis and release of ACh, histamine, catecholamines, and GABA. We also detected differential expression of the somatostatin receptor (SSTR1) indicating that the mature MN population can secrete neurotransmitters [NR3C1) was upregulated in MNPs and MNs and two new classes of neuroactive receptors were upregulated in the last stage of MN maturation: hypocretin/orexin (HCRTR2) and prostaglandin E receptor 4 (PTGER4). Our transcriptomic data indicate that the replacement of ionotropic nAChR (CHRNA3) and dopamine receptor (DRD2) expression with adrenergic receptor (ADRA1A) and mAChRs (CHRM2 and CHRM3) marked the formation of mature MNs. The controversial activation of mAChRs and genes encoding excitatory neurotransmitters, including glutamate and catecholamines (ADRA1A), could stem from the formation of a heterogeneous population consisting of parasympathetic and sympathetic MNs.Formation of eMNs was marked by reactivation of genes encoding glutamate receptors which were downregulated at the NSC stage. Gene activity of various ionotropic glutamate receptors peaked when eMNs became mature MNs (D18-D28). Activation of genes regulating the synthesis of various neurotransmitters defined the transition from eMNs to MNs. The transcript levels of the neuropeptide smitters . The gluPathways regulating axon guidance and synapse development were upregulated during iPSC-to-MN differentiation (Tables NTNG1) to be initially downregulated as iPSCs transitioned into NSC, but then upregulated in eMNs and MNs from D13–28. Deleted in colorectal carcinoma (DCC) was also significantly upregulated as iPSCs transition through NSCs to become MNPs, indicating its critical role in prompting axon extension. SLIT1 was upregulated at D7 and both SLIT1 and SLIT2 were upregulated as the NSCs became MNPs (D7-D13). HB9 tightly regulates Robo2 expression to regulate motor axon guidance in ventrally-projecting MNs [ROBO2 downregulation in NSCs, during which time HB9 transcripts were initially accumulating  was significantly increased late in MNs maturation (D18-D28), which encodes an enzyme responsible for the hydrolysis of ACh into choline and acetic acid, a critical step in ACh recycling in the cholinergic synapse. Membrane transporter solute carrier family 5 member 7 (SLC5A7) is then able to import choline back into a cholinergic neuron for subsequent ACh synthesis. We found gene expression of SLC5A7 to be increased in both the eMNs and mature MNs.In vivo as MN axons extend, they have the potential to terminate on a muscle fiber at a cholinergic synapse, the site of ACh neurotransmission. In the pre-synaptic neuron, ACh synthesis is driven by choline O-acetyltransferase (ChAT) and we observed +/HB9+ MN populations reported by Thiry et al [p < 1e-5) activation (log2 > 2) of key molecular markers specific to various MN subtypes ranging from the preganglionic column (SMAD1 and ZEB2), spinal accessory column (RUNX1 and PHOX2B), and the lateral LMC (FOXP2 and ALDH1A2). Taken together, our results  of MN maturation when we were able to detect neuronal synaptic activity via recordings of spontaneous AP firings.This comprehensive study documents transcriptomic and morphological changes of iPSCs as they differentiate into motor neurons (MNs) in vitro. We performed RNA-Seq meta-transcriptome analysis of human iPSC and the four cell types representing stages of spinal motor neuron differentiation  and maturation (MN). The results from our genome-wide transcriptome study revealed basic developmental principles of in vitro neurogenesis from iPSC that have not been elucidated by previous studies while confirming the regulatory role of TGFβ, Notch, and Shh signaling pathways in the formation of adult spinal motor neurons. We further corroborated the findings from the next-generation RNA-Seq analysis with RT-qPCR gene expression assays and immunohistochemistry profiles of key pluripotency and MN markers. Applying novel OPaver software we found strict temporal correlation between the formation of functional neuronal network connections on a MEA chip and the expression of genes involved in the regulation of Ca2+ levels, known as calcium transients, regulate neuronal subtype and neurotransmitter specifications [2+ entry, specifically voltage-sensitive calcium channels and ionotropic glutamate receptors, not sooner than at the eMN developmental stage  entry and direct interaction with phospholipase C-coupled (PLC) and inositol trisphosphate (IP3) receptors [CXCR4 by 16-fold at D0-D7 underlines the importance of intracellular Ca2+ signaling inhibition as the neuronal cell lineage commitment process begins. The CXCR4/CXCR7 complex recruits β-arrestin to trigger the canonical GPCR pathway activating ERK1/2, p38, and SAPK, while inhibiting both Ca2+ mobilization and cAMP signaling [NMUR2 and APLNR) in NSCs suggest a role for phosphoinositide (PI) signaling pathways that inhibit cAMP production upon Ca2+ mobilization and possibly in regulating cytoskeleton dynamics, cell growth, and hormone release [Intracellular Caications , 37. Ouring GPCR . Signifieceptors . Upregulignaling . Upregul release , 42.2+ signaling in motor neuron development in vitro. It has been previously demonstrated that voltage-gated Ca2+ influx activated by cAMP is instrumental in the maturation of neuronal progenitor cells into functional neurons [2+ homeostasis (RYR2) were immediately inhibited upon transition of iPSC into NSC and were re-activated in mature MN. Collectively, our data suggests that inhibition of pathways regulating the Ca2+ transients is required for the successful transition from pluripotency to neuronal progenitor cells. Our findings corroborate previous studies that have demonstrated that low cellular excitability is vital for cell migration while increase in the Ca2+ transients stops migration and promotes dendrite formation in cortical neurons [Our findings underline the role of temporal gene regulation of Ca neurons . The com neurons .SKIL gene expression throughout the neuronal cell differentiation process suggests a regulatory feed-back loop that balances survival and cell differentiation programs. PTCH1, a Shh receptor gene, is consistently and significantly upregulated from D7 to D28 suggesting a role as a positive regulator in neuronal cell division. Binding of Shh to PTCH1 results in the release of the smoothened protein initiating cell proliferation. Upon ligand binding, PTCH1 is trafficked away from the Shh positive regulator, G-coupled receptor SMO, resulting in downstream signal transduction [PTCH1 by D28 supports the possible inhibition of cell proliferation and subsequent differentiation to neurons. ARHGAP36 and CRMP1, both of which are downregulated simultaneously, encode for Rho GTPase activating protein 36 and collapsin response mediator protein 1, are implicated in semaphorin-induced growth cone and axon guidance [The transcriptome analysis revealed the underlying molecular changes in differentiation of iPSC to mature MNs. Specifically, we observed that short-term inhibition of TGFβ signaling was sufficient to push iPSCs into NSCs and that continuous activation of Notch and Shh signaling pathways ensured morphogenesis and cell survival throughout the MN differentiation and maturation process. While the majority of changes were characteristic of neuronal development, deeper analysis revealed the involvement of more unexpected genes as well as unique temporal changes. For example, the oscillatory pattern of sduction . Downregguidance , 47 and ChAT, a marker of mature neurons, was upregulated as soon as iPSC committed to a neuronal lineage (D7). In addition, the well-orchestrated expression of key genes such as Nestin, Pax6, NgN2, MAP2, Isl1 and HB9 corroborated the phenotypic changes to MNs. The patterns of gene expression reported here are consistent with similar studies using small molecules to drive neuron reprogramming from astrocytes [Applying quantitative gene transcript analysis and immunohistochemistry, we observed that trocytes  or fibrotrocytes .CHRNA3) and dopamine receptor (DRD2) were downregulated while adrenergic receptor (ADRA1A) and mAChRs (CHRM2 and CHRM3) gene expression was activated. Since mAChRs are found exclusively on the neurons from the parasympathetic system [While Shh drives motor neuron formation over intermediate neurons, there are multiple subtypes of MNs. In the final stage of MN maturation, we observed that genes encoding ionotropic nAChR , were significantly upregulated in the population of mature motor neurons. This could stem from the formation of a heterogeneous population consisting of parasympathetic and sympathetic MNs. Previous studies based on single-cell RNA-Seq analysis of iPSC-derived spinal MN have demonstrated that the protocol for in vitro differentiation produces a mixed population of MN subtypes with a predominant 58%) sub-population of lateral MNs and several minority sub-populations, including hypaxial motor column (19%) and median motor column (6%) MNs [% sub-popThe electrophysiological activity of mature MN reflects their ability to form functional neuronal network connections. Neuronal precursors and neurons are capable of spontaneous electrical activity , and we MNs are often characterized by their role in forming cholinergic synapses at NMJs. MN axons terminate on muscle fibers and nerve impulses are translated into muscle contractions as the neurotransmitter ACh is released from the presynaptic MN terminal for uptake by postsynaptic ACh receptors on the target muscle cell . Underst2+ signaling are downregulated throughout the cell differentiation process and are activated in mature MN. Differentiation of iPSC into MN in vitro monocultures leads to formation of both parasympathetic and sympathetic neuronal types. Downregulation of tumor suppressing genes upon chemical conversion of iPSC to mature MN underlines the danger from direct application of the technology in vivo. Understanding the underlying molecular and cellular cues involved in MN differentiation of iPSCs has the potential to enable the discovery of novel treatments for neural injuries.This study revealed significant changes to the transcriptome, protein expression, and electrical function as iPSCs differentiated into mature MNs. To ensure cellular mobility that is essential for tissue layer formation, genes regulating CaHuman iPSCs  were cultured and maintained on vitronectin (ThermoFisher Scientific) treated culture plates in Essential8 medium (ThermoFisher Scientific). Differentiation of iPSCs into MNs was directed as previously described  with sliCells were lifted with accutase, pelleted by centrifugation, and stored at − 20 °C. Total RNA was extracted from cell pellets using RNeasy Mini Kit (Qiagen), following the recommendations of the manufacturer. After DNase digestion by Turbo DNA-free kit (ThermoFisher Scientific), samples were quantified and divided for qPCR and transcriptomic analyses.Extracted and DNase-treated RNA was quantified using the Qubit 4 Fluorometer (ThermoFisher) with the High Sensitivity RNA reagents and Bioanalyzer (Agilent) with RNA 6000 Pico reagents. Ribosomal depletion, DNA conversion, and library preparation was performed on all samples using the Illumina TruSeq Stranded Total RNA kit. 151 base pair reads were sequenced on the Illumina NextSeq. Across fifteen samples (three independent experiments x five time points) the total number of reads generated for each sample ranged from approximately 26 million to 40 million reads. Sequencing data was quality trimmed using FaQCs  with a qThree independent experiments were run in duplicate using a 7500 Fast Real-Time PCR System (Applied Biosciences). Fifty ng of each RNA sample were probed for motor neuron differentiation markers using Taqman RNA-to-CT 1-Step Kit (Applied Bioscience) in a 25 μl volume according to the manufacturer’s instructions. Taqman probes included NEUROG2 (Hs00935087_g1), ChAT (Hs00758143_m1), Isl1 (Hs01099686_m1), PAX6 (Hs01088114_m1), MAP2 (Hs00258900_m1), Nestin (Hs04187831_g1), Oct4 (Hs00999632_g1), and HB9 (Hs00907365_m1). Two endogenous controls, actin (Hs99999903_m1) and GAPDH (Hs01922876_u1), were analyzed by RT-qPCR and no significant differences were observed in their expression levels (data not shown); thus, all data shown are normalized to GAPDH.Nunc Lab-Tek chamber slides (ThermoFisher Scientific) were coated with vitronectin, seeded, and subsequently fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, blocked with 3% BSA in PBS for at least 1 h. Samples were incubated overnight at 4 °C with primary antibody solutions Table  diluted Cells were seeded on MEA chips, either in 60MEA200/30iR-Ti arrays or 24-well Plate with PEDOT Electrodes on Glass, 24 W300/30G-288 (Multichannel Systems). MEA’s were coated with poly-D-lysine and vitronectin. Before recording, the MEA chips were moved to the MEA2100 system (MultiChannel Systems) equipped with temperature control and allowed to equilibrate for 10 min before recording. The data were acquired using Multi Channel Experimenter or Multiwell Screen (MultiChannel Systems) at a sampling rate of 20 kHz for 2 min at 37 °C. Data were filtered using Butterworth band pass filter with 200 Hz cutoff frequency and threshold of 5 x SD were set to minimize both false-positive and missed detection. The representative electrodes were selected for analysis of mean spike frequency and percentage of spikes in the burst.https://david.ncifcrf.gov. The raw transcriptomic data of D0-D28 significant DEGs (p < 0.05) included 2242 upregulated and 1438 downregulated terms, available in the supplemental material. Each list of Homo sapiens genes was independently analyzed by DAVID, to generate an analysis of associated gene ontology (GO) terms.DAVID Functional Annotation Bioinformatics Microarray Analysis can be accessed at p < 0.05) identified in the DEseq2 analysis performed by PiRet. Differential expression calculation from DEseq2 in Log2 fold change and associated genes  were used as input for the OPaver software.OPaver , is a web-based tool to integrate multiple types  and time series of data to KEGG biochemical pathways maps. This software analysis tool was developed at Los Alamos National Laboratory. In this case, OPaver was utilized to map significantly differentially expressed genes  at each stage of MN development that were found to be significantly (p < 0.001) up or downregulated compared to the transcriptome profile of iPSC (D0). Additionally, DEG between two consecutive cell types are included.Additional file 2. Snapshot of the Ontology Pathway at the Glutamatergic synapse showing regulatory genes in cAMP and Ca2+ signaling pathways with DGE during the transition from iPSC to NSC, MNP (D13 vs D07), early MNs (D18 vs D13) and mature MNs (D28 vs D13). From left to right the color-coded legend shows DGE (p < 0.001) upon transition from iPSC to MNs. OPaver software was used to link the DGE profiles to the metabolic pathways.


MaACS1 and MaACO1 gene expression were analyzed. Result showed that the optimum condition to extend shelf life and maintain fruit quality was by treating the banana fruits with 1.5% carrageenan and storing them at a cool temperature (20°C). In addition, the result obtained from this study suggested that carrageenan can be used as edible coating to extend the shelf life of banana fruits (Musa acuminata AAA group).Banana is very important for both food and economic securities in many tropical and subtropical countries, because of its nutritional values. However, banana fruit is a climacteric fruit which has short shelf life, so an alternative method to delay its ripening is needed. Our group has used carrageenan as an edible coating to delay banana fruit ripening. In this study, the effect of different concentrations of carrageenan and storage temperatures on Cavendish banana shelf life and fruit quality was evaluated. The fruits were treated with 0.5%, 1.0%, and 1.5% carrageenan and stored at two different temperatures, 26°C and 20°C. Carrageenan functional groups in banana peel samples as well as changes in surface structure of banana peel, color, weight loss, pulp to peel ratio, total soluble solid, and levels of  Musa acuminata aminocyclopropane-1-carboxylic acid synthase (MaACS1) and Musa acuminata aminocyclopropanecarboxylate oxidase (MaACO1). These genes encode ACC (1 aminocyclopropane-1-carboxylic acid) synthase (ACS) and ACC oxidase (ACO), respectively, and catalyze the synthesis of ethylene from its precursor S-AdoMet (S-adenosyl-methionine/SAM) and ACC (1 aminocyclopropane-1-carboxylic acid) [2 and CO2 during fruit respiration [Banana is one of the most popular fruits that are in great demand. The Cavendish banana cultivar is commonly consumed as a quick dessert. Its high nutrient content makes it attractive to be consumed daily. Banana fruit is a climacteric fruit that will quickly ripen after harvest . The ripic acid) . This prpiration . The prepiration , respirapiration .2 penetration into the fruit and inhibit microbial growth [One alternative method to increase the fruit shelf life is by using edible coating. Edible coating could prevent Ol growth . The usel growth . The usel growth . Our prel growth , 11.Eucheuma cottonii (Rhodophyceae), had been reported to be used as edible packaging material for fruits [2 and CO2 gas [MaACO1 and MaACS1) was analyzed with quantitative polymerase chain reaction (qPCR). The expression profile of these two genes could become a molecular marker for carrageenan treatment in banana fruit ripening.Carrageenan, extracted from the red alga r fruits –14. The  CO2 gas . This po CO2 gas . Carrage CO2 gas . Howeverw/v), 1% (w/v), and 1.5% (w/v) carrageenan or not coated combined with storage either at room temperature (26 ± 1°C) or in an air-conditioned room (20°C). Each treatment had three replicates.The Cavendish Ambon banana cultivar (AAA) was obtained from PT Sewu Segar Indonesia . The green matured bananas in this study had been exposed to 100 ppm ethylene at 14°C for 24 hours. The criteria for the experimental banana fruits were absence of physical defects on the skin or the pulp. The experimental fruits all had the same physiological age, color, and size. The banana fruits were separated into fingers from the side and randomly grouped into four treatments: coated with 0.5% (w/v). Carrageenan powder was added to the solution followed by glycerol to a final concentration of 0.5% (v/v). The solution was stirred at 80°C for 30 more minutes until homogeneous, then stored at room temperature until it reached 50°C.The carrageenan solution was prepared according to the method of edible coating with modification . DistillCoating was performed by dipping the fruits in 0.5%, 1.0%, and 1.5% carrageenan solutions for around 30 seconds at 50°C. After dipping, the bananas were hanged to air-dry at room temperature. The bananas were then placed in storage racks at either 26± 1°C or 20°C. As control, banana fruits that had not been coated with carrageenan were stored at 26 ± 1°C and 20°C.The presence of 1.5% carrageenan on the surface of the banana fruit peel was confirmed by attenuated total reflection (ATR) spectroscopy using an ALPHA FTIR Spectrometer .Square pieces (1 cm × 1 cm) of carrageenan-coated and uncoated peels of 5 mm thick were freeze-dried for seven hours and analyzed using SEM  [Physical and biochemical analyses during fruit ripening  were perConversion of starch into sugar during ripening was assessed by measuring the starch percentage of the pulp. The starch-iodine staining solution consisted of 1% potassium iodide and 0.25% iodine. The banana was cut transversely about 2-3 cm thick at midpoint, and the peel was separated from the pulp. The surface of the cut banana was dipped 5 mm deep in the starch-iodine staining solution for 5 seconds. The starch pattern of each fruit was analyzed by comparing it to a starch iodine staining chart for bananas .U test.Statistical analyses were performed at all the results, in order to determine which treatment groups had significant differences of TSS, weight loss, and pulp to peel ratio, compared to another groups. Tests of normal distribution were conducted using Kolmogorov-Smirnov test. Data with normal distribution were then analyzed using the ANOVA test and further analyzed using the Tukey post hoc test. Data that were not normally distributed were analyzed using Kruskal-Wallis test and further analyzed using Mann-Whitney μL preheated extraction buffers at 65°C. In addition, 30 μL β-mercaptoethanol was added to the buffer immediately before use.Total RNA was isolated from the pulp of the banana fruit following the protocol used in the previous study . RNA isoμL) of chloroform, isoamylalcohol (24 : 1), was added after the solution reached room temperature. Subsequently, the solution was vortexed for 10 minutes and centrifuged at 8000 rpm for 10 minutes at 4°C. The aqueous phase (500 μL) was transferred into a new micro tube using a micropipette. Then, the process from adding chloroform, isoamylalcohol (24 : 1), until taking the aqueous phase was repeated. 500 μL of the supernatant was obtained, and 1/3 volume (167 μL) of 7.5 M lithium chloride (LiCl) was added. The solution was homogenized slowly by inverting the tube 10 times and then incubated for 16-18 hours at 4°C. The incubated solution was centrifuged at 8000 rpm for 30 minutes at 4°C. The supernatant was discarded, and the obtained pellet was dissolved in 500 μL of diethylpyrocarbonate- (DEPC-) treated water, and 1/5 volume (63.3 μL) of 3 M sodium acetate (pH 5.2) and 2 volumes (1 mL) of 100% ethanol were added and slowly homogenized by inverting the tube. The solution was incubated at -20°C for 2 hours. The incubated solution was centrifuged at 8000 rpm for 10 minutes at 4°C. Then, the supernatant was removed, and the obtained RNA pellet was washed with 70% ethanol and centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was subsequently discarded, and the RNA sample was dried by turning the micro tube over on dry tissue for approximately 25 minutes. The RNA sample was resuspended in 30 μL DEPC-treated water and stored at -80°C until use. Concentration and purity of the RNA sample was measured using NanoDrop™ 2000C , at wavelengths of 260, 280, 230, and 320 nm. Contaminating genomic DNA was removed from the total RNA by digestion using DNAse I kit . The purified total RNA was used as template for cDNA synthesis using iScript™ cDNA synthesis kit .The sample and extraction buffer were homogenized in the micro tube using vortex for 1 minute. Then, the mixed solution was incubated at 65°C for 15 minutes with vortex for 1 minute every 5-minute interval. An equal volume  that was connected to IQ™5 Real-Time PCR Detection Systems . Primers for  studies , 21, 22:FTIR analysis was performed to confirm the coating of banana peel with 1.5% carrageenan. Samples of banana peel coated with carrageenan as well as uncoated banana peel as control were analyzed with FTIR. Changes in transmittance intensity in the spectrum are shown in .−1 for the S=O bond of sulfate ester, 1030 cm−1 for glycoside bonds, a typical peak for galactose-4-sulfate at 845 cm−1, and a typical peak for 3,6-anhydro-D-galactose at 925 cm−1 [The FTIR spectrum of the coated banana samples showed several specific peaks for functional groups present in carrageenan. There were very strong peaks at wavenumber 1241 cm925 cm−1 .Scanning Electron Microscopy (SEM) was performed on the banana peels to view the morphology of the banana peels with their carrageenan edible coatings. Samples of banana peel without treatment and banana peel treated with 0.5% and 1.5% carrageenan solutions were analyzed. The electron micrographs with 500x and 1000x magnifications are shown in .Banana peels coated with carrageenan Figures  had a smChanges in the color of banana peel and starch percentage of the pulp during 13 days of storage are shown in .There were differences in the changes of the banana peel color and the starch percentage of the pulp between the treatments. Banana that did not get any coating and stored at low temperature took two days longer to ripen compared to the control but had the same starch conversion rate with complete conversion on day 7. Within treatments that were stored at room temperature, the 1.5% carrageenan treatment took the longest time to ripen, with a shelf life two days longer than the control. Within treatments stored at low temperature, the one treated with 1.5% carrageenan took the longest time to ripen and the shelf life was six days longer than the control.Results of the total soluble solid (TSS) analysis in  showed sIn the control group, there was a high increase in TSS at the beginning followed by a lesser increase up till day 11; then, the TSS decreased. The pattern of TSS changed on 0.5% and 0.1% carrageenan edible coating where the peak TSS was reached earlier followed by a decrease in TSS. The lowest TSS was obtained from 1.5% carrageenan edible coating stored at low temperature.Weight loss during ripening of the banana fruits from each treatment is shown in .The level of weight loss at each observation point varied between each treatment, with a tendency to increase from the beginning to the end of the treatment. The longer the storage time of the fruit, the higher the weight loss. In all the treatments, there were occasional decrease and increase of weight loss at certain observation points. The weight loss value was obtained from the average of three replicates that started with banana fruits with different initial weight, and this could cause variations in the data measurement.The pulp to peel ratio of banana fruits from each treatment is shown in .w/v) carrageenan edible coating stored at low temperature had the lowest pulp to peel ratio compared to the other treatments.The general pattern of the pulp to peel ratio tended to increase in each treatment, with increasing and decreasing ratios at a few observation points. The change in the values of the ratios can originate from the fact that the average of the three replicates comes from bananas that do not have the same initial weight which caused variations in the data measurement. Based on the average pulp to peel ratio, it could be concluded that treatment with 1.5%  and control at room temperature (uncoated and storage at room temperature), on days 1, 7, and 13. Two genes involved in the biosynthesis of ethylene, MaACS1 and MaACO1 gene expression in the pulp of the control and the optimum treatment  carrageenan edible coating and stored at low temperature (20°C).The results also showed that lower temperature could also delay ripening compared to room temperature. Ethylene biosynthesis was affected by temperature, increasing with the increase in ripening temperature until a certain point . In thisIt had been known previously that during banana fruit ripening, there was a gradual conversion of amylum into simple sugars. That is why the more advanced stage of ripening had higher TSS because the starch had been converted into sugar . The sam2 supply to the banana fruit [2 and low concentration of O2 could inhibit degradation of chlorophyll in the banana peel [w/v) carrageenan edible coating stored at low temperature could optimally reduce water transpiration from the banana fruit compared to the other treatments.The average weight loss of the low temperature (9.46%) treatment was lower than the room temperature one (20.48%). This could be caused by the slower metabolism at low temperature which would then decrease the respiration and transpiration rate of the fruit, and thus, there would be less water loss . Variatina fruit . High coana peel  and ethyana peel . Based oTreatment with carrageenan edible coating and low temperature could decrease the pulp to peel ratio of banana fruit. The edible coating could become a shield for the banana fruit. Storage of fruits at low temperature could decrease the rate of respiration and transpiration of the fruit, thus preserving the water content of the pulp and peel. During fruit ripening, the pulp to peel ratio of the banana fruit would consistently increase . The preMaACS1 gene expression in the optimum treatment tended to be lower. In contrast to MaACS1 gene expression, the pattern of MaACO1 gene expression of the control was different from the optimum treatment. In the control sample, the expression of MaACO1 gene increased from the beginning to the end of the observation period on day 13, while in the optimum treatment, expression decreased from the beginning followed by increase and decrease to the end of the observation period. The level of MaACO1 gene expression of the treated sample was lower than that of the control.This was in agreement with the results of previous studies , 10, 21,MaACS1 and MaACO1 expression. The high increase in MaACS1 in fruit ripening indicated a pattern of specific ethylene interaction during the ripening process of climacteric plants. Ethylene production would increase then decrease in a relatively short period of time at the beginning of the increase in respiration that coincided with the beginning of fruit ripening, which was observed through the expression of ripening genes [MaACS1 gene that would increase ACC synthase synthesis. High ACC synthase would result in the increase in ACC synthesis which would cause an increase of ethylene synthesis by the fruit.In climacteric fruits such as banana, there is a significant increase in respiration and production of endogenous ethylene at the beginning of ripening, concomitant with the increase in ng genes . The higMaACO1 gene expression tended to be lower than the expression of MaACS1 gene. This was in agreement with a previous study [MaACS1 expression during ripening; meanwhile, expression of the MaACO1 gene since the preclimacteric period was maintained until the end of ripening [MaACO1 gene was affected by the presence of O2 in the cell. O2 in the cell would help ACC oxidase convert ACC into ethylene. ACO enzyme was reported to be the limiting factor in ethylene biosynthesis [MaACS1 gene expression increased during the experiment, MaACO1 expression remained low.us study  that staripening , 32. ACCynthesis ; therefo2 concentration in the cell as the consequence of the presence of a gas barrier. As mentioned above, the expression of MaACO1 gene decreased from the beginning due to carrageenan coating. Previous studies using carrageenan as edible coating on strawberries had reported that it affected O2 permeability [2 to produce ethylene [2 and then to ACC [MaACO1 gene increased at the end of ripening which might due to ethylene accumulation that results in autocatalytic reaction and accumulation of ethylene due to a gas barrier. It has been reported that LeACO1 gene expression may be enhanced during autocatalytic ethylene production [Carrageenan edible coating affected both gene expressions probably due to the limited Oeability . In the ethylene . The kinn to ACC . Howeveroduction .MaACS1 and MaACO1. Results from this study indicated that carrageenan edible coating has the potential to be used as an alternative material to lengthen the shelf life of Cavendish banana fruit.In conclusion, treatment with 1.5% carrageenan edible coating and storage at low temperature (20°C) could lengthen the shelf life for six days compared to the control. Treatment of banana fruit with carrageenan edible coating and storage at room temperature could lengthen the shelf life for two days compared to the control, while storage at low temperature without edible coating could also lengthen the shelf life for two days compared to the control. Not only was it optimum for shelf life, treatment with 1.5% carrageenan edible coating and storage at low temperature (20°C) was also optimum for its effect on the expression of genes related to ethylene biosynthesis,


Wnt/β-catenin signaling plays a critical role in colorectal cancer (CRC) tumorigenesis and the homeostasis of colorectal cancer stem cells (CSCs), but its molecular mechanism remains unclear. B-cell lymphoma 3 (Bcl-3), a member of the IκB family, is overexpressed in CRC and promotes tumorigenicity. Here, we report a novel function of Bcl-3 in maintaining colorectal CSC homeostasis by activating Wnt/β-catenin signaling. Silencing Bcl-3 suppresses the self-renewal capacity of colorectal CSCs and sensitizes CRC cells to chemotherapeutic drugs through a decrease in Wnt/β-catenin signaling. Moreover, our data show that Bcl-3 is a crucial component of Wnt/β-catenin signaling and is essential for β-catenin transcriptional activity in CRC cells. Interestingly, Wnt3a increases the level and nuclear translocation of Bcl-3, which binds directly to β-catenin and enhances the acetylation of β-catenin at lysine 49 (Ac-K49-β-catenin) and transcriptional activity. Bcl-3 depletion decreases the Ac-K49-β-catenin level by increasing the level of histone deacetylase 1 to remove acetyl groups from β-catenin, thus interrupting Wnt/β-catenin activity. In CRC clinical specimens, Bcl-3 expression negatively correlates with the overall survival of CRC patients. A significantly positive correlation was found between the expression of Bcl-3 and Ac-K49-β-catenin. Collectively, our data reveal that Bcl-3 plays a crucial role in CRC chemoresistance and colorectal CSC maintenance via its modulation of the Ac-K49-β-catenin, which serves as a promising therapeutic target for CRC. Subsequently, β-catenin in the cytoplasm is released from the β-catenin destruction complex, and stabilized β-catenin translocates to the nucleus to form a transcriptional complex with LEF/TCF.6 In the absence of Wnt ligand treatment, β-catenin in the cytoplasm is rapidly degraded by a multiprotein complex that includes APC (adenomatous polyposis coli), AXIN, and GSK-3 (glycogen synthase kinase-3) kinase. One of the core components of the Wnt/β-catenin destruction complex is GSK-3. Under normal circumstances, GSK-3 phosphorylates β-catenin on its N-terminal Thr41, Ser37, and Ser33 residues.10 Thereafter, phosphorylated β-catenin is ubiquitinated and degraded by the proteasome.11Colorectal CSCs can be identified by a variety of cell surface markers, including CD133, CD44, and CD24.13 Bcl-3 promotes breast cancer metastasis14 by stabilizing Smad3/TGFβ signaling.15 In glioblastoma cells, Bcl-3 significantly facilitated proliferation by stabilizing the expression of STAT3, p-STAT3, and STAT3 signaling target genes, including Bcl-2, MCL-1, and cyclin D1.16 Recently, the function of Bcl-3 in CRC has been reported by various research groups. First, Bcl-3 was found to promote CRC cell growth by stabilizing the c-Myc protein level and regulating ERK signaling.17 In addition, Bcl-3 promotes CRC tumorigenesis based on the activation of AKT signaling.18 Finally, Bcl-3 suppresses inflammation-associated colon tumorigenesis in epithelial cells by dampening tumorigenic NF-κB signaling.19 Moreover, previously uncovered evidence demonstrated the function of Bcl-3 in pluripotency maintenance. Bcl-3 functions as a downstream molecule of LIF/STAT3 signaling and positively regulates pluripotency genes, including Oct4, Sox2, and Nanog, in mouse embryonic stem cells (mESCs).21B-cell lymphoma 3 (Bcl-3) is a member of the IκB family, and it is an important transcriptional regulator that forms complexes with p50 and p52 on DNA.Due to the overactivation of Bcl-3 and β-catenin that occurs in many cancers, we hypothesize that there is a potential functional relationship between these two transcriptional regulators. In this study, we explored the possibility of Bcl-3 as a component in Wnt/β-catenin signaling and determined the biological role of Bcl-3 in CRC progression, as well as in colorectal CSCs.17 as well as the research that Bcl-3 is necessary for pluripotency and self-renewal of mESCs,21 we hypothesized that Bcl-3 may play an important role in maintaining the homeostasis of colorectal CSCs. The in vitro stemness capability of cells was assessed by flow cytometry to examine the percentage of cells coexpressing CD44 and CD133.24 The knockdown (KD) efficiency of two different shRNA sequences targeting Bcl-3 is shown in Supplementary Fig. +CD133+ cells in CRC cells . Linear regression analyses showed that the mRNA level of Bcl-3 was positively correlated with CD133 and SOX2 25  and oxaliplatin  treatment of HCT116 and SW620 cells. There was a significant increase in the mRNA level of nes Fig. . The sam33 which prompted us to examine whether Bcl-3 responds to Wnt ligands. The expression of Bcl-3 increased after 1 h of Wnt3a treatment and reached maximal induction after 4 h  analysis Fig. . As show36 We used a TOP/Flash reporter to determine whether Bcl-3 affects β-catenin–TCF-4/LEF transcriptional activity. Interestingly, Bcl-3 KD strongly inhibited TOP/Flash reporter expression in the HCT116 cell line with or without Wnt3a treatment  into HEK293T cells, followed by IP of the cell lysates with an antibody against the HA tag. Immunoblot assays revealed that both WT and dN89 β-catenin could bind to Bcl-3  and Ac-K49 β-catenin is required for activation of Wnt target genes,lls Fig. . The samlls Fig. . When dells Fig. . Similarlls Fig. . Moreovells Fig. .Fig. 6Bc41 As shown in Supplementary Fig. CD133 and SOX2 genes indicated the same effect of ICG-001 on endogenous target gene expression  and niacinamide , and analyzed the protein level of Ac-K49 β-catenin in Bcl-3 KD and control cells. As shown in Supplementary Fig. 34 We hypothesized that Bcl-3 alters the interaction between β-catenin and HDACs. Co-IP assays showed that β-catenin bound to endogenous Bcl-3, HDAC-1, HDAC-6 25 and found that Bcl-3 mRNA in adenocarcinoma tissues was much higher than that in normal colorectal tissues . Patients with higher levels of Bcl-3 mRNA (n = 173) showed significantly lower relapse-free survival than patients whose tumors expressed lower levels of Bcl-3 . Whether Bcl-3 influences the binding of acetylated H3 to the Wnt target gene promoter remains to be determined.In the process of oligodendrocyte differentiation, HDAC-1 competes with β-catenin for TCF7L2 interaction to negatively regulate Wnt target gene expression.Because of the crucial role of Wnt signaling in CRC progression and colorectal CSC maintenance, inhibition of Wnt/β-catenin signaling by targeting Bcl-3 may offer a therapeutic approach in CRC treatment.Supplementary material


Although this terminology is widely used, it may not reflect the expression of CX3CR1 on Mo subsets. Using an unsupervised multiparametric analysis of blood Mo in steady state and after sterile peritonitis, we observed that CX3CR1 expression did not discriminate the CMo from the NCMo subsets. Our results highlight that despite being a reliable reporter to discriminate Mo subpopulations, EGFP level in Cx3cr1EGFP+/ mice does not reflect CX3CR1 expression measured by a fluorescently-labeled CX3CL1 chemokine and a CX3CR1 specific antibody. In conclusion, authors should be cautious not to identify murine classical and non-classical Mo as CX3CR1low and CX3CR1high but rather use alternative markers such as the combination of Ly6C and CD43.In mice, monocytes (Mo) are conventionally described as CX3CR1 Chemokine receptors are key G protein-coupled receptors for immune cell trafficking in inflammation and physiological conditions. They are critical for lymphocytes homing, for normal lymphoid tissue development, for Mo egress from bone marrow (BM) and facilitate organ infiltration of immune cells . BecauseCx3cr1 gene was replaced with the gene encoding the enhanced green fluorescent protein (EGFP) and analyzed it in heterozygote (Cx3cr1EGFP+/) or homozygote (Cx3cr1EGFP/EGFP) configuration. This approach allowed the examination of the CX3CR1 expression pattern and migration of cells that normally express this receptor. Based on the green fluorescence and the use of a Fractalkine/NTN-Fc fusion peptide, they confirmed the presence of CX3CR1 on the surface of Mo, part of NK cells, circulating and skin resident DC and microglia. However, the CX3CL1 receptor was absent in resting tissue macrophages , astrocytes, and oligodendrocytes, neutrophils and eosinophils, B lymphocytes, resting and concanavalin A activated T cells, unlike what has been observed in humans. Nonetheless, recent works clearly demonstrated that terminally differentiated cytotoxic CD8+ T cells express CX3CR1   –15.Cx3cr1EGFP+/ mice   marker in 3CR1high . This st (>24 h) . Green f (>24 h)  observedCx3cr1EGFP+/, Cx3cr1EGFP/EGFP . GFP/EGFP  and Nr4aa1+/EGFP  mice werLPS was administered intraperitoneally at 300 ng/kg in 100 μl phosphate-buffered saline (PBS). LPS-free PBS was administered for control group.− cells were considered to be parenchymal.Intravascular (i.v.) CD45 labeling was performed, as previously described . Mice weBlood was drawn via retro-orbital puncture with heparin and directly stained with antibodies.The spleen and the lung were digested in RPMI-1640 medium  containing 1 mg/mL collagenase IV  for 30 min at 37°C. After digestion, tissues were mashed through a 30 μm pore cell strainer  and washed in PBS. Cell surface staining was performed by incubating for 20 min the freshly prepared cells or whole blood with Panel-1 antibodies . After sCx3cr1EGFP/EGFP mice or incubating CX3CR1-proficient Mo in the presence of unlabeled CX3CL1 at 1μM before staining.For surface CX3CR1 staining on circulating monocytes, lysed blood cells were incubated or not with 50, 100, or 200 nM murine CX3CL1-AF647  for 30 min at 37°C. Cell were washed once in PBS and cell surface staining was performed using Panel-2 . When ciSample acquisitions were performed on the LSRFortessa X-20 Flow cytometry (BD) using FACSDIVA software (BD), and data were analyzed with FlowJo software (BD) and Cytobank analysis platform   was used to compare differences in mean fluorescence intensity (MFI) between two groups. Nonparametric two-tailed Wilcoxon signed-rank test with a significance threshold of alpha (α = 0.05) was used to compare differences in MFI from two matched samples. Statistical tests were performed using commercial statistics software Prism .Nr4a1 reporter, and CX3CR1  provides a reliable staining of CX3CR1 in Cx3cr1+/EGFP compared to circulating Mo from Cx3cr1EGFP/EGFP mice  from tissue-resident cells (CD45−)  as well as 4 h after LPS inoculation with a higher proportion in BM vasculature when compared to steady state   at homeo (CD45−)  and infl (CD45−) . Blood ts before  and 91.7njection ]. While njection , 19, 25.low and CX3CR1high, respectively, should not be used in mice. In the absence of EGFP reporter, we propose to refer to an alternative phenotypic strategy using Ly6C and CD43 to identify Mo subsets at homeostasis and inflammation, in blood and tissues.In conclusion, we show that CMo express higher level of CX3CR1than NCMo hence the definition of these subsets as CX3CR1The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Comité d'éthique en expérimentation animale Charles Darwin N°5.This manuscript has been released as a pre-print at bioRxiv, Meghraoui-Kheddar et al. .AM-K, AB, and CC designed the study and wrote the manuscript. AM-K performed experimental work, compiled the data, and performed data analysis. SB performed the genotyping of the mice. AB and CC co-authored the manuscript and provided financial support. All authors contributed in reviewing the manuscript.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.


And covalent bonding and hydrogen-bond interaction models were presented for analyzing the thermal conductivity and dielectric properties.Dielectric materials with good thermal transport performance and desirable dielectric properties have significant potential to address the critical challenges of heat dissipation for microelectronic devices and power equipment under high electric field. This work reported the role of synergistic effect and interface on through-plane thermal conductivity and dielectric properties by intercalating the hybrid fillers of the alumina and boron nitride nanosheets (BNNs) into epoxy resin. For instance, epoxy composite with hybrid fillers at a relatively low loading shows an increase of around 3 times in through-plane thermal conductivity and maintains a close dielectric breakdown strength compared to pure epoxy. Meanwhile, the epoxy composite shows extremely low dielectric loss of 0.0024 at room temperature and 0.022 at 100 ℃ and 10 These energy storage and conversion devices working at high electric field, probably suffer from not only high electric field intensity but also a great number of heat fluxes4. In this regard, dielectric polymer composites with high thermal conductivity and high glass transition temperature (Tg) and outstanding thermal stability are necessary to maintain the good performance and reliability for energy conversion devices8. Thermosetting epoxy resins have been widely used as insulating materials in electrical engineering because of their excellent thermal stability, dielectric properties and low cost, which show significant potential in the application of energy storage and conversion at high temperature. For instance, Chen et al.9 presented a kind of epoxy resin films by grafting asymmetric alicyclic amine-polyether amine molecular chain structure, which enhanced dielectric breakdown strength, thermal stability and charge–discharge efficiency of epoxy film capacitors for their application in electric power systems and electrical automobile industry. However, this kind of epoxy resin still has low thermal conductivity due to its amorphous structure10, possibly leading to the problem of heat dissipation and the partially interior high temperature in energy conversion devices. Although liquid crystal epoxy resins are obtaining more attention due to their relatively high thermal conductivity12, there are some limitations such as lower thermostability, therefore the nanocomposites are necessary to obtain high thermal conductivity.Dielectric polymer composites with high dielectric constant and high dielectric breakdown strength are needed for many power electronic devices and power equipment under high electric field, such as energy storage and conversion devices13. BNNs has a large band gap (5.7–6 eV), high thermal conductivity in (002) or (004) crystal plane15, which is a good candidate for obtaining a material with high in-plane thermal conductivity and desirable dielectric properties18. However, through-plane thermal conductivity of BNNs is relatively low (around 30 W/m K). And the BNNs is easier to form horizontal distribution and the horizontal orientation of (002) or (004) in polymer matrix due to its two-dimensional structure and large aspect ratio19, benefit for the enhancement of in-plane thermal conductivity. Therefore, the enhancement of through-plane thermal conductivity is more difficult because of the much lower intrinsic through-plane thermal conductivity of BNNs and the interface thermal resistance between BNNs and epoxy matrix. In fact, some strategies have been employed to enhance thermal conductivity and maintain dielectric breakdown strength as high as possible, such as the alignment of fillers along through-plane direction20, design of multilayered structures21, and core–shell structures23, and so on. Alumina also has outstanding dielectric properties and an acceptable thermal conductivity of around 30 W/m K25. Alumina spheres and BNNs are expected to have a synergistic effect on increasing through-plane thermal conductivity by forming partial networks in through-plane direction.Carbon materials have high thermal conductivity, but their are also electrically conductive30. For example, Li et al. presented a kind of BNNs/c-BCB nanocomposites by using BNNs as fillers, obtaining significantly increasing dielectric breakdown strength and energy storage density at high temperature26. Compared to c-BCB, epoxy resins have lower cost and higher dielectric breakdown strength9. In particular, its low cost is more competitive for the practical application in energy storage and conversion devices. Moreover, some other strategies have been developed for high energy storage performances35.Meanwhile, alumina and BNNs are also widely used as inorganic fillers to enhance the dielectric properties of polymers because of their desirable dielectric breakdown properties at high electric filed strengthIn this work, epoxy composites were prepared by using epoxy resin as polymer matrix and two dimensional BNNs and zero dimensional alumina as inorganic fillers, which are expected to improve thermal conductivity, dielectric constant and maintain the dielectric breakdown strength of composites. The zero dimensional alumina in epoxy resin matrix can offer deep trap, effectively restricting the migration of free carriers, resulting in a further increase in dielectric breakdown strength of composites. Moreover, the interface interaction between fillers and epoxy matrix is analyzed to explain the improvement of dielectric properties and thermal transport performance due to the formation of hydrogen bonds and carbon boron oxygen bonds. This study is expected to obtain a deeper understanding about the effects of the embedded fillers with different dimensions including zero dimensional fillers and two dimensional fillers into polymer matrix and interface characteristic on thermal conductivity and dielectric properties of the polymer composites with various fillers.BNNs with lateral size of around 1 μm was provided by XFNANO Co. Ltd, Jiangsu. Alumina microspheres with a average diameter of 200 nm were provided by Aladdin Reagent Co. Ltd, Shanghai. The bisphenol-A epoxy resin  were commercially available from Hexion Specialty Chemicals Co. Ltd, Shanghai. And methyl tetrahydrophthalic anhydrite and benzyldimethylamine were employed as the hardener and catalytic agent, respectively, which were purchased from Aladdin Reagent Co. Ltd, Shanghai.The epoxy composites with hybrid fillers were prepared by the following experimental procedures. Alumina and BNNs of a certain proportion were mixed into hardener solution and sonicated for forming a homogeneously dispersed suspension, which had no visible agglomerations and there is no residual fillers on the edges of beaker. Subsequently, epoxy monomers were mixed into the suspension of fillers and hardener solution and effectively dispersed by ball milling in a via planetary mixer with Zirconia balls of various sizes, which their diameters are 3 mm and 5 mm, respectively. And then the catalytic agent was dispersed by an extra ball milling for a short time. After that, the obtained suspensions were carried out a degassing treatment via a vacuum equipment at around 60 °C until no bubbles removed . Then the final suspensions were infunded into a mould for curing process by a precuring process at 100 °C for 120 min and subsequently post curing at 150 °C for 600 min. In this study, alumina, BNNs and epoxy were abbreviated as A, B and Ep, respectively. B/Ep composites were prepared by using various filler loading from 2 to 10% (percent by volume), which were defined as A2, A4, A6, A8 and A10, respectively. For A/B/Ep, the loading alumina was same at 6%, and those of BNNs were 2 and 4%, respectively (defined as A6 + B2 and A6 + B4).α) and specific heat (Cp) were tested by using laser flash analyzer . The densities (ρ) of specimens were obtained by measuring their volume and mass. Thermal conductivity (TC) values of specimens were obtained by a typical equation: TC = α × Cp × ρ. The specimen was sliced into quadrate with the length of around 1 cm on the side. Subsequently, a layer of graphite was sprayed onto the surface of samples and measured at different temperature. For dielectric measurements, a layer of gold were sputtered onto the surface of samples used as electrodes via an auto sputtering equipment. Dielectric constant, dielectric loss and alternating current conductivity of samples were measured by broadband dielectric analyzer . The values of dielectric breakdown strength were tested by a classical breakdown system with two ball–ball electrodes. The slice of specimens was held by two spherical electrodes and then was immersed into electrically insulating oil. Subsequently, step-voltage measurement with a rate of 2 kV/s was carried out until breakdown. Thermal gravimetric analysis (TGA) was carried out from room temperature to 800 °C at the temperature rate of 10 °C/min in nitrogen atmosphere. Differential scanning calorimetry  was carried out at temperature from 25 to 200 °C at a heating rate of 10 °C/min under a nitrogen atmosphere to study glass transition temperature (Tg) of samples. Infrared spectrums of the alumina and BNNs were obtained by a fourier transform infrared spectrometer  for further analyzing the groups on the surface of fillers. Surface morphologies of fillers in the epoxy composites were observed by a SEM equipment (FEI QUANTA F250). The phase structure of materials was examined by X-ray diffraction using Bruker D2 PHASER diffractometer.Thermal diffusivity , the peak of hydroxyl group on the surface of the alumina spheres can be observed at 3452 cm−1. The thermogravimetric analysis (TGA)  spectrum of the alumina spheres and the BNNs powder is shown in Fig. Figure Moreover, alumina microspheres will be a connecting bridge among separated BNNs, forming some partial networks to increase thermal transport performance. More importantly, the alternating distribution structure of various fillers with different dimension can show a synergistic effect on the improvement of dielectric breakdown and discharge. Figure 41. Experimental values and theoretical evaluation of through-plane TC of the pure epoxy and its composites at room temperature were shown in Fig. 43. Through-plane TC of the A2 is 0.20 W/m K, rather close to that of pure epoxy. It is worth noting that the through-plane TCof A/B/Ep composites are distinctly higher than those of the A/Ep composites. For instance, through-plane TC of the A8 and A10 are 0.32 and 0.35 W/m K, respectively. And those of the A6 + B2 and A6 + B4 are 0.38 and 0.47 W/m K, respectively.Generally speaking, all of the Morphology, structure and dispersion of filler and interface resistance have a significant effect on thermal transport performance of polymer nanocompositesThe results may be ascribed to the following reasons. (1) Intrinsic thermal conductivity of BNNs is higher than that of alumina microspheres. (2) Alumina microspheres and BNNs have a synergistic effect on the enhancement of through-plane TC. More precisely, alumina microspheres serve as connecting bridges for separated BNNs, favorable for the formation of partial thermal transport networks due to the difference of their morphologies. (3) Interface interaction between epoxy and BNNs, forming covalent bonds in addition to hydrogen bonds, is stronger than that between epoxy and alumina.44. Bruggeman model was shown as follows:Kc, Km, and Kf are the thermal conductivity of the epoxy composites, epoxy matrix, and alumina filler, respectively, and Φf are the volume fraction of the filler. As seen from Fig. In order to further analyze thermal transport performance of the epoxy composites, Bruggeman model was used to predict their thermal conductivity valuesg) of epoxy composites, as shown in the Fig. Figure –1 to 106 Hz at room temperature (25 ℃). The dielectric constant and dielectric loss of epoxy composites with different fillers and loading were shown in Fig. 46, effecting on the migration and accumulation of free carriers at the interface between fillers and polymer matrix. BNNs have a large band gap and high electrical resistivity, suppressing migration of carriers and charges, leading to the lower electronic displacement polarization.As expected, the dielectric constant increased with increasing filler content in epoxy resin matrix from 103 to 106 Hz. In contrast, A/B/Ep composites show a relatively slow increase in dielectric loss with the increment of frequency. Meanwhile, the dielectric loss of A/B/Ep composites are also distinctly lower than those of A/Ep composites. For instance, the dielectric loss of A6 + B2 is only 0.0024 at 0.1 Hz, which is lower than that of neat epoxy, as shown in Fig. –1 to 106 Hz. It can be attributed to the high intrinsic resistance of BNNs and the interface layer between epoxy matrix and fillers based on the mutil-core model47, restraining the direct migration of carriers and prolonging the transfer router of carriers in composites. Meanwhile, the intercalation of BNNs introduces great number of electron traps, resulting in the formation of peak valley of electrical condution. Therefore, it declares that the intercalating BNNs play key role in blocking the conduction of carries in the epoxy composites.The dependence of dielectric loss of epoxy composites on frequency was shown in Fig. P(E) is the cumulative probability of electrical failure, E is the experimental breakdown strength, β is the shape parameter which evaluates the scattering of data, and E0 represents the characteristic dielectric breakdown strength at the cumulative failure probability of 63.2%. P is calculated as follows:i is the i-th result when E values are sorted in ascending order of breakdown strength data, and n is the number of total data points.Dielectric breakdown strength of materials is an important parameter to evaluate the insulation characteristic of dielectric materials. The dielectric breakdown strength values are analyzed by two-parameter Weibull distribution given by Eqs.  and 3)::3):2\docFigure 2 Hz) when the tested temperature is at 100 ℃ or more. In particular, the dielectric constant and loss of pure epoxy at 125 ℃ and 10–1 Hz are 7.7 and 0.45, respectively. Compared to the pure epoxy, A6 + B2 shows no obvious increase in dielectric constant and loss at 100 ℃ and still remains relatively low dielectric constant (6) and dielectric loss (0.16) at 125 ℃ and 10–1 Hz, as shown in Fig. –1 Hz, which is desirable for energy storage and energy conversion in capacitors and the practical application at high temperature.To further study the dielectic properties and explore their potential for application at high temperatures, the temperature dependence of dielectric properties of pure epoxy and A/B/Ep composites were measured. As shown in Fig. 51, the edges of BNNs have a large number of unstable and broken B–N bonds. These broken chemical bonds especially for B atoms are identified as B free radicals51, which are rather active to combine the hydroxyl or amine groups. Therefore, th edges of BNNs exist some hydroxyl groups by absorbing water molecular. More notably, the broken chemical bonds or B free radicals could be obtained during the process of epoxy composites by sonicating and ball milling. These new B free radicals is feasible to combine the hydroxyl or amine groups in epoxy resin matrix and hardener during pre-curing and post-curing process under high temperature, as illustrated in Fig. In addition to the synergistic effect of alumina and BNNs, the other important mechanism for enhancing dielectric breakdown strength and high-temperature dielectric properties is the interface interaction between fillers and polymer matrix. In the work, the surface of alumina microspheres have a large number of hydroxyl groups see Fig. a,b, whic52, the interface issues between nanofillers and polymer matrix have already obtained lots of attention and extensive study53. And some theoretical and empirical models about the interface were presented54. In particular, multi-core model presented by T Tanaka was generally accepted by research scholars47. In this work, a similar multi-core model is constructed by utilizing the hydrogen bond and C–O–B chemical bond, as illustrated in Figs. 60. In this regard, the study on the interface characteristic of nanocomposite dielectric materials is very promising for addressing the challenges in interface thermal resistance and improving dielectric properties.Since nanodielectric material was proposed by LewisUed = 0.5 ε0εrE2. Therefore, the energy density (Ued) of materials mainly depends on the dielectric constant (εr) and dielectric breakdown strength (E). Compared to pure epoxy, the E of A6 + B2 is rather close and its εr is relatively higher. The energy density of A6 + B2 shows an increasing by 11.3% compared to that of pure epoxy, and other epoxy composites show slight decrease in energy storage capability due to the decrease of dielectric breakdown strength. Even though, these epoxy composites still shows strong application potential due to the distinct increase of through-plane thermal conductivity, which can be capable of working at the high temperature. Moreover, it is observed that the both pure epoxy and its composites show extremely low dielectric loss characteristic, which is very benefit for the charge–discharge efficiency and good stability61. It is clear that epoxy and its composites by intercalating BNNs and alumina are promising for the energy storage and energy conversion devices at relatively high temperature because of its extremely low dielectric loss and ultra-high dielectric breakdown strength. It’s worth noting that energy density of the epoxy and its composites with hybrid fillers could be enhanced significantly by preparing the much thinner epoxy films9 due to the significant increase in dielectric breakdown strength for thin-film materials, and its composites films with high energy density and thermal conductivity are expected in our future work.The energy density of capacitors with linear dielectrics can be expressed by the following equation –1 Hz compared to that (0.05) of pure epoxy under same testing condition. Moreover, the glass transition temperature and thermal decomposition temperature of A6 + B2 also is much higher than that of pure epoxy and A/Ep composites, explaining that the A/B/Ep composites have lower dielectric loss at high temperature. Therefore, the simple strategies of epoxy with hybrid fillers show good synergistic effect, interface interaction and outstanding comprehensive properties, which is desirable for applications in microelectronic devices and power equipments under high electric field such as energy storage and conversion devices, insulated gate bipolar translator (IGBT) and gas insulated substation (GIS) etc.In summary, we prepared two epoxy-based composites using alumina or the hybrid fillers of alumina and BNNs as fillers. The thermal conductivity and dielectric properties of epoxy composites were studied. Compared to pure epoxy and A/Ep composites, the A/B/Ep show good synergistic effect on the enhancement of thermal transport property and maintaining high dielectric breakdown strength and low dielectric loss. In particular, the A6 + B2 exhibits relatively higher thermal conductivity and energy density. For instance, through-plane thermal conductivity of A6 + B2 increase of around 3 times compared to those of pure epoxy. Meanwhile, the A6 + B2 shows ultra-low dielectric loss (0.022) at high temperature (100 ℃) and 10Supplementary Information.


GBA1—a gene that encodes the lysosomal hydrolase β-glucocerebrosidase (GCase). The neuronopathic forms of GD (nGD) are characterized by severe neurological abnormalities that arise during gestation or early in infancy. Using GD-induced pluripotent stem cell (iPSC)-derived neuronal progenitor cells (NPCs), we have previously reported that neuronal cells have neurodevelopmental defects associated with the downregulation of canonical Wnt signaling. In this study, we report that GD NPCs display elevated levels of Dkk1, which is a secreted Wnt antagonist that prevents receptor activation. Dkk1 upregulation in mutant NPCs resulted in an increased degradation of β-catenin, and there was a concomitant reduction in lysosomal numbers. Consistent with these results, incubation of the mutant NPCs with recombinant Wnt3a (rWnt3a) was able to outcompete the excess Dkk1, increasing β-catenin levels and rescuing lysosomal numbers. Furthermore, the incubation of WT NPCs with recombinant Dkk1 (rDkk1) phenocopied the mutant phenotype, recapitulating the decrease in β-catenin levels and lysosomal depletion seen in nGD NPCs. This study provides evidence that downregulation of the Wnt/β-catenin pathway in nGD neuronal cells involves the upregulation of Dkk1. As Dkk1 is an extracellular Wnt antagonist, our results suggest that the deleterious effects of Wnt/β-catenin downregulation in nGD may be ameliorated by the prevention of Dkk1 binding to the Wnt co-receptor LRP6, pointing to Dkk1 as a potential therapeutic target for GBA1-associated neurodegeneration.Gaucher Disease (GD), which is the most common lysosomal storage disorder, is caused by bi-allelic mutations in  GBA1 gene. GBA1 encodes β-glucocerebrosidase (GCase), which is a 60 KDa lysosomal hydrolase that breaks down glucosylceramide (GlcCer). Mutations in GBA1 affect GCase protein folding, resulting in endoplasmic reticulum retention and subsequent degradation [GBA1 are the most common genetic risk factor for Parkinson’s disease (PD), with a 5–20 fold increased risk of developing PD in GD patients or carriers of GBA1 mutations [Gaucher Disease (GD) is an autosomal recessive disease caused by monogenic mutations in the radation . The decradation ,4,5,6. Gradation . Patientradation ,13,14,15radation ,17,18. Autations ,22,23,24GBA1 mutations cause lysosomal abnormalities, defects in autophagic clearance, and the accumulation of protein aggregates in neurons [Neurodegeneration in neuronopathic GD (nGD) has been associated with dysfunction in the autophagy and lysosomal (ALP) pathway, which is essential for the survival of post-mitotic neurons ,26. Many neurons ,27,28,29 neurons . TFEB is neurons ,32. Furt neurons ,34,35, sWnt genes, receptors, cofactors, and regulators. Among the different Wnt signaling mechanisms identified, the “canonical” Wnt pathway is the most studied and well-characterized. This signaling cascade begins with the interaction between Wnt ligands (secreted glycoproteins) and the receptor complex, consisting of Frizzled (Fz) seven-pass transmembrane receptor and single pass low density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors [The Wnt pathway is an important regulator of biological phenomena such as gastrulation, cell fate decisions, cell polarity, embryonic patterning, organogenesis, and the maintenance of stem cell pluripotency ,37,38,39eceptors . This ineceptors ,50. Actieceptors .DKK1 gene expression in fibroblasts from type 1 GD patients [Wnt signaling can be modulated both intracellularly and extracellularly. Various extracellular antagonists of the canonical Wnt pathway have been identified, such as the secreted frizzled-related protein (sFRP), Wnt inhibitory factor 1 (Wif1), and Dickkopf (Dkk) family of secreted proteins. Dickkopf-1 (Dkk1), which is one of the four known Dkk family members, is a negative regulator of the Wnt pathway. Dkk1 inhibits Wnt signaling by binding to the co-receptor LRP6, thereby disrupting the LRP6-Fz complex and promoting LRP6 receptor internalization, hence limiting the availability of the receptor complex to Wnt ligands ,52. It hpatients . HoweverUsing GD patient-derived iPSCs, we previously showed that this in vitro system recapitulated pathological hallmarks of the disease in all of the cell types that we tested . GD-macrv/v) N2 supplement (Life Technologies) and 20 ng/mL basic fibroblast growth factor (bFGF) . The EBs adhered to the Matrigel and started the formation of neuronal rosettes within 7–10 days in culture. The rosettes were manually picked and expanded as NPCs in Neurobasal Medium (Life Technologies) containing 1X (v/v) GlutaMAX (Life Technologies), 1X (v/v) non-essential amino acids (Life Technologies), 1X (v/v) B27 supplement (Life Technologies), 1X (v/v) Pen/Strep, and 20 ng/mL bFGF (Peprotech), with a change of media every other day. The work with the human iPSC lines used in this study is considered non-human research; these iPSC lines are exempt under 45 CFR Part 46. This work was approved by the University of Maryland School of Medicine Institutional Review Board (IRB) on 15 July 2009 (HP-42545).The nGD and control (MJ) iPSC lines utilized in this study have been previously described ,59. The rWnt3a (5036-WN) and rDkk1 (5439-DK) were purchased from R&D Systems  and reconstituted as per the manufacturer’s instructions. Stocks of rWnt3a (200 μg/mL) and rDkk1 (100 μg/mL) were prepared in sterile PBS containing 0.2% bovine serum albumin. The final concentration used for both recombinant proteins was 100 ng/mL.GAPDH and the relative gene expression was calculated using the 2(−ΔΔCt) method. The primer sequences used in this study were as follows:To analyze gene expression in NPCs, mRNA was isolated using an RNA isolation kit , and cDNA was synthesized using the iScript kit . Utilizing SYBR Green PCR Master Mix , the gene expression was determined by qPCR  in duplicate or triplicate wells. The mRNA expression for every gene was normalized to the corresponding values of CYCD1Forward: 5′- CCG TCC ATG CGG AAG ATC- 3′Reverse: 5′- GAA GAC CTC CTC CTC GCA CT- 3′AXIN2Forward: 5′- AGT GTG AGG TCC ACG GAA AC- 3′Reverse: 5′- TGG CTG GTG CAA AGA CAT AG- 3′DKK1Forward: 5′- GAT CAT AGC ACC TTG GAT GGG- 3′Reverse: 5′- GGC ACA GTC TGA TGA CCG G- 3′GAPDHForward: 5′- CAA GAT CAT CAC GAA TGC CTC- 3′Reverse: 5′- GCA TGG ACT GTG GTC ATG AGT C- 3′v/v) paraformaldehyde  for 15 min, washed thrice with DPBS, and blocked in Buffer A (PBS containing 8% FBS (v/v)) for 30 min at room temperature. Primary antibodies were prepared in Buffer B (Buffer A with 2 mg/mL saponin) and incubated for 1–2 h at room temperature or overnight at 4 °C. The cells were then incubated with the appropriate fluorochrome-conjugated secondary antibodies for 1 h at room temperature. Nuclei were labeled using 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium .NPCs were plated in 8-well chamber slides (Thermo Fisher Scientific) and cultured as described above. When the slides were ready to be stained, media were aspirated from the chambers and the cells were washed once with Dulbecco’s Phosphate-Buffered Saline (DPBS) . The cells were then fixed with 4% (Lysotracker staining was performed by adding 1 μM Lysotracker Red DND-99 (ThermoFisher Scientific) directly to cell culture medium and incubating it for 45 min to 1 h at 37 °C. The cells were then processed as described above. The following antibodies were used in this study: Primary antibodies; Cell Signaling Technology : pLRP6 (Ser1490) (#2568), non-phospho (Active) β-catenin (#8814), and pGSK-3β (Ser9) (#9323); U. Iowa Developmental Hybridoma Bank : LAMP1 (H4A3); Santa Cruz: Dkk1 (sc-374574) and total β-catenin (sc-7199). Additionally, the secondary antibodies were Goat anti-rabbit Alexa Fluor 488 and Donkey anti-mouse Cy3 .Confocal immunofluorescence images were taken using an inverted Nikon Eclipse Ti2 microscope attached to a spinning disk unit  and Hamamatsu sCMOS camera . A Nikon oil immersion objective  was used for all imaging experiments. The excitation wavelengths used were 405, 488, and 561 nm for blue, green, and red fluorophores, respectively. Further image processing and analysis was conducted using Fiji software . The fluorescence intensity of the respective signal or Lysotracker counts was obtained from at least three independent replicates (3–5 different fields/replicate). The mean fluorescence intensity (MFI) and average puncti count were calculated accordingly.NPCs were cultured in 12-well plates  and treated as indicated in the text. The cells were lysed directly in SDS sample buffer, sonicated, and denatured by heating at 95 °C for 5 min. The samples were then loaded onto 4–20% SDS/polyacrylamide gels , and electrophoresis was performed at 100 V for 2 h. This was followed by the transfer of protein onto a nitrocellulose membrane, blocking with 5% milk in Tris-buffered saline Tween (TBS-T), and incubation with the indicated primary antibodies overnight at 4 °C. Anti-mouse or anti-rabbit HRP-conjugated secondary antibodies were added to the membrane for 1 h at room temperature. Membranes were developed with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific), and imaged using the Chemidoc system and Imagelab software (BioRad). Densitometry analysis was conducted using the Imagelab software (BioRad) and all proteins were normalized to β-actin.t-tests for comparing two groups with a confidence interval of 95%. Results are expressed as the mean ± standard error of the mean (SEM).Data were analyzed using Prism software version 7.0a . Significance was assessed using two-tailed unpaired Student’s GBA1 mutations downregulate the Wnt/β-catenin pathway in nGD iPSC-derived NPCs. In this system, the level of β-catenin, which is the main mediator of canonical Wnt signaling, was lower in nGD NPCs when compared to wild-type (WT) cells. This was attributed to the destabilization of β-catenin through proteasomal degradation since treatment with a proteasomal inhibitor was able to rescue the levels of both total and active (non-phosphorylated) β-catenin [AXIN2 and CYCD1 by qRT-PCR analysis, there was a significant reduction in AXIN2 and CYCD1 transcript levels in the mutant NPCs, consistent with reduced β-catenin activity in these cells  and the lysosomal compartment. In this study, we report that Dkk1 plays a key role in downregulation of the canonical Wnt pathway in nGD iPSC-derived NPCs, and that Wnt downregulation interferes with the lysosomal function. Dkk1 elevation in the mutant cells led to lysosomal depletion, reducing the sequestration of GSK3β into these organelles, thereby facilitating the phosphorylation and subsequent degradation of β-catenin. A critical role of Dkk1 in this process was confirmed by demonstrating that the addition of rDkk1 to WT NPCs recapitulated the mutant phenotype. Importantly, the phenotypic abnormalities of the nGD neuronal cells were reversed by incubation with rWnt3a. The addition of this ligand to mutant NPCs outcompeted the elevated levels of endogenous Dkk1, enabling GSK3β sequestration into the lysosomal compartment, protecting β-catenin from degradation, and preventing lysosomal depletion.DKK1 mRNA expression in type 1 GD patient fibroblasts [The Dkk family consists of four secreted proteins (Dkk1-4). Although Dkk2 has been shown to activate Wnt signaling, the other members of the family play mostly inhibitory roles . Dkk1 waroblasts . It willWe have previously shown that in nGD NPCs, a loss of GCase activity downregulates the canonical Wnt pathway, leading to an increased degradation of β-catenin . This waWhen nGD NPCs were treated with rWnt3a, the mutant phenotype was reversed, showing that this extracellular ligand was capable of overcoming the inhibitory effects of elevated, endogenous Dkk1. There was an increased activation of LRP6, β-catenin stabilization through GSK3β lysosomal sequestration, and an increase in lysosome numbers. The strong link between the Wnt and ALP pathways we observed suggests the existence of a bi-directional feedback loop, so that alterations in one pathway may affect the other. This idea is in line with other reports on the ability of the endo-lysosomal system to modulate Wnt signaling and in turn, the Wnt pathway regulating the lysosomal compartment ,73,74,75As Wnt agonists  and antagonists  act extracellularly, these are important pharmacological targets. For instance, the inhibition of Dkk1 and sclerostin using a bi-specific antibody has been shown to stimulate bone formation, increase the bone mass density and strength, and improve fracture healing in pre-clinical models of bone disease . SimilarGBA1-associated neuropathology, and suggests that the canonical Wnt pathway is a potential therapeutic target for GBA1-associated neurodegeneration.In conclusion, this study suggests that the elevation of Dkk1 observed in nGD NPCs is responsible for downregulation of the canonical Wnt pathway and deregulation of the lysosomal compartment. We also found that exogenous rWnt3a was able to outcompete the deleterious effects of elevated Dkk1 in the mutant cells. This study implicates Dkk1 in