Notch Signaling Pathway Research Articles

Paralemmin-3 Augments Lipopolysaccharide-Induced Acute Lung Injury with M1 Macrophage Polarization via the Notch Signaling Pathway

BackgroundAcute lung injury (ALI) involves severe lung damage and respiratory failure, which are accompanied by alveolar macrophage (AM) activation. The aim of this article is to verify the influence of paralemmin-3 (PALM3) on alveolar macrophage (AM) polarization in ALI and the underlying mechanism of action. MethodsAn ALI rat model was established by successive lipopolysaccharide (LPS) inhalations. The influence of PALM3 on the survival rate, severity of lung injury, and macrophage polarization was analyzed. Furthermore, we explored the underlying mechanism of PALM3 in regulating macrophage polarization. ResultsPALM3 overexpression increased mortality of ALI rats, augmented lung pathological damage, and promoted AM polarization toward M1 cells. Conversely, PALM3 knockdown had the opposite effects. Mechanistically, PALM3 might promote M1 polarization by acting as an adaptor to facilitate transduction of Notch signaling. ConclusionPALM3 aggravates lung injury and induces macrophage polarization toward M1 cells by activating the Notch signaling pathway in LPS-induced ALI, which may shed light on ALI/ARDS treatments.

Changes in transcriptome regulations of a marine rotifer Brachionus plicatilis under methylmercury stress

Mercury (Hg), a heavy metal pollutant worldwide, can be transformed into methylmercury (MeHg) by various aquatic microorganisms in water, thus accumulating along the aquatic food chain and posing a particular challenge to human health. Zooplankton plays a crucial role in aquatic ecosystems and serves as a major component of the food chain. To evaluate the effects of MeHg on the rotifer Brachionus plicatilis and reveal the underlying mechanism of these effects, we exposed B. plicatilis to MeHg by either direct immersion or by feeding with MeHg-poisoned Chlorella pyrenoidesa, respectively, and conducted a transcriptomic analysis. The results showed that B. plicatilis directly exposed to MeHg by immersion showed significant enrichment of the glutathione metabolism pathway for detoxification of MeHg. In addition, the exposure to MeHg by feeding induced a significant enrichment of lysosome and notch signaling pathways of rotifers, supporting the hypothesis that MeHg can induce autophagy dysfunction in cells and disturb the nervous system of rotifers. In two different routes of MeHg exposure, the pathway of cytochrome P450 in rotifers showed significant enrichment for resisting MeHg toxicity. Our results suggest further studies on the potential mechanism and biological responses of MeHg toxicity in other links of the aquatic food chain.

Effect of humanine on the Notch signaling pathway in myocardial infarction.

By applying humanin (HN) before myocardial infarction (MI), its protection in myocardial injury and the possible roles of its cellular mechanism in the Notch pathway were investigated. The study was carried out at Fırat University Experimental Research Center (12/24/2018-12/23/2019). Spraque-Dawley rats were divided into 10 groups: I (control) (n = 6), II (HN 6 h) (n = 6), III (HN 24 h) (n = 6), IV (HN day 7) (n = 6), V (MI 6 h) (n = 7), VI (MI 24 h) (n = 7), VII (MI day 7) (n = 7), VIII (MI+HN 6 h) (n = 7), IX (MI+HN 24 h) (n = 7), and X (MI+HN day 7) (n = 7). To create MI, 200 mg/kg of isoproterenol (ISO) was administered to the rats subcutaneously. Moreover, 252 μg/kg of HN was given intraperitoneally (ip) to the rats on its own and before MI. Molecular parameters Notch1, Notch2, Hes1, Hes2, Jagged1, Jagged2, DLL1, and DLL4 were examined using polymerase chain reaction in the heart tissue, Notch1, Hes1, and DLL4 were examined using western blot, while heart tissue was taken for histochemical examinations. The mRNA expression levels of the Notch signaling members (Notch1, Notch2, Hes1, Hes2, Jagged1, Jagged2, DLL1, and DLL4) tended to decrease after MI. The Notch signaling members increased more significantly, especially toward day 7 after HN application before MI. In the western blot anylyses, the Notch1, Hes1, and DLL4 protein levels increased significantly toward day 7 in the groups given HN before MI. Moreover, the serum AST, LDH, CK-MB, and troponin I levels tended to decrease with the application of HN before MI and there was a significant decrease in edema, hemorrhage, and mononuclear cells in the heart tissue at 24 h post-MI and fibrosis on day 7 post-MI. HN administration before MI has a cardioprotective effect on rats via the Notch signaling pathway.

Colorectal Cancer Stem Cells and Targeted Agents.

Since their discovery, cancer stem cells have become a hot topic in cancer therapy research. These cells possess stem cell-like self-renewal and differentiation capacities and are important factors that dominate cancer metastasis, therapy-resistance and recurrence. Worse, their inherent characteristics make them difficult to eliminate. Colorectal cancer is the third-most common cancer and the second leading cause of cancer death worldwide. Targeting colorectal cancer stem cells (CR-CSCs) can inhibit colorectal cancer metastasis, enhance therapeutic efficacy and reduce recurrence. Here, we introduced the origin, biomarker proteins, identification, cultivation and research techniques of CR-CSCs, and we summarized the signaling pathways that regulate the stemness of CR-CSCs, such as Wnt, JAK/STAT3, Notch and Hh signaling pathway. In addition to these, we also reviewed recent anti-CR-CSC drugs targeting signaling pathways, biomarkers and other regulators. These will help researchers gain insight into the current agents targeting to CR-CSCs, explore new cancer drugs and propose potential therapies.

Exosome-mediated Delivery of miR-519e-5p Promotes Malignant Tumor Phenotype and CD8+ T-Cell Exhaustion in Metastatic PTC.

Distant metastases are the primary cause of therapy failure and mortality in patients with papillary thyroid carcinomas (PTCs). However, the underlying mechanism responsible for the initiation of tumor cell dissemination and metastasis in PTCs has rarely been investigated. The aim of this study was to investigate effects and underlying molecular mechanisms of circulating exosomal microRNAs (miRNAs) in distant metastatic PTCs. The most relevant circulating exosomal miRNA to distant metastatic PTCs were verified between distant metastatic PTCs and nondistant metastatic PTCs by miRNA microarray, quantitative real-time polymerase chain reaction (qRT-PCR) assays and receiver operating characteristic (ROC) curves. The parental and recipient cells of that circulating exosomal miRNA were then explored. In vitro and in vivo experiments were further performed to elucidate the function and potential mechanisms of circulating exosomal miRNAs that contribute to the development of distant metastases. We determined that PTC-derived exosomal miR-519e-5p was significantly upregulated in the circulatory system in distant metastatic PTCs. Further tests demonstrated that PTC cells can acquire a more malignant phenotype via hnRNPA2B1-mediated sorting of tumor suppressor miR-519e-5p into exosomes to activate Wnt signaling pathway via upregulating PLAGL2. Furthermore, miR-519e-5p included in PTC-derived exosomes can be transferred to recipient CD8+ T cells and aid in tumor immune escape in distant organs through inhibiting Notch signaling pathway by downregulating NOTCH2. Our findings highlight the dual role of PTC-derived exosomal miR-519e-5p in distant metastasis, which may improve our understanding of exosome-mediated distant metastatic mechanisms.

Loss of the centrosomal protein ALMS1 alters lipid metabolism and the regulation of extracellular matrix-related processes

BackgroundAlström syndrome (ALMS) is a rare autosomal recessive disease that is associated with mutations in ALMS1 gene. The main clinical manifestations of ALMS are retinal dystrophy, obesity, type 2 diabetes mellitus, dilated cardiomyopathy and multi-organ fibrosis, characteristic in kidneys and liver. Depletion of the protein encoded by ALMS1 has been associated with the alteration of different processes regulated via the primary cilium, such as the NOTCH or TGF-β signalling pathways. However, the cellular impact of these deregulated pathways in the absence of ALMS1 remains unknown.MethodsIn this study, we integrated RNA-seq and proteomic analysis to determine the gene expression profile of hTERT-BJ-5ta ALMS1 knockout fibroblasts after TGF-β stimulation. In addition, we studied alterations in cross-signalling between the TGF-β pathway and the AKT pathway in this cell line.ResultsWe found that ALMS1 depletion affects the TGF-β pathway and its cross-signalling with other pathways such as PI3K/AKT, EGFR1 or p53. In addition, alterations associated with ALMS1 depletion clustered around the processes of extracellular matrix regulation and lipid metabolism in both the transcriptome and proteome. By studying the enriched pathways of common genes differentially expressed in the transcriptome and proteome, collagen fibril organisation, β-oxidation of fatty acids and eicosanoid metabolism emerged as key processes altered by the absence of ALMS1. Finally, an overactivation of the AKT pathway was determined in the absence of ALMS1 that could be explained by a decrease in PTEN gene expression.ConclusionALMS1 deficiency disrupts cross-signalling between the TGF-β pathway and other dependent pathways in hTERT-BJ-5ta cells. Furthermore, altered cross-signalling impacts the regulation of extracellular matrix-related processes and fatty acid metabolism, and leads to over-activation of the AKT pathway.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

The Jagged-1/Notch1 Signaling Pathway Promotes the Construction of Tissue-Engineered Heart Valves.

Background: Tissue-engineered heart valves (TEHVs) are promising new heart valve substitutes for valvular heart disease. The Notch signaling pathway plays a critical role in the development of congenital heart valves. Objective: To investigate the role of the Notch signaling pathway in the construction of TEHVs. Methods: The induced endothelial cells, which act as seed cells, were differentiated from adipose-derived stem cells and were treated with Jagged-1 (JAG-1) protein and γ-secretase inhibitor (DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycine t-butyl ester), respectively. Cell phenotypic changes, the expression of proteins relating to the epithelial-mesenchymal transition (EMT), and changes in paxillin expression were detected. Decellularized valve scaffolds were produced from decellularized porcine aortic valves. The seed cells were them inoculated into Matrigel-coated flap scaffolds for complex culture and characterization. Results: JAG-1 significantly reduced apoptosis and promoted the seeded cells' proliferation and migration ability, in contrast to the treatment of DAPT. In addition, the expression of EMT-related proteins, E-cadherin and N-cadherin, was significantly increased after treatment with JAG-1 and was reduced after the application of DAPT. Meanwhile, the adhesive-related expression of paxillin and fibronectin proteins was increased after the activation of Notch1 signaling and vice versa. Of interest, activation of the Notch1 signaling pathway resulted in more closely arranged cells on the valve surface after recellularization. Conclusion: Activation of the JAG-1/Notch1 signaling pathway increased seeded cells' proliferation and migratory ability and promoted the EMT and adhesion of seed cells, which was conducive to binding to the matrix, facilitating accelerated endothelialization of TEHVs.

Transcriptome Dynamics and Cell Dialogs Between Oocytes and Granulosa Cells in Mouse Follicle Development.

The development and maturation of follicles is a sophisticated and multistage process. The dynamic gene expression of oocytes and their surrounding somatic cells and the dialogs between these cells are critical to this process. In this study, we accurately classified the oocyte and follicle development into nine stages and profiled the gene expression of mouse oocytes and their surrounding granulosa cells and cumulus cells. The clustering of the transcriptomes showed the trajectories of two distinct development courses of oocytes and their surrounding somatic cells. Gene expression changes precipitously increased at Type 4 stage and drastically dropped afterward within both oocytes and granulosa cells. Moreover, the number of differentially expressed genes between oocytes and granulosa cells dramatically increased at Type 4 stage, most of which persistently passed on to the later stages. Strikingly, cell communications within and between oocytes and granulosa cells became active from Type 4 stage onward. Cell dialogs connected oocytes and granulosa cells in both unidirectional and bidirectional manners. TGFB2/3, TGFBR2/3, INHBA/B, and ACVR1/1B/2B of TGF-β signaling pathway functioned in the follicle development. NOTCH signaling pathway regulated the development of granulosa cells. Additionally, many maternally DNA methylation- or H3K27me3-imprinted genes remained active in granulosa cells but silent in oocytes during oogenesis. Collectively, Type 4 stage is the key turning point when significant transcription changes diverge the fate of oocytes and granulosa cells, and the cell dialogs become active to assure follicle development. These findings shed new insights on the transcriptome dynamics and cell dialogs facilitating the development and maturation of oocytes and follicles.

Gambogic acid inhibits HBx-mediated hepatitis B virus replication by targeting the DTX1-Notch signaling pathway

Background & AimsCurrent antiviral drugs, including nucleoside analogs and interferon, fail to eliminate the HBV covalently closed circular DNA (cccDNA), which serves as a transcript template in infected hepatocytes. Silencing the HBV X protein, which plays a crucial role in cccDNA transcription, is a promising approach to inhibit HBV replication. Therefore, the identification of novel compounds that can inhibit HBx-mediated cccDNA transcription is critical. MethodsInitially, a compound library consisting of 715 monomers derived from traditional Chinese medicines known for their liver-protecting properties was established. Then, MTT assays were used to determine the cytotoxicity of each compound. The effect of candidates on Flag-HBx expression was examined by real-time PCR and western blotting in Flag-HBx transfected HepG2-NTCP cells. Ultimately, the antiviral effect of gambogic acid (GA) on HBV was observed in HBV-infected HepG2-NTCP cells. Mechanistically, the functional role of DTX1 in GA-induced HBV inhibition was examined using RNA-seq. Finally, the antiviral effect of GA was estimated in vivo. ResultsGambogic acid (GA), a natural bioactive compound with a myriad of biological activities, markedly reduced Flag-HBx expression. Potent and dose-dependent reductions in extracellular HBV RNAs, HBV DNA, HBsAg, HBeAg and HBc protein were discovered three days after GA treatment in HBV-infected cells, accompanied by the absence of significant cytotoxicity. Furthermore, our research revealed that GA exhibited a dose-dependent inhibition of HBx expression, which is a pleiotropic protein required for HBV infection in vivo. We explored the mechanisms underlying GA-mediated inhibition of HBV and confirmed that this inhibition is accomplished by upregulating the expression of the DTX1 gene and boosting the Notch signaling pathway. Finally, the inhibitory effect of GA on HBV replication was tested in vivo using a mouse model of hepatitis B virus recombinant cccDNA. ConclusionsHerein, we discovered GA, which is a natural bioactive compound that targets HBx to inhibit hepatitis B virus replication by activating the DTX1-Notch signaling pathway.

The role of estrogen receptors (ERs)-Notch pathway in thyroid toxicity induced by Di-2-ethylhexyl phthalate (DEHP) exposure: Population data and in vitro studies

BackgroundThis study aimed to assess the exposure level and risk of Di-2-ethylhexyl Phthalate (DEHP) among adults in Jilin Province, China, clarify the impact of DEHP on human thyroid function, and to explore the role of estrogen receptors (ERs)-Notch signaling pathway in the effect of DEHP metabolites on thyroid hormones based on population data and in vitro experiments. Methods312 adults participated in this study. Urinary DEHP metabolites were determined by high performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS). Two pharmacokinetic models were used to evaluate the estimated daily intake (EDI) and hazard quotient (HQ) of the adults. Multiple linear regression and mediating effect models were used to evaluate the target associations. In cell experiments, thyroid follicular epithelial (Nthy-ori3–1) cells were exposed to mono (2-ethylhexyl) phthalate (MEHP) for testing. The inhibitions of ERα and Notch pathway were conducted by siRNA and Notch pathway inhibitor DAPT. ResultsThe detection rate of five DEHP metabolites was 97.1∼100.0%. The HQ value of 0.3% of adults was higher than 1. The levels of urinary DEHP metabolites were significantly correlated with thyrotropin (TSH), thyrotropin-releasing hormone (TRH), total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3) and free thyroxine (FT4) and gene (estrogen receptor α (ERα), Notch1, Dll4) levels. The ERα-Notch pathway played a mediating role in the association between DEHP metabolite levels and FT4. The cell results showed, the levels of FT3 and FT4 in cell supernatant decreased after MEHP exposure, and the downward trend was reversed after ERα and notch pathways were inhibited, notch pathway genes also decreased after ERα inhibition. ConclusionAdults in the Jilin Province of China were widely exposed to DEHP. ERs-Notch pathway played an important role in the effect of DEHP metabolites on thyroid hormones.

Effect of Notch Signal Pathway on Steroid Synthesis Enzymes in TM3 Cells.

Studies have indicated that the conservative Notch pathway contributes to steroid hormone synthesis in the ovaries; however, its role in hormone synthesis of the testis remains unclear. We have previously reported Notch 1, 2, and 3 to be expressed in murine Leydig cells and that inhibition of Notch signaling caused G0/G1 arrest in TM3 Leydig cells. In this study, we have further explored the effect of different Notch signal pathways on key steroidogenic enzymes in murine Leydig cells. TM3 cells were treated with Notch signaling pathway inhibitor MK-0752, and different Notch receptors were also overexpressed in TM3 cells. We evaluated the expression of key enzymes of steroid synthesis, including p450 cholesterol side-chain cleavage enzyme (P450Scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and steroidogenic acute regulatory protein (StAR), and key transcriptional factors for steroid synthesis, including steroidogenic factor 1 (SF1), GATA-binding protein 4 (GATA4) and GATA6. We found the level of P450Scc, 3β-HSD, StAR and SF1 to be decreased after treatment with MK-0752, while overexpression of Notch1 up-regulated the expression of 3β-HSD, P450Scc, StAR and SF1. MK-0752 and overexpression of different Notch members had no influence on the expression of GATA4 and GATA6. In conclusion, Notch1 signaling may contribute to the steroid synthesis in Leydig cells through regulating SF1 and downstream steroidogenic enzymes (3β-HSD, StAR and P450Scc).

The Notch1 signaling pathway directly modulates the human RANKL-induced osteoclastogenesis

Notch signaling is an evolutionary conserved pathway with a key role in tissue homeostasis, differentiation and proliferation. It was reported that Notch1 receptor negatively regulates mouse osteoclast development and formation by inhibiting the expression of macrophage colony-stimulating factor in mesenchymal cells. Nonetheless, the involvement of Notch1 pathway in the generation of human osteoclasts is still controversial. Here, we report that the constitutive activation of Notch1 signaling induced a differentiation block in human mononuclear CD14+ cells directly isolated from peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation to osteoclasts. Additionally, using a combined approach of single-cell RNA sequencing (scRNA-Seq) simultaneously with a panel of 31 oligo-conjugated antibodies against cell surface markers (AbSeq assay) as well as unsupervised learning methods, we detected four different cell stages of human RANKL-induced osteoclastogenesis after 5 days in which Notch1 signaling enforces the cell expansion of specific subsets. These cell populations were characterized by distinct gene expression and immunophenotypic profiles and active Notch1, JAK/STAT and WNT signaling pathways. Furthermore, cell–cell communication analyses revealed extrinsic modulators of osteoclast progenitors including the IL7/IL7R and WNT5a/RYK axes. Interestingly, we also report that Interleukin-7 receptor (IL7R) was a downstream effector of Notch1 pathway and that Notch1 and IL7R interplay promoted cell expansion of human RANKL-induced osteoclast progenitors. Taken together, these findings underline a novel cell pattern of human osteoclastogenesis, outlining the key role of Notch1 and IL-7R signaling pathways.

Ex vivo identification of transcriptional regulation landscape and potential therapeutic targets against oxidative stress using Alzheimer’s disease patient‐derived dermal fibroblast: findings from BICWALZS cohort

AbstractBackgroundReactive oxygen species (ROS) is considered as a central pathophysiology for neurodegeneration in Alzheimer’s disease (AD). Many studies using in vitro cell line or in vivo mouse model have shown the possible association between ROS defense mechanisms and cellular damage. However, that connection was not elucidated well in AD patient‐derived cell or brain organoids. In this study, we aimed to identify transcriptional regulation landscape and potential therapeutic targets against oxidative stress using AD dementia patient‐derived dermal fibroblastMethodsFor the purposes of ex vivo research, we performed skin biopsy and stabilized dermal fibroblasts in vitro. Total of 20 AD dementia patients and 22 cognitively normal older adults were assessed. All of AD dementia patients were amyloid positive on PET imaging, APOE e4 carrier, and cognition was imparied. On the contrary, all of cognitively normal older adults were amyloid negative on PET imaging, and APOE e4 non‐carrier. We applied H2O2 (600 uM) to dermal fibroblast for 24 hours as an oxidant agent to promote oxidative stress. After that, to identify transcriptional regulation landscape against oxidative stress, RNA sequencing was performed.ResultsTotal of 406 transcripts (up = 61, down = 345) were commonly differentially expressed in both of AD dementia and cognitively normal older adults derived dermal fibroblast by ROS‐H2O2. These transcripts were enriched in GO categories related to cell division, cell cycle, and DNA repair. Interestingly, 133 transcripts (up = 41, down = 92) were only differentially expressed in AD dementia dermal fibroblast by ROS‐H2O2. These transcripts were enriched in GO categories related to DNA duplex unwinding, cell division, and DNA replication. Total of 147 transcripts (up = 67, down = 80) were only differentially expressed in cognitively normal older adults dermal fibroblast by ROS‐H2O2. These transcripts were enriched in GO categories related to negative regulation of cell proliferation, notch signaling pathway, and NMDA glutamate receptor signaling pathway.ConclusionWe identified transcriptional regulation landscape by ROS‐H2O2 in AD dementia and cognitively normal older adults dermal fibroblast. Further bioinformatic analysis with computational tools and validation experiments will be performed to find potential therapeutic targets against oxidative stress.

The epigenetics orchestra of Notch signaling: a symphony for cancer therapy.

The aberrant regulation of the Notch signaling pathway, which is a fundamental developmental pathway, has been implicated in a wide range of human cancers. The Notch pathway can be activated by both canonical and noncanonical Notch ligands, and its role can switch between acting as an oncogene or a tumor suppressor depending on the context. Epigenetic modifications have the potential to modulate Notch and its ligands, thereby influencing Notch signal transduction. Consequently, the utilization of epigenetic regulatory mechanisms may present novel therapeutic opportunities for both single and combined therapeutics targeted at the Notch signaling pathway. This review offers insights into the mechanisms governing the regulation of Notch signaling and explores their therapeutic potential.

Sex‐specific CSF proteomic profiling of amyloid/tau positivity identifies distinctive sex‐specific alternation of multiple proteins involved in Alzheimer’s disease

AbstractBackgroundSporadic Alzheimer’s disease (AD) is a highly heterogeneous multifactorial disease in which females have a higher risk. Among 5.3 million senior Americans diagnosed with AD, over 60% are female. Despite the well‐established sex differences, sex‐specific molecular findings in AD are still limited. We therefore examined the role of sex in the proteomic alterations due to AD to elucidate potential protein markers of AD heterogeneity.MethodIn the discovery cohort, 599 preclinical AD and 435 controls underwent lumbar puncture. Over 7,000 proteins in cerebrospinal fluid (CSF) were measured using SomaLogic assay. Amyloid/tau positivity (AT classification) was determined using ELISA Aβ42 and pTau181 levels in CSF. Proteins with sex‐specific effects were identified through linear models focusing on the interaction term between AT classification and sex. Results were tested in the independent cohort (N = 732) using the identical approaches. We analyzed brain proteomics and transcriptomics data to validate the replicated sex‐specific proteins. We performed pathway enrichment, interaction network using ConsensusPathDB and drug‐target prediction.ResultWe identified 594 proteins with sex‐specific effects in discovery (P<0.05 and permutation FDR <0.05). Among them, 36 were validated in replication (P<0.05 and consistent direction). These proteins were found to be involved in neurologic disorders and highly enriched in axon regeneration (fold enrichment = 9.38 and FDR = 2.8×10−4). Among the 36 proteins identified, 13 were classified as female‐specific and 23 as male‐specific. These 36 proteins strongly predicted amyloid/tau positivity (AUC = 0.926) for all individuals as well as 13 female‐specific proteins for females (AUC = 0.929). The interaction network identified CCN2 as a hub and contained 10 additional proteins including JAG1 and DLL4 of Notch signaling pathway and CTSB, an amyloid precursor protein secretase, among others. The results of 36 proteins in brain proteomics and transcriptomics confirmed the sex‐specific effect of CCN2 and others on AD in different tissues. Drug target prediction suggests minocycline and etoposide as potential treatments for females as well amitriptyline and verteporfin for males.ConclusionWe robustly identified 36 proteins that showed significant sex‐specific alterations by preclinical AD. Several proteins involved in AD and neurodegeneration were highlighted. Our findings facilitate mechanistic understanding of sex differences for AD risk and progression.

Abstract A020: Reversing triple negative breast cancer stem cells by disrupting the Notch signaling pathway via BCL6 targeting

Abstract Triple negative breast cancer (TNBC) is a heterogeneous molecular breast cancer (BC) subtype that still has a very poor prognosis. Genetic, molecular and clinical tumor heterogeneity represent major pathobiological factors involved in poor clinical outcome. The sequence specific transcriptional repressor and proto-oncogene B cell lymphoma protein 6 (BCL6) has emerged as a suitable therapeutic target as it plays broad and critical roles in many hematological and solid cancers. BCL6 exerts control over cellular proliferation and survival by binding to specific responsive elements located within the regulatory regions of target genes and recruiting of chromatin modifying co-repressor complexes such as silencing mediator for retinoid and thyroid receptors (SMRT), BCL6 corepressor (BCoR) and histone deacetylases (HDACs). Moreover, in hematological malignancies, BCL6 transcriptional activity is also associated to the recruitment of the chromatin remodeling factor, Enhancer of Zeste 2 (EZH2). The role of BCL6 in BC is still not well defined; however emerging studies report its involvement in tumorigenesis, disease progression and poor survival. We recently showed that BCL6 is expressed at significantly higher levels in TNBC mammospheres, 3D multicellular structures enriched in cancer stem cells (CSCs), than in their differentiated bulk cell counterparts. Additionally, we found that the functional role of BCL6 is crucial for mammosphere formation, as its loss of function decreased this process. Intriguingly, we uncovered a functional interplay occurring between BCL6 and EZH2, which triggers the BCL6/Notch stemness signaling axis by inhibiting Numb transcription in both SUM149 and SUM159 TNBC cell lines. We exploited the molecular mechanisms responsible for this specific interplay, taking advantage of two molecules, FX1, a small molecule inhibitor able to block BCL6 recruiting activity and EED226, a potent Polycomb Repressive Complex 2 (PRC2) inhibitor. In order to define the role of both BCL6, as epigenetic regulator, and NUMB, as inhibitor of NOTCH stemness pathway in TNBC, SUM149 and SUM159 cell lines were stably transduced with lentiviral shRNA to derive stable NUMB-silenced matched cells. The expression of Notch downstream target genes, such as HES1/2 and HEY1/2, was assessed by qRT PCR analysis in the engineered TNBC cell models and resulted only slightly increased following NUMB downmodulation. Upon FX1 or EED226 administration, a strongest effect was observed, indicating that NUMB is not the only player in regulating the Notch pathway. Further experiments are currently ongoing aiming at identify additional players and molecular pathway(s). Collectively, our preliminary results provide additional preclinical evidences on the role exerted by BCL6 as epigenetic regulator of TNBC and underlie its interplay with EZH2thus paving the way for a novel potential therapeutic strategy targeting BCL6/Notch signaling axis. Citation Format: Francesca De Santis, Francesca Putti, Giorgia Bravin, Carla Portulano, Federica Zonca, Lorenzo Castagnoli, Michele Magni, Filippo de Braud, Serenella M Pupa, Massimo Di Nicola. Reversing triple negative breast cancer stem cells by disrupting the Notch signaling pathway via BCL6 targeting [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A020.

Comparative Transcriptome Analysis Reveals Sexually Dimorphic Gene Expression in the Gonads of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is an endangered cold-water salmonid fish native to China. This study aimed to identify sex-related genes and biological pathways via gonadal transcriptome sequencing of B. tsinlingensis Li. A total of 167,904 unigenes were identified with an average length of 836 bp and an N50 of 1452 bp, of which 84,977 (50.61%) unigenes were successfully annotated in six major databases. Comparative transcriptome analysis identified 22,864 differentially expressed genes (DEGs), of which 17,231 were up-regulated (male-biased genes, mDEGs) and 5633 were down-regulated (female-biased genes, fDEGs). Several DEGs associated with gonadal development were found through Gene Ontology enrichment analysis, such as ccnb1, zp3, bmp15, dmrt1, and psmc3ip. Signaling pathways related to gonadal development were found to be enriched through analysis using the Kyoto Encyclopedia of Genes and Genomes Pathway database, such as genes involves in base excision repair, the notch signaling pathway, neuroactive ligand-receptor interaction, the VEGF signaling pathway, and the estrogen signaling pathway. In addition, mRNA expression levels of 19 DEGs were determined to validate the reliability of the transcriptomic data by quantitative real-time polymerase chain reaction. These results revealed genes and signaling pathways potentially involved in gonadal development in B. tsinlingensis Li and provided basic molecular data for future research on reproductive regulation and breeding of B. tsinlingensis Li.

Targeting Microglia/Macrophages Notch1 Protects Neurons from Pyroptosis in Ischemic Stroke.

The immune-inflammatory cascade and pyroptosis play an important role in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). The maintenance of immune homeostasis is inextricably linked to the Notch signaling pathway, but whether myeloid Notch1 affects microglia polarization as well as neuronal pyroptosis in CIRI is not fully understood. This study was designed to clarify the role of myeloid Notch1 in CIRI, providing new therapeutic strategies for ischemic stroke. Myeloid-specific Notch1 knockout (Notch1M-KO) mice and the floxed Notch1 (Notch1FL/FL) mice were subjected to middle cerebral artery occlusion (MCAO). After 3 days of CIRI, we evaluated the neurological deficit score and cerebral infarction volume. Immunofluorescence staining was used to detect the expression of Notch1 and microglial subtype markers. Cerebral infiltrating macrophages were detected by flow cytometry. RT-qPCR was used to detect pro-inflammatory cytokines. Western blot was used to detect the expression of pyroptosis related proteins. The Notch1-siRNA transfected BV2 cells were co-cultured with HT22 cells to investigate the potential mechanisms by which microglial Notch1 affects neuronal pyroptosis induced by anoxia/reoxygenation in vitro. We found that Notch1 was activated in cerebral microglia/macrophages after CIRI. Myeloid Notch1 deficiency decreased the cerebral infarct volume (24.17 ± 3.29 vs. 36.17 ± 2.27, p < 0.001), neurological function scores (2.33 ± 0.47 vs. 3.17 ± 0.37, p < 0.001) and the infiltration of peripheral monocytes/macrophages (3.26 ± 0.53 vs. 5.67 ± 0.57, p < 0.01). Strikingly, myeloid-specific Notch1 knockout alleviated pyroptosis. Compared with microglia M1, increased microglia M2 were detected in the ischemic penumbra. In parallel in vitro co-culture experiments, we found that Notch1 knockdown in microglial BV2 cells inhibited anoxia/reoxygenation-induced JAK2/STAT3 activation and pyroptosis in hippocampal neuron HT22 cells. Our findings elucidate the underlying mechanism of the myeloid Notch1 signaling pathway in regulating neuronal pyroptosis in CIRI, suggesting that targeting myeloid-specific Notch1 is an effective strategy for the treatment of ischemic stroke.

Jiawei Taohe Chengqi decoction inhibition of the notch signal pathway affects macrophage reprogramming to inhibit HSCs activation for the treatment of hepatic fibrosis

Ethnopharmacological relevanceJiawei Taohe Chengqi Tang (JTCD) is a modified formulation of Traditional Chinese Medicine (TCM) known as Taohe Chengqi Decoction, which has been described in the ancient TCM literature “Treatise on Febrile Diseases”. As a formula that can activate blood circulation and eliminate blood stasis and regulate Yin and Yang in traditional Chinese medicine applications, JTCD has been reported to be effective in the treatment of chronic liver disease and hepatic fibrosis (HF). Aim of studyThe current study aimed to evaluate the effectiveness of JTCD in modulating hepatic macrophages by regulating the Notch signal pathway, and to further investigate the mechanisms underlying macrophage reprogramming that leads to HF. Materials and methodsMolecular assays were performed using in vitro cultures of human mononuclear THP-1 cells and human-derived hepatic stellate cells LX-2. CCl4-induced mice were utilized as an in vivo model to simulate HF. ResultsOur results demonstrated that JTCD exhibited dual effects by inhibiting hepatic stellate cell (HSCs) activation and modulating the polarisation of macrophages towards the M2 phenotype while decreasing the M1 phenotype. Network pharmacological analyses and molecular docking studies revealed that the Notch signal pathway was significantly enriched and played a crucial role in the therapeutic response of JTCD against HF. Moreover, through the establishment of a co-culture model, we validated that JTCD inhibited the Notch signal pathway in macrophages, leading to alterations in macrophage reprogramming, subsequent inhibition of HSC activation, and ultimately exerting anti-HF effects. ConclusionIn conclusion, our findings provide solid evidence for JTCD in treating HF, as it suppresses the Notch signal pathway in macrophages, regulates macrophage reprogramming, and inhibits HSC activation.