Gene Expression Differentiation Research Articles

Amyloid and tau‐driven signatures in hippocampal GFAP‐positive astrocytes

AbstractBackgroundDespite the known heterogeneity of astrocyte responses to injuries, Alzheimer’s disease (AD) pre‐clinical and clinical research relies on the glial fibrillary acidic protein (GFAP) as a proxy of astrocyte reactivity. Recently, it was demonstrated that plasma GFAP levels differentially relate to AD hallmarks, associating with amyloid‐β (Aβ), but not with tau pathology. However, the molecular underpinnings behind this specificity are still unexplored. Here, we aim to uncover the molecular signatures of GFAP‐positive astrocytes driven by either by Aβ or tau pathology.MethodHippocampal GFAP‐positive astrocytes data from 6 month‐old TauP301S (n = 5), 11.5 months‐old APP/PS2 (n = 5) and their wild‐type littermates (WT, n = 5) were acquired from GEO datasets (GSE129770;GSE129797) and used to identify differentially expressed genes (DEGs) and enriched biological processes. DEGs were evaluated using the DESeq2 method. Further data analysis included Gene Ontology (GO) functional evaluation.ResultPrincipal component analysis clearly differentiated gene expression of GFAP‐positive astrocytes from APP/PS2, TauP301S and their WT controls (Fig.1A). Interestingly, only 30 DEGs overlapped among GFAP‐positive astrocytes from these two transgenic mouse models, revealing multiple DEGs specifically associated with Tau or Aβ (Fig.1B). The functional enrichment analysis revealed 36 GO terms enriched with DEGs altered in the PS2APP mice compared to WT. The most significantly enriched terms include processes such as “protein localization to cell periphery and plasma membrane”, “interleukin−6 production and regulation”, “exocytosis” and “neuron death” (Fig.1C). For the comparison of the TauP301S mice versus WT controls, the functional enrichment analysis using DEGs revealed 95 terms. Among the most significantly enriched terms, we found “learning”, “cognition”, and “small GTPase mediated signal transduction” (Fig.1C).ConclusionOverall, we observed a small overlap of DEGs between the Aβ and tau mouse models, indicating the unique nature of GFAP‐positive astrocytic responses to different aspects of AD pathology. The findings presented in this study allowed the identification of functional categories and biological processes particular to Aβ or tau burden. Characterizing how these upstream triggers influence GFAP‐positive astrocyte phenotypes may contribute to the biological interpretation of GFAP as a biomarker of AD pathology and pave the way to unravel novel mechanisms and context‐specific astrocyte biomarkers for AD.

Obesity alters the mouse endometrial transcriptome in a cell context-dependent manner

Obesity impacts fertility and is positively correlated with endometrial hyperplasia and endometrial cancer occurrence. Endometrial epithelia often harbor disease driver-mutations, while endometrial stroma are highly regulative of neighboring epithelia. Here, we sought to determine distinct transcriptome changes occurring in individual cell types in the obese mouse uterus. Outbred CD-1 mice were fed high-fat or control diets for 18 weeks, estrous cycle staged, and endometrial epithelia, macrophages, and stroma isolated for transcriptomic analysis. High-fat diet mice displayed increased body mass and developed glucose intolerance, hyperinsulinemia, and fatty liver. Obese mouse epithelia displayed differential gene expression for genes related to innate immunity and leukocyte chemotaxis. The obese mouse stroma differentially expressed factors related to circadian rhythm, and expression of these genes correlated with glucose tolerance or body mass. We observed correlations between F4/80 + macrophage numbers, Cleaved Caspase 3 (CC3) apoptosis marker staining and glucose intolerance among obese mice, including a subgroup of obese mice with high CC3 + luminal epithelia. This subgroup displayed differential gene expression among all cell types, with pathways related to immune escape in epithelia and macrophages, while the stroma dysregulated pathways related to regulation of epithelia. These results suggest an important role for differential response of both the epithelia and stroma in their response to obesity, while macrophages are dysregulated in the context of apoptotic epithelia. The obesity-related gene expression programs in cells within the uterine microenvironment may influence the ability of the endometrium to function during pregnancy and influence disease pathogenesis.

Synthesis and characterization of a calcium phosphate bone cement with quercetin-containing PEEK/PLGA microparticles

This study aimed to describe the synthesis and characterization of a calcium phosphate cement (CPC) with polyetheretherketone/poly (lactic-co-glycolic) acid (PEEK/PLGA) micro-particles containing quercetin. CPC powder was synthesized by mixing dicalcium phosphate anhydrate and tetracalcium phosphate. To synthesize PEEK/PLGA microparticles, PLGA85:15 was mixed with 90 wt% PEEK. The weight ratio of quercetin/PLGA/PEEK was 1:9:90 wt%. PEEK/PLGA/quercetin microparticles with 3, 5, and 6 wt% was added to CPC. The setting time, compressive strength, drug release profile, solubility, pH, and porosity of synthesized cement were evaluated. The morphology and physicochemical properties of particles was analyzed by scanning electron microscopy, Fourier-transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), and inductively coupled plasma. Cytotoxicity was assessed by the methyl thiazolyl tetrazolium assay using dental pulp stem cells. Expression of osteoblastic differentiation genes was evaluated by real-time polymerase chain reaction. Data were analyzed by one-way ANOVA and Tukey’s test (alpha = 0.05). The setting time of 3 wt% CPC was significantly longer than 5 and 6 wt% CPC (P < 0.001). The 6 wt% CPC had significantly higher compressive strength than other groups (P = 0.001). The release of quercetin from CPCs increased for 5 d, and then reached a plateau. XRD and FTIR confirmed the presence of hydroxyapatite in cement composition. Significantly higher expression of osteocalcin (OCN) and osteopontin (OPN) was noted in 3 wt% and 6 wt% CPCs. Addition of quercetin-containing PEEK/PLGA microparticles to CPC enhanced its compressive strength, decreased its setting time, enabled controlled drug release, and up-regulated OPN and OCN.

Metabolic Abnormalities Associated with T-Cell Exhaustion in CLL Eµ-TCL1 Murine Model

Introduction. T-cell differentiation and immune responses are associated with major metabolic changes that control cell fate upon antigen exposure. Chronic lymphocytic leukemia (CLL) is characterized by a marked T-cell dysfunction that plays a major role in disease progression and failure of adoptive cell therapy. A handful of recent studies have demonstrated mitochondrial and metabolic defects in the T-cell compartment in CLL patients. However, to date, no studies have been done to validate such metabolic abnormalities in the Eµ-TCL1 model, the most established and reliable murine model for studying CLL. Therefore, we hypothesized that CD8+ T-cells derived from the Eµ-TCL1 adoptive transfer model exhibit metabolic and mitochondrial disturbance that might contribute to the overall dysfunctional state of these cells. Methods. To investigate the influence of leukemic burden on the differentiation phenotypes, metabolic state and gene expression profile of CD8+ T-cells, C57BL/6 mice adoptively transferred with Eµ-TCL1 leukemic splenocytes were euthanized at varying time points. Once disease was established, spleens were collected for analysis. Mice were divided into 3 groups; early, intermediate, and late disease, based on the peripheral blood leukemic burden at endpoint. Multicolor flow cytometry was used for identification of T-cell phenotypes and measurement of mitochondrial mass, membrane potential, reactive oxygen species (ROS) and cellular glucose uptake. For T-cell activation studies, we cultured splenocytes in the presence of CD3/CD28 dynabeads and 30IU/ml hIL-2 for 72 hours. Nanostring metabolic pathways gene expression analysis was done on splenic CD8+ T-cells isolated from early and late-stage Eµ-TCL1 adoptive transfer mice via fluorescence-activated cell sorting. Differential gene expression was measured using ROSALIND cloud-based software platform. Results. Unsupervised t-distributed stochastic neighborhood embedding (tSNE) and Xshift clustering analyses revealed a gradual enrichment of specific CD8+ T-cell populations along disease progression. These populations upregulated exhaustion-related markers (EOMES, TOX, PD-1 and KLRG-1) and significantly downregulated TCF-1 expression, a transcription factor responsible for cell self-renewal capacity. Further analysis demonstrated a significant downregulation of PGC-1α expression, a master regulator of mitochondrial function, in Eµ-TCL1 CD8+ T-cells with high EOMES and low TCF-1 expression levels (EOMEShi TCF-1lo) compared to EOMEShi TCF-1hi cells. At late disease stage, we noticed significant mitochondrial depolarization, an increase in mitochondrial ROS and marked reduction in glucose uptake capacity in the Eµ-TCL1 CD8+ T-cells. Post in vitro activation, there was a significant increase in Eµ-TCL1 CD8+ T-cell mitochondrial biogenesis, membrane potential and ROS production compared to wild type (WT) controls. However, Eµ-TCL1 cultures showed significant enrichment in EOMEShi TCF-1lo CD8+ T-cell population, that was not detected in the activated WT cells. Interestingly, this population exhibited marked downregulation of PGC-1α, SOD-2 and NRF-2 expression compared to the populations maintaining TCF-1 expression. Moreover, we found that Eµ-TCL1 CD8+ T-cells had increased frequency of effector memory phenotypes compared to more central memory cells in WT cultures. These effector memory populations accumulated significant levels of mitochondrial ROS and depolarized mitochondria compared to the central memory counterparts. Nanostring analysis revealed disease stage-dependent changes in gene expression, where glycolysis was the top differentially expressed pathway in the late versus early-stage CD8+ T-cells as indicated by the gene set global significance score. On the other hand, negative significance scores were assigned to AMPK and autophagy pathways indicating a probable downregulation of these pathways during late phases of the disease. Conclusion. Our results demonstrate mitochondrial and metabolic abnormalities in Eµ-TCL1-derived CD8+ T-cells and point to possible defects in cellular stress signaling response that might lead to accumulation of dysfunctional mitochondria and push the cells towards exhausted states.

Targeting HIF-Hydroxylases Compromises Disease Initiation and Propagation, and Synergises with Current Therapies to Eliminate Cancer Stem Cells in AML

Acute myeloid leukaemia (AML) is an aggressive disorder of haematopoietic stem and progenitor cells (HSPCs), in which they acquire mutations, resulting in generation of disease fuelling, and ultimately treatment-resistant, leukaemic stem cells (LSCs). Given the hypoxic nature of the bone marrow microenvironment, the role, and pharmacological manipulation of the Hypoxia Inducible Factor (HIF) pathway in normal and malignant haematopoiesis is an area of significant interest. We, and others, previously found that HIF-α, the master regulator of the pathway, is not essential for normal haematopoiesis and its deletion promotes leukaemogenesis. Here we reveal through genetic, pharmacological and omics-based investigations that HIF stabilisation, through inhibition of its regulator PHD2, is a novel potent therapeutic strategy in AML. First, to genetically dissect to role of PHD2 in the initiation of AML, we transformed Phd2cKO HSPCs with AML-associated oncogenes and found that AML cells lacking Phd2 were compromised in vitro, established a weaker leukemic burden and initiated AML with a significantly longer latency in vivo. Moreover, employing our iMLL-AF9;shPhd2 mice, which upon DOX activate MLL-AF9 and a short-hairpin targeting Phd2, we found that Phd2 knockdown in vivo significantly delayed the onset of AML. Next, to investigate acute Phd2 depletion in newly diagnosed disease, we transplanted transformed shPhd2 cells, and upon AML detection induced expression of shPhd2. Notably, Phd2 knockdown in established AML cells compromised disease progression and significantly improved survival of leukaemic mice. Following this, we examined the impact of Phd2 inactivation on HSC function and multilineage haematopoiesis. Phd2cKO mice displayed no adverse phenotypes, and Phd2-deficient HSCs were able to successfully sustain multilineage haematopoiesis upon transplantation. Moreover, global DOX-inducible Phd2 knockdown had little effect on haematopoiesis. Thus, PHD2 inactivation compromises AML development and maintenance, but does not impact normal haematopoiesis, suggesting an ample therapeutic window for its inhibition. Given our genetic evidence that PHD2 is a promising target in AML, we explored the potential of its inhibition. We employed a commercially available clinical trial grade PHD inhibitor, as well as our own novel, selective, and potent PHD inhibitor, both of which work through distinct mechanisms. Significantly, both inhibitors compromised the growth and viability of murine AML cells, as well as multiple established human AML cell-lines and primary patient samples with diverse mutational backgrounds. Moreover, we have also discovered that PHD inhibition markedly synergises with AML treatments currently used in the clinic, thus enhancing their anti-leukaemic effect. Investigating the molecular mechanisms behind our findings, we found HIF-α stabilisation across multiple AML human cell lines following PHD inhibition. Furthermore, we utilised our HifαcKO leukaemic cells which, unlike control cells, were not affected by PHD inhibition, indicating that the anti-leukaemic effect of PHD inhibition is indeed HIF-dependent. Next, we performed RNA-seq and proteomic analyses following genetic and pharmacological PHD inhibition. These experiments revealed increased expression of key myeloid differentiation genes, glycolytic pathway, and a consistent upregulation of HIF target genes, including the pro-apoptotic BNIP3. Finally, pharmacological stimulation of apoptosis combined with PHD inhibition displayed a further and profound anti-leukaemic effect. Taken together, our data strongly indicate that genetic deletion and specific potent pharmacological inhibition of PHD2 can upregulate HIF-dependent pathways, eradicating AML disease in vitro and in vivo, thus unveiling a novel promising therapeutic target.

TIF1β Enhances Self-Renewal Capacity of BCR-ABL Leukemic Stem Cells and Inhibits the Myeloid Differentiation

Chronic myeloid leukemia (CML), characterized by expression of p210-BCR-ABL, is a clonal myeloproliferative disease initiated by malignant transformation of hematopoietic stem cells (HSC). Blastic crisis phase (BC) of CML that shows expansion of therapy-refractory and differentiation-arrested blasts, remains a therapeutic challenge. Transcription intermediary factor TIF1β/TRIM28/KAP1 modulates chromatin structure to repress or activate transcription of target genes. Tif1β is essential for the proliferation and differentiation of normal hematopoietic stem cells, but the role of Tif1β in myeloid leukemia is unclear. Recently, high expression of TIF1β was correlated with poor prognosis in patients with various types of cancer. By analyzing datasets of gene expression in bone marrow (BM) cells in CML patients, we found that TIF1β expression levels were robustly elevated in CML cells at the BC stage. Thus, we hypothesized that TIF1β functions as an oncogene for the development of BCR-ABL leukemia. To determine whether TIF1β promoted the development of BCR-ABL-induced myeloid leukemia in vivo, we generated BCR-ABL;Tif1βf/f;Cre-ERT2 compound mice by crossing Rosa26 locus BCR-ABL conditional knock-in mice and Tif1βf/f;Cre-ERT2 conditional KO mice. We transplanted BM cells isolated from Cre-ERT2 (WT), Tif1βf/f;Cre-ERT2 (Tif1β KO), BCR-ABL;Cre-ERT2 (BCR-ABL), and BCR-ABL;Tif1βf/f;Cre-ERT2 (BCR-ABL;Tif1β KO) mice into lethally irradiated CD45.1+ WT recipient mice, and injected tamoxifen 4 weeks after BM transplantation. While BCR-ABL KI mice showed rapid expansion of leukemic blast cells and died in a short period of time, the deletion of Tif1β gene resulted in a significantly longer survival with reduced tumor burden in BM, liver and spleen in BCR-ABL KI mice. BCR-ABL KI mice mostly developed myeloid leukemia, in contrast, BCR-ABL;Tif1β KO mice dominantly showed B-cell leukemia or bone marrow failure diseases. A subset of BCR-ABL;Tif1β KO mice survived without development of lethal leukemia in the primary and the secondary transplantation settings, indicating that TIF1β maintained the self-renewal capacity of leukemic stem cells (LSCs) but also controlled the myeloid/lymphoid lineage commitment. To elucidate the mechanisms underlying the impaired development of leukemia in BCR-ABL;Tif1βKO mice, we performed RNA sequencing analyses of HSCs isolated from WT, Tif1β KO, BCR-ABL, BCR-ABL;Tif1βKO mice. Gene set enrichment analysis revealed that the loss of Tif1β repressed BCR-ABL-induced activation of proliferative stem cell signatures, but increased expression of myeloid differentiation genes including PU.1 gene in HSCs. We found that BCR-ABL;Tif1βKO HSCs reduced expression levels of Myc and Myc-target genes including Fra1 transcription factor (TF), which was known to promote tumor cell proliferation. Notably, human CML-CP and CML-BC CD34+ cells markedly increased expression levels of MYC and FRA1 genes. To clarify how the Tif1β protein controlled self-renewal of LSCs, we performed ATAC-sequencing in murine HSCs and TIF1β-ChIP-sequencing using BCR-ABL-expressing human leukemia cells. ATAC-seq revealed that BCR-ABL KI HSCs showed enrichments of binding motif of Fra1 in open chromatin and that of PU.1 in closed chromatin, compared to WT HSCs, while BCR-ABL;Tif1βKO HSCs cancelled those enrichments of binding motifs of TFs. TIF1β-ChIP-seq revealed that the TIF1β protein directly bound to a promoter region of the FRA1 gene. TIF1β binding sites were enriched with canonical PU.1- and JUN/FRA1-binding motifs. We also found that treatment with imatinib decreased enrichment of FRA1-binding motif among TIFβ-binding regions in BCR-ABL+ cells, indicating that TIFβ opened chromatin accessibility to activate expression of and transcriptional function of the Fra1 gene in a BCR-ABL dependent manner, but repressed the accessibility of myeloid differentiation regulator TFs. Furthermore, knockdown of Fra1 significantly impaired the proliferation of BCR-ABL stem cells in an in vitro condition. In conclusion, we demonstrate that the Tif1β gene was required for the propagation of BCR-ABL-induced myeloid leukemia stem cell in mice. Tif1β enhanced chromatin accessibility of Fra1 TF to promote transcription of LSC signature genes, but also reduced chromatin accessibility at the PU.1-binding motif, resulting in the enhanced proliferation and limited differentiation of LSCs.

CNSC-12. NEURONAL DIFFERENTIATION DRIVES THE ANTI-TUMOR EFFICACY OF MEK INHIBITION IN GLIOBLASTOMA

Abstract Multiple genomic aberrations and distinct molecular subtypes of glioblastoma (GBM) have been identified through multi-omics analyses including single-cell genomic and epigenomic analysis. Epidermal Growth Factor Receptor (EGFR) amplification is found in nearly 50% of GBM tumors. Several EGFR inhibitors have been tested in GBM but failed to demonstrate long-term therapeutic benefit, presumably due to target heterogeneity, escape signal pathways, and acquired resistance. Therefore, we investigated downstream signaling, mitogen-activated protein kinase (MEK1/2) as a potential therapeutic target for GBM. We utilized an unbiased high-throughput screening with a panel of glioma stem-like cells (GSCs) and identified that GBM cells harboring EGFR amplification showed anti-proliferative and apoptotic cell death (sensitive) to multiple MEK inhibitors. RNA-seq analysis of cells pre- and post-treatment with trametinib, a potent MEK inhibitor, revealed an upregulation of neurogenesis and neuronal differentiation genes such as β-III tubulin, ASCL1, DLL1, DLL3, NeuroD4, PAX6 and NCAM1. In addition, downregulation of the several MEK targets including erythroblast transformation specific (ETS) family genes, particularly ETV5 was observed. We demonstrated that MEK inhibition increased neuronal differentiation demonstrated by immunofluorescence, western blot, and RT-qPCR experiments using selected differentiation markers (β-III tubulin, ASCL1, DLL3, and NeuroD4). In addition, ETV-5 knockout in GSCs by CRISPR/Cas-9 promoted the expression of neuronal differentiation genes. Oral treatment of trametinib in an orthotopic GSC xenograft model significantly improved animal survival, in which 25-30% of mice are long-term survivors. This was accompanied by decreased MEK/ERK signal and cell proliferation in tumor tissues. Thus, we demonstrated here that MEK1/2 inhibition by trametinib induces neuronal differentiation mediated through downregulation of ETV-5 in GBM, a potential novel mechanism of action of MEK1/2 signal inhibition.

Exercise facilitates regeneration after severe nerve transection and further modulates neural plasticity

Patients with severe traumatic peripheral nerve injury (PNI) always suffer from incomplete recovery and poor functional outcome. Physical exercise-based rehabilitation, as a non-invasive interventional strategy, has been widely acknowledged to improve PNI recovery by promoting nerve regeneration and relieving pain. However, effects of exercise on chronic plastic changes following severe traumatic PNIs have been limitedly discussed. In this study, we created a long-gap sciatic nerve transection followed by autograft bridging in rats and tested the therapeutic functions of treadmill running with low intensity and late initiation. We demonstrated that treadmill running effectively facilitated nerve regeneration and prevented muscle atrophy and thus improved sensorimotor functions and walking performance. Furthermore, exercise could reduce inflammation at the injured nerve as well as prevent the overexpression of TRPV1, a pain sensor, in primary afferent sensory neurons. In the central nervous system, we found that PNI induced transcriptive changes at the ipsilateral lumber spinal dorsal horn, and exercise could reverse the differential expression for genes involved in the Notch signaling pathway. In addition, through neural imaging techniques, we found volumetric, microstructural, metabolite, and neuronal activity changes in supraspinal regions of interest (i.e., somatosensory cortex, motor cortex, hippocampus, etc.) after the PNI, some of which could be reversed through treadmill running. In summary, treadmill running with late initiation could promote recovery from long-gap nerve transection, and while it could reverse maladaptive plasticity after the PNI, exercise may also ameliorate comorbidities, such as chronic pain, mental depression, and anxiety in the long term.

RF15 | PMON293 Metabolic activation of tachysterol to biologically active hydroxyderivatives that act on VDR, AhR, LXRs and PPARγ receptors

Abstract Absorption of ultraviolet B (UVB) radiation by the B ring of 7-dehydrocholesterol (7-DHC) leads to the transformation of 7-DHC to previtamin D3, which after absorption of additional UVB isomerizes to tachysterol3 (T3) and lumisterol3 (L3). Previously we demonstrated that CYP11A1 can hydroxylate the side chain of vitamin D3 (D3), 7-DHC and L3. Similarly CYP27A1 can hydroxylate the side chain of L3 to biologically active hydroxyderivatives. In a continuation of these studies, we report that CYP11A1 and CYP27A1 hydroxylate T3 to 20S(OH)T3 and 25(OH)T3, respectively, plus minor unidentified hydroxyderivatives. Both 20S(OH)T3 and 25(OH)T3were detected in the human epidermis and serum. T3 was also detected in human serum and was present at a concentration of 7.3±2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 . They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR-coupled GFP (VDR-GFP) from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay revealed marked activation of AhR by 20S(OH)T3, a smaller effect by 25(OH)T3 and only minimal activation by T3. The T3 hydroxyderivatives showed high affinity binding to the ligand binding domain (LBD) of the liver X receptor (LXR) α and β, and to the peroxisome proliferator-activated receptor γ (PPARg) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3, comparable to their previously characterized ligands. The scores for binding to the non-genomic site of the VDR were very low indicating a lack of interaction with hydroxy-T3 ligands. In conclusion, we have identified 20S(OH)T3 and 25(OH)T3 in the human epidermis and serum, identified CYP enzymes responsible for their production, demonstrated phenotypic effects on skin cells, and identified VDR, AhR, LXRs and PPARγ, and possibly RORs, as their genomic receptor targets. Thus CYP11A1, aside from its role in steroidogenesis, not only metabolizes vitamin D3, 7-DHC and L3 to biologically active metabolites, but also activates T3 to biologically active 20S(OH)T3. Similarly, CYP27A1, previously reported to act on L3, D3 and 7DHC, also activates T3 via hydroxylation at C25. We believe that these novel findings open new areas for future studies on the role of active forms of T3, not only in the skin, but also in systemic physiology and pathology. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

In toto imaging of early enteric nervous system development reveals that gut colonization is tied to proliferation downstream of Ret.

The enteric nervous system is a vast intrinsic network of neurons and glia within the gastrointestinal tract and is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation, and these are pertinent subjects for study due to their relevance to the human congenital disease Hirschsprung disease (HSCR). For the first time, we performed in toto gut imaging and single-cell generation tracing of ENCC migration in wild type and a novel ret heterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamics in vivo. We observed that retwmr1/+ zebrafish produced fewer ENCCs localized along the gut, and these ENCCs failed to reach the hindgut, resulting in HSCR-like phenotypes. Specifically, we observed a proliferation-dependent migration mechanism, where cell divisions were associated with inter-cell distances and migration speed. Lastly, we detected a premature neuronal differentiation gene expression signature in retwmr1/+ ENCCs. These results suggest that Ret signaling may regulate maintenance of a stem state in ENCCs.

New synthetic PEKK/bioceramic hybrids and their surface sulfonation counterparts have increased cellular osteogenic capacity and promoted osseointegration

PEKK has good biocompatibility as an implantable material, but its mechanical properties and osseointegration are not satisfied enough. Aluminum oxide (Al2O3) is highly wear-resistant, while hydroxyapatite (HA) is a biologically active. However all previous biocomposites made via mechanical mixing of these two bioceramic materials have not been able to meet clinical standards. The aim of this work is to resolve these problems by using newly synthesized bioceramic hybrids. In vitro and in vivo experiments have demonstrated that these new biomaterials have significantly improved biocompatibility and biomechanical properties when compared to neat PEKK. Surface sulfonation of the hybrids also leads to further improvements of these two properties. S-PEKK/Al2O3 and S-PEKK/HA promote MC3T3-E1 adhesion. PEKK/HA and S-PEKK/HA significantly promote cell proliferation by upregulating both mRNA and protein levels of cell cycle genes to facilitate cells shift from G0/G1 phase to S phase. PEKK/HA hybrids and S-PEKK/Al2O3 promote cell differentiation by upregulating the expression of osteogenic differentiation genes and proteins. PEKK/HA, S-PEKK/Al2O3 and S-PEKK/HA are able to strongly bind to the new bone and exhibit good in vivo osseointegration properties. We conclude that PEKK/HA, S-PEKK/Al2O3 and S-PEKK/HA show good osteogenic ability, especially S-PEKK/HA, and could be promising materials for bone implants.

Ganglioside Microdomains on Cellular and Intracellular Membranes Regulate Neuronal Cell Fate Determination.

Gangliosides are sialylated glycosphingolipids (GSLs) with essential but enigmatic functions in brain activities and neural stem cell (NSC) maintenance. Our group has pioneered research on the importance of gangliosides for growth factor receptor signaling and epigenetic regulation of NSC activity and differentiation. The primary localization of gangliosides is on cell-surface microdomains and the drastic dose and composition changes during neural differentiation strongly suggest that they are not only important as biomarkers, but also are involved in modulating NSC fate determination. Ganglioside GD3 is the predominant species in NSCs and GD3-synthase knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal. Since morphological changes during neurogenesis require a huge amount of energy, mitochondrial functions are vital for neurogenesis. We discovered that a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein, and GD3 regulates mitochondrial dynamics. Furthermore, we discovered that GM1 ganglioside promotes neuronal differentiation by an epigenetic regulatory mechanism. Nuclear GM1 binds with acetylated histones on the promoters of N-acetylgalactosaminyltransferase (GalNAcT; GM2 synthase) as well as on the NeuroD1 genes in differentiated neurons. In addition, epigenetic activation of the GalNAcT gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. GM1 is indeed localized in the nucleus where it can interact with transcriptionally active histones. Interestingly, GM1 could induce epigenetic activation of the tyrosine hydroxylase (TH) gene, with recruitment of nuclear receptor related 1 (Nurr1, also known as NR4A2), a dopaminergic neuron-associated transcription factor, to the TH promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression. GM1 interacts with active chromatin via acetylated histones to recruit transcription factors at the nuclear periphery, resulting in changes in gene expression for neuronal differentiation. The significance is that multifunctional gangliosides modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides could regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains.

Effects of sodium hypochlorite and ethylenediaminetetraacetic acid on proliferation, osteogenic/odontogenic differentiation, and mechanosensitive gene expression of human dental pulp stem cells

Sodium Hypochlorite (NaOCl) and Ethylene Diamine Tetraacetic Acid (EDTA) can change the biochemical and biophysical properties of dentin. However, the response of human dental pulp stem cells (hDPSCs) to NaOCl and EDTA-treated dentin remains unknown. This study was conducted to investigate the effect of NaOCl and EDTA on cell proliferation, osteogenic/odontogenic differentiation, and the response to mechanosensitive gene expression in hDPSCs. Dentin slices were treated with 5.25% NaOCl, 17% EDTA, and saline (0.9% NaCl) separately. The cell viability and osteogenic/odontogenic differentiation of hDPSCs were analyzed using scanning electron microscopy, cell counting assay, alkaline phosphatase (ALP) staining, and quantitative polymerase chain reaction (qPCR). Besides, the hardness was measured by a Vickers microhardness tester. The expression of mechanosensitive genes was detected by the qPCR assay. All the irrigant-treated dentin allowed cell attachment. The EDTA-treated dentin significantly boosted the ALP and osteogenic/odontogenic differentiation, followed by NaCl and NaOCl groups. Remarkably, these trends were similar to the expression of mechanosensitive genes but were different from the trends of hardness values. The effect of irrigant-treated dentin on regulating hDPSCs differentiation might correlate with mechanosensitive signals. Whereas, the hardness changes between groups might not produce significant roles in regulating osteogenic/odontogenic differentiation of stem cells on dentin surfaces.

Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures.

Melanoma is a highly aggressive skin cancer, which, although highly immunogenic, frequently escapes the body’s immune defences. Immune checkpoint inhibitors (ICI), such as anti-PD1, anti-PDL1, and anti-CTLA4 antibodies lead to reactivation of immune pathways, promoting rejection of melanoma. However, the benefits of ICI therapy remain limited to a relatively small proportion of patients who do not exhibit ICI resistance. Moreover, the precise mechanisms underlying innate and acquired ICI resistance remain unclear. Here, we have investigated differences in melanoma tissues in responder and non-responder patients to anti-PD1 therapy in terms of tumour and immune cell gene-associated signatures. We performed multi-omics investigations on melanoma tumour tissues, which were collected from patients before starting treatment with anti-PD1 immune checkpoint inhibitors. Patients were subsequently categorized into responders and non-responders to anti-PD1 therapy based on RECIST criteria. Multi-omics analyses included RNA-Seq and NanoString analysis. From RNA-Seq data we carried out HLA phenotyping as well as gene enrichment analysis, pathway enrichment analysis and immune cell deconvolution studies. Consistent with previous studies, our data showed that responders to anti-PD1 therapy had higher immune scores (median immune score for responders = 0.1335, median immune score for non-responders = 0.05426, p-value = 0.01, Mann-Whitney U two-tailed exact test) compared to the non-responders. Responder melanomas were more highly enriched with a combination of CD8+ T cells, dendritic cells (p-value = 0.03) and an M1 subtype of macrophages (p-value = 0.001). In addition, melanomas from responder patients exhibited a more differentiated gene expression pattern, with high proliferative- and low invasive-associated gene expression signatures, whereas tumours from non-responders exhibited high invasive- and frequently neural crest-like cell type gene expression signatures. Our findings suggest that non-responder melanomas to anti-PD1 therapy exhibit a de-differentiated gene expression signature, associated with poorer immune cell infiltration, which establishes a gene expression pattern characteristic of innate resistance to anti-PD1 therapy. Improved understanding of tumour-intrinsic gene expression patterns associated with response to anti-PD1 therapy will help to identify predictive biomarkers of ICI response and may help to identify new targets for anticancer treatment, especially with a capacity to function as adjuvants to improve ICI outcomes.

R-spondin-3 is an oncogenic driver of poorly differentiated invasive breast cancer.

R-spondins (RSPOs) are influential signaling molecules that promote the Wnt/β-catenin pathway and self-renewal of stem cells. Currently, RSPOs are emerging as clinically relevant oncogenes, being linked to cancer development in multiple organs. Although this has instigated the rapid development and testing of therapeutic antibodies targeting RSPOs, functional evidence that RSPO causally drives cancer has focused primarily on the intestinal tract. Here, we assess the oncogenic capacity of RSPO in breast cancer in a direct fashion by generating and characterizing a novel mouse model with conditional Rspo3 expression in the mammary gland. We also address the prevalence of RSPO gene alterations in breast cancer patients. We found that a quarter of breast cancer patients harbor RSPO2/RSPO3 copy number amplifications, which are associated with lack of steroid hormone receptor expression and reduced patient survival. Foremost, we demonstrate the causal oncogenic capacity of RSPO3 in the breast, as conditional Rspo3 overexpression consistently drives the development of mammary adenocarcinomas in our novel Rspo3 breast cancer model. RSPO3-driven mammary tumors typically show poor differentiation, areas of epithelial-to-mesenchymal transition, and metastatic potential. Given the reported interplay in the Wnt/β-catenin pathway, we comparatively analyzed RSPO3-driven mouse mammary tumors versus classical WNT1-driven analogues. This revealed that RSPO3-driven tumors are distinct, as the poorly differentiated tumor morphology and metastatic potential were observed in RSPO3-driven tumorigenesis exclusively, further substantiated by differentiating gene expression profiles. Co-expression of Rspo3 and Wnt1 transduced mammary tumors with a mixed phenotype harboring morphological features characteristic of both transgenes. In summary, we report that a quarter of breast cancer patients harbor RSPO2/RSPO3 copy number gains, and these patients have a worse prognosis, whilst providing in vivo evidence that RSPO3 drives poorly differentiated invasive breast cancer in mice. Herewith, we establish RSPO3 as a driver of breast cancer with clinical relevance, proposing RSPO3 as a novel candidate target for therapy in breast cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Long-term culture of feline oviduct epithelial cells on permeable filter supports

Basic knowledge about cellular and molecular mechanisms underlying feline reproduction is required to improve reproductive biotechnologies in endangered felids. Commonly, the domestic cat (Felis catus) is used as a model species, but many of the fine-tuned, dynamic reproductive processes can hardly be observed in vivo. This necessitates the development of in vitro models. The oviduct is a central reproductive organ hosting fertilization in the ampulla and early embryonic development in the isthmus part, which also functions as a sperm reservoir before fertilization. In other species, culturing oviduct epithelial cells in compartmentalized culture systems has proven useful to maintain oviduct epithelium polarization and functionality. Therefore, we made the first attempt to establish a compartmentalized long-term culture system of feline oviduct epithelial cells from both ampulla and isthmus. Cells were isolated from tissue samples (n = 33 animals) after routine gonadectomy, seeded on permeable filter supports and cultured at the liquid–liquid or air–liquid interface. Cultures were harvested after 21 days and microscopically evaluated for epithelial differentiation (monolayer formation with basal–apical polarization) and protein expression of marker genes (oviduct-specific glycoprotein, acetylated tubulin). Due to the heterogeneous and undefined native tissue material available for this study, the applied cell culture approach was only successful in a limited number of cases (five differentiated cultures). Even though the protocol needs optimization, our study showed that the compartmentalized culture approach is suitable for maintaining differentiated epithelial cells from both isthmus and ampulla of the feline oviduct.

315.4: CREB May Regulate Gene Expression in MDSC Differentiation by Influencing DNA Methylation

315.4: CREB May Regulate Gene Expression in MDSC Differentiation by Influencing DNA Methylation

Sex-Dependent Role of Adipose Tissue HDAC9 in Diet-Induced Obesity and Metabolic Dysfunction.

Obesity is a major risk factor for both metabolic and cardiovascular disease. We reported that, in obese male mice, histone deacetylase 9 (HDAC9) is upregulated in adipose tissues, and global deletion of HDAC9 protected against high fat diet (HFD)-induced obesity and metabolic disease. Here, we investigated the impact of adipocyte-specific HDAC9 gene deletion on diet-induced obesity in male and female mice. The HDAC9 gene expression was increased in adipose tissues of obese male and female mice and HDAC9 expression correlated positively with body mass index in humans. Interestingly, female, but not male, adipocyte-specific HDAC9 KO mice on HFD exhibited reduced body weight and visceral adipose tissue mass, adipocyte hypertrophy, and improved insulin sensitivity, glucose tolerance and adipogenic differentiation gene expression. Furthermore, adipocyte-specific HDAC9 gene deletion in female mice improved metabolic health as assessed by whole body energy expenditure, oxygen consumption, and adaptive thermogenesis. Mechanistically, compared to female mice, HFD-fed male mice exhibited preferential HDAC9 expression in the stromovascular fraction, which may have offset the impact of adipocyte-specific HDAC9 gene deletion in male mice. These results suggest that HDAC9 expressed in adipocytes is detrimental to obesity in female mice and provides novel evidence of sex-related differences in HDAC9 cellular expression and contribution to obesity-related metabolic disease.

Harpagoside attenuates local bone Erosion and systemic osteoporosis in collagen-induced arthritis in mice

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes local bone erosion and systemic osteoporosis. Harpagoside (HAR), an iridoid glycoside, has various pharmacological effects on pain, arthritis, and inflammation. Our previous study suggests that HAR is more deeply involved in the mechanism of bone loss caused by inflammatory stimuli than hormonal changes. Here, we identified the local and systemic bone loss inhibitory effects of HAR on RA and its intracellular mechanisms using a type 2 collagen-induced arthritis (CIA) mouse model.MethodsThe anti-osteoporosis and anti-arthritic effects of HAR were evaluated on bone marrow macrophage in vitro and CIA in mice in vivo by obtaining clinical scores, measuring hind paw thickness and inflammatory cytokine levels, micro-CT and histopathological assessments, and cell-based assay.ResultsHAR markedly reduced the clinical score and incidence rate of CIA in both the prevention and therapy groups. Histological analysis demonstrated that HAR locally ameliorated the destruction of bone and cartilage and the formation of pannus. In this process, HAR decreased the expression of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1β in the serum of CIA mice. Additionally, HAR downregulated the expression of receptor activator of nuclear factor-κB ligand and upregulated that of osteoprotegerin. HAR suppressed systemic bone loss by inhibiting osteoclast differentiation and osteoclast marker gene expression in a CIA mouse model.ConclusionsTaken together, these findings show the beneficial effect of HAR on local symptoms and systemic bone erosion triggered by inflammatory arthritis.

Supplementation of articular cartilage-derived chondroprogenitors with bone morphogenic protein-9 enhances chondrogenesis without affecting hypertrophy.

Chondroprogenitors (CPCs) have emerged as a promising cellular therapy for cartilage-related pathologies due to their inherent primed chondrogenic potential. Studies report that the addition of growth factors such as parathyroid hormone (PTH) and Bone Morphogenic Protein (BMP) enhance the chondroinducive potential in chondrocytes and mesenchymal stem cells. This study evaluated if supplementation of the standard culture medium for cell expansion with 1-34 PTH and BMP-9 would enhance the chondrogenic potential of CPCs and reduce their hypertrophic tendency. Human chondrocytes were isolated from patients undergoing total knee replacement for osteoarthritis (n = 3). Following fibronectin adhesion assay, passage 1 CPCs were divided and further expanded under three culture conditions (a) control, i.e., cells continued under standard culture conditions, (b) 1-34 PTH group, additional intermittent 6h exposure with 1-34 PTH and (c) BMP-9 group, additional BMP-9 during culture expansion. All the groups were evaluated for population-doubling, cell cycle analysis, surface marker and gene expression for chondrogenesis, hypertrophy, multilineage differentiation and GAG (glycosaminoglycan)/DNA following chondrogenic differentiation. Concerning growth kinetics, the BMP-9 group exhibited a significantly lower S-phase and population-doubling when compared to the other two groups. Qualitative analysis for chondrogenic potential (Alcian blue, Safranin O staining and Toluidine blue for GAG) revealed that the BMP-9 group exhibited the highest uptake. The BMP-9 group also showed significantly higher COL2A1 expression than the control group, with no change in the hypertrophy marker expression. BMP-9 can potentially be used as an additive for CPCs expansion, to enhance their chondrogenic potential without affecting their low hypertrophic tendency. The mitigating effects of 1-34PTH on hypertrophy would benefit further investigation when used in combination with BMP-9 to enhance chondrogenesis whilst reducing hypertrophy.