Domain Containing Research Articles

Molecular interplay between the upregulated levels of Sad1 and UNC84 Domain Containing 2 (SUN2) and gene expression in medulloblastoma cells.

SUN2 is a nuclear envelope protein associated with the nuclear lamina and with proteins linked to nuclear export, splicing, and nucleo-cytoskeleton communication. Studies of SUN2 in cancer have been limited but have suggested that it has tumor-suppressive activity in some carcinomas. Medulloblastoma is a pediatric tumor that develops in the cerebellum and is currently classified intofour molecular groups: WNT (Wingless), SHH (Sonic Hedgehog), 3, and 4. SUN2 expression profiles appear to be altered in brain cancer but have not been previously evaluated in medulloblastoma. The University of Alabama at Birmingham Cancer (UALCAN) data analysis portal, Gene Expression Profiling Interactive Analysis (GEPIA), the Oncopression gene expression compendium, and the R2 genomics analysis and visualization platform were used to analyze SUN2 expression in cancer, which was found to vary by cancer type; in particular, SUN2 expression was found to be upregulated in medulloblastoma. We also explored the effects of reduced SUN2 protein levels (by RNA interference) on gene expression profiles using a cDNA microarray in DAOY medulloblastoma-derived cells. We found that SUN2 protein is upregulated in medulloblastoma, mainly in the SHH group, which correlates with poor survival. Furthermore, the reduced SUN2 expression in medulloblastoma cells is associated with the downregulation of the expression of other genes, including members of the bitter taste-sensing type 2 receptor (TAS2R) family. This study shows that SUN2 is upregulated in medulloblastoma-with molecular interplay in gene expression-which has group-dependent implications for medulloblastoma development. In particular, the upregulation of SUN2 is associated with a progression of the SHH group of medulloblastoma.

Helicobacter pylori promotes gastric cancer progression by activating the TGF-β/Smad2/EMT pathway through HKDC1

Helicobacter pylori (H. pylori) infection is widely acknowledged as the primary risk factor for gastric cancer, facilitating its progression via the Correa cascade. Concurrently, Hexokinase Domain Containing 1 (HKDC1) has been implicated in the mediation of aerobic glycolysis, contributing to tumorigenesis across various cancers. However, the precise role of HKDC1 in the inflammatory transformation associated with H. pylori-induced gastric cancer remains elusive. In this study, transcriptome sequencing revealed a significant correlation between HKDC1 and H. pylori-induced gastric cancer. Subsequent validation using qRT-PCR, immunohistochemistry, and Western blot analysis confirmed elevated HKDC1 expression in both human and murine gastritis and gastric tumors. Moreover, in vitro and in vivo experiments demonstrated that H. pylori infection up-regulates TGF-β1 and p-Smad2, thereby activating the epithelial-mesenchymal transition (EMT) pathway, with HKDC1 playing a pivotal role. Suppression of HKDC1 expression or pharmacological inhibition of TGF-β1 reversed EMT activation, consequently reducing gastric cancer cell proliferation and metastasis. These results underscore HKDC1’s essential contribution to H. pylori-induced gastric cancer progression via EMT activation.Graphical

Abstract 4140121: Lipoprotein(a) Local Sybthesis And Plasma Uptake Via MDFS5 in Human Calcified Valve

Background: High circulating levels of lipoprotein(a) [Lp(a)] are associated with increased risk of calcific aortic stenosis (CAS). Targeting Lp(a) metabolism holds promise for therapeutic interventions. However, the precise mechanisms by which Lp(a) accumulates on aortic valves still not fully understood. Hypothesis: Lp(a) could be synthesized in situ in human aortic valves under specific stimuli, such as calcification, in addition to being taken up from plasma via membrane receptors, including the newly identified receptor Major Facilitator Superfamily Domain Containing 5 (MFSD5). Methods: Immunohistochemistry was used to detect the Lp(a) and its receptor MFSD5 in human aortic valves from CAS patients or controls, which were non-CAS patients. Plasma Lp(a) levels were measured in individuals undergoing aortic valve resection. Human valvular interstitial cells (VICs) were cultured in procalcifying medium, with or without Lp(a) (10uM) for 14 and 21 days. Von-kossa or Alizarin Red staining were used to assess valve calcification. Expression of Lp(a) and MFSD5 was analyzed with quantitative RT-PCR and western blot. Results: Lp(a) was nearly absent in control valve site, whereas it was abundantly present in valve site from CAS patients (p＜0.05). The higher the level of Lp(a) detected, the greater the extent of calcification noted in valve site. The mRNA and protein levels of Lp(a) extracted from calcified human valves were significantly higher compared to controls. However, the concentration of plasma Lp(a) did not affect the expression of Lp(a) in valve site (p＞0.05). In vitro studies showed that Lp(a) mRNA was minimally detectable in normal VICs, but was significantly upregulated following exposure to procalcifying medium (p < 0.05). Lp(a) incubation led to severer calcification than that induced by procalcifying medium alone (p < 0.05). As MFSD5 was a newly identified receptor of Lp(a), we also test its expression. MDFS5 was increased in human calcified valve (p＜0.05) and calcified VICs (p＜0.05). These results showed that the Lp(a) in valve could be increased by either uptake from serum or by synthesized locally by proper stimuli. Conclusion: Our study has shown that the degree of aortic valve calcification is correlated with the level of Lp(a) in the valve site rather than in serum. We also verified MFSD5 was the receptor of Lp(a) on human valve. Targeting Lp(a) accumulation at the valve site may offer a more effective strategy for the prevention or treatment of CAS.

M6A-related genes and their role in Parkinson's disease: Insights from machine learning and consensus clustering.

Parkinson disease (PD) is a chronic neurological disorder primarily characterized by a deficiency of dopamine in the brain. In recent years, numerous studies have highlighted the substantial influence of RNA N6-methyladenosine (m6A) regulators on various biological processes. Nevertheless, the specific contribution of m6A-related genes to the development and progression of PD remains uncertain. In this study, we performed a differential analysis of the GSE8397 dataset in the Gene Expression Omnibus database and selected important m6A-related genes. Candidate m6A-related genes were then screened using a random forest model to predict the risk of PD. A nomogram model was built based on the candidate m6A-related genes. By employing a consensus clustering method, PD was divided into different m6A clusters based on the selected significant m6A-related genes. Finally, we performed immune cell infiltration analysis to explore the immune infiltration between different clusters. We performed a differential analysis of the GSE8397 dataset in the Gene Expression Omnibus database and selected 11 important m6A-related genes. Four candidate m6A-related genes (YTH Domain Containing 2, heterogeneous nuclear ribonucleoprotein C, leucine-rich pentatricopeptide repeat motif containing protein and insulin-like growth factor binding protein-3) were then screened using a random forest model to predict the risk of PD. A nomogram model was built based on the 4 candidate m6A-related genes. The decision curve analysis indicated that patients can benefit from the nomogram model. By employing a consensus clustering method, PD was divided into 2 m6A clusters (cluster A and cluster B) based on the selected significant m6A-related genes. The immune cell infiltration analysis revealed that cluster A and cluster B exhibit distinct immune phenotypes. In conclusion, m6A-related genes play a significant role in the development of PD and our study on m6A clustering may potentially guide personalized treatment strategies for PD in the future.

Targeting DDX3X eliminates leukemia stem cells in chronic myeloid leukemia by blocking NT5DC2 mRNA translation.

Tyrosine kinase inhibitors (TKIs) are highly effective in the treatment of patients with chronic myeloid leukemia (CML), but fail to eliminate leukemia stem cells (LSCs), which can lead to disease relapse or progression. It is urgently need to identify the regulators specifically driving LSCs. In this study, we identified DEAD-box helicase 3 X-linked (DDX3X), a ubiquitously expressed RNA helicase, as a critical regulator for CML LSCs by using patient samples and BCR-ABL-driven CML mouse model. We found that DDX3X enhanced the survival, serially plating and long-term engraftment abilities of human primary CML CD34+ cells. Inhibition of DDX3X reduced leukemia burden, eradicated LSCs and extended the survival of CML mice. Mechanistically, we uncovered that DDX3X protein bound to 5'-Nucleotidase Domain Containing 2 (NT5DC2) mRNA and promoted its translation in CML cells. NT5DC2 was a functional mediator in DDX3X regulation of LSCs. Collectively, our findings provide new evidence for RNA helicase facilitating the translation of specific mRNA in LSCs. Targeting DDX3X may represent a promising therapeutic strategy for eradication of LSCs in CML patients.

Epileptic encephalopathy linked to a DALRD3 missense variant that impairs tRNA modification

Epileptic encephalopathies are severe epilepsy syndromes characterized by early onset and progressive cerebral dysfunction. A nonsense variant in the DALR anticodon binding domain containing 3 (DALRD3) gene has been implicated in epileptic encephalopathy, but no other disease-associated variants in DALRD3 have been described. In human cells, the DALRD3 protein forms a complex with the METTL2 methyltransferase to generate the 3-methylcytosine (m3C) modification in specific arginine tRNAs. Here, we identify an individual with a homozygous missense variant in DALRD3 who displays developmental delay, cognitive deficiencies, and multifocal epilepsy. The missense variant substitutes an arginine residue to cysteine (R517C) within the DALR domain of the DALRD3 protein that is required for binding tRNAs. Cells derived from the individual homozygous for the DALRD3-R517C variant exhibit reduced levels of m3C modification in arginine tRNAs, indicating that the R517C variant impairs DALRD3 function. Notably, the DALRD3-R517C protein displays reduced association with METTL2 and loss of interaction with substrate tRNAs. Our results uncover another loss-of-function variant in DALRD3 linked to epileptic encephalopathy disorders. Importantly, these findings underscore DALRD3-dependent tRNA modification as a key contributor to proper brain development and function.

Serum Levels of Protein Z, Laminin Subunit Alpha 2, Mixed Lineage Leukemia 4, and Plexin Domain Containing 2 in Newly Diagnosed and Metformin-Treated Type 2 Diabetes Patients

Diabetes mellitus is a globally prevalent disease with several parameters that may be involved in diagnosis or altered throughout disease progression. In this study, 120 patients with type 2 diabetes and healthy controls were recruited, with ages ranging from 30-65 years old. The research focused on determining levels of Protein Z (PROZ), Laminin Subunit Alpha 2 (LAMA2), Mixed Lineage Leukemia 4 (MLL4), and Plexin Domain Containing 2 (PLXDC2). Participants were divided into four groups: group I (n=30) with long-term metformin treatment, group II (n=30) with short-term metformin treatment, group III (n=30) newly diagnosed without medication, and group IV (n=30) healthy controls. Protein markers were measured by ELISA, while fasting blood glucose, lipid profiles, and HbA1c were determined by spectrophotometry and HPLC. ROC curve analysis revealed PLXDC2 had high sensitivity and specificity as a potential biomarker for diabetes diagnosis. LAMA2, MLL4, and PROZ also distinguished between diabetes patients and controls. Overall, these results identify promising biomarkers for diabetes detection and monitoring.

The impacts of tobacco and nicotine on HIV-1 infection, inflammation, and the blood-brain barrier in the central nervous system.

Human immunodeficiency virus (HIV-1) remains a persistent global health crisis. Even while successfully virologically suppressed, people with HIV (PWH) experience a higher risk for inflammatory disorders such as HIV-associated neurocognitive disorder (HAND). Tobacco use puts PWH at higher risk for neurocognitive symptoms resulting from HIV-associated neuroinflammation. The NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome has been implicated as a driver of HIV-associated inflammation, including HAND. Nicotine, the psychoactive component of tobacco smoke, has also been shown to signal through the NLRP3 inflammasome and modulate inflammatory signaling in the CNS. Here, we explore the impacts of nicotine and tobacco on the complex neurobiology of HAND, including effects on cognition, inflammation, viral latency, and blood-brain barrier integrity. We outline nicotine's role in the establishment of active and latent infection in the brain and posit the NLRP3 inflammasome as a common pathway by which HIV-1 and nicotine promote neuroinflammation in PWH.

CircFndc3b Mediates Exercise-Induced Neuroprotection by Mitigating Microglial/Macrophage Pyroptosis via the ENO1/KLF2 Axis in Stroke Mice.

Circular RNA (circRNA) plays a pivotal role in regulating neurological damage post-ischemic stroke. Previous researches demonstrated that exercise mitigates neurological dysfunction after ischemic stroke, yet the specific contributions of circRNAs to exercise-induced neuroprotection remain unclear. This study reveals that mmu_circ_0001113 (circFndc3b) is markedly downregulated in the penumbral cortex of a mouse model subjected to middle cerebral artery occlusion (MCAO). However, exercise increased circFndc3b expression in microglia/macrophages, alleviating pyroptosis, reducing infarct volume, and enhancing neurological recovery in MCAO mice. Mechanistically, circFndc3b interacted with Enolase 1 (ENO1), facilitating ENO1's binding to the 3' Untranslated Region (3'UTR) of Krüppel-like Factor 2 (Klf2) mRNA, thereby stabilizing Klf2 mRNA and increasing its protein expression, which suppressed NOD-like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome-mediated microglial/macrophage pyroptosis. Additionally, circFndc3b enhanced ENO1's interaction with the 3'UTR of Fused in Sarcoma (FUS) mRNA, leading to increased FUS protein levels and promoting circFndc3b cyclization. These results suggest that circFndc3b mediates exercise-induced anti-pyroptotic effects via the ENO1/Klf2 axis, and a circFndc3b/ENO1/FUS positive feedback loop may potentiate exercise's neuroprotective effects. This study unveils a novel mechanism underlying exercise-induced neuroprotection in ischemic stroke and positions circFndc3b as a promising therapeutic target for stroke management, mimicking the beneficial effects of exercise.

Quercetin supplementation in metabolic syndrome: nutrigenetic interactions with the Zbtb16 gene variant in rodent models

BackgroundQuercetin is a promising phytochemical in treating abnormalities associated with metabolic syndrome (MetS). This study aimed to explore the morphometric, metabolic, transcriptomic, and nutrigenetic responses to quercetin supplementation using two genetically distinct MetS models that only differ in the variant of the MetS-related Zbtb16 gene (Zinc Finger And BTB Domain Containing 16).ResultsQuercetin supplementation led to a significant reduction in the relative weight of retroperitoneal adipose tissue in both investigated strains. A decrease in visceral (epididymal) fat mass, accompanied by an increase in brown fat mass after quercetin treatment, was observed exclusively in the SHR strain. While the levels of serum triglycerides decreased within both strains, the free fatty acids levels decreased in SHR-Zbtb16-Q rats only. The total serum cholesterol levels were not affected by quercetin in either of the two tested strains. While there were no significant changes in brown adipose tissue transcriptome, quercetin supplementation led to a pronounced gene expression shift in white retroperitoneal adipose tissue, particularly in SHR-Zbtb16-Q.ConclusionQuercetin administration ameliorates certain MetS-related features; however, the efficacy of the treatment exhibits subtle variations depending on the specific variant of the Zbtb16 gene.

SARS-CoV-2 spike S1 protein induces microglial NLRP3-dependent neuroinflammation and cognitive impairment in mice

Cognitive impairment is often found at the acute stages and sequelae of coronavirus disease 2019 (COVID-19), and the underlying mechanisms remain unclear. The S1 protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be a cause of cognitive impairment associated with COVID-19. The nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome and neuroinflammation play important roles in Alzheimer's disease (AD) with cognitive impairment. However, their roles remain unknown in COVID-19 with cognitive impairment. We stimulated BV2 cells with S1 protein in vitro and injected the hippocampi of wild-type (WT) mice, NLRP3 knockout (KO), and microglia NLRP3 KO mice in vivo with S1 protein to induce cognitive impairment. We assessed exploratory behavior as associative memory using novel object recognition and Morris water maze tests. Neuroinflammation was analyzed using immunofluorescence and western blotting to detect inflammatory markers. Co-localized NLRP3 and S1 proteins were investigated using confocal microscopy. We found that S1 protein injection led to cognitive impairment, neuronal loss, and neuroinflammation by activating NLRP3 inflammation, and this was reduced by global NLRP3 KO and microglia NLRP3 KO. Furthermore, TAK 242, a specific inhibitor of Toll-like receptor-4, resulted in a significant reduction in NLRP3 and pro-IL-1β in BV2 cells with S1 protein stimulation. These results reveal a distinct mechanism through which the SARS-CoV-2 spike S1 protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines

Glioblastoma is a deadly tumor which possesses glioblastoma stem cell populations involved in temozolomide (TMZ) resistance. To gain insight into the mechanisms of self-renewing and therapy-resistant cancer stem cells, subcellular proteomics was utilized to identify proteins whose expression is enriched in U251-derived glioblastoma stem-like cells. The KH RNA Binding Domain Containing, Signal Transduction Associated 3, KHDRBS3, was successfully identified as a gene up-regulated in the cancer stem cell population compared with its differentiated derivatives. Depletion of KHDRBS3 by RNA silencing led to a decrease in cell proliferation, neurosphere formation, migration, and expression of genes involved in glioblastoma stemness. Importantly, TMZ sensitivity can be induced by the gene knockdown. Collectively, our results highlight KHDRBS3 as a novel factor associated with self-renewal of glioblastoma stem-like cells and TMZ resistance. As a consequence, targeting KHDRBS3 may help eradicate glioblastoma stem-like cells.

Unraveling potential gene biomarkers for dengue infection through RNA sequencing.

Dengue virus hijacks host cell mechanisms and immune responses in order to replicate efficiently. The interaction between the host and the virus affects the host's gene expression, which remains largely unexplored. This pilot study aimed to profile the host transcriptome as a potential strategy for identifying specific biomarkers for dengue prediction and detection. High-throughput RNA sequencing (RNA-seq) was employed to generate host transcriptome profiles in 16 dengue patients and 10 healthy controls. Differentially expressed genes (DEGs) were identified in patients with severe dengue and those with dengue with warning signs compared to healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to elucidate the functions of upregulated and downregulated genes. Compared to healthy controls, 6466 genes were significantly differentially expressed (p < 0.05) in the dengue with warning signs group and 3082 genes in the severe dengue group, with over half being upregulated. The major KEGG pathways implicated included transport and catabolism (14.4%-16.3%), signal transduction (6.6%-7.3%), global and overview maps (6.7%-7.1%), viral diseases (4.6%-4.8%), and the immune system (4.4%-4.6%). Several genes exhibited consistent and significant upregulation across all dengue patients, regardless of severity: Interferon alpha inducible protein 27 (IFI27), Potassium Channel Tetramerization Domain Containing 14 (KCTD14), Syndecan 1 (SDC1), DCC netrin 1 receptor (DCC), Ubiquitin C-terminal hydrolase L1 (UCHL1), Marginal zone B and B1 cell-specific protein (MZB1), Nestin (NES), C-C motif chemokine ligand 2 (CCL2), TNF receptor superfamily member 17 (TNFSF17), and TNF receptor superfamily member 13B (TNFRSF13B). Further analysis revealed potential biomarkers for severe dengue prediction, including TNF superfamily member 15 (TNFSF15), Plasminogen Activator Inhibitor-2 (SERPINB2), motif chemokine ligand 7 (CCL7), aconitate decarboxylase 1 (ACOD1), Metallothionein 1G (MT1G), and Myosin Light Chain Kinase (MYLK2), which were expressed 3.5 times, 2.9 times, 2.3 times, 2.1 times, 1.7 times, and 1.4 times greater, respectively, than dengue patients exhibiting warning signs. The identification of these host biomarkers through RNA-sequencing holds promising implications and potential to augment existing dengue detection algorithms, contributing significantly to improved diagnostic and prognostic capabilities.

Effect of JMJD3-IRF4 Signaling Pathway-Mediated Macrophage Polarization on the Malignant Biological Behavior of Multiple Myeloma Cells

To investigate the effect of macrophage polarization mediated by Jumonji domain containing-3 (JMJD3)-interferon regulatory factor 4 (IRF4) signaling pathway on the malignant biological behavior of multiple myeloma (MM) cells. THP-1 monocytes were induced to differentiate into macrophages by phorbol myristate acetate (PMA). THP-1 macrophages were divided into control group (normal culture), M2 induction group ［added recombinant human interleukin (IL) -4, IL-13 proteins］, M2+JMJD3 protein group (added recombinant human IL-4, IL-13 and JMJD3 proteins) and M2+JMJD3 inhibitor group (added recombinant human IL-4, IL-13 proteins and JMJD3 inhibitor), the proportion of CD206+ cells was detected by flow cytometry, the levels of IL-10 and transforming growth factor-β (TGF-β) in the culture supernatant were detected by ELISA assay, the expression levels of arginase-1 (Arg-1), JMJD3 and IRF4 mRNA were detected by real-time quantitative PCR (qRT-PCR), and the expression levels of Arg-1, JMJD3 and IRF4 proteins were detected by Western blot. Correspondingly, human MM cells U266 were cultured with THP-1 macrophage culture supernatant of each group, Methyl thiazolyl tetrazolium (MTT) method and plate colony formation assay were used to detect cell proliferation, cell apoptosis was detected by flow cytometry, Western blot was used to detect the expression levels of apoptosis-promoting protein Bcl-2-associated X protein (Bax) and cleaved caspase-3 in cells, and Transwell assay was used to detect cell migration and invasion. Compared with the control group, the proportion of CD206+ cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-β in the cell culture supernatant in M2 induction group were significantly increased (P <0.001), meanwhile, the proliferation activity and the number of clones of U266 cells were significantly increased (P <0.01), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were significantly decreased (P <0.001), the numbers of migrated cells and invasive cells were increased (P <0.001). Compared with M2 induction group, the proportion of CD206+ cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-β in the cell culture supernatant in M2+JMJD3 protein group were further increased (P <0.01), meanwhile, the proliferation activity and the number of clones of U266 cells were further increased (P <0.05), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were further decreased (P <0.01), the numbers of migrated cells and invasive cells were further increased (P <0.001); However, the change trends of the above indexes in M2+JMJD3 inhibitor group were opposite to those in M2+JMJD3 protein group. M2 polarization of macrophages mediated by JMJD3-IRF4 signaling pathway can promote the proliferation, migration and invasion of MM cells, and inhibit cell apoptosis.

Abnormal Promoter Methylation of Nucleotide-Binding Oligomerization Domain Containing 2 (NOD2) Gene in the Pathogenesis of Crohn’s Disease

Background: Changes in the expression of nucleotide-binding oligomerization domain containing 2 (NOD2) play an important role in the pathogenesis of a variety of autoimmune diseases including inflammatory bowel diseases (IBDs). Epigenetic modifications, including DNA methylation, are considered an important mechanism in the suppression of gene activity. In this study, we investigated the relationship between DNA methylation patterns of the promoter region of the NOD2 gene and the pathogenesis of Crohn’s disease (CD). Methods: Colonic mucosa samples were obtained from 15 Iranian patients with IBD and 15 age- and sexmatched healthy controls with no history of autoimmune disease. After the bisulfite conversion of genomic DNA, the DNA methylation status of three CpG sites in the promoter region of the NOD2 gene was determined by the real-time quantitative multiplex methylation-specific PCR (QM-MSP) assay. Results: Using this approach, we identified that IBD patients showed a decreased level of methylation of the NOD2 promoter in the colonic mucosa than did the healthy controls. (Unmethylated DNA in Crohn's disease vs. healthy controls; 0.128±0.093 vs. 0.025±0.016, P&lt;0.000). Conclusion: According to our findings, promoter hypomethylation of the NOD2 gene in the colonic mucosa might contribute to the development and severity of CD. Furthermore, aberrant DNA methylation levels are expected to serve as a clinically useful risk marker.

Parthenolide ameliorates 3-nitropropionic acid-induced Huntington’s disease-like aberrations via modulating NLRP3 inflammasome, reducing microglial activation and inducing astrocyte shifting

BackgroundHuntington’s disease (HD) is a progressive neurodegenerative disease that causes motor, cognitive, and psychiatric abnormalities, with no satisfying disease-modifying therapy so far. 3-nitropropionic acid (3NP) induces behavioural deficits, together with biochemical and histological alterations in animals’ striata that mimic HD. The role of nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome in HD pathogenesis remains largely uncharacterized. Parthenolide (PTL), a naturally occurring nuclear factor kappa B (NF-κB) inhibitor, is also known to inhibit NLRP3 inflammasome. Whether PTL is beneficial in HD has not been established yet.AimThis study evaluated the possible neuroprotective effects of PTL against 3NP-induced behavioural abnormalities, striatal biochemical derangements, and histological aberrations.MethodsMale Wistar rats received PTL (0.5 mg/kg/day, i.p) for 3 weeks and 3NP (10 mg/kg/day, i.p) was administered alongside for the latter 2 weeks to induce HD. Finally, animals were subjected to open-field, Morris water maze and rotarod tests. Rat striata were examined histologically, striatal protein expression levels of glial fibrillary acidic protein (GFAP), cluster of differentiation 45 (CD45) and neuron-specific enolase (NSE) were evaluated immunohistochemically, while those of interleukin (IL)-1β, IL-18, ionized calcium-binding adapter molecule-1 (Iba1) and glutamate were determined by ELISA. Striatal nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein (Keap1), NF-κB, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, S100 calcium-binding protein A10 (S100A10) and complement-3 (C3) were assessed by gene expression analysis.ResultsPTL improved motor, locomotor, cognitive and anxiety-like behaviours, restored neuronal integrity, upregulated Nrf2, and inhibited NLRP3 inflammasome, NF-κB and microglial activation. Additionally, PTL induced astrocyte shifting towards the neuroprotective A2 phenotype.ConclusionPTL exhibits neuroprotection against 3NP-induced HD, that might be ascribed, at least in part, to its modulatory effects on Keap1/Nrf2 and NF-κB/NLRP3 inflammasome signaling.

Melatonin alleviates high glucose-induced cardiomyocyte injury through suppressing mitochondrial FUNDC1-DRP1 axis.

To use H9c2 cardiomyocytes to establish a diabetic cardiomyopathic model by exposing these cells to high glucose (HG), followed by treating them with melatonin (MEL) or plasmid vectors overexpressing FUN14 Domain Containing 1 (FUNDC1). We employed quantitative real-time PCR, mitochondrial staining, and biochemical assays to measure the activity of various antioxidant and mitochondrial complex functions under various treatment conditions. Our results showed that HG induced the expression of FUNDC1 and increased mitochondrial oxidative stress and fragmentation, while MEL treatment reversed most of these pathological effects. Moreover, HG exposure activated dynamin-related protein 1 expression and its translocation to mitochondria. Modulation of AMP-activated protein kinase level was found to be another pathological hallmark. In silico molecular docking, analysis revealed that MEL could directly bind the catalytic groove of FUNDC1 through Van der Waal's force and hydrogen bonding. Finally, MEL ameliorated diabetic cardiomyopathy-induced mitochondrial injury through FUNDC1 in vivo. Hyperglycemia induced mitochondrial fragmentation and altered electron transport chain complex functions, which could be ameliorated by MEL treatment, suggesting its potential as a cardiovascular therapeutic.

TDRD3 functions as a selective autophagy receptor with dual roles in autophagy and modulation of stress granule stability.

Tudor Domain Containing 3 (TDRD3) is a methylarginine-reader protein that functions as a scaffold in the nucleus facilitating transcription, however TDRD3 is also recruited to stress granules (SGs) during the Integrated Stress Response (ISR) although its function therein remains largely unknown. We previously showed that TDRD3 is a novel antiviral restriction factor that is cleaved by virus 2A protease, and plays complex modulatory roles in both interferon and inflammatory signaling during stress and enterovirus infections. Here we have found that TDRD3 contains structural motifs similar to known selective autophagy receptors such as p62/SQSTM1, sharing ubiquitin associated domains (UBA) and LC3 interacting regions (LIR) that anchor cargo destined for autophagosomes to activated LC3 protein coating autophagosome membranes. This is of interest since enteroviruses hijack autophagy machinery to facilitate formation of viral replication factories, virus assembly and egress from the infected cell. Here we explored possible roles of TDRD3 in autophagy, hypothesizing that TDRD3 may function as a specialized selective autophagy receptor. We found that KO of TDRD3 in HeLa cells significantly reduces starvation induced autophagy, while its reintroduction restores it in a dose-dependent manner. Autophagy receptors are degraded during autophagy and expression levels decrease during this time. We found that TDRD3 levels decrease to the same extent as the autophagy receptor p62/SQSTM1 during autophagy, indicating autophagy-targeted turnover in that role. Knockout of TDRD3 or G3BP1 did not make significant changes in overall cell localization of LC3B or p62/SQSTM1, but did result in greater concentration of Lamp2 phagosome marker for phagosomes and phagolysosomes. To test the potential roles of TDRD3 in autophagic processes, we created a series of deletion mutants of TDRD3 lacking either UBA domain or the various LIR motifs that are predicted to interact with LC3B. Microscopic examination of starved cells expressing these variants of TDRD3 showed ΔLIR-TDRD3 had defects in colocalization with LC3B or Lamp2. Further, super resolution microscopy revealed ring structures with TDRD3 interfacing with p62/SQSTM1. In examination of arsenite induced stress granules we found recruitment of TDRD3 variants disrupted normally tight SG condensation, altered the decay rate of SGs upon release from stress and the kinetics of SG formation. We found evidence that the LIR3 motif on TDRD3 is involved in TDRD3 interaction with LC3B in coIP experiments, colocalization studies, and that this motif plays a key role in TDRD3 recruitment to SGs and SG resolution. Overall, these data support a functional role of TDRD3 in selective autophagy in a mode similar to p62/SQSTM1, with specific roles in SG stability and turnover. Enterovirus cleavage of TDRD3 likely affects both antiviral and autophagic responses that the virus controls for replication.

Abstract B083: The splicing factor SMNDC1 facilitates alternative RNA splicing, contributing to therapy resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and lethal cancers, mainly due to its high resistance to existing treatments. The standard treatment for PDAC involves chemotherapy, either with Gemcitabine (GEM) combined with paclitaxel or the FOLFIRINOX regimen (5-fluorouracil (5FU), leucovorin, irinotecan, oxaliplatin). However, PDACs rapidly develop resistance to these therapies. Similar resistance is observed with new treatments targeting the KRAS-MAPK pathway, which is crucial in driving PDAC. Our preliminary studies have shown that Survival Motor Neuron Domain Containing 1 (SMNDC1) promotes PDAC tumor growth by facilitating exon retention, specifically cassette exon 4 (E4) of MAPK3, linking RNA splicing to KRAS-MAPK signaling. To uncover the mechanisms underlying PDAC therapeutic resistance, we have developed novel in vitro models of resistance to chemotherapy and MAPK inhibitors (MAPKi). Using these models, we found that SMNDC1 and MAPK3 E4 retention is significantly increased in GEM, 5FU (chemotherapy agents), Adagrasib, Sotorasib (KRAS inhibitors; KRASi G12C), and Selumetinib (MEK inhibitor; MEKi) resistant PDAC cells. Additionally, PDAC lines acutely treated (24h) with trametinib or selumetinib (MEKi) showed a doubling in the expression of SMNDC1 and MAPK3 E4 retention. Protein expression analysis via western blotting revealed that GEM, 5FU, Adagrasib, MRTX1133 (KRASi G12D), and Selumetinib resistant PDAC cells upregulated SMNDC1 expression compared to their sensitive counterparts. To determine the role of SMNDC1 in MAPK resistance, we tested SMNDC1 proficient and deficient PDAC cells with KRASi or MEKi. SMNDC1-deficient PDAC cells were more sensitive to these inhibitors than isogenic cells expressing SMNDC1. Furthermore, to show that increased E4 retention of MAPK3 drives resistance to KRASi or MEKi, we treated SMNDC1 proficient PDAC cells with these inhibitors and MAPK3 E4 specific antisense morpholino oligonucleotide (ASO), which increased sensitivity compared to cells treated with the inhibitor and control ASO. To determine if increased E4 retention is a resistance mechanism specific to therapies targeting upstream of ERK1, we treated PDAC cells, proficient and deficient in SMNDC1, with ERK inhibitors (MK8353, Temuterkib). No changes in sensitivity were observed, as these inhibitors target the ERK1 protein downstream of MAPK3 splicing. Additionally, in an orthogonal model, we found that mutant EGFR lung adenocarcinoma cells resistant to the third-generation tyrosine kinase EGFR inhibitor (Osimertinib) also had higher incorporation of E4 of MAPK3. This suggests that upstream inhibition of ERK in the MAPK pathway leads to increased MAPK3 E4 retention. These findings indicate that PDACs use a "splicing switch" to enhance ERK kinase activity, thereby resisting targeted therapies and chemotherapy in cancers driven by mutant KRAS-MAPK signaling pathways. Citation Format: Md Afjalus Siraj, Yushan Zhang, Deanne Yugawa, Gilbert Giri, Prabir Chakraborty, Grant Goda, Geeta Rao, Daniel Dominguez, Stefan Kubicek, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. The splicing factor SMNDC1 facilitates alternative RNA splicing, contributing to therapy resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B083.

E3 ubiquitin ligase BTBD3 inhibits tumorigenesis of colorectal cancer by regulating the TYRO3/Wnt/β-catenin signaling axis

Clinical trials and studies have implicated that E3 ubiquitin ligase BTBD3 (BTB Domain Containing 3) is a cancer-associated gene. However, the role and underlying mechanism of BTBD3 in colorectal cancer (CRC) is not fully understood yet. Herein, our study demonstrated that the mRNA and protein levels of BTBD3 were decreased in CRC tissues and associated with TYPO3 and Wnt/β-catenin pathway. Our results showed that circRAE1 knockdown and TYRO3 overexpression activated Wnt/β-catenin signaling pathway and the EMT process-associated markers, indicating that circRAE1/miR-388-3p/TYRO3 axis exacerbated tumorigenesis of CRC by activating Wnt/β-catenin signaling pathway. In addition, overexpression of BTBD3 reduced CRC cell migration and invasion in vitro and inhibited tumor growth in vivo. Our data demonstrated that BTBD3 suppressed CRC progression through negative regulation of the circRAE1/miR-388-3p/TYRO3 axis and the Wnt/β-catenin pathway. Our data further confirmed that BTBD3 bound and ubiquitinated β-catenin and led to β-catenin degradation, therefore blocked the Wnt/β-catenin pathway and suppressed the CRC tumorigenesis. This study explored the mechanism of BTBD3 involved in CRC tumorigenesis and provided a new theoretical basis for the prevention and treatment of CRC.