Search Results (109)

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by cerebellar ataxia and retinal degeneration, caused by an abnormal expansion of CAG repeats at the ATXN7 gene. Disease onset and progression vary among patients, underscoring the need for novel tools to improve disease monitoring. Circulating miRNAs represent a promising prognostic tool, due to their minimally invasive sampling and high stability. The aim of this study was to assess the expression of twelve circulating miRNAs associated with neurodegeneration in plasma samples from SCA7 patients and in an inducible SCA7 glial cell model. A comparison of SCA7 patients and controls revealed that nine miRNAs exhibited significantly higher expression. Furthermore, comparison of patients with different SCA7 phenotypes to controls revealed that most miRNAs were overexpressed in plasma from early-onset patients corresponding to the clinically more severe phenotype. Regarding the cell model, we identified three miRNAs that were dysregulated; however, only hsa-miR-342-5p displayed a pattern consistent with that observed in the plasma of patient. Our findings indicate that hsa-miR-342-5p is differentially expressed in the plasma of patients and the SCA7 cellular model, implying that it can serve as a biomarker and facilitate the identification of novel processes involved in SCA7. Full article

Background: Atherosclerosis is the pathological basis for lethal cardio-cerebral vascular diseases, such as coronary artery disease and stroke. Fructus Choerospondiatis (FC) has demonstrated cardiac protective effects in multiple ethnomedicine. Whether these protective effects are attributed to the prevention of vascular atherosclerosis, however, remains unknown. We aim to examine the anti-atherosclerotic effect of FC aqueous extract and elucidate the underlying mechanism. Methods: FC was separated into peel and pulp, and the aqueous extract was obtained separately by boiling in water to mimic decocting. Atherosclerosis model was established in ApoE^−/− mice fed with a high-fat diet, and histological analysis were utilized to evaluate the development of atherosclerosis. Various inflammatory models were constructed in mice to evaluate the anti-inflammatory effect of FC extract systemically, including acute local inflammation induced by traumatic injury (ear/foot swelling), acute systemic inflammation triggered by pathogenic infection (LPS- and POLY (I:C)-induced), as well as chronic inflammatory conditions associated with oxidative stress (D-galactose-induced), metabolic disorder (db/db mice), and aging. LC-MS and network pharmacology identified bioactive components and targets. Western blotting, ELISA, qPCR, and immunofluorescence were utilized to analyze the key genes involved in the mechanisms. Results: FC peel extract reduced serum IL-6 level, atherosclerotic plaque area, and macrophage content in the plaque, while pulp extract showed no protective effects. Peel extract exhibits anti-inflammatory effects in all models. The integrative application of LC-MS and network pharmacology identified ellagic acid as the major bioactive component and AKT as its target protein. Mechanistically, FC peel extract inhibits AKT phosphorylation, suppresses c-FOS expression and nuclear translocation, reduces IL-6 transcription and inflammation, and thus alleviates atherosclerosis. Conclusions: FC peel aqueous extract exerts anti-atherosclerotic effect by inhibiting inflammation through AKT/c-FOS/IL-6 axis. This study provides novel insights into the protective effects against atherosclerosis of FC peel and highlights its potential application in the prevention and treatment of coronary artery diseases. Full article

Background: Breast cancer remains the most common malignancy among women worldwide. Current systemic treatment strategies include chemotherapy, immunotherapy, bone-stabilizing agents, endocrine therapy for hormone receptor-positive disease, anti-HER2 therapy for HER2-positive disease, and poly (ADP-ribose) polymerase (PARP) inhibitors for BRCA mutation cases. However, effectively overcoming drug resistance and reducing recurrence and metastasis rates remain major therapeutic challenges. Methods: To investigate the underlying mechanism of RSL3 in PARPi-resistant breast cancer cells, we treated several PARPi-resistant breast cancer cells with varying doses of RSL3. The regulatory proteins of STAT3 were analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Immunoprecipitation and ubiquitination assay were performed to identify the STAT3 ubiquitination levels. Results: Recently, we identified that RSL3, a ferroptosis activator, exhibits potent antitumor activity against PARPi-resistant breast cancer. Yet, its underlying mechanism remains unclear. Here, we demonstrate that RSL3 directly targets STAT3 and promotes its degradation via the ubiquitination pathway, leading to increased LC3-II levels and decreased p62 expression. These changes ultimately enhance autophagy, which at least partially contributes to elevated apoptosis. Rescue experiments confirmed that STAT3 overexpression reverses RSL3-induced autophagy and apoptosis. Conclusions: Our findings highlight RSL3 as a promising therapeutic agent and STAT3 as a potential target for treating PARPi-resistant breast cancer. Full article

Ataxin-2 (Atx2) is a general RNA-binding protein involved in processes such as RNA processing and metabolism in cells. Atx2 is also a polyglutamine (polyQ) tract-containing protein; its abnormal expansion can lead to protein aggregation associated with neurodegenerative diseases. Previous studies have shown that the C-terminal intrinsically disordered regions (c-IDRs) of Atx2 participate in its condensation and aggregation processes. To elucidate the role of polyQ expansion in biomolecular condensation and aggregation, we studied the N-terminal fragments of Atx2 (namely, Atx2-N317 and Atx2-N81) that preserve a polyQ tract and compared their molecular behaviors in cells to those of the full-length Atx2. We found that the molecular mobility of the N-terminal fragments decreases with the increasing length of polyQ, indicating that polyQ expansion promotes a gradual phase transition to an irreversible and insoluble state. Moreover, the molecular state and mobility of Atx2-N317 are not distinct from those of Atx2-N81, regardless of the presence of other domains, demonstrating that the polyQ tract is a direct and sufficient element for protein condensation and aggregation, while the Like Sm (LSm) and LSm-associated (LSmAD) domains and their interactions with RNA are not necessary for these processes. This result is also validated through the in vitro investigation of Atx2-N81 with different polyQ expansions. This study reveals that polyQ expansion controls the biomolecular condensation and aggregation of the N-terminal fragments of Atx2 and is thus thought to modulate the dynamic behaviors of the full-length protein as well, which is implicated in the pathological accumulation of Atx2 in cells. Full article

Huntingtin (HTT) is a large, ubiquitously expressed scaffold protein that participates in multiple cellular processes, including vesicular transport, transcriptional regulation, and energy metabolism. The mutant form of HTT (mHTT), characterized by an abnormal polyglutamine (polyQ) expansion in its N-terminal region, is the causative agent of Huntington’s disease (HD), a progressive neurodegenerative disorder. Current therapeutic efforts for HD have primarily focused on lowering HTT levels through gene silencing or promoting mHTT degradation. However, accumulating evidence suggests that post-translational modifications (PTMs) of HTT—such as phosphorylation, ubiquitination, acetylation, and SUMOylation—play pivotal roles in modulating HTT’s conformation, aggregation propensity, subcellular localization, and degradation pathways. These modifications regulate the balance between HTT’s physiological functions and pathological toxicity. Importantly, dysregulation of PTMs has been linked to mHTT accumulation and selective neuronal vulnerability, highlighting their relevance as potential therapeutic targets. A deeper understanding of how individual PTMs and their crosstalk regulate HTT homeostasis may not only provide mechanistic insights into HD pathogenesis but also uncover novel, more specific strategies for intervention. In this review, we summarize recent understanding on HTT PTMs, discuss their implications for disease modification, and outline critical knowledge gaps that remain to be addressed. Full article

Several genetic diseases affecting the human nervous system are incurable and insufficiently understood. Among them, nine rare diseases form the polyglutamine (polyQ) family: Huntington’s disease (HD), spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17, dentatorubral pallidoluysian atrophy, and spinal and bulbar muscular atrophy. In most patients, these diseases progress over decades to cause severe movement incoordination and neurodegeneration. Although their inherited genes with tandem-repeat elongations and the encoded polyQ-containing proteins have been extensively studied, the neuronal-type-specific pathologies and their long pre-symptomatic latency await further investigations. However, recent advances in detecting the single-nucleus transcriptome, alongside the length of tandem repeats in HD post-mortem brains, have enabled the identification of very high CAG repeat sizes that trigger transcriptional dysregulation and cell death in specific projection neurons. One challenge is to better understand the complexity of movement coordination circuits, including the basal ganglia and cerebellum neurons, which are most vulnerable to the high CAG expansion in each disease. Another challenge is to detect dynamic changes in CAG repeat length and their effects in vulnerable neurons at single-cell resolution. This will offer a platform for identifying pathological events in vulnerable long projection neurons and developing targeted therapies for all tandem-repeat expansions affecting the CNS projection neurons. Full article

The retinal pigment epithelium (RPE) is strongly involved in the pathogenesis of several retinal diseases, such as age-related macular degeneration (AMD). RPE models addressing specific pathological pathways are of high importance for understanding cellular pathomechanisms and pre-clinical screening of potential new therapeutics. The goal of this study is to establish standard operation protocols for single-eye porcine RPE preparation for AMD-relevant models of oxidative stress (RPE-Ox) and inflammation (RPE-Inf). Porcine primary RPE were prepared from one eye and seeded into one well of 12-well plates or, for polar differentiation, in transwell inserts. Different coatings (Poly-ᴅ-Lysine and laminin) and serum content of media (10%, 5%, and 1%) were tested to determine optimal culture parameters. For RPE-Ox, cells were treated with NaIO₃, CoCl₂, or erastin; cell viability (thiazolyl blue tetrazolium bromide, MTT), and gene expression (RT-qPCR) were determined. For RPE-Inf, cells were treated with lipopolysaccharide (LPS), polyinosinic/polycytidylic acid (Poly I:C), or tumor necrosis factor alpha (TNF-α); cell viability (MTT), cytokine secretion (ELISA), and gene expression (RT-qPCR) were determined. For transwell plates in RPE-Inf, cell viability (MTT), polar cytokine secretion (ELISA), gene expression (RT-qPCR), and transepithelial electrical resistance (TEER) for barrier assessment were conducted. For RPE-Ox, effective LD₅₀ could be achieved by using 24 h stimulation with 25 µm erastin, seven days after preparation in 5% serum cultures, without coating. For gene expression assessment, the use of Poly-ᴅ-Lysine is recommended. For RPE-Inf, three days of LPS stimulation (1 µg/mL) showed effective cytokine activation with 5% serum on uncoated 12-well plates. Transwell plates are not recommended for cytokine secretion assessment. It can be used for cell barrier assays in which LPS also showed effective cell barrier decrease and gene expression assays. Two specific best practice protocols for the use of porcine single-eye cultures in AMD research concerning oxidative stress and inflammation with optimized parameters were established and are provided. Full article

Huntington’s Disease (HD) originates from the expansion of a polyglutamine (PolyQ) tract in the huntingtin protein (Htt), which can assume a coiled-coil fold (Cc). We previously found that Cc structures mediate the aggregation and toxicity of polyQ Htt. Since polyQ Htt aggregates were previously found to be internalized by cells, here we hypothesize that Cc structures might be implicated in the intercellular propagation of Htt aggregates. To test this hypothesis, we performed experiments using human cell lines expressing Htt proteins with different probabilities to acquire a Cc fold. We found that Htt with reduced Cc structures were released significantly less compared to Htt with intact Cc structures. We also found that Cc structures mediate the internalization of Htt proteins in recipient cells. Together, these results underline the importance of the Cc structure in the process of intercellular propagation of Htt polyQ aggregates and suggest that interfering with Cc formation might be a therapeutic strategy for HD. Full article

Sjögren’s disease (SjD) is an autoimmune disease of exocrine tissues. Prior research has shown that ETS proto-oncogene 1 (ETS1), STAT1, and IL33 may contribute to the disease’s pathology. However, the regulatory mechanisms of these genes remain poorly characterized. Our objective was to explore the mechanisms of SjD pathology and to identify dysfunctional regulators of these genes by immunostimulation of SjD and sicca relevant cell lines. We used immortalized salivary gland epithelial cell lines (iSGECs) from Sjögren’s disease (pSS1) and sicca (nSS2) patients, previously developed in our lab, and control cell line A253 to dose with immunostimulants IFN-γ or poly(I:C) (0 to 1000 ng/mL and 0 to 1000 µg/mL, respectively) over a 72 h time course. Gene expression was determined using qRT-PCR delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for mRNA and U6 small nuclear RNA 1 (U6) for miRNA, using normalized relative fold changes 48 h post-immunostimulation. Protein expression was quantified 72 h post-stimulation by Western blotting. Reference-based RNA-seq of immunostimulated pSS1 and nSS2 cells was performed to characterize the reactome of genes conserved across all used doses. The expression of ETS1 and STAT1 protein was upregulated (p < 0.05) in IFN-γ-treated pSS1 and nSS2, as compared to A253 cells. IFN-γ-treated nSS2 cell showed significant IL33 upregulation. Also, IL33 had a correlated (p < 0.01) U-shaped response for low-mid-range doses for IFN-γ- and poly(I:C)-treated pSS1 cells. RNA-seq showed 175 conserved differentially expressed (DE) genes between nSS2 and pSS1 immunostimulated cells. Of these, 44 were shown to interact and 39 were more abundant (p < 0.05) in pSS1 cells. Western blotting demonstrated nSS2 cells expressing ETS1 uniformly across treatments compared to pSS1 cells, despite similar mRNA abundance. miR-145b and miR-193b were significantly under-expressed in IFN-γ-treated nSS2 cells compared to pSS1 cells (p < 0.01). ETS1 and IL33 showed disproportionate mRNA and protein abundances between immunostimulated Sjögren’s disease-derived (pSS1), and sicca-derived (nSS2) cell lines. Such differences could be explained by higher levels of miR-145b and miR-193b present in pSS1 cells. Also, RNA-seq results suggested an increased sensitivity of pSS1 cells to immunostimulation. These results reflect current pathobiology aspects, confirming the relevance of immortalized salivary gland epithelial cell lines. Full article

Background: Huntington’s Disease (HD) is a neurodegenerative disorder caused by an abnormal extension of a CAG repeat stretch located in the exon 1 of the HTT (IT15) gene, leading to production of a mutated and misfolded Huntingtin protein (muHTT) with an abnormally elongated polyglutamine (polyQ) region. This mutation causes muHTT to oligomerize and aggregate in the brain, particularly in the striatum and cortex, causing alterations in intracellular trafficking, caspase activation, and ganglioside metabolism, ultimately leading to neuronal damage and death and causing signs and symptoms such as chorea and cognitive dysfunction. Currently, there is no available cure for HD patients; hence, there is a strong need to look for effective therapies. Methods: This study aims to investigate the efficacy of siRNA-containing nano-engineered lipopolymers in selectively silencing the HTT expression in a neuronal model expressing a chimeric protein formed by the human mutated exon 1 of the HTT gene, tagged with GFP. Toxicity of lipopolymers was assessed using MTT assay, while efficacy of silencing was monitored using qRT-PCR, as well as Western blotting/flow cytometry. Changes in muHTT-GFP aggregation were observed using fluorescence microscopy and image analyses. Results: Here, we show that engineered lipopolymers can be used as delivery vehicles for specific siRNAs, decreasing the transcription of the mutated gene, as well as the muHTT protein production and aggregation, with Leu-Fect C being the most effective candidate amongst the assessed lipopolymers. Conclusions: Our findings have profound implications for genetic disorder therapies, highlighting the potential of nano-engineered materials for silencing mutant genes and facilitating molecular transfection across cellular barriers. This successful in vitro study paves the way for future in vivo investigations with preclinical models, offering hope for previously considered incurable diseases such as HD. Full article

Huntington disease’s (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine region (PolyQ) within the huntingtin protein (HTT). Mutated huntingtin (mHTT) is cytotoxic, particularly for striatal medium spiny neurons (MSNs), whose degeneration is the hallmark of HD. Autophagy inducers currently available promote the clearance of toxic proteins. However, due to their low selectivity and the possibility that prolonged autophagy hampers essential processes in unaffected cells, researchers have questioned their benefits in neurodegenerative diseases. Since MSNs express dopamine receptors D2 (DRD2) and D3 (DRD3) and DRD2/DRD3 agonists may activate autophagy, here, we explored how healthy and mHTT-challenged cells respond to prolonged DRD2/DRD3 agonist treatment. Autophagy activation and its effects on mHTT/polyQ clearance were studied in R6/1 mice (a genetic model of HD), their wild-type littermates, and DRD2- and DRD3-HEK cells expressing a pathogenic (Q74) and a non-pathogenic (Q23) polyQ fragment of mHTT treated with the DRD2/DRD3 agonist pramipexole. Two forms of DRD3-mediated autophagy were found: a transient mTORC1-dependent in WT mice and Q23-DRD3-HEK cells and a persistent AMPK-ULK1-activated in R6/1 mice and Q74-DRD3-HEK cells. This also promoted a robust clearance of soluble mHTT/polyQ and neuroprotection in striatal neurons and DRD3-HEK cells. The findings indicate that DRD3-induced autophagy may be a safe, disease-modifying intervention in HD patients. Full article

This study investigates the role of the Opnod1, Opnod2, and Optbk1 genes in antiviral and antibacterial immunity of spotted knifejaw (Oplegnathus punctatus). The expression patterns of these genes were analyzed using qRT-PCR in different tissues and at different time points. The open reading frame (ORF) of the Opnod1 gene was 2757 bp in length and encoded 918 amino acids, the ORF of the Opnod2 gene was 2970 bp in length and encoded 990 amino acids, while the Optbk1 gene was 2172 bp in length and encoded 723 amino acids. The Opnod1 and Opnod2 proteins contained three conserved domains (CARD, NOD, and LRR), and Optbk1 contained an STKc domain. The Opnod1, Opnod2, and Optbk1 genes were mainly expressed in immune-related tissues of spotted knifejaw, with the highest relative expression of the Opnod1 in the skin, the Opnod2 in the gill, and the Optbk1 in the liver. The expression of these genes changed significantly in the immune tissues following infection with SKIV-SD and Vibrio harveyi. In kidney cells, the Opnod1, Opnod2, and Optbk1 expression was up-regulated after stimulation by poly I:C and LPS in vitro. The results suggest that the NOD1/2-TBK1 signal pathway may play an important role in the resistance of the spotted knifejaw to virus and bacteria, providing valuable insights for disease-resistant breeding. Full article

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease characterized by cerebellar ataxia and retinal degeneration, caused by an abnormal expansion of the CAG trinucleotide in the coding region of the ATXN7 gene. Currently, in silico analysis is used to explore mechanisms and biological processes through bioinformatics predictions in various neurodegenerative diseases. Therefore, the aim of this study was to identify candidate human gene targets of four miRNAs (hsa-miR-29a-3p, hsa-miR-132-3p, hsa-miR-25-3p, and hsa-miR-92a-3p) involved in pathways that could play an important role in SCA7 pathogenesis through comprehensive in silico analysis including the prediction of miRNA target genes, Gen Ontology enrichment, identification of core genes in KEGG pathways, transcription factors and validated miRNA target genes with the mouse SCA7 transcriptome data. Our results showed the participation of the following pathways: adherens junction, focal adhesion, neurotrophin signaling, endoplasmic reticulum processing, actin cytoskeleton regulation, RNA transport, and apoptosis and dopaminergic synapse. In conclusion, unlike previous studies, we highlight using a bioinformatics approach the core genes and transcription factors involved in the different biological pathways and which ones are targets for the four miRNAs, which, in addition to being associated with neurodegenerative diseases, are also de-regulated in the plasma of patients with SCA7. Full article

Dysfunctional mitochondria are present in many neurodegenerative diseases, such as spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease (MJD). SCA3/MJD, the most frequent neurodegenerative ataxia worldwide, is caused by the abnormal expansion of the polyglutamine tract (polyQ) at ataxin-3. This protein is known to deubiquitinate key proteins such as Parkin, which is required for mitophagy. Ataxin-3 also interacts with Beclin1 (essential for initiating autophagosome formation adjacent to mitochondria), as well as with the mitochondrial cristae protein TBK1. To identify other proteins of the mitophagy pathway (according to the KEGG database) that can interact with ataxin-3, here we developed a pipeline for in silico analyses of protein–protein interactions (PPIs), called auto-p2docking. Containerized in Docker, auto-p2docking ensures reproducibility and reduces the number of errors through its simplified configuration. Its architecture consists of 22 modules, here used to develop 12 protocols but that can be specified according to user needs. In this work, we identify 45 mitophagy proteins as putative ataxin-3 interactors (53% are novel), using ataxin-3 interacting regions for validation. Furthermore, we predict that ataxin-3 interactors from both Parkin-independent and -dependent mechanisms are affected by the polyQ expansion. Full article

It is known that a number of neurodegenerative diseases, also called diseases of waiting, are associated with the expansion of the polyQ tract in the first exon of the ATXN2 gene. In the expanded polyQ tract, the probability of occurrence of non-canonical configurations (hairpins, G-quadruplexes, etc.) is significantly higher than in the normal one. Obviously, for their formation, the occurrence of open states (OSs) is necessary. Calculations were made for these processes using the angular mechanical model of DNA. It has been established that the probability of the large OS zones genesis in a DNA segment depends not only on the “strength” of the nucleotide sequence but also on the factors determining the dynamics of DNA; localization of the energy in the DNA molecule and the potential energy of interaction between pairs of nitrogenous bases also depend on environmental parameters. The potential energy of hydrogen bonds does not remain constant, and oscillatory movements lead to its redistribution and localization. In this case, OSs effectively dissipate the energy of oscillations. Thus, mathematical modeling makes it possible to calculate the localization of mechanical energy, which is necessary for the OSs formation, and to predict the places of their origin, taking into account the mechanical oscillations of the DNA molecule. Full article