Search Results (7)

The primary function of microglia is to maintain brain homeostasis. In neurodegenerative diseases like Alzheimer’s, microglia contribute to neurotoxicity and inflammation. In this study, we exposed neonatal murine primary microglial cultures to stimuli mimicking pathogens, injury, or toxins. Treatment with benzoyl ATP (bzATP) and lipopolysaccharide (LPS) triggered a coordinated increase in interleukin and chemokine expression. We analyzed statistically significant differentially expressed microRNAs (DEMs) at 3 and 8 h post-activation, identifying 33 and 57 DEMs, respectively. Notably, miR-155, miR-132, miR-3473e, miR-222, and miR-146b showed strong temporal regulation, while miR-3963 was sharply downregulated by bzATP. These DEMs regulate inflammatory pathways, including TNFα and NFκB signaling. We also examined the effect of ladostigil, a neuroprotective agent known to reduce oxidative stress and inflammation. At 8 h post-activation, ladostigil induced upregulation of anti-inflammatory miRNAs, such as miR-27a, miR-27b, and miR-23b. Our findings suggest that miRNA profiles reflect microglial responses to inflammatory cues and that ladostigil modulates these responses. This model of controlled microglial activation offers a powerful tool with which to study inflammation in the aging brain and the progression of neurodegenerative diseases. Full article

Treatment of aging rats for 6 months with ladostigil (1 mg/kg/day) prevented a decline in recognition and spatial memory and suppressed the overexpression of gene-encoding pro-inflammatory cytokines, TNFα, IL1β, and IL6 in the brain and microglial cultures. Primary cultures of mouse microglia stimulated by lipopolysaccharides (LPS, 0.75 µg/mL) and benzoyl ATPs (BzATP) were used to determine the concentration of ladostigil that reduces the secretion of these cytokine proteins. Ladostigil (1 × 10⁻¹¹ M), a concentration compatible with the blood of aging rats in, prevented memory decline and reduced secretion of IL1β and IL6 by ≈50%. RNA sequencing analysis showed that BzATP/LPS upregulated 25 genes, including early-growth response protein 1, (Egr1) which increased in the brain of subjects with neurodegenerative diseases. Ladostigil significantly decreased Egr1 gene expression and levels of the protein in the nucleus and increased TNF alpha-induced protein 3 (TNFaIP3), which suppresses cytokine release, in the microglial cytoplasm. Restoration of the aberrant signaling of these proteins in ATP/LPS-activated microglia in vivo might explain the prevention by ladostigil of the morphological and inflammatory changes in the brain of aging rats. Full article

The primary role of microglia is to maintain homeostasis by effectively responding to various disturbances. Activation of transcriptional programs determines the microglia’s response to external stimuli. In this study, we stimulated murine neonatal microglial cells with benzoyl ATP (bzATP) and lipopolysaccharide (LPS), and monitored their ability to release pro-inflammatory cytokines. When cells are exposed to bzATP, a purinergic receptor agonist, a short-lived wave of transcriptional changes, occurs. However, only combining bzATP and LPS led to a sustainable and robust response. The transcriptional profile is dominated by induced cytokines (e.g., IL-1α and IL-1β), chemokines, and their membrane receptors. Several abundant long noncoding RNAs (lncRNAs) are induced by bzATP/LPS, including Ptgs2os2, Bc1, and Morrbid, that function in inflammation and cytokine production. Analyzing the observed changes through TNF (Tumor necrosis factor) and NF-κB (nuclear factor kappa light chain enhancer of activated B cells) pathways confirmed that neonatal glial cells exhibit a distinctive expression program in which inflammatory-related genes are upregulated by orders of magnitude. The observed capacity of the microglial culture to activate a robust inflammatory response is useful for studying neurons under stress, brain injury, and aging. We propose the use of a primary neonatal microglia culture as a responsive in vitro model for testing drugs that may interact with inflammatory signaling and the lncRNA regulatory network. Full article

Neurodegenerative disorders, brain injury, and the decline in cognitive function with aging are accompanied by a reduced capacity of cells in the brain to cope with oxidative stress and inflammation. In this study, we focused on the response to oxidative stress in SH-SY5Y, a human neuroblastoma cell line. We monitored the viability of the cells in the presence of oxidative stress. Such stress was induced by hydrogen peroxide or by Sin1 (3-morpholinosydnonimine) that generates reactive oxygen and nitrogen species (ROS and RNS). Both stressors caused significant cell death. Our results from the RNA-seq experiments show that SH-SY5Y cells treated with Sin1 for 24 h resulted in 94 differently expressed long non-coding RNAs (lncRNAs), including many abundant ones. Among the abundant lncRNAs that were upregulated by exposing the cells to Sin1 were those implicated in redox homeostasis, energy metabolism, and neurodegenerative diseases (e.g., MALAT1, MIAT, GABPB1-AS1, NEAT1, MIAT, GABPB1-AS1, and HAND2-AS1). Another group of abundant lncRNAs that were significantly altered under oxidative stress included cancer-related SNHG family members. We tested the impact of ladostigil, a bifunctional reagent with antioxidant and anti-inflammatory properties, on the lncRNA expression levels. Ladostigil was previously shown to enhance learning and memory in the brains of elderly rats. In SH-SY5Y cells, several lncRNAs involved in transcription regulation and the chromatin structure were significantly induced by ladostigil. We anticipate that these poorly studied lncRNAs may act as enhancers (eRNA), regulating transcription and splicing, and in competition for miRNA binding (ceRNA). We found that the induction of abundant lncRNAs, such as MALAT1, NEAT-1, MIAT, and SHNG12, by the Sin1 oxidative stress paradigm specifies only the undifferentiated cell state. We conclude that a global alteration in the lncRNA profiles upon stress in SH-SY5Y may shift cell homeostasis and is an attractive in vitro system to characterize drugs that impact the redox state of the cells and their viability. Full article

A hallmark of the aging brain is the robust inflammation mediated by microglial activation. Pathophysiology of common neurodegenerative diseases involves oxidative stress and neuroinflammation. Chronic treatment of aging rats by ladostigil, a compound with antioxidant and anti-inflammatory function, prevented microglial activation and learning deficits. In this study, we further investigate the effect of ladostigil on undifferentiated SH-SY5Y cells. We show that SH-SY5Y cells exposed to acute (by H₂O₂) or chronic oxidative stress (by Sin1, 3-morpholinosydnonimine) induced apoptotic cell death. However, in the presence of ladostigil, the decline in cell viability and the increase of oxidative levels were partially reversed. RNA-seq analysis showed that prolonged oxidation by Sin1 resulted in a simultaneous reduction of the expression level of endoplasmic reticulum (ER) genes that participate in proteostasis. By comparing the differential gene expression profile of Sin1 treated cells to cells incubated with ladostigil before being exposed to Sin1, we observed an over-expression of Clk1 (Cdc2-like kinase 1) which was implicated in psychophysiological stress in mice and Alzheimer’s disease. Ladostigil also suppressed the expression of Ccpg1 (Cell cycle progression 1) and Synj1 (Synaptojanin 1) that are involved in ER-autophagy and endocytic pathways. We postulate that ladostigil alleviated cell damage induced by oxidation. Therefore, under conditions of chronic stress that are observed in the aging brain, ladostigil may block oxidative stress processes and consequently reduce neurotoxicity. Full article

MicroRNAs (miRNAs) act as negative regulators of gene expression in the cytoplasm. Previous studies have identified the presence of miRNAs in the nucleus. Here we study human breast cancer-derived cell-lines (MCF-7 and MDA-MB-231) and a non-tumorigenic cell-line (MCF-10A) and compare their miRNA sequences at the spliceosome fraction (SF). We report that the levels of miRNAs found in the spliceosome, their identity, and pre-miRNA segmental composition are cell-line specific. One such miRNA is miR-7704 whose genomic position overlaps HAGLR, a cancer-related lncRNA. We detected an inverse expression of miR-7704 and HAGLR in the tested cell lines. Specifically, inhibition of miR-7704 caused an increase in HAGLR expression. Furthermore, elevated levels of miR-7704 slightly altered the cell-cycle in MDA-MB-231. Altogether, we show that SF-miR-7704 acts as a tumor-suppressor gene with HAGLR being its nuclear target. The relative levels of miRNAs found in the spliceosome fractions (e.g., miR-100, miR-30a, and let-7 family) in non-tumorigenic relative to cancer-derived cell-lines was monitored. We found that the expression trend of the abundant miRNAs in SF was different from that reported in the literature and from the observation of large cohorts of breast cancer patients, suggesting that many SF-miRNAs act on targets that are different from the cytoplasmic ones. Altogether, we report on the potential of SF-miRNAs as an unexplored route for cancerous cell state. Full article

ClanTox (classifier of animal toxins) was developed for identifying toxin-like candidates from complete proteomes. Searching mammalian proteomes for short toxin-like proteins (coined TOLIPs) revealed a number of overlooked secreted short proteins with an abundance of cysteines throughout their sequences. We applied bioinformatics and data-mining methods to infer the function of several top predicted candidates. We focused on cysteine-rich peptides that adopt the fold of the three-finger proteins (TFPs). We identified a cluster of duplicated genes that share a structural similarity with elapid neurotoxins, such as α-bungarotoxin. In the murine proteome, there are about 60 such proteins that belong to the Ly6/uPAR family. These proteins are secreted or anchored to the cell membrane. Ly6/uPAR proteins are associated with a rich repertoire of functions, including binding to receptors and adhesion. Ly6/uPAR proteins modulate cell signaling in the context of brain functions and cells of the innate immune system. We postulate that TOLIPs, as modulators of cell signaling, may be associated with pathologies and cellular imbalance. We show that proteins of the Ly6/uPAR family are associated with cancer diagnosis and malfunction of the immune system. Full article