Ladostigil Reduces the Adenoside Triphosphate/Lipopolysaccharide-Induced Secretion of Pro-Inflammatory Cytokines from Microglia and Modulate-Immune Regulators, TNFAIP3, and EGR1.

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, IL1ß, 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 IL1ß 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.

Coordinated Transcriptional Waves Define the Inflammatory Response of Primary Microglial Culture.

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-1ß), 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.

Challenging Cellular Homeostasis: Spatial and Temporal Regulation of miRNAs.

Mature microRNAs (miRNAs) are single-stranded non-coding RNA (ncRNA) molecules that act in post-transcriptional regulation in animals and plants. A mature miRNA is the end product of consecutive, highly regulated processing steps of the primary miRNA transcript. Following base-paring of the mature miRNA with its mRNA target, translation is inhibited, and the targeted mRNA is degraded. There are hundreds of miRNAs in each cell that work together to regulate cellular key processes, including development, differentiation, cell cycle, apoptosis, inflammation, viral infection, and more. In this review, we present an overlooked layer of cellular regulation that addresses cell dynamics affecting miRNA accessibility. We discuss the regulation of miRNA local storage and translocation among cell compartments. The local amounts of the miRNAs and their targets dictate their actual availability, which determines the ability to fine-tune cell responses to abrupt or chronic changes. We emphasize that changes in miRNA storage and compactization occur under induced stress and changing conditions. Furthermore, we demonstrate shared principles on cell physiology, governed by miRNA under oxidative stress, tumorigenesis, viral infection, or synaptic plasticity. The evidence presented in this review article highlights the importance of spatial and temporal miRNA regulation for cell physiology. We argue that limiting the research to mature miRNAs within the cytosol undermines our understanding of the efficacy of miRNAs to regulate cell fate under stress conditions.

Oxidative Stress and Its Modulation by Ladostigil Alter the Expression of Abundant Long Non-Coding RNAs in SH-SY5Y Cells.

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.

Turning Data to Knowledge: Online Tools, Databases, and Resources in microRNA Research.

MicroRNAs (miRNAs) provide a fundamental layer of regulation in cells. miRNAs act posttranscriptionally through complementary base-pairing with the 3'-UTR of a target mRNA, leading to mRNA degradation and translation arrest. The likelihood of forming a valid miRNA-target duplex within cells was computationally predicted and experimentally monitored. In human cells, the miRNA profiles determine their identity and physiology. Therefore, alterations in the composition of miRNAs signify many cancer types and chronic diseases. In this chapter, we introduce online functional tools and resources to facilitate miRNA research. We start by introducing currently available miRNA catalogs and miRNA-gateway portals for navigating among different miRNA-centric online resources. We then sketch several realistic challenges that may occur while investigating miRNA regulation in living cells. As a showcase, we demonstrate the utility of miRNAs and mRNAs expression databases that cover diverse human cells and tissues, including resources that report on genetic alterations affecting miRNA expression levels and alteration in binding capacity. Introducing tools linking miRNAs with transcription factor (TF) networks reveals miRNA regulation complexity within living cells. Finally, we concentrate on online resources that analyze miRNAs in human diseases and specifically in cancer. Altogether, we introduce contemporary, selected resources and online tools for studying miRNA regulation in cells and tissues and their utility in health and disease.

Ladostigil Attenuates Induced Oxidative Stress in Human Neuroblast-like SH-SY5Y Cells.

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.

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2.

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

Facilitating In Situ Cross-Linking and Mass Spectrometry by Antibody-Based Protein Enrichment.

Cross-linking of living cells followed by mass spectrometry identification of cross-linked peptides (in situ CLMS) is an emerging technology to study protein structures in their native environment. One of the inherent difficulties of this technology is the high complexity of the samples following cell lysis. Currently, this difficulty largely limits the identification of cross-links to the more abundant proteins in the cell. Here, we describe a targeted approach in which an antibody is used to purify a specific protein-of-interest out of the cell lysate. Mass spectrometry analysis of the protein material that binds to the antibody can then identify considerably more cross-links on the target protein. By using an antibody against the CCT chaperonin, we identified over 200 cross-links that provide in situ evidence for the subunit arrangement of the CCT particle and its interactions with prefoldin. Similar targeting with an antibody against tubulin provided in situ evidence for the structure of the microtubule. Finally, the approach was also successful in identifying cross-links within a protein that expresses at a low level. These results demonstrate the general utility of antibody-based sample simplification for in situ CLMS and greatly expand the scope of protein systems that are amenable to in situ structural studies.

Spliceosome-Associated microRNAs Signify Breast Cancer Cells and Portray Potential Novel Nuclear Targets.

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.