Novel SK channel positive modulators prevent ferroptosis and excitotoxicity in neuronal cells.

Small conductance calcium-activated potassium (SK) channel activity has been proposed to play a role in the pathology of several neurological diseases. Besides regulating plasma membrane excitability, SK channel activation provides neuroprotection against ferroptotic cell death by reducing mitochondrial Ca2+ uptake and reactive oxygen species (ROS). In this study, we employed a multifaceted approach, integrating structure-based and computational techniques, to strategically design and synthesize an innovative class of potent small-molecule SK2 channel modifiers through highly efficient multicomponent reactions (MCRs). The compounds' neuroprotective activity was compared with the well-studied SK positive modulator, CyPPA. Pharmacological SK channel activation by selected compounds confers neuroprotection against ferroptosis at low nanomolar ranges compared to CyPPA, that mediates protection at micromolar concentrations, as shown by an MTT assay, real-time cell impedance measurements and propidium iodide staining (PI). These novel compounds suppress increased mitochondrial ROS and Ca2+ level induced by ferroptosis inducer RSL3. Moreover, axonal degeneration was rescued by these novel SK channel activators in primary mouse neurons and they attenuated glutamate-induced neuronal excitability, as shown via microelectrode array. Meanwhile, functional afterhyperpolarization of the novel SK2 channel modulators was validated by electrophysiological measurements showing more current change induced by the novel modulators than the reference compound, CyPPA. These data support the notion that SK2 channel activation can represent a therapeutic target for brain diseases in which ferroptosis and excitotoxicity contribute to the pathology.

Novel crosstalk mechanisms between GluA3 and Epac2 in synaptic plasticity and memory in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease which accounts for the most cases of dementia worldwide. Impaired memory, including acquisition, consolidation, and retrieval, is one of the hallmarks in AD. At the cellular level, dysregulated synaptic plasticity partly due to reduced long-term potentiation (LTP) and enhanced long-term depression (LTD) underlies the memory deficits in AD. GluA3 containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are one of key receptors involved in rapid neurotransmission and synaptic plasticity. Recent studies revealed a novel form of GluA3 involved in neuronal plasticity that is dependent on cyclic adenosine monophosphate (cAMP), rather than N-methyl-d-aspartate (NMDA). However, this cAMP-dependent GluA3 pathway is specifically and significantly impaired by amyloid beta (Aß), a pathological marker of AD. cAMP is a key second messenger that plays an important role in modulating memory and synaptic plasticity. We previously reported that exchange protein directly activated by cAMP 2 (Epac2), acting as a main cAMP effector, plays a specific and time-limited role in memory retrieval. From electrophysiological perspective, Epac2 facilities the maintenance of LTP, a cellular event closely associated with memory retrieval. Additionally, Epac2 was found to be involved in the GluA3-mediated plasticity. In this review, we comprehensively summarize current knowledge regarding the specific roles of GluA3 and Epac2 in synaptic plasticity and memory, and their potential association with AD.

Lipid profile in breast cancer: From signaling pathways to treatment strategies.

Breast cancer is the most prevalent cancer in women. Metabolic abnormalities, particularly increased lipid synthesis and uptake, impact the onset and progression of the disease. However, the influence of lipid metabolism in breast cancer varies according to the disease stage and patient's hormone status. In postmenopausal patients, obesity is associated with a higher risk and poor prognosis of luminal tumors, while in premenopausal individuals, it is correlated to BRCA mutated tumors. In fact, the tumor's lipid profile may be used to distinguish between HER2+, luminal and BRCA-mutated tumors. Moreover, drug resistance was associated with increased fatty acid synthesis and alterations in membrane composition, impacting its fluidity and spatial subdomains such as lipid rafts. Here, we discuss the subtype-specific lipid metabolism alterations found in breast cancer and the potentiality of its modulation in a clinical setting.

Negative modulation of mitochondrial calcium uniporter complex protects neurons against ferroptosis.

Ferroptosis is an iron- and reactive oxygen species (ROS)-dependent form of regulated cell death, that has been implicated in Alzheimer's disease and Parkinson's disease. Inhibition of cystine/glutamate antiporter could lead to mitochondrial fragmentation, mitochondrial calcium ([Ca2+]m) overload, increased mitochondrial ROS production, disruption of the mitochondrial membrane potential (ΔΨm), and ferroptotic cell death. The observation that mitochondrial dysfunction is a characteristic of ferroptosis makes preservation of mitochondrial function a potential therapeutic option for diseases associated with ferroptotic cell death. Mitochondrial calcium levels are controlled via the mitochondrial calcium uniporter (MCU), the main entry point of Ca2+ into the mitochondrial matrix. Therefore, we have hypothesized that negative modulation of MCU complex may confer protection against ferroptosis. Here we evaluated whether the known negative modulators of MCU complex, ruthenium red (RR), its derivative Ru265, mitoxantrone (MX), and MCU-i4 can prevent mitochondrial dysfunction and ferroptotic cell death. These compounds mediated protection in HT22 cells, in human dopaminergic neurons and mouse primary cortical neurons against ferroptotic cell death. Depletion of MICU1, a [Ca2+]m gatekeeper, demonstrated that MICU is protective against ferroptosis. Taken together, our results reveal that negative modulation of MCU complex represents a therapeutic option to prevent degenerative conditions, in which ferroptosis is central to the progression of these pathologies.

Unravelling the signaling power of pollutants.

Exposure to environmental pollutants contributes to diverse pathologies, including pulmonary disease, lower respiratory infections, cancer, and stroke. Pollutants' entry can occur through inhalation, traversing endothelial and epithelial barriers, and crossing the blood-brain barrier, leading to a wide distribution throughout the human body via systemic circulation. Pollutants cause cellular damage by multiple mechanisms encompassing oxidative stress, mitochondrial dysfunction, (neuro)inflammation, and protein instability/proteotoxicity. Sensing pollutants has added a new dimension to disease progression and drug failure. Understanding the molecular pathways and potential receptor binding/signaling that underpin 'sensing' could contribute to ways to combat the detrimental effects of pollutants. We highlight key points of pollutant signaling, crosstalk with receptors acting as drug targets for chronic diseases, and discuss the potential for future therapeutics.

Identification and characterization of a small molecule that activates thiosulfate sulfurtransferase and stimulates mitochondrial respiration.

The enzyme Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), is a positive genetic predictor of diabetes type 2 and obesity. As increased TST activity protects against the development of diabetic symptoms in mice, an activating compound for TST may provide therapeutic benefits in diabetes and obesity. We identified a small molecule activator of human TST through screening of an inhouse small molecule library. Kinetic studies in vitro suggest that two distinct isomers of the compound are required for full activation as well as an allosteric mode of activation. Additionally, we studied the effect of TST protein and the activator on TST activity through mitochondrial respiration. Molecular docking and molecular dynamics (MD) approaches supports an allosteric site for the binding of the activator, which is supported by the lack of activation in the Escherichia coli. mercaptopyruvate sulfurtransferase. Finally, we show that increasing TST activity in isolated mitochondria increases mitochondrial oxygen consumption.

Mitochondrial transplantation rescues neuronal cells from ferroptosis.

Ferroptosis is a type of oxidative cell death that can occur in neurodegenerative diseases and involves damage to mitochondria. Previous studies demonstrated that preventing mitochondrial dysfunction can rescue cells from ferroptotic cell death. However, the complexity of mitochondrial dysfunction and the timing of therapeutic interventions make it difficult to develop an effective treatment strategy against ferroptosis in neurodegeneration conditions. In this study, we explored the use of mitochondrial transplantation as a novel therapeutic approach for preventing ferroptotic neuronal cell death. Our data showed that isolated exogenous mitochondria were incorporated into both healthy and ferroptotic immortalized hippocampal HT-22 cells and primary cortical neurons (PCN). The mitochondrial incorporation was accompanied by increased metabolic activity and cell survival through attenuating lipid peroxidation and mitochondrial superoxide production. Further, the function of mitochondrial complexes I, III and V activities contributed to the neuroprotective activity of exogenous mitochondria. Similarly, we have also showed the internalization of exogenous mitochondria in mouse PCN; these internalized mitochondria were found to effectively preserve the neuronal networks when challenged with ferroptotic stimuli. The administration of exogenous mitochondria into the axonal compartment of a two-compartment microfluidic device induced mitochondrial transportation to the cell body, which prevented fragmentation of the neuronal network in ferroptotic PCN. These findings suggest that mitochondria transplantation may be a promising therapeutic approach for protecting neuronal cells from ferroptotic cell death.

The old second messenger cAMP teams up with novel cell death mechanisms: potential translational therapeutical benefit for Alzheimer's disease and Parkinson's disease.

Alzheimer's disease (AD) and Parkinson's disease (PD) represent the most prevalent neurodegenerative disorders severely impacting life expectancy and quality of life of millions of people worldwide. AD and PD exhibit both a very distinct pathophysiological disease pattern. Intriguingly, recent researches, however, implicate that overlapping mechanisms may underlie AD and PD. In AD and PD, novel cell death mechanisms, encompassing parthanatos, netosis, lysosome-dependent cell death, senescence and ferroptosis, apparently rely on the production of reactive oxygen species, and seem to be modulated by the well-known, "old" second messenger cAMP. Signaling of cAMP via PKA and Epac promotes parthanatos and induces lysosomal cell death, while signaling of cAMP via PKA inhibits netosis and cellular senescence. Additionally, PKA protects against ferroptosis, whereas Epac1 promotes ferroptosis. Here we review the most recent insights into the overlapping mechanisms between AD and PD, with a special focus on cAMP signaling and the pharmacology of cAMP signaling pathways.

The TNFR1 antagonist Atrosimab reduces neuronal loss, glial activation and memory deficits in an acute mouse model of neurodegeneration.

Tumor necrosis factor alpha (TNF-α) and its key role in modulating immune responses has been widely recognized as a therapeutic target for inflammatory and neurodegenerative diseases. Even though inhibition of TNF-α is beneficial for the treatment of certain inflammatory diseases, total neutralization of TNF-α largely failed in the treatment of neurodegenerative diseases. TNF-α exerts distinct functions depending on interaction with its two TNF receptors, whereby TNF receptor 1 (TNFR1) is associated with neuroinflammation and apoptosis and TNF receptor 2 (TNFR2) with neuroprotection and immune regulation. Here, we investigated the effect of administering the TNFR1-specific antagonist Atrosimab, as strategy to block TNFR1 signaling while maintaining TNFR2 signaling unaltered, in an acute mouse model for neurodegeneration. In this model, a NMDA-induced lesion that mimics various hallmarks of neurodegenerative diseases, such as memory loss and cell death, was created in the nucleus basalis magnocellularis and Atrosimab or control protein was administered centrally. We showed that Atrosimab attenuated cognitive impairments and reduced neuroinflammation and neuronal cell death. Our results demonstrate that Atrosimab is effective in ameliorating disease symptoms in an acute neurodegenerative mouse model. Altogether, our study indicates that Atrosimab may be a promising candidate for the development of a therapeutic strategy for the treatment of neurodegenerative diseases.

Collagen type I alters the proteomic signature of macrophages in a collagen morphology-dependent manner.

Idiopathic pulmonary fibrosis is a progressive lung disease that causes scarring and loss of lung function. Macrophages play a key role in fibrosis, but their responses to altered morphological and mechanical properties of the extracellular matrix in fibrosis is relatively unexplored. Our previous work showed functional changes in murine fetal liver-derived alveolar macrophages on fibrous or globular collagen morphologies. In this study, we applied differential proteomics to further investigate molecular mechanisms underlying the observed functional changes. Macrophages cultured on uncoated, fibrous, or globular collagen-coated plastic were analyzed by liquid chromatography-mass spectrometry. The presence of collagen affected expression of 77 proteins, while 142 were differentially expressed between macrophages grown on fibrous or globular collagen. Biological process and pathway enrichment analysis revealed that culturing on any type of collagen induced higher expression of enzymes involved in glycolysis. However, this did not lead to a higher rate of glycolysis, probably because of a concomitant decrease in activity of these enzymes. Our data suggest that macrophages sense collagen morphologies and can respond with changes in expression and activity of metabolism-related proteins. These findings suggest intimate interactions between macrophages and their surroundings that may be important in repair or fibrosis of lung tissue.

Extending the analogy between intracellular motion in mammalian cells and glassy dynamics.

How molecules, organelles, and foreign objects move within living cells has been studied in organisms ranging from bacteria to human cells. In mammalian cells, in particular, cellular vesicles move across the cell using motor proteins that carry the vesicle down the cytoskeleton to their destination. We have recently noted several similarities between the motion of such vesicles and that in disordered, "glassy", systems, but the generality of this observation remains unclear. Here we follow the motion of mitochondria, the organelles responsible for cell energy production, in mammalian cells over timescales from 50 ms to 70 s. Qualitative observations show that single mitochondria remain within a spatially limited region for extended periods of time, before moving longer distances relatively quickly. The displacement distribution is roughly Gaussian for shorter distances (≲0.05 µm) but exhibits exponentially decaying tails at longer distances (up to 0.40 µm). This behaviour is well-described by a model developed to describe the motion in glassy systems. These observations are extended to in total 3 different objects (mitochondria, lysosomes and nano-sized beads enclosed in vesicles), 3 different mammalian cell types (HEK 293, HeLa, and HT22), from 2 different organisms (human and mouse). Further evidence that supports glass-like characteristics of the motion is a difference between the time it takes to move a longer distance for the first time and subsequent times, as well as a weak ergodicity breaking of the motion. Overall, we demonstrate the ubiquity of glass-like motion in mammalian cells, providing a different perspective on intracellular motion.

Mitochondrial dysfunction mediates neuronal cell response to DMMB photodynamic therapy.

Photodynamic therapy (PDT) is a process in which a photosensitizer (PS) is exposed to specific wavelengths and generates reactive oxygen species (ROS) which act within nanometers. The low invasive nature and directed cytotoxicity of this approach render it attractive to the treatment of different conditions, including the ones that affect the central nervous system (CNS). The effect of PDT on healthy neurons is one main concern over its use in the CNS, since neuronal-like cells were shown to be particularly sensitive to certain PSs. Among available PSs, 1,9-dimethyl-methylene blue (DMMB) stands out as being resistant to reduction to its inactive leuco form and by being able to produce high levels of singlet­oxygen. In this study, we aimed to investigate DMMB photodamage mechanisms in the hippocampal cell line HT22. Our results demonstrate that DMMB-PDT decrease in cell viability was linked with an increase in cell death and overall ROS production. Besides, it resulted in a significant increase in mitochondrial ROS production and decreased mitochondria membrane potential. Furthermore, DMMB-PDT significantly increased the presence of acidic autolysosomes, which was accompanied by an increase in ATG1 and ATG8 homologue GaBarap1 expression, and decreased DRAM1 expression. Taken together our results indicated that mitochondrial and autophagic dysfunction underlie DMMB-PDT cytotoxicity in neuronal cells.

Characterization of Lipopolysaccharide Effects on LRRK2 Signaling in RAW Macrophages.

Dysfunction of the immune system and mitochondrial metabolism has been associated with Parkinson's disease (PD) pathology. Mutations and increased kinase activity of leucine-rich repeat kinase 2 (LRRK2) are linked to both idiopathic and familial PD. However, the function of LRRK2 in the immune cells under inflammatory conditions is contradictory. Our results showed that lipopolysaccharide (LPS) stimulation increased the kinase activity of LRRK2 in parental RAW 264.7 (WT) cells. In addition to this, LRRK2 deletion in LRRK2 KO RAW 264.7 (KO) cells altered cell morphology following LPS stimulation compared to the WT cells, as shown by an increase in the cell impedance as observed by the xCELLigence measurements. LPS stimulation caused an increase in the cellular reactive oxygen species (ROS) levels in both WT and KO cells. However, WT cells displayed a higher ROS level compared to the KO cells. Moreover, LRRK2 deletion led to a reduction in interleukin-6 (IL-6) inflammatory cytokine and cyclooxygenase-2 (COX-2) expression and an increase in lactate production after LPS stimulation compared to the WT cells. These data illustrate that LRRK2 has an effect on inflammatory processes in RAW macrophages upon LPS stimulation.

Chemical structure and biological activity of the (1 → 3)-linked ß-D-glucan isolated from marine diatom Conticribra weissflogii.

Several polysaccharides are considered to be "biological response modifiers" (BRM) - these refer to biomolecules that augment immune responses and can be derived from a variety of sources. Microalgae produce a diverse range of polysaccharides and could be an excellent source of BRM. Here, we describe the chemical structure and biological activity of water-soluble polysaccharide isolated from the marine diatom Conticribra weissflogii. Using chemical and NMR spectroscopic methods, the polysaccharide was identified as a (1 â†’ 3)-linked ß-D-glucan with a low proportion of C-6 substitution by single ß-glucose units. The biological activity of this low molecular weight ß-glucan (11.7 kDa) was investigated with respect to glioblastoma cell lines (U87 MG and U251) and macrophages (RAW 264.7). We observed that this ß-D-glucan did not exhibit cytotoxic activity against glioblastoma cells, but did enhance the phagocytic activity of macrophages, suggesting that it possesses immunomodulatory properties.

LRRK2 protects immune cells against erastin-induced ferroptosis.

Ferroptosis is an iron-dependent regulated cell death pathway characterized by excessive lipid peroxidation. It is implicated in many neurodegenerative diseases, including Parkinson's Disease (PD). Mutations and increased leucine-rich repeat kinase 2 (LRRK2) kinase activity are associated with both familial and idiopathic PD pathology. Increased iron deposition was observed in the substantia nigra of LRRK2 mutation-carrying PD patients compared to healthy individuals, suggesting a potential link between LRRK2 and ferroptosis. However, the role of LRRK2 in the immune cells is still not well-understood. This study aims to investigate the effect of LRRK2 on ferroptosis-induced cell death in immune cells. We used LRRK2 parental (WT) and LRRK2 KO (KO) RAW 264.7 murine macrophages. Cells were challenged with the ferroptosis inducer, erastin, and the kinase activity was investigated using the LRRK2 kinase inhibitor, MLi2. Cell metabolism and viability analysis showed that WT cells were more resistant to ferroptosis than the KO cells. Lipid peroxidation and cellular reactive oxygen species (ROS) generation were significantly elevated in the KO cells. Furthermore, mitochondrial membrane potential and mitochondrial respiration were decreased in the KO cells after erastin treatment compared to the WT cells. Inhibition of the LRRK2 kinase function resulted in increased cell sensitivity to erastin. Cell and mitochondrial substrates utilization were altered in the KO and kinase inhibited WT cells compared to WT cells. These results indicate a protective role of LRRK2 against erastin-induced ferroptosis in RAW macrophages and point towards the importance of LRRK2 kinase function in the protective mechanism.

The multifaceted role of LRRK2 in Parkinson's disease: From human iPSC to organoids.

Parkinson's disease (PD) is the second most common neurodegenerative disease affecting elderly people. Pathogenic mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the most common cause of autosomal dominant PD. LRRK2 activity is enhanced in both familial and idiopathic PD, thereby studies on LRRK2-related PD research are essential for understanding PD pathology. Finding an appropriate model to mimic PD pathology is crucial for revealing the molecular mechanisms underlying disease progression, and aiding drug discovery. In the last few years, the use of human-induced pluripotent stem cells (hiPSCs) grew exponentially, especially in studying neurodegenerative diseases like PD, where working with brain neurons and glial cells was mainly possible using postmortem samples. In this review, we will discuss the use of hiPSCs as a model for PD pathology and research on the LRRK2 function in both neuronal and immune cells, together with reviewing the recent advances in 3D organoid models and microfluidics.

Thiosulfate sulfurtransferase prevents hyperglycemic damage to the zebrafish pronephros in an experimental model for diabetes.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also found to be a genetic predictor of resistance to obesity-related type 2 diabetes. Diabetes type 2 is characterized by progressive loss of adequate ß-cell insulin secretion and onset of insulin resistance with increased insulin demand, which contributes to the development of hyperglycemia. Diabetic complications have been replicated in adult hyperglycemic zebrafish, including retinopathy, nephropathy, impaired wound healing, metabolic memory, and sensory axonal degeneration. Pancreatic and duodenal homeobox 1 (Pdx1) is a key component in pancreas development and mature beta cell function and survival. Pdx1 knockdown or knockout in zebrafish induces hyperglycemia and is accompanied by organ alterations similar to clinical diabetic retinopathy and diabetic nephropathy. Here we show that pdx1-knockdown zebrafish embryos and larvae survived after incubation with thiosulfate and no obvious morphological alterations were observed. Importantly, incubation with hTST and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros. Activation of the mitochondrial TST pathway might be a promising option for therapeutic intervention in diabetes and its organ complications.

Transcriptomic and epigenomic landscapes of Alzheimer's disease evidence mitochondrial-related pathways.

Alzheimers disease (AD) is the main cause of dementia and it is defined by cognitive decline coupled to extracellular deposit of amyloid-beta protein and intracellular hyperphosphorylation of tau protein. Historically, efforts to target such hallmarks have failed in numerous clinical trials. In addition to these hallmark-targeted approaches, several clinical trials focus on other AD pathological processes, such as inflammation, mitochondrial dysfunction, and oxidative stress. Mitochondria and mitochondrial-related mechanisms have become an attractive target for disease-modifying strategies, as mitochondrial dysfunction prior to clinical onset has been widely described in AD patients and AD animal models. Mitochondrial function relies on both the nuclear and mitochondrial genome. Findings from omics technologies have shed light on AD pathophysiology at different levels (e.g., epigenome, transcriptome and proteome). Most of these studies have focused on the nuclear-encoded components. The first part of this review provides an updated overview of the mechanisms that regulate mitochondrial gene expression and function. The second part of this review focuses on evidence of mitochondrial dysfunction in AD. We have focused on published findings and datasets that study AD. We analyzed published data and provide examples for mitochondrial-related pathways. These pathways are strikingly dysregulated in AD neurons and glia in sex-, cell- and disease stage-specific manners. Analysis of mitochondrial omics data highlights the involvement of mitochondria in AD, providing a rationale for further disease modeling and drug targeting.

Cytochrome c Oxidase Inhibition by ATP Decreases Mitochondrial ROS Production.

This study addresses the eventual consequence of cytochrome c oxidase (CytOx) inhibition by ATP at high ATP/ADP ratio in isolated rat heart mitochondria. Earlier, it has been demonstrated that the mechanism of allosteric ATP inhibition of CytOx is one of the key regulations of mitochondrial functions. It is relevant that aiming to maintain a high ATP/ADP ratio for the measurement of CytOx activity effectuating the enzymatic inhibition as well as mitochondrial respiration, optimal concentration of mitochondria is critically important. Likewise, only at this concentration, were the differences in ΔΨm and ROS concentrations measured under various conditions significant. Moreover, when CytOx activity was inhibited in the presence of ATP, mitochondrial respiration and ΔΨm both remained static, while the ROS production was markedly decreased. Consubstantial results were found when the electron transport chain was inhibited by antimycin A, letting only CytOx remain functional to support the energy production. This seems to corroborate that the decrease in mitochondrial ROS production is solely the effect of ATP binding to CytOx which results in static respiration as well as membrane potential.

Corrigendum: The Potential of Ferroptosis-Targeting Therapies for Alzheimer's Disease: From Mechanism to Transcriptomic Analysis.

[This corrects the article DOI: 10.3389/fnagi.2021.745046.].