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A study of the distribution of two fluorescent phospholipid analogs in an acetylcholine receptor (AChR)-rich membrane in Torpedo marmorata shows that 95% of NBD-phosphatidylcholine (NBD-PC) and rhodamine-phosphatidylethanolamine (Rho-PE) can be induced into AChR-rich membranes and liposomes. The experimental technique involves a combination of nonradiative fluorescence resonance energy transfer and quenching. The quenching experiments show that NBD-PC and Rho-PE are preferentially located on the same exofacial leaflet of the bilayer and attain high energy transfer efficiencies.

Coenzyme Q is one of the most reactive molecules against hydroxyl radicals present in mammalian cells. Theoretical studies indicate that the reaction of ubiquinol and ubiquinone with hydroxyl radicals leads to intermediate states of coenzyme Q that should have different reduction potentials.

Cytochrome b reductase (C bR) and cytochrome b (C b) form an enzymatic redox system that plays many roles in mammalian cells. In the last 15 years, it has been proposed that this system is involved in the recycling of ascorbate, a vital antioxidant molecule in the brain and that its deregulation can lead to the production of reactive oxygen species that play a major role in oxidative-induced neuronal death. In this work, we have performed a regional and cellular distribution study of the expression of this redox system in adult rat brain by anti-C bR isoform 3 and anti-C b antibodies. We found high expression levels in cerebellar cortex, labeling heavily granule neurons and Purkinje cells, and in structures such as the fastigial, interposed and dentate cerebellar nuclei. A large part of C bR isoform 3 in the cerebellum cortex was regionalized in close proximity to the lipid raft-like nanodomains, labeled with cholera toxin B, as we have shown by fluorescence resonance energy transfer imaging. In addition, vestibular, reticular and motor nuclei located at the brain stem level and pyramidal neurons of somatomotor areas of the brain cortex and of the hippocampus have been also found to display high expression levels of these proteins. All these results point out the enrichment of C bR isoform 3/C b system in neuronal cells and structures of the cerebellum and brain stem whose functional impairment can account for neurological deficits reported in type II congenital methemoglobinemia, as well as in brain areas highly prone to undergo oxidative stress-induced neurodegeneration. [ABSTRACT FROM AUTHOR]

The Preyssler-type polyoxotungstate ({P 5 W 30 }) belongs to the family of polyanionic metal-oxides formed by group V and VI metal ions, such as V, Mo and W, commonly known as polyoxometalates (POMs). POMs have demonstrated inhibitory effect on a significant number of ATP-binding proteins in vitro. Purinergic P2 receptors, widely expressed in eukaryotic cells, contain extracellularly oriented ATP-binding sites and play many biological roles with health implications. In this work, we use the immortalized mouse hippocampal neuronal HT-22 cells in culture to study the effects of {P 5 W 30 } on the cytosolic Ca 2+ concentration. Changes in cytosolic Ca 2+ concentration were monitored using fluorescence microscopy of HT-22 cells loaded with the fluorescent Ca 2+ indicator Fluo3. 31 P-Nuclear magnetic resonance measurements of {P 5 W 30 } indicate its stability in the medium used for cytosolic Ca 2+ measurements for over 30 min. The findings reveal that addition of {P 5 W 30 } to the extracellular medium induces a sustained increase of the cytosolic Ca 2+ concentration within minutes. This Ca 2+ increase is triggered by extracellular Ca 2+ entry into the cells and is dose-dependent, with a half-of-effect concentration of 0.25 ± 0.05 μM {P 5 W 30 }. In addition, after the {P 5 W 30 }-induced cytosolic Ca 2+ increase, the transient Ca 2+ peak induced by extracellular ATP is reduced up to 100% with an apparent half-of-effect concentration of 0.15 ± 0.05 μM {P 5 W 30 }. Activation of metabotropic purinergic P2 receptors affords about 80% contribution to the increase of Fluo3 fluorescence elicited by {P 5 W 30 } in HT-22 cells, whereas ionotropic receptors contribute, at most, with 20%. These results suggest that {P 5 W 30 } could serve as a novel agonist of purinergic P2 receptors., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Annette Rompel, Nadiia I. Gumerova reports financial support was provided by Austrian Science Fund. Manuel Aureliano reports financial support was provided by Foundation for Science and Technology. Carlos Gutierrez-Merino, Ana M. Mata reports financial support was provided by Spanish State Research Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Kaempferol, a flavonoid present in many food products, has chemical and cellular antioxidant properties that are beneficial for protection against the oxidative stress caused by reactive oxygen and nitrogen species. Kaempferol administration to model experimental animals can provide extensive protection against brain damage of the striatum and proximal cortical areas induced by transient brain cerebral ischemic stroke and by 3-nitropropionic acid. This article is an updated review of the molecular and cellular mechanisms of protection by kaempferol administration against brain damage induced by these insults, integrated with an overview of the contributions of the work performed in our laboratories during the past years. Kaempferol administration at doses that prevent neurological dysfunctions inhibit the critical molecular events that underlie the initial and delayed brain damage induced by ischemic stroke and by 3-nitropropionic acid. It is highlighted that the protection afforded by kaempferol against the initial mitochondrial dysfunction can largely account for its protection against the reported delayed spreading of brain damage, which can develop from many hours to several days. This allows us to conclude that kaempferol administration can be beneficial not only in preventive treatments, but also in post-insult therapeutic treatments.

Lipid membrane nanodomains or lipid rafts are 10-200 nm diameter size cholesterol- and sphingolipid-enriched domains of the plasma membrane, gathering many proteins with different roles. Isolation and characterization of plasma membrane proteins by differential centrifugation and proteomic studies have revealed a remarkable diversity of proteins in these domains. The limited size of the lipid membrane nanodomain challenges the simple possibility that all of them can coexist within the same lipid membrane domain. As caveolin-1, flotillin isoforms and gangliosides are currently used as neuronal lipid membrane nanodomain markers, we first analyzed the structural features of these components forming nanodomains at the plasma membrane since they are relevant for building supramolecular complexes constituted by these molecular signatures. Among the proteins associated with neuronal lipid membrane nanodomains, there are a large number of proteins that play major roles in calcium signaling, such as ionotropic and metabotropic receptors for neurotransmitters, calcium channels, and calcium pumps. This review highlights a large variation between the calcium signaling proteins that have been reported to be associated with isolated caveolin-1 and flotillin-lipid membrane nanodomains. Since these calcium signaling proteins are scattered in different locations of the neuronal plasma membrane, i.e., in presynapses, postsynapses, axonal or dendritic trees, or in the neuronal soma, our analysis suggests that different lipid membrane-domain subtypes should exist in neurons. Furthermore, we conclude that classification of lipid membrane domains by their content in calcium signaling proteins sheds light on the roles of these domains for neuronal activities that are dependent upon the intracellular calcium concentration. Some examples described in this review include the synaptic and metabolic activity, secretion of neurotransmitters and neuromodulators, neuronal excitability (long-term potentiation and long-term depression), axonal and dendritic growth but also neuronal cell survival and death.

Amyloid β (Aβ) oligomers are the most neurotoxic forms of Aβ, and Aβ(1-42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer's disease patients. Aβ(25-35) is the shortest peptide that retains the toxicity of Aβ(1-42). Aβ oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aβ(1-42) concentration range. Aβ and histidine-rich proteins have a high affinity for transition metal ions Cu 2+ , Fe 3+ and Zn 2+ . In this work, we show that the fluorescence of Aβ(1-42) HiLyte TM -Fluor555 can be used to monitor hexa-histidine peptide (His 6 ) interaction with Aβ(1-42). The formation of His 6 /Aβ(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His 6 block the increase in the fluorescence of Aβ(1-42) HiLyte TM -Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His 6 -tag provides a high-affinity site for the binding of Aβ(1-42) and Aβ(25-35) peptides to the human recombinant cytochrome b 5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His 6 -tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets.

Amyloid β (Aβ) oligomers have been linked to Alzheimer's disease (AD) pathogenesis and are the main neurotoxic forms of Aβ. This review focuses on the following: (i) the Aβ(1-42):calmodulin interface as a model for the design of antagonist Aβ peptides and its limitations; (ii) proteolytic degradation as the major source of highly hydrophobic peptides in brain cells; and (iii) brain peptides that have been experimentally demonstrated to bind to Aβ monomers or oligomers, Aβ fibrils, or Aβ plaques. It is highlighted that the hydrophobic amino acid residues of the COOH-terminal segment of Aβ(1-42) play a key role in its interaction with intracellular protein partners linked to its neurotoxicity. The major source of highly hydrophobic endogenous peptides of 8-10 amino acids in neurons is the proteasome activity. Many canonical antigen peptides bound to the major histocompatibility complex class 1 are of this type. These highly hydrophobic peptides bind to Aβ and are likely to be efficient antagonists of the binding of Aβ monomers/oligomers concentrations in the nanomolar range with intracellular proteins. Also, their complexation with Aβ will protect them against endopeptidases, suggesting a putative chaperon-like physiological function for Aβ that has been overlooked until now. Remarkably, the hydrophobic amino acid residues of Aβ responsible for the binding of several neuropeptides partially overlap with those playing a key role in its interaction with intracellular protein partners that mediates its neurotoxicity. Therefore, these latter neuropeptides are also potential candidates to antagonize Aβ peptides binding to target proteins. In conclusion, the analysis performed in this review points out that hydrophobic endogenous brain neuropeptides could be valuable biomarkers to evaluate the risk of the onset of sporadic AD, as well as for the prognosis of AD.

Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid β protein precursor (APP) and neurotoxic amyloid β (Aβ) peptides in reactive astrocytes. Also, aberrant Ca 2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer's disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aβ peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca 2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca 2+ entry after complete Ca 2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca 2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca 2+ from the ER, preventing a rise in the resting cytosolic Ca 2+ concentration.

Caveolin-2 is a protein suitable for the study of interactions of caveolins with other proteins and lipids present in caveolar lipid rafts. Caveolin-2 has a lower tendency to associate with high molecular weight oligomers than caveolin-1, facilitating the study of its structural modulation upon association with other proteins or lipids. In this paper, we have successfully expressed and purified recombinant human caveolin-2 using E. coli . The structural changes of caveolin-2 upon interaction with a lipid bilayer of liposomes were characterized using bioinformatic prediction models, circular dichroism, differential scanning calorimetry, and fluorescence techniques. Our data support that caveolin-2 binds and alters cholesterol-rich domains in the membranes through a CARC domain, a type of cholesterol-interacting domain in its sequence. The far UV-CD spectra support that the purified protein keeps its folding properties but undergoes a change in its secondary structure in the presence of lipids that correlates with the acquisition of a more stable conformation, as shown by differential scanning calorimetry experiments. Fluorescence experiments using egg yolk lecithin large unilamellar vesicles loaded with 1,6-diphenylhexatriene confirmed that caveolin-2 adsorbs to the membrane but only penetrates the core of the phospholipid bilayer if vesicles are supplemented with 30% of cholesterol. Our study sheds light on the caveolin-2 interaction with lipids. In addition, we propose that purified recombinant caveolin-2 can provide a new tool to study protein-lipid interactions within caveolae.

Plasma membrane calcium ATPases (PMCA) and sarco(endo) reticulum calcium ATPases (SERCA) are key proteins in the maintenance of calcium homeostasis. Herein, we compare for the first time the inhibition of SERCA and PMCA calcium pumps by several polyoxotungstates (POTs), namely by Wells-Dawson phosphotungstate anions [P 2 W 18 O 62 ] 6- (intact, {P 2 W 18 }), [P 2 W 17 O 61 ] 10- (monolacunary, {P 2 W 17 }), [P 2 W 15 O 56 ] 12- (trilacunary, {P 2 W 15 }), [H 2 P 2 W 12 O 48 ] 12- (hexalacunary, {P 2 W 12 }), [H 3 P 2 W 15 V 3 O 62 ] 6- (trivanadium-substituted, {P 2 W 15 V 3 }) and by Preyssler-type anion [NaP 5 W 30 O 110 ] 14- ({P 5 W 30 }). The speciation in the solutions of tested POTs was investigated by 31 P and 51 V NMR spectroscopy. The tested POTs inhibited SERCA Ca 2+ -ATPase activity, whereby the Preyssler POT showed the strongest effect, with an IC 50 value of 0.37 μM. For {P 2 W 17 } and {P 2 W 15 V 3 } higher IC 50 values were determined: 0.72 and 0.95 μM, respectively. The studied POTs showed to be more potent inhibitors of PMCA Ca 2+ -ATPase activity, with lower IC 50 values for {P 2 W 17 }, {P 5 W 30 } and {P 2 W 15 V 3 }., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer's disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-β peptides (Aβ) in AD. In recent works, we have proposed that Aβ-calmodulin (CaM) complexation may play a major role in neuronal Ca 2+ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 μM Aβ(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aβ(1-42), respectively. Internalized Aβ(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aβ. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aβ(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aβ(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aβ(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca 2+ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 μM Aβ(1-42) oligomers. We conclude that Aβ(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca 2+ -leak channels.