Microencapsulation of sunscreen reduces toxicity of its components to A. salina: Biochemical, behavioral and morphological studies.

Sunscreens contain several substances that cause damage to species where they are disposed. New formulations have been created to prevent such marine environmental damages. One promising formulation is the microencapsulated sunscreen. The objective of this study was to evaluate the possible safety to marine environment of one microencapsulated sunscreen formulation. The animal model Artemia salina (cists and nauplii) was tested with two sunscreen formulations (microencapsulated and non-microencapsulated) and toxicological, behavioral, morphological parameters as well as biochemical assays (lipoperoxidation and carbonylation tests) were analyzed. Results showed that microencapsulated sunscreen impeded some toxic effects caused by the release of the substances within the microcapsule in the highest concentration, reestablishing the mortality and hatching rates to control levels, while removing the sunscreen microcapsule by adding 1 % DMSO reduced the cyst hatching rate, increasing the nauplii mortality rate and decreased locomotor activity in higher concentrations. Finally, nauplii with 24 hours of life and exposed to sunscreen without the microcapsule showed an increase in mitochondrial activity (assessed at 48 hours after exposure) and presented malformations when exposed to the highest concentration non-microencapsulated concentration (assessed by SEM at 72 hours after exposure), when compared to the control group. These results together allow us to conclude that the microencapsulation process of a sunscreen helps protecting A. salina from the harmful effects of higher concentrations of said sunscreens. However, long-term studies must be carried out as it is not known how long a microencapsulated sunscreen can remain in the environment without causing harmful effects to the marine ecosystem and becoming an ecologically relevant pollutant.

Cellular prion protein offers neuroprotection in astrocytes submitted to amyloid ß oligomer toxicity.

The cellular prion protein (PrPC), in its native conformation, performs numerous cellular and cognitive functions in brain tissue. However, despite the cellular prion research in recent years, there are still questions about its participation in oxidative and neurodegenerative processes. This study aims to elucidate the involvement of PrPC in the neuroprotection cascade in the presence of oxidative stressors. For that, astrocytes from wild-type mice and knockout to PrPC were subjected to the induction of oxidative stress with hydrogen peroxide (H2O2) and with the toxic oligomer of the amyloid ß protein (AßO). We observed that the presence of PrPC showed resistance in the cell viability of astrocytes. It was also possible to monitor changes in basic levels of metals and associate them with an induced damage condition, indicating the precise role of PrPC in metal homeostasis, where the absence of PrPC leads to metallic unbalance, culminating in cellular vulnerability to oxidative stress. Increased caspase 3, p-Tau, p53, and Bcl2 may establish a relationship between a PrPC and an induced damage condition. Complementarily, it has been shown that PrPC prevents the internalization of AßO and promotes its degradation under oxidative stress induction, thus preventing protein aggregation in astrocytes. It was also observed that the presence of PrPC can be related to translocating SOD1 to cell nuclei under oxidative stress, probably controlling DNA damage. The results of this study suggest that PrPC acts against oxidative stress activating the cellular response and defense by displaying neuroprotection to neurons and ensuring the functionality of astrocytes.

The Impact of Hydroxytyrosol on the Metallomic-Profile in an Animal Model of Alzheimer's Disease.

It is undeniable that as people get older, they become progressively more susceptible to neurodegenerative illnesses such as Alzheimer's disease (AD). Memory loss is a prominent symptom of this condition and can be exacerbated by uneven levels of certain metals. This study used inductively coupled plasma mass spectrometry (ICP-MS) to examine the levels of metals in the blood plasma, frontal cortex, and hippocampus of Wistar rats with AD induced by streptozotocin (STZ). It also tested the effects of the antioxidant hydroxytyrosol (HT) on metal levels. The Barnes maze behavior test was used, and the STZ group showed less certainty and greater distance when exploring the Barnes maze than the control group. The results also indicated that the control group and the STZ + HT group exhibited enhanced learning curves during the Barnes maze training as compared to the STZ group. The ICP-MS analysis showed that the STZ group had lower levels of cobalt in their blood plasma than the control group, while the calcium levels in the frontal cortex of the STZ + HT group were higher than in the control group. The most important finding was that copper levels in the frontal cortex from STZ-treated animals were higher than in the control group, and that the STZ + HT group returned to equivalent levels to the control group. The antioxidant HT can restore copper levels to their basal physiological state. This finding may help explain HT's potential beneficial effect in AD-patients.

Selective cytotoxicity of ent-kaurene diterpenoids isolated from Baccharis lateralis and Baccharis retusa (Asteraceae).

This study presents the cytotoxic activity evaluation of the natural diterpenes ent-kaurenoic acid (1) and its 15ß-hydroxy (2), 15ß-senecioyloxy (3), and 15ß-tiglinoyloxy (4) derivatives, isolated from Brazilian native plants, Baccharis retusa and B. lateralis (Asteraceae). Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay, it was observed that compound 1 displayed in vitro activity towards the aggressive MDA-MB-231 adenocarcinoma cell line and reduced toxicity against MCF-10A nontumorigenic epithelial cells, indicating expressive selectivity. On the contrary, compounds 2-4 exhibited reduced toxicity and selectivity in both tested cell lines. Based on the chemical structures of compounds 1-4, it is suggested that the presence of additional functional groups at the C-15 position-a hydroxyl group in compound 2 and isomeric isoprene units in compounds 3 and 4-might be responsible for the reduction in the potential/selectivity. In silico studies show, for compounds 1-4, good predictions regarding bioavailability and ADME (absorption, distribution, metabolism, and excretion) properties as well as no alerts for PAINS (pan-assay structures interference). In conclusion, ent-kaurenoic acid (1), a common diterpenoid isolated in high amounts from different plants belonging to the Baccharis genus, has been shown to be a promising cytotoxic agent against an aggressive adenocarcinoma cell line (MDA-MB-23) and, if well exploited, could be used as a scaffold in the development of molecular prototypes for the treatment of breast cancer.

Is There a Connection between the Metabolism of Copper, Sulfur, and Molybdenum in Alzheimer's Disease? New Insights on Disease Etiology.

Alzheimer's disease (AD) and other forms of dementia was ranked 3rd in both the Americas and Europe in 2019 in a World Health Organization (WHO) publication listing the leading causes of death and disability worldwide. Copper (Cu) imbalance has been reported in AD and increasing evidence suggests metal imbalance, including molybdenum (Mo), as a potential link with AD occurrence.We conducted an extensive literature review of the last 60 years of research on AD and its relationship with Cu, sulfur (S), and Mo at out of range levels.Weanalyzed the interactions among metallic elements' metabolisms;Cu and Mo are biological antagonists, Mo is a sulfite oxidase and xanthine oxidase co-factor, and their low activities impair S metabolism and reduce uric acid, respectively. We found significant evidence in the literature of a new potential mechanism linking Cu imbalance to Mo and S abnormalities in AD etiology: under certain circumstances, the accumulation of Cu not bound to ceruloplasmin might affect the transport of Mo outside the blood vessels, causing a mild Mo deficiency that might lowerthe activity of Mo and S enzymes essential for neuronal activity. The current review provides an updated discussion of the plausible mechanisms combining Cu, S, and Mo alterations in AD.

Cellular prion protein activates Caspase 3 for apoptotic defense mechanism in astrocytes.

The cellular prion protein (PrPC) is anchored in the plasma membrane of cells, and it is highly present in cells of brain tissue, exerting numerous cellular and cognitive functions. The present study proves the importance of PrPC in the cellular defense mechanism and metal homeostasis in astrocytes cells. Through experimental studies using cell lines of immortalized mice astrocytes (wild type and knockout for PrPC), we showed that PrPc is involved in the apoptosis cell death process by the activation of Caspase 3, downregulation of p53, and cell cycle maintenance. Metal homeostasis was determined by inductively coupled plasma mass spectrometry technique, indicating the crucial role of PrPC to lower intracellular calcium. The lowered calcium concentration and the Caspase 3 downregulation in the PrPC-null astrocytes resulted in a faster growth rate in cells, comparing with PrPC wild-type one. The presence of PrPC shows to be essential to cell death and healthy growth. In conclusion, our results show for the first time that astrocyte knockout cells for the cellular prion protein could modulate apoptosis-dependent cell death pathways.

Net charge tuning modulates the antiplasmodial and anticancer properties of peptides derived from scorpion venom.

VmCT1, a linear helical antimicrobial peptide isolated from the venom of the scorpion Vaejovis mexicanus, displays broad spectrum antimicrobial activity against bacteria, fungi, and protozoa. Analogs derived from this peptide containing single Arg-substitutions have been shown to increase antimicrobial and antiparasitic activities against Trypanossoma cruzi. Here, we tested these analogs against malaria, an infectious disease caused by Plasmodium protozoa, and assessed their antitumoral properties. Specifically, we tested VmCT1 synthetic variants [Arg]3 -VmCT1-NH2 , [Arg]7 -VmCT1-NH2 , and [Arg]11 -VmCT1-NH2 , against Plasmodium gallinaceum sporozoites and MCF-7 mammary cancer cells. Our screen identified peptides [Arg]3 -VmCT1-NH2 and [Arg]7 -VmCT1-NH2 as potent antiplasmodial agents (IC50 of 0.57 and 0.51 µmol L-1 , respectively), whereas [Arg]11 -VmCT1-NH2 did not show activity against P. gallinaceum sporozoites. Interestingly, all peptides presented activity against MCF-7 and displayed lower cytotoxicity toward healthy cells. We demonstrate that increasing the net positive charge of VmCT1, through arginine substitutions, modulates the biological properties of this peptide family yielding novel antiplasmodial and antitumoral molecules.

Natural and redesigned wasp venom peptides with selective antitumoral activity.

About 1 in 8 U.S. women (≈12%) will develop invasive breast cancer over the course of their lifetime. Surgery, chemotherapy, radiotherapy, and hormone manipulation constitute the major treatment options for breast cancer. Here, we show that both a natural antimicrobial peptide (AMP) derived from wasp venom (decoralin, Dec-NH2), and its synthetic variants generated via peptide design, display potent activity against cancer cells. We tested the derivatives at increasing doses and observed anticancer activity at concentrations as low as 12.5 µmol L-1 for the selective targeting of MCF-7 breast cancer cells. Flow cytometry assays further revealed that treatment with wild-type (WT) peptide Dec-NH2 led to necrosis of MCF-7 cells. Additional atomic force microscopy (AFM) measurements indicated that the roughness of cancer cell membranes increased significantly when treated with lead peptides compared to controls. Biophysical features such as helicity, hydrophobicity, and net positive charge were identified to play an important role in the anticancer activity of the peptides. Indeed, abrupt changes in peptide hydrophobicity and conformational propensity led to peptide inactivation, whereas increasing the net positive charge of peptides enhanced their activity. We present peptide templates with selective activity towards breast cancer cells that leave normal cells unaffected. These templates represent excellent scaffolds for the design of selective anticancer peptide therapeutics.

A Trishistidine Pseudopeptide with Ability to Remove Both CuΙ and CuΙΙ from the Amyloid-ß Peptide and to Stop the Associated ROS Formation.

The pseudopeptide L, derived from a nitrilotriacetic acid scaffold and functionalized with three histidine moieties, is reminiscent of the amino acid side chains encountered in the Alzheimer's peptide (Aß). Its synthesis and coordination properties for CuΙ and CuΙΙ are described. L efficiently complex CuΙΙ in a square-planar geometry involving three imidazole nitrogen atoms and an amidate-Cu bond. By contrast, CuΙ is coordinated in a tetrahedral environment. The redox behavior is irreversible and follows an ECEC mechanism in accordance with the very different environments of the two redox states of the Cu center. This is in line with the observed resistance of the CuΙ complex to oxidation by oxygen and the CuΙΙ complex reduction by ascorbate. The affinities of L for CuΙΙ and CuΙ at physiological pH are larger than that reported for the Aß peptide. Therefore, due to its peculiar Cu coordination properties, the ligand L is able to target both redox states of Cu, redox silence them and prevent reactive oxygen species production by the CuAß complex. Because reactive oxygen species contribute to the oxidative stress, a key issue in Alzheimer's disease, this ligand thus represents a new strategy in the long route of finding molecular concepts for fighting Alzheimer's disease.

Flow of essential elements in subcellular fractions during oxidative stress.

Essential trace elements are commonly found in altered concentrations in the brains of patients with neurodegenerative diseases. Many studies in trace metal determination and quantification are conducted in tissue, cell culture or whole brain. In the present investigation, we determined by ICP-MS Fe, Cu, Zn, Ca, Se, Co, Cr, Mg, and Mn in organelles (mitochondria, nuclei) and whole motor neuron cell cultured in vitro. We performed experiments using two ways to access oxidative stress: cell treatments with H2O2 or Aß-42 peptide in its oligomeric form. Both treatments caused accumulation of markers of oxidative stress, such as oxidized proteins and lipids, and alteration in DNA. Regarding trace elements, cells treated with H2O2 showed higher levels of Zn and lower levels of Ca in nuclei when compared to control cells with no oxidative treatments. On the other hand, cells treated with Aß-42 peptide in its oligomeric form showed higher levels of Mg, Ca, Fe and Zn in nuclei when compared to control cells. These differences showed that metal flux in cell organelles during an intrinsic external oxidative condition (H2O2 treatment) are different from an intrinsic external neurodegenerative treatment.

Photochemical and Photophysical Properties of Phthalocyanines Modified with Optically Active Alcohols.

Three phthalocyanine derivatives were synthesized and characterized: one modified with a racemic mixture of 1-(4-bromophenyl)ethanol and two other macrocycles modified with each one of the enantioenriched isomers (R)-1-(4-bromophenyl)ethanol and (S)-1-(4-bromophenyl)ethanol. The compounds were characterized by 1H-NMR spectroscopy, mass spectrometry, UV-Vis absorption, and excitation and emission spectra. Additionally, partition coefficient values and the quantum yield of the generation of oxygen reactive species were determined. Interestingly, the phthalocyanine containing a (R)-1-(4-bromophenyl)ethoxy moiety showed higher quantum yield of reactive oxygen species generation than other compounds under the same conditions. In addition, the obtained fluorescence microscopy and cell viability results have shown that these phthalocyanines have different interactions with mammary MCF-7 cells. Therefore, our results indicate that the photochemical and biological properties of phthalocyanines with chiral ligands should be evaluated separately for each enantiomeric species.

Synthesis, characterization, and cytotoxic effects of new copper complexes using Schiff-base derivatives from natural sources.

Copper(II) complexes are interesting for cancer treatment due to their unique properties, including their redox potential, possible coordination structures with different ligands, the most diverse geometries, and different biomolecule reactivity. The present work synthesized new copper(II) complexes with Schiff-base (imine) type ligands using natural aldehydes such as cinnamaldehyde, vanillin, or ethyl vanillin. The ligands were obtained through the reaction of these aldehydes with the amines 1,3-diaminopropane, 2,2-dimethyl-1,3-propanediamine, or 1,3-diamino-2-propanol and characterized by 1H and 13C NMR, FTIR and ESI-HRMS. The complexation reaction used copper(II) as perchlorate salt, obtaining six new copper(II) complexes. The complexes were characterized using FTIR, UV-vis, elemental analysis, ESI-HRMS, and EPR. In addition, the interaction with the copper(II) complexes and serum albumin was investigated by electronic absorption, showing complex incorporation in the albumin structure. The cytotoxicity of the complexes was evaluated using MTT assay in neuroblastoma cell lines SH-SY5Y, CHP 212, and glioblastoma LN-18, and presented EC50 values between 90 and 300 µM. Based on our results, a square-planar copper(II) complex derived from Schiff-base cinnamaldehyde was found here to possess significant potential as an anti-cancer treatment. Further investigation is required to explore this compound's benefits in cancer co-treatment approaches fully.

Dehydrodieugenol isolated from Ocotea cymbarum induces cell death in human breast cancer cell lines by dysregulation of intracellular copper concentration.

In this work, two neolignans - dehydrodieugenol (1) and dehydrodieugenol B (2) - were isolated from leaves of Ocotea cymbarum (H. B. K.) Ness. (Lauraceae). When tested against two human breast cancer cell lines (MCF7 and MDA-MB-231), compound 1 was inactive (IC50 > 500 µM) whereas compound 2 displayed IC50 values of 169 and 174 µM, respectively. To evaluate, for the first time in the literature, the synergic cytotoxic effects of compounds 1 and 2 with ion Cu2+, both cell lines were incubated with equimolar solutions of these neolignans and Cu(ClO4)2·6H2O. Obtained results revealed no differences in cytotoxicity upon the co-administration of compound 2 and Cu2+. However, the combination of compound 1 and Cu2+ increases the cytotoxicity against MCF7 and MDA-MB-231 cells, with IC50 values of 165 and 204 µM, respectively. The activity of compound 1 and Cu2+ in MCF7 spheroids regarding the causes/effects considering the tumoral microenvironment were accessed using fluorescence staining and imaging by fluorescence microscopy. This analysis enabled the observation of a higher red filter fluorescence intensity in the quiescence zone and the necrotic core, indicating a greater presence of dead cells, suggesting that the combination permeates the spheroid. Finally, using ICP-MS analysis, the intracellular copper disbalance caused by mixing compound 1 and Cu2+ was determined quantitatively. The findings showcased a 50-fold surge in the concentration of Cu2+ compared with untreated cells (p > 0.0001) - 18.7 ng of Cu2+/mg of proteins and 0.37 ng of Cu2+/mg of protein, respectively. Conversely, the concentration of Cu2+ in cells treated with compound 1 was similar to values of the negative control group (0.29 ng of Cu2+/mg of protein). This alteration allowed us to infer that compound 1 combined with Cu2+ induces cell death through copper homeostasis dysregulation.

Molecular hybridization strategy for tuning bioactive peptide function.

The physicochemical and structural properties of antimicrobial peptides (AMPs) determine their mechanism of action and biological function. However, the development of AMPs as therapeutic drugs has been traditionally limited by their toxicity for human cells. Tuning the physicochemical properties of such molecules may abolish toxicity and yield synthetic molecules displaying optimal safety profiles and enhanced antimicrobial activity. Here, natural peptides were modified to improve their activity by the hybridization of sequences from two different active peptide sequences. Hybrid AMPs (hAMPs) were generated by combining the amphipathic faces of the highly toxic peptide VmCT1, derived from scorpion venom, with parts of four other naturally occurring peptides having high antimicrobial activity and low toxicity against human cells. This strategy led to the design of seven synthetic bioactive variants, all of which preserved their structure and presented increased antimicrobial activity (3.1-128 µmol L-1). Five of the peptides (three being hAMPs) presented high antiplasmodial at 0.8 µmol L-1, and virtually no undesired toxic effects against red blood cells. In sum, we demonstrate that peptide hybridization is an effective strategy for redirecting biological activity to generate novel bioactive molecules with desired properties.

PrPC displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrPC knockout mice.

The PrP(C) protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP(C) in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP(C) decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu(2+)/ascorbate/H(2)O(2)), which was demonstrated by approximately 70% less DMPO/OH(). In cultured PrP(C)-knockout astrocytes from mice, the absence of PrP(C) caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3h of H(2)O(2) treatment. This rapid increase in ROS disrupted the cell cycle in the PrP(C)-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP(C) in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

Cytotoxicity towards Breast Cancer Cells of Pluronic F-127/Hyaluronic Acid Hydrogel Containing Nitric Oxide Donor and Silica Nanoparticles Loaded with Cisplatin.

The incorporation of both nitric oxide (NO) donor (S-nitrosoglutathione, GSNO) and silica nanoparticles loaded with cisplatin (SiO2@CisPt NPs) into a polymeric matrix represents a suitable approach to creating a drug-delivery system with sustained and localized drug release against tumor cells. Herein, we report the synthesis, characterization, and cytotoxicity evaluation of Pluronic F-127/hyaluronic acid hydrogel containing GSNO and SiO2@CisPt NPs against breast cancer cells. SiO2@CisPt NPs were successfully synthesized, revealing a spherical morphology with an average size of 158 ± 20 nm. Both GSNO and SiO2@CisPt NPs were incorporated into the thermoresponsive Pluronic/hyaluronic hydrogel for sustained and localized release of both NO and cisplatin. The kinetics of NO release from a hydrogel matrix revealed spontaneous and sustained release of NO at the millimolar range for 24 h. The MTT assay showed concentration-dependent cytotoxicity of the hydrogel. The combination of GSNO and SiO2@CisPt incorporated into a polymeric matrix decreased the cell viability 20% more than the hydrogel containing only GSNO or SiO2@CisPt. At 200 µg/mL, this combination led to a critical cell viability of 30%, indicating a synergistic effect between GSNO and SiO2@CisPt NPs in the hydrogel matrix, and, therefore, highlighting the potential application of this drug-delivery system in the field of biomedicine.

Iron in Alzheimer's Disease: From Physiology to Disease Disabilities.

Reactive oxygen species (ROS) play a key role in the neurodegeneration processes. Increased oxidative stress damages lipids, proteins, and nucleic acids in brain tissue, and it is tied to the loss of biometal homeostasis. For this reason, attention has been focused on transition metals involved in several biochemical reactions producing ROS. Even though a bulk of evidence has uncovered the role of metals in the generation of the toxic pathways at the base of Alzheimer's disease (AD), this matter has been sidelined by the advent of the Amyloid Cascade Hypothesis. However, the link between metals and AD has been investigated in the last two decades, focusing on their local accumulation in brain areas known to be critical for AD. Recent evidence revealed a relation between iron and AD, particularly in relation to its capacity to increase the risk of the disease through ferroptosis. In this review, we briefly summarize the major points characterizing the function of iron in our body and highlight why, even though it is essential for our life, we have to monitor its dysfunction, particularly if we want to control our risk of AD.

Copper in tumors and the use of copper-based compounds in cancer treatment.

Copper homeostasis is strictly regulated by protein transporters and chaperones, to allow its correct distribution and avoid uncontrolled redox reactions. Several studies address copper as involved in cancer development and spreading (epithelial to mesenchymal transition, angiogenesis). However, being endogenous and displaying a tremendous potential to generate free radicals, copper is a perfect candidate, once opportunely complexed, to be used as a drug in cancer therapy with low adverse effects. Copper ions can be modulated by the organic counterpart, after complexed to their metalcore, either in redox potential or geometry and consequently reactivity. During the last four decades, many copper complexes were studied regarding their reactivity toward cancer cells, and many of them could be a drug choice for phase II and III in cancer therapy. Also, there is promising evidence of using 64Cu in nanoparticles as radiopharmaceuticals for both positron emission tomography (PET) imaging and treatment of hypoxic tumors. However, few compounds have gone beyond testing in animal models, and none of them got the status of a drug for cancer chemotherapy. The main challenge is their solubility in physiological buffers and their different and non-predictable mechanism of action. Moreover, it is difficult to rationalize a structure-based activity for drug design and delivery. In this review, we describe the role of copper in cancer, the effects of copper-complexes on tumor cell death mechanisms, and point to the new copper complexes applicable as drugs, suggesting that they may represent at least one component of a multi-action combination in cancer therapy.

VDAC1 regulates neuronal cell loss after retinal trauma injury by a mitochondria-independent pathway.

The voltage-dependent anion channel 1 (VDAC1) was first described as a mitochondrial porin that mediates the flux of metabolites and ions, thereby integrating both cell survival and death signals. In the nervous system, the functional roles of VDAC1 remain poorly understood. Herein, the rat retina was employed to study VDAC1. First, it was observed that even subtle changes in VDAC1 levels affect neuronal survival, inducing severe alterations in the retinal morphology. We next examined the regulation of VDAC1 after traumatic retinal injury. After mechanical trauma, SOD1 translocates towards the nucleus, which is insufficient to contain the consequences of oxidative stress, as determined by the evaluation of protein carbonylation. Using in vitro models of oxidative stress and mechanical injury in primary retinal cell cultures, it was possible to determine that inhibition of VDAC1 oligomerization by 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) rescues cell viability, impacting microglial cell activation. We next focused on the regulation of VDAC1 after retinal mechanical injury. VDAC1 was promptly upregulated 2 h after lesion in the plasma membrane and endoplasmic reticulum rather than in the mitochondria, and multimers of VDAC1 were assembled after lesion. DIDS intraocular application decreased apoptosis and prevented microglial polarization, which confirmed in vitro observations. Considering the role of microglia in neuroinflammation, multiplex evaluation of cytokines showed that DIDS application disorganized the inflammatory response 2 h after the lesion, matching the fast regulation of VDAC1. Taken together, data disclosed that fine regulation of VDAC1 influences neuronal survival, and pharmacological inhibition after trauma injury has neuroprotective effects. This protection may be attributed to the effects on VDAC1 abnormal accumulation in the plasma membrane, thereby controlling the activation of microglial cells. We concluded that VDAC1 is a putative therapeutic target in neuronal disorders since it integrates both death and survival cellular signaling.

Structural effects of stabilization and complexation of a zinc-deficient superoxide dismutase.

The activity of the erythrocyte Cu2,Zn2-superoxide dismutase (SOD1) is altered in Alzheimer's disease (AD) patients. These patients, compared to healthy subjects, exhibit low plasmatic zinc (Zn) levels in the presence of high plasmatic levels of copper (Cu). SOD1 is an antioxidant enzyme characterized by the presence of two metal ions, Cu and Zn, on its active site. On the SOD1, Cu exerts a catalytic role, and Zn serves a structural function. In this study, we generated a modified SOD1 characterized by an altered capacity to complex Zn. The study investigates the metal-binding dynamics of the enzyme, estimating the stability of a SOD1 protein lacking the appropriate Zn site complexation. Our mutant SOD1 possesses a double amino acid mutation (T135S and K136E) that interferes with the correct Zn site complexation. We found that the protein mutations produce unstable Zn coordination and lower enzymatic activity even when complexed with Cu. Analysis with circular dichroism (CD) spectra on metal titration showed a considerable difference between the two Zn entries in the native dimeric enzyme, and Cu presents a simultaneous entrance in the protein. Otherwise, the mutant T135S,K136E-SOD1 exhibited Zn and Cu complexation instability, being a useful in vitro model to study the SOD1 behavior in AD patients.