Focal adhesion kinase, RhoA, and p38 mitogen-activated protein kinase modulates apoptosis mediated by angiotensin II AT2 receptors.

Apoptosis plays an important role in cellular processes such as development, differentiation, and homeostasis. Although the participation of angiotensin II (Ang II) AT2 receptors (AT 2 R) in cellular apoptosis is well accepted, the signaling pathway involved in this process is not well established. We evaluated the participation of signaling proteins focal adhesion kinase (FAK), RhoA, and p38 mitogen-activated protein kinase (p38MAPK) in apoptosis induced by Ang II via AT 2 R overexpressed in HeLa cells. Following a short stimulation time (120 to 240 minutes) with Ang II, HeLa-AT 2 cells showed nuclear condensation, stress fibers disassembly and membrane blebbing. FAK, classically involved in cytoskeleton reorganization, has been postulated as an early marker of cellular apoptosis. Thus, we evaluated FAK cleavage, detected at early stimulation times (15 to 30 minutes). Apoptosis was confirmed by increased caspase-3 cleavage and enzymatic activity of caspase-3/7. Participation of RhoA was evaluated. HeLa-AT 2 cells overexpressing RhoA wild-type (WT) or their mutants, RhoA V14 (constitutively active form) or RhoA N19 (dominant-negative form) were used to explore RhoA participation. HeLa-AT 2 cells expressing the constitutively active variant RhoA V14 showed enhanced apoptotic features at earlier times as compared with cells expressing the WT variant. RhoA N19 expression prevented nuclear condensation/caspase activation. Inhibition of p38MAPK caused an increase in nuclear condensation and caspase-3/7 activation, suggesting a protective role of p38MAPK. Our results clearly demonstrated that stimulation of AT 2 R induce apoptosis with participation of FAK and RhoA while p38MAPK seems to play a prosurvival role.

Amyotrophic lateral sclerosis-immunoglobulins selectively interact with neuromuscular junctions expressing P/Q-type calcium channels.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a gradual loss of motoneurons. The majority of ALS cases are associated with a sporadic form whose etiology is unknown. Several pieces of evidence favor autoimmunity as a potential contributor to sporadic ALS pathology. To gain understanding concerning possible antigens interacting with IgGs from sporadic ALS patients (ALS-IgGs), we studied immunoreactivity against neuromuscular junction (NMJ), spinal cord and cerebellum of mice with and without the Ca(V) 2.1 pore-forming subunit of the P/Q-type voltage-gated calcium (Ca(2+)) channel. ALS-IgGs showed a strong reactivity against NMJs of wild-type diaphragms. ALS-IgGs also increased muscle miniature end-plate potential frequency, suggesting a functional role for ALS-IgGs on synaptic signaling. In support, in mice lacking the Ca(V) 2.1 subunit ALS-IgGs showed significantly reduced NMJ immunoreactivity and did not alter spontaneous acetylcholine release. This difference in reactivity was absent when comparing N-type Ca(2+) channel wild-type or null mice. These results are particularly relevant because motoneurons are known to be early pathogenic targets in ALS. Our findings add further evidence supporting autoimmunity as one of the possible mechanisms contributing to ALS pathology. They also suggest that serum autoantibodies in a subset of ALS patients would interact with NMJ proteins down-regulated when P/Q-type channels are absent.

Silicon nanoparticle photophysics and singlet oxygen generation.

The effect of molecular oxygen and water on the blue photoluminescence of silicon nanoparticles synthesized by anodic oxidation of silicon wafers and surface functionalized with 2-methyl 2-propenoic acid methyl ester is investigated. The particles of 3 +/- 1 nm diameter and a surface composition of Si(3)O(6)(C(5)O(2)H(8)) exhibit room-temperature luminescence in the wavelength range 300-600 nm upon excitation with 300-400 nm light. The luminescence shows vibronic resolution and high quantum yields in toluene suspensions, while a vibronically unresolved spectrum and lower emission quantum yields are observed in aqueous suspensions. The luminescence intensity, though not the spectrum features, depends on the presence of dissolved O(2). Strikingly, the luminescence decay time on the order of 1 ns does not depend on the solvent or on the presence of O(2). To determine the mechanisms involved in these processes, time-resolved and steady-state experiments are performed. These include low-temperature luminescence, heavy atom effect, singlet molecular oxygen ((1)O(2)) phosphorescence detection, reaction of specific probes with (1)O(2), and determination of O(2) and N(2) adsorption isotherms at 77 K. The results obtained indicate that physisorbed O(2) is capable of quenching nondiffusively the particle luminescence at room temperature. The most probable mechanism for (1)O(2) generation involves the energy transfer from an exciton singlet state to O(2) to yield an exciton triplet of low energy (<0.98 eV) and (1)O(2). In aqueous solutions, excited silicon nanoparticles are able to reduce methylviologen on its surface.

Microglial autophagy is impaired by prolonged exposure to ß-amyloid peptides: evidence from experimental models and Alzheimer's disease patients.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of misfolded proteins, amyloid-ß (Aß) aggregates, and neuroinflammation in the brain. Microglial cells are key players in the context of AD, being capable of releasing cytokines in response to Aß and degrading aggregated proteins by mechanisms involving the ubiquitin-proteasome system and autophagy. Here, we present in vivo and in vitro evidence showing that microglial autophagy is affected during AD progression. PDAPPJ20 mice-murine model of AD-exhibited an accumulation of the autophagy receptor p62 and ubiquitin+ aggregates in Iba1+ microglial cells close to amyloid deposits in the hippocampus. Moreover, cultured microglial BV-2 cells showed an enhanced autophagic flux during a 2-h exposure to fibrillar Aß, which was decreased if the exposure was prolonged to 24 h, a condition analogous to the chronic exposure to Aß in the human pathology. The autophagic impairment was also associated with lysosomal damage, depicted by membrane permeabilization as shown by the presence of the acid hydrolase cathepsin-D in cytoplasm and altered LysoTracker staining. These results are compatible with microglial exhaustion caused by pro-inflammatory conditions and persistent exposure to aggregated Aß peptides. In addition, we found LC3-positive autophagic vesicles accumulated in phagocytic CD68+ microglia in human AD brain samples, suggesting defective autophagy in microglia of AD brain. Our results indicate that the capacity of microglia to degrade Aß and potentially other proteins through autophagy may be negatively affected as the disease progresses. Preserving autophagy in microglia thus emerges as a promising approach for treating AD. Graphical abstract.

Comparative toxicity of PEG and folate-derived blue-emitting silicon nanoparticles: in vitro and in vivo studies.

AIM: Amino functionalization is a first step modification aiming to achieve biomedical applications of silicon nanoparticles, for example, for photodynamic therapy or radiotherapy. Nevertheless, toxicity and low quantum yields due to the positive charge of amino groups emerge as a problem that could be solved with subsequent derivatizations. MATERIALS & METHODS: Folic and PEG-conjugated nanoparticles were obtained from amino-functionalized silicon nanoparticle (NH2SiNP). Cytotoxicity was determined on a tumor cell line at low and high concentrations. Four end points of in vivo toxicity were evaluated on zebrafish (Danio rerio). RESULTS: Folic acid functionalization reduced the cytotoxicity in comparison to amino and PEG-functionalized nanoparticles. In zebrafish, folic functionalization lowered toxicity in general while PEG increased it. CONCLUSION: Functionalization of NH2SiNP with folic acid reduced the toxic effects in vitro and in vivo. This could be useful for therapeutic applications. PEG functionalization did not lower the toxicity.

Manganese activates the mitochondrial apoptotic pathway in rat astrocytes by modulating the expression of proteins of the Bcl-2 family.

Manganese induces the central nervous system injury leading to manganism, by mechanisms not completely understood. Chronic exposure to manganese generates oxidative stress and induces the mitochondrial permeability transition. In the present study, we characterized apoptotic cell death mechanisms associated with manganese toxicity in rat cortical astrocytes and demonstrated that (i) Mn treatment targets the mitochondria and induces mitochondrial membrane depolarization followed by cytochrome c release to the cytoplasm, (ii) Mn induces both effector caspases 3/7 and 6 as well as PARP-1 cleavage and (iii) Mn shifts the balance of cell death/survival of Bcl-2 family proteins to favor the apoptotic demise of astrocytes. Our model system using cortical rat astrocytes treated with Mn would emerge as a good tool for investigations aimed to elucidate the role of apoptosis in manganism.

Toward biomedical application of amino-functionalized silicon nanoparticles.

Silicon blue-emitting nanoparticles (NPs) are promising effectors for photodynamic therapy and radiotherapy, because of their production of reactive oxygen species (ROS) upon irradiation. RESULTS: Amino-functionalized silicon NPs (NH2SiNP) were intrinsically nontoxic below 100 µg/ml in vitro (on two tumor cell lines) and in vivo (zebrafish larvae and embryos). NH2SiNP showed a moderate effect as a photosensitizer for photodynamic therapy and reduced ROS generation in radiotherapy, which could be indicative of a ROS scavenging effect. Encapsulation of NH2SiNP into ultradeformable liposomes improved their skin penetration after topical application, reaching the viable epidermis where neoplastic events occur. CONCLUSION: Subsequent derivatizations after amino-functionalization and incorporation to nanodrug delivery systems could expand the spectrum of the biomedical application of these kind of silicon NPs.

From the Cover: Vulnerability of C6 Astrocytoma Cells After Single-Compound and Joint Exposure to Type I and Type II Pyrethroid Insecticides.

A primary mode-of-action of all pyrethroid insecticides (PYRs) is the disruption of the voltage-gated sodium channel electrophysiology in neurons of target pests and nontarget species. The neurological actions of PYRs on non-neuronal cells of the nervous system remain poorly investigated. In the present work, we used C6 astrocytoma cells to study PYR actions (0.1-50 µM) under the hypothesis that glial cells may be targeted by and vulnerable to PYRs. To this end, we characterized the effects of bifenthrin (BF), tefluthrin (TF), α-cypermethrin (α-CYP), and deltamethrin (DM) on the integrity of nuclear, mitochondrial, and lysosomal compartments. In general, 24- to 48-h exposures produced concentration-related impairment of cell viability. In single-compound, 24-h exposure experiments, effective concentration (EC)15s 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT assay) were computed as follows (in µM): BF, 16.1; TF, 37.3; α-CYP, 7.8; DM, 5.0. We found concentration-related damage in several C6-cell subcellular compartments (mitochondria, nuclei, and lysosomes) at ≥ 10-1 µM levels. Last, we examined a mixture of all PYRs (ie, Σ individual EC15) using MTT assays and subcellular analyses. Our findings indicate that C6 cells are responsive to nM levels of PYRs, suggesting that astroglial susceptibility may contribute to the low-dose neurological effects caused by these insecticides. This research further suggests that C6 cells may provide relevant information as a screening platform for pesticide mixtures targeting nervous system cells by expected and unexpected toxicogenic pathways potentially contributing to clinical neurotoxicity.

Deregulation of mitochondria-shaping proteins Opa-1 and Drp-1 in manganese-induced apoptosis.

Mitochondria are dynamic organelles that undergo fusion and fission processes. These events are regulated by mitochondria-shaping proteins. Changes in the expression and/or localization of these proteins lead to a mitochondrial dynamics impairment and may promote apoptosis. Increasing evidence correlates the mitochondrial dynamics disruption with the occurrence of neurodegenerative diseases. Therefore, we focused on this topic in Manganese (Mn)-induced Parkinsonism, a disorder associated with Mn accumulation preferentially in the basal ganglia where mitochondria from astrocytes represent an early target. Using MitoTracker Red staining we observed increased mitochondrial network fission in Mn-exposed rat astrocytoma C6 cells. Moreover, Mn induced a marked decrease in fusion protein Opa-1 levels as well as a dramatic increase in the expression of fission protein Drp-1. Additionally, Mn provoked a significant release of high MW Opa-1 isoforms from the mitochondria to the cytosol as well as an increased Drp-1 translocation to the mitochondria. Both Mdivi-1, a pharmacological Drp-1 inhibitor, and rat Drp-1 siRNA reduced the number of apoptotic nuclei, preserved the mitochondrial network integrity and prevented cell death. CsA, an MPTP opening inhibitor, prevented mitochondrial Δψm disruption, Opa-1 processing and Drp-1 translocation to the mitochondria therefore protecting Mn-exposed cells from mitochondrial disruption and apoptosis. The histological analysis and Hoechst 33258 staining of brain sections of Mn-injected rats in the striatum showed a decrease in cellular mass paralleled with an increase in the occurrence of apoptotic nuclei. Opa-1 and Drp-1 expression levels were also changed by Mn-treatment. Our results demonstrate for the first time that abnormal mitochondrial dynamics is implicated in both in vitro and in vivo Mn toxicity. In addition we show that the imbalance in fusion/fission equilibrium might be involved in Mn-induced apoptosis. This knowledge may provide new therapeutic tools for the treatment of Manganism and other neurodegenerative diseases.

Manganese induces mitochondrial dynamics impairment and apoptotic cell death: a study in human Gli36 cells.

Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment.

The extrinsic and intrinsic apoptotic pathways are involved in manganese toxicity in rat astrocytoma C6 cells.

Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, chronic exposure to high levels of Mn in occupational or environmental settings can lead to its accumulation in the brain resulting in a degenerative brain disorder referred to as Manganism. Astrocytes are the main Mn store in the central nervous system and several lines of evidence implicate these cells as major players in the role of Manganism development. In the present study, we employed rat astrocytoma C6 cells as a sensitive experimental model for investigating molecular mechanisms involved in Mn neurotoxicity. Our results show that C6 cells undergo reactive oxygen species-mediated apoptotic cell death involving caspase-8 and mitochondrial-mediated pathways in response to Mn. Exposed cells exhibit typical apoptotic features, such as chromatin condensation, cell shrinkage, membrane blebbing, caspase-3 activation and caspase-specific cleavage of the endogenous substrate poly (ADP-ribose) polymerase. Participation of the caspase-8 dependent pathway was assessed by increased levels of FasL, caspase-8 activation and Bid cleavage. The involvement of the mitochondrial pathway was demonstrated by the disruption of the mitochondrial membrane potential, the opening of the mitochondrial permeability transition pore, cytochrome c release, caspase-9 activation and the increased mitochondrial levels of the pro-apoptotic Bcl-2 family proteins. In addition, our data also shows for the first time that mitochondrial fragmentation plays a relevant role in Mn-induced apoptosis. Taking together, these findings contribute to a deeper elucidation of the molecular signaling mechanisms underlying Mn-induced apoptosis.