Kinetic and protective role of autophagy in manganese-exposed BV-2 cells.

Manganese (Mn) plays an important role in many physiological processes. Nevertheless, Mn accumulation in the brain can cause a parkinsonian-like syndrome known as manganism. Unfortunately, the therapeutic options for this disease are scarce and of limited efficacy. For this reason, a great effort is being made to understand the cellular and molecular mechanisms involved in Mn toxicity in neuronal and glial cells. Even though evidence indicates that Mn activates autophagy in microglia, the consequences of this activation in cell death remain unknown. In this study, we demonstrated a key role of reactive oxygen species in Mn-induced damage in microglial cells. These species generated by Mn2+ induce lysosomal alterations, LMP, cathepsins release and cell death. Besides, we described for the first time the kinetic of Mn2+-induced autophagy in BV-2 microglial cells and its relevance to cell fate. We found that Mn promotes a time-dependent increase in LC3-II and p62 expression levels, suggesting autophagy activation. Possibly, cells trigger autophagy to neutralize the risks associated with lysosomal rupture. In addition, pre-treatment with both Rapamycin and Melatonin enhanced autophagy and retarded Mn2+ cytotoxicity. In summary, our results demonstrated that, despite the damage inflicted on a subset of lysosomes, the autophagic pathway plays a protective role in Mn-induced microglial cell death. We propose that 2 h Mn2+ exposure will not induce disturbances in the autophagic flux. However, as time passes, the accumulated damage inside the cell could trigger a dysfunction of this mechanism. These findings may represent a valuable contribution to future research concerning manganism therapies.

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.

Toxicity of blue led light and A2E is associated to mitochondrial dynamics impairment in ARPE-19 cells: implications for age-related macular degeneration.

Age-related macular degeneration (AMD) is a multifactorial retinal disease characterized by a progressive loss of central vision. Retinal pigment epithelium (RPE) degeneration is a critical event in AMD. It has been associated to A2E accumulation, which sensitizes RPE to blue light photodamage. Mitochondrial quality control mechanisms have evolved to ensure mitochondrial integrity and preserve cellular homeostasis. Particularly, mitochondrial dynamics involve the regulation of mitochondrial fission and fusion to preserve a healthy mitochondrial network. The present study aims to clarify the cellular and molecular mechanisms underlying photodamage-induced RPE cell death with particular focus on the involvement of defective mitochondrial dynamics. Light-emitting diodes irradiation (445 ± 18 nm; 4.43 mW/cm2) significantly reduced the viability of both unloaded and A2E-loaded human ARPE-19 cells and increased reactive oxygen species production. A2E along with blue light, triggered apoptosis measured by MC540/PI-flow cytometry and activated caspase-3. Blue light induced mitochondrial fusion/fission imbalance towards mitochondrial fragmentation in both non-loaded and A2E-loaded cells which correlated with the deregulation of mitochondria-shaping proteins level (OPA1, DRP1 and OMA1). To our knowledge, this is the first work reporting that photodamage causes mitochondrial dynamics deregulation in RPE cells. This process could possibly contribute to AMD pathology. Our findings suggest that the regulation of mitochondrial dynamics may be a valuable strategy for treating retinal degeneration diseases, such as AMD.

Nanohydroxyapatite Exerts Cytotoxic Effects and Prevents Cellular Proliferation and Migration in Glioma Cells.

Hydroxyapatite (Ca10(PO4)6(OH)2; HAP) is an essential component of the human bone inorganic phase. At the nanoscale level, nano-HAP (nHAP) presents marked emergent properties differing substantially from those of the bulk counterpart. Interestingly, these properties depend on nanoparticle characteristics. In this study, we investigated the cytotoxicity of rod-shaped crystalline nHAP (10-20 nm × 50-100 nm) in both normal (ARPE-19, BV-2) and tumoral (HepG2, HEp-2, A549 and C6) cells. We found that nHAP was cytotoxic in tumor HEp-2, A549, and C6 cells. Moreover, it induced an expansion of the lysosomal compartment at sublethal concentrations in different cell lines, while lysosomal membrane damage was not detected. In C6 glioma cells, the most sensitive cell line to nHAP, these nanoparticles increased reactive oxygen species (ROS) production and induced DNA damage measured by γ-H2AX phosphorylation. Interestingly, our data also show for the first time that nHAP affects both cell unlimited proliferative capacity and cell migration, two of the major pathways involved in cancer progression. The present results showed the cytotoxic and antiproliferative effects of nHAP and suggest its potential as an alternative agent for glioma therapy.

Regulated Necrosis Orchestrates Microglial Cell Death in Manganese-Induced Toxicity.

Microglia, the brain resident immune cells, play prominent roles in immune surveillance, tissue repair and neural regeneration. Despite these pro-survival actions, the relevance of these cells in the progression of several neuropathologies has been established. In the context of manganese (Mn) overexposure, it has been proposed that microglial activation contributes to enhance the neurotoxicity. However, the occurrence of a direct cytotoxic effect of Mn on microglial cells remains controversial. In the present work, we investigated the potential vulnerability of immortalized mouse microglial cells (BV-2) toward Mn2+, focusing on the signaling pathways involved in cell death. Evidence obtained showed that Mn2+ induces a decrease in cell viability which is associated with reactive oxygen species (ROS) generation. In this report we demonstrated, for the first time, that Mn2+ triggers regulated necrosis (RN) in BV-2 cells involving two central mechanisms: parthanatos and lysosomal disruption. The occurrence of parthanatos is supported by several cellular and molecular events: (i) DNA damage; (ii) AIF translocation from mitochondria to the nucleus; (iii) mitochondrial membrane permeabilization; and (iv) PARP1-dependent cell death. On the other hand, Mn2+ induces lysosomal membrane permeabilization (LMP) and cathepsin D (CatD) release into the cytosol supporting the lysosomal disruption. Pre-incubation with CatB and D inhibitors partially prevented the Mn2+-induced cell viability decrease. Altogether these events point to lysosomes as players in the execution of RN. In summary, our results suggest that microglial cells could be direct targets of Mn2+ damage. In this scenario, Mn2+ triggers cell death involving RN pathways.

Heme Oxygenase-1 protects astroglia against manganese-induced oxidative injury by regulating mitochondrial quality control.

Heme Oxygenase-1 (HO-1), a stress- responsive enzyme which catalyzes heme degradation into iron, carbon monoxide, and biliverdin, exerts a neuroprotective role involving many different signaling pathways. In Parkinson disease patients, elevated HO-1 expression levels in astrocytes are involved in antioxidant defense. In the present work, employing an in vitro model of Mn2+-induced Parkinsonism in astroglial C6 cells, we investigated the role of HO-1 in both apoptosis and mitochondrial quality control (MQC). HO-1 exerted a protective effect against Mn2+ injury. In fact, HO-1 decreased both intracellular and mitochondrial reactive oxygen species as well as the appearance of apoptotic features. Considering that Mn2+ induces mitochondrial damage and a defective MQC has been implicated in neurodegenerative diseases, we hypothesized that HO-1 could mediate cytoprotection by regulating the MQC processes. Results obtained provide the first evidence that the beneficial effects of HO-1 in astroglial cells are mediated by the maintenance of both mitochondrial fusion/fission and biogenesis/mitophagy balances. Altogether, our data demonstrate a pro-survival function for HO-1 in Mn2+-induced apoptosis that involves the preservation of a proper MQC. These findings point to HO-1 as a new therapeutic target linked to mitochondrial pathophysiology in Manganism and probably Parkinson´s disease.

Drp-1 dependent mitochondrial fragmentation and protective autophagy in dopaminergic SH-SY5Y cells overexpressing alpha-synuclein.

Parkinson's disease is a neurodegenerative movement disorder caused by the loss of dopaminergic neurons from substantia nigra. It is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. Additional common features of this disease are the mitochondrial dysfunction and the activation/inhibition of autophagy both events associated to the intracellular accumulation of α-synuclein. The mechanism by which these events contribute to neural degeneration remains unknown. In the present work we investigated the effect of α-synuclein on mitochondrial dynamics and autophagy/mitophagy in SH-SY5Y cells, an in vitro model of Parkinson disease. We demonstrated that overexpression of wild type α-synuclein causes moderated toxicity, ROS generation and mitochondrial dysfunction. In addition, α-synuclein induces the mitochondrial fragmentation on a Drp-1-dependent fashion. Overexpression of the fusion protein Opa-1 prevented both mitochondrial fragmentation and cytotoxicity. On the other hand, cells expressing α-synuclein showed activated autophagy and particularly mitophagy. Employing a genetic strategy we demonstrated that autophagy is triggered in order to protect cells from α-synuclein-induced cell death. Our results clarify the role of Opa-1 and Drp-1 in mitochondrial dynamics and cell survival, a controversial α-synuclein research issue. The findings presented point to the relevance of mitochondrial homeostasis and autophagy in the pathogenesis of PD. Better understanding of the molecular interaction between these processes could give rise to novel therapeutic methods for PD prevention and amelioration.

Developmental changes in Ca2+ homeostasis and contractility in gallbladder smooth muscle.

Relatively little is known about the contribution of Ca(2+)-dependent and -independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We therefore studied Ca(2+) homeostasis and Ca(2+) sensitization mechanisms in 10-day-old and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, intracellular Ca(2+) concentration was estimated by epifluorescence microscopy of fura-2-loaded isolated cells, and protein expression and phosphorylation were assessed by Western blot analysis. The neonatal gallbladder contracted significantly less to CCK than adult tissue, but this correlated with an increased Ca(2+) mobilization, suggesting immaturity of Ca(2+) sensitization mechanisms. The enhanced Ca(2+) release in the newborn gallbladder was the result of the increase in the size of the releasable Ca(2+) pool. Moreover, in neonatal smooth muscle cells, neither the plasma membrane Ca(2+) pump nor the Na(+)/Ca(2+) exchanger collaborate in the extrusion of Ca(2+). In contrast, in these cells, there is an increase in phospholamban phosphorylation, which could drive to an overactivity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase pump. The reduced Ca(2+) sensitivity in neonatal tissues was demonstrated by the lack of effect to Y-27362, an inhibitor of Rho kinase (ROCK), and GF-109203X, an inhibitor of PKC, on agonist-induced contraction. In addition, the neonatal gallbladder showed lower levels of RhoA, ROCK, PKC, and two effectors [C-kinase-dependent inhibitor of 17 kDa (CPI-17) and myosin phosphatase targetting 1 (MYPT1)] as well as an absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation.

Coactivation of capacitative calcium entry and L-type calcium channels in guinea pig gallbladder.

We have evaluated the presence of capacitative Ca(2+) entry (CCE) in guinea pig gallbladder smooth muscle (GBSM), including a possible relation with activation of L-type Ca(2+) channels. Changes in cytosolic Ca(2+) concentration induced by Ca(2+) entry were assessed by digital microfluorometry in isolated, fura 2-loaded GBSM cells. Application of thapsigargin, a specific inhibitor of the Ca(2+) store pump, induced a transient Ca(2+) release followed by sustained entry of extracellular Ca(2+). Depletion of the stores with thapsigargin, cyclopiazonic acid, ryanodine and caffeine, high levels of the Ca(2+)-mobilizing hormone cholecystokinin octapeptide, or simple removal of external Ca(2+) resulted in a sustained increase in Ca(2+) entry on subsequent reapplication of Ca(2+). This entry was attenuated by 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockade, pinacidil, and Gd(3+). Accumulation of the voltage-sensitive dye 3,3'-dipentylcarbocyanine and direct intracellular recordings showed that depletion of the stores is sufficient for depolarization of the plasma membrane. Contractility studies in intact gallbladder muscle strips showed that CCE induced contractions. The CCE-evoked contraction was sensitive to 2-aminoethoxydiphenylborane, L-type Ca(2+) channel blockers, and Gd(3+). We conclude that, in GBSM, release of Ca(2+) from internal stores activates a CCE pathway and depolarizes plasma membrane, allowing coactivation of voltage-operated L-type Ca(2+) channels. This process may play a role in excitation-contraction coupling in GBSM.

Contribution of different phospholipases and arachidonic acid metabolites in the response of gallbladder smooth muscle to cholecystokinin.

Guinea pig gallbladder muscle strips were used to investigate the contribution of different sources of diacylglicerol (DAG) in the cholecystokinin (CCK)-induced contraction. The involvement of arachidonic acid (AA) in this response was also investigated. Three distinct pathways for DAG production were investigated with specific phospholipase (PL) inhibitors. U-73122 (10 microM) was used for inhibition of phosphoinositide-specific-PLC (PI-PLC), D-609 (100 microM) for phosphatidylcholine specific-PLC (PC-PLC), and propranolol (100 microM) for phospholipase D (PLD). Separate or combined inhibition of each of these enzymes showed that the CCK-induced output of DAG involves the parallel activation of each of these phospholipases. Thus, after inhibition of a PL subtype, the remaining subtypes were able to functionally compensate in mediating CCK-induced contraction. Inhibition of AA production via DAG-lipase or phospholipase A(2) (PLA(2)) was accomplished using RHC-80267 (40 microM), mepacrine (100 microM) and 4-BPB (100 microM). These inhibitors diminished contractile response, indicating that AA is an important modulator of CCK-induced contraction. Indomethacin (10 microM) and nordihydroguaiaretic acid (NDGA, 100 microM), which inhibit subsequent steps in AA metabolism through the cyclooxygenase and 5-lipooxygenase pathways, also inhibited contractions. Taken together, these results show that CCK redundantly activates PC-PLC, PI-PLC and PLD, to produce DAG, which in turn stimulates PKC and provides a substrate for the generation of AA. sPLA(2) is also a source of AA, whose metabolites are, in part, responsible for determining the magnitude of the CCK-evoked contraction.