Roxana Mayra Gorojod

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.

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.

The autophagic- lysosomal pathway determines the fate of glial cells under manganese- induced oxidative stress conditions.

Manganese (Mn) overexposure is frequently associated with the development of a neurodegenerative disorder known as Manganism. The Mn-mediated generation of reactive oxygen species (ROS) promotes cellular damage, finally leading to apoptotic cell death in rat astrocytoma C6 cells. In this scenario, the autophagic pathway could play an important role in preventing cytotoxicity. In the present study, we found that Mn induced an increase in the amount and total volume of acidic vesicular organelles (AVOs), a process usually related to the activation of the autophagic pathway. Particularly, the generation of enlarged AVOs was a ROS- dependent event. In this report we demonstrated for the first time that Mn induces autophagy in glial cells. This conclusion emerged from the results obtained employing a battery of autophagy markers: a) the increase in LC3-II expression levels, b) the formation of autophagic vesicles labeled with monodansylcadaverine (MDC) or LC3 and, c) the increase in Beclin 1/ Bcl-2 and Beclin 1/ Bcl-X(L) ratio. Autophagy inhibition employing 3-MA and mAtg5(K130R) resulted in decreased cell viability indicating that this event plays a protective role in Mn- induced cell death. In addition, mitophagy was demonstrated by an increase in LC3 and TOM-20 colocalization. On the other hand, we proposed the occurrence of lysosomal membrane permeabilization (LMP) based in the fact that cathepsins B and D activities are essential for cell death. Both cathepsin B inhibitor (Ca-074 Me) or cathepsin D inhibitor (Pepstatin A) completely prevented Mn- induced cytotoxicity. In addition, low dose of Bafilomycin A1 showed a similar effect, a finding that adds evidence about the lysosomal role in Mn cytotoxicity. Finally, in vivo experiments demonstrated that Mn induces injury and alters LC3 expression levels in rat striatal astrocytes. In summary, our results demonstrated that autophagy is activated to counteract the harmful effect caused by Mn. These data is valuable to be considered in future research concerning Manganism therapies.

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 Parkinsons 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.

Glial alterations from early to late stages in a model of Alzheimer's disease: Evidence of autophagy involvement in Aβ internalization.

Alzheimers disease (AD) is a progressive neurodegenerative disease without effective therapy. Brain amyloid deposits are classical histopathological hallmarks that generate an inflammatory reaction affecting neuronal and glial function. The identification of early cell responses and of brain areas involved could help to design new successful treatments. Hence, we studied early alterations of hippocampal glia and their progression during the neuropathology in PDAPP‐J20 transgenic mice, AD model, at 3, 9, and 15 months (m) of age. At 3 m, before deposits formation, microglial Iba1+ cells from transgenic mice already exhibited signs of activation and larger soma size in the hilus, alterations appearing later on stratum radiatum. Iba1 immunohistochemistry revealed increased cell density and immunoreactive area in PDAPP mice from 9 m onward selectively in the hilus, in coincidence with prominent amyloid Congo red + deposition. At pre‐plaque stages, GFAP+ astroglia showed density alterations while, at an advanced age, the presence of deposits was associated with important glial volume changes and apparently being intimately involved in amyloid degradation. Astrocytes around plaques were strongly labeled for LC3 until 15 m in Tg mice, suggestive of increased autophagic flux. Moreover, β‐Amyloid fibrils internalization by astrocytes in in vitro conditions was dependent on autophagy. Co‐localization of Iba1 with ubiquitin or p62 was exclusively found in microglia contacting deposits from 9 m onward, suggesting torpid autophagy. Our work characterizes glial changes at early stages of the disease in PDAPP‐J20 mice, focusing on the hilus as an especially susceptible hippocampal subfield, and provides evidence that glial autophagy could play a role in amyloid processing at advanced stages.

Interplay between lysosomal, mitochondrial and death receptor pathways during manganese-induced apoptosis in glial cells

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

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

&NA; Parkinsons disease is a neurodegenerative movement disorder caused by the loss of dopaminergic neurons from substantia nigra. It is characterized by the accumulation of aggregated &agr;‐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 &agr;‐synuclein. The mechanism by which these events contribute to neural degeneration remains unknown. In the present work we investigated the effect of &agr;‐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 &agr;‐synuclein causes moderated toxicity, ROS generation and mitochondrial dysfunction. In addition, &agr;‐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 &agr;‐synuclein showed activated autophagy and particularly mitophagy. Employing a genetic strategy we demonstrated that autophagy is triggered in order to protect cells from &agr;‐synuclein‐induced cell death. Our results clarify the role of Opa‐1 and Drp‐1 in mitochondrial dynamics and cell survival, a controversial &agr;‐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. HighlightsAlpha‐synuclein induces mitochondrial fragmentation.Opa‐1 protects against mitochondrial disruption and cytotoxicity.Mdivi‐1 inhibits alpha‐ synuclein‐ induced fragmentation.Autophagy protects SH‐SY5HY cells against alpha‐ synuclein induced damage.Mitochondrial fragmentation is associated with superoxide anion generation.

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.