PEP-1-PEA-15 protects against toxin-induced neuronal damage in a mouse model of Parkinson's disease

Background

PEA-15 is abundantly expressed in both neurons and astrocytes throughout the brain. It is a multifunctional protein with the ability to increase cell survival via anti-apoptotic and anti-proliferative properties. However, the function of PEA-15 in neuronal diseases such as Parkinson's disease (PD) remains unclear. In this study, we investigated the protective effects of PEA-15 on neuronal damage induced by MPP⁺ in neuroblastoma SH-SY5Y and BV2 microglia cells and in a MPTP-induced PD mouse model using cell-permeable PEP-1-PEA-15.

Methods

PEP-1-PEA-15 was purified using affinity chromatography. Cell viability and DNA fragmentation were examined by MTT assay and TUNEL staining. Dopaminergic neuronal cell death in the animal model was examined by immunohistochemistry.

Results

PEP-1-PEA-15 transduced into the SH-SY5Y and BV2 cells in a time- and dose-dependent manner. Transduced PEP-1-PEA-15 protected against MPP⁺-induced toxicity by inhibiting intracellular ROS levels and DNA fragmentation. Further, it enhanced the expression levels of Bcl-2 and caspase-3 while reducing the expression levels of Bax and cleaved caspase-3. We found that PEP-1-PEA-15 transduced into the substantia nigra and prevented dopaminergic neuronal cell death in a MPTP-induced PD mouse. Also, we showed the neuroprotective effects in the model by demonstrating that treatment with PEP-1-PEA-15 ameliorated MPTP-induced behavioral dysfunctions and increased dopamine levels in the striatum.

Conclusions

PEP-1-PEA-15 can efficiently transduce into cells and protects against neurotoxin-induced neuronal cell death in vitro and in vivo.

General significance

These results demonstrate the potential for PEP-1-PEA-15 to provide a new strategy for protein therapy treatment of a variety of neurodegenerative diseases including PD.     

Glycyrrhizic Acid Alleviates 6-Hydroxydopamine and Corticosterone-Induced Neurotoxicity in SH-SY5Y Cells Through Modulating Autophagy

Recent researches have shown that autophagy is associated with the pathogenesis of neurodegenerative disorders, but there is no paper to investigate the effects of autophagy modulation on Parkinson’s disease depression (PDD). In addition, glycyrrhizic acid (GA), the major bioactive ingredient of Radix glycyrrhizae, can induce autophagy and ease rotenone-induced Parkinson’s disease (PD). However, there is also no paper to study the action and molecular mechanisms of GA on PDD. In this research, we built the injury model of SH-SY5Y cells through 6-hydroxydopamine (6-OHDA) and corticosterone (CORT). Then, our results showed that GA markedly increased the viability and decreased the apoptosis in SH-SY5Y cells after pre-treating with 6-OHDA and CORT. Moreover, GA notably decreased the expressions of α-Syn and p-S1292-LRRK2 proteins, and significantly increased the levels of CREB and BDNF proteins. Previous papers have suggested that CORT contributed to dopaminergic neurodegeneration via the glucocorticoid (GC)/glucocorticoid receptor (GR) interaction, and our results showed that GA reduced GC level and hypothalamic–pituitary–adrenal (HPA) activity in SH-SY5Y cells by regulating GR signaling pathway. Furthermore, mechanism investigations also showed that GA had the ability to up-regulate the conversion of LC3B II/I and the expression of Beclin-1, and induce autophagy in SH-SY5Y cells, which were reversed by the autophagy inhibitor 3-methyladenine (3-MA). Collectively, these findings proved that GA exerted efficient activity against neurotoxicity in SH-SY5Y cells induced by 6-OHDA and CORT via activation of autophagy, which should be developed as an efficient candidate for treating PDD in the future.     

Changes in iron-regulatory gene expression occur in human cell culture models of Parkinson's disease

Background

Neuronal iron accumulation is thought to be relevant to the pathogenesis of Parkinson’s disease (PD), although the mechanism remains elusive. We hypothesized that neuronal iron uptake may be stimulated by functional mitochondrial iron deficiency.

Objective

To determine firstly whether the mitochondrial toxin, 1-methyl-4-phenylpyridinium iodide (MPP⁺), results in upregulation of iron-import proteins and transporters of iron into the mitochondria, and secondly whether similar changes in expression are induced by toxins with different mechanisms of action.

Methods

We used quantitative PCR and Western blotting to investigate expression of the iron importers, divalent metal transporter, transferrin receptor 1 and 2 (TfR1 and TfR2) and mitoferrin-2 and the iron exporter ferroportin in differentiated SH-SY5Y cells exposed to three different toxins relevant to PD, MPP⁺, paraquat (a free radical generator) and lactacystin (an inhibitor of the ubiquitin-proteasome system (UPS)).

Results

MPP⁺ resulted in increased mRNA and protein levels of genes involved in cellular iron import and transport into the mitochondria. Similar changes occurred following exposure to paraquat, another inducer of oxidative stress. Lactacystin also resulted in increased TfR1 mRNA levels, although the other changes were not found.

Conclusion

Our results support the hypothesis of a functional mitochondrial iron deficit driving neuronal iron uptake but also suggest that differences exist in neuronal iron handling induced by different toxins.     

Tissue transglutaminase cross-links beclin 1 and regulates autophagy in MPP-treated human SH-SY5Y cells

Tissue transglutaminase (tTG) is a cross-linking enzyme involved in protein aggregation during Parkinson’s disease (PD) pathogenesis. Autophagy is inhibited by tTG activation via a mechanism in which cross-linking of beclin 1, an autophagy initiator at the level of the endoplasmic reticulum (ER), has been implicated. We reported increased tTG protein levels and activity at the ER in both PD brain and in a PD-mimicking cell system. Here we characterized the interaction between tTG and beclin 1 at the ER membrane and the role of tTG in reduced autophagy in an in vitro model of PD, using differentiated SH-SY5Y neurons treated with the PD-mimic MPP⁺. We found that under PD-mimicking conditions, beclin 1 and tTG partially colocalized at the ER, beclin 1 levels increased at the ER, and tTG readily cross-linked beclin 1 which was prevented by enzymatic blockade of tTG. Under these conditions, accumulation of beclin 1 at the ER was enhanced by inhibition of tTG activity. In line with these observations and the role of beclin 1 in autophagy, levels of the autophagy marker protein LC3II in MPP⁺-treated cells, were significantly increased by inhibition of tTG activity. Our data provide first evidence for a role of tTG-mediated regulation of beclin 1 and autophagy in MPP⁺-treated human SH-SY5Y cells.     

Detoxified Extract of <Emphasis Type="Italic">Rhus verniciflua</Emphasis> Stokes Inhibits Rotenone-Induced Apoptosis in Human Dopaminergic Cells,SH-SY5Y

Rhus verniciflua Stokes (RVS), traditionally used as a food supplement and in traditional herbal medicine for centuries in Korea, is known to possess various pharmacological properties. Environmental neurotoxins such as rotenone, a specific inhibitor of complex I provide models of Parkinson’s disease (PD) both in vivo and in vitro. In this study, we investigated the neuroprotective effect of RVS against rotenone-induced toxicity in human dopaminergic cells, SH-SY5Y. Cells exposed to rotenone for 24 h-induced cellular injury and apoptotic cell death. Pretreatment of cells with RVS provided significant protection to SH-SY5Y cells. Further, RVS offered remarkable protection against rotenone-induced oxidative stress and markedly inhibited mitochondrial membrane potential (MMP) disruption. RVS also attenuated the up-regulation of Bax, Caspase-9 and Caspase-3 and down-regulation of Bcl-2. Moreover, pretreatment with RVS prevented the decrease in tyrosine hydroxylase (TH) levels in SH-SY5Y cells. Interestingly, RVS conferred profound protection to human dopaminergic cells by preventing the downregulation of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). These results suggest that RVS may protect dopaminergic neurons against rotenone-induced apoptosis by multiple functions and contribute to neuroprotection in neurodegenerative diseases, such as PD.     

AMP-activated protein kinase is activated in Parkinson’s disease models mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

The selective loss of dopaminergic neurons in the substantia nigra pars compacta is a feature of Parkinson’s disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is the most common experimental model used to investigate the pathogenesis of PD. Administration of MPTP in mice produces neuropathological defects as observed in PD and 1-methyl-4-pyridinium (MPP⁺) induces cell death when neuronal cell cultures are used. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. In the present study, we demonstrated that AMPK is activated by MPTP in mice and MPP⁺ in SH-SY5Y cells. The inhibition of AMPK by compound C resulted in an increase in MPP⁺-induced cell death. We further showed that overexpression of AMPK increased cell viability after exposure to MPP⁺ in SH-SY5Y cells. Based on these results, we suggest that activation of AMPK might prevent neuronal cell death and play a role as a survival factor in PD.     

Rutin from Dendropanax morbifera Leveille Protects Human Dopaminergic Cells Against Rotenone Induced Cell Injury Through Inhibiting JNK and p38 MAPK Signaling

Dendropanax morbifera Leveille (Araliaceae) is well known in Korean traditional medicine for a variety of diseases. Rotenone is a commonly used neurotoxin to produce in vivo and in vitro Parkinson’s disease models. This study was designed to elucidate the processes underlying neuroprotection of rutin, a bioflavonoid isolated from D. morbifera Leveille in cellular models of rotenone-induced toxicity. We found that rutin significantly decreased rotenone-induced generation of reactive oxygen species levels in SH-SY5Y cells. Rutin protected the increased level of intracellular Ca²⁺ and depleted level of mitochondrial membrane potential (ΔΨm) induced by rotenone. Furthermore, it prevented the decreased ratio of Bax/Bcl-2 caused by rotenone treatment. Additionally, rutin protected SH-SY5Y cells from rotenone-induced caspase-9 and caspase-3 activation and apoptotic cell death. We also observed that rutin repressed rotenone-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinase phosphorylation. These results suggest that rutin may have therapeutic potential for the treatment of neurodegenerative diseases associated with oxidative stress.     

Circulating hormone adrenomedullin and its binding protein protect neural cells from hypoxia-induced apoptosis

Background

Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. Neural cells exposed to ischemia can undergo apoptosis. Adrenomedullin (AM) in combination with its enhancing binding protein, AMBP-1, has been shown to reduce tissue damage in inflammation.

Methods

To evaluate a beneficial effect of AM/AMBP-1 administration in brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma SH-SY5Y cells.

Results

After exposure to 1% O₂ for 20 h, neural cells were injured with decreased ATP levels and increased LDH release. Pre-administration of AM/AMBP-1 significantly reduced hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and activation of caspase-3, compared to cells exposed to hypoxia alone. AM/AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia exposure. Correspondingly, an elevation of cAMP levels by forskolin protected neural cells from hypoxia-induced injury. Inhibition of PKA by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced apoptosis.

Conclusions

AM/AMBP-1 elevates cAMP levels, followed by activating PKA, to protect neural cells from the injury caused by hypoxia.

General significance

AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.     

p38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells

Aims

This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells.

Main methods

CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit.

Key findings

Among the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC₅₀ of 45 μM. Ft1 not only arrested the cell cycle at S, G₂/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1.

Significance

Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.     

Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells

Background

Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease.

Methods

We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging.

Results

Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90 nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional.

Conclusions

We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells.

General significance

The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomer distribution and properties, and the study of their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms of Parkinson's disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models.     

Glutaredoxin 1 is a major player in copper metabolism in neuroblastoma cells

Background

Glutaredoxin 1 (Grx1), a small protein belonging to the thioredoxin family, is involved in redox-regulation since it catalyzes the reduction of protein disulfides and that of mixed disulfides. It was reported to modulate active copper extrusion from cells, by affecting the function of the pumps ATP7A and B. These are components of the network of protein chaperones involved in the control of the homeostasis of copper, an essential, though harmful, metal. However, the effect of Grx1 on copper levels, copper chaperones and copper-elicited cell toxicity was never investigated.

Methods

In order to investigate the effect of Grx1 on copper metabolism, we constitutively overexpressed Grx1 in human neuroblastoma SH-SY5Y cells (SH-Grx1 cells) and assessed a number of copper-related parameters.

Results

SH-Grx1 cells show a basal intracellular copper level higher than control cells, accumulate more copper upon CuSO₄ treatment, but are more resistant to copper-induced toxicity. Grx1 shows copper-binding properties and copper overload produces a decrease of Grx1 enzyme activity in SH-Grx1 cells. Finally, Grx1 overexpression decreases copper accumulation in mitochondria upon copper overload and modulates the expression of copper transporter 1 (Ctr1).

Conclusion

Altogether, these data demonstrate that Grx1 is a major player in copper metabolism in neuronal cells.     

Sleep Disorders in Parkinson’s Disease: Clinical Features,Iron Metabolism and Related Mechanism

Objective

To investigate clinical features, iron metabolism and neuroinflammation in Parkinson’s disease (PD) patients with sleep disorders (SD).

Methods

211 PD patients were evaluated by Pittsburgh Sleep Quality Index (PSQI) and a body of scales for motor symptoms and non-motor symptoms. 94 blood and 38 cerebral spinal fluid (CSF) samples were collected and iron and its metabolism-relating proteins, neuroinflammatory factors were detected and analyzed.

Results

136 cases (64.5%) of PD patients were accompanied by SD. Factor with the highest score in PSQI was daytime dysfunction. Depression, restless leg syndrome, autonomic symptoms and fatigue contributed 68.6% of the variance of PSQI score. Transferrin level in serum and tumor necrosis factor–α level in CSF decreased, and the levels of iron, transferrin, lactoferrin and prostaglandin E₂ in CSF increased in PD patients with SD compared with those without SD. In CSF, prostaglandin E₂ level was positively correlated with the levels of transferrin and lactoferrin, and tumor necrosis factor–α level was negatively correlated with the levels of iron, transferrin and lactoferrin in CSF.

Conclusions

Depression, restless leg syndrome, autonomic disorders and fatigue are the important contributors for the poor sleep in PD patients. Abnormal iron metabolism may cause excessive iron deposition in brain and be related to SD in PD patients through dual potential mechanisms, including neuroinflammation by activating microglia and neurotoxicity by targeting neurons. Hence, inhibition of iron deposition-related neuroinflammation and neurotoxicity may cast a new light for drug development for SD in PD patients.     

Celastrol protects human neuroblastoma SH-SY5Y cells from rotenone-induced injury through induction of autophagy

Celastrol, an active component found in the Chinese herb tripterygium wilfordii has been identified as a neuroprotective agent for neurodegenerative diseases including Parkinson’s disease (PD) through unknown mechanism. Celastrol can induce autophagy, which plays a neuroprotective role in PD. We tested the protective effect of celastrol on rotenone-induced injury and investigated the underlying mechanism using human neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were treated with celastrol before rotenone exposure. The cells survival, apoptosis, accumulation of α-synuclein, oxidative stress and mitochondrial function, and autophagy production were analyzed. We found celastrol (500 nM) pre-treatment enhanced cell viability (by 28.99%, P < 0.001), decreased cell apoptosis (by 54.38%, P < 0.001), increased SOD and GSH (by 120.53% and 90.46%, P < 0.01), reduced accumulation of α-synuclein (by 35.93%, P < 0.001) and ROS generation (by 33.99%, P < 0.001), preserved MMP (33.93 ± 3.62%, vs. 15.10 ± 0.71% of JC-1 monomer, P < 0.001) and reduced the level of cytochrome C in cytosol (by 45.57%, P < 0.001) in rotenone treated SH-SY5Y cells. Moreover, celastrol increased LC3-II/LC3 I ratio by 60.92% (P < 0.001), indicating that celastrol activated autophagic pathways. Inhibiting autophagy by 3-methyladenine (3-MA) abolished the protective effects of celastrol. Our results suggested that celastrol protects SH-SY5Y cells from rotenone induced injuries and autophagic pathway is involved in celastrol neuroprotective effects.     

Effects of chronic low dose rotenone treatment on human microglial cells

Background

Exposure to toxins/chemicals is considered to be a significant risk factor in the pathogenesis of Parkinson's disease (PD); one putative chemical is the naturally occurring herbicide rotenone that is now used widely in establishing PD models. We, and others, have shown that chronic low dose rotenone treatment induces excessive accumulation of Reactive Oxygen Species (ROS), inclusion body formation and apoptosis in dopaminergic neurons of animal and human origin. Some studies have also suggested that microglia enhance the rotenone induced neurotoxicity. While the effects of rotenone on neurons are well established, there is little or no information available on the effect of rotenone on microglial cells, and especially cells of human origin. The aim of the present study was to investigate the effects of chronic low dose rotenone treatment on human microglial CHME-5 cells.

Methods

We have shown previously that rotenone induced inclusion body formation in human dopaminergic SH-SY5Y cells and therefore used these cells as a control for inclusion body formation in this study. SH-SY5Y and CHME-5 cells were treated with 5 nM rotenone for four weeks. At the end of week 4, both cell types were analysed for the presence of inclusion bodies, superoxide dismutases and cell activation (only in CHME-5 cells) using Haematoxylin and Eosin staining, immunocytochemical and western blotting methods. Levels of active caspases and ROS (both extra and intra cellular) were measured using biochemical methods.

Conclusion

The results suggest that chronic low dose rotenone treatment activates human microglia (cell line) in a manner similar to microglia of animal origin as shown by others. However human microglia release excessive amounts of ROS extracellularly, do not show excessive amounts of intracellular ROS and active caspases and most importantly do not show any protein aggregation or inclusion body formation. Human microglia appear to be resistant to rotenone (chronic, low dose) induced damage.

     

Chronic hypoxia leads to a glycolytic phenotype and suppressed HIF-2 signaling in PC12 cells

Background

Along with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated.

Methods

Proliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O₂ and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O₂.

Results

Hypoxic PC12 cells (H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O₂ deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected.

Conclusions

In chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia.

General significance

Demonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other.     

Increase in anti-apoptotic molecules,nucleolin, and heat shock protein 70, against upregulated LRRK2 kinase activity

Leucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson’s disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression.

Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson’s disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species     

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7

Background

Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses.

Methods

In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~ 30 nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD₅₀ doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM.

Results

This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis.

Conclusions

A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells.

General significance

CuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.     

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.     

Autophagy in the Human Placenta throughout Gestation

Background

Autophagy has been reported to be essential for pre-implantation development and embryo survival. However, its role in placental development and regulation of autophagy during pregnancy remain unclear. The aims of this study were to (1) study autophagy by characterizing changes in levels of beclin-1, DRAM, and LC3B in human placenta throughout gestation; (2) determine whether autophagy is involved in regulation of trophoblast invasion in JEG-3 cells (a choriocarcinoma cell line); (3) examine the effects of reduced oxygen and glucose on the autophagic changes; and (4) investigate the effect of reoxygenation and supplementation of glucose after oxygen-glucose deprivation (OGD) on the autophagic changes in primary cytotrophoblasts obtained from normal term pregnancy.

Methodology/Principal Findings

An analysis of 40 placental samples representing different gestational stages showed (1) no significant differences in beclin-1, DRAM, and LC3B-II levels in placentas between early and mid-gestation, and late gestation with vaginal delivery; (2) placentas from late gestation with cesarean section had lower levels of LC3B-II compared to early and mid-gestation, and late gestation with vaginal delivery; levels of DRAM were also lower compared to placentas from early and mid-gestation; and (3) using explant cultures, villous tissues from early and late gestation had similar rates of autophagic flux under physiological oxygen concentrations. Knockdown of BECN1, DRAM, and LC3B had no effects on viability and invasion activity of JEG-3 cells. On the other hand, OGD caused a significant increase in the levels of LC3B-II in primary cytotrophoblasts, while re-supplementation of oxygen and glucose reduced these changes. Furthermore, there were differential changes in levels of beclin-1, DRAM, and LC3B-II in response to changes in oxygen and glucose levels.

Conclusions/Significance

Our results indicate that autophagy is involved in development of the human placenta and that changes in oxygen and glucose levels participate in regulation of autophagic changes in cytotrophoblast cells.     

Interleukin-10 blocks in vitro replication of human cytomegalovirus by inhibiting the virus-induced autophagy in MRC5 cells

Background

Interleukin-10 is an important cytokine that regulates immune response. Previous studies have shown that human cytomegalovirus can trigger cell autophagy during the early stages of infection. To our knowledge, whether IL-10 inhibits HCMV-induced autophagy and virus replication has not been studied previously.

Objectives

We investigated whether IL-10 affects cell viability and autophagy under the conditions of starvation and HCMV infection by using the MRC5 cell line. We also explored the role of IL-10-mediated autophagy on HCMV replication.

Results

Our data showed that IL-10 inhibited the autophagic flux of the MRC5 cells irrespective of starvation or HCMV infection, and suppressed HCMV replication. The promotion of autophagy with either a pharmacological inducer (rapamycin), or a technique to over-express the BECN1 gene reversed the effect of IL-10 on virus replication. Furthermore, the PI3K/Akt signal pathway was activated when the cells were pretreated with IL-10.

Conclusions

Our results indicated that IL-10 can suppress HCMV replication by inhibiting autophagy in host cells during the early stages of infection.