Autophagy In Astrocytes Research Articles

HIV-1 differentially modulates autophagy in neurons and astrocytes

Autophagy, a lysosomal degradative pathway that maintains cellular homeostasis, has emerged as an innate immune defense against pathogens. The role of autophagy in the deregulated HIV-infected central nervous system (CNS) is unclear. We have found that HIV-1-induced neuro-glial (neurons and astrocytes) damage involves modulation of the autophagy pathway. Neuro-glial stress induced by HIV-1 led to biochemical and morphological dysfunctions. X4 HIV-1 produced neuro-glial toxicity coupled with suppression of autophagy, while R5 HIV-1-induced toxicity was restricted to neurons. Rapamycin, a specific mTOR inhibitor (autophagy inducer) relieved the blockage of the autophagy pathway caused by HIV-1 and resulted in neuro-glial protection. Further understanding of the regulation of autophagy by cytokines and chemokines or other signaling events may lead to recognition of therapeutic targets for neurodegenerative diseases.

Heme oxygenase‐1 modulates microRNA expression in cultured astroglia: Implications for chronic brain disorders

Over-expression of the heme-degrading enzyme, heme oxygenase-1 (HO-1) promotes iron deposition, mitochondrial damage, and autophagy in astrocytes and enhances the vulnerability of nearby neuronal constituents to oxidative injury. These neuropathological features and aberrant brain microRNA (miRNA) expression patterns have been implicated in the etiopathogeneses of various neurodevelopmental and aging-related neurodegenerative disorders. To correlate glial HO-1 overexpression with altered miRNA patterns, which have been linked to the aforementioned "core" neuropathological features. miRNA microchip assays were performed on HMOX1- and sham-transfected primary rat astroglia and affected miRNAs were further validated by qPCR. The roles of the heme degradation products, carbon monoxide (CO), iron (Fe) and bilirubin on miRNA expression were assessed and salient mRNA targets of the impacted miRNAs were ascertained. In HMOX1-transfected astrocytes, rno-miR-140*, rno-miR-17, and rno-miR-16 were significantly up-regulated, and rno-miR-297, rno-miR-206, rno-miR-187, rno-miR-181a, rno-miR-138 and rno-miR-29c were down-regulated, compared to sham-transfected controls. CO and Fe were implicated in the HMOX1 effects, whereas bilirubin was inert or counteracted the HMOX1-related changes. mRNA levels of Ngfr, Vglut1, Mapk3, Tnf-α, and Sirt1, known targets of the down-regulated miRNAs and abnormal in various human brain disorders, were significantly increased in the HMOX-1-transfected astrocytes. In chronic CNS disorders, altered expression of salient miRNAs and their mRNA targets may contribute to the neural damage accruing from the over-expression of glial HO-1.

Autophagy Mediates Astrocyte Death During Zinc-Potentiated Ischemia--Reperfusion Injury.

Pathological release of excess zinc ions and the resultant increase in intracellular zinc has been implicated in ischemic brain cell death, although the underlying mechanisms are not fully understood. Since zinc promotes the formation of the autophagic signal, reactive oxygen species (ROS), and increases autophagy, a known mechanism of cell death, we hypothesized that autophagy is involved in zinc-induced hypoxic cell death. To study this hypothesis, we determined the effect of zinc on autophagy and ROS generation in C8-D1A astrocytes subjected to hypoxia and rexoygenation (H/R), simulating ischemic stroke. C8-D1A astrocytes subjected to 3-h hypoxia and 18-h reoxygenation exhibited dramatically increased autophagy and astrocyte cell death in the presence of 100 μM zinc. Pharmacological inhibition of autophagy decreased zinc-potentiated H/R-induced cell death, while scavenging ROS reduced both autophagy and cell death caused by zinc-potentiated H/R. These data indicate that zinc-potentiated increases in ROS lead to over-exuberant autophagy and increased cell death in H/R-treated astrocytes. Furthermore, our elucidation of this novel mechanism indicates that modulation of autophagy, ROS, and zinc levels may be useful targets in decreasing brain damage during stroke.

Autophagy is differently regulated in astrocytes and microglia exposed to environmental toxic molecules

Autophagy is generally considered a degradation pathway involved in many neurodegenerative processes. It can be observed in different stress conditions such as starvation generally improving cell survival. Our previous results described the occurrence of autophagy in neuronal cultures exposed to the toxic compound trimethyltin (TMT) (1). TMT belongs to a family of organotin compounds with wide industrial and agricultural applications, especially as heat stabilizers in PVC production and as biocides. In the nervous system TMT determines the selective destruction of neurons in specific brain regions such as the olfactory bulb and the hippocampus. When this toxic molecule was administered to glial cells we observed in astrocytes a rapid block of the autophagic flux and a consequent increased expression of LC3 and p62 which can be observed both in cultured astrocytes and in the brain of intoxicated animals. Conversely, in microglia autophagy was not impaired in the same conditions and p62 accumulation was not observed neither in vitro primary cultures, nor in brain sections of TMT-treated rats. The protein p62 (also known as SQTM1) is known to be selectively degraded through autophagy and its accumulation activates the transcription factor Nrf2 by sequestering Keap1 (2). To note the block of autophagy has been reported to exert an immunosopressive effect in macrophages (3). Thus, the impairment of autophagy in astrocytes could be related to their limited production of pro-inflammatory cytokines and nitric oxide respect to microglia observed after TMT treatment.

Ginsenoside compound K promotes β-amyloid peptide clearance in primary astrocytes via autophagy enhancement.

The aim of the present study was to investigate the effect of ginsenoside compound K on β-amyloid (Aβ) peptide clearance in primary astrocytes. Aβ degradation in primary astrocytes was determined using an intracellular Aβ clearance assay. Aggregated LC3 in astrocyte cells, which is a marker for the level of autophagy, was detected using laser scanning confocal microscope. The effect of compound K on the mammalian target of rapamycin (mTOR)/autophagy pathway was determined using western blot analysis, and an enzyme-linked immunosorbent assay was used for Aβ detection. The results demonstrated that compound K promoted the clearance of Aβ and enhanced autophagy in primary astrocytes. In addition, it was found that phosphorylation of mTOR was inhibited by compound K, which may have contributed to the enhanced autophagy. In conclusion, compound K promotes Aβ clearance by enhancing autophagy via the mTOR signaling pathway in primary astrocytes.

Human stefin B role in cell's response to misfolded proteins and autophagy.

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

AMP-activated protein kinase is involved in induction of protective autophagy in astrocytes exposed to oxygen-glucose deprivation.

AMP-activated kinase (AMPK) acts as the intracellular ATP depletion sensor, which detects and limits increases in the AMP/ATP ratio. AMPK may be significantly activated under stress conditions that deplete cellular ATP levels such as ischemia/hypoxia or glucose deprivation. Recent studies strongly suggest that AMPK participates in autophagy regulation, but it is not known whether AMPK activated by ischemia regulates autophagy in astrocytes and the consequence of autophagy activation in ischemic astrocytes are unclear. We have investigated the contribution of AMPK to autophagy activation in rat primary astrocyte cultures subjected to ischemia-simulating conditions (combined oxygen glucose deprivation, OGD) and its potential effects on astrocyte damage induced by OGD (1-12 h). The evidence supports the conclusion that AMPK activation at early stages of OGD is involved in induction of protective autophagy in astrocytes. Inhibition of AMPK, either by siAMPKα1 or by compound C, significantly attenuated the expression of autophagy-related proteins and decrease of astrocyte viability following OGD. The findings provide additional data about the role of AMPK in ischemic astrocytes and downstream responses that may be involved in OGD-induced protective autophagy.

A Proautophagic Antiviral Role for the Cellular Prion Protein Identified by Infection with a Herpes Simplex Virus 1 ICP34.5 Mutant

The cellular prion protein (PrP) often plays a cytoprotective role by regulating autophagy in response to cell stress. The stress of infection with intracellular pathogens can stimulate autophagy, and autophagic degradation of pathogens can reduce their replication and thus help protect the infected cells. PrP also restricts replication of several viruses, but whether this activity is related to an effect on autophagy is not known. Herpes simplex virus 1 (HSV-1) effectively counteracts autophagy through binding of its ICP34.5 protein to the cellular proautophagy protein beclin-1. Autophagy can reduce replication of an HSV-1 mutant, Δ68H, which is incapable of binding beclin-1. We found that deletion of PrP in mice complements the attenuation of Δ68H, restoring its capacity to replicate in the central nervous system (CNS) to wild-type virus levels after intracranial or corneal infection. Cultured primary astrocytes but not neurons derived from PrP(-/-) mice also complemented the attenuation of Δ68H, enabling Δ68H to replicate at levels equivalent to wild-type virus. Ultrastructural analysis showed that normal astrocytes exhibited an increase in the number of autophagosomes after infection with Δ68H compared with wild-type virus, but PrP(-/-) astrocytes failed to induce autophagy in response to Δ68H infection. Redistribution of EGFP-LC3 into punctae occurred more frequently in normal astrocytes infected with Δ68H than with wild-type virus, but not in PrP(-/-) astrocytes, corroborating the ultrastructural analysis results. Our results demonstrate that PrP is critical for inducing autophagy in astrocytes in response to HSV-1 infection and suggest that PrP positively regulates autophagy in the mouse CNS.

Evodiamine Induces Transient Receptor Potential Vanilloid-1-Mediated Protective Autophagy in U87-MG Astrocytes

Cerebral ischemia is a leading cause of mortality and morbidity worldwide, which results in cognitive and motor dysfunction, neurodegenerative diseases, and death. Evodiamine (Evo) is extracted from Evodia rutaecarpa Bentham, a plant widely used in Chinese herbal medicine, which possesses variable biological abilities, such as anticancer, anti-inflammation, antiobesity, anti-Alzheimer's disease, antimetastatic, antianoxic, and antinociceptive functions. But the effect of Evo on ischemic stroke is unclear. Increasing data suggest that activation of autophagy, an adaptive response to environmental stresses, could protect neurons from ischemia-induced cell death. In this study, we found that Evo induced autophagy in U87-MG astrocytes. A scavenger of extracellular calcium and an antagonist of transient receptor potential vanilloid-1 (TRPV-1) decreased the percentage of autophagy accompanied by an increase in apoptosis, suggesting that Evo may induce calcium-mediated protective autophagy resulting from an influx of extracellular calcium. The same phenomena were also confirmed by a small interfering RNA technique to knock down the expression of TRPV1. Finally, Evo-induced c-Jun N-terminal kinases (JNK) activation was reduced by a TRPV1 antagonist, indicating that Evo-induced autophagy may occur through a calcium/c-Jun N-terminal kinase (JNK) pathway. Collectively, Evo induced an influx of extracellular calcium, which led to JNK-mediated protective autophagy, and this provides a new option for ischemic stroke treatment.

The neurosteroids, allopregnanolone and progesterone, induce autophagy in cultured astrocytes

Recent studies have suggested that neurosteroids such as pregnenolone, progesterone (PG) and their derivatives, have a role in activating autophagy in addition to diverse other functions. In our previous studies, we demonstrated that cellular free Zn2+ is involved in oxidative stress-induced autophagy and autophagic cell death in astrocytes. In the present study, we examined the possibility that neurosteroids, allopregnanolone (Allo) and PG, also activate autophagy in cultured mouse astrocytes through modulation of intracellular Zn2+.Exposure of astrocytes to 250nM Allo or 500nM PG caused cytosolic vacuoles to appear within a few hours of treatment onset. Live-cell confocal microscopy of astrocytes transfected with red fluorescent protein-conjugated LC3 (RFP-LC3), a marker for autophagic vacuoles (AVs), as well as transmission electron microscopy, revealed that these vacuoles were AVs. In addition, Western blots showed increases in LC3-II levels. Interestingly, mTOR and Akt were concurrently activated, and their blockade further increased LC3-II levels and caused some cell death. These results indicate that co-activation of mTOR and Akt may act to limit neurosteroid-induced autophagy and thus inhibit autophagic cell death. As in other cases of autophagy, cellular Zn2+ levels increased after treatment with neurosteroids. The neurosteroid-induced increase in LC3-II levels was inhibited by addition of the Zn2+ chelator TPEN. Both the increase in LC3-II levels and activation of Akt and mTOR by neurosteroids were all mediated by PG receptors, as the effects were blocked by the addition of RU-486, a PG receptor antagonist. Moreover, mutant huntingtin (mHtt) aggregates in GFP-mHttQ74-transfected astrocytes were substantially reduced by neurosteroid treatment, indicating that neurosteroid-induced autophagy may be functional.Present results demonstrate that Allo and PG activate autophagy in astrocytes. Notably, unlike several other autophagy inducers that, in excess, may cause autophagic cell death, Allo and PG are relatively non-toxic, possibly because of concurrent Akt and mTOR activation. Thus, as natural endogenous brain substances, Allo and PG may have a potential as therapeutic agents in neurodegenerative conditions in which abnormal protein aggregates are involved.

Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

Autophagy is a highly regulated cellular mechanism that leads to degradation of long-lived proteins and dysfunctional organelles. The process has been implicated in a variety of physiological and pathological conditions relevant to neurological diseases. Recent studies show the existence of autophagy in cerebral ischemia, but no consensus has yet been reached regarding the functions of autophagy in this condition. This article highlights the activation of autophagy during cerebral ischemia and/or reperfusion, especially in neurons and astrocytes, as well as the role of autophagy in neuronal or astrocytic cell death and survival. We propose that physiological levels of autophagy, presumably caused by mild to modest hypoxia or ischemia, appear to be protective. However, high levels of autophagy caused by severe hypoxia or ischemia and/or reperfusion may cause self-digestion and eventual neuronal and astrocytic cell death. We also discuss that oxidative and endoplasmic reticulum (ER) stresses in cerebral hypoxia or ischemia and/or reperfusion are potent stimuli of autophagy in neurons and astrocytes. In addition, we review the evidence suggesting a considerable overlap between autophagy on one hand, and apoptosis, necrosis and necroptosis on the other hand, in determining the outcomes and final morphology of damaged neurons and astrocytes.

Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia

The present study evaluated autophagy activation in astrocytes and its contribution to astrocyte injury induced by cerebral ischemia and hypoxia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. In vitro hypoxia in cultured primary astrocytes was induced by the oxygen-glucose deprivation (OGD). Alterations of astrocytes were evaluated with astroglia markers glial fibrillary acidic protein (GFAP). The formation of autophagosomes in astrocytes was examined with transmission electron microscopy (TEM). The expression of autophagy-related proteins were examined with immunoblotting. The role of autophagy in OGD or focal cerebral ischemia-induced death of astrocytes was assessed by pharmacological inhibition of autophagy with 3-methyladenine (3-MA) or bafilomycin A1 (Baf). The results showed that GFAP staining was reduced in the infarct brain areas 3-12 h following pMCAO. Cerebral ischemia or OGD induced activation of autophagy in astrocytes as evidenced by the increased formation of autophagosomes and autolysosomes and monodansylcadaverine (MDC)-labeled vesicles; the increased production of microtubule-associated protein 1 light chain 3 (LC3-II); the upregulation of Beclin 1, lysosome-associated membrane protein 2 (LAMP2) and lysosomal cathepsin B expression; and the decreased levels of cytoprotective Bcl-2 protein in primary astrocytes. 3-MA inhibited OGD-induced the increase in LC3-II and the decline in Bcl-2. Furthermore, 3-MA and Baf slightly but significantly attenuated OGD-induced death of astrocytes. 3-MA also significantly increased the number of GFAP-positive cells and the protein levels of GFAP in the ischemic cortex core 12 h following pMCAO. These results suggest that ischemia or hypoxia-induced autophagic/lysosomal pathway activation may at least partly contribute to ischemic injury of astrocytes.

NF-κB as a common signaling pathway in ganglioside-induced autophagic cell death and activation of astrocytes

We have previously shown that gangliosides induce autophagic cell death of brain astrocytes. As gangliosides are also known to induce inflammatory activation of astrocytes, we hypothesized that a canonical inflammatory signaling pathway NF-κB might be involved in the ganglioside-induced astrocyte cell death and activation. Using cultured mouse astrocytes and C6 rat glioma cell line, we determined the role of NF-κB in autophagic cell death and nitric oxide (NO) production in astrocytes. Gangliosides induced iNOS/GFAP expression and NF-κB activation. IKK inhibitor SC-514 and NF-κB inhibitor PDTC reduced ganglioside-induced astrocyte activation and cell death. Moreover, inhibition of NF-κB pathway also attenuated autophagy of astrocytes. Rho subfamily of small G proteins antagonized the ganglioside-induced astrocyte cell death as well as activation pathways. Taken together, IKK/NF-κB may constitute one of the common signaling pathways in ganglioside-induced astrocyte activation and autophagic cell death, and may play an important role in the ganglioside intracellular signaling that regulates astrocyte physiology and pathology.

Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes

Zinc dyshomeostasis has been recognized as an important mechanism for cell death in acute brain injury. An increase in the level of free or histochemically reactive zinc in astrocytes and neurons is considered one of the major causes of death of these cells in ischemia and trauma. Although zinc dyshomeostasis can lead to cell death via diverse routes, the major pathway appears to involve oxidative stress.Recently, we found that a rise of zinc in autophagic vacuoles, including autolysosomes, is a prerequisite for lysosomal membrane permeabilization and cell death in cultured brain cells exposed to oxidative stress conditions. The source of zinc in this process is likely redox-sensitive zinc-binding proteins such as metallothioneins, which release zinc under oxidative conditions. Of the metallothioneins, metallothionein-3 is especially enriched in the central nervous system, but its physiologic role in this tissue is not well established. Like other metallothioneins, metallothionein-3 may function as metal detoxicant, but is also known to inhibit neurite outgrowth and, sometimes, promote neuronal death, likely by serving as a source of toxic zinc release. In addition, metallothionein-3 regulates lysosomal functions. In the absence of metallothionein-3, there are changes in lysosome-associated membrane protein-1 and -2, and reductions in certain lysosomal enzymes that result in decreased autophagic flux. This may have dual effects on cell survival. In acute oxidative injury, zinc dyshomeostasis and lysosomal membrane permeabilization are diminished in metallothionein-3 null cells, resulting in less cell death. But over the longer term, diminished lysosomal function may lead to the accumulation of abnormal proteins and cause cytotoxicity.The roles of zinc and metallothionein-3 in autophagy and/or lysosomal function have just begun to be investigated. In light of evidence that autophagy and lysosomes may play significant roles in the pathogenesis of various neurological diseases, further insight into the contribution of zinc dynamics and metallothionein-3 function may help provide ways to effectively regulate these processes in brain cells.

Defective autophagy in neurons and astrocytes from mice deficient in PI(3,5)P2

Mutations affecting the conversion of PI3P to the signaling lipid PI(3,5)P2 result in spongiform degeneration of mouse brain and are associated with the human disorders Charcot–Marie–Tooth disease and amyotrophic lateral sclerosis (ALS). We now report accumulation of the proteins LC3-II, p62 and LAMP-2 in neurons and astrocytes of mice with mutations in two components of the PI(3,5)P2 regulatory complex, Fig4 and Vac14. Cytoplasmic inclusion bodies containing p62 and ubiquinated proteins are present in regions of the mutant brain that undergo degeneration. Co-localization of p62 and LAMP-2 in affected cells indicates that formation or recycling of the autolysosome is impaired. These results establish a role for PI(3,5)P2 in autophagy in the mammalian central nervous system (CNS) and demonstrate that mutations affecting PI(3,5)P2 can contribute to inclusion body disease.

Oxidative injury triggers autophagy in astrocytes: The role of endogenous zinc

We have recently demonstrated that the accumulation of labile zinc in lysosomes during oxidative stress causes lysosomal membrane permeabilization (LMP) in cultured hippocampal neurons. Since autophagy involves fusion of autophagic vacuoles (AVs) with lysosomes, zinc accumulation may start in AVs. In the present study, we examined the role of endogenous zinc in H2O2-induced autophagy and cell death in mouse astrocyte cultures. Live-cell confocal imaging of astrocytes transfected with GFP-LC3 revealed that the number of AVs positive for LC3 (microtubule-associated protein 1 light chain 3) increased following exposure to H2O2 or ferrous chloride (FeCl2). Staining of RFP-LC3-transfected astrocytes with FluoZin-3 indicated that the levels of labile zinc increased in AVs as well as in the cytosol and nuclei. The majority of AVs were double-stained with LysoTracker, indicating that they were fused with lysosomes. Chelation of zinc with tetrakis [2-pyridylmethyl]ethylenediamine (TPEN) decreased the number of AVs in H2O2-treated astrocytes, whereas exposure to zinc increased their number, suggesting that dysregulation of zinc homeostasis is mechanistically linked to autophagy. Unexpectedly, inhibition of autophagy blocked the rise in labile zinc levels. Astrocytic death induced by H2O2) was ccompanied by LMP. Autophagy inhibitors (3-methyladenine, bafilomycin-1) or TPEN attenuated LMP and cell death in astrocytes. These results support the possibility that endogenous zinc plays a key role in autophagy under oxidative stress in astrocytes, and suggest that autophagy is a necessary preceding event for LMP and cell death in oxidative injury.

Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H(2)O(2)) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H(2)O(2) or 2-ME-induced cell death. H(2)O(2) and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H(2)O(2) or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H(2)O(2)- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H(2)O(2) or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells.