Potential compensatory responses through autophagic/lysosomal pathways in neurodegenerative diseases

Intracellular protein degradation decreases with age, altering the important balance between protein synthesis and breakdown. Slowly, protein accumulation events increase causing axonopathy, synaptic deterioration, and subsequent cell death. As toxic species accumulate, autophagy-lysosomal protein degradation pathways are activated. Responses include autophagic vacuoles that degrade damaged cellular components and long-lived proteins, as well as enhanced levels of lysosomal hydrolases. Although such changes correlate with neuronal atrophy in age-related neurodegenerative disorders and in related models of protein accumulation, the autophagic/lysosomal responses appear to be compensatory reactions. Recent studies indicate that protein oligomerization/ aggregation induces autophagy and activates lysosomal protein degradation in an attempt to clear toxic accumulations. Such compensatory responses may delay cell death and account for the gradual nature of protein deposition pathology that can extend over months/years in model systems and years/decades in the human diseases. Correspondingly, enhancement of compensatory pathways shifts the balance from pathogenesis to protection. Positive modulation of protein degradation processes represents a strategy to promote clearance of toxic accumulations and to slow the synaptopathogenesis and associated cognitive decline in aging-related dementias.     

Neurodegenerative lysosomal disorders: a continuum from development to late age

Neuronal survival requires continuous lysosomal turnover of cellular constituents delivered by autophagy and endocytosis. Primary lysosomal dysfunction in inherited congenital "lysosomal storage" disorders is well known to cause severe neurodegenerative phenotypes associated with accumulations of lysosomes and autophagic vacuoles (AVs). Recently, the number of inherited adult-onset neurodegenerative diseases caused by proteins that regulate protein sorting and degradation within the endocytic and autophagic pathways has grown considerably. In this Perspective, we classify a group of neurodegenerative diseases across the lifespan as disorders of lysosomal function, which feature extensive autophagic-endocytic-lysosomal neuropathology and may share mechanisms of neurodegeneration related to degradative failure and lysosomal destabilization. We highlight Alzheimer's disease as a disease within this group and discuss how each of the genes and other risk factors promoting this disease contribute to progressive lysosomal dysfunction and neuronal cell death.     

Increased autophagy accelerates colchicine-induced muscle toxicity

Colchicine treatment is associated with an autophagic vacuolar myopathy in human patients. The presumed mechanism of colchicine-induced myotoxicity is the destabilization of the microtubule system that leads to impaired autophagosome-lysosome fusion and the accumulation of autophagic vacuoles. Using the MTOR inhibitor rapamycin we augmented colchicine’s myotoxic effect by increasing the autophagic flux; this resulted in an acute myopathy with muscle necrosis. In contrast to myonecrosis induced by cardiotoxin, myonecrosis induced by a combination of rapamycin and colchicine was associated with accumulation of autophagic substrates such as LC3-II and SQSTM1; as a result, autophagic vacuoles accumulated in the center of myofibers, where LC3-positive autophagosomes failed to colocalize with the lysosomal protein marker LAMP2. A similar pattern of central LC3 accumulation and myonecrosis is seen in human patients with colchicine myopathy, many of whom have been treated with statins (HMGCR/HMG-CoA reductase inhibitors) in addition to colchicine. In mice, cotreatment with colchicine and simvastatin also led to muscle necrosis and LC3 accumulation, suggesting that, like rapamycin, simvastatin activates autophagy. Consistent with this, treatment of mice with four different statin medications enhanced autophagic flux in skeletal muscle in vivo. Polypharmacy is a known risk factor for toxic myopathies; our data suggest that some medication combinations may simultaneously activate upstream autophagy signaling pathways while inhibiting the degradation of these newly synthesized autophagosomes, resulting in myotoxicity.     

Lysosomal-Associated Protein Multispanning Transmembrane 5 Gene (LAPTM5) Is Associated with Spontaneous Regression of Neuroblastomas

Background

Neuroblastoma (NB) is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD) or mature through differentiation into benign ganglioneuroma (GN). In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors.

Principal Findings

We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5) was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP) in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells.

Conclusions

We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy, not cell death by autophagy, so-called autophagic cell death. Thus LAPTM5-mediated PCD is closely associated with the spontaneous regression of NBs and opens new avenues for exploring innovative clinical interventions for this tumor.     

Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis

Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

GFP‐LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy

Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP‐LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP‐LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP‐Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin‐positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP‐LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP‐LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP‐LC3, a fusion protein widely used as a marker for autophagic vesicles and pre‐autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley‐Liss, Inc.     

The Anticancer Agent Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Prosurvival Autophagy by Two Mechanisms: PERSISTENT INDUCTION OF AUTOPHAGOSOME SYNTHESIS AND IMPAIRMENT OF LYSOSOMAL INTEGRITY

Autophagy functions as a survival mechanism during cellular stress and contributes to resistance against anticancer agents. The selective antitumor and antimetastatic chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) causes lysosomal membrane permeabilization and cell death. Considering the integral role of lysosomes in autophagy and cell death, it was important to assess the effect of Dp44mT on autophagy to further understand its mechanism of action. Notably, Dp44mT affected autophagy by two mechanisms. First, concurrent with its antiproliferative activity, Dp44mT increased the expression of the classical autophagic marker LC3-II as a result of induced autophagosome synthesis. Second, this effect was supplemented by a reduction in autophagosome degradation as shown by the accumulation of the autophagic substrate and receptor p62. Conversely, the classical iron chelator desferrioxamine induced autophagosome accumulation only by inhibiting autophagosome degradation. The formation of redox-active iron or copper Dp44mT complexes was critical for its dual effect on autophagy. The cytoprotective antioxidant N-acetylcysteine inhibited Dp44mT-induced autophagosome synthesis and p62 accumulation. Importantly, Dp44mT inhibited autophagosome degradation via lysosomal disruption. This effect prevented the fusion of lysosomes with autophagosomes to form autolysosomes, which is crucial for the completion of the autophagic process. The antiproliferative activity of Dp44mT was suppressed by Beclin1 and ATG5 silencing, indicating the role of persistent autophagosome synthesis in Dp44mT-induced cell death. These studies demonstrate that Dp44mT can overcome the prosurvival activity of autophagy in cancer cells by utilizing this process to potentiate cell death.     

Glucocerebrosidase deficits in sporadic Parkinson disease

Parkinson disease (PD) is a progressive neurodegenerative movement disorder characterized pathologically by abnormal SNCA/α-synuclein protein inclusions in neurons. Impaired lysosomal autophagic degradation of cellular proteins is implicated in PD pathogenesis and progression. Heterozygous GBA mutations, encoding lysosomal GBA/glucocerebrosidase (glucosidase, β, acid), are the greatest genetic risk factor for PD, and reduced GBA and SNCA accumulation are related in PD models. Here we review our recent human brain tissue study demonstrating that GBA deficits in sporadic PD are related to the early accumulation of SNCA, and dysregulation of chaperone-mediated autophagy (CMA) pathways and lipid metabolism.     

Autophagy: many paths to the same end

Different mechanisms lead to the degradation of intracellular proteins in the lysosomal compartment. Activation of one autophagic pathway or another, under specific cellular conditions, plays an important role in the ability of the cell to adapt to environmental changes. Each form of autophagy has its own individual characteristics, but it also shares common steps and components with the others. This interdependence of the autophagic pathways confers to the lysosomal system, both specificity and flexibility on substrate degradation. We describe in this review some of the recent findings on the molecular basis and regulation for each of the different autophagic pathways. We also discuss the cellular consequences of their interdependent function. Malfunctioning of the autophagic systems has dramatic consequences, especially in non-dividing differentiated cells. Using the heart as an example of such cells, we analyze the relevance of autophagy in aging and cell death, as well as in different pathological conditions.     

Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling

Reactive oxygen and nitrogen species change cellular responses through diverse mechanisms that are now being defined. At low levels, they are signalling molecules, and at high levels, they damage organelles, particularly the mitochondria. Oxidative damage and the associated mitochondrial dysfunction may result in energy depletion, accumulation of cytotoxic mediators and cell death. Understanding the interface between stress adaptation and cell death then is important for understanding redox biology and disease pathogenesis. Recent studies have found that one major sensor of redox signalling at this switch in cellular responses is autophagy. Autophagic activities are mediated by a complex molecular machinery including more than 30 Atg (AuTophaGy-related) proteins and 50 lysosomal hydrolases. Autophagosomes form membrane structures, sequester damaged, oxidized or dysfunctional intracellular components and organelles, and direct them to the lysosomes for degradation. This autophagic process is the sole known mechanism for mitochondrial turnover. It has been speculated that dysfunction of autophagy may result in abnormal mitochondrial function and oxidative or nitrative stress. Emerging investigations have provided new understanding of how autophagy of mitochondria (also known as mitophagy) is controlled, and the impact of autophagic dysfunction on cellular oxidative stress. The present review highlights recent studies on redox signalling in the regulation of autophagy, in the context of the basic mechanisms of mitophagy. Furthermore, we discuss the impact of autophagy on mitochondrial function and accumulation of reactive species. This is particularly relevant to degenerative diseases in which oxidative stress occurs over time, and dysfunction in both the mitochondrial and autophagic pathways play a role.     

Autophagy: Many paths to the same end

Different mechanisms lead to the degradation of intracellular proteins in the lysosomal compartment. Activation of one autophagic pathway or another, under specific cellular conditions, plays an important role in the ability of the cell to adapt to environmental changes. Each form of autophagy has its own individual characteristics, but it also shares common steps and components with the others. This interdependence of the autophagic pathways confers to the lysosomal system, both specificity and flexibility on substrate degradation. We describe in this review some of the recent findings on the molecular basis and regulation for each of the different autophagic pathways. We also discuss the cellular consequences of their interdependent function. Malfunctioning of the autophagic systems has dramatic consequences, especially in non-dividing differentiated cells. Using the heart as an example of such cells, we analyze the relevance of autophagy in aging and cell death, as well as in different pathological conditions. (Mol Cell Biochem 263: 55–72, 2004)     

Autophagy impairment as a key feature for acetaminophen-induced ototoxicity

Macroautophagy/autophagy is a highly conserved self-digestion pathway that plays an important role in cytoprotection under stress conditions. Autophagy is involved in hepatotoxicity induced by acetaminophen (APAP) in experimental animals and in humans. APAP also causes ototoxicity. However, the role of autophagy in APAP-induced auditory hair cell damage is unclear. In the present study, we investigated autophagy mechanisms during APAP-induced cell death in a mouse auditory cell line (HEI-OC1) and mouse cochlear explant culture. We found that the expression of LC3-II protein and autophagic structures was increased in APAP-treated HEI-OC1 cells; however, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence, and the activity of lysosomal enzymes decreased in APAP-treated HEI-OC1 cells. The degradation of p62 protein and the expression of lysosomal enzymes also decreased in APAP-treated mouse cochlear explants. These data indicate that APAP treatment compromises autophagic degradation and causes lysosomal dysfunction. We suggest that lysosomal dysfunction may be directly responsible for APAP-induced autophagy impairment. Treatment with antioxidant N-acetylcysteine (NAC) partially alleviated APAP-induced autophagy impairment and apoptotic cell death, suggesting the involvement of oxidative stress in APAP-induced autophagy impairment. Inhibition of autophagy by knocking down of Atg5 and Atg7 aggravated APAP-induced ER and oxidative stress and increased apoptotic cell death. This study provides a better understanding of the mechanism responsible for APAP ototoxicity, which is important for future exploration of treatment strategies for the prevention of hearing loss caused by ototoxic medications.Subject terms: Macroautophagy, Hair cell     

Participation of autophagy in renal ischemia/reperfusion injury

Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation, and it results in renal tubular epithelial cells undergoing cell death. We observed an increase in autophagosomes in the tubular epithelial cells of I/R-injured mouse models, and in biopsy specimens from human transplanted kidney. However, it remains unclear whether autophagy functions as a protective pathway, or contributes to I/R-induced cell death. Here, we employed the human renal proximal tubular epithelial cell line HK-2 in order to explore the role of autophagy under hypoxia (1% O₂) or activation of reactive oxygen species (500 μM H₂O₂). When compared to normoxic conditions, 48 h of hypoxia slightly increased LC3-labeled autophagic vacuoles and markedly increased LAMP2-labeled lysosomes. We observed similar changes in the mouse IR-injury model. We then assessed autophagic generation and degradation by inhibiting the downstream lysosomal degradation of autophagic vacuoles using lysosomal protease inhibitor. We found that autophagosomes increased markedly under hypoxia in the presence of lysosomal protease inhibitors, thus suggesting that hypoxia induces high turnover of autophagic generation and degradation. Furthermore, inhibition of autophagy significantly inhibited H₂O₂-induced cell death. In conclusion, high turnover of autophagy may lead to autophagic cell death during I/R injury.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

Impairment of Autophagic Flux Promotes Glucose Reperfusion-Induced Neuro2A Cell Death after Glucose Deprivation

Hypoglycemia-induced brain injury is a common and serious complication of intensive insulin therapy experienced by Type 1 diabetic patients. We previously reported that hypoglycemic neuronal death is triggered by glucose reperfusion after hypoglycemia rather than as a simple result of glucose deprivation. However, the precise mechanism of neuronal death initiated by glucose reperfusion is still unclear. Autophagy is a self-degradation process that acts through a lysosome-mediated trafficking pathway to degrade and recycle intracellular components, thereby regulating metabolism and energy production. Recent studies suggest that autophagic and lysosomal dysfunction leads to abnormal protein degradation and deposition that may contribute to neuronal death. Here, we focused on the relationship between autophagy and lysosomal dysfunction in hypoglycemia-induced neuronal death. In neuronal cells, glucose reperfusion after glucose deprivation resulted in inhibition of autophagy, which may promote cell death. This cell death was accompanied with activation of caspase3 and the lysosomal proteases cathepsin B and D, which indicated impairment of autophagic flux. Taken together, these results suggest that interplay of autophagy, caspase3 activation and lysosomal proteases serve as a basis for neuronal death after hypoglycemia. Thus, we provide the molecular mechanism of neuronal death by glucose reperfusion and suggest some clues for therapeutic strategies to prevent hypoglycemia-induced neuronal death.     

Lysosomal dysfunction–induced autophagic stress in diabetic kidney disease

The catabolic process that delivers cytoplasmic constituents to the lysosome for degradation, known as autophagy, is thought to act as a cytoprotective mechanism in response to stress or as a pathogenic process contributing towards cell death. Animal and human studies have shown that autophagy is substantially dysregulated in renal cells in diabetes, suggesting that activating autophagy could be a therapeutic intervention. However, under prolonged hyperglycaemia with impaired lysosome function, increased autophagy induction that exceeds the degradative capacity in cells could contribute toward autophagic stress or even the stagnation of autophagy, leading to renal cytotoxicity. Since lysosomal function is likely key to linking the dual cytoprotective and cytotoxic actions of autophagy, it is important to develop novel pharmacological agents that improve lysosomal function and restore autophagic flux. In this review, we first provide an overview of the autophagic‐lysosomal pathway, particularly focusing on stages of lysosomal degradation during autophagy. Then, we discuss the role of adaptive autophagy and autophagic stress based on lysosomal function. More importantly, we focus on the role of autophagic stress induced by lysosomal dysfunction according to the pathogenic factors (including high glucose, advanced glycation end products (AGEs), urinary protein, excessive reactive oxygen species (ROS) and lipid overload) in diabetic kidney disease (DKD), respectively. Finally, therapeutic possibilities aimed at lysosomal restoration in DKD are introduced.     

Adaptive autophagy in Alexander disease-affected astrocytes

The ubiquitin-proteasome and autophagy-lysosomal pathways are the two main routes of protein and organelle clearance in eukaryotic cells. The proteasome system is responsible for unfolded, short-lived proteins, which precludes the clearance of oligomeric and aggregated proteins, whereas macroautophagy, a process generally referred to as autophagy, mediates mainly the bulk degradation of long-lived cytoplasmic proteins, large protein complexes or organelles.¹ Recently, the autophagy-lysosomal pathway has been implicated in neurodegenerative disorders as an important pathway for the clearance of abnormally accumulated intracellular proteins, such as huntingtin, tau, and mutant and modified α-synuclein.^1-6 Our recent study illustrated the induction of adaptive autophagy in response to mutant glial fibrillary acidic protein (GFAP) accumulation in astrocytes, in the brains of patients with Alexander disease (AxD), and in mutant GFAP knock-in mouse brains.⁷ This autophagic response is negatively regulated by mammalian target of rapamycin (mTOR). The activation of p38 MAPK by GFAP accumulation is responsible for mTOR inactivation and the induction of autophagy. We also found that the accumulation of GFAP impairs proteasome activity.⁸ In this commentary we discuss the potential compensatory relationship between an impaired proteasome and activated autophagy, and propose that the MLK-MAPK (mixed lineage kinase–mitogen-activated protein kinase) cascade is a regulator of this crosstalk.

Addendum to: Tang G, Yue Z, Talloczy, Z, Hagemann T, Cho W, Sulzer D, Messing A, Goldman JE. Alexander disease-mutant GFAP accumulation stimulates autophagy through p38 MAPK and mTOR signaling pathways. Hum Mol Genetics 2008; In press.     

Apoptosis and Autophagy Function Cooperatively for the Efficacious Execution of Programmed Nurse Cell Death During Drosophila virilis Oogenesis

Programmed cell death consists of two major types, apoptotic and autophagic, both of which are mainly defined by morphological criteria. Our findings indicate that both types of programmed cell death occur in the ovarian nurse cells during middle and late oogenesis of Drosophila virilis. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain condensed chromatin and fragmented DNA, whereas active caspase assays and immunostaining procedures demonstrate the presence of highly activated caspases. Distinct features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an accumulation of lysosomal proteases. At the late stages of D. virilis oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones described above, being also escorted by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during D. virilis oogenesis for a more efficient elimination of the degenerated nurse cells.

Addendum to:

Mechanisms of Programmed Cell Death During Oogenesis in Drosophila virilis

A.D. Velentzas, I.P. Nezis, D.J. Stravopodis, I.S. Papassideri and L.H. Margaritis

Cell Tissue Res 2006; doi: 10.1007/s00441-006-0298-x     

Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.