Sleep Breath DOI 10.1007/s11325-013-0930-4
REVIEW
Autophagy and hippocampal neuronal injury Lulu Li & Qiang Zhang & Jin Tan & Yunyun Fang & Xu An & Baoyuan Chen
Received: 18 September 2013 / Revised: 11 December 2013 / Accepted: 16 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract Background Autophagy is a specific universal biological phenomenon in eukaryotic cells, which is characterized by cytoplasmic vacuoles in the process of degrading cellular contents in lysosomes. The hippocampus plays an important role in higher nervous activities such as emotional integration, cognition, and memory. As an area closely related to learning and memory functions of the brain, the hippocampus is particularly sensitive to injuries caused by various reasons. Purpose Autophagy has certain links with a variety of causes of hippocampal neuronal injury. This short review discusses and summarizes this correlation with a focus on the possible role of autophagy and mechanisms in it. Conclusion The current correlation between autophagy and hippocampal neuronal injury has not been completely determined by the general public alike. Further studies are needed to determine special effects of autophagy on hippocampal neuronal injury, which might accelerate the development of therapeutic interventions in hippocampal neuronal injury in many neurological disorders. Keywords Autophagy . Hippocampal neurons . Alzheimer’s disease (AD) . Hypoxia–ischemia . Excitotoxicity . Vitamin E Autophagy is the homeostatic and degradative cellular pathway for intracellular recycling and autodigesting of bulk proteins and aging organelles under certain physiological or pathophysiological conditions. It has been described as a physiological and dynamic process essential for cellular health and survival [1]. Studies have demonstrated that autophagy is involved in hippocampal neuronal injury caused by many L. Li : Q. Zhang (*) : J. Tan : Y. Fang : X. An Tianjin Institute of Geriatrics, Tianjin Medical University General Hospital, Anshan Road, Tianjin 300052, China e-mail: [email protected] B. Chen Department of Respiration, Tianjin Medical University General Hospital, Tianjin, China
reasons, such as Alzheimer’s disease (AD), hypoxia–ischemia brain injury and excitotoxicity [2]. In the present review, we particularly discuss the roles of autophagy in hippocampal neuronal survival and death in the corresponding diseases.
Autophagy Soon after the discovery of the presence of cytoplasmic organelles within membrane-limited vacuoles by electron microscopists Clark and Ashford in the 1950s, de Duve named this process “autophagy”. Autophagy was gradually taken seriously with the establishment of yeast model and development of gene technology in the 1990s, and recently it has become a new hotspot in the field of biology. The degree of evolution of autophagy is highly conserved. Homologous genes involved in autophagy can be found from yeast, fruit flies to vertebrates, and humans. The ubiquitin-proteasome system is the most important for degrading short-lived proteins, whereas autophagy is the main mechanism for degrading long-lived proteins and organelles [3, 4]. Autophagy is a class of programmed cell death independent on caspase family, which is different from apoptosis, thus it is also known as type II programmed cell death.
Classification and formation process of autophagy Mammal autophagy exists in three major subtypes: microautophagy, macroautophagy, and chaperone-mediated autophagy according to different ways of intracellular substrates’ transportation to lysosomes [5]. Of these classifications, macroautophagy has been widely referred to as autophagy. It can be artificially divided into three stages of specific formation process of autophagy [6]: (1) induction of autophagy—autophagy is an important cellular response to environmental “stresses”. Eukaryotic cells must adapt to fluctuations in external conditions, including physical parameters (temperature), chemical cues (pH, oxygen tension, redox
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potentials, and metabolite concentrations), extracellular signals (contact-dependent signals, hormones, cytokines, and neurotransmitters) and microbial pathogens. Beyond a certain threshold, such fluctuations are considered “stresses”. One of the key pathways that mediate environmental stress-induced metabolic adaptation and damage control is autophagy; (2) autophagosome formation—the core pathway of autophagy always begin with the formation of an isolation membrane (also called a phagophore) and involves at least five molecular components, including (a) the Atg1/unc-51-like kinase complex; (b) the Beclin 1/class III phosphatidylinositol 3-kinase (PI3K) complex; (c) two transmembrane proteins, Atg9, and vacuole membrane protein 1; (d) two ubiquitin-like protein (Atg12 and Atg8/LC3) conjugation systems; and (e) proteins that mediate fusion between autophagosomes and lysosomes [1], then complete sequestration of degradation cytoplasmic material by the edges of the phagophore fuse to form the autophagosome, which is typically a double-membraned vesicle. The origin of the sequestering membrane is unknown, but for mammalian cells, it is generally thought to be the endoplasmic reticulum; (3) autophagosome transportation and degradation—autophagosome fuses with lysosome to form the autolysosome in which cytosolic components are degraded. Degradation products are cyclically used, therefore, autophagy can be considered as the degradation/recirculation system widely existing in eukaryotic cells. Signal transduction molecules and regulation involved in autophagy process are very complex, which mainly include two pathways. The protein kinase mammalian target of rapamycin (mTOR) is a central actor at the crossroads of different pathways regulating autophagy, and the class I phosphatidylinositol 3-kinase (class I PI3K)AKT/PKB (activator) or AMPK (inhibitor) pathways are indirectly involved in mTOR activity and autophagy regulation. In addition, amino acids, certain hormones such as insulin, calcium can also participate in the activation of autophagy.
Physiological/pathophysiological roles of autophagy Studies have confirmed that as an ancient and evolutionarily conserved mechanism for maintaining cellular homeostasis [10], autophagy has a dual nature role in different physiological/pathophysiological conditions. Autophagy plays an important role in developmental processes, human diseases, and cellular response to nutrient deprivation. In the period of bioenergetic stress imposed by decreased extracellular supply of nutrients, autophagy can degrade unnecessary macromolecules and organelles into nutrients that can be reused for generating energy and for rebuilding essential cell structures. For example, the amino acids generated by autophagy can be reused to support the tricarboxylic acid cycle, which maintains cell metabolic viability [11]. In addition to its role in optimizing metabolic conditions in stressed cells, autophagy can also eliminate toxic metabolites, damaged proteins, and organelles accumulated from the cytoplasm, all of which are injurious to the cell. As a “waste disposal” system, autophagy preferentially removes aging or oxidatively damaged organelles and proteins, both of which have the potential to cause or aggravate cell injury [12]. However, as a type II programmed cell death, autophagy also has a close and complex link with necrosis and apoptosis. Under some pathological conditions, the induction of excessive autophagy can lead to cell death and promote pathological processes [13]. Currently, researches in this respect remain to be unclear. In the nervous system, autophagy also plays such various roles. Studies have found that autophagy not only presents physiological expression in the hippocampus neurons [14], but also participates in the hippocampal neuronal injury, which may include chronic neurodegenerative diseases, hypoxic–ischemic brain injury, excitotoxicity, and so on.
Autophagy and hippocampal neuronal injury in AD Detection of autophagy Relevance Detection of autophagy includes the following methods [7]: (1) transmission electron microscopy—it is currently the only reliable method for detecting autophagy in situ and is the “gold standard” for detection of autophagy; (2) detection of marker proteins on autophagic membrane, such as autophagy regulatory genes 12-autophagy regulatory genes 5 (Atg12-Atg5) complexes and microtubule-associated protein light chain-3-II (LC3-II, a good indicator of autophagy induction) [8]; (3) monodansylcadaverine (MDC) [9]—it is used for monitoring the labeling of autophagic vacuoles in vivo. The specific binding of Atg-8 and MDC can achieve specific detection through staining; (4) analysis of autophagic flux—it includes conversion from LC3-I to LC3-II, degradation of the autophagic receptor protein and the detection of colocalization between LC3-II and lysosomal transmembrane proteins-2.
AD, as an important chronic neurodegenerative disease has been characterized in age-related brain degeneration, eventually leading to progressive cognitive and behavioral disorders. The main histopathological changes in AD include the presence of “senile plaques” consisting of β-amyloid deposits, neurofibrillary tangles, foci of degenerating axons, and dendrites and aggregates of the abnormal hyperphosphorylated microtubule-associated tau protein. All of these changes can cause a lot of progressive and selective neuronal loss, mainly existing in larger neurons, especially in the hippocampus, temporal lobe, and frontal cortex [15]. Neuronal death pattern in AD does not conform to be conventional and may vary among apoptosis, necrosis, and autophagic/autolysosomal pathology [16]. In recent years,
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many studies have focused on autophagic/autolysosomal pathophysiology in hippocampal impaired neurons in AD. Current studies support that all the fibrillar amyloid deposits in the hippocampus of transgenic mice are identified as human-like neuritic plaques, and dystrophic axons in AD are particularly abundant in the hippocampus originating from the subiculum, CA1, as well as the entorhinal cortex. Synaptic loss in the hippocampus is, so far, the best pathological correlate of early cognitive decline [17]. Therefore, studies focus on the hippocampus would be more clinical significant to other neurons. Hara et al. [18] found the accumulation of cytoplasmic inclusion bodies and the diffuse, abnormal intracellular proteins in neurons by electron microscopy in mice deficient for Atg5, which was accompanied by deficits in motor function. Caccamo et al. [19] also established mouse model of AD and detected that in hippocampal neurons mTOR signaling was significantly enhanced, which was accompanied by the buildup of β-amyloid (Aβ), whereas in the rapamycintreated AD mice, mTOR activity and Aβ levels were significantly reduced, levels of Atg7 and the Atg5/Atg12 complex, and LC3-II were significantly increased in the brains, cognitive deficits in rapamycin-treated AD mice were also rescued. These studies all indicate that there is a close link between autophagy and hippocampal neuronal damage in AD. The role of autophagy in hippocampal neuronal injury in AD For the specific role of autophagy in hippocampal neuronal injury in AD, the current studies inclined to be neuroprotective. Autophagy can reduce the level of mutant or toxic proteins such as Aβ and Tau protein in neurons. At the earliest disease stage, a progressive dysfunction of autophagy could be detected in damaged hippocampal neurons [20, 21], mainly including disorders of autophagic membrane formation and autophagy flux transportation, the former can be manifested in the significant reduction of the autophagy-related proteins such as Beclin 1 and LC3-II, the latter occurs mainly in dysfunction of autolysosomal protein degradation, which may affect neuroprotective role of autophagy in AD. Pickford et al. [22] labeled LC3 in hippocampal neurons of Beclin-1+/− and Beclin-1+/+ mice by immunofluorescence and detected that Beclin 1-deficient neurons had fewer than half the number of autophagosomes in Beclin-1+/− group than in Beclin-1+/+ group, which indicated that heterozygous deletion of Beclin-1 in mice may decrease neuronal autophagy. They further established Beclin-1 deficient and amyloid precursor protein (APP) heterozygous transgenic mice model and detected that Beclin-1 protein levels were reduced by half in APP+ Beclin-1+/− group compared with APP+ Beclin-1+/+ group, which was also accompanied by a nearly twofold increase in extracellular Aβ deposition. By injecting the lentiviral vector expressing Beclin-1, they detected the high level of GFP-LC3 in hippocampal neurons, and both
intracellular and extracellular amyloid pathology were reduced. These studies suggest that Beclin-1 deficiency can disrupt autophagy and induce disorders of autophagic membrane formation in hippocampal neurons. Reduced expression of Beclin-1 may modulate APP metabolism, leading to abnormal accumulation of extracellular Aβ and abnormal nerve cell ultrastructure, which finally promote neurodegeneration and the occurrence of AD. Conversely, increased expression of Beclin-1 may promote the formation of autophagosomes, which can reduce abnormal deposition of Aβ, delay the development of AD, and thus may play a protective role in hippocampal neuronal injury in AD. Studies show that disorders of autophagy flux transportation may also increase hippocampal neuronal injury, leading to cognitive impairment. Yang et al. [23] established TgCRND8 mouse model overexpressing a version of APP695 and producing more Aβ. It was similar to the pathological mechanisms previously described in AD brains and immunolabelled with an rabbit polyclonal antibody against mouse cathepsin D, the results detected that in the hippocampal CA1 sector, including the pyramidal cell layer, giant cathepsin D-positive lysosomal compartments were most abundant, along with large numbers of reduced normal-sized lysosomes, indicating abnormal accumulation of ubiquitinated proteins within these compartments, which were incompletely degraded. They detected by immunoblot analysis that there were abnormally high levels of LC3 and very high proportions of LC3-II in two autophagic vacuole fractions and lysosome fractions in TgCRND8 mice than in control groups. These analyses suggest that disruption of autophagy flux transportation may exist in hippocampal neurons in AD, leading to the accumulation of Aβ in autophagic vacuoles and autolysosomes. To examine and confirm the inherent relationship between autophagy flux transportation and accumulation of Aβ, they further deleted cystatin B in TgCRND8 mouse model to establish CBKO/TgCRND8 mouse model, cystatin B can selectively enhance the activities of cysteine proteases and improve lysosomal proteolytic function. They found that the number of giant autolysosomes seen in TgCRND8 mice was largely reduced in CBKO/TgCRND8 mice, while normal-sized lysosomes increased extensively, enzymatic activities of cathepsin B, cathepsin L, and cathepsin D were higher in CBKO/TgCRND8 brains than in CBKO brains. In addition, intracellular Aβ and LC3-II immunoreactivity seen by immunocytochemistry and western blotting were markedly reduced in the autophagic vacuole and lysosome fractions in CBKO/TgCRND8 brains, these mice may improve contextual memory deficits those found in TgCRND8 mice. These results consistently demonstrate that the incidence of AD may influence downstream of autophagy flux transportation in hippocampal neurons and reduce hydrolytic activity of autolysosomess as well as long-term excessive Aβ deposition, which may induce the degeneration and death
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of hippocampal neurons, eventually leading to hippocampal neuronal dysfunction. In addition, the process of autophagy may be influenced by different pathological stages of AD. Yu et al. [24] studied that autophagy was induced very early in AD; elevation of LC3-II in AD brains at preclinical stages of AD suggested that autophagic induction was an early response to the disease process. They also analyzed autophagic vacuoles (AVs) levels in PS1/APP mice that could simulate pathological changes of AD including Aβ deposition. The result showed that AVs were visible in the cell bodies. These observations demonstrate that autophagy is induced at a prepathological stage of disease in PS1/APP mice, as in AD. Sun et al. [25] also found that expression of mTOR was expressed in the control and AD hippocampus. The expression levels of the corresponding signaling molecules were significantly increased in the hippocampus in the severe stages of AD, compared with controls and other stages of AD. They also indicated that mTORC1, but not mTORC2, was activated in AD brains and that the level of mTOR signaling activation was correlated with cognitive severity of AD patients. Therefore, in different pathological stages of AD, the activation of autophagy and the expression of the corresponding labeled molecules may also be influenced by varying degrees. In short, appropriate level of autophagy could be essential in delaying degeneration of hippocampal neurons and preventing the development of AD, which can clear the abnormal deposition of intracellular and extracellular proteins in AD. Additionally, targeted therapies for disorders of autophagic membrane formation and autophagy flux transportation may be expected to reduce the abnormal accumulation of proteins, which could become a potential clinical treatment to prevent or retard the development of AD.
Autophagy and hippocampal neuronal injury in cerebral ischemia/hypoxia Relevance Autophagy is not only widely involved in generation and the development of hippocampal neuronal injury in neurodegenerative diseases such as AD, a large number of studies have confirmed that there are also closely internal relations between autophagy and hypoxia/ischemia-induced hippocampal neuronal injury [26–28]. And studies on neuron death after hypoxia/ischemia brain injury using various animals have also shown that the hippocampus is the most vulnerable to ischemic insult to other neurons [29]. As early as 1995, Nitatori et al. [30] detected autophagy in ischemic brain injury for the first time, they observed a large number of autophagic vacuoles and cathepsin B in hippocampal CA1 neurons by electron microscopy 3 days after
treatment against hypoxic stimulation; Koike et al. [29] established the mice model of acute ischemic brain injury by ligating the left common carotid artery and detected large numbers of LC3 particles appeared in left hippocampus pyramidal cells by immunofluorescence after 8 h ischemic stimulation, suggesting that autophagy activity exist in damaged hippocampal neurons. They further established Atg7-deficient rats model and detected by Western blotting that conversion from LC3-I to LC3-II in hippocampal neurons was significantly reduced at 1 day after ischemic treatment. These studies indicate that autophagy plays a crucial role in hippocampal neuronal injury induced by cerebral ischemia. The dual role of autophagy Recent studies have found that the role of autophagy in hippocampal neuronal injury induced by cerebral ischemia seemed to be duality, which not only could be cytoprotective, but also could lead to cell death and increase hippocampal neuronal injury under certain conditions. This paradoxical role of autophagy always depends on the time of ischemia stimulation, the extent of ischemic reperfusion, and induction of autophagy [31]. Hippocampal neuroprotective actions of autophagy Studies have confirmed that transient ischemia can promote mild to moderate activation of autophagy in hippocampal neurons. Moderate autophagic activity may help to delete the abnormal sediment in impaired neurons and promote neuronal survival, which can protect ischemic hippocampal neurons. Carloni et al. [32] established Sprague–Dawley rats model which was anesthetized and subjected to ligation of the right common carotid artery followed by 2.5 h hypoxia (92 % nitrogen and 8 % oxygen) to observe the expression of autophagy in the cortex and hippocampal neurons after transient ischemic stimulation. The results showed that Beclin-1positive cells were sparsely detectable at 4 h after hypoxia– ischemia (HI) in the injured side in the cerebral cortex. In the hippocampus, conversely, the number of Beclin-1-positive cells was higher and cells were mainly present in the CA1 area. terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were found mainly in the superficial layers of the cerebral cortex, in which most TUNEL-positive cells were also Beclin-1-positive. They further evaluated through 3MA/WT/rapamycin treatments if modulation of autophagy could modify this pattern of cell demise, the results showed that the reduction of Beclin-1positive cells and activated caspase-3 expression and simultaneously intact PARP-1were detectable after treatment with the two inhibitors. As expected, treatment with rapamycin increased Beclin-1 expression and reduced the expression of activated caspases-3; accordingly, cell loss appeared markedly attenuated both in the cerebral cortex and in the hippocampus.
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These results suggested that inhibition of autophagy may transform mode of cell death from apoptosis to necrosis. In contrast, enhanced autophagy activity can reduce the progression of cells towards necrotic cell death, resulting, therefore, in decreased ischemic hippocampal neuronal injury. These studies indicate that activation of autophagy may be a component of the hippocampal neuronal survival mechanisms turned on after brain ischemia. If autophagy is inhibited, neurons may quickly proceed towards necrotic degeneration through interrupting the apoptotic program. Conversely, limited overactivation autophagic pathways may delay the progression of cells towards death, simultaneously indicating that moderate activation of autophagic pathways may represent a potential, protective mechanism in hippocampal neuronal injury induced by cerebral ischemia. Liu et al. [33] also detected through the establishment of Wistar rats HI model the abnormal accumulation of denatured organelles in the hippocampal CA1 region after transient ischemic stimulation. They also found that intracellular LC3II expression was significantly increased, which suggested that autophagy may protect hippocampal neurons from being eroded by harmful substances through degradating abnormal protein deposition and denatured organelles. Hippocampal neuronal injury induced by overactivations of autophagy However, some other studies took different opinions that increased autophagic activity may conversely aggravate hippocampal neuronal injury after transient ischemia. Zheng et al. [34] established adult male mice model of transient middle cerebral artery occlusion and nicotinamide adenine dinuleotide (NAD(+)) was administered intraperitoneally at the same time that reperfusion started, detecting that LC3-II expression was significantly lower in the cell cortex and hippocampus. In addition, administration with NAD(+) led to significant decreases in neurological deficits at 48 h after ischemia. The results suggest that NAD(+) may inhibit autophagy and reduce hippocampal neuronal injury. NAD(+) can reduce cell death caused by excessive activation of oxidative stress, speculating that in ischemic stimulation, autophagy may aggravate cellular oxidative damage via activation of oxidative stress and thus aggravate ischemic neuronal damage. Further studies have found that in the long term of cerebral ischemia especially after reperfusion, autophagy occurs to be overexpression, which not only degrades denatured proteins, but also degrades normal cellular components such as mitochondria and other organelles, resulting in the depletion of energy needed for cells growth and normal metabolism; all of these would ultimately aggravate neuronal damage [35]. Shi et al. [36] administrated rat neurons exposed to oxygen and glucose deprivation (OGD) and reperfusion (RP), respectively, and observed numerous autophagic vacuoles with
characteristic morphological features of autophagosomes and autolysosomal structures in neurons after OGD/RP injury. Meanwhile, a large number of nuclear condensation, swelling, and dilated mitochondria and acidophilic denatured protein were also frequently found. They also observed that the level of LC3 punctuate staining in the rat neurons after OGD increased dramatically according to the elongation of RP time, which indicated an intense and excessive activation of autophagy after HI and RP injury. They further detected that the number of cell death was significantly reduced at RP 72 h after inhibiting autophagosome formation by 3-methyladenine (a specific autophagy inhibitor), suggesting that the increase of autophagy was accompanied with an progressively increase of autophagic neuronal death. On the contrary, inhibition of autophagy significantly reduced autophagic neuronal death. These data demonstrate that excessive activation of autophagy may be a contributing factor of neuronal death and can further aggravate the neuronal injury in cerebral ischemia. In short, the specific role that autophagy plays in hypoxic– ischemic induces hippocampal neuronal injury, which should be very complex. Both transient hypoxia–ischemia and longterm cerebral ischemia such as ischemia–reperfusion could induce neuronal damage. Autophagy, to some extent, could contribute to duality. A certain degree of autophagic activity may be beneficial to the deletion of denatured proteins and aging organelles; however, excessive activation of autophagy might actually lead to neuronal self-digestion and even necrosis, which may further aggravate the hippocampus ischemic neuronal injury.
Others In recent years, some other studies have reported that neural excitotoxicity factors, varying degrees of deficiency in essential nutrients such as vitamins or trace elements can also lead to hippocampal neuronal injury by inducing autophagy. Excitotoxicity, a phenomenon induced usually by the overactivation of excitatory amino acid receptors, is the main mediator of neuronal death including traumatic brain injury and cerebral ischemia [37]. Studies have confirmed that toxic substances can stimulate actions of autophagy in hippocampal neurons in several acute excitotoxicity models such as the application of N-methyl-D-aspartate onto organotypic hippocampal slices [38] or the direct injection of kainate into the brains of mice in vivo [39]. Meanwhile, autophagy has also been reported in some chronic excitotoxicity models [40]. Moreover, the lack of essential nutrients in vivo can also lead to a significant reduction in the number of cells and even cell death. Fukui et al. [41] found that diffuse axonal injury occurred in hippocampal CA1 neural fibers in vitamin Edeficient mice, and hippocampal CA1 neurons were significantly lower than the control group. Expressions of collapsin
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response mediator protein-2 (CRMP-2), playing a crucial role in the maintenance of axonal conditions, were significantly lower in the cerebral cortex and hippocampus of vitamin Edeficient mice than both the regions of normal ones. In addition, the appearance of MAP-LC3, a major index of autophagy, was higher in the cerebral cortex and hippocampus of vitamin E-deficient mice than in normal mice. These results demonstrate that changes in CRMP-2 and MAP-LC3 may underlie vitamin E-deficiency-related axonal degeneration by inducing activities of autophagy. In short, many reasons can lead to hippocampal neuronal injury. The degree of expression of autophagy may play a key regulatory role in the process of neuronal damage.
Concluding remarks In summary, the significance of autophagy in neurological disorders and its importance in physiological and pathological situations have been established for several decades. Nevertheless, the factors that govern the neuroprotective/neurotoxic functions of autophagy and how autophagy contributes to neuronal death are unknown. On the one hand, as an energy supplier, autophagy can be a physiological mechanism indispensable for the health of neurons by its essential role in quality control for proteins and organelles; however, the activation of autophagy exceeding a certain level may induce neuronal death by initiating the self-digestion of dying neurons. Thus, whether autophagy is a prosurvival strategy or a step in the cell death program still needs further examination and a profound understanding of the neuronal autophagy process will be expected to contribute to the future development of therapeutic interventions in chronic and acute neuronal disorders. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (no. 81370183) and a grant from Natural Science Foundation of Tianjin (no. 13JCYBJC23700).
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Autophagy and hippocampal neuronal injury Lulu Li & Qiang Zhang & Jin Tan & Yunyun Fang & Xu An & Baoyuan Chen
Received: 18 September 2013 / Revised: 11 December 2013 / Accepted: 16 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract Background Autophagy is a specific universal biological phenomenon in eukaryotic cells, which is characterized by cytoplasmic vacuoles in the process of degrading cellular contents in lysosomes. The hippocampus plays an important role in higher nervous activities such as emotional integration, cognition, and memory. As an area closely related to learning and memory functions of the brain, the hippocampus is particularly sensitive to injuries caused by various reasons. Purpose Autophagy has certain links with a variety of causes of hippocampal neuronal injury. This short review discusses and summarizes this correlation with a focus on the possible role of autophagy and mechanisms in it. Conclusion The current correlation between autophagy and hippocampal neuronal injury has not been completely determined by the general public alike. Further studies are needed to determine special effects of autophagy on hippocampal neuronal injury, which might accelerate the development of therapeutic interventions in hippocampal neuronal injury in many neurological disorders. Keywords Autophagy . Hippocampal neurons . Alzheimer’s disease (AD) . Hypoxia–ischemia . Excitotoxicity . Vitamin E Autophagy is the homeostatic and degradative cellular pathway for intracellular recycling and autodigesting of bulk proteins and aging organelles under certain physiological or pathophysiological conditions. It has been described as a physiological and dynamic process essential for cellular health and survival [1]. Studies have demonstrated that autophagy is involved in hippocampal neuronal injury caused by many L. Li : Q. Zhang (*) : J. Tan : Y. Fang : X. An Tianjin Institute of Geriatrics, Tianjin Medical University General Hospital, Anshan Road, Tianjin 300052, China e-mail: [email protected] B. Chen Department of Respiration, Tianjin Medical University General Hospital, Tianjin, China
reasons, such as Alzheimer’s disease (AD), hypoxia–ischemia brain injury and excitotoxicity [2]. In the present review, we particularly discuss the roles of autophagy in hippocampal neuronal survival and death in the corresponding diseases.
Autophagy Soon after the discovery of the presence of cytoplasmic organelles within membrane-limited vacuoles by electron microscopists Clark and Ashford in the 1950s, de Duve named this process “autophagy”. Autophagy was gradually taken seriously with the establishment of yeast model and development of gene technology in the 1990s, and recently it has become a new hotspot in the field of biology. The degree of evolution of autophagy is highly conserved. Homologous genes involved in autophagy can be found from yeast, fruit flies to vertebrates, and humans. The ubiquitin-proteasome system is the most important for degrading short-lived proteins, whereas autophagy is the main mechanism for degrading long-lived proteins and organelles [3, 4]. Autophagy is a class of programmed cell death independent on caspase family, which is different from apoptosis, thus it is also known as type II programmed cell death.
Classification and formation process of autophagy Mammal autophagy exists in three major subtypes: microautophagy, macroautophagy, and chaperone-mediated autophagy according to different ways of intracellular substrates’ transportation to lysosomes [5]. Of these classifications, macroautophagy has been widely referred to as autophagy. It can be artificially divided into three stages of specific formation process of autophagy [6]: (1) induction of autophagy—autophagy is an important cellular response to environmental “stresses”. Eukaryotic cells must adapt to fluctuations in external conditions, including physical parameters (temperature), chemical cues (pH, oxygen tension, redox
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potentials, and metabolite concentrations), extracellular signals (contact-dependent signals, hormones, cytokines, and neurotransmitters) and microbial pathogens. Beyond a certain threshold, such fluctuations are considered “stresses”. One of the key pathways that mediate environmental stress-induced metabolic adaptation and damage control is autophagy; (2) autophagosome formation—the core pathway of autophagy always begin with the formation of an isolation membrane (also called a phagophore) and involves at least five molecular components, including (a) the Atg1/unc-51-like kinase complex; (b) the Beclin 1/class III phosphatidylinositol 3-kinase (PI3K) complex; (c) two transmembrane proteins, Atg9, and vacuole membrane protein 1; (d) two ubiquitin-like protein (Atg12 and Atg8/LC3) conjugation systems; and (e) proteins that mediate fusion between autophagosomes and lysosomes [1], then complete sequestration of degradation cytoplasmic material by the edges of the phagophore fuse to form the autophagosome, which is typically a double-membraned vesicle. The origin of the sequestering membrane is unknown, but for mammalian cells, it is generally thought to be the endoplasmic reticulum; (3) autophagosome transportation and degradation—autophagosome fuses with lysosome to form the autolysosome in which cytosolic components are degraded. Degradation products are cyclically used, therefore, autophagy can be considered as the degradation/recirculation system widely existing in eukaryotic cells. Signal transduction molecules and regulation involved in autophagy process are very complex, which mainly include two pathways. The protein kinase mammalian target of rapamycin (mTOR) is a central actor at the crossroads of different pathways regulating autophagy, and the class I phosphatidylinositol 3-kinase (class I PI3K)AKT/PKB (activator) or AMPK (inhibitor) pathways are indirectly involved in mTOR activity and autophagy regulation. In addition, amino acids, certain hormones such as insulin, calcium can also participate in the activation of autophagy.
Physiological/pathophysiological roles of autophagy Studies have confirmed that as an ancient and evolutionarily conserved mechanism for maintaining cellular homeostasis [10], autophagy has a dual nature role in different physiological/pathophysiological conditions. Autophagy plays an important role in developmental processes, human diseases, and cellular response to nutrient deprivation. In the period of bioenergetic stress imposed by decreased extracellular supply of nutrients, autophagy can degrade unnecessary macromolecules and organelles into nutrients that can be reused for generating energy and for rebuilding essential cell structures. For example, the amino acids generated by autophagy can be reused to support the tricarboxylic acid cycle, which maintains cell metabolic viability [11]. In addition to its role in optimizing metabolic conditions in stressed cells, autophagy can also eliminate toxic metabolites, damaged proteins, and organelles accumulated from the cytoplasm, all of which are injurious to the cell. As a “waste disposal” system, autophagy preferentially removes aging or oxidatively damaged organelles and proteins, both of which have the potential to cause or aggravate cell injury [12]. However, as a type II programmed cell death, autophagy also has a close and complex link with necrosis and apoptosis. Under some pathological conditions, the induction of excessive autophagy can lead to cell death and promote pathological processes [13]. Currently, researches in this respect remain to be unclear. In the nervous system, autophagy also plays such various roles. Studies have found that autophagy not only presents physiological expression in the hippocampus neurons [14], but also participates in the hippocampal neuronal injury, which may include chronic neurodegenerative diseases, hypoxic–ischemic brain injury, excitotoxicity, and so on.
Autophagy and hippocampal neuronal injury in AD Detection of autophagy Relevance Detection of autophagy includes the following methods [7]: (1) transmission electron microscopy—it is currently the only reliable method for detecting autophagy in situ and is the “gold standard” for detection of autophagy; (2) detection of marker proteins on autophagic membrane, such as autophagy regulatory genes 12-autophagy regulatory genes 5 (Atg12-Atg5) complexes and microtubule-associated protein light chain-3-II (LC3-II, a good indicator of autophagy induction) [8]; (3) monodansylcadaverine (MDC) [9]—it is used for monitoring the labeling of autophagic vacuoles in vivo. The specific binding of Atg-8 and MDC can achieve specific detection through staining; (4) analysis of autophagic flux—it includes conversion from LC3-I to LC3-II, degradation of the autophagic receptor protein and the detection of colocalization between LC3-II and lysosomal transmembrane proteins-2.
AD, as an important chronic neurodegenerative disease has been characterized in age-related brain degeneration, eventually leading to progressive cognitive and behavioral disorders. The main histopathological changes in AD include the presence of “senile plaques” consisting of β-amyloid deposits, neurofibrillary tangles, foci of degenerating axons, and dendrites and aggregates of the abnormal hyperphosphorylated microtubule-associated tau protein. All of these changes can cause a lot of progressive and selective neuronal loss, mainly existing in larger neurons, especially in the hippocampus, temporal lobe, and frontal cortex [15]. Neuronal death pattern in AD does not conform to be conventional and may vary among apoptosis, necrosis, and autophagic/autolysosomal pathology [16]. In recent years,
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many studies have focused on autophagic/autolysosomal pathophysiology in hippocampal impaired neurons in AD. Current studies support that all the fibrillar amyloid deposits in the hippocampus of transgenic mice are identified as human-like neuritic plaques, and dystrophic axons in AD are particularly abundant in the hippocampus originating from the subiculum, CA1, as well as the entorhinal cortex. Synaptic loss in the hippocampus is, so far, the best pathological correlate of early cognitive decline [17]. Therefore, studies focus on the hippocampus would be more clinical significant to other neurons. Hara et al. [18] found the accumulation of cytoplasmic inclusion bodies and the diffuse, abnormal intracellular proteins in neurons by electron microscopy in mice deficient for Atg5, which was accompanied by deficits in motor function. Caccamo et al. [19] also established mouse model of AD and detected that in hippocampal neurons mTOR signaling was significantly enhanced, which was accompanied by the buildup of β-amyloid (Aβ), whereas in the rapamycintreated AD mice, mTOR activity and Aβ levels were significantly reduced, levels of Atg7 and the Atg5/Atg12 complex, and LC3-II were significantly increased in the brains, cognitive deficits in rapamycin-treated AD mice were also rescued. These studies all indicate that there is a close link between autophagy and hippocampal neuronal damage in AD. The role of autophagy in hippocampal neuronal injury in AD For the specific role of autophagy in hippocampal neuronal injury in AD, the current studies inclined to be neuroprotective. Autophagy can reduce the level of mutant or toxic proteins such as Aβ and Tau protein in neurons. At the earliest disease stage, a progressive dysfunction of autophagy could be detected in damaged hippocampal neurons [20, 21], mainly including disorders of autophagic membrane formation and autophagy flux transportation, the former can be manifested in the significant reduction of the autophagy-related proteins such as Beclin 1 and LC3-II, the latter occurs mainly in dysfunction of autolysosomal protein degradation, which may affect neuroprotective role of autophagy in AD. Pickford et al. [22] labeled LC3 in hippocampal neurons of Beclin-1+/− and Beclin-1+/+ mice by immunofluorescence and detected that Beclin 1-deficient neurons had fewer than half the number of autophagosomes in Beclin-1+/− group than in Beclin-1+/+ group, which indicated that heterozygous deletion of Beclin-1 in mice may decrease neuronal autophagy. They further established Beclin-1 deficient and amyloid precursor protein (APP) heterozygous transgenic mice model and detected that Beclin-1 protein levels were reduced by half in APP+ Beclin-1+/− group compared with APP+ Beclin-1+/+ group, which was also accompanied by a nearly twofold increase in extracellular Aβ deposition. By injecting the lentiviral vector expressing Beclin-1, they detected the high level of GFP-LC3 in hippocampal neurons, and both
intracellular and extracellular amyloid pathology were reduced. These studies suggest that Beclin-1 deficiency can disrupt autophagy and induce disorders of autophagic membrane formation in hippocampal neurons. Reduced expression of Beclin-1 may modulate APP metabolism, leading to abnormal accumulation of extracellular Aβ and abnormal nerve cell ultrastructure, which finally promote neurodegeneration and the occurrence of AD. Conversely, increased expression of Beclin-1 may promote the formation of autophagosomes, which can reduce abnormal deposition of Aβ, delay the development of AD, and thus may play a protective role in hippocampal neuronal injury in AD. Studies show that disorders of autophagy flux transportation may also increase hippocampal neuronal injury, leading to cognitive impairment. Yang et al. [23] established TgCRND8 mouse model overexpressing a version of APP695 and producing more Aβ. It was similar to the pathological mechanisms previously described in AD brains and immunolabelled with an rabbit polyclonal antibody against mouse cathepsin D, the results detected that in the hippocampal CA1 sector, including the pyramidal cell layer, giant cathepsin D-positive lysosomal compartments were most abundant, along with large numbers of reduced normal-sized lysosomes, indicating abnormal accumulation of ubiquitinated proteins within these compartments, which were incompletely degraded. They detected by immunoblot analysis that there were abnormally high levels of LC3 and very high proportions of LC3-II in two autophagic vacuole fractions and lysosome fractions in TgCRND8 mice than in control groups. These analyses suggest that disruption of autophagy flux transportation may exist in hippocampal neurons in AD, leading to the accumulation of Aβ in autophagic vacuoles and autolysosomes. To examine and confirm the inherent relationship between autophagy flux transportation and accumulation of Aβ, they further deleted cystatin B in TgCRND8 mouse model to establish CBKO/TgCRND8 mouse model, cystatin B can selectively enhance the activities of cysteine proteases and improve lysosomal proteolytic function. They found that the number of giant autolysosomes seen in TgCRND8 mice was largely reduced in CBKO/TgCRND8 mice, while normal-sized lysosomes increased extensively, enzymatic activities of cathepsin B, cathepsin L, and cathepsin D were higher in CBKO/TgCRND8 brains than in CBKO brains. In addition, intracellular Aβ and LC3-II immunoreactivity seen by immunocytochemistry and western blotting were markedly reduced in the autophagic vacuole and lysosome fractions in CBKO/TgCRND8 brains, these mice may improve contextual memory deficits those found in TgCRND8 mice. These results consistently demonstrate that the incidence of AD may influence downstream of autophagy flux transportation in hippocampal neurons and reduce hydrolytic activity of autolysosomess as well as long-term excessive Aβ deposition, which may induce the degeneration and death
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of hippocampal neurons, eventually leading to hippocampal neuronal dysfunction. In addition, the process of autophagy may be influenced by different pathological stages of AD. Yu et al. [24] studied that autophagy was induced very early in AD; elevation of LC3-II in AD brains at preclinical stages of AD suggested that autophagic induction was an early response to the disease process. They also analyzed autophagic vacuoles (AVs) levels in PS1/APP mice that could simulate pathological changes of AD including Aβ deposition. The result showed that AVs were visible in the cell bodies. These observations demonstrate that autophagy is induced at a prepathological stage of disease in PS1/APP mice, as in AD. Sun et al. [25] also found that expression of mTOR was expressed in the control and AD hippocampus. The expression levels of the corresponding signaling molecules were significantly increased in the hippocampus in the severe stages of AD, compared with controls and other stages of AD. They also indicated that mTORC1, but not mTORC2, was activated in AD brains and that the level of mTOR signaling activation was correlated with cognitive severity of AD patients. Therefore, in different pathological stages of AD, the activation of autophagy and the expression of the corresponding labeled molecules may also be influenced by varying degrees. In short, appropriate level of autophagy could be essential in delaying degeneration of hippocampal neurons and preventing the development of AD, which can clear the abnormal deposition of intracellular and extracellular proteins in AD. Additionally, targeted therapies for disorders of autophagic membrane formation and autophagy flux transportation may be expected to reduce the abnormal accumulation of proteins, which could become a potential clinical treatment to prevent or retard the development of AD.
Autophagy and hippocampal neuronal injury in cerebral ischemia/hypoxia Relevance Autophagy is not only widely involved in generation and the development of hippocampal neuronal injury in neurodegenerative diseases such as AD, a large number of studies have confirmed that there are also closely internal relations between autophagy and hypoxia/ischemia-induced hippocampal neuronal injury [26–28]. And studies on neuron death after hypoxia/ischemia brain injury using various animals have also shown that the hippocampus is the most vulnerable to ischemic insult to other neurons [29]. As early as 1995, Nitatori et al. [30] detected autophagy in ischemic brain injury for the first time, they observed a large number of autophagic vacuoles and cathepsin B in hippocampal CA1 neurons by electron microscopy 3 days after
treatment against hypoxic stimulation; Koike et al. [29] established the mice model of acute ischemic brain injury by ligating the left common carotid artery and detected large numbers of LC3 particles appeared in left hippocampus pyramidal cells by immunofluorescence after 8 h ischemic stimulation, suggesting that autophagy activity exist in damaged hippocampal neurons. They further established Atg7-deficient rats model and detected by Western blotting that conversion from LC3-I to LC3-II in hippocampal neurons was significantly reduced at 1 day after ischemic treatment. These studies indicate that autophagy plays a crucial role in hippocampal neuronal injury induced by cerebral ischemia. The dual role of autophagy Recent studies have found that the role of autophagy in hippocampal neuronal injury induced by cerebral ischemia seemed to be duality, which not only could be cytoprotective, but also could lead to cell death and increase hippocampal neuronal injury under certain conditions. This paradoxical role of autophagy always depends on the time of ischemia stimulation, the extent of ischemic reperfusion, and induction of autophagy [31]. Hippocampal neuroprotective actions of autophagy Studies have confirmed that transient ischemia can promote mild to moderate activation of autophagy in hippocampal neurons. Moderate autophagic activity may help to delete the abnormal sediment in impaired neurons and promote neuronal survival, which can protect ischemic hippocampal neurons. Carloni et al. [32] established Sprague–Dawley rats model which was anesthetized and subjected to ligation of the right common carotid artery followed by 2.5 h hypoxia (92 % nitrogen and 8 % oxygen) to observe the expression of autophagy in the cortex and hippocampal neurons after transient ischemic stimulation. The results showed that Beclin-1positive cells were sparsely detectable at 4 h after hypoxia– ischemia (HI) in the injured side in the cerebral cortex. In the hippocampus, conversely, the number of Beclin-1-positive cells was higher and cells were mainly present in the CA1 area. terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were found mainly in the superficial layers of the cerebral cortex, in which most TUNEL-positive cells were also Beclin-1-positive. They further evaluated through 3MA/WT/rapamycin treatments if modulation of autophagy could modify this pattern of cell demise, the results showed that the reduction of Beclin-1positive cells and activated caspase-3 expression and simultaneously intact PARP-1were detectable after treatment with the two inhibitors. As expected, treatment with rapamycin increased Beclin-1 expression and reduced the expression of activated caspases-3; accordingly, cell loss appeared markedly attenuated both in the cerebral cortex and in the hippocampus.
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These results suggested that inhibition of autophagy may transform mode of cell death from apoptosis to necrosis. In contrast, enhanced autophagy activity can reduce the progression of cells towards necrotic cell death, resulting, therefore, in decreased ischemic hippocampal neuronal injury. These studies indicate that activation of autophagy may be a component of the hippocampal neuronal survival mechanisms turned on after brain ischemia. If autophagy is inhibited, neurons may quickly proceed towards necrotic degeneration through interrupting the apoptotic program. Conversely, limited overactivation autophagic pathways may delay the progression of cells towards death, simultaneously indicating that moderate activation of autophagic pathways may represent a potential, protective mechanism in hippocampal neuronal injury induced by cerebral ischemia. Liu et al. [33] also detected through the establishment of Wistar rats HI model the abnormal accumulation of denatured organelles in the hippocampal CA1 region after transient ischemic stimulation. They also found that intracellular LC3II expression was significantly increased, which suggested that autophagy may protect hippocampal neurons from being eroded by harmful substances through degradating abnormal protein deposition and denatured organelles. Hippocampal neuronal injury induced by overactivations of autophagy However, some other studies took different opinions that increased autophagic activity may conversely aggravate hippocampal neuronal injury after transient ischemia. Zheng et al. [34] established adult male mice model of transient middle cerebral artery occlusion and nicotinamide adenine dinuleotide (NAD(+)) was administered intraperitoneally at the same time that reperfusion started, detecting that LC3-II expression was significantly lower in the cell cortex and hippocampus. In addition, administration with NAD(+) led to significant decreases in neurological deficits at 48 h after ischemia. The results suggest that NAD(+) may inhibit autophagy and reduce hippocampal neuronal injury. NAD(+) can reduce cell death caused by excessive activation of oxidative stress, speculating that in ischemic stimulation, autophagy may aggravate cellular oxidative damage via activation of oxidative stress and thus aggravate ischemic neuronal damage. Further studies have found that in the long term of cerebral ischemia especially after reperfusion, autophagy occurs to be overexpression, which not only degrades denatured proteins, but also degrades normal cellular components such as mitochondria and other organelles, resulting in the depletion of energy needed for cells growth and normal metabolism; all of these would ultimately aggravate neuronal damage [35]. Shi et al. [36] administrated rat neurons exposed to oxygen and glucose deprivation (OGD) and reperfusion (RP), respectively, and observed numerous autophagic vacuoles with
characteristic morphological features of autophagosomes and autolysosomal structures in neurons after OGD/RP injury. Meanwhile, a large number of nuclear condensation, swelling, and dilated mitochondria and acidophilic denatured protein were also frequently found. They also observed that the level of LC3 punctuate staining in the rat neurons after OGD increased dramatically according to the elongation of RP time, which indicated an intense and excessive activation of autophagy after HI and RP injury. They further detected that the number of cell death was significantly reduced at RP 72 h after inhibiting autophagosome formation by 3-methyladenine (a specific autophagy inhibitor), suggesting that the increase of autophagy was accompanied with an progressively increase of autophagic neuronal death. On the contrary, inhibition of autophagy significantly reduced autophagic neuronal death. These data demonstrate that excessive activation of autophagy may be a contributing factor of neuronal death and can further aggravate the neuronal injury in cerebral ischemia. In short, the specific role that autophagy plays in hypoxic– ischemic induces hippocampal neuronal injury, which should be very complex. Both transient hypoxia–ischemia and longterm cerebral ischemia such as ischemia–reperfusion could induce neuronal damage. Autophagy, to some extent, could contribute to duality. A certain degree of autophagic activity may be beneficial to the deletion of denatured proteins and aging organelles; however, excessive activation of autophagy might actually lead to neuronal self-digestion and even necrosis, which may further aggravate the hippocampus ischemic neuronal injury.
Others In recent years, some other studies have reported that neural excitotoxicity factors, varying degrees of deficiency in essential nutrients such as vitamins or trace elements can also lead to hippocampal neuronal injury by inducing autophagy. Excitotoxicity, a phenomenon induced usually by the overactivation of excitatory amino acid receptors, is the main mediator of neuronal death including traumatic brain injury and cerebral ischemia [37]. Studies have confirmed that toxic substances can stimulate actions of autophagy in hippocampal neurons in several acute excitotoxicity models such as the application of N-methyl-D-aspartate onto organotypic hippocampal slices [38] or the direct injection of kainate into the brains of mice in vivo [39]. Meanwhile, autophagy has also been reported in some chronic excitotoxicity models [40]. Moreover, the lack of essential nutrients in vivo can also lead to a significant reduction in the number of cells and even cell death. Fukui et al. [41] found that diffuse axonal injury occurred in hippocampal CA1 neural fibers in vitamin Edeficient mice, and hippocampal CA1 neurons were significantly lower than the control group. Expressions of collapsin
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response mediator protein-2 (CRMP-2), playing a crucial role in the maintenance of axonal conditions, were significantly lower in the cerebral cortex and hippocampus of vitamin Edeficient mice than both the regions of normal ones. In addition, the appearance of MAP-LC3, a major index of autophagy, was higher in the cerebral cortex and hippocampus of vitamin E-deficient mice than in normal mice. These results demonstrate that changes in CRMP-2 and MAP-LC3 may underlie vitamin E-deficiency-related axonal degeneration by inducing activities of autophagy. In short, many reasons can lead to hippocampal neuronal injury. The degree of expression of autophagy may play a key regulatory role in the process of neuronal damage.
Concluding remarks In summary, the significance of autophagy in neurological disorders and its importance in physiological and pathological situations have been established for several decades. Nevertheless, the factors that govern the neuroprotective/neurotoxic functions of autophagy and how autophagy contributes to neuronal death are unknown. On the one hand, as an energy supplier, autophagy can be a physiological mechanism indispensable for the health of neurons by its essential role in quality control for proteins and organelles; however, the activation of autophagy exceeding a certain level may induce neuronal death by initiating the self-digestion of dying neurons. Thus, whether autophagy is a prosurvival strategy or a step in the cell death program still needs further examination and a profound understanding of the neuronal autophagy process will be expected to contribute to the future development of therapeutic interventions in chronic and acute neuronal disorders. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (no. 81370183) and a grant from Natural Science Foundation of Tianjin (no. 13JCYBJC23700).
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