Autophagy and Its Possible Roles in Nervous System Diseases,Damage and Repair

Increased numbers of autophagosomes are seen in a variety of physiological and pathological states in the nervous system. In many cases, it is unclear if this phenomenon is the result of increased autophagic activity or decreased autophagosome-lysosome fusion. The functional significance of autophagy and its relationship to cell death in the nervous system is also poorly understood. In this review, we have considered these issues in the context of acute neuronal injury and a range of chronic neurodegenerative conditions, including the Lurcher mouse, Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases. While many issues remain unresolved, these conditions raise the possibility that autophagy can have either deleterious or protective effects depending on the specific situation and stage in the pathological process.     

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.     

Regulation of neuronal autophagy in axon: implication of autophagy in axonal function and dysfunction/degeneration

Autophagy has recently emerged as potential drug target for prevention of neurodegeneration. However, the details of autophagy process and regulation in the central nervous system (CNS) are unclear. By using a neuronal excitotoxicity model mice, we engineered expression of a fluorescent autophagic marker and systematically investigated autophagic activity under neurodegenerative condition. The study reveals an early response of Purkinje cells to excitotoxic insult by induction of autophagy in axon terminals, and that axonal autophagy is particularly robust in comparison to the cell body and dendrites. The accessibility of axons to rapid autophagy induction suggests local biogenesis of autophagosomes in axons. Characterization of functional interaction between autophagosome protein LC3 and microtubule-associated protein 1B (MAP1B), which is involved in axonal growth, injury and transport provides evidence for neuron or axon-specific regulation of autophagosomes. Furthermore, we propose that p62/SQSTM1, a putative autophagic substrate can serve as a marker for evaluating impairment of autophagic degradation, which helps resolve the controversy over autophagy levels under various pathological conditions. Future study of the relationship between autophagy and axonal function (e.g., transport) will provide insight into the mechanism underlying axonopathy which is directly linked to neurodegeneration.     

The variability of autophagy and cell death susceptibility

Impaired autophagic machinery is implicated in a number of diseases such as heart disease, neurodegeneration and cancer. A common denominator in these pathologies is a dysregulation of autophagy that has been linked to a change in susceptibility to cell death. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain. How does the metabolic contribution of autophagy connect with the cell’s history and how does its current autophagic flux affect metabolic status and susceptibility to undergo cell death? How does autophagic flux operate to switch metabolic direction and what are the underlying mechanisms in metabolite and energetic sensing, metabolite substrate provision and metabolic integration during the cellular stress response? In this article we focus on unresolved questions that address issues around the role of autophagy in sensing the energetic environment and its role in actively generating metabolite substrates. We attempt to provide answers by explaining how and when a change in autophagic pathway activity such as primary stress response is able to affect cell viability and when not. By addressing the dynamic metabolic relationship between autophagy, apoptosis and necrosis we provide a new perspective on the parameters that connect autophagic activity, severity of injury and cellular history in a logical manner. Last, by evaluating the cell’s condition and autophagic activity in a clear context of regulatory parameters in the intra- and extracellular environment, this review provides new concepts that set autophagy into an energetic feedback loop, that may assist in our understanding of autophagy in maintaining healthy cells or when it controls the threshold between cell death and cell survival.     

Regulation of Neuronal Autophagy in Axon: Implication of Autophagy in Axonal Function and Dysfunction/Degeneration

Autophagy has recently emerged as potential drug target for prevention of neurodegeneration. However, the details of the autophagy process and regulation in the central nervous system (CNS) are unclear. By using a neuronal excitotoxicity model in mice, we engineered expression of a fluorescent autophagic marker and systematically investigated autophagic activity under neurodegenerative conditions. The study reveals an early response of Purkinje cells to excitotoxic insult by induction of autophagy in axon terminals, and that axonal autophagy is particularly robust in comparison to the cell body and dendrites. The accessibility of axons to rapid autophagy induction suggests local biogenesis of autophagosomes in axons. Characterization of functional interaction between autophagosome protein LC3 and microtubule-associated protein 1B (MAP1B), which is involved in axonal growth, injury and transport provides evidence for neuron- or axon-specific regulation of autophagosomes. Furthermore, we propose that p62/SQSTM1, a putative autophagic substrate, can serve as a marker for evaluating impairment of autophagic degradation, which helps resolve the controversy over autophagy levels under various pathological conditions. Future study of the relationship between autophagy and axonal function (e.g., transport) will provide insight into the mechanism underlying axonopathy which is directly linked to neurodegeneration.

Addendum to:

Induction of Autophagy in Axonal Dystrophy and Degeneration

Q.J. Wang, Y. Ding, Y. Zhong, D.S. Kohtz, N. Mizushima, I.M. Cristea, M.P. Rout, B.T. Chait, N. Heintz and Z. Yue

J Neurosci 2006; 26:8057-68     

自噬与血管新生相关细胞因子

血管新生发生于机体多种生理病理过程中,已成为诸多病理过程的标志之一。自噬参与调节机体血管新生。在病变组织中,自噬不仅与血管形成密切相关,而且经调节血管新生向病理组织提供必要的氧与能量。通过抑制自噬可以抑制缺氧、能量缺乏等刺激诱导的血管新生。血管新生过程中相关细胞因子参与调节自噬而影响新生血管的形成。通过二者的作用,既可以促进血管新生,也可抑制血管新生,这种机制在机体生理和病理过程中具有重要的作用。本文从自噬通过血管新生细胞因子促进血管新生以及自噬通过血管新生细胞因子抑制血管新生两个方面概述了自噬在血管新生过程中的作用,为疾病的治疗提供新的思路与方法。     

A new method to measure autophagy flux in the nervous system

A current need in the neuroscience field is a simple method to monitor autophagic activity in vivo in neurons. Until very recently, most reports have been based on correlative and static determinations of the expression levels of autophagy markers in the brain, generating conflicting interpretations. Autophagy is a fundamental process mediating the degradation of diverse cellular components, including organelles and protein aggregates at basal levels, whereas alterations in the process (i.e., autophagy impairment) operate as a pathological mechanism driving neurodegeneration in most prevalent diseases. We have recently described a new simple method to deliver and express an autophagy flux reporter through the peripheral and central nervous system of mice by the intracerebroventricular delivery of adeno-associated viruses (AAV) into newborn mice. We obtained a wide expression of a monomeric tandem mCherry-GFP-LC3 construct in neurons through the nervous system and demonstrated efficient and accurate measurements of LC3 flux after pharmacological stimulation of the pathway or in disease settings of axonal damage. Here we discuss the possible applications of this new method to assess autophagy activity in neurons in vivo.     

A new method to measure autophagy flux in the nervous system

A current need in the neuroscience field is a simple method to monitor autophagic activity in vivo in neurons. Until very recently, most reports have been based on correlative and static determinations of the expression levels of autophagy markers in the brain, generating conflicting interpretations. Autophagy is a fundamental process mediating the degradation of diverse cellular components, including organelles and protein aggregates at basal levels, whereas alterations in the process (i.e., autophagy impairment) operate as a pathological mechanism driving neurodegeneration in most prevalent diseases. We have recently described a new simple method to deliver and express an autophagy flux reporter through the peripheral and central nervous system of mice by the intracerebroventricular delivery of adeno-associated viruses (AAV) into newborn mice. We obtained a wide expression of a monomeric tandem mCherry-GFP-LC3 construct in neurons through the nervous system and demonstrated efficient and accurate measurements of LC3 flux after pharmacological stimulation of the pathway or in disease settings of axonal damage. Here we discuss the possible applications of this new method to assess autophagy activity in neurons in vivo.     

Selective autophagy against membranous compartments: Canonical and unconventional purposes and mechanisms

Selective autophagic degradation of cellular components underlies many of the important physiological and pathological implications that autophagy has for mammalian cells. Cytoplasmic vesicles, just like other intracellular items, can be subjected to conventional autophagic events where double-membrane autophagosomes specifically isolate and deliver them for lysosomal destruction. However, intracellular membranes appear to constitute common platforms for unconventional versions of the autophagic pathway, a notion that has become apparent during the past few years. For instance, in many cases of autophagy directed against bacterial phagosomes, subversion of the process results in multimembrane vacuoles that promote bacterial replication instead of the usual degradative outcome. In a different atypical modality, single-membrane vesicles can be labeled with LC3 to direct their contents for lysosomal degradation. In fact, single-membrane compartments of various kinds often provide an assembly site for the autophagic machinery to perform unanticipated nondegradative activities that range from localized secretion of lysosomal contents to melanosome function. Interestingly, many of these unconventional processes seem to be initiated through engagement of relevant nodes of the autophagic signaling network that, once activated, promote LC3 decoration of the targeted membrane, and some cases of inducer/receptor proteins that specifically engage those important signaling hubs have recently been described. Here we review the available examples of all autophagic variants involving membranous compartments, with a main focus on the more recently discovered unconventional phenomena where the usual degradation purpose of autophagy or its canonical mechanistic features are not completely conserved.     

Autophagy for tissue homeostasis and neuroprotection

Although autophagy has frequently been viewed as a cell death mechanism in the mammalian system, it is now considered as indispensable for the homeostasis of cells, tissues, and organisms. Basal or stress-induced autophagy plays essential and diverse roles in a variety of tissues, due to its cytoprotective properties. In this review, we briefly discuss the different homeostatic functions of autophagy that have been finely dissected in mammals through the generation and characterization of animal models with tissue-specific autophagic alterations. In addition, and given the importance of constitutive autophagy in neuronal tissues, we describe in more detail the specific roles of autophagy in the central nervous system (CNS). Finally, we discuss the contribution of autophagy malfunctions to the development of several common neurological disorders and the potential benefits of pharmacologically induced autophagy for the avoidance of neurodegeneration.     

Measurement of autophagy flux in the nervous system in vivo

Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings.     

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.     

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.     

Selective autophagy against membranous compartments

Selective autophagic degradation of cellular components underlies many of the important physiological and pathological implications that autophagy has for mammalian cells. Cytoplasmic vesicles, just like other intracellular items, can be subjected to conventional autophagic events where double-membrane autophagosomes specifically isolate and deliver them for lysosomal destruction. However, intracellular membranes appear to constitute common platforms for unconventional versions of the autophagic pathway, a notion that has become apparent during the past few years. For instance, in many cases of autophagy directed against bacterial phagosomes, subversion of the process results in multimembrane vacuoles that promote bacterial replication instead of the usual degradative outcome. In a different atypical modality, single-membrane vesicles can be labeled with LC3 to direct their contents for lysosomal degradation. In fact, single-membrane compartments of various kinds often provide an assembly site for the autophagic machinery to perform unanticipated nondegradative activities that range from localized secretion of lysosomal contents to melanosome function. Interestingly, many of these unconventional processes seem to be initiated through engagement of relevant nodes of the autophagic signaling network that, once activated, promote LC3 decoration of the targeted membrane, and some cases of inducer/receptor proteins that specifically engage those important signaling hubs have recently been described. Here we review the available examples of all autophagic variants involving membranous compartments, with a main focus on the more recently discovered unconventional phenomena where the usual degradation purpose of autophagy or its canonical mechanistic features are not completely conserved.     

MicroRNA-modulated autophagic signaling networks in cancer

MicroRNAs (miRNAs) are small, non-coding endogenous RNAs ～22 nucleotides (nt) in length that may play the essential roles for regulation of programed cell death, referring to apoptosis and autophagy. Of note, autophagy is an evolutionarily conserved, multi-step lysosomal degradation process in which a cell degrades long-lived proteins and damaged organelles. Accumulating evidence has recently revealed that miRNAs can modulate the autophagic pathways in many pathological processes, most notably cancer. In this review, we focus on highlighting the dual functions of miRNAs as either oncogenes (e.g., miRNA-183, miRNA-376b, miRNA-106a, miRNA-221/222, miRNA-31 and miRNA-34c) or tumor suppressors (e.g., miRNA-30a, miRNA-101 and miRNA-9*) via mediating several autophagic signaling pathways in cancer pathogenesis. Taken together, these findings may uncover the regulatory mechanisms of oncogenic and tumor suppressive miRNAs in autophagy, which would provide a better understanding of miRNA-modulated autophagic signaling networks for future cancer therapeutics.     

MicroRNAs在心血管自噬中的调节作用

自噬作为一种进化上高度保守的细胞降解途径,其调节异常与心血管疾病的发生、发展密切相关.研究显示,在心血管系统中,基础水平自噬对维持心肌正常收缩和传导至关重要,而在缺血/再灌注损伤和心力衰竭等心血管病理状态下,自噬水平明显增强.细胞自噬是一种多基因参与的复杂过程,近年来越来越多的证据表明,microRNAs(miRNAs)在心血管系统发育、正常生理功能维持以及不同心血管疾病(cardiovascular disease,CVDs)自噬中具有重要调节作用.本文通过对miRNAs与CVDs自噬调节方面的进展进行归纳,针对miRNAs对CVDs自噬的潜在机制进行总结,望为心血管疾病的诊断和治疗提供新的方向.     

Autophagic cell death exists

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor's Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor's Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.     

The crosstalk between autophagy and apoptosis: where does this lead?

Recent advances in the understanding of the molecular processes contributing to autophagy have provided insight into the relationship between autophagy and apoptosis. In contrast to the concept of “autophagic cell death,” accumulating evidence suggests that autophagy serves a largely cytoprotective role in physiologically relevant conditions. The cytoprotective function of autophagy is mediated in many circumstances by negative modulation of apoptosis. Apoptotic signaling, in turn, serves to inhibit autophagy. While the mechanisms mediating the complex counter-regulation of apoptosis and autophagy are not yet fully understood, important points of crosstalk include the interactions between Beclin-1 and Bcl-2/Bcl-xL and between FADD and Atg5, caspase- and calpain-mediated cleavage of autophagy-related proteins, and autophagic degradation of caspases. Continued investigation of these and other means of crosstalk between apoptosis and autophagy is necessary to elucidate the mechanisms controlling the balance between survival and death both under normal conditions and in diseases including cancer.     

Autophagy receptors in developmental clearance of mitochondria

Recent discoveries of autophagy receptors, which specifically recognize different cellular cargo destined for degradation, have opened a new chapter in the autophagy field. Selective cargo recognition by autophagic machinery is important in the context of cellular homeostasis and survival. One of the crucial homeostasis events involving autophagy is the removal of damaged or excessive mitochondria through mitophagy. Future studies on mitochondrial receptors and proteins associated with mitochondrial clearance will help us better understand the role of mitophagy in normal physiological processes as well as in diverse pathological conditions.     

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.