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.     

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.     

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.     

Evaluation of autophagy using mouse models of brain injury

Autophagy is a homeostatic, carefully regulated, and dynamic process for intracellular recycling of bulk proteins, aging organelles, and lipids. Autophagy occurs in all tissues and cell types, including the brain and neurons. Alteration in the dynamics of autophagy has been observed in many diseases of the central nervous system. Disruption of autophagy for an extended period of time results in accumulation of unwanted proteins and neurodegeneration. However, the role of enhanced autophagy after acute brain injury remains undefined. Established mouse models of brain injury will be valuable in clarifying the role of autophagy after brain injury and are the topic of discussion in this review.     

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.     

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.     

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     

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.     

Chemical modulators of autophagy as biological probes and potential therapeutics

Autophagy is an evolutionarily conserved mechanism for protein degradation that is critical for the maintenance of homeostasis in man. Autophagy has unexpected pleiotropic functions that favor survival of the cell, including nutrient supply under starvation, cleaning of the cellular interior, defense against infection and antigen presentation. Moreover, defective autophagy is associated with a diverse range of disease states, including neurodegeneration, cancer and Crohn's disease. Here we discuss the roles of mammalian autophagy in health and disease and highlight recent advances in pharmacological manipulation of autophagic pathways as a therapeutic strategy for a variety of pathological conditions.     

Complex inhibitory effects of nitric oxide on autophagy

Autophagy, a major degradation process for long-lived and aggregate-prone proteins, affects various human processes, such as development, immunity, cancer, and neurodegeneration. Several autophagy regulators have been identified in recent years. Here we show that nitric oxide (NO), a potent cellular messenger, inhibits autophagosome synthesis via a number of mechanisms. NO impairs autophagy by inhibiting the activity of S-nitrosylation substrates, JNK1 and IKKβ. Inhibition of JNK1 by NO reduces Bcl-2 phosphorylation and increases the Bcl-2-Beclin 1 interaction, thereby disrupting hVps34/Beclin 1 complex formation. Additionally, NO inhibits IKKβ and reduces AMPK phosphorylation, leading to mTORC1 activation via TSC2. Overexpression of nNOS, iNOS, or eNOS impairs autophagosome formation primarily via the JNK1-Bcl-2 pathway. Conversely, NOS inhibition enhances the clearance of autophagic substrates and reduces neurodegeneration in models of Huntington's disease. Our data suggest that nitrosative stress-mediated protein aggregation in neurodegenerative diseases may be, in part, due to autophagy inhibition.     

Functions of autophagy in normal and diseased liver

Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases.     

Dismantling the autophagic arsenal when it is time to die

Under stress conditions cells activate different response pathways which result in cell survival or apoptosis depending on: (1) the nature of the insults, (2) the type, if acute or chronic stress, and (3) how long the stress persists. Generally, autophagy is induced early to sustain cell survival and inhibit cell death. However, adverse conditions are able to overcome autophagy to promote cell death. Increasing evidence suggests that the inhibition of autophagy by the apoptotic machinery has been proposed as one of the crucial events responsible for the irreversible switch from survival to death. The mechanism seems to be related to the selective apoptotic protease-mediated degradation of key autophagic proteins. We recently found that AMBRA1, an important regulator of the autophagic process mediating the initial steps of autophagosome formation, is also irreversibly degraded by the combined activity of caspases and calpains. This phenomenon is not merely a consequence of apoptosis execution but represents a key step required to efficiently promote the autophagic vs apoptosis switch.     

Neuronal autophagy: going the distance to the axon

Autophagy, a regulated cellular degradation process responsible for the turnover of long-lived proteins and organelles, has been increasingly implicated in neurological disorders. Although autophagy is mostly viewed as a stress-induced process, recent studies have indicated that it is constitutively active in central nervous system (CNS) neurons and is protective against neurodegeneration. Neurons are highly specialized, post-mitotic cells that are typically composed of a soma (cell body), a dendritic tree and an axon. The detailed process of autophagy in such a highly differentiated cell type remains to be characterized. To elucidate the physiological role of neuronal autophagy, we generated mutant mice containing a neural cell type-specific deletion of Atg7, an essential gene for autophagy. Establishment of these mutant mice allowed us to examine cell-autonomous events in cerebellar Purkinje cells deficient in autophagy. Our data reveal the indispensability of autophagy in the maintenance of axonal homeostasis and the prevention of axonal dystrophy and degeneration. Furthermore, our study implicates dysfunction of axonal autophagy as a potential mechanism underlying axonopathy, which is linked to neurodegeneration associated with numerous human neurological disorders. Finally, our study has raised a possibility that "constitutive autophagy" in neurons involves processes that are not typical of autophagy in other cell types, but rather is highly adapted to local physiological function in the axon, which is projected in a distance from one neuron to another for transducing neural signals.     

Autophagic-lysosomal dysfunction and neurodegeneration in Niemann-Pick Type C mice: lipid starvation or indigestion?

Increasing evidence shows that autophagy, particularly macroautophagy, plays a Dr. Jekyll and Mr. Hyde role in determining cell fate; autophagic activity can be protective under certain conditions, whereas it may lead to cell death under others. Niemann-Pick Type C (NPC) disease is an early onset autosomal recessive disorder characterized by accumulation of cholesterol and other lipids in late endosomes/lysosomes. About 95% of the cases are caused by mutations in the NPC1 gene, whereas the remaining 5% are due to mutations in the NPC2 gene. Severe neurodegeneration that accompanies NPC is likely the fatal cause in this disease, although the underlying mechanism remains unclear. Our study shows that autophagic activity is enhanced in Npc1-/- mice, as evidenced by increased levels of LC3-II and the number of autophagic vacuole-like structures. Interestingly, LC3 immunoreactivity co-localizes with filipin-labeled cholesterol clusters inside Purkinje cells. Furthermore, increases in autophagic activity are closely associated with alteration in lysosomal function and protein ubiquitination. In this article, these results are further discussed in the context of autophagic-lysosomal function and neuronal survival and degeneration.     

Silencing DJ-1 reveals its contribution in paraquat-induced autophagy

The role of autophagy as a survival strategy of cells constitutes an emerging topic in the study of the pathogenesis of several diseases with autophagic changes being described in a number of age-related neurodegenerative disorders, including Parkinson's disease (PD). Although the etiology of PD is still unknown, both environmental (for example, paraquat exposure) and genetic factors have been investigated as putative causes of the disease. In the latter case, mutations or changes in the protein DJ-1 have been reported to be associated with autosomal recessive, early-onset parkinsonism. In this paper we established a model system to study the involvement of the DJ-1 protein in paraquat-induced autophagy. When human neuroblastoma SH-SY5Y cells were transfected with DJ-1-specific small interfering RNAs and exposed to paraquat, we observed (i) sensitization additive with paraquat-induced apoptotic cell death, (ii) inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles, (iii) exacerbation of apoptotic cell death in the presence of the autophagy inhibitor 3-methyladenine, and (iv) an increase in mammalian target of rapamycin phosphorylation. Taken together, these findings suggest an active role for DJ-1 in the autophagic response produced by paraquat, providing evidence for the role of PD-related proteins in the autophagic degradation pathway, a factor that should be considered in the design of potential therapies for the treatment of the disease.     

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.     

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.     

A novel method for autophagy detection in primary cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8⁺ T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.     

A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.