Programmed cell death, together with proliferation and differentiation, is an essential process during the development of the nervous system. During neurogenesis, neurons and glia are generated in large numbers and, subsequently, they die in a process that depends on trophic signalling that refines the cytoarchitecture and connectivity of the nervous system. In addition, programmed cell death also affects proliferating neuroepithelial cells and recently differentiated neuroblasts. Autophagy is a lysosomal degradative pathway that allows the recycling of cell constituents, and seems to be able to play a dual role. It may serve to protect the cell by preventing the accumulation of deleterious products and organelles and supplying energy and amino acids. On the other hand, it has been considered a type of cell death. The role of autophagy during development is little characterized. The retina provides an excellent model system to study autophagy in the context of neural development, and to establish its relationship with proliferation, differentiation and cell death. In the present review, we summarize recent findings showing that autophagy contributes to the development of the nervous system by providing energy for cell corpse removal after physiological cell death, a process associated with retinal neurogenesis.
PCD (programmed cell death)
PCD is a highly regulated cellular self-destruction process that enables an organism to complete its morphogenesis and to eliminate damaged cells that could jeopardize its survival. Thus PCD is a physiological process that occurs both during embryonic development and throughout adult life . Classically, cell death has been morphologically classified as type I (apoptosis), type II (autophagic cell death) and type III (necrosis) . Apoptosis is the process that is most widely associated with physiological PCD.
At the cellular level, apoptosis is characterized by a reduction in cell size, chromatin condensation and the formation of blebs in the plasma membrane. At the biochemical level, the leakage of pro-apoptotic proteins from the mitochondria induces the activation of caspases, as well as other proteases and nucleases, and provokes a cascade that leads to the degradation of DNA and other cellular components . Apoptotic cells expose PtdSer (phosphatidylserine) residues in the outer leaflet of the plasma membrane, a signal that triggers their engulfment by phagocytes or neighbouring cells . Consequently, apoptotic cells are eliminated efficiently from tissues and organs by phagocytosis and lysosomal degradation, thereby avoiding the accumulation of cellular components in the extracellular space and the ensuing inflammatory reaction .
Autophagic cell death is characterized by the appearance of multiple autophagosomes in the cytosol before chromatin condensation and the degradation of intracellular organelles [6,7]. This phenotype has been observed during development in several tissues, including the nervous system [2,8,9]. In addition, autophagy has been associated with myopathies and neurodegenerative diseases [10–12], where this type of cell death seems to participate in the pathological process. However, the involvement of the autophagic machinery in bona fide physiological PCD still remains unclear.
Autophagy as a cytoprotective mechanism
Autophagy is a lysosomal pathway that permits the degradation and recycling of cell constituents, including long-lived proteins and organelles . During the autophagic process, a double membrane envelops parts of the cytosol, including complete organelles, which finally closes to form an autophagosome. The fusion of these autophagosomes with lysosomes leads to the degradation of the engulfed cellular material into amino acids and other building blocks, which can then be recycled .
Autophagy has recently gained much attention owing to the discovery of its molecular regulators, the Atg proteins, present across evolution from yeast to humans [13,14]. Yeast strains with mutations in the Atg proteins die under conditions of nutrient deprivation, indicating that autophagy promotes cell survival under such circumstances . In mammals, autophagy is involved in supplying amino acids in situations of nutrient deprivation and during the perinatal period . In addition, pharmacological or genetic inhibition of the Atg proteins induces apoptotic PCD , and, under conditions of metabolic stress, cancer cells also rely on autophagy to survive . Together, these observations indicate that, in situations of stress (e.g. a lack of nutrients, hypoxia), autophagy probably promotes cell survival by recycling cellular components to maintain proper cell function.
The cytoprotection associated with autophagy is not simply limited to obtaining nutrients under conditions of deprivation . Autophagy also degrades damaged organelles (e.g. mitochondria), as well as intracellular bacteria and viruses [20,21], and it is implicated in longevity during situations of caloric restriction . Furthermore, autophagy is also involved in eliminating protein aggregates that often appear in a variety of neurodegenerative diseases . In animal models of Huntington's disease, the activation of autophagy by a rapamycin derivate has a protective effect, reducing the number of protein aggregates and thereby augmenting cell survival . In this respect, brain-specific knockout mice for Atg5 and Atg7 show symptoms of neurodegeneration and the accumulation of ubiquitinated proteins in their brains [24,25]. Consequently, autophagy has a cytoprotective function during metabolic stress, aging and intracellular infection, as well as in certain neurodegenerative diseases.
Functions of autophagy during development
Recent studies indicate that autophagy may play an important role during embryonic development [26,27]. In Arabidopsis, Dictyostelium, Drosophila and Caenorhabditis elegans, mutants of the Atg proteins display developmental alterations that may even be lethal [28–31]. In mammals, knocking out the Beclin protein in mice (the orthologue of Atg6) produces embryonic lethality very early during development owing to severe malformations of the visceral endoderm [32,33]. Conversely, mice in which Atg5 is knocked out develop normally, but they die much faster than wild-type mice under conditions of food deprivation .
Ambra1 is a protein recently implicated in autophagy that binds to Beclin1 . This protein is highly conserved in vertebrates, where it is expressed throughout development and in the postnatal brain. Homozygous Ambra1 mutations cause embryonic lethality around E (embryonic day) 14.5, with neural tube defects such as midbrain/hindbrain exencephaly or spina bifida . In Ambra1-mutant mice, there is extensive overgrowth of the proliferative neuroepithelium associated with an increase in the number of mitotic and apoptotic cells. Thus Ambra1 is necessary to control cell proliferation and to guarantee cell survival during the development of the nervous system .
PCD and autophagy during retinal neurogenesis
Autophagy was shown recently to participate in the early development of the nervous system . Indeed, disrupting the autophagic machinery alters the correct orchestration of proliferation, differentiation and cell death necessary to establish the complex cytoarchitecture of the nervous system. To gain an insight into the role of autophagy during the early stages of neural development, we have used the chick retina, since it is a well-characterized model system for developmental studies . The chick retina can be easily grown in organotypic cultures with defined culture medium and, under such conditions, developmental processes are reproduced accurately [36,37]. In addition, this highly controlled model system permits short-term pharmacological approaches to be employed, with the aim of defining the hierarchy of the ongoing processes. We have analysed the role of autophagy in the chick retinal neuroepithelium at E4, a stage at which the earliest neuronal cell type, the RGCs (retinal ganglion cells), differentiate from the neuroepithelium following a centroperipheral gradient . This differentiation coincides with the intense proliferation of neuroepithelial cells and with PCD in the central part of the retina . Thus the early embryonic chick retina provides a good model system to characterize the possible relationship of autophagy with proliferation, neural differentiation and PCD.
To study the effect of blocking autophagy on the PCD associated with neurogenesis during retinal development, we initially used a well-known pharmacological inhibitor of autophagy, 3-MA (3-methyladenine). Inhibition of autophagy induced the accumulation of numerous TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells in a well-defined area in the central retina (Figure 1A, upper panels), where RGCs are generated at this embryonic stage [37,38]. The cell morphology in this area was examined by electron microscopy and there were numerous apoptotic cells in 3-MA-treated retinas, with small condensations at the periphery of the nucleus and a reduced cytoplasm (Figure 1B). During apoptosis, PtdSer is exposed at the outer leaflet of the plasma membrane of dying cells , which serves as a signal for their engulfment by phagocytes or neighbouring cells . When E4 chick retinas are deprived of insulin, which is a physiological survival factor in the neural retina at this stage [37,39], the number of TUNEL-positive cells increases compared with in untreated retinas (Figure 2). As expected, this increase in PCD is accompanied by an increase in annexin V staining, indicating that PtdSer is presented by the apoptotic cell in the outer leaflet of the plasma membrane (Figure 2). Conversely, when autophagy is inhibited by 3-MA and the number of TUNEL-positive cells increases, no annexin V staining is observed, indicating that apoptosis and PtdSer exposure become dissociated following inhibition of autophagy. Consequently, the increase in TUNEL-positive cells appears to be the consequence of failing to engulf apoptotic cells owing to the absence of the PtdSer ‘eat-me’ signal. Indeed, phospho-L-serine, which binds the PtdSer receptor in the engulfing cell, blocks phagocytosis and mimics the accumulation of cell corpses in the retinal neuroepithelium .
Inhibition of autophagy induces the accumulation of TUNEL-positive cells in the E4 chick retina
E4 retinas respond to growth factor deprivation but not to the inhibition autophagy by increasing annexin V and LysoTracker staining
After engulfment, apoptotic bodies are degraded inside lysosomes, a process that can be visualized using acid lysosomotropic probes such as LysoTracker . Double-labelled LysoTracker- and TUNEL-positive apoptotic bodies are present in control retinas and insulin-deprived retinas (Figure 2), confirming that lysosomes degrade the engulfed apoptotic bodies. In accordance with our previous observations, we do not detect TUNEL-positive apoptotic bodies in acidic organelles in the central part of 3-MA-treated retinas where apoptotic cells accumulate (Figure 2).
It has been demonstrated that autophagy can provide ATP for different cell functions by recycling amino acids in the tricarboxylic acid cycle . Indeed, we find that inhibiting autophagy decreases ATP levels in the E4 chick retina . To determine whether the reduction in ATP causes the accumulation of apoptotic cells, we treated retinas with MP (methylpyruvate), a cell-permeant substrate for ATP production. In the presence of MP, no increase in TUNEL staining is detected upon exposure to 3-MA (Figure 1A, lower panels) and, in addition, PtdSer exposure is restored, along with engulfment and lysosomal degradation .
Together, our data reveal a new role of autophagy with respect to PCD. Autophagy seems to be necessary to provide ATP for PtdSer exposure in apoptotic cells, a prequisite for the correct engulfment and degradation of dying cells during early stages of neural development (Figure 3). Our observations that autophagy drives the engulfment of dying cells during retinal neurogenesis is correlated with the proposed role of autophagy during cavitation . This activity may also underlie the increase in apoptosis observed in Ambra1-knockout mice at stages when neurogenesis occurs . Our short-term pharmacological approach (a mere 6 h exposure) suggests that autophagy has a primary and basal role during neural development, providing ATP for the correct removal of apoptotic cells. ATP production seems to be the key step for this role, a process relying on the homoeostatic degradative function of autophagy. A similar scenario has been found in situations of nutrient deprivation, damage to organelles, accumulation of deleterious proteins and hypoxia, where the degradative function of autophagy is cytoprotective. Further work is required to evaluate whether or not this primary degradative role underlies most of the observations on the implication of autophagy in physiological, as well as pathological processes.
Schematic representation of cell corpse removal during chick retina development and of the effect of inhibiting autophagy
Third Intracellular Proteolysis Meeting: A joint Biochemical Society and INPROTEOLYS Network Focused Meeting held at Auditorio de Tenerife, Santa Cruz de Tenerife, Canary Islands, Spain, 5–7 March 2008. Organized and Edited by Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (Barcelona, Spain), Manuel Rodríguez (CICbioGUNE, Derio, Spain), Eduardo Salido (La Laguna, Tenerife, Spain) and Dimitris Xirodimas (Dundee, U.K.).
Research in our laboratory is supported by grants from the Spanish Ministerio de Educación y Ciencia (BFU2006-00508 and BFU2006-26073-E to P.B. and SAF2007-66175 to E.J. de la R.). M.A.M. is an FPU (Formación de Profesorado Universitario) Fellow and P.B. is a Ramón y Cajal Fellow (both programmes are financed by the Ministerio de Educación y Ciencia). We thank Teresa Suárez for her comments on the manuscript.