The process of ageing compromises the age-associated decrease in fertility, gradual loss of function, and increased vulnerability to disease, which progressively diminishes the capability of an organism to survive [1-3]. Unsurprisingly, in the past years it has been of great interest to understand which factors influence this inevitable and complex process. As a result a wide array of molecular and cellular damages has been identified and shown to accumulate during ageing. The lifelong accumulation of such damages will eventually result in frailty and disease . The variety of identified age-dependent damages has given rise to different theories for molecular ageing mechanisms. These mechanisms include decreased cellular capacity to deal with DNA damage, and decline in cellular division capacity, which is linked to the progressive shortening of telomeres upon each cell cycle. Also an increased accumulation of damaged mitochondria and the involved increase in reactive oxygen species (ROS) production and decline in ATP synthesis has been shown to occur over time (reviewed in ). One of the phenotypic hallmarks of aged cells is the intracellular accumulation of damaged proteins and therefore protein turnover/protein degradation has attracted attention over the last years .
At the same time, forward genetics have allowed to investigate single gene alterations and their influence on lifespan of whole organisms. Even though the ageing process is without doubt influenced by stochastic and environmental factors, single gene mutations were shown to extend lifespan in worms, flies, and mice, suggesting the existence of a central process of ageing [6, 7]. Many of the genetic manipulations that alter longevity affect metabolism, nutrient sensing and stress response pathways. As all these pathways are connected to autophagy (an important player also in protein turnover), the question about the role of autophagy in ageing has come more and more to the fore. In this chapter we will focus on how research conducted in the excellent genetic model system
Autophagy, which literally means “self-eating” (coined by Nobel Laureate Christian de Duve in 1963), allows cells to digest cytosolic components via lysosomal degradation. Autophagy and the Ubiquitin Proteasome System (UPS) constitute together the main cellular pathways for protein and organelle turnover [8, 9]. Today, three different classes of autophagy are distinguished: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy.
During microautophagy, which is mainly studied in yeast (containing vacuoles instead of lysosomes), cytoplasmic material is delivered to the vacuolar lumen by direct invagination of the vacuolar boundary membrane and budding of autophagic bodies into the vacuolar lumen . The molecular mechanisms underlying microautophagy in eukaryotic cells are largely unknown. However, Cuervo and colleagues described a microautophagy-like process (named endosomal microautophagy, e-MI) in mammalian cells, whereby soluble cytosolic proteins are selectively taken up by late endosomes/multivesicular bodies (MVBs). The cargo selection in e-MI depends on the chaperone Hsc70 and electrostatic interactions with the endosomal membrane . Hsc70 is also involved in chaperone-mediated autophagy (CMA), in which cytosolic cargo is selectively recognized, bound by the lysosome-associated membrane type protein 2A (LAMP-2A) and finally taken up by the lysosome, thereby allowing for direct lysosomal degradation of cytosolic proteins. The requirement of protein unfolding and the binding of LAMP2-A is characteristic for CMA and thereby distinguishes CMA from e-MI [11, 12]. So far, CMA has not been investigated in
The formation of a cytosolic double membrane structure called the phagophore (also called isolation membrane) is an important step of autophagy initiation. It is subject of discussion about the origin of this initial autophagic membrane. Independent experiments identified ER, Golgi, or the outer membrane of mitochondria to contribute to the phagophore double membrane [17, 18]. Cytosolic components are enwrapped during the growth of the phagophore. Closure of the phagophore completes this engulfment and gives rise to a new structure called the autophagosome. These newly formed autophagosomes will further mature and subsequently fuse with lysosomes where the captured cytosolic constituents will be degraded. Autophagy can achieve several purposes; it scavenges the cytosol from macromolecules and organelles but also provides a way to supply the cells with amino acids and if necessary with energy once the recycled amino acids are converted into intermediates of the tricarboxylic acid cycle (TCA) [15, 18-20]. It is therefore of little surprise that the autophagic machinery, which under normal conditions is running on low basal levels, can be set in motion by several intra- and extracellular stress factors, such as starvation, ER-stress, hypoxia and pathogen invasion . Besides non-selective cytosolic bulk-degradation, autophagy is also implicated in selective turnover in yeast, a pathway known as the cytoplasm-to-vacuole targeting (CVT) pathway . In analogy, cargo selective degradation of aggregated proteins (aggrephagy ), mitochondria (mitophagy ), ribosomes (ribophagy ), peroxisomes (pexophagy ), endoplasmic reticulum (reticulophagy ) and many more have been reported for mammalian systems . The role of selective autophagy in ageing will be further addressed in a separate section of this chapter.
Several protein complexes are involved along the path from initiation to completion of autophagy. Induction of autophagy in
It is believed that stepwise fusion of autophagosomes with different endosomal populations account for maturation and culminates in the fusion with lysosomes, the organelle responsible for degradation . Such stepwise fusion is supported by the findings that impairment of ESCRT machinery results in reduced autolysosome formation, measured as decrease in lysotracker staining and accumulation of Atg8 positive punctate respectively [34, 35]. Similar accumulation of autophagosomes can be seen in flies with mutant
3. Role of autophagy in development, homeostasis and ageing
In general, the role of autophagy is predominantly described as cytoprotective. Intensive research over the last decade has increased our understanding of multiple cellular events that involve autophagy, e.g. dealing with low nutrient levels, development and morphogenesis, response to oxidative stress, turnover of protein aggregates and damaged organelles, immune response and lately also cell signaling (figure 2). Altogether the picture has emerged that the role and regulation of autophagy is extremely dependent on the cellular context [20, 38, 39]. With this review we therefore want to highlight what is known from research conducted in
More than ten years ago, both the class I phosphatidylinositol 3-kinase (PI3K) and the serine/threonine kinase Target of Rapamycin (TOR) have been shown to control a signaling network that is important for development (reviewed in ). The growth of cells and tissues does require energy and building blocks. Hormones, such as insulin have been identified as important signals in order to meet these requirements by e.g. upregulation of protein synthesis. Already in 2003, Tom Neufeld speculated about the role of catabolic processes, such as autophagy, to be important in development. This idea was supported by previous findings that established a connection between reduced basal autophagic protein turnover and cellular growth as well as that Apg6p, the yeast homologue of the tumor suppressor gene
A very different aspect of autophagy during development has been revealed in the context of programmed cell death. Autophagy is upregulated during the reorganisation of the salivary gland and gut [49, 50]. Inhibition of autophagy in salivary glands by activating the class I PI3K pathway reduces salivary gland cell degradation. In contrast, induction of autophagy in salivary gland cells results in premature cell death and it was shown that this cell death is dependent on both caspases and autophagy . Similar events can be seen in the midgut. Even though caspases are highly expressed, the canonical apoptotis pathway is not required for midgut removal. Inhibition of autophagy on the other hand, impairs midgut degradation and simultaneously decreases caspase activity . Additional ways how cell death and autophagy are connected are pointed out by the findings that autophagy can selectively degrade survival factors and thereby initiate cell death. During late oogenesis, autophagy is necessary to degrade the apoptosis inhibitor dBruce in nurse cells. Nurse cells lack the, under normal conditions typical, fragmentation of DNA and caspase-3 activity in the absence of autophagy . A similar principle for cell death control is suggested by the finding that the valosin-containing protein (vcp), a ubiquitin-selective AAA chaperone, is required for degradation of the apoptosis inhibitor DIAP1 during regulated degeneration of dendrites of class IV dendritic arborisation neurons . It was already shown before that vcp is necessary for autophagy . Altogether, this implies a role for autophagy in activating apoptosis by selective degradation of apoptosis inhibitors. It will be interesting to see if such a mechanism is limited to the programmed reorganization events during development or if this is a strategy employed even in other cellular contexts. If this is a general mechanism to initiate cell death, autophagic degradation of apoptosis inhibitors might become an interesting strategy for developing drugs aimed for cancer treatment.
The role of autophagy in
A different putative way for autophagy to protect cells from oxidative stress is given by its involvement in the selective degradation of damaged mitochondria, termed mitophagy. So far, mitophagy has not been directly shown to occur in
Without doubt autophagy is crucial for cellular homeostasis and it is therefore of no surprise that autophagy is also induced upon viral or bacterial infections as both lead to changes in the intracellular environment. Flies with impaired autophagy are hampered in their immune defence. Even though this role of autophagy is much more studied in mammalian system, there are 4 different reports that highlight an involvement of autophagy in the
Autophagy does not only protect against bacterial but also against viral infection as shown in the case of the mammalian viral pathogen vesicular stomatitis virus (VSV) . Autophagy protects flies against VSV by decreasing viral replication. Repression of autophagy has the contrary effect, increased viral replication and pathogenesis. The authors of this study were able to pinpoint the PI3K/Akt pathway to be responsible for autophagy regulation upon VSV infection . Flies infected with
An additional involvement of autophagy in immunity was found in the cortical remodelling of hemocytes (
Another example for the necessity of functional selective autophagic degradation of proteins for proper homeostasis is given in muscle tissue maintenance. There, chaperone-assisted selective autophagy is necessary to remove contraction-induced damaged filamin from Z-discs in order to prevent Z disk disintegration and progressive muscle weakness in flies .
Autophagy also serves several functions in neuron plasticity and homeostasis. An interesting finding was that synapse development is controlled by autophagy via the E3 ubiquitin ligase highwire. Highwire inhibits neuromuscular junction growth and is itself a substrate for selective autophagic turnover, indicating that autophagy activity might lead to synaptic overgrowth . Tian et al. identified Rae1 to bind to highwire and thereby protecting highwire from autophagic degradation . The link between autophagy and synaptic growth at the neuromuscular junction is further strengthened by the observation that ROS can act as signaling molecules and mediate synaptic growth. At the same time, high ROS levels activate the JNK pathway, a previously reported activator of autophagy. As impairment of autophagy results in decreased synaptic size in a
Off course, autophagy also plays a major role in simply keeping the cells “clean” by enabling the cells to turn over the cytosol. This recycling effect is especially pronounced in post-mitotic cells. Flies that are mutant for Atg8a are severely hampered in their efficiency to eliminate cellular material, which can be observed as an increase in ubiquitinated proteins and the increased presence of electron dense protein aggregates in young fly brains when investigated with transmission electron microscopy. Moreover, such Atg8a mutant flies display a drastic decrease of lifespan . In addition, a very recent report by Fouillet et al., reveals an autophagy-mediated decrease of apoptosis in neurons upon mild ER-stress and further underscore a cytoprotective role of autophagy, that potentially can prolong survival of the whole organism . Taken together, these data outline the versatile role of autophagy in homeostasis and normal survival of flies.
4. Autophagy and neurodegeneration
Ageing is a major risk factor for the development of neurodegenerative diseases and over the last decade autophagy has been implicated in many neurodegenerative diseases, such as Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), or Alzheimer’s disease (AD). Many neurodegenerative diseases share the common phenotype of accumulations of protein aggregates . Before reviewing the role of autophagy in
Both, HD and PD are connected to elevated autophagy. In case of HD, autophagy can only be triggered by a mutant form of huntingtin that is prone to aggregate but not by wildtype huntingtin. Cytosolic aggregates of α-synuclein, the protein involved in PD, can be degraded by macroautophagy and CMA [74-77]. In ALS loss of motor neurons deprives patients of voluntary controlled muscle movements. The disease is associated with ubiquitinated, p62 positive protein inclusions of TDP-43 (TAR DNA binding protein 43) or SOD1 (superoxide dismutase 1) or rare mutations in a subunit of the ESCRT complex [78, 79]. A defective ESCRT complex in its turn has been shown to result in autophagosome accumulation , but also point mutations of the p150 subunit of dynactin resulting in defects in the transport machinery along microtubules have been implicated in ALS. Transport along microtubules is necessary for autophagosome-lysosome fusion and therefore crucial for functional autophagy [81, 82]. Extensive alterations in macroautophagy can also be found in patients with AD. An immuno-electron microscopy study on neocortical biopsies from AD patients identified autophagosomes, multivesicular bodies, multilamellar bodies, and cathepsin-containing autophagolysosomes as the predominant organelles that occupied most of the cytosol of dystrophic neurites. Autophagy was detected in cell bodies with neurofibrillary pathology and associated with a relative depletion of mitochondria and other organelles. The authors of this study speculated that the accumulation of immature autophagic vacuoles results from impaired transport to and fusion with lysosomes thereby hampering the protective effects of autophagy . Disruption of lysosomal proteolysis in primary mouse cortical neurons by inhibiting cathepsins, or by supressing lysosomal acidification, impairs transport of autolysosomes, endosomes and lysosomes, and leads to accumulation of these structures within dystrophic axonal swellings. Such a phenotype can also be seen in numerous mouse models of AD. The phenotype is not caused by general disruption of the axonal transport machinery, as mitochondria and cathepsin-lacking organelles were not influenced in their movements. Axonal dystrophy is reversed once lysosomal function is restored .
In the past, several independent groups have established
Macroautophagy in flies can also be upregulated by Rab5 over-expression and this approach also mitigates polyQ-huntingtin mediated degeneration in the eye . However, there is a fine line between beneficial and detrimental consequences of autophagy activation in the context of neurodegeneration as shown in a dentatorubralpallidoluysian atrophy (DRPLA) fly model . This model is built upon the expression of atrophin with a polyQ expansion and is characterized by lysosomal dysfunction and blocked autophagosome-lysosome fusion, hence reduced autophagic flux . Even though introduction of a mutant form of Atg1 intensified the neurodegenerative phenotype, upregulation of autophagy in this system had no rescuing effect but, in some case, even had the opposite outcome and increased neurodegeneration . In other words, autophagy plays an important role in scavenging polyQ atrophin from the cytosol, but is only of beneficial nature as long as autophagy can proceed all the way to lysosomal degradation. Reaching a rate-limiting step in the autophagy cycle can have negative effects on the outcome of autophagy initiation. Work from the same lab also identified a mechanism how polyQ atrophin itself impairs autophagic flux. PolyQ-atrophin inhibits the tumor suppressor fat, which under normal conditions protects from neurodegeneration through the Hippo kinase cascade and subsequent increases autophagy . How Hippo exactly activates autophagy is not completely understood yet . Data obtained from studies in the salivary gland suggests that the phosphorylation of Warts (wts), a substrate of the Hippo kinase, acts upstream of TOR and thereby regulates autophagy . It also has been reported that the Hippo pathway can directly interact with LC3 (the mammalian homolog of Atg8) and thereby initiate autophagy .
An additional, interesting link between polyQ sequence derived neurodegeneration and macroautophagy is given by puromycin-sensitive aminopeptidase (PSA). PSA is the only cytosolic enzyme capable of digesting polyQ sequences and it is therefore not surprising that there is inverse correlation between PSA expression and severity of neurodegeneration, e.g. over-expression of PSA has protective effects in cells expressing polyQ expanded ataxin-3, mutant α-synuclein and mutant superoxide dismutase (SOD) . It comes as a surprise though that this beneficial role of PSA is mediated by its activation of macroautophagy rather than its role in degrading polyQ aggregates and thereby making them available for proteasomal degradation, although the putative involvement of the proteasome in cell protection in this process remains to be further understood .
A different way to induce neurodegeneration is to inhibit proteasomal function. Interestingly, proteasome impairment can be compensated for by autophagy, a rescue that depends on the histone deacetylase 6 (HDAC6) . A protective role of autophagy in context of neurodegeneration was also demonstrated in a genetic screen conducted in
The Alzheimer’s disease related peptide Aβ1-42 also induces neurodegeneration, mediated by age-dependent autophagy-lysosomal injury in a
5. Autophagy and its role in lifetime extension
The rate of ageing is reciprocally linked to lifespan and therefore are interventions that extend longevity of an organism the most direct indication that ageing is slowed down . One well established, and long known intervention that extends lifespan is dietary restriction (DR), the limitation of food intake below the
Simonsen and co-workers showed that downregulation of autophagy genes in
The question if IUPs are cause or a consequence of the ageing process remains to be answered though. Albeit, the age-dependent accumulation of ubiquitinated proteins that are positive for Ref(2)P, a protein necessary for cargo recognition in selective autophagy, can be employed as conserved marker of neuronal ageing and progressive autophagic defects .
Also Atg7 was recently reported to extend life span when over-expressed in neuronal tissues of flies . The life-extending effect of Atg7 is not as pronounced when compared to Atg8. This might be due to different capabilities in inducing autophagic turnover, or non-autophagy related side effects of either Atg7 or Atg8a.
Proteostasis is not only important in neuronal tissues but also in muscles of flies. With increasing age polyubiquitinated proteins accumulate that co-localise with Ref(2)P in muscles and the cumulative appearance of such aggregates has been demonstrated to impair muscle fitness . The build-up of such aggregates can be reverted in muscles by the constitutive activation of the transcription factor FOXO and its target 4E-BP (eukaryotic translation initiation factor 4E binding protein). Interestingly, the activation of FOXO/4E-BP signaling in muscles is sufficient to extend lifespan of the whole organism . Furthermore it has been shown that the FOXO/4E-BP dependent delay in protein aggregate accumulation in muscles depends on functional autophagy, suggesting promotion of basal autophagy upon FOXO/4E-BP signaling . The autophagy dependent beneficial effect of FOXO is well in line with earlier findings that revealed FOXO to be capable to upregulate autophagy . In addition, the translational repressor 4E-BP is known to be upregulated upon DR and to mediate enhanced mitochondrial function and life span extension in
Taken together, all these data indicate an anti-ageing effect of autophagy, however caution is advised in trying to merely upregulate autophagy pharmacologically in order to counter-act ageing. Autophagy is essential for the recycling of cellular content, which can serve two general purposes: autophagy can unburden cells from hazards by removal of those and autophagy can provide cells with new building blocks for cellular survival. During the lifetime of an organism, autophagy will most certainly switch forth and back between those roles. In order to completely understand the complex role of autophagy in ageing it is therefore important to understand the regulation and cellular outcome of autophagy in a tissue and time dependent manner.
6. Selective autophagy and ageing
In the following section we want to shed some light on the current knowledge about the selective removal of cellular contents by autophagy in
Selective autophagy in the form of CVT has been known in yeast for a long time and has gained major attention in mammalian systems over the last years. Selectivity requires crucial, additional steps to the above described autophagy process: cargo has to be recognized by specific receptors and must be delivered to the autophagic machinery.
Ubiquitin has emerged as a molecule to tag proteins that are determined for degradation . Conjugation of ubiquitin depends on a complex reaction cascade that requires activation of ubiquitin (by E1 enzymes), conjugation (E2 ubiquitin conjugating enzyme), and ligation of ubiquitin with a target substrate (E3 ubiquitin ligase). As a result, ubiquitin is covalently bound via an isopeptide bond between the C-terminal glycine of ubiquitin and the ε-amino group of a lysine residue on the substrate protein. Substrate specificity is given by the E3 ubiquitin ligase that specifically recognizes a protein substrate and brings it to the E2 ubiquitin conjugating enzyme. A wide spectrum of E1, E2, and E3 enzymes provide cells with selectivity for this signaling machinery . Ubiquitin itself contains seven lysine residues enabling ubiquitin to self-attach, thereby forming a polyubiquitin tag. The best-characterised linkages occur via K48, targeting the substrate for proteasomal degradation, and via K63, which is preferred by ubiquitin-binding autophagy receptors. Furthermore, K63 ubiquitination has been reported to be a potent enhancer of inclusion formation and leads to substrate degradation via the autophagy/lysosome degradation pathway [116, 118-120]. Also more atypical sites for polyubiquitination, such as K6 or K29, have been reported but the exact role of these ubiquitin chains is still poorly understood .
Taken together, ubiquitin conjugation offers several possibilities to flag proteins and organelles in different ways by variation of chain length and various sites for ubiquitin self-attachment and thereby act as a signal for distinct subsequent cellular processing. Molecular links between ubiquitinated proteins and autophagy were identified in form of the cargo receptors sequestosome marker SQSTM1/p62 and NBR1 (neighbour of BRCA1 gene) . The conserved functional homologue for p62/NBR1 in
Filimonenko et al. were able to identify the mammalian phosphatidylinositol-3-phosphate (PI3P) binding protein Alfy (PI3P-binding Autophagy-linked FYVE domain protein) to be actively involved in autophagic degradation of polyglutamine (polyQ) expanded, aggregated proteins . Albeit harbouring a FYVE domain Alfy is usually not found on endosomes but instead resides in the nucleus decorating the nuclear membrane. The presence of ubiquitinated, aggregated proteins in the cytosol leads to relocalization of Alfy to these aggregates . Alfy can directly interact with p62 and Atg5 [125, 127].
Accumulation of damaged mitochondria and increased production of ROS are generally believed to account for age associated pathologies . The efficiency of selective removal of damaged mitochondria, mitophagy, might therefore play a major role in the outcome of the ageing process. Although several lines of evidence suggest the existence of mitophagy in
7. Summary and outlook
The cytoprotective role of autophagy has been shown in many different cellular contexts and induction of autophagy by either pharmacological or genetical means has life extending effects. However, research conducted in
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