Recent evidence suggests that the autophagic turnover of amyloid beta precursor protein (APP) may underlie the generation of toxic amyloid-β species [61].
In addition, inducing autophagy in an TOR-independent manner using lithium [53] or trehalose [54–56] has been shown to accelerate clearance of disease proteins in vitro [56] and protect against neurodegeneration in mouse and Drosophila models of Huntington’s disease [53,54].
This role in recycling is complementary to that of the UPS, which degrades proteins to generate oligopeptides that are subsequently degraded into amino acids while replenishing the cell’s supply of free ubiquitin.
Pharmacological upregulation of autophagy can be accomplished using the drug rapamycin, which works by inhibiting TOR (target of rapamycin), a pleiotropic molecule that negatively regulates autophagy, among other functions
Indeed, treatment with rapamycin ameliorates the degenerative phenotype in a Drosophila model of SBMA, as well as in Drosophila and mouse models of Huntington’s disease [48,50,52].
p62 localizes to a variety of ubiquitin-positive neuropathological inclusions including Lewy bodies in Parkinson’s disease, neurofibrillary tangles in tauopathies, polyglutamine-expanded huntingtin aggregates in Huntington’s disease, and aggregates of mutant SOD1 in familial amyotrophic lateral sclerosis [85–87].
It has been inferred that aggresome formation in vitro is a cytoprotective response in cultured cells since their formation correlates inversely with cell death, whereas interventions that block aggresome formation enhance cytotoxicity and slow the rate of turnover of misfolded proteins [27,64,70–72]
In mammals, autophagosomes first fuse with endosomes and multivesicular bodies to form amphisomes, which subsequently fuse with lysosomes to create degradative vacuoles termed autolysosomes [17].
In Huntington’s disease, affected neurons show accumulation of huntingtin in cathepsin-D-positive vacuoles [24]. Cathepsin-D is a lysosomal protease enriched in neuronal tissues, suggesting that these are autolysosomes
CLN3-related neurodegeneration appears to be a consequence of reduced autophagosome-lysosome fusion [42].
The initial step in autophagy involves expansion of a membranous structure called the “isolation membrane” or “phagophore” that engulfs a portion of the cell; the membrane eventually fuses to form a new double-membraned structure known as an autophagosome (Figure 1).
Once formed, new autophagosomes move through a stepwise maturation process that culminates with fusion to a lysosome permitting degradation of the lumenal contents.
LC3-II staining is also used as a primary histological marker of autophagosomes.
This suggestion originates from the observed accumulation of autophagic vacuoles in neurons from affected brain regions in a number of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Creutzfeldt- Jakob disease, and many of the polyglutamine diseases [21–24].
Alzheimer’s disease pathology features massive accumulation of autophagic vacuoles within large swellings along dystrophic and degenerating neurites in neocortical and hippocampal pyramidal neurons [21].
The consequences of impaired lysosome function, for example, may be observed in cathepsin D knockout mice and Drosophila melanogaster cathepsin D mutants which show neurodegeneration and associated accumulation of autophagosomes and lysosomes [33–35].
Indeed, a mouse model of distal-SBMA that expresses mutant p150/dynactin is characterized by accumulation of ubiquitin-positive aggregates and autophagic vacuoles in affected neurons [49].
Indeed, primary lysosomal dysfunction in inherited congenital “lysosomal storage disorders” has long been recognized to cause severe neurodegenerative phenotypes characterized pathologically by accumulations of lysosomes and autophagic vacuoles [38]
For example, the neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of inherited, neurodegenerative disorders with onset ranging from infancy to late adulthood that are caused by a variety of defects in lysosomal function.
Microtubule-based vesicular trafficking is essential for delivery of autophagosomes to lysosomes and subsequent fusion [46], and impaired dynein-mediated trafficking is associated with impaired autophagosome/ lysosome fusion and reduced protein turnover [47,48].
The substrate-chaperone complex is then targeted to the lysosome by binding to lysosome-associated membrane protein 2A (LAMP-2A) which carries out receptor-mediated translocation of the substrate into the lysosome for degradation [11,13]
HDAC6 activity was also reported to regulate chaperone expression in response to heat shock by deacetylating Hsp90 leading to release and activation of the transcription factor HSF-1 [79].
Recent advances have demonstrated that autophagy also serves a surprisingly diverse array of additional functions, including organelle clearance, antigen presentation, elimination of microbes, as well as regulation of development and cell death [9].
Several lines of evidence suggest that there is an impairment of autophagy resulting from impaired autophagosome-lysosome fusion combined with decreasing efficiency of the lysosomal system [60].
Considering the importance of protein catabolism in maintaining cell homeostasis, it is not surprising that dysregulation of protein turnover is associated with myriad disease states such as cancer and neurodegeneration [5].
Cellular stresses such as polyQ expression, proteasome impairment, oxidative stress, and increased misfolded protein burden activate transcription and translation of p62, suggesting that it functions broadly in stress situations [83,84]
Microautophagy consists of direct engulfment of small volumes of cytosol by lysosomes [10], whereas chaperone-mediated autophagy (CMA) involves selective, receptor-mediated translocation of proteins into the lysosomal lumen [11].
CMA has also been found to contribute to the degradation of α-synuclein [29].
For example, α-synuclein is degraded at least in part by CMA [29].
The neuronal protein α-synuclein, for example, can be degraded by the UPS, macroautophagy and chaperone-mediated autophagy [26,29].
UPS-mediated catabolism is also essential to mainten amino acid pools in acute starvation and contributes significantly to the degradation of defective proteins [1,2,7].
Indeed, experimental evidence indicates that neurodegeneration is frequently associated with impaired UPS function, although whether this is a cause or consequence of neurodegeneration is a contested issue, as is reviewed elsewhere in this special issue
In the case of α-synuclein, for example, Webb et al. concluded that soluble forms of the disease protein are efficiently degraded by the UPS, while aggregated or oligomeric α-synuclein require autophagy for clearance [26].
In addition to aggresome formation, impairment of the UPS in vitro has been found to induce autophagy [63,64].
In cells that are exposed to stressors such as starvation or UPS inhibition, Alfy relocalizes from the nuclear envelope to filamentous cytoplasmic structures that are near autophagic membranes and ubiquitinated protein inclusions, as well as within autophagosomes [77].
Specifically, it is suggested that K63-linked polyubiquitin chains recruit p62 and HDAC6 providing a signal for autophagic degradation [92,93].
This view was initially challenged by the observation that monoubiquitination operates as a key signal in endocytosis, a process important for numerous cell functions including lysosomal biogenesis [62]
The process of autophagy is controlled by parallel activation cascades that involve ubiquitin-like (UBL) protein modification, strikingly similar to the activation cascade that regulates the UPS (Figure 2a).
Autophagy, by contrast, is primarily responsible for degrading long-lived proteins and maintaining amino acid pools in the setting of chronic starvation, although its contribution to the degradation of defective proteins may equal that of the UPS.
“Autophagy”, literally “self-eating”, describes a catabolic process in which cell constituents such as organelles and proteins are delivered to the lysosomal compartment for degradation.
Autophagy is an evolutionarily conserved process whose primary task in lower organisms is the maintenance of metabolic homeostasis in the face of changing nutrient availability [8].
It has also been suggested that autophagy plays a role in the initiation or progression of some neurodegenerative diseases [20].
Insight into the role of autophagy in neurodegeneration has been provided by studies indicating that: 1) some neurodegenerative disease-related proteins are degraded by autophagy, 2) impairment of autophagy promotes neurodegeneration in animal models and several human neurodegenerative diseases, and 3) manipulation of autophagy modifies phenotypes in animal models of neurodegeneration.
That neurodegenerative disease-causing proteins are frequently degraded by autophagy was demonstrated by a series of in vitro studies which showed that pharmacological induction or inhibition of macroautophagy alters the rate of turnover of a number of disease-related proteins including polyglutamine-expanded proteins, polyalanine-expanded proteins, as well as wild type and mutant forms of α-synuclein [25,26]
It is becoming increasingly evident that the autophagy-lysosomal system is essential to neuronal homeostasis, and may in some settings be neuroprotective
Similarly, in transgenic mice expressing amyloid precursor protein, a mouse model of Alzheimer’s disease, genetic inhibition of autophagy by heterozygous depletion of beclin-1 results in enhancement of neurodegeneration [51].
For example, in a Drosophila model of X-linked spinobulbar muscular atrophy (SBMA), a polyglutamine disease, degeneration was strongly enhanced by genetic inhibition of autophagy [50].
It has now been established that clearance of misfolded proteins from aggresomes is mediated at least in part by autophagy, implicating this pathway as a compensatory mechanism for degrading misfolded proteins when the proteasome is impaired [27,64,71,73].
Similar induction of autophagy is observed in response to genetic impairment of the proteasome in Drosophila [50].
These processes are distinguished from macroautophagy, in which an isolation membrane expands to engulf a portion of the cell, eventually fusing to form a new autophagic vacuole that subsequently fuses with a lysosome [12].
There have been fewer efforts to manipulate UPS function for therapeutic benefit in neurodegenerative disease, but it was recently shown that use of a proteasome activator enhanced survival in an in vitro model of Huntington’s disease [58], suggesting that augmenting other routes of protein degradation may also provide neuroprotection.
While GABARAP and GATE-16 may also be conjugated to PE in experimental systems, at present MAP-LC3 (typically abbreviated LC3) is the only protein that is known to remain associated with the autophagosome in higher eukaryotes.
LC3-I becomes conjugated to PE to form “LC3-II” and thereby covalently associates with the phagophore
CMA is a process in which proteins harboring a pentapeptide motif related to the sequence KFERQ are specifically recognized by a cytosolic chaperone, the heat shock cognate protein of 70 kDa (hsc70).
These Atg12-Atg5 conjugates are further cross-linked to Atg16 to form a large (~350 kDa) multimeric complex, which has been thought to act as a structural support for membrane expansion [15].
As mentioned above, dramatic illustration of the interrelatedness of the UPS and autophagy was provided by characterizations of mice with conditional knockout of the essential autophagy genes Atg5 or Atg7 in the central nervous system, which resulted in neurodegeneration with accumulation of ubiquitin-positive pathology [36,37].
More recent work has demonstrated that the Atg12-Atg5 conjugate can function as an E3-like enzyme in the second arm of the Atg conjugation cascade to promote lipidation of Atg8 [16]
In a second arm of the Atg conjugation system, Atg4 cleaves the UBL protein Atg8 to promote interaction with Atg7.
Similar results have recently been observed in vitro using the proteasome inhibitor lactacystin, as pre-treatment with rapamycin attenuates lactacystin-induced apoptosis and reduces lactacystin-induced ubiquitinated protein aggregation [74].
In the first arm of the Atg conjugation system, phagophore membrane elongation is triggered through the sequential action of an E1-like protein (Atg7) and an E2-like protein (Atg10) leading to an isopeptide linkage between the C-terminal glycine the UBL protein Atg12 and a lysine residue of Atg5 (Figure 2b).
Atg8 is then conjugated with the phospholipid phosphotidylethanolamine (PE) by the concerted action of the E2-like Atg3 and the E3-like Atg12-Atg5 conjugate
Diseasecausing mutations in ATP13A2 result in protein retention in the endoplasmic reticulum and enhanced proteasomal degradation, suggesting that neurodegeneration could be caused by overwhelming the UPS and/or loss of function in lysosomal protein degradation [39].
Mutations in CLN3, a transmembrane protein that localizes to the late endosomal/lysosomal membrane, cause a form of NCL
Although the mechanism whereby autophagy and UPS function are coordinated is little understood, several regulators have emerged as important players in mediating this crosstalk, including histone deacetylase 6 (HDAC6) [50,64,75], p62/sequestosome 1 (p62) [76], and the FYVE-domain containing protein Alfy [77]; notably, these proteins have all been found to regulate or be essential for aggresome formation
Recent models propose that p62 and HDAC6 function analogously to facilitate autophagic degradation of proteins that display specific polyubiquitin topology.
HDAC6 activity appears to be important for trafficking ubiquitinated proteins and lysosomes in vitro and this has led to the suggestion that HDAC6 coordinates delivery of substrates to autophagic machinery [64,70,78].
Rab7 participates in trafficking autophagosomes and fusion with lysosomes and disease-causing mutations are predicted to impair this process [43–45].
Mutations in α-synuclein that are causative of familial Parkinson’s disease are poorly transferred to the lysosomal lumen and accumulate on the lysosomal surface, resulting in blockade of receptor-mediated translocation.
Thus, it has been suggested that p62 provides a key link between autophagy and the UPS by facilitating autophagic degradation of ubiquitinated proteins.
This model is consistent with an older study showing that inactivation of the ubiquitin-activating enzyme E1 leads to a defect in autolysosomal degradation and to an absence of ubiquitin-positive proteins within lysosomes [68].
Mutations in blue cheese, the Drosophila homology of human Alfy, lead to reduced longevity and the accumulation of ubiquitinated neural aggregates, suggesting that its role in autophagic degradation may be involved in the clearance of ubiquitin aggregates [77,94]
Many neurodegenerative diseases are characterized by accumulation of misfolded protein deposits in affected brain regions, suggesting a failure in the cell’s degradative capacity [19].
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If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.