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1.6.1 Introduction to autophagy

Autophagy is an evolutionarily conserved process of cellular “self-eating” that degrades aged organelles, protein aggregates, and ubiquitylated cargo by the formation of autophagosomes, double-membrane vesicles, for fusion with lysosomes reviewed [151]. Under resting conditions, basal levels of autophagy are very low [151]. However, multiple forms of stimuli and cell stress can induce autophagy (Figure 1.5), including cellular development and differentiation, nutrient deprivation, oxidative and endoplasmic reticulum (ER) stress, accumulation of aged organelles, hypoxia, and pathogen infection [151-154]. Under normal cellular conditions, various molecules function as inhibitors of autophagy, such as the class I PI3K/Akt signaling pathway, nutrient abundance, and cytoplasmic p53 [155, 156]. Downstream of PI3K/Akt, mTOR (mammalian target of rapamycin) is a potent negative regulator of autophagy [157]. mTOR forms a complex with ULK1, Atg13, and FIP200 – known as mTORC1 (mammalian target of rapamycin complex-1) – to inhibit autophagy [155]. ULK1 and Atg13 are hyperphosphorylated by mTOR, thus maintaining repression of the autophagy pathway [155].

During autophagy, structures known as autophagosomes are formed, which are characterized by double- or multi- membranes (approximately 300-900 nm) [158]. There are four distinct phases of ATG (autophagy-related gene) activity throughout the progression of autophagic flux, reviewed [159]. The first stage is known as the initiation phase (or the nucleation step) of autophagosome formation and involves the mTORC1, the ULK1/2, and the Class III PI3K complexes, reviewed [156, 159]. However, the source(s) of the autophagosome membrane has been under constant debate with various groups citing mitochondrial origin [160],

endoplasmic reticulum, plasma membrane, and/or Golgi, reviewed [161]. In the canonical pathway of autophagy (under conditions such as nutrient deprivation), mTOR association with ULK1-Atg13-Fip200 is limited and ULK1 and Atg13 remain hypophosphorylated, allowing for membrane expansion and the generation of the isolation membrane, or phagophore [159, 161].

Along different arms of the pathway, eukaryotic initiation factor-2 (eIF2), c-jun-N-terminal kinase-1 (JNK1), various GTPases, and intracellular calcium act upstream to induce autophagy [159]. This activates the Class III PI3K complex, composed of vacuolar protein-sorting-34 (VPS34), VPS15, Beclin-1, ATG14, AMBRA1, UVRAG (ultraviolet radiation resistance-associated gene protein), and Rubicon [156, 159]. The complex generates phosphatidylinositol-3-phosphate (PI3P), possibly on the ER membrane [156, 159]. Rubicon negatively regulates the fusion of autophagosomes and lysosomes by interacting with UVRAG [156]. Its direct binding with BCL2 and BCL-XL negatively regulates beclin-1. At the ER, the factor DFCP1 prepares for the expansion of the phagophore into a structure known as the omegasome [156, 161].

Additional proteins such as ATG9, WIPI1-4, and VMP1 are present on the preliminary autophagic membrane [156].

In the second stage of autophagy (also known as the elongation step) vesicle extension and completion occurs through two separate, conserved ubiquitin-like conjugation systems [156, 159]. The first system requires the ATG12-conjugation system involving a heterotrimeric complex ATG16L1-ATG12-ATG5, which is localized to the isolation membrane and crucial for LC3-PE (phosphatidylethanolamine) conjugation [156, 159]. The ATG12-complex is required for proper elongation [156]. ATG7, an E1-like enzyme, and ATG10, an E2-like enzyme, promote the association of the heterotrimeric ATG12-complex on the autophagosome membrane [156, 159].

The second system – the LC3-conjugation system – is indispensable for membrane expansion and closure [156]. The LC3-conjugation system is comprised of LC3 (microtubule-associated protein light chain-3) and additional autophagy related factors – ATG7, ATG4A-D, and ATG3 [156, 159]. LC3 is present at both the inner and outer autophagosome membrane with adapter functions for other substrates (including p62, NBR1, and NDP52) at the inner membrane [156]. LC3-I (18-kDa molecular weight protein) is converted to its secondary form LC3-II (16-kDa molecular weight protein) by ATG7- and ATG4-mediated cleavage, followed by ATG3-mediated PE (phosphatidylethanolamine)-lipidation at a conserved glycine residue at the LC3 C-terminus [159]. The completion of the autophagosome is followed by an ATG9 and ATG19 concerted removal of the ATG12 complex and outer membrane-associated LC3. The third phase consists of autophagosome docking and fusion with lysosomes, to form autolysosomes. The final step is the degradation of the autolysosome vesicle and its cargo, which is completed by lysosomal cathepsins B, D, and L. As autophagy is an evolutionarily conserved cell survival and recycling pathway, the residual by-products of lysosomal degradation – such as amino acids and lipids – are exported to the cytoplasm for the anabolic generation of new macromolecules [162, 163]. More recently, alternatives of the canonical pathway have been identified (e.g. independent of „required‟ factors [164, 165]) and variations (such as mitophagy, lipophagy, virophagy, pexophagy, etc.) further complicate an already convoluted and inherently complex primordial biological mechanism.

Figure 1.5. Multiple pathways to autophagy. Shown is a schematic demonstrating two of the myriad of pathways leading to the autophagic response. (Left) Autophagy as an antiviral innate immune response downstream of TLR

signaling following the recognition of viral nucleic acids. (Right) Autophagy is induced in response to cellular nutrient deprivation.

1.6.2 Autophagy as an antiviral host defense mechanism

The autophagy pathway also participates in antiviral host defense by: targeting cytoplasmic viruses for lysosomal degradation (known as xenophagy or virophagy) [166], limiting viral replication [74, 167], and/or interacting with innate immune components such as toll-like receptors (TLRs) and associated adaptors [168, 169]. Previous work has indicated that autophagy may be required for the activation of antiviral IFN-mediated signaling by certain viruses [73, 170, 171]. Autophagosome-mediated sequestration of viral antigens was required for recognition and innate immune signaling by endosomal toll-like receptor-7 (TLR7) [73, 171].

Several viruses have been identified to be susceptible to host-induced antiviral autophagy including Sindbis virus [167], tobacco mosaic virus [172], and VSV [73-75]. Other viruses have

evolved mechanisms to overcome autophagy such as HSV-1 [173-175], human cytomegalovirus (hCMV) [176, 177], Kaposi‟s sarcoma herpesvirus (KSHV), and human immunodeficiency virus (HIV), reviewed [159]. However, certain viruses usurp the host autophagy machinery to promote and enhance their replication such as HCV [178], the picornaviruses poliovirus (PV) [179] and CVB [180, 181], VZV, EBV, (Epstein-Barr virus), hepatitis B virus (HBV), HPV-16, parvovirus B19, simian virus-40 (SV40), influenza A virus, and dengue virus reviewed [159].

1.6.3 Autophagy as a post-birth survival mechanism

Autophagy has been recognized as a critical post-birth survival mechanism during the early neonatal starvation period [154, 182-184]. This is especially important in the time immediately following birth when the supply of nutrients, which had been driven by the placenta, is no longer available [154, 182]. Mouse pups deficient in an essential autophagy factor Atg5 (and thus could not undergo autophagy) died within one day post-delivery compared to wild-type littermates [182]. Numerous studies have indicated that loss of vital pro-autophagy genes including Atg3, Atg7, Atg9, Atg16L1, Beclin-1, and FIP-200 lead to early embryonic lethality, reviewed [154].

Autophagy is essential for maintaining survival immediately following birth, prior to the availability of mother‟s milk for neonatal nourishment.