The Autophagy-Lysosomal Pathway in Neurodegeneration: A TFEB Perspective

Abstract

The autophagy-lysosomal pathway (ALP) is involved in the degradation of long-lived proteins. Deficits in the ALP result in protein aggregation, the generation of toxic protein species, and accumulation of dysfunctional organelles, which are hallmarks of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and prion disease. Decades of research have therefore focused on enhancing the ALP in neurodegenerative diseases. More recently, transcription factor EB (TFEB), a major regulator of autophagy and lysosomal biogenesis, has emerged as a leading factor in addressing disease pathology. We review the regulation of the ALP and TFEB and their impact on neurodegenerative diseases. We also offer our perspective on the complex role of autophagy and TFEB in disease pathogenesis and its therapeutic implications through the examination of prion disease.

Keywords: TFEB; autophagy–lysosomal pathway; neurodegenerative disease; prion disease.

Figure 1. Simplified Diagram of Autophagy Pathways

Macroautophagy is initiated by recruitment of ULK and PI3K complex to the phagophore assembly site (PAS). The ULK complex is composed of ULK1/2, FIP200, and ATG13. The PI3K complex is made up of Vps34, Vps15, Beclin 1 (ATG6), and Barkor (ATG14). In elongation, two ubiquitin-like conjugating systems are involved. In the first, ATG12 is linked to ATG5 through ATG7 and ATG10, then the ATG5–ATG12 conjugate forms a complex with ATG16. In the second ubiquitin-like reaction, LC3 is cleaved at its C-terminus by ATG4B to form LC3-I. LC3-I is then conjugated to phosphatidylethanolamine (PE) by ATG7 and ATG3 to form LC3-II. The ATG12–ATG5–ATG16 complex can also conjugate PE to LC3-I to form LC3-II. LC3-II localizes to the autophagosome and remains on the membrane from elongation to lysosome fusion. In selective autophagy, LC3-II binds to p62, which delivers ubiquitinated protein aggregates for autophagic degradation. At completion of elongation, a complete bubble-like autophagosome surrounding the cargo has formed. The autophagosome can travel along microtubules via motor proteins, and eventually fuses with the lysosome to form an autolysosome. Within the autolysosome, the lysosomal proteases degrade the cargo and inner membrane of the former autophagosome. Chaperone-mediated autophagy (CMA) differs from macroautophagy in that HSC70 binds to protein substrates containing a KFERQ motif. The substrate–HSC complex interacts with LAMP-2A which multimerizes in response to binding to form an active transport complex through which substrates pass into the lysosome after unfolding. With microautophagy, the lysosome directly engulfs cytoplasmic cargo, such as protein aggregates and lysosomal proteases degrade the material.

Figure 2. mTOR Regulatory Pathways of the Autophagy–Lysosomal Pathway (ALP)

mTORC1 is composed of mTOR, regulatory associated protein of mTOR (Raptor), G protein β-subunit-like protein (GβL), proline-rich Akt substrate of 40 kDa (PRAS40), and DEP domain-containing mTOR-interacting protein (Deptor). PRAS40 is phosphorylated (P) by Akt and dissociates from Raptor to activate mTORC1. Activated mTORC1 (in nutrient-rich conditions or in response to growth factors, etc.) phosphorylates ULK1 to inhibit its role in autophagosome formation. AMPK, however, phosphorylates ULK1 at different sites to activate autophagy. mTORC2 is composed of mTOR, rapamycin-insensitive companion of mTOR (Rictor), GβL, stress-activated protein kinase-interacting protein (SIN) 1, and protein observed with Rictor (PROTOR). mTORC2 can also participate in autophagy regulation through the FOXO3 pathway. mTORC2 phosphorylates Akt, followed by Akt phosphorylation of FOXO3. Phosphorylated FOXO3 binds to 14-3-3 protein, which retains it in the cytoplasm, preventing activation of autophagy gene transcription.

Figure 3. Regulation and Activity of TFEB

In conditions of normal nutrient availability, no lysosomal stress, and no V-ATPase inhibition, the V-ATPase, Ragulator, and Rag GTPases form an active complex that binds to mTORC1 at the lysosomal surface and activates it. mTORC1 then phosphorylates (P) TFEB which sequesters it in the cytoplasm. Phosphorylated TFEB is also bound by 14-3-3 protein in the cytoplasm. In conditions of starvation, lysosomal stress, or V-ATPase inhibition, the Rag GTPases are turned off, which releases mTORC1 from the lysosomal surface and inactivates it. Because mTORC1 can no longer phosphorylate TFEB, the unphosphorylated TFEB translocates to the nucleus to bind to the CLEAR sequence of its target genes, leading to the upregulation of autophagy and lysosomal genes. The TFEB gene also has numerous CLEAR sequences in its promoter, and thus TFEB upregulates its own expression in an autoregulatory loop. Another positive feedback loop deals with MCOLN1, a lysosomal calcium efflux channel that is a transcriptional target of TFEB. MCOLN1 creates a microdomain of high Ca²⁺ concentration near the lysosomal surface. The higher Ca²⁺ concentration then activates the phosphatase calcineurin (CN), which dephosphorylates TFEB, promoting its activation.