Brain regions affected by Alzheimer disease (AD) display well-recognized early neuropathologic features in the endolysosomal and autophagy systems of neurons, including enlargement of endosomal compartments, progressive accumulation of autophagic vacuoles, and lysosomal dysfunction. Although the primary causes of these disturbances are still under investigation, a growing body of evidence suggests that the amyloid precursor protein (APP) intracellular C-terminal fragment Î² (C99), generated by cleavage of APP by Î²-site APP cleaving enzyme 1 (BACE-1), is the primary cause of the endosome enlargement in AD and the earliest initiator of synaptic plasticity and long-term memory impairment. The aim of the present study was to evaluate the possible relationship between the endolysosomal degradation pathway and autophagy on the proteolytic processing and turnover of C99. We found that pharmacologic treatments that either inhibit autophagosome formation or block the fusion of autophagosomes to endolysosomal compartments caused an increase in C99 levels. We also found that inhibition of autophagosome formation by depletion of Atg5 led to higher levels of C99 and to its massive accumulation in the lumen of enlarged perinuclear, lysosomal-associated membrane protein 1 (LAMP1)-positive organelles. In contrast, activation of autophagosome formation, either by starvation or by inhibition of the mammalian target of rapamycin, enhanced lysosomal clearance of C99. Altogether, our results indicate that autophagosomes are key organelles to help avoid C99 accumulation preventing its deleterious effects.—González, A. E., Muñoz, V. C., Cavieres, V. A., Bustamante, H. A., Cornejo, V.-H., Januário, Y. C., González, I., Hetz, C., daSilva, L. L., Rojas-Fernández, A., Hay, R. T., Mardones, G. A., Burgos, P. V. Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway.
Alzheimer disease (AD) is the most common form of dementia. However, the identification of cell biologic processes that cause AD remains a challenge. An important feature of brain regions of patients affected by AD is the accumulation of protein aggregates, namely Ï„ tangles and amyloid Î² (AÎ²) plaques (1, 2). AÎ² plaques are formed during the so-called amyloidogenic cleavage of the amyloid precursor protein (APP), which starts with the Î²-site APP cleaving enzyme 1 (BACE-1) that generates a C-terminal fragment (CTF) named C99 (also called CTFÎ²). In contrast, nonamyloidogenic cleavage of APP by Î±-secretases generates a shorter CTF called C83 (also known as CTFÎ±). The transmembrane domain of each CTF is further cleaved by Î³-secretase, which generates additional fragments: AÎ² and APP intracellular domain (AICDÎ³) for C99, or p3 and AICDÎ³ for C83 (3, 4).
Although enhancement of amyloidogenic proteolytic processing of APP is considered crucial to AD pathogenesis, several reports indicate that defects in autophagy and the endolysosomal pathway of protein degradation are also critical (5–7). Autophagy can proceed via 3 mechanistically distinct pathways: macroautophagy, microautophagy, and chaperone-mediated autophagy (8). Macroautophagy deficiency in mice brain results in neurodegeneration and is characterized by accumulation of ubiquitylated-protein aggregates (9), highlighting the importance of this pathway in neuronal homeostasis. Central to macroautophagy is the existence of transitory organelles called autophagosomes. Formation of autophagosomes occurs constitutively under normal nutrient conditions (9, 10), but it is highly inducible by cellular stress, such as amino acid starvation and inhibition of the key cell regulator mammalian target of rapamycin (mTOR) (11). Autophagosomes sequester cytoplasmic constituents and fuse with lysosomes to form autolysosomes, promoting degradation of sequestered content to recycle macromolecules, a process known as autophagic flux (12). Ultrastructural studies of the brain of postmortem AD patients revealed that autophagic organelles including autophagosomes and autophagolysosomes gradually accumulate in dystrophic neurites, a neuropathologic hallmark of AD, having important implications in AÎ² peptide generation and neuronal survival in later stages of AD (13, 14). Although these findings explain the lower rates of protein degradation in AD neurons, it is still under study how to manipulate these pathways to increase protein clearance in neurons (15, 16).
Autophagosomes can also fuse to prelysosomal endocytic organelles, such as late endosomes, also called multivesicular bodies (MVBs). Fusion of autophagosomes to MVBs generates a hybrid organelle called the amphisome, which ultimately also fuses to lysosomes (17, 18). Biogenesis of MVBs involves the formation of intraluminal vesicles (ILVs), which requires the activity of the 4 multisubunit endosome-sorting complexes required for transport (ESCRTs 0 to III), collectively called the ESCRT machinery (19, 20). Macroautophagy is abrogated in cells depleted of ESCRT subunits (21, 22), indicating a close interaction between these 2 pathways (10, 23).
Intracellular trafficking of APP includes its targeting to endolysosomal membranes, either from the trans-Golgi network sorted by the AP-4 adaptor complex (24, 25) or from the plasma membrane by endocytosis (26, 27). Several studies have demonstrated that APP is normally incorporated into the ILVs of MVBs in a process dependent on the ESCRT machinery (26, 28, 29). However, it is still under debate if the MVB pathway prevents amyloidogenic processing of APP (28, 29) or increases AÎ² production (26). Moreover, it is not clear how the ESCRT machinery affects both APP processing and CTF levels, and whether its effects are a consequence of ILV formation or via autophagic flux (21, 22, 30). Positive regulators of macroautophagy, such as SMER28 (31) and the transcription factor EB (32), reduce the levels of APP and its CTFs. However, the mechanism that explains how autophagosomes impact the turnover of APP and its CTFs remains unknown.
In the present study, we found that inhibition of either autophagosome formation or the fusion of autophagosomes with endolysosomal compartments caused a dramatic increase in CTF levels. Surprisingly, microscopy revealed that these CTFs accumulated in structures resembling ILVs of MVBs. In agreement with these findings, we found that activation of autophagosome formation, either by starvation or treatment with the mTOR inhibitor Torin-1, enhanced C99 lysosomal clearance. Therefore, we propose that autophagosomes are key organelles that connect with the MVB/lysosomal pathway for efficient turnover of CTFs.