The convergence of endosomal and autophagosomal pathways

Abstract

We have reported previously that autophagy is responsible for amyloid precursor protein-C-terminal fragment (APP-CTF) degradation and therefore Aβ clearance. To elucidate the underlying mechanism, using LC3 affinity purification and mass spectrometry analysis, immunoprecipitation (IP), as well as live imaging analysis, we identified and demonstrated that the adaptor-related protein complex 2 (AP2) and PICALM (phosphatidylinositol binding clathrin assembly protein) are in a complex with LC3 and APP-CTF. Taken together, this new set of data suggests that the AP2-PICALM complex functions as an autophagic cargo receptor for the recognition and shipment of APP-CTF from the endocytic pathway to the LC3-dependent autophagic degradation pathway. Interestingly this AP2-LC3 connection seems to be involved in chemically-induced APP-CTF clearance as we observed using the small compound SMER28. The effect observed following SMER28 was significantly reduced after silencing AP2. While more work is required to elucidate the detailed molecular mechanisms involved, our actual data suggest that there is some level of specificity in the steps mentioned above.

Aging and related dementias have constituted an extremely difficult challenge for conventional therapeutic interventions. While highly targeted approaches (e.g., gamma-secretase inhibitors) have proven to be rather inefficient, some of the nascent strategies might bring some new hope. Aging is a global and complex phenomenon, involving multifaceted processes affecting most biological functions and pathways, such as protein folding, protein trafficking, and protein degradation among others. Protein degradation can follow various cellular routes. We will focus here on autophagy, a phenomenon relatively recently associated with Alzheimer disease.

While the gamma-secretase strategy has not yet been fruitful, the amyloid plaques made of aggregated amyloid β (Aβ) peptides remain a hallmark pathological feature of Alzheimer disease (AD). Aβ peptides are generated via sequential proteolysis of APP during the course of its intracellular trafficking. Others and we have recently reported that the clearance of Aβ peptides via autophagy results from the degradation of the precursor APP-CTF. However, the molecular mechanism by which autophagy leads to the downregulation of the membrane-bound APP-CTF is not known. In order to address this question, we sought to identify factors that interact with LC3 and that might be involved in the targeting of APP-CTFs to phagophores.

Using LC3 affinity purification and mass spectrometry analysis, we identified, among others, AP2, an adaptor protein involved in the proteolysis and maturation of Aβ via clathrin-mediated endocytosis. The association of AP2 with LC3 was confirmed through IP experiments in different systems including in vivo using AD double-transgenic mouse brain lysates (APPswe/PS1dE9). Silencing an AP2 subunit (AP2A1) through RNAi leads to a significant increase in the levels of APP-CTF and Aβ40 peptide. Through IP experiments, we found that AP2 interacts with APP-CTF (βCTF) in addition to binding to LC3, suggesting that AP2 may serve as a linking molecule as recently shown for SQSTM1/p62.

Investigating the putative AP2 recognition signal located on APP, we demonstrated that after mutating the “YKFF” motif, APP-CTF is no longer able to interact with AP2, nor with LC3. Likewise, when an LC3-interacting region containing the “WTHL” motif found in AP2A1 is mutated, LC3 no longer co-immunoprecipitates AP2A1. Collectively, our biochemical analysis indicates a crucial role of AP2 as an intermediate to connect APP-CTF and LC3, therefore potentially facilitating the fusion of APP-CTF-containing vesicles with autophagosomes. This was further supported by live imaging studies performed in cultured cells. Indeed, punctate structures are visible throughout the cytoplasm for both eGFP-LC3 and mCherry-tagged AP2A1. While moderate levels of colocalization of mCherry-AP2A1 and eGFP-LC3 are observed under normal conditions, serum starvation yields a significant increase in mCherry-AP2A1 and eGFP-LC3 colocalization. Through time-lapse imaging, we were able to follow the synchronized movements of mCherry-AP2A1 and eGFP-LC3 for a substantial period of time, indicating that the increased colocalization is not random and likely due to the stable association of vesicles that carry mCherry-AP2A1 and eGFP-LC3.

Interestingly, in addition to AP2, we found that PICALM, a protein encoded by a gene identified through GWAS studies as a risk factor for AD and a known AP2 binding partner, is also recruited to LC3-marked phagophores. Through IP experiments, we showed enhanced binding of PICALM, as well as AP2, to LC3 upon starvation. Therefore, we speculate that PICALM might have an important role through its function in the clearance of APP-CTF via autophagy. The precise role of PICALM in relation to APP-CTF and autophagosomes remains to be elucidated, but one can propose, based on the results summarized here, that APP-CTF can be removed from the cell surface via a complex involving LC3, AP2, and PICALM, which allows autophagosomes and endosomes to come together, leading to their fusion and ultimately their lysosomal degradation (see model Fig. 1). While it makes sense energy-wise that cell surface proteins and receptors would be removed from the surface when not needed (e.g., in the case of starvation) and then targeted to autophagic degradation to compensate for missing nutrients, it is not clear what level of specificity is involved in this process.

Figure 1. AP2-PICALM serve as intermediary factors to facilitate the interaction of LC3 and APP-CTF, an interaction required for APP-CTF degradation and Aβ clearance. A schematic model of the involvement of AP2 and PICALM in the regulation of APP-CTF and Aβ levels. Through simultaneous association with LC3 and APP-CTF, located at the surface of autophagosomes and endosomes, respectively, AP2 participates in the fusion of the 2 types of vesicles, which then fuse with lysosomes leading to the degradation of APP-CTF, and ultimately to the clearance of Aβ peptide.

Finally, through RNA silencing experiments, we demonstrated that AP2 is required for APP-CTF degradation and Aβ clearance mediated by enhanced autophagy induced by starvation. We obtained similar results using a compound that we previously described as increasing autophagy and APP-CTF clearance. Indeed, the small molecule enhancers of rapamycin 28 (SMER28) effect is significantly reduced in parallel to lowering AP2 expression. We have shown previously that SMER28 has no effect on the level of processed NOTCH and APLP1. All together these results seem to indicate that there is some specificity associated at least with some of the steps described above. In the future, it will be of interest to elucidate the mechanism underlying this selectivity and also identify the components responsible for the recognition of APP-CTF-containing membranes/vesicles.

Tian Y, Chang JC, Fan EY, Flajolet M, Greengard P. Adaptor complex AP2/PICALM, through interaction with LC3, targets Alzheimer’s APP-CTF for terminal degradation via autophagy. Proc Natl Acad Sci U S A. 2013;110:17071–6. doi: 10.1073/pnas.1315110110.