The time of residence associated with APP traversing the endosomal pathway is critical to its processing and appears to correlate with Abeta levels and AD pathogenesis [28, 58, 59]. Endosomes are known sorting stations, crucial to understanding AD [21–24, 37], but the molecular mechanisms underlying endosomal APP sorting and trafficking are not clearly defined [4, 28]. In the work here described, we observed that endocytosed APP molecules can be sorted for rapid retrieval to the TGN, in a retromer-mediated manner. Although we have found vesicle tubulation outside the TGN vicinity, our results suggest that tubulation occurs to a higher extent in APP-containing intermediate endosomes near the TGN (Fig. 2). Hence, the nascent tubule appears to be directly responsible for the delivery of retrieved APP cargo to the TGN. As previously reported, at the protein level, the intermediate endosomes are positive for clathrin and for the early endocytic marker Rab5, although with apparent minor differences in their distribution (Fig. 3). We have also shown that, at least at the photonic level of resolution used, Rab5 appears not to be sorted to the emerging tubule, while clathrin was present in this nascent structure (ROIs in Fig. 3). Other early endosome markers, such as EEA1 , present a distribution similar to that observed by us for Rab5 in the intermediate endosomes destined for the TGN. The co-localization of APP with a retromer component related to cargo recognition, VPS35, strongly suggested it as a retromer-mediated pathway.
Since components of the retromer-mediated pathway and endocytic APP fate have been associated with AD pathology, we found it particularly important to address the regulatory signals determining retrieval of APP to the TGN. Transmembrane protein trafficking in the post-TGN membrane system may contain several sorting signals regulating protein transport between the various compartments . For example, CIMPR undergoes retromer-dependent retrieval to the TGN and its cytoplasmic tail has both an YXXϕ and a DXXLL motif. These are motifs known to be involved in retromer-dependent BACE-1 and sortilin retrieval to the TGN, respectively [41, 42]. APP also has a characteristic YXXϕ sorting signal,653YTSI656, well positioned in the juxtamembrane region of the cytoplasmic tail  that could support both sorting at the TGN and TGN retrieval, as recently observed for the YXXϕ motif in sortilin . The653YTSI656 functional motif was first related to APP endocytosis and post-TGN degradation [61–66], and lately to AP-1-binding dependent APP basolateral sorting in epithelial cells . Further, protein cargo phosphorylation near or at the sorting motif could be a positive modulator for its retrieval to the TGN. Indeed, this occurs with CIMPR and BACE phosphorylation at serine residues near their cytoplasmic sorting DXXLL motif [41, 67–69].
Protein phosphorylation is a major regulatory process, and APP phosphorylation is known to alter its subcellular processing [16, 46, 60, 64, 65, 70, 71]. Although phosphoS655 APP molecules, within the APP653YTSI656 motif, have been reported in AD brains [65, 72, 73], a clear physiological role for S655 phosphorylation, first proposed to regulate APP sorting by Gandy et al. , has not been forthcoming. We have recently observed that APP phosphorylation at S655 enhances the protein exit from the TGN to the PM and increased its cleavage to αsAPP . Together with the data described here, one can conclude that S655 phosphorylation is important in regulating APP traffic from the TGN to the PM and in recycling APP back to the TGN. In fact, as we demonstrated, S655 phosphorylation has a key modulatory role in the sorting fate of endocytosed APP molecules. The phosphomimetic APPS655E, undergoes faster and enhanced retrieval to the TGN (Fig. 5). Further characterization of S655-dependent sorting at endosomes revealed that endocytosed APPS655A was preferentially targeted to the lysosomal default route (Fig. 4). Similarly, retromer impairment has also been observed to promote Sortilin and the Shiga toxin B-subunit targeting to the lysosomal pathway [33, 42]. The differential sorting of the APP mutants at endosomes, for TGN retrieval or lysosomal delivery, were reflected in the half-lives of their APP-GFP mature forms (Fig. 4). This confirmed a correlation between S655 phospho-state dependent endosomal sorting and APP-GFP turnover rates. The validation that S655 phosphorylation dependent APP retrieval to the TGN occurred in a retromer-mediated manner is confirmed by the VPS35 siRNA downregulation assays, where APP retrieval to the TGN and APP half-life were significantly reduced (Fig. 8). Importantly, we have observed a tendency for less Abeta production for APPS655E that may be a result of its shorter time of residence in endosomes due to more rapid retrieval to the TGN. This agrees with reports inversely correlating components of this pathway with Abeta production and AD [21, 36–38, 59, 74].
From a molecular mechanistic perspective, APP S655 phosphorylation appears to lead to an increase in its binding to sorting proteins, as occurs with the phosphorylation of BACE-1 and CI-MRP, wich enhance their binding to GGA, a protein involved in this transport [41, 67–69, 75, 76]. In agreement with this, NMR analysis of S655 phosphorylated APP was found to induce significant local conformational changes in the APP C-terminus at and downstream the653YTSI656 motif . Accordingly, we have observed more VPS35 immunoreactivity when VPS35 was co-immunoprecipitated with the S655E mutant (Fig. 7c). Nonetheless, Wt and S655A co-immunoprecipitated with VPS35 to similar extents, suggesting that increased S655A targeting to lysosomes involves not a default passive but a mediated active process, involving lysosomal sorting molecules. Other reports have indicated that retromer binding to cargo proteins most likely occurs via its VPS10-containing sorting receptor proteins, such as SorLA, and not via direct binding of cargo to VPS35 . Noticeably, SorLA can bind GGA , and both can bind the clathrin AP-1/2 adaptor proteins , and all are reported to play roles in retrograde retrieval of cargo [21, 27, 75]. In light of our results, we speculate that S655 phosphorylation enhances APP binding affinity for sorting proteins such as SorLA  and/or the AP-1 adaptor, which function to retrieve APP to the TGN in a complex containing SorLA, AP-1, GGAs and the retromer.