In Alzheimer’s disease, enlarged endosomes hamper waste disposal pathways in neurons. In the July 21 Molecular Psychiatry, researchers led by Ralph Nixon at the Nathan Kline Institute in Orangeburg, New York, extend previous research that implicated the β-C-terminal fragment of amyloid precursor protein in this process. The researchers drilled down into the mechanism in cell culture models, finding that excess β-CTF recruits a protein called APPL1 to early endosomes. APPL1 helps rev up endocytosis, causing vesicles to swell and get stuck as they travel along axons. Knockdown of APPL1 reversed these effects, suggesting this mechanism could be targeted therapeutically. The findings shed light on one of the earliest pathologic features of Alzheimer’s, say the authors.
Commenters found the data intriguing. Greg Cole at the University of California, Los Angeles, wrote, “This is a novel mechanism and potentially very important … it seems likely to offer new therapeutic targets.” (See full comment below.) Stefan Lichtenthaler at the German Center for Neurodegenerative Diseases, Bonn, noted that the finding helps elucidate the role of β-CTF in toxicity. “We long thought that β-CTF may have an ‘unspecific’ neurotoxic function when it accumulates, but now this paper shows a specific mechanism,” he wrote to Alzforum. He noted that in general, BACE-derived CTFs may not just be fragments on the path of membrane protein degradation, but have specific physiological roles (see full comment below).
Previous studies by Nixon and others suggest that neuronal endocytic and lysosomal systems become gummed up early in Alzheimer’s disease. Endocytosis speeds up, lysosomal degradation can't keep pace and grinds to a halt, and endosomal vesicles become swollen and accumulate in neurites (see Feb 2005 news; Jun 2010 news; May 2011 news). The small GTPase rab5, which switches on in AD, regulates endosome trafficking and drives these changes (see Stenmark, 2009; Cataldo et al., 2008). Nixon reported that the β-CTF fragment of APP also contributed, but it was not clear how (see Jan 2010 news).
Since β-CTF did not directly bind rab5, Nixon and colleagues looked for molecules that might mediate their interaction. They focused on APPL1, aka adaptor protein containing pleckstrin homology domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif. Despite its acronym, this protein is not related to APP or to amyloid precursor-like protein 1 (APLP1). As an adaptor protein, APPL1 brings other proteins together. It binds and stabilizes active rab5 on early endosomes inside the cell (see Miaczynska et al., 2004). Because amyloid precursor protein is known to bind PTB domains, the authors considered the β-CTF cytosolic domain of APP a good candidate to interact with APPL1 as well (see Tamayev et al., 2009).
Using co-immunoprecipitations, first author Seonil Kim found that β-CTF bound directly to APPL1, and that he could abolish this by mutating the interaction domains on either protein. To investigate the consequences of binding, the authors turned to a neuroblastoma cell line that overexpressed human wild-type APP. Because APP is processed in endosomes, excessive β-CTF should accumulate there, allowing APPL1 to be recruited in turn. Consistent with this, the authors saw more APPL1 decorating early endosomes in these cells than in a control neuroblastoma line. Moreover, the APP line had oversized vesicles, 50 percent more activated rab5, and twofold more endocytosis overall. Knockdown of APPL1 shrank vesicles back to normal, lowered active rab5, and slowed endocytosis.
In Nixon’s 2010 study, he found that BACE inhibitors, which lower β-CTF levels, restored normal endocytosis, while γ-secretase inhibitors, which pump up β-CTF levels, worsened it. Those studies were done in fibroblasts from people with Down’s syndrome, who carry an extra copy of APP and develop Alzheimer’s-like pathology in middle age. Evidence suggests that endosomes malfunction decades earlier in DS (see Cataldo et al., 2000). The authors wanted to see if APPL1 mediated β-CTF’s effects in DS endosomes as well. Consistent with this, they saw more APPL1 on enlarged endosomes in DS fibroblasts compared to controls. Knockdown of APPL1 restored vesicles to normal size.
Endosomal dysfunction has numerous downstream effects, including slowing endosome transport along axons. Would APPL1 mediate these also? Watching vesicle movements in mouse cortical neuron cultures via time-lapse photography, the authors saw that in cells overexpressing wild-type APP and rab5, endosomes crawled along axons and made frequent stops, whereas in control cells they moved quickly and steadily. The largest endosomes, those with a surface area greater than 0.5 square microns, halted most frequently. These big vesicles may get stuck trying to push through cell cytoplasm, the authors suggested. Again, knockdown of APPL1 shrank endosomes and completely reversed these transport defects.
Endosomes also act as platforms for cell signaling, with rab5 activating numerous downstream molecules. These include NF-κB, which regulates inflammation and apoptosis and is implicated in both Alzheimer’s disease and Down’s syndrome (see Engidawork et al., 2001; Tilstra et al., 2011). In Down’s syndrome cells, levels of a downstream activator of NF-κB signaling were up 75 percent in the nucleus compared with control fibroblast levels. Knockdown of APPL1 restored levels to normal, again demonstrating the key role of APPL1 in endosome dysfunction.
While these cell culture results implicate APPL1 in numerous Alzheimer’s pathologies, does any of this translate to human brain? Preliminary results suggest they might. The authors found elevated β-CTF in 13 postmortem AD brains compared to 13 controls, as might be expected since BACE activity has been found to be high in AD (see Sep 2002 news). AD patients also had twofold more APPL1 on enlarged endosomes in cortical neurons than controls (see image above). Other studies have also reported abnormal distribution of APPL1 in AD brains (see Ogawa et al., 2013).
Numerous questions remain. In future work, Nixon will examine the effects of APPL1 on downstream late endosome and lysosome function, and whether this contributes to the failure of autophagy in AD. Because ApoE4 and cholesterol also activate rab5, and cholesterol binds β-CTF, he will investigate whether ApoE4 and cholesterol play a role in endosome regulation as well. One possibility is that ApoE4 and cholesterol might increase the concentration of β-CTF in endosomes, said Nixon.
The results suggest that lowering APPL1 or β-CTF might be therapeutic. Knockdown of APPL1 did not harm normal endocytosis in cultured cells, hinting that it might be safe to target this protein, Nixon noted. Commentators pointed out that BACE inhibitors might provide a double benefit to AD patients because they suppress production of both Aβ and β-CTF. “We can hope BACE inhibitors may have some therapeutic benefits that are not shared by γ-secretase inhibitors or most Aβ immunotherapy approaches,” Cole wrote to Alzforum. Several such inhibitors are currently in clinical trials (see Dec 2013 news; Oct 2014 news; Apr 2015 conference news).—Madolyn Bowman Rogers
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