New Insights on the Role of Residue 673 of APP in Alzheimer's Disease
Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disorder and its two major pathological hallmarks are neuritic plaques and neurofibrillary tangles (Benilova et al., 2012; Ward et al., 2012). The formation of neuritic plaques through the aggregation of amyloid-β (Aβ) oligomers is thought to be foundational to the pathophysiology of AD according to the amyloid cascade hypothesis (Hardy and Selkoe, 2002). Amyloid precursor protein (APP), existing as three major isoforms (APP695, APP751, and APP770), can be processed through two major pathways (Nalivaeva and Turner, 2013) (Fig. 1). In the non-amyloidogenic pathway, APP is first cleaved by α-secretase at the Leu17 site, resulting in membrane-bound C83 and a secreted soluble APP fragment, s-APPα (Esch et al., 1990). Next, C83 is cleaved by γ-secretase to produce a C-terminal fragment (CTFγ) and P3 (Vardy et al., 2005). In the amyloidogenic pathway, APP is initially cleaved by β-secretase, also known as β-site APP cleaving enzyme 1 (BACE1), which can cleave APP at one of two distinct sites, the major Glu682 or the minor Asp672 site, which are commonly referred to as the Glu11 and Asp1 sites because of their corresponding positions on C99/C89 and Aβ. This results in secreted sAPPβ and a membrane-bound C-terminal fragment β (CTFβ). Cleavage at the Glu11 site forms a C-terminal fragment that is 89 residues in length (C89), which, after γ-secretase cleavage, results in a truncated form of Aβ. Cleavage by BACE1 at the Asp1 residue, however, creates a C-terminal fragment that is 99 residues long (C99), which generates Aβ after cleavage by γ-secretase.
APP can be cleaved through amyloidogenic or non-amyloidogenic pathways. In the non-amyloidogenic pathway, α-secretase cleaves APP to form sAPPα and CTF83. In the amyloidogenic pathway, APP is cleaved by β-secretase (BACE1) at either the Glu11 site to form CTF89, resulting in a truncated form of Aβ, or the Asp1 site to make CTF99, which is cleaved by γ-secretase to form Aβ (gray). The Ala673 site of APP becomes the Ala2 site (red) of Aβ.
Most cases of AD are considered sporadic, but some cases (<1%) are classified as familial AD (FAD). Some cases of FAD are caused by point mutations in the genes that code for APP (Bekris et al., 2010). A well studied FAD-causing mutation is the dominantly inherited Swedish mutation, APPSwe, which leads to increased C99 levels. Another FAD mutation, found in an Italian family, is inherited recessively and characterized by an alanine to valine missense mutation at residue 673 (A673V) of APP770 (APPrIta). Upon cleavage by BACE1 and γ-secretase, the A673V mutation can be found on C99 and Aβ on the second residue, which is referred to as an A2V mutation (Fig. 1). There is currently limited knowledge about the molecular processes that link these APP mutations to the pathogenic manifestation of AD. A recent study by Zhang et al. (2017) published in the The Journal of Neuroscience addressed this question by investigating the role of residue 673 of APP in BACE1 cleavage site selection and APP processing in the context of amyloidogenesis in AD.
Zhang et al. (2017) investigated whether APPrIta led to increased C99 production that translated into increased levels of Aβ. Adeno-associated viruses containing wild-type APP (APPWT) or APPrIta were injected into brains of neonatal mice. An antibody specific for the last 20 residues of the APP C terminus was used to examine the relative levels of C83, C89, and C99. The APPrIta A673V mutation resulted in higher C99 levels and C99/C89 ratios compared with APPWT. This suggests that the A673V mutation shifted the dominant BACE1 cleavage in APP from the Glu11 site to the Asp1 site, a result consistent with previous research (Kimura et al., 2016). Zhang et al. (2017) then examined the effects of APPrIta and APPSwe in vitro using PC12 rat pheochromocytoma cells and a BACE1 stable cell line, 293B2. They showed that APPrIta cleavage generated more C99 fragment than APPWT and APPSwe and therefore had a stronger effect than APPSwe in shifting the preferential β-cleavage site from Glu11 to Asp1.
Despite APPrIta cleavage producing more C99 than the APPSwe mutation, Zhang et al. (2017) discovered that the APPrIta cleavage pathway produced less Aβ40 and Aβ42 than APPSwe (although still more than APPWT). To determine why this is the case, the researchers investigated the fate of C99 after BACE1 cleavage. HEK293 cells expressing either C99WT or C99A2V, the C99 fragment resulting from cleavage of the APPrIta mutation, were treated with a γ-secretase inhibitor, a lysosome inhibitor, or a proteasome inhibitor. The γ-secretase inhibitor resulted in an ∼184% increase of C99WT, whereas C99A2V only increased by ∼53%. In contrast, the proteasome inhibitor resulted in a 25-fold increase in C99A2V compared with only an eightfold increase in C99WT. These findings suggest that the A673V mutation enhances C99 degradation by the proteasome and decreases cleavage by γ-secretase compared with C99WT.
Finally, Zhang et al. (2017) investigated the overall effect of APP residue 673 on BACE1 cleavage. Another mutation at the same residue of APP, A673T, was previously discovered to be protective against AD in the Icelandic population (Jonsson et al., 2012). Knowing that mutation of this site could be either protective or FAD causing, the investigators substituted the hydrophobic alanine on site 673 of APP770 with a range of amino acids including negatively charged aspartic acid, structurally rigid proline, positively charged arginine, hydrophobic leucine, and polar uncharged glutamine and serine. They then used immunoblotting to examine the CTF cleavage profiles after β-cleavage. These point mutations all reduced C99 levels and increased C89 and C83 levels compared with WT, with the exception of leucine, which shares characteristics with valine and alanine in that they are short-chain alkane residues. In general, these findings imply that cleavage of APP by α-secretase is enhanced by these mutations and that the preferential BACE1 cleavage site is shifted to the Glu682 site of APP.
Compared with the APPSwe mutation, APPrIta results in less Aβ generation; however, patients still develop severe FAD pathology. A reason for this discrepancy might be the difference in Aβ composition: the APPrIta mutation occurs in the Aβ peptide (AβA2V), but the APPSwe mutation does not (it produces AβWT). Giaccone et al. (2010) compared these peptides and discovered that AβA2V in a homozygous form had higher aggregation kinetics and more stable Aβ assemblies than AβWT (Giaccone et al., 2010). The rIta mutation is thought to form amyloid aggregates more rapidly, thus triggering the downstream cascade of effects leading to neurotoxicity.
The findings of Zhang et al. (2017) provide insight into the role of APP residue 673 in FAD. They show that a single mutation from alanine to valine can affect BACE1 cleavage site selection, resulting in disproportionately high C99 production but lower Aβ production relative to APPSwe. Regarding the fate of C99A2V after initial β-cleavage, the investigators revealed that the fragment has reduced γ-secretase cleavage and increased proteasomal degradation. Together, the results presented by Zhang et al. (2017) suggest site 673 of APP as a therapeutic target. Zhang et al. (2017) showed the 673-residue site to be a key determinant between the amyloidogenic and non-amyloidogenic pathways, as demonstrated in the A673V and A673T mutations, respectively. To determine why heterozygous carriers of APPrIta did not develop FAD, Di Fede et al. (2009) investigated the interaction between AβA2V generated as a result of the APPrIta cleavage pathway and AβWT. They showed that this interaction in a heterozygous form had a protective effect against Aβ aggregation compared with both AβA2V and AβWT on their own (Di Fede et al., 2012, 2016). Using this information, Di Fede et al. (2016) developed a synthetic rIta peptide (Aβ1–6A2V). The peptide was then tested in mouse and in vitro models, where it interacted with AβWT and showed beneficial interference with Aβ oligomer production (Di Fede et al., 2016). The methods discussed here and results by Zhang et al. (2017) provide the groundwork for future investigations into how APP mutations have broader implications for AD and for the development of treatment strategies.
Footnotes
- Received September 19, 2017.
- Revision received November 12, 2017.
- Accepted November 20, 2017.
-
Editor's Note: These short reviews of recent JNeurosci articles, written exclusively by students or postdoctoral fellows, summarize the important findings of the paper and provide additional insight and commentary. If the authors of the highlighted article have written a response to the Journal Club, the response can be found by viewing the Journal Club at www.jneurosci.org. For more information on the format, review process, and purpose of Journal Club articles, please see http://jneurosci.org/content/preparing-manuscript#journalclub.
-
We thank Dr. Rebecca MacPherson and Dr. Aleksandar Necakov for comments on the manuscript.
-
The authors declare no competing financial interests.
- Correspondence should be addressed to Matthew C. Mueller, Department of Biological Sciences, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, Ontario, Canada L2S 3A1. mm13zk{at}brocku.ca
- Copyright © 2018 the authors 0270-6474/18/380515-03$15.00/0
