Researchers have a new entrant to the pantheon of pathogenic APP mutations. The Uppsala mutation, identified in one Swedish family, is the first known multi-codon deletion in the gene to lead to Alzheimer’s disease. It snips six amino acids, residues 19-24, from the middle of the Aβ peptide. In the August 11 Science Translational Medicine, researchers led by Dag Sehlin and Martin Ingelsson at Uppsala University, Sweden, describe how this deletion alters APP processing, boosting production of the pathogenic AβUpp42 peptide. Moreover, this shortened peptide is far more prone to aggregate than is wild-type Aβ42, resulting in rapid plaque deposition and symptom onset around age 40 in the three known carriers. The researchers previously presented the mutation at the 2019 International Conference on Alzheimer’s and Parkinson’s Diseases in Lisbon, Portugal, but didn’t say what it was (Apr 2019 conference news).
- The six-amino-acid Uppsala deletion in APP spurs Aβ42 production.
- AβUpp42 is short, fibrillizes rapidly, and leads to very early onset AD.
- Fibrils are different than wild-type, barely registering on PET scans.
“This is a thorough and extremely convincing story,” Sangram Sisodia at the University of Chicago wrote to Alzforum, lauding the researchers’ use of multiple state-of-the-art mass spectrometry and analytical techniques to characterize amyloid pathology in the mutation carriers. Robert Vassar of Northwestern University, Chicago, was struck by the way this mutation married increased Aβ production with aggressive aggregation. “In a sense, it is like combining the Swedish mutation that makes APP a better substrate for BACE1 together with the Arctic mutation that increases fibrillogenesis,” he wrote (full comment below).
Before Uppsala, there were 31 known pathogenic mutations in APP; most are missense mutations, with the only prior deletion being the Osaka mutation, which snips a single glutamic acid from position 693, or residue 22 of Aβ. This lies within the Uppsala deletion region. Many of these known mutations alter APP processing. Typically, in the amyloidogenic pathway, the protein is cleaved sequentially by β-secretase (BACE1) to leave a C-terminal fragment in the cell membrane, and then γ-secretase to produce Aβ40 or Aβ42. In the non-amyloidogenic pathway, initial cleavage by α-secretase prevents the formation of toxic peptides (see image above).
Second author Vilmantas Giedraitis identified the deletion in two siblings and a cousin in Uppsala. When they came to the university’s memory clinic, all three were symptomatic, having problems with executive function, speaking, and basic math, and they scored from 20 to 22 on the MMSE. They were still in their early 40s. The two siblings had neuropsychiatric symptoms, including apathy and anxiety, but their cousin did not. Brain scans showed typical features of Alzheimer’s in all three, with atrophy in the mediotemporal and frontoparietal regions and reduced FDG uptake in temporal and parietal lobes. However, their amyloid PiB PET scans were only weakly positive (see image below). Cerebrospinal fluid findings were also puzzling, with all three carriers having elevated total tau and p-tau181 typical of AD, but Aβ42 levels similar to those of healthy controls. One of the siblings died at age 49 and donated his brain for autopsy; the other two are still alive.
Despite the CSF and PiB PET data to throw them off track, the researchers suspected some form of early onset AD in the family and sequenced targeted exomes from the three affected members and unaffected relatives. That’s when the APP deletion popped out. It seems to be unique to this family; it has not cropped up in an admittedly small study of around 500 DNA samples from Swedish sporadic AD patients and healthy controls.
To study its effects, first author María Pagnon de la Vega transfected a human kidney cell line with Uppsala APP. Compared to cells transfected with wild-type protein, the Uppsala mutation boosted β-secretase cleavage, and thus Aβ40 and Aβ42 production, by about 50 percent. This high production may explain the CSF Aβ findings, the authors speculated. Aβ normally drops in CSF as the peptide becomes sequestered in plaques in the brain, but high levels of AβUpp42 could mask this effect. Supporting this, only the mutant peptide was high in CSF from the three relatives; wild-type Aβ42 levels were low, similar to those in other AD patients.
In contrast to heightened β-secretase cleavage, no α-secretase cleavage could be detected in these cell cultures, as seen by a lack of the sAPPα fragment. The Uppsala deletion occurs only two amino acids away from the α-secretase cleavage site between amino acids 16 and 17 of Aβ, in effect pulling this site much closer to the cell membrane. This led the authors to wonder if α-cleavage might simply be shifted toward the N-terminus of the protein, further from the membrane. Hinting at this, the authors found high levels of AβUpp5-40 in transfected cultures, indicating the presence of a new cleavage site between residues 4 and 5, 12 amino acids upstream of the normal α-secretase spot. However, it remains to be shown whether α-secretase or some other enzyme creates this fragment. Sisodia suggested that pulse-chase experiments, in which cells briefly take up radiolabeled amino acids, might shed more light on the processing and metabolism of APPUpp.
Not only is more AβUpp made, it also aggregates more readily than wild-type Aβ42. In cell-free assays, synthetic AβUpp42 reached half-maximum fibril concentration in less than an hour, compared to eight hours for wild-type. This is even faster than Aβ42 carrying the Arctic mutation, which takes 1.3 hours. The authors analyzed the two most dominant AβUpp42 fibril species by cryo-electron microscopy (see image below). At 5 Å, the resolution was too low to determine the amino acid sequence, but the shapes of these fibrils roughly resembled previously reported structures (Sep 2015 news; Sep 2017 news). They were not identical, however, suggesting subtle structural differences. Michel Goedert at the MRC Laboratory of Molecular Biology, Cambridge, U.K., noted that the relatively low resolution makes it difficult to compare structures, but the six residues missing from AβUpp42 form the beginning of an S-shaped domain in wild-type fibrils (May 2015 news). Thus, their lack might lead to changes (full comment below).
What happens in the brain? In the brain that came to autopsy, the authors found numerous amyloid plaques throughout all regions, including cerebellum, with the pathology corresponding to Thal stage 5. Mass spectrometry showed that these plaques contained mostly AβUpp42, but also had some AβUpp5-42. If the latter is in fact a product of α-secretase cleavage, that would suggest the non-amyloidogenic pathway is completely abolished by this mutation, with α-cleavage now contributing to aggregation.
With plaque burden high, why is the amyloid PET signal low? The authors speculate that PiB might bind more weakly to AβUpp fibrils than to wild-type, similar to how the Arctic mutation abolishes tracer binding. William Klunk at the University of Pittsburgh suggested another possibility, noting that many early onset AD mutations lead to high amyloid deposition in the basal ganglia, deep in the brain. These deposits tend not to show up on PET scans, which favor the cortical surface. Klunk suggested examining PiB binding to purified AβUpp42 fibrils to resolve this. He also thought the use of cerebellum as a reference region could have clouded the data, given the presence of plaques there, and suggested recalculating the scans using a white-matter reference region (full comment below).
In other respects, the autopsied brain was typical of AD, with tau tangles at Braak stage VI and extensive microgliosis in limbic areas and neocortex. Colin Masters at the University of Melbourne, Australia, noted that like other pathogenic APP mutations, the Uppsala deletion drives tau aggregation, again highlighting the mechanistic connection between the pathologies. Vassar agreed, writing, “This interesting new mutation offers yet additional compelling evidence in favor of the amyloid cascade hypothesis.”
One curiosity, however, was that there were few Aβ oligomers and protofibrils in the Uppsala brain, far less than in most AD brains and instead comparable to the amount found in age-matched healthy controls. Because the mutation carriers developed severe impairment early in life, this may suggest that aggregated forms of AβUpp are highly toxic by themselves, Klunk noted.
The authors plan to study this and other details of APPUpp processing and metabolism in mice. They have generated a model that expresses APP containing the Uppsala and Swedish mutations. They will examine how mutant AβUpp interacts with wild-type, and how this affects aggregation. “We think this might be important for how the amyloid structure forms, and how seeding and propagation occur,” Sehlin told Alzforum.
Meanwhile, Masters believes the mutation highlights the roles of both increased Aβ production and aggregation in disease. “As we enter the era of disease-modifying therapies, the pathogenic mechanisms revealed in this study reinforce the need to develop combination strategies that tackle both Aβ clearance and production,” he wrote to Alzforum (full comment below).—Madolyn Bowman Rogers
- APP Upp: Mutation Nixes Six Amino Acids from Aβ, Spurs Aggregation
- Electron Microscope Yields Finer Structure of α-Synuclein, Aβ Fibrils
- Amyloid-β Fibril Structure Bares All
- Danger, S-Bends! New Structure for Aβ42 Fibrils Comes into View
Mutation Interactive Images Citations
- Pagnon de la Vega M, Giedraitis V, Michno W, Kilander L, Güner G, Zielinski M, Löwenmark M, Brundin R, Danfors T, Söderberg L, Alafuzoff I, Nilsson LN, Erlandsson A, Willbold D, Müller SA, Schröder GF, Hanrieder J, Lichtenthaler SF, Lannfelt L, Sehlin D, Ingelsson M. The Uppsala APP deletion causes early onset autosomal dominant Alzheimer's disease by altering APP processing and increasing amyloid β fibril formation. Sci Transl Med. 2021 Aug 11;13(606) PubMed.