Alzheimer’s disease: Aβ38 beta-amyloid can help people live longer


Findings from a new study on Alzheimer’s disease (AD), led by researchers at the University of Saskatchewan (USask), could eventually help clinicians identify people at highest risk for developing the irreversible, progressive brain disorder and pave the way for treatments that slow or prevent its onset.

The research, published in the journal Scientific Reports in early January, has demonstrated that a shorter form of the protein peptide believed responsible for causing AD (beta-amyloid 42, or Aβ42) halts the damage-causing mechanism of its longer counterpart.

“While Aβ42 disrupts the mechanism that is used by brain cells to learn and form memories, Aβ38 completely inhibits this effect, essentially rescuing the brain cells,” said molecular neurochemist Darrell Mousseau, professor in USask’s Department of Psychiatry and head of the Cell Signalling Laboratory.

Previous studies have hinted that Aβ38 might not be as bad as the longer form, said Mousseau, but their research is the first to demonstrate it is actually protective.

“If we can specifically take out the Aβ42 and only keep the Aβ38, maybe that will help people live longer or cause the disease to start later, which is what we all want.”

Aβ42 is toxic to cells, disrupts communication between cells, and over time accumulates to form deposits called plaques. This combination of factors is believed responsible for causing AD. Experts have long thought that all forms of Aβ peptides cause AD, despite the fact that clinical trials have shown removing these peptides from the brains of patients does not prevent or treat the disease.

Mousseau said the idea behind the study was simple enough: If two more amino acids is bad, what about two less?

“We just thought: Let’s compare these three peptides, the 40 amino acid one that most people have, the 42 amino acid that we think is involved in Alzheimer’s, and this 38 one, the slightly shorter version,” said Mousseau, who is Saskatchewan Research Chair in Alzheimer disease and related dementias, a position co-funded by the Saskatchewan Health Research Foundation and the Alzheimer Society of Saskatchewan.

The project confirmed the protective effects of the shorter protein across a variety of different analyses: in synthetic versions of the protein in test tubes; in human cells; in a worm model widely used for studying aging and neurodegeneration; in tissue preparations used to study membrane properties and memory; and in brain samples from autopsies.

In the brain samples, they also found that men with AD who had more Aβ42 and less Aβ38 died at an earlier age. The fact that they didn’t see this same pattern in samples from women suggests the protein peptide behaves differently in men and women.

The USask team also included Maa Quartey and Jennifer Nyarko from the Cell Signalling Lab (Department of Psychiatry), Jason Maley at the Saskatchewan Structural Sciences Centre, Carlos Carvalho in the Department of Biology, and Scot Leary in the Department of Biochemistry, Microbiology and Immunology.

Joseph Buttigieg at the University of Regina and Matt Parsons at Memorial University of Newfoundland were also part of the research team.

While Mousseau wasn’t surprised to see that the shorter form prevents the damage caused by the longer version, he said he was a little taken aback at how significant an effect it had.

“As soon as you put Aβ38 into it, it brings it back up to control levels, completely inhibiting the toxic effects of Aβ42. That’s what was pleasantly surprising.”

Alzheimer’s disease (AD), the most common form of dementia, is a devastating neurodegenerative disorder that affects perhaps 30 million people worldwide, with demographic projections suggesting this will increase substantially in the coming decades [1].

Cerebral neurodegeneration typically takes place first in the hippocampus, a region below the neocortex that is critical for consolidating long-term memories. Neuronal loss spreads to other cortical areas, leading to progressive cognitive decline.

By the end stages of the disease, patients lose cognitive function to the point of requiring constant care, often institutionalized. Although a number of risk factors have been associated with AD, disease onset correlates best with age, and the large majority of cases occur in the elderly. Among people over age 85, over a third are afflicted.

Two types of protein deposits are found in the AD brain: amyloid plaques and neurofibrillary tangles [2]. The former are extraneuronal and primarily composed of the 4 kDa amyloid β-peptide (Aβ), whereas the latter are intraneuronal filaments of the normally microtubule-associated protein tau. Neuroinflammation is a third pathological feature of AD, in which microglia—phagocytic brain immune cells that release cytokines—become overactivated [3].

The role of each of these features in AD etiology and pathogenesis are not well understood. However, Aβ aggregation—in the form of oligomers, protofibrils, fibrils, and plaques—is generally observed as the earliest pathology, followed by tau tangle formation and neurodegeneration [4].

For this reason and those mentioned in the next section, pathological Aβ is widely considered the initiator of AD, triggering downstream tau pathology and neuroinflammation, and Aβ has been the primary target for the development of AD therapeutics for over 25 years [5].

Familial AD and Genetics

As mentioned above, the “amyloid hypothesis” of AD pathogenesis has reigned for decades, and AD drug development has largely focused on inhibiting Aβ production, blocking Aβ aggregation, or facilitating Aβ clearance from the brain [6]. The primary basis for this dogma is the discovery in the 1990s of dominant genetic mutations associated with early-onset AD [7,8,9,10].

This familial AD (FAD) has a disease onset before age 60 and can occur even before age 30. Other than the monogenetic cause and mid-life onset, FAD is closely similar to the sporadic AD of old age with respect to pathology, presentation, and progression. The most parsimonious explanation is that similar molecular and cellular events are involved in the pathogenesis and progression of both forms of the disease.

The first genetic mutations associated with FAD were in the amyloid precursor protein (APP) [7]. This gene encodes a single-pass membrane protein that is initially cleaved by β-secretase, a membrane-tethered aspartyl protease in the pepsin family, to release the large APP ectodomain [11] (Figure 1).

The remnant C-terminal fragment (APP CTF-β) is then proteolyzed within its transmembrane domain (TMD) by γ-secretase to produce Aβ, which is then secreted from the cell [12]. Most Aβ is 40 residues in length (Aβ40), but a small portion is the much more aggregation-prone 42-residue form (Aβ42). Although Aβ42 is a minor Aβ variant produced through APP CTF-β processing by γ-secretase, it is the major form deposited in the characteristic cerebral plaques of AD [13].

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Figure 1
Amyloid precursor protein (APP) processing by β- and γ-secretases. The single-pass membrane protein APP is proteolyzed just outside the transmembrane domain (TMD) by β-secretase. The remaining membrane-bound C-terminal fragment (APP CTF-β) is then cleaved within the TMD to produce the amyloid β-peptide (Aβ) and the APP intracellular domain (AICD).

The 27 known APP mutations associated with FAD [14], each devastating different families, are all missense mutations in and around the small Aβ region of the large APP. These mutations either alter Aβ production or increase the aggregation tendency of the peptide [15].

A double mutation just outside the N-terminus of the Aβ region in APP increases proteolysis by β-secretase, leading to elevated APP CTF-β and therefore elevated Aβ overall. Mutations in the TMD near γ-secretase cleavage sites elevate Aβ42/Aβ40, and mutations within the Aβ region itself make the peptide more prone to aggregation.

FAD mutations were then discovered in presenilin-1 (PSEN1) [8] and presenilin-2 (PSEN2) [9], genes encoding multi-pass membrane proteins that at the time had no known function. These missense mutations—now with over 200 known [14], all but a dozen or so in PSEN1—are located throughout the sequence of the protein but mostly within its nine TMDs [16,17].

Presenilin FAD mutations were soon found to increase Aβ42/Aβ40 [18,19,20], further strengthening the idea that this ratio is critical to pathogenesis. Moreover, these findings indicated that presenilins can modulate γ-secretase cleavage of APP substrate, as the FAD mutations altered the preference for cleavage sites by the protease. Soon after came the observation that knockout of PSEN1 dramatically reduced Aβ production at the level of γ-secretase [21], with the remaining Aβ production attributed to PSEN2 (later verified [22,23]). Thus, presenilins are required for γ-secretase processing of APP CTF-β to Aβ peptides.

Presenilin and the γ-Secretase Complex

Meanwhile, the design of substrate-based peptidomimetic inhibitors suggested that γ-secretase is an aspartyl protease [24,25,26]. Peptide analogs, based on the γ-secretase cleavage site in the APP TMD leading to Aβ production and containing difluoroketone or difluoroalcohol moieties—mimetics of the transition state of aspartyl protease catalysis—were effective inhibitors of γ-secretase activity in cell-based assays.

Given the requirement of presenilin for γ-secretase activity, the site of proteolysis of APP within its TMD, the multi-TMD nature of presenilin, and the evidence that γ-secretase is an aspartyl protease, the possibility was raised that presenilin could be a novel membrane-embedded protease.

Indeed, two conserved TMD aspartates were found in presenilins, and both aspartates were required for γ-secretase activity [27]. Subsequent reports that affinity-labeling reagents based on the transition-state analog inhibitors of γ-secretase bound directly to presenilin cemented the idea that presenilin is an unprecedented aspartyl protease with its active site located within the lipid bilayer [28,29].

Although presenilins appeared to be unusual aspartyl proteases, it was clear that they did not have this activity on their own. Presenilins themselves undergo proteolysis within the large loop between TMD6 and TMD7 to form an N-terminal fragment (NTF) and C-terminal fragment (CTF) [30] (Figure 2). The formation of PSEN NTF and CTF is gated by limiting cellular factors [31], and these two presenilin subunits assemble into a larger complex [32,33].

Biochemical analysis and genetic screening ultimately identified three other components of what became known as the γ-secretase complex [34,35,36]. These three components, membrane proteins nicastrin, Aph-1, and Pen-2, assemble with presenilin, activating an autoproteolytic function of presenilin to form PSEN NTF and CTF. [The two essential TMD aspartates of presenilins are also required for PSEN NTF/CTF formation [27].]

This assembly with cleaved presenilin is the active form of γ-secretase. Indeed, the transition-state analog affinity labeling reagents that tag presenilins specifically bound to PSEN NTF and CTF [28,29], suggesting that the active site of the protease resides at the interface between these two presenilin subunits. This idea is consistent with the observation that one of the essential aspartates is in TMD6 in the PSEN NTF, and the other is in TMD7 in the PSEN CTF.

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Figure 2
Presenilin and other components of the γ-secretase complex. Presenilin is a 9-TMD protein that contains two TMD aspartates (D) essential to catalysis. Assembly of presenilin with the three other components—nicastrin, Aph-1, and Pen-2—triggers autoproteolysis of presenilin into an N-terminal fragment (NTF) and C-terminal fragment (CTF) to form the active γ-secretase complex.

Soon after the discovery of presenilin as the catalytic component of γ-secretase, analysis of the other proteolytic product of γ-secretase cleavage of APP (AICD), revealed that the APP TMD was proteolyzed at two different sites [37,38,39,40,41]. Cleavage at the second (ε) site releases AICD products composed of residues 49–99 or 50–99 of the 99-residue APP CTF-β substrate for γ-secretase (Figure 3). With secreted Aβ peptides ranging from 38–43 residues (Aβ38-Aβ43), this left 5 to 11 APP TMD residues unaccounted for. Subsequent discovery of Aβ45, Aβ46, Aβ48, and Aβ49, but no N-terminally extended AICD peptides, led to the hypothesis that ε proteolysis occurs first [42,43,44].

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Figure 3
Processive proteolysis of APP substrate by γ-secretase. Initial endoproteolysis at the ε cleavage site of APP substrate C99 results in Aβ48 or Aβ49 and corresponding APP intracellular domain fragments AICD49-99 and AICD50-99. The carboxypeptidase activity of γ-secretase then trims the initial Aβ peptides along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38.

The generated Aβ48 and Aβ49 (counterparts of AICD49-99 and AICD50-99, respectively) were postulated to undergo tripeptide trimming along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38 (this last cleavage step generating a tetrapeptide coproduct). Mass spectrometric analysis of the small peptide products supported this notion [45], as did the finding that synthetic Aβ49 is primarily processed to Aβ40 and Aβ48 is primarily trimmed to Aβ42 by purified γ-secretase [46]. Kinetic analysis of trimming of synthetic Aβ48 and Aβ49 by five different FAD-mutant γ-secretase complexes revealed that all five were dramatically deficient in this carboxypeptidase trimming activity [46].

Presenilin and the γ-secretase complex have many more substrates besides APP [47]. Indeed, so many substrates have been identified that the γ-secretase complex has been called the proteasome of the membrane [48], implying that one of its major functions is to clear out membrane protein stubs that remain after ectodomain release by sheddases. While membrane protein clearance may be an important function of γ-secretase, the protease also plays essential roles in certain cell signaling pathways. The most important of these is signaling from the Notch family of receptors [49].

Notch receptors are single-pass membrane proteins like APP, and proteolytic processing of Notch, triggered by interaction with cognate ligands on neighboring cells, leads to release of its Notch intracellular domain (NICD) (Figure 4). The NICD translocates to the nucleus and interacts with specific transcription factors that control the expression of genes involved in cell differentiation.

These signaling pathways, particularly from Notch1 receptors, are essential to proper development in all multi-cellular animals. Knockout of presenilin genes in mice is lethal and leads to phenotypes that are virtually identical with those observed upon knockout of the Notch1 gene [50,51], findings that, as explained later, have major implications for the potential of γ-secretase inhibitors as AD therapeutics.

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Figure 4
Notch receptor processing and signaling. Interaction with a cognate ligand on a neighboring cell triggers Notch ectodomain shedding by the metalloprotease ADAM-10 and then cleavage of the Notch extracellular truncation (NEXT) fragment with its single TMD by γ-secretase. Release of the Notch intracellular domain (NICD) leads to translocation to the nucleus, activation of transcriptor factors, and gene expression that controls cell differentiation.

reference link:

More information: Maa O. Quartey et al, The Aβ(1–38) peptide is a negative regulator of the Aβ(1–42) peptide implicated in Alzheimer disease progression, Scientific Reports (2021). DOI: 10.1038/s41598-020-80164-w


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