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If amyloid drives Alzheimer disease, why have anti-amyloid therapies not yet slowed cognitive decline? [1]

['Christian Haass', 'German Center For Neurodegenerative Diseases', 'Dzne', 'Munich', 'Metabolic Biochemistry', 'Biomedical Center', 'Bmc', 'Faculty Of Medicine', 'Ludwig-Maximilians University', 'Munich Cluster For Systems Neurology']

Date: 2022-08 

If increased Aβ production, as observed in patients with Down syndrome or the Swedish APP mutation, drives AD pathogenesis, what about reduced Aβ production? After all, the genetic evidence would strongly imply that Aβ lowering strategies may be therapeutically useful—a goal that is being actively pursued in clinical research (see below). In a large genetic project involving a substantial portion of the Icelandic population, geneticists searched for gene variants that protect against AD and age-related cognitive decline. Strikingly, a mutation within APP was found right at the β-secretase cleavage site that decreased Aβ production by approximately 40% throughout life [ 30 ]. Moreover, this variant not only protected from AD but also seemed to prevent age-dependent cognitive decline. Reduced Aβ deposition was confirmed in a Finnish family [ 31 ], in which the same mutation in APP was identified in a patient who died at 104 years of age with very little amyloid pathology. Thus, a lifelong decrease in Aβ production protects from AD. Together with the effects of the familial AD-causing mutations that increase Aβ production and aggregation, this work strongly supports a disease-triggering role of excessive Aβ. Moreover, these findings recommend Aβ as a primary target for disease modifying strategies, since nature has shown that a lifelong lowering of Aβ by approximately 40% could enable humans to live for extended periods without being affected by cognitive decline.

What about the much more common AD-causing mutations in the homologous presenilin-1 (PS1) and presenilin-2 (PS2) genes? Strikingly, all these mutations increase the relative levels of the longer Aβ 42 and Aβ 43 species, similar to the carboxyl-terminal APP mutations ( Fig 1A ) [ 24 , 25 ]. But how do these mutations affect APP processing so selectively? It turned out that PS1 and PS2 encode the catalytically active subunit of γ-secretase [ 26 , 27 ], the protease that effects APP carboxyl-terminal cleavage and thus the final liberation of Aβ. Mechanistically, it is now well described that PS1 and PS2 mutations slow the proteolytic performance of γ-secretase [ 28 ]. This exceptional protease mediates stepwise cleavages every 3 or 4 amino acids (termed “processivity”), starting several amino acids carboxyl-terminal to the final cleavage site ( Fig 1A ). The AD-causing mutations in PS1 and PS2 reduce this processive cleavage of APP by the PS1/PS2–γ-secretase complexes (because the mutant presenilin falls off the APP substrate too soon) and therefore increase the likelihood that longer, more hydrophobic Aβ with higher aggregation propensities are produced. Moreover, in patients with sporadic late-onset disease, higher levels of the short Aβ 38 , which arises from more efficient processivity of PS1/PS2–γ-secretase ( Fig 1A ), are associated with a lower degree of AD-related changes [ 29 ]. Strikingly, there are more and less aggressive PS1 and PS2 mutations, which now can be explained mechanistically by a correlation of greater reduction of processive APP cleavage with an earlier age of onset of symptoms [ 28 ]. Thus, early-onset familial AD is caused by mutations in the substrate for Aβ production (APP) or the proteases (presenilins) performing its release, and all these mutations enhance the aggregation propensity of Aβ and thus drive AD via increased amyloid pathology. Genetic evidence thus unequivocally positions Aβ at the top of the cascade that triggers familial AD ( Fig 1B ).

The movie “Still Alice” caused greater public awareness of a rare form of AD that is strictly inherited. In families with inherited AD, certain gene mutations cause a very aggressive, early-onset variant of AD which is transmitted to children with a 50% probability. Moreover, patients, who have such mutations in their genome invariably develop the disease. Although it is argued that these familial variants of AD are rare and may not be related to the “sporadic” late-onset form of AD, the amyloid plaque and tangle pathologies are practically identical ( Fig 2 ). Not surprisingly, the pathogenic mechanisms of the genes that are mutated in familial AD represent a holy grail for deciphering AD, and a worldwide hunt for the causative genes was initiated in the early 1990s. Today, these genes have been identified, and indeed, they hold the key not only for understanding AD mechanistically, but also for the development of therapeutic strategies. Only 3 distinct genes have been identified to harbor disease-causing mutations in dominantly inherited AD [ 6 ]. The first to be recognized was APP itself [ 16 , 17 ]. One of the early APP variants identified contained the so-called “Swedish” mutation [ 18 ], which is located right at the cleavage site recognized by β-secretase, the enzyme that first cuts APP to initiate Aβ production ( Fig 1A ). Strikingly, the Swedish mutation changes the cleavage site in a way that makes it more readily cleaved by β-secretase. As a consequence, an approximately 3-fold overproduction of Aβ is observed in patients with this mutation [ 19 , 20 ]. Here, we have a second example, beside the triplication of APP in Down syndrome, where simple overproduction of Aβ is causative of AD. APP variants caused by mutations at the other end of the Aβ region ( Fig 1A ) are more complicated. Such mutations do not simply increase Aβ production but can result in the generation of longer Aβ species that are 42 or even 43 amino acids long (Aβ 42 and Aβ 43 , respectively), rather than the more common 40-residue Aβ [ 21 ]. Such longer, more hydrophobic peptides facilitate self-aggregation of Aβ and therefore enhance the formation of neurotoxic oligomeric aggregates (see below) and the deposition of fibrillar amyloid plaques. Mutations within the central region of Aβ seem to change the structure of Aβ in a way that drives its self-aggregation [ 22 ]. Thus, AD-causing mutations in APP either increase total production of Aβ or generate more aggregation-prone peptides. Since peptide aggregation is a concentration dependent event [ 23 ], all AD-linked mutations in APP promote amyloidogenesis.

Human genetics revolutionized AD research by identifying genetic risk factors for AD, as well as mutations in genes that definitively cause AD. In the latter cases, these genetic causes lower the conventional age of symptom onset by decades, leading to familial forms of AD. But even before the dawn of modern AD genetics, a pivotal finding pointed to the importance of Aβ as a cause of the disease. When scientists cloned APP, they quickly learned that the gene is located on chromosome 21 [ 11 ], a finding that was important because patients with trisomy 21 have Down syndrome and invariably develop neuropathological and clinical signs of early-onset AD, starting in their 40s or earlier. The reason for this early-onset AD could not be simpler, as the extra copy of APP leads to a lifelong overproduction of Aβ [ 12 , 13 ]. This interpretation is supported by the finding that, in rare cases of translocation Down syndrome in which only parts of chromosome 21 are duplicated, if the translocated parts contained APP, the patients developed AD [ 14 ], but when the duplicated part did not contain APP, they did not develop the disease [ 15 ]. Moreover, close study of Down syndrome enabled the discovery of temporal stages in AD pathology [ 13 ]. Young people with Down syndrome who lacked AD symptoms already had so-called diffuse plaques, largely nonfilamentous deposits of Aβ that are not associated with visible surrounding cytotoxicity. However, starting around age 20 to 30 years, these plaques slowly became more fibrillar, resembling precisely the amyloid plaques that occur in patients with AD. Thus, early on, it was apparent that a lifelong increase in Aβ production could cause AD.

Aβ exists in many conformers (assembly states), but what are the most disease-relevant species and do they trigger tau pathology? In an analogy to fulfilling Koch’s postulates for infectious diseases, the injection of soluble Aβ oligomers (oAβ) isolated from the brains of patients with AD into healthy rodents consistently induces memory impairment and altered microglia (see below) resembling those in human AD [ 32 , 33 ]. Likewise, applying human oAβ to rodent or human neurons induces neuritic dystrophy and hyperphosphorylation of tau at epitopes relevant to AD [ 34 , 35 ]. These cellular abnormalities can be prevented by monoclonal antibodies directed at human Aβ, demonstrating the molecular specificity of the AD-like effects [ 34 , 35 ]. Such studies using bona fide human oAβ suggest that diffusible oligomers that are in a complex equilibrium with fibrillar Aβ in plaques are a principal pathogenic species in AD. This concept is in accord with the development of AD-like neuropathology in mice in which AD-causing mutations in human Aβ have been inserted (knocked-in) to their APP gene [ 36 ]. Indeed, these so-called “APP-humanized” mice accumulate oAβ and develop amyloid fibrils that are structurally like those from the brains of patients with familial AD, based on the latest cryo-electron microscopy analyses [ 37 ]. The APP knock-in mice and earlier transgenic mouse models of AD that express familial AD mutations in APP all develop not only amyloid, neuritic, astrocytic, and microglial pathologies, but also cognitive abnormalities and even biomarker evidence of neurodegeneration [ 38 , 39 ]. Although mice expressing mutant APP do not spontaneously develop bona fide tau pathology, a deficiency that is sometimes used to criticize the amyloid cascade hypothesis, they do show biomarker evidence of neurodegeneration, including increased phospho-tau in the brain. Moreover, a mutant APP transgenic mouse model was used to discover the amyloid-clearing effects of anti-Aβ immunotherapy, leading directly to current clinical trials [ 40 , 41 ].

Role of immune cells

Due to the unequivocal preclinical evidence supporting a pivotal role of altered Aβ in triggering AD, numerous laboratories have focused their research on the mechanisms of Aβ generation and its metabolism. Moreover, since APP is strongly expressed in neurons, and since AD pathology is exclusively observed within the brain, AD research became rather neurocentric over the years. This narrow focus changed dramatically when geneticists identified a set of risk variants for sporadic (late-onset) AD that were principally expressed within microglia, innate immune cells of the brain, but not in neurons or other brain cells [42]. This realization caused a landslide-like shift in AD research away from neurons to microglia, and a fundamentally new topic, sometimes referred to as the cellular phase of AD, was scrutinized [10]. The cellular phase now describes a more holistic view, in which multiple cell types in addition to neurons contribute to disease onset and pathogenesis. So, the cascade may not be as linear as it was originally depicted. However, one should not forget that even Alois Alzheimer had described apparent microglial pathology [1], although he did not know the identity of these cells, and work by other pioneers in the field suggested that microglia might be invariably involved in AD pathology [43].

One of the key microglial genes identified was TREM2 [44,45], variants of which significantly increase the risk for late-onset AD. AD risk variants in TREM2 have surprising and unexpected functional consequences. Microglia are a very dynamic population of functionally different types of cells, ranging from a resting homeostatic population that constantly surveil the brain, to a responsive population, so-called disease-associated microglia (DAM), which actively fight brain pathology [46]. TREM2 is a receptor on the surface of microglia that senses pathological changes in the brain and is responsible for initiating the switch in gene expression pattern to allow microglia to adopt a defensive disease response [46]. Strikingly, certain loss-of-function mutations in TREM2 prevent this switch of microglia and arrest them in a homeostatic state, even under disease conditions, whereas other mutations reduce ligand binding and signaling [47–49]. Thus, microglial activation is, at least to some extent, a protective event, and reduced activation results in a significantly enhanced risk for AD [42]. This can be directly observed in the brains of patients with AD, in which microglia are attracted to amyloid plaques, but such microglial clustering is reduced by AD risk variants in TREM2 and almost absent upon a complete genetic loss of TREM2 in mice [50,51]. Strikingly, in humans, the TREM2-dependent protective response is triggered by deposition of amyloid seeds (small aggregates of Aβ), which drive the further aggregation and deposition of Aβ [23,52,53]; this can happen >20 years before familial AD patients show cognitive decline [54]. Moreover, amyloid seeding is clearly enhanced in the absence of functional TREM2 [50]. Thus, the earliest detectable amyloid deposition seems to drive a TREM2-dependent protective response (Fig 1B). Moreover, microglia seem to interact with all other steps of the amyloid cascade such as diffuse (nonfibrillar) and mature (largely fibrillar) plaques [50,55] and tau-triggered cell death [56] (Fig 1B). One may even speculate that there might be individual microglial populations that are specialized to deal with the different pathological challenges of AD. Thus, microglial activation is an integral component of the amyloid cascade and not just an alternate disease-causing pathway unrelated to amyloid-driven events. Finally, Aβ-triggered high levels of TREM2 have even been associated with reduced brain shrinkage and a better cognitive outcome in patients [54,57]. Major attempts are currently underway to develop TREM2-modulating therapeutic strategies. It is likely that they would be most beneficial if employed together with anti-amyloid treatments in combinatorial clinical trials [42].

Microglia may also link another key AD risk factor to amyloid-related pathology, namely apolipoprotein E (ApoE). ApoE comes in 3 variants, ApoE2, ApoE3, and ApoE4, of which ApoE4 considerably increases the risk for sporadic (late onset) AD [58]. ApoE is a component of amyloid plaques [59] and is believed to be involved in Aβ aggregation and clearance [59–61]. Blocking ApoE expression in APP transgenic mice therefore leads to disruption of plaques, which can then liberate cytotoxic oAβ (see below) and/or cause local microvascular damage by Aβ accumulating in blood vessels [62]. ApoE expression is part of the DAM response (see above), and ApoE levels are greatly increased in microglia as soon as they respond to damage [46]. In AD, this occurs in microglia clustering around amyloid plaques [50] and, since the ApoE protein is secreted, its local extracellular concentration may then increase at sites of injury. Therefore, it is conceivable that ApoE may temporarily help to keep plaques compacted to avoid liberation of oAβ that could confer local synaptotoxicity [63]. However, the ApoE4 variant is a major risk factor of AD and believed to enhance Aβ aggregation much more than the 2 other variants. Thus, it seems that very early on when plaques are seeded by oligomeric forms of Aβ, ApoE4 may efficiently facilitate Aβ deposition and therefore initiation of the disease cascade, whereas later on, when plaques have matured, microglial-derived ApoE may help to keep plaques compacted. This possibility also implies that Aβ-lowering treatment approaches are confronted with multiple and maybe even opposite consequences of Aβ accumulating in deposits. Moreover, the very early response of microglia in patients with AD and the ability of these cells to remove amyloid seeds also implies that presymptomatic treatment with amyloid-lowering drugs is likely to be most successful. In fact, aducanumab, the only currently Food and Drug Administration (FDA)-approved anti-Aβ antibody (see below), prevents oAβ seeding in a mouse model more effectively than certain other candidate anti-Aβ antibodies that do not bind to newly formed plaques [53].

Taken together, the above overwhelming preclinical evidence supports Aβ as the major pathogenic factor that triggers AD and strongly suggests that Aβ-lowering therapies should be beneficial. Then why are the clinical findings apparently so disappointing?

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001694

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