The “amyloid cascade hypothesis”1
has dominated translational research on Alzheimer's disease for over 20 years. As originally stated, this hypothesis placed emphasis on the deposition of β-amyloid as the initiating event in the neuronal dysfunction and death that occurs in brain.
Implicit in the arguments for this hypothesis is that excess production of β-amyloid occurs at some point in the disease process, although this has only rarely been demonstrated. The major arguments in favor of the hypothesis are genetic. Mutations in the gene encoding the precursor of β-amyloid (the amyloid precursor protein, or APP) are a very rare cause of familial Alzheimer's disease.2
The most common causes of familial Alzheimer's disease are mutations in the presenilin 1 gene,3
and presenilin 1 (as part of a multisubunit proteolytic enzyme called y secretase) clearly plays an important role in cleavage of APP to produce β-amyloid.4
Less common are mutations in the presenilin 2 gene,5
and again this appears to function as part of a y secretase complex. Thus all three genes in which mutation causes familial Alzheimer's disease are involved with proteolytic processing of the amyloid precursor protein.6
The discovery of amyloid deposits in both diffuse and neuritic plaques as a major characteristic of Alzheimer's disease pathology has been interpreted to mean that there is increased amyloid production. However, deposition could clearly be the result of decreased clearance, degradation, or of some other process occurring in the tissue. Recent data from three different groups has suggested that most of the familial Alzheimer's disease mutations in APP and presenilins 1 and 2 actually result in reductions in the rate of cleavage of the APP, and reduced rates of β-amyloid production.7-9
This is clearlydifficult to reconcile with the huge increase in amyloid deposits in brain tissue, and has led to modifications in the original pathogenic cascade model.
Indeed, over the last 10 years, more and more groups have moved away from the original formulation of the amyloid cascade hypothesis, in large measure because it is clear that there is only very limited neurotoxicity associated with deposition of β-amyloid. This is especiallytrue in mice. A large number of transgenic mice have been made in which overexpression of mutant human APP (sometimes combined with a mutant presenilinl gene) drives deposition of large amounts of β-amyloid in the brain. The vast majority of these transgenic mice do not have evidence of neuronal degeneration or cell death, nor do they feature neurofibrillary tangle formation. This result is not what would be expected if the original proposal of the amyloid cascade hypothesis were correct. These and other results have led to modifications of the original hypothesis that propose that it is not deposition of β-amyloid that is the initiating event in pathology, but the formation of a soluble “toxic species” of βamyloid peptides.10,11
Along this line of reasoning, some have suggested that the deposition of β-amyloid may in fact be neuroprotective,12,13
with resultant sequestration of potentially toxic species. These toxic species are proposed to be oligomers, small aggregates of 2 to 12 peptide molecules, usually of the 42 amino acid long β-amyloid peptide.11,14
There remains considerable controversy about the precise molecular nature of the toxic species, and about the mechanism by which this species produces detrimental effects on neurons. The most common explanation is that synaptic disruption is the immediate toxic event,15
although precisely how this happens in the Alzheimer's disease brain remains poorly understood. Whether amyloid deposits or some soluble species is considered to be the initiating factor in the disease, these approaches are considered as “toxic gain of function models,” in which disease is proposed to be caused by the formation of novel molecular entities that cause toxicity. There is now a fairly vocal minority of researchers who have proposed that it is not actually the formation of any β-amyloid species that is the problem. All of the known familial Alzheimer's disease mutations disrupt proteolytic processing of the amyloid precursor protein, and probably several other proteins normally cleaved by the y secretase complex. If the production of a toxic β-amyloid species could be considered as a “toxic gain of function” in the majority view, the minority view would regard familial Alzheimer's disease mutations as “loss of γ secretase function.” While this view would appear consistent with the apparent reductions in the rate of cleavage of the APP (and some other substrates) noted with mutant APP or presenilin 1, a major problem is to provide an explanation for the abundant deposition of β-amyloid in the Alzheimer brain. If less amyloid is made, why is there so much deposition?
Regardless of the position taken on the molecular details of APP processing in Alzheimer's disease, it remains true that the vast majority of attempts at therapy for Alzheimer's disease to date are directed at reducing the amount of γ-amyloid in brain. These attempts fall into four different groups, depending on the approach.
Use of inhibitors of amyloid aggregation
The first interventional amyloid approach, based on the unmodified amyloid cascade hypothesis, was an attempt to prevent amyloid aggregation and/or to disrupt preformed amyloid aggregates. Enthusiasm for this mechanism of intervention has waned somewhat, in tandem with the original version of the amyloid cascade hypothesis. Although a major clinical trial of an aggregation inhibitor, called Alzhemed16,17
was carried out recently, results appear to have been negative, although some debate about variability between clinical trial sites has prevented a clear statement on this issue. Given the possibility that deposition of β-amyloid in tissues sequesters toxic species, and that disruption of deposition may increase toxic effects, further attempts along these lines appear unlikely.
Use of inhibitors of β-secretase
The proteolytic enzyme that cuts APP to liberate the Nterminus of the β-amyloid peptide, β secretase or BACE1, was identified and cloned by several groups, and it appears to be a single protein that cleaves APP and only a few other protein substrates.18,19
Mice in which the BACE1
gene is knocked out appear relatively normal, surviving into adulthood with subtle, if any, neuronal defects.20
BACE1 appears to be essential for generation of β-amyloid, such that mice overexpressing mutant human APP do not generate any measurable β-amyloid in the absence of the mouse BAC El
Clearly, the generation of specific inhibitors of BACE1 is an obvious and attractive prospect for prevention of production of β-amyloid. X-ray crystallography has been used to determine the precise structure of BACE1, and this should facilitate the development of inhibitors.22
The nature of the active site of this enzyme presents significant challenges to the development of small molecule inhibitors that can cross the blood-brain barrier,23,24
but it is very likely that such compounds will be forthcoming. Given the absence of a major detrimental effect of the knockout of the BACE1
gene, inhibition of BACE1 appears unlikely to result in severe side effects (but see ref 25). It is important to emphasize that success with BACE1 inhibitors will be dependent, to a large extent, on the validity of the “toxic gain of function” model, as suppression of BACE1 activity seems certain to reduce rates of production of β-amyloid by reducing rates of cleavage of APP. The challenge here is that if most mutations in APP and presenilin 1 also result in reduced rates of cleavage, and produce disease by this mechanism, one would expect an acceleration of disease progression on inhibition of either BACE1 (or γ secretase - see below). One of the most significant problems here is the absence of appropriate animal models. As mentioned above, mice with extensive amyloid deposition driven by overexpression of a mutant human APP gene do not develop a significant neurodegeneration. Thus while studies with BACE1 inhibitors could readily be performed in these mice to show reductions in amyloid deposition, few of the other features of Alzheimer's disease are evident in these mice, so that the effects of these compounds on the pathology and/or clinical features of Alzheimer's disease will remain untested until human trials are conducted.
Use of inhibitors of γ secretase
The problems with the use of γ secretase inhibitors are somewhat similar to those of inhibiting BACE1, although there are some notable distinctions. Knockout of vital components of γ secretase (presenilin 1, for example) does not produce viable mice unless the knockout is conditional26
(effectively unless the knockout is engineered to occur only in adult mice). The problem here is that γ secretase cleaves numerous proteins as well as APP, and at least some of these proteins (eg, Notch127
) play critical roles in brain development. Their role in the adult animal is less clear, although knockout of both presenilins 1 and 2 in adult animals results in a striking neurodegeneration.28,29
However, complete inhibition of y secretase is not what is intended by therapeutics, and the question still remains about whether the production of β-amyloid can be reduced without unacceptable consequences, these resulting presumably from reductions in the rate of processing of other γ secretase substrates. Preliminary reports appear to suggest that this is possible,30,31
and it appears that a large-scale phase 3 clinical trial of a γ secretase inhibitor is now underway. Again, success would seem to be dependent largely on the validity of the “toxic gain of function” model. There is perhaps the more direct concern here that again, the treatment exacerbates rather than interrupts the disease as reductions and not increases in the activity of γ secretase appear to result from mutations, particularly in presenilin 1.
Finally, much has been made of the effects of mutations in presenilin 1 (and perhaps presenilin 2) on the ratio of β-amyloid 40 to β-amyloid 42 produced by APP cleavage.7
These two peptides both appear to be produced by normal γ-secretase function, and it is true that many of the mutations shift the pattern of cleavage of APP so that despite the overall reduction in APP cleavage, relatively more of the 42 amino acid peptide is produced, decreasing the 40/42 ratio.4
The β-amyloid 42 peptide does aggregate more readily than β-amyloid 40, and is more neurotoxic in in vitro assays.32,33
The ”toxic gain of function“ model suggests that this is critical to the cascade of events that ensue. Precisely what inhibitors of y secretase do to this ratio is unclear, although at least some published data indicates that suppression of γ secretase activity can occur without a significant change in the 40/42 ratio.7
Perhaps this will prove critical to the success - or failure - of secretase inhibition in general. Only the clinical trials seem likely to provide this answer.
Use of antibodies, presumably to remove amyloid from the brain
Antibody approaches to reducing β amyloid in brain began with the spectacular studies of Schenk and colleagues, who immunized mutant human APP transgenic mice with β-amyloid peptides, and reported very significant reductions in amyloid deposition in these mice.34
Several others have confirmed and extended this early work,35,36
and human trials of “amyloid vaccination” have already been carried out. This is not the forum for discussing the controversial nature of these studies: suffice it to say that the results were far from the ideal. A number of patients developed an encephalitis,37
and in some cases this appeared to be disastrous. Whether or not there was any benefit remains highly dubious,38,39
but from a mechanistic viewpoint this approach raises a fundamental question: just how is an immune response to amyloid peptides supposed to reduce β amyloid concentrations in the brain?
Active immunization of transgenic mice with human amyloid peptides can produce the full range of B- and Tcell responses, in part because the human and mouse peptides differ in sequence - the human peptide is “foreign” to mice. Presumably the T-cell responses are what led to the encephalitis in humans immunized with human peptides, consistent with the induction of an autoimmune response.36,40,41
But why would a B cell - an antibody-producing response - be helpful?
Generally, antibodies in the circulation penetrate into the brain in only low concentrations.42
However, studies again in transgenic mice suggested that passive immunization, in which antibodies to β-amyloid were injected into the mice, have also been reported to cause significant reductions in the deposition of β-amyloid in the brains of the mice.43,44
There are two basic ideas of how this might work. First, it seems possible that while only a very small fraction of the injected antibodies makes it across the blood-brain barrier, this is sufficient to bind enough β-amyloid to reduce deposition.45,46
Antibody binding to β-amyloid in the brain may also activate the microglial (and possibly astrocytic) mechanisms that can reduce amyloid deposition.44,47
Critical in this formulation is the penetration of antibody into the brain.
A second proposed mechanism is what has been called the “peripheral sink hypothesis.” In this case, antibody binding to β-amyloid in the blood is thought to result in a sharp concentration gradient between the blood and the brain, such that β-amyloid movement from brain to blood is accelerated, and β-amyloid concentrations drop sharply and thus reduce the rate of deposition.48
Although this mechanism initially seems highly unlikely, there is evidence for transport of β-amyloid from brain to blood, at least under some circumstances.49
Perhaps it is unnecessary for the antibody to reach the brain at all.
The first clinical trials of “passive immunization” as a treatment for Alzheimer's disease appear to be underway, and preliminary results were reported in mid-2008. In passive immunization of transgenic mice, at least some antibodies appear to cause a shift in the localization of β-amyloid from deposits in the tissue to deposition in vessel walls, with some microhemorrhages reported.43
Human trials reported some vasculitis as a side effect in groups receiving the highest doses of antibody, although effects on rates of cognitive decline did not appear to be large, if measurable at all. Further trials of passive immunization are underway, in some cases using intravenous immunoglobulin G (IgG) fractions, with the presumption that natural IgG fractions - prepared by isolation of IgG from many thousands of donors - contain sufficient concentrations of anti-β-amyloid antibodies to reduce amyloid deposition.50,51
Whether this will prove a viable approach to therapy is as yet unclear.