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Mutations in both the amyloid precursor protein (APP) and the presenilin (PSEN) genes cause familial Alzheimer's disease (FAD) with autosomal dominant inheritance and early onset of disease. The clinical course and neuropathology of FAD and sporadic Alzheimer's disease are highly similar, and patients with FAD constitute a unique population in which to conduct treatment and, in particular, prevention trials with novel pharmaceutical entities. It is critical, therefore, to exactly defi ne the molecular consequences of APP and PSEN FAD mutations. Both APP and PSEN mutations drive amyloidosis in FAD patients through changes in the brain metabolism of amyloid-β (Aβ) peptides that promote the formation of pathogenic aggregates. APP mutations do not seem to impair the physiological functions of APP. In contrast, it has been proposed that PSEN mutations compromise γ-secretase-dependent and -independent functions of PSEN. However, PSEN mutations have mostly been studied in model systems that do not accurately refl ect the genetic background in FAD patients. In this review, we discuss the reported cellular phenotypes of APP and PSEN mutations, the current understanding of their molecular mechanisms, the need to generate faithful models of PSEN mutations, and the potential bias of APP and PSEN mutations on therapeutic strategies that target Aβ.

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Alzheimer's disease (AD) is the most common agerelated neurodegenerative disorder, currently affecting 20 to 30 million individuals worldwide [1]. The cardinal symptom of the disease is progressive memory loss due to the degeneration of neurons and synapses in the cerebral cortex and subcortical regions of the brain. Comprehensive evidence supports the amyloid hypothesis of AD, which argues that accumulation and aggregation of amyloid-β (Aβ) peptides in the brain is causal in its pathogenesis. Aβ is a proteolytic fragment generated through sequential cleavage of the amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase. Cells produce Aβ peptides of variable length. A peptide of 40 amino acids (Aβ40) is the most prevalent species secreted by cells, whereas the longer Aβ42 isoform appears to be the key pathogenic species and the most abundant species deposited in the brain. Major support for the amyloid hypothesis is drawn from cases of earlyonset familial AD (FAD). As used in this review, the term FAD is confined to familial cases with an autosomal-dominant inheritance pattern. Missense mutations that are causative of FAD have been identified in three genes that are essential for the generation of Aβ peptides: the APP gene and two homologous genes that encode the catalytic subunit of γ-secretase, PSEN1 (encoding presenilin-1) and PSEN2 (encoding presenilin-2) [2, 3]. Overall, the clinical presentation of FAD patients with APP and PSEN mutations is very similar to that of sporadic AD, which is supported by neuroimaging, biomarker and post-mortem neuropathology studies. Recently, the clinical findings in FAD mutation carriers have been summarized in an excellent review by Bateman and colleagues [4] and will not be further discussed here.

Amyloid precursor protein (APP) mutations. (a) The APP transmembrane domain (TMD) extends from the glycine in position 700 to the lysine in position 723. The Aβ42 peptide isoform is highlighted in yellow. Depicted by arrows are the β-secretase (BACE1) cleavage site, the γ-secretase cleavage sites generating Aβ40 and Aβ42, and the ε-cleavage sites. According to the sequential cleavage model, ε-cleavage is the initiating event for the stepwise generation of Aβ peptides, which proceeds from the ε-site to the γ-cleavage sites and reflects the periodicity of the APP TMD α-helix. Amino acid exchanges causative of either familial Alzheimer's disease (FAD) or cerebral amyloid angiopathy (CAA) are shown below the peptide sequence. (b) Timeline of the discovery of APP mutations (see also [55]).

When comparing the APP FAD and CAA mutations it is evident that these cluster around hot spots in the APP protein sequence (Figure 1a). The mutations causative of CAA are located in the central region of the peptide. At the molecular level, these mutations change the charge distribution and thereby likely affect the peptide structure, ultimately promoting fibril formation [15, 16]. Most data to date have been generated for the E22Q (E693Q) Dutch peptide. Limited proteolysis and NMR has identified a turn in the V24-K28 region, which appears to be critical for folding of the monomer and is stabilized, in part, by electrostatic interactions between E22 and K28. E22 and D23 mutations destabilize this turn and thereby promote oligomer formation [17, 18]. Accordingly, in biological systems, increased toxicity in human leptomeningeal smooth muscle cells and enhanced neurotoxicity have been reported for the Dutch peptide [19, 20]. This peptide also appears to be less efficiently degraded by the prototypical Aβ-degrading enzyme insulin-degrading enzyme [21]. Transgenic animal models expressing Dutch APP recapitulate the human pathology, with the vasculature being the main site of amyloid deposition [22]. With respect to Aβ production itself, no coherent phenotype has been observed for the CAA mutants. The A692G Flemish mutation enhances Aβ40 and Aβ42 production whereas the Dutch mutation does not appear to affect Aβ production at all [23]. The enhanced Aβ production of the Flemish mutation was reported in 1994 [24]. Many years later, a systematic analysis of the domain surrounding A692 has identified a substrate inhibitory domain (ASID) in APP [25]. This domain appears to exert a negative control over the activity of γ-secretase. Intriguingly, only the A692G amino acid exchange introduced by the Flemish mutation, but none of the other CAA/FAD-associated mutations in the ASID domain, lowered its inhibitory potency, thus raising Aβ production [25]. This adds another facet to the mechanistic understanding of the Flemish CAA mutation. It is reasonable to assume, however, that the main driver for the CAA pathology is the change of the Aβ peptide sequence itself, since increasing total Aβ production will lead to FAD and not CAA, as shown for the APP Swedish mutation (see below).

Taken together, this leaves some important questions unanswered, such as why CAA mutations specifically target Aβ deposition to the brain capillaries. One common hypothesis is that the aggregation kinetics of the CAA peptides reduce their clearance across the blood-brain barrier [22]. A major contributor could be the specific cellular environment in the vasculature since smooth muscle cell surfaces in particular have been shown to promote CAA Aβ aggregation [34].

The key question of how exactly these FAD mutations promote the elevation of the Aβ42/Aβ40 ratio still remains unresolved. The answer may lie in the way γ-secretase cleaves its substrates. γ-Secretase cleaves at multiple sites within the APP TMD, and various Aβ peptide species have been identified in cell supernatants (Aβ33, 34, 37, 38, 39, 40, 42, 43) and cell lysates (Aβ45, 46, 48, 49). Recent data suggest a stepwise mode of cleavage with initiation at the ε-cleavage site [2, 50]. This initial processing event is followed by successive tripeptide generation, which proceeds from the ε-cleavage site to the γ-cleavage sites and reflects the periodicity of the α-helix. According to this model, the initiation site for Aβ42 and Aβ40 would be at positions 48 (APP T719) and 49 (APP L720), respectively, in the Aβ domain. An increase in the efficiency to initiate the Aβ42 lineage of peptides at T719 or in turn a decrease at initiating the Aβ40 lineage at L720 would lead to an elevation of the Aβ42/Aβ40 ratio. In this respect, the region comprising residues T714 to V717 must harbor critical structural determinants governing enzyme binding and positioning for lineage initiation [51]. Mechanistically, one could view these mutations as quasi loss-of-function variants. If the enzyme had evolved to efficiently convert the APP substrate into Aβ40, any mutation forcing the enzyme towards the less efficient Aβ42 lineage would fit this definition.

The vast majority of FAD cases harbor heterozygous mutations in the PSEN1 gene on chromosome 14. Sherrington and colleagues [52] identified the first mutations in PSEN1 in 1995. In the same year, mutations in the homologous gene PSEN2, on chromosome 1, were described [53, 54]. Since then, more than 180 different pathogenic mutations in more than 400 families have been identified in PSEN1 and an additional 13 mutations in PSEN2 (see the Alzheimer Disease and Frontotemporal Dementia Mutation Database [55, 56] for a complete list of mutations). Individuals with PSEN1 mutations typically become symptomatic between the ages of 30 and 50 years.

PSEN proteins have been proposed to exert both γ-secretase-dependent and -independent functions. While it is far beyond the scope of this review to discuss all known physiological functions of PSEN proteins, we will briefly summarize PSEN activities that might be impaired by FAD mutations.

Tissue culture models of presenilin (PSEN) mutations. In most studies, PSEN mutants have been stably overexpressed either in permanent cells lines (left) or in PSEN1/PSEN2-/- double-knockout cell lines (middle). Due to the replacement phenomenon or the lack of endogenous wild type (WT) PSEN proteins, functional γ-secretase complexes in both of these tissue culture models contain predominantly or solely the exogenously expressed PSEN mutants. This situation is different from familial Alzheimer's disease (FAD) patients with heterozygous PSEN1 (or PSEN2) mutations that express mutant and WT PSEN1 (or PSEN2) in an equal ratio in the background of two WT PSEN2 (or PSEN1) alleles (right). CMV, cytomegalovirus.

Initially, measurements of steady-state Aβ levels in transfected cells, transgenic mice and primary cells of FAD patients with PSEN1 or PSEN2 mutations suggested that the common pathogenic mechanism of PSEN mutations was to selectively elevate the absolute amount of cellular Aβ42 production, which was interpreted as a gain-of-toxic function mechanism [70, 74]. However, subsequent experiments have demonstrated that many FAD PSEN mutations when overexpressed display reduced overall γ-secretase activity compared to WT PSEN proteins. This was first recognized by Song and colleagues, who showed that overexpression of the PSEN1 mutations C410Y and G384A in PSEN1-/- knockout cells resulted in reduced NICD production [75]. These findings correlated closely with results from in vivo experiments in PSEN-deficient Caenorhabditis elegans and Drosophila that reported a complete rescue of NOTCH phenotypes after transgenic expression of human WT PSEN1 but only partial rescue with FAD PSEN1 mutants [76, 77].