APP

Definition: Amyloid precursor protein (APP) is an integral membrane protein with a single membrane-spanning domain, a large extracellular N terminus and a shorter cytoplasmic C terminus. The normal functions of APP in the body are only partly understood.

In Alzheimer disease (AD), APP is abnormally processed and converted into amyloid β peptide (Aβ). Mutations in the APP gene account for 1 out of 10 of all early onset AD families with known mutations.

The transmembrane protein APP (membrane indicated in green) can be processed along two main pathways, the alpha-secretase pathway and the amyloid-forming beta-secretase pathway.

In the alpha-secretase pathway, alpha-secretase cleaves in the middle of the amyloid-beta region (orange) to release a large soluble APP-fragment, alpha-APPs.

The carboxy (C)-terminal C83 peptide is metabolized to p3 by gamma-secretase. In the amyloid-forming beta-secretase pathway, beta-secretase releases a large soluble fragment, beta-APPs.

The C-terminal C99 peptide is then cleaved by gamma-secretase at several positions, leading to the formation of amyloid-40 (A40) and the pathogenic amyloid-42 (A42).

Gamma-Secretase cleavage also releases the APP intracellular domain (AICD), which could have a role in transcriptional regulation.

The effects of beta- and gamma-secretase inhibitors can be distinguished in secondary assays: both inhibitor classes block the formation of pathogenic A42, but beta-secretase inhibitors also block the formation of beta-APPs and C99, whereas gamma-secretase inhibitors also block the formation of p3 and the APP C-terminal fragment, leading to accumulation of C99 and C83.

Alzheimer disease

Current understanding of the principal events in the pathogenesis of AD is centered on the properties of Aβ. This peptide aggregates readily, forms β-pleated sheets and binds Congo red, is relatively resistant to degradation, elicits a response from astrocytes and microglia, and can be directly neurotoxic. The Aβ peptides are derived through processing of APP.

APP is a protein of uncertain cellular function that is synthesized with a single transmembrane domain and expressed on the cell surface.

A soluble form of APP can be released from the cell surface by proteolytic cleavage, by an enzymatic activity termed α-secretase; at least three distinct enzymes have been shown to have α-secretase activity. Molecules of APP that have undergone this cleavage cannot give rise to the Aβ fragment.

However, surface APP can also be endocytosed and may then undergo processing to generate Aβ peptides that are less soluble and tend to aggregate into amyloid fibrils. These are generated through cleavage at a site N-terminal to the start of the transmembrane domain by an enzyme called β-secretase (BACE-1) and cleavage within the transmembrane domain by γ-secretase.

This process is constitutively active in cells, and γ-secretase appears to perform other important intramembranous proteolysis events, including cleavage of Notch, a cell fate-determining molecule. The cleavage of Notch results in release into the cell of a portion of the molecule that is involved in cell signaling and transcriptional regulation.147 Both by inference and by direct experimentation, it has been suggested that a similar function can be attributed to a fragment of the C-terminal portion of APP that is generated by the same cleavages that generate Aβ.

Several gene loci have been identified for familial Alzheimer disease. The first of these was the gene for APP on chromosome 21. The pathogenic mutations in the APP gene all result in increased generation of Aβ.

Furthermore, the development of Alzheimer disease in individuals with trisomy 21 has been related to a gene dosage effect with increased production of APP and subsequently Aβ.

Two other genetic loci linked to early-onset familial Alzheimer disease have been identified on chromosomes 14 and 1; these probably account for the majority of early-onset familial Alzheimer disease pedigrees.

The genes on these two chromosomes encode highly related intracellular proteins, presenilin-1 (PS1) and presenilin-2 (PS2). Even before these genes were cloned, it was recognized that the cellular phenotype of these mutations was an increased level of Aβ generation, particularly Aβ42.

It has now become clear from studies of knockout mice, from directed mutatagenesis of PS1 and PS2, from pharmacologic studies, and from biochemical purifications that the presenilins are a component of γ-secretase and possibly are the portion of a multiprotein complex containing the active proteolytic site. Thus, the genetic evidence strongly supports the notion that the underlying pathogenetic event in AD is the accumulation of Aβ.

How Aβ is related to the neurodegeneration of AD, how it is linked to the other pathologic features of AD such as tangles and abnormal hyperphosphorylation of tau, and what controls the stereotypic pattern of involvement of brain regions and the pattern of progression all remain open questions.

There are various lines of evidence indicating that the small aggregates of Aβ as well as larger fibrils are directly neurotoxic and can elicit various cellular responses, including oxidative damage and alterations in calcium homeostasis.

In addition, the reactions of other cell types in the brain influence the disease. There is evidence that the inflammatory response that accompanies Aβ deposition may have both protective effects (through assisting clearance of the aggregated peptide) and injurious effects.

APP Function

APP is a type-1 integral transmembrane protein in which the C-terminal portion of Aβ is embedded within the cell membrane. APP processing results in or precludes formation of the amyloidogenic Aβ peptide.

APP is a member of an evolutionary conserved family of type I membrane proteins that contain a large N-terminal extracellular domain, a single transmembrane region and a short C-terminal cytoplasmic tail.

Besides APP, the amyloid precursor-like proteins APLP1 and APLP2 are members of the APP gene family in mammals. The evolutionary conservation of this gene family also extends to invertebrates; for example, there is the Drosophila melanogaster homologue known as APPL and the Caenorhabditis elegans gene product named APL-1.

The function of the APP holoprotein is not yet established and mice lacking the APP gene show relatively minor neurological impairments. This subtle phenotype is probably due to compensatory effects mediated by two other members of the APP gene family (APLPs): amyloid-precursor-like protein-1 and -2 (APLP1 and APLP2).

The combined ablation of APP and APLP2, both APLPs genes or all three family members leads to early postnatal lethality. Nevertheless, no consensus has been reached regarding a functional role for APP.

APP functions

The ubiquitious expression of APP in many tissues as well as the presence of homologues in a variety of species, including mammals and invertebrates, argue for an important physiological function of APP.

 A receptor-like function ?

On the basis of its structural features, APP has been proposed to have a receptor-like function on the cell surface. Indeed, an extracellular APP ligand, F-spondin, has been identified.

F-spondin, a secreted signalling molecule implicated in neuronal development and repair, binds to the conserved extracellular domain of APP and inhibits β-secretase cleavage of APP.

The low-density lipoprotein receptor-related protein (LRP), a member of the low-density lipoprotein-receptor (LDLR) gene family, has been shown to interact with APP, further supporting the hypothesis that APP might function as a receptor at the cell surface. LRP is a major receptor for apolipoprotein E (APOE), and the 4 allele of APOE is a strong genetic risk factor for late-onset AD.

Localized cell-surface APP has also been shown to be involved in cell–cell and cell–matrix adhesion. The short cytoplasmic tail of APP interacts with different binding partners, including Fe65, disabled homology (Dab) 1, Dab2, X11, Numb, c-Jun N-terminal kinase interacting protein 1b (Jip1b) and autosomal recessive hypercholesterolemia (ARH).

The interaction of the cytoplasmic tail of APP with these proteins is mediated via the YENPTY sequence near the C terminus of APP and the phosphotyrosine-binding (PTB) domains of the corresponding adaptor proteins.

The importance of the YENPTY domain of APP in the physiological functions of APP is further substantiated by the fact that this is the only region that is completely conserved in the cytoplasmic domain from C. elegans to humans.

Most of these adaptor proteins (ARH, Numb and Dab2) might be involved in internalization of APP from the cell surface, whereas Dab1 is a downstream messenger of the Reelin pathway, which is involved in neuronal migration to cortical layers during development.

Dab1 binds to APP, but it binds even more strongly to the YENPTY sequence of the APP homologue APLP1. The interaction of Dab1 with APP and APLP1 might therefore represent an intersection of the APP and Reelin intracellular signalling pathways, further supporting a role for APP and its homologues in neuronal development.

A potential role for APP as a cargo receptor for the axonal transport of vesicles is supported by the binding of APP via Jip1b to the kinesin light chain 1, which is a component of the axonal transport machinery.

Furthermore, Jip1b interaction with APP enhances JNK-mediated threonine-668 phosphorylation, indicating a possible role of APP in kinase-mediated signalling cascades.

Such a role is further supported by binding of Abl or Shc to APP, when the tyrosine in the YENP sequence is phosphorylated. G-protein-mediated signal transduction via APP is substantiated by the binding of G0, a major GTP-binding protein in the brain, to the cytoplasmic domain of APP.

Most research interest, however, has focused on the interaction of APP with Fe65, which is a multimodular adaptor that modulates the trafficking and processing of APP. The initial binding of Fe65 to the YENPTY motif of APP occurs before γ-secretase liberates the APP intracellular domain (AICD).

It has been proposed that once γ-secretase has cleaved APP the Fe65/AICD complex is transported to the nucleus, where a transcriptionally active complex with the histone acetyltransferase Tip60 is formed.

Target genes, which have been proposed to be regulated via the Fe65/AICD/Tip60 complex, are APP itself, BACE, tetraspanin KAI1/CD82, glycogen synthase kinase-3β and neprilysin. However, recent findings show that the proposed target genes are at best indirectly and weakly influenced by AICD transcriptional activation.

Besides the possible functional roles of the APP holoprotein or the released AICD fragment in gene transcription, the soluble APP (sAPP) ectodomain is proposed to be involved in different physiological processes.

sAPP is predicted to regulate neuronal processes such as neurite outgrowth, synaptogenesis, synaptic plasticity and neuronal excitability and exerts neurotrophic and protective effects, indicating a potential role in learning and memory. Furthermore, sAPP seems to play a role in neuronal stem cell division.

APP-processing pathways

Amyloid precursor protein [APP] is endoproteolytically processed by BACE1 and gamma-secretase to release amyloid peptides [Abeta40 and 42] that aggregate to form senile plaques in the brains of patients with Alzheimer’s disease [AD].

The C-terminus of Abeta40/42 is generated by gamma-secretase, whose activity is dependent upon presenilin 1 and presenilin 2 [PS 1 or 2].

Cleavage by the membrane-associated metalloprotease α-secretase, the activity of which is probably manifested by several distinct enzymes, occurs within the Aβ domain (between residues 16 and 17), thereby preventing the formation of Aβ, and instead results in the release of the large soluble extracellular N-terminal portion of APP (APPsα) and a C-terminal fragment consisting of 83 residues (C83). C83 might undergo further processing by γ-secretase to release the p3 peptide, which is considered non-amyloidogenic, although it is deposited in diffuse plaques.

To form Aβ, APP must undergo two sequential endoproteolytic steps, elaborated by distinct enzymatic activities known as β- and γ-secretase. β-Secretase, which is the enzyme known as BACE1 (β-site APP-cleaving enzyme), cleaves APP at the N-terminal region of the Aβ sequence.

Cleavage by β-secretase generates a slightly shorter soluble N-terminus (APPsβ) and the amyloidogenic C-terminal fragment (C99). The cleavage of C99 by γ-secretase liberates the C-terminal 50 residues of APP, which is known as APP intracellular domain (AICD) and Aβ.

Pathology (APP diseases)

 Alzheimer disease (AD)

One important pathologic feature of Alzheimer disease (AD) is the formation of extracellular senile plaques (amyloid plaques) in the brain, whose major components are small peptides called beta-amyloid (Abeta) that are derived from beta-amyloid precursor protein (APP) through sequential cleavages by beta-secretases and gamma-secretases.

Three secretases (alpha-secretases, beta-secretases, and gamma-secretases) are involved in APP processing and various molecular and cellular mechanisms underlying intracellular trafficking of APP.

Intracellular trafficking of APP is a highly regulated process and its disturbance has direct impacts on the production of Abeta.

APP germline mutations are observed in the different forms of Alzheimer disease:
 in familial Alzheimer disease
 in early-onset Alzheimer disease
 in late-onset Alzheimer disease

 duplication of the APP locus on chromosome 21 in autosomal dominant early-onset Alzheimer disease (ADEOAD) and cerebral amyloid angiopathy (CAA)

• Brains from individuals with APP duplication showed abundant parenchymal and vascular deposits of amyloid-beta peptides.
• Duplication of the APP locus, resulting in accumulation of amyloid-beta peptides, causes ADEOAD with CAA.

 germline mutations in dutch type cerebroarterial amyloidosis (cerebral amyloid angiopathies or CAA)

• in dutch type cerebroarterial amyloidosis with presenile dementia
• in artic type cerebroarterial amyloidosis
• in italian type cerebroarterial amyloidosis
• in Iowa type cerebroarterial amyloidosis

 germline mutations in the familial occipital calcifications, hemorrhagic strokes, leukoencephalopathy, arterial dysplasia, and dementia (FOCCHS-LADD) syndrome (MIM.605714)

 variant in chronic schizophrenic with cognitive defects

APP mutations

Since the identification of the first AD-causing mutation in APP at 21q21, 18 different causative APP mutations have now been reported (http://www.molgen.ua.ac.be/ADMutations).

All of these mutations cluster at, or are near to, sites within APP that are normally cleaved by proteases called the α-secretases, β-secretases and γ-secretases. These enzymatic activities regulate the metabolism of APP including the generation of the Aβ peptide, which is the major constituent of the amyloid plaques.

The phenotypic outcome of APP mutations is strongly dependent on which cleavage site is mutated. For example, mutations affecting the α-cleavage site promote the self-aggregation of mutated Aβ peptides, leading to severe amyloid deposition within the cerebral vessel walls or cerebral amyloid angiopathy (CAA) and hence strongly predispose affected individuals to haemorrhagic strokes.

The Dutch APP E693Q mutation causes a pure CAA-related haemorrhagic stroke phenotype. Other mutations at the α-cleavage site also lead to the deposition of amyloid plaques and tau tangles in the brain parenchyma in addition to CAA, which together result in a combined neurodegenerative and haemorrhagic stroke disorder as illustrated by the Flemish APP A692G mutation.

Mutations affecting the β-secretase and γ-secretase sites favor the release of amyloidogenic Aβ42 from its precursor and mostly result in typical AD phenotypes characterized by amyloid plaques and tau tangles with CAA being less prominent.

The Austrian APP T714I mutation, which is located at the γ-secretase-cleavage site of APP, results in an extremely aggressive AD phenotype with an onset age of 35 years and is characterized by tau tangles and extensive deposition of nonfibrillar ‘cotton wool’ amyloid plaques.

Genotype–phenotype correlation studies of APP mutations strongly implicate various forms of Aβ deposition, ranging from vascular CAA and fibrillar core-containing plaques to non-fibrillar ‘cotton wool’ plaques, as an essential characteristic of AD.

Morphological studies of APP mutations in human and mouse have strongly implicated the vascular system in the formation of core-containing amyloid plaques suggesting that vascular damage might be an important contributing factor to AD pathogenesis.

Importantly, and with the possible exception of the Dutch APP mutation, tau deposits (MAPT) in the form of tangles are a consistent but downstream consequence throughout the APP spectrum of disorders.

References

 Regulatory RNA goes awry in Alzheimer’s disease. Peter St George-Hyslop, Christian Haass. Nature, 2008, 14, 711-12.

 Zhang YW, Xu H. Molecular and cellular mechanisms for Alzheimer’s disease: understanding APP metabolism. Curr Mol Med. 2007 Nov;7(7):687-96. PMID: 18045146

 Adalbert R, Gilley J, Coleman MP. Abeta, tau and ApoE4 in Alzheimer’s disease: the axonal connection. Trends Mol Med. 2007 Apr;13(4):135-42. PMID: 17344096

 Alzheimer’s APP mangles mitochondria. Michael T Lin & M Flint Beal. Nature Medicine - 12, 1241 - 1243 (2006)

 Rovelet-Lecrux A, Hannequin D, Raux G, Le Meur N, Laquerriere A, Vital A, Dumanchin C, Feuillette S, Brice A, Vercelletto M, Dubas F, Frebourg T, Campion D. APP locus duplication causes autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy. Nat Genet. 2006 Jan;38(1):24-6. PMID: 16369530

 Revesz T, Ghiso J, Lashley T, Plant G, Rostagno A, Frangione B, Holton JL. Cerebral amyloid angiopathies: a pathologic, biochemical, and genetic view. J Neuropathol Exp Neurol. 2003 Sep;62(9):885-98. PMID: 14533778

 Vardy ER, Catto AJ, Hooper NM. Proteolytic mechanisms in amyloid-beta metabolism: therapeutic implications for Alzheimer’s disease. Trends Mol Med. 2005 Oct;11(10):464-72. PMID: 16153892

 Laferla FM, Oddo S. Alzheimer’s disease: Abeta, tau and synaptic dysfunction. Trends Mol Med. 2005 Apr;11(4):170-6. PMID: 15823755

 Nixon RA. Niemann-Pick Type C disease and Alzheimer’s disease: the APP-endosome connection fattens up. Am J Pathol. 2004 Mar;164(3):757-61. PMID: 14982829