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Progress in Neurobiology 82 (2007) 11–32

Structure and functions of the human amyloid precursor protein:

The whole is more than the sum of its parts

Matthias Gralle 1,*, Sergio T. Ferreira *

Instituto de Bioquımica Medica, Programa de Bioquımica e Biofısica Celular, Universidade Federal do Rio de Janeiro,

Rio de Janeiro, RJ 21944-590, Brazil

Received 15 September 2006; received in revised form 26 October 2006; accepted 1 February 2007

Abstract

The amyloid precursor protein (APP) is a transmembrane protein that plays major roles in the regulation of several important cellular functions,

especially in the nervous system, where it is involved in synaptogenesis and synaptic plasticity. The secreted extracellular domain of APP, sAPPa,

acts as a growth factor for many types of cells and promotes neuritogenesis in post-mitotic neurons. Alternative proteolytic processing of APP

releases potentially neurotoxic species, including the amyloid-b (Ab) peptide that is centrally implicated in the pathogenesis of Alzheimer’s

disease (AD). Reinforcing this biochemical link to neuronal dysfunction and neurodegeneration, APP is also genetically linked to AD. In this

review, we discuss the biological functions of APP in the context of tissue morphogenesis and restructuring, where APP appears to play significant

roles both as a contact receptor and as a diffusible factor. Structural investigation of APP, which is necessary for a deeper understanding of its roles

at a molecular level, has also been advancing rapidly. We summarize recent progress in the determination of the structure of isolated APP fragments

and of the conformations of full-length sAPPa, in both monomeric and dimeric states. The potential role of APP dimerization for the regulation of

its biological functions is also discussed.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Alzheimer’s disease; Amyloid precursor protein; Adhesion protein; Structural determination; Dimerization; Functions

Contents

1. Morphoregulatory molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.1. Adhesion proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.2. Secreted factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2. APP in brain function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.1. Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.2. Adult phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.3. Secreted APP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3. APP in Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.1. Genesis of the amyloid-b peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.2. Familial Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.3. APP and caspases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.4. Sporadic Alzheimer’s disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4. Determination of protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5. APP structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Abbreviations: APP, b-amyloid precursor protein; APLP, APP-like protein; HCZ, heparin-, copper- and zinc-binding fragment of APP; KPI, Kunitz-type

protease inhibitor domain of APP; LRP, low density lipoprotein receptor-related protein; NGF, nerve growth factor; NMR, nuclear magnetic resonance; sAPP,

secreted APP; SAXS, small-angle X-ray scattering

* Corresponding authors. Tel.: +55 21 2562 6789; fax: +55 21 2270 8647.

E-mail addresses: mgralle@gwdg.de (M. Gralle), ferreira@bioqmed.ufrj.br (S.T. Ferreira).1 Present address: Cell Biophysics Group, European Neuroscience Institute, Waldweg 33, 37073 Gottingen, Germany.

0301-0082/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pneurobio.2007.02.001

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–3212

5.1. Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5.2. Protease inhibitory domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5.3. N-Terminal domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5.4. Central domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5.5. Interactions between APP domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

6. Structure of full-length sAPPa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

6.1. Direct measurements on full-length sAPPa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

6.2. Topology and positioning of APP fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

7. Dimerization of sAPPa and APP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

8. Functional implications from structural studies with sAPPa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

9. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

9.1. Possible questions to be addressed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

9.2. APP and cholinergic transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

9.3. Implications for APLP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

9.4. Unexpected functions of APLP2 and APP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

10. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

1. Morphoregulatory molecules

Most of the research on the amyloid precursor protein (APP)

has aimed to understand its physiological and pathological roles

in the central nervous system (CNS). Because there are reasons

to believe that APP may have important functions in

intercellular communication in the brain, we initially present

a brief discussion of the molecular basis of tissue organization

and function, first in general and then in the specific case of the

CNS.

Because of the complexity of human tissues, a large part of

what is known about their functions, regulation and develop-

ment has been generated by studies carried out using

dissociated cellular models. Despite the wealth of information

such studies have provided, it is necessary to recognize the

limits posed by the use of cellular models and the types of

questions such models cannot answer. In the same way as the

large number of different molecular components of a cell,

connected by multiple non-linear relations, leads to the

emergence of new properties at the cellular level, the large

number of different cells in a tissue, connected by multiple

signaling pathways, can also give rise to new properties that

cannot be easily or at all derived from studies of isolated cells;

these so-called emergent properties are often among the most

interesting ones, both to the researcher and to the physician

(Nelson et al., 2005). The most complex human tissue is the

CNS, as it contains an immense number of cell types of distinct

morphologies, biochemical and electrophysiological properties

(Toledo-Rodriguez et al., 2004). In contrast to the immune

system, which also comprises a very large number of

functionally distinct cells, the nervous system additionally

has an exact spatiotemporal organization: each of its cells is

defined not only by its characteristic phenotype but also by

specific coordinates within the tissue and by precise temporally

regulated communication with distinct partners.

The most interesting properties of the CNS, among them the

formation and storage of memories and their later retrieval or

extinction, arise from interactions between specific nerve cells

localized in different parts of the brain. Although a vast number

of studies have examined different electrophysiological or

biochemical markers of neuronal plasticity as surrogates of

memory formation, tissue-level emergent phenomena such as

memory are not immediately intelligible from the study of

isolated neurons or glia cells—the effect of a given protein in

dissociated cells may often appear very distant from that in the

functional brain. Hence, two of the most important questions in

structural neuroscience—necessary for understanding complex

functional phenomena such as memory—are as follows: (1)

How do the cells initially organize to give rise to the exact

three-dimensional structure of the brain? (2) How do

intercellular connections restructure and reorganize during

life, responding to environmental stimuli?

During both development and adult phases, external

information in the form of sensorial stimuli interacts with

genetically encoded information in brain cells in order to guide

the (re)organization of the tissue. Two classes of molecules that

appear to play key roles in the development of the CNS and in

its plasticity are the cell adhesion and the substrate adhesion

molecules (Edelman and Cunningham, 1990; Washbourne

et al., 2004), while another important class is represented by

secreted molecules.

1.1. Adhesion proteins

In many cell types, from unicellular organisms to human

epithelia, adhesion regulates proliferation. In fact, considering

their diverse biological functions, the term ‘‘contact receptors’’

has been suggested as a substitute for ‘‘adhesion proteins’’,

which might suggest a merely mechanical role (Gebbink et al.,

1995). In the developing nervous system, as well as in the

neurogenesis that continues on a reduced scale in the adult

human brain, contact with other cells and with extracellular

molecules is an important regulator of proliferation. A well-

known example is the division of the neuroblasts of the

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–32 13

ventricular zone, which give rise to the cerebral cortex: some of

the adhesion proteins expressed in the apical membrane of the

neuroblast, probably at adhesion junctions, maintain the

neuroblast and its apical descendents in a proliferative state.

Thus, adhesion proteins play an important role in tissue

morphogenesis (Lien et al., 2006), a role that would be difficult

to infer from the study of neuroblast development in an in vitro

two-dimensional culture only.

Contact receptors, which in the proliferative state mediate

cell adhesion, continue to function in post-mitotic neurons.

Interaction between developing neurons and radial glia cells is

mediated by contact receptors and guides the future neurons

while they travel through the embryonic cortex (Trapp and

Hauer, 1994). After the developing neuronal cell reaches its

correct localization in the tissue, small processes arise from the

soma. Adhesion to the substrate will cause one of these

processes to become an axon, while the others later become

dendrites (Heidemann, 1996). At the tip of the processes,

specialized structures known as growth cones explore the

environment and guide the extension of the process. While

adhesion of the cell body inhibits cell migration, adhesion of

the tips of the filopodia in the growth cone to some types of

substrate promotes and is necessary for the extension of the

process, while repulsion by other types of substrate leads to

retraction or degeneration of the process. These different types

of cellular responses are not due to a simple biomechanical

effect of adhesion, but rather to signaling cascades set in motion

by contact receptors present in the growth cone (Heidemann,

1996). Finally, when the growth cone of an axon encounters an

adequate target cell, a synapse is formed. The central role of

adhesion molecules for general synaptogenesis, for the

specificity of synapses and for their continuing plasticity is

only now beginning to be understood (Washbourne et al.,

2004). Some of the same adhesion molecules that regulate the

cell cycle in mitotic cells regulate the extension and retraction

of axons and dendrites and the formation of synapses as well

(Goda, 2002).

While neuritogenesis and synaptogenesis reach a peak in

the perinatal period (Dunaevsky et al., 1999; Moya et al.,

1994), the extension and retraction of dendrites and axon

collaterals, the formation of synapses and the modification of

existing synapses continue during our whole life. Even in the

adult phase, there is considerable plasticity: in some areas of

the human brain, the mean total length of all dendrites of a

neuron increases 10% with age when individuals in their 30s

and 40s are compared to those in their 50s and 60s (Arendt

et al., 1998). This overall increase results from continuous

modifications, both extension and retraction, that take place in

a time scale of minutes (Dunaevsky et al., 1999; Engert and

Bonhoeffer, 1999). The formation and modification of long-

term memories and their extinction seem to depend on the

formation and modification of synapses, and, once again,

adhesion molecules, or rather contact receptors, contribute to

synaptic plasticity (Goda, 2002; Washbourne et al., 2004). It is

clear from this short discussion that contact receptors will

generally not function to generate a clear, environment-

independent phenotype; rather, they function as ‘‘resources’’,

i.e. they contribute to the ability of each cell in a tissue to

interact with its surroundings (Moss, 2003).

1.2. Secreted factors

Morphoregulatory communication between the cells of the

nervous system depends not only on adhesion molecules but

also on secreted factors. The latter include both proteins known

as growth factors and small molecule neurotransmitters. While

some secreted molecules diffuse freely through the extra-

cellular space and act over considerable distances, especially in

the embryonic nervous system, many others are released at the

synaptic cleft and act only in the immediate neighborhood.

Every synaptic transmission event may, in addition to its

immediate effect of transmitting information, also strengthen

(or sometimes weaken) the morphological connection between

pre- and postsynaptic cells, thereby allowing information

storage for longer times. While the effect of a secreted molecule

on a cell, even an isolated one, may be intense and obvious, it is

important to keep in mind that the effect of that molecule in the

functioning cellular network of the tissue may be more

complex.

2. APP in brain function

2.1. Development

The functions of APP are much better defined at the tissue

level than at the cellular and subcellular levels, and serve as

examples for many of the functions of contact receptors and

secreted factors described above. Full-length APP contains a

large extracellular domain (corresponding to �88% of the total

protein mass for the main neuronal isoform), a single

transmembrane region and a small cytoplasmic tail (Fig. 1).

This type of structure led its discoverers to suggest that APP

might function as a receptor (Kang et al., 1987). Though

functions, ligands and intracellular pathways have been well

established for many cellular receptors studied in vitro, this

kind of knowledge has been slow to emerge for APP, despite the

fact that an impressive body of immunohistochemical and

genetic data indicate the functional importance of APP in

nervous tissue.

Many studies using cells in culture have consistently shown

a role of APP in adhesion to other cells or to extracellular

matrix components (Kibbey et al., 1993; Qiu et al., 1995; Sabo

et al., 2001; Schubert et al., 1989; Soba et al., 2005). For

example, using an immortalized neuronal cell line, an early

study showed that APP increases adhesion to the substrate, to

other cells of the same line and to glia cells (Breen et al., 1991).

While these findings are interesting in themselves and

immediately suggest a role of APP in tissue maintenance, an

interesting aspect of the dynamic function of the neuron-glia

adherence promoted by APP emerged only from tissue-level

analysis: The discovery that APP is expressed at high

concentrations in the radial glia, which guides future neurons

to their correct positions in the embryonic cortex, suggests that

the adhesion of neurons to glia cells promoted by APP plays an

Fig. 1. Domain structure of APP. Grey ellipses: APP domains for which complete or partial structures are known. 1mwp, 1owt, 1aap, 1rw6, 1tkn correspond to the

PDB access codes for the fragments of known structure. (DE)n, RC: Intervening non-conserved stretches (Asp- and Glu-rich; random coil) suggested to be largely

unstructured or to have non-standard secondary structures (see Sections 5.3 and 3.4). KPI: Kunitz-type protease inhibitory domain (see Section 5.2). HBD1, HBD2:

Heparin-binding domains (see Sections 5.3 and 5.4). CuBD: Copper-binding domain (see Section 5.3). Ab: Amyloidogenic sequence (see Section 3.1). TM:

transmembrane domain. Cyt: Cytoplasmic domain. Cleavage by a-secretase (vertical arrow) releases sAPPa and the membrane-bound C-terminal fragment (CTFa),

as indicated (see Section 2.3). Amino acid numbering is for APP770 (see Section 5.1). Figure reprinted with permission from Gralle et al. (2006).

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–3214

important role in brain development (Trapp and Hauer, 1994).

Based on the findings described above, it seems warranted to

describe APP as a contact receptor. Indeed, several high-

specificity ligands of APP in the extracellular matrix have been

identified, though the mechanisms of signal transduction

triggered by such interactions are still unclear (Beher et al.,

1996; Ho and Sudhof, 2004; Kibbey et al., 1993; Williamson

et al., 1996; see also Sections 5 and 8).

As is also the case for other contact receptors, the adhesion-

promoting activity of APP in migratory cells is accompanied by

a neuritogenic activity in stationary, immature neurons in vitro

and in vivo (Allinquant et al., 1995; LeBlanc et al., 1992;

Majocha et al., 1994; Milward et al., 1992; Ohsawa et al., 1997;

Perez et al., 1997; Qiu et al., 1995; Small et al., 1994). The

biochemical basis for the involvement of APP in axonal growth

has recently been elucidated: The cytoplasmic tail of APP binds

to the actin regulatory proteins Fe65 and Mena, and this ternary

complex is localized to the dynamic adhesion sites in the

growth cone (Sabo et al., 2003). In non-neuronal cells, co-

expression of APP and Fe65 drastically increases their

migration velocity (Sabo et al., 2001). While studies with

cultured cells alone cannot fully reveal the spatial and temporal

importance of the involvement of APP, in vivo studies have shed

some light on where and when in brain development APP is

especially needed. A role of APP in the formation of long-

distance connections is supported indirectly by the fact that its

levels rise in the perinatal period, when axons rapidly extend in

the olfactory system of the rat and fall again after this period

(Clarris et al., 1995). During the peak period of axon extension,

APP and its homologues, the APP-like proteins APLP1 and

APLP2, comprise most of the protein mass that is rapidly

transported by the optical nerve (Lyckman et al., 1998; Moya

et al., 1994). Furthermore, the importance of APP for axon

arborization was demonstrated in Drosophila, where the

absence of the APP homologue leads to a failure of arborization

and, in consequence, to a reduced reproductive fitness of the

organism (Leyssen et al., 2005).

APP is also important for the next step of neuronal

morphogenesis, i.e., the formation of functional synapses. The

expression of APP in cultured hippocampal neurons increases

their response to glutamate (Tominaga-Yoshino et al., 2001),

and other studies have demonstrated electrophysiological

consequences of the lack of APP (Priller et al., 2006). On

the other hand, in vivo studies have focused on the importance

of APP for both morphological and electrophysiological

maturity of specific synapses. In double knock-out mice for

APP and its nearest homologue, APLP2, motoneuron axons

bypass their targets in muscle fibers and do not form synaptic

terminals at the correct sites, leading to a dramatic increase in

transmission failure on the day of birth (Wang et al., 2005) and

to the death of these animals a few days later (Heber et al.,

2000; Herms et al., 2004; Wang et al., 2005; Yang et al., 2005).

In the central nervous system, APP seems to be important for

the maturation of specific subtypes of neurons (Salbaum and

Ruddle, 1994).

2.2. Adult phase

In the adult brain, APP is expressed at high levels in those

regions that undergo more intense synaptic modifications

(Loffler and Huber, 1992; Ouimet et al., 1994), and its

expression level increases in animals reared in an enriched

environment (Huber et al., 1997). In an interesting example of

APP function in live, awake adult animals, specific knock-down

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–32 15

of APP in retinal ganglion cells leads to a reduction in

metabolic activity in the area innervated by those cells (Herard

et al., 2005). It is, therefore, not surprising that its permanent

absence in knock-out animals induces memory deficits

(Dawson et al., 1999).

Only a small fraction of the APP transported to the neuronal

plasma membrane remains there for any longer period of time

(Allinquant et al., 1994; Storey et al., 1999). This pool of APP

present at the plasma membrane probably mediates the

physiological functions described above. However, APP

metabolism in mature neurons is quite complex. Upon

emerging from the trans-Golgi network, APP is initially

inserted into fast axonal transport vesicles (Koo et al., 1990;

Sisodia et al., 1993). The hypothesis that APP itself anchors

these vesicles to kinesin, the molecular motor that transports

them along the axon to the synapses (Kamal et al., 2000), has

been broadly questioned (Lazarov et al., 2005). The details of

axonal transport of APP were very recently investigated using

tau mutant cells (Goldsbury et al., 2006). After the vesicles fuse

with the axonal or synaptic plasma membrane and APP appears

at the surface of the neuron, part of it is cleaved by proteases

collectively known as secretases and the extracellular domain is

secreted (see below); another fraction of APP returns via

endocytic vesicles to the cell body and to the dendrites

(Marquez-Sterling et al., 1997; Yamazaki et al., 1995). The

function of this return journey of APP is still unclear, though the

regulated endocytosis of other contact receptors has been

shown to be necessary for their functions (Schaefer et al.,

2002). The combination of partial proteolysis by the secretases

and re-internalization of uncleaved APP by the endocytic

pathway shortens the half-life of APP at the plasma membrane

to just �10 min (Koo and Squazzo, 1994).

2.3. Secreted APP

While the trafficking of transmembrane APP is still

incompletely understood, however, the biological effects of

the cleaved and secreted form of APP (known as sAPP, for

secreted APP) have been extensively investigated. Several

extracellular proteases of the ADAM (‘‘a disintegrin and

metalloproteinase’’) family cleave APP with the specificity

expected for the major secretase (‘‘a-secretase’’), 12 amino

acid residues upstream from the extracellular face of the

membrane, releasing the extracellular domain termed sAPPa

(Fig. 1). Among these proteases, ADAM10 is necessary for the

secretion of sufficient amounts of sAPPa in vivo (Postina et al.,

2004). In neuronal precursors in the adult brain, as well as in

non-neuronal cells, sAPPa acts as a proliferative factor

(Pietrzik et al., 1998; Saitoh et al., 1989; Caille et al., 2004).

In addition to stimulating proliferation, sAPPa also increases

the motility of keratinocytes (Kirfel et al., 2002) and of

melanocyte processes (Quast et al., 2003). Interestingly, sAPPa

increases neurite outgrowth in immortalized neuronal cell lines

(Milward et al., 1992). Not only the full-length transmembrane

form of APP but also sAPPa is transported through the axon

during the perinatal period (Lyckman et al., 1998; Moya et al.,

1994). At the synapse, sAPPa modulates transmission and is

neuroprotective against ischemic and excitotoxic injury

(Furukawa et al., 1996a,b; Mattson et al., 1993; Morimoto

et al., 1998; Smith-Swintosky et al., 1994). Probably as a

consequence of synaptic modulation, neuroprotection or both,

intracerebral injection of sAPPa enhances memory perfor-

mance in adult rats (Meziane et al., 1998).

Today it is clear that APP, acting both as a contact receptor in

its transmembrane form and as a proliferation and motility

factor in its secreted form, has important morphoregulatory

functions. The importance of APP is underscored by the

existence of two homologous proteins with partly redundant

functions that can compensate for part of the effects of eventual

APP loss (Heber et al., 2000). A more complete view of the

development of the brain and of synaptic plasticity will have to

await further advances in our knowledge of the dynamics of

neural circuits, on the one hand (Sakurai, 1999), and of the

exact modifications and relations of the participating mole-

cules, on the other. Elucidation of the structural basis of the

interactions of APP with its physiological partners will provide

important insight towards such an integrated view.

3. APP in Alzheimer’s disease

3.1. Genesis of the amyloid-b peptide

Retrospectively, it may now seem strange that APP was not

initially discovered because of its morphoregulatory roles in the

healthy brain, but rather because of the characteristic deposition

of plaques (known as senile plaques) that contain the amyloid-b

peptide (Ab), a proteolytic fragment derived from APP, in the

brains of demented patients (Glenner and Wong, 1984; Masters

et al., 1985). Following the elucidation of the amino acid

sequence of the Ab peptide, the cDNA of the principal neuronal

isoform of human APP (APP695) was cloned (Kang et al., 1987)

and, in the following year, the two isoforms that predominate in

other cell types (APP751 and APP770) were characterized

(Kitaguchi et al., 1988; Ponte et al., 1988; Tanzi et al., 1988).

The Ab peptide is derived from a quantitatively less important

cleavage pathway of APP (carried out by b-secretase; see

below) than the cleavage by a-secretase mentioned above. Ab

may serve neurotrophic and signaling functions when present at

low physiological concentrations (Kamenetz et al., 2003;

Yankner et al., 1990). However, amyloid aggregates that

accumulate in vivo in the brains of AD patients and that also

form in vitro in aqueous solutions of Ab are potent neurotoxins

(for an early study, see Pike et al., 1991; reviewed in Klein et al.,

2006). In spite of the notable presence of senile plaques in aged

brains and, particularly, in the brains of individuals affected by

Alzheimer’s disease, the degree of dementia in the patients

correlates better with the brain levels of soluble Ab than with

the quantity of Ab aggregated in plaques (McLean et al., 1999;

Lue et al., 1999). This important neuropathological observation

supports the notion that the species responsible for synaptic

dysfunction in AD patients may be soluble oligomers of Ab,

which interfere with synaptic plasticity (Lambert et al., 1998;

Walsh et al., 2002; Wang et al., 2002), rather than the fibrillar

amyloid aggregates that were initially considered to be the main

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–3216

neurotoxic species. Soluble Ab oligomers are present at much

higher concentrations in AD brains than in unaffected brains

(Gong et al., 2003), and they bind specifically to a

subpopulation of dendritic spines (Lacor et al., 2004).

The Ab peptide is proteolytically generated by the

successive action of b-secretase at the N-terminus of the so-

called amyloidogenic sequence, and g-secretase, which cleaves

APP at one of several different peptide bonds in its

transmembrane domain to form the C-terminus of Ab (for

an overview of the secretases, see De Strooper and Annaert,

2000; Turner et al., 2003). b-Secretase was identified as an

aspartic protease (Vassar, 2002). On the other hand, g-secretase

activity is associated with a high-molecular-weigh protein

complex containing four necessary constituents (presenilin 1 or

2, nicastrin, Aph-1 and Pen-2), which cleave a large number of

substrates besides APP (De Strooper et al., 1999; Edbauer et al.,

2003; Kimberly et al., 2003; Wolfe et al., 1999). The existence

of other, accessory factors that might regulate the activity of g-

secretase and thereby potentially influence the genesis of Ab

was inferred from studies in Drosophila (Loewer et al., 2004);

two of these accessory factors have been recently reported to be

CD147 (Zhou et al., 2005) and TMP21 (Chen et al., 2006b).

Within these high-molecular-weight complexes, the active site

is localized in the presenilins (Esler et al., 2000; Tolia et al.,

2006; Wolfe et al., 1999). g-Secretase does not act on integral

APP, but only on fragments not containing the extracellular

domain, and therefore it cleaves APP following its initial

processing by a- or b-secretase (Goo and Park, 2004; Struhl

and Adachi, 2000).

3.2. Familial Alzheimer’s disease

A small percentage of AD patients develop clinical

symptoms before the age of 60 and show mutations in the

APP gene. This indicates that alterations in this protein are

sufficient to cause the entire spectrum of disease-associated

clinical symptoms and pathological hallmarks (Goate et al.,

1991). In those cases with mutations near the b-cleavage site,

dementia appears to be causally related to a large increase in the

production of Ab (Citron et al., 1992). In other cases, such as

mutations in amino acids 716 and 717 of APP, there is no

increase in total Ab concentration, but only in that of its longer

and more amyloidogenic form, Ab42, leading to the proposal

that pathogenesis in those patients is due to the higher

proportion of Ab42 relative to Ab40 (Suzuki et al., 1994).

Another proposed explanation, however, involves pathological

signaling by mutant transmembrane APP, without necessarily

involving processing of the protein by the secretases (Yamatsuji

et al., 1996b).

No cases of Alzheimer’s disease caused by mutations or

dysfunction of b-secretase, or by lack of a-secretase, are known

to date. On the other hand, mutations in the presenilin genes are

known to be causally associated with familial Alzheimer’s

disease, and these mutations are much more frequent than those

in APP (De Strooper and Annaert, 2000).

g-Secretase cleavage of the membrane-bound fragment of

APP releases not only the Ab peptide but also the C-terminal

cytoplasmic domain of APP. The cytoplasmic domain, which

retains the sites of interaction with signaling proteins present in

transmembrane APP, has been reported to translocate to the

nucleus and regulate gene transcription in vitro (Cao and

Sudhof, 2001). However, the in vivo target genes of this

possible regulatory pathway have not yet been discovered and,

therefore, the consequences of this signaling pathway for the

physiological actions of APP and/or for the genesis of

Alzheimer’s disease are still unclear (see also Section 8).

3.3. APP and caspases

Another interesting aspect of APP biology is its cytoplasmic

cleavage by proteases of the caspase family, which leads to the

release of fragments different from those produced by the g-

secretase complex (De Strooper and Annaert, 2000). As the

primary and best-known function of caspases is to promote cell

death by apoptosis, a connection between neuronal death by

apoptosis and neuronal death in the advanced stages of

Alzheimer’s disease has been the usual interpretation of these

data (De Strooper and Annaert, 2000). It is noteworthy,

however, that apoptosis induced by caspase activation typically

proceeds on a time scale of hours to days, whereas neuronal

death in AD takes place over the course of many years. It is also

important to remember that the highest expression level of APP

is during the development of the brain, and that selective

neuronal death by apoptosis is necessary for correct develop-

ment (Kuida et al., 1996). Therefore, if cleavage of APP by

caspases really causes nerve cell death, it will be important to

first understand the role of this signaling event in normal

development before discussing its possible role in slow

neurodegeneration.

Over the past few years, it has become clear that apoptotic

signaling is not the only function of caspases and that their

activation can lead to other cellular events as well (Arama et al.,

2003; Kennedy et al., 1999). In the specific case of neurons,

caspase signaling may have functions in synaptic plasticity and

axonal growth; for example, caspase 1 can negatively regulate

long-term potentiation (Lu et al., 2006), and caspase 3 must be

activated in order for the growth cone to change direction

(Campbell and Holt, 2003).

The recent evidence for functions of the caspases in synaptic

plasticity, one of the areas of physiological action suggested for

APP, suggests that it would be interesting to re-examine the

published results on caspase cleavage of APP. It is possible that,

rather than killing neurons, cleavage of APP by caspases might

be another of the signaling pathways used to regulate neuronal

plasticity. Still, very recent evidence indicates that cleavage of

APP by caspases might be necessary for the progression of AD

(Galvan et al., 2006).

3.4. Sporadic Alzheimer’s disease

In spite of considerable advances in understanding the

pathogenesis of AD, a still unanswered question is why soluble

Ab levels, and specifically Ab oligomer levels (Lacor et al.,

2004), rise in the brains of the great majority of AD patients that

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–32 17

have no mutations in the APP or secretase genes. While we will

not attempt to solve this question here, a few observations on

the etiology of sporadic AD are summarized in the following.

One suggestion that is completely independent of the

metabolism of APP itself is a deficiency in the proteases that

physiologically degrade Ab (such as neprilysin and insulin-

degrading enzyme), causing accumulation of the peptide

(Selkoe, 2001). In another series of observations, a link between

cholesterol and late-onset AD has been observed: lowering

plasma cholesterol levels can contribute to decreasing the risk

of dementia (Jick et al., 2000), and certain alleles of the lipid

transport protein ApoE (alleles e2 and e4) are associated with

an increase or decrease, respectively, of the age of onset of

dementia (Corder et al., 1994; Strittmatter et al., 1993a). These

findings, again, have been sometimes explained in terms of a

direct interaction of cholesterol and/or ApoE4 with Ab, without

taking into account possible changes in APP function or

neuronal physiology (Strittmatter et al., 1993b; Kakio et al.,

2001). Cholesterol may also affect the trafficking or the affinity

of APP for the secretases and thereby influence the release of

Ab (Kojro et al., 2001). On the other hand, the importance of

cholesterol and of different ApoE isoforms for neuronal

plasticity (Arendt et al., 1997; Mauch et al., 2001) indicates that

disturbances of neuronal plasticity lead to an adaptive change in

APP metabolism, which ultimately has deleterious conse-

quences, culminating, whether because of increased Ab

production or otherwise, in dementia. Cerebral traumatism is

also a risk factor for dementia (Jellinger, 2004), which suggests

that the regeneration of neurons after an insult may cause

neurodegeneration. In this regard, a relevant observation is that

trauma leads directly to a (possibly adaptive) increase in the

expression of APP and to the deposition of Ab (Blasko et al.,

2004; Roberts et al., 1991). Finally, in different mouse models,

a deficiency in nerve growth factor (NGF) has been reported to

cause neuronal degeneration and, as a secondary consequence

of this degeneration, the deposition of Ab (for a review, see

Isacson et al., 2002).

In conclusion, there can be no doubt that mutations in the

APP gene are sufficient to cause the entire spectrum of clinical

symptoms and neuropathology of Alzheimer’s disease. On the

other hand, it is unclear why so many different risk factors (age,

plasma cholesterol level, ApoE4 status, trauma, deficiency in

growth factors and other environmental factors) should lead to

neurotoxicity in certain brain regions. One or more of the

functions of APP that are important for synaptic plasticity may

be activated in response to insults, leading to pathological

deregulation of APP metabolism. Possibly, the ensuing

accumulation of soluble oligomers of Ab in the synaptic cleft

then causes synaptic dysfunction, neuronal damage and

dementia in all of those cases.

4. Determination of protein structure

As mentioned above, a deeper understanding of the actions

of APP in the healthy and demented brain will require both

identifying the dynamic neuronal circuits which depend on

APP and understanding the molecular/structural basis of the

interactions of APP with other molecules. With respect to

normal neuronal physiology, this may be obvious. As noted in

the previous section, however, in AD, too, and especially in

sporadic forms of the disease, the possibility that adaptive

interactions between APP and its physiological binding

partners may lead to dementia by Ab-dependent or Ab-

independent mechanisms highlights the need for understanding

the structure of APP outside the amyloidogenic domain. In the

following, we briefly outline a few basic principles that guide

protein structural investigation and review recent advances in

the structural biology of both full-length and secreted forms of

human APP.

In general, structure/function relationships for any given

protein cannot be completely understood by simply elucidating

the structures of isolated fragments. The temptation to regard a

protein as an object equivalent to the sum of small parts

possibly arises from our intuitive nature of comparing

biological molecules to macroscopic machines made of metal

and/or plastic pieces. In such macroscopic machines, each

constituent part is usually hard (i.e., it does not deform

significantly during work) and often moves independently from

neighbouring parts. In contrast, however, the final folded

structure of a polypeptide chain depends on a large number of

attractive and repulsive molecular interactions that very nearly

cancel out each other. As a result, the stability of proteins is

often quite small compared to thermal energy at physiological

temperatures. Consequently, proteins are not static objects, but

rather exhibit considerable conformational dynamics (or

plasticity) ranging from very fast (subnanosecond) residue

motions (for early examples, see Ferreira, 1989; Ferreira et al.,

1994) to larger domain movements in the millisecond to second

time scales (reviewed in Weber, 1992). In fact, even in the case

of proteins that yield well-diffracting crystals suitable for high-

resolution structural determination by X-ray crystallography,

the apparently rigid crystallographic structures hide substantial

and significant fluctuations and should be actually viewed as

time-averaged structures (DePristo et al., 2004).

Methods to determine protein structures in solution by

nuclear magnetic resonance (NMR) spectroscopy or small-

angle X-ray scattering (SAXS) offer the possibility to model

protein dynamics explicitly (Arai et al., 2004; Lindorff-Larsen

et al., 2005). In addition to the low-amplitude movements

present in all proteins, experiments on dynamics have revealed

the existence of protein domains, or even entire polypeptide

chains, that exhibit the large mobility typical of unfolded states

even in their biologically active states (Wright and Dyson,

1999). Such proteins (or protein domains) have been described

as natively unstructured, or natively unfolded, and they usually

have the capacity to acquire well-defined structures upon

interactions with other proteins or other cellular components

(e.g., lipid bilayers) (Wright and Dyson, 1999; McMahon and

Gallop, 2005).

In the case of large proteins that contain several distinct

structural domains, the acquisition of the biologically active

structure by one domain may depend on the presence of another

domain, even if the two domains are apart in the amino acid

sequence (Fedorov and Baldwin, 1999; Jansens et al., 2002), or

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–3218

it may depend on the participation of other protein(s)

(Szepanski et al., 1994). In this way, different domains and/

or polypeptide chains cooperate to create proteins with more

complex structures and functions. Consequently, determining

the structure of an isolated fragment or domain of a protein does

not guarantee that that fragment maintains the same structure

when inserted into the full-length protein, and certainly does

not offer reliable information on the topological relations

between different domains in the full-length protein.

The energetics that rule protein folding are also very

important for understanding the interactions between proteins

and other molecules (ligands, substrates). In many cases,

intramolecular interactions are so weak and/or distributed over

so many residues that the entire protein moves and changes

conformation when it contacts a partner. For example,

mutational studies have shown that protease specificity towards

substrates is not necessarily localized to the handful of residues

present at the active site, but rather can be broadly distributed

throughout the protease (Hedstrom et al., 1992). Similarly,

studies on protein dimers have shown that the energy of

interaction between the monomers is not the sum of the

interactions of each residue at the dimer interface. Groups of

amino acids interact strongly and non-additively to build up the

interface (Reichmann et al., 2005).

5. APP structure

5.1. Overview

APP is a protein of up to 770 amino acids (in its longest

isoform). To date, it has not been crystallized satisfactorily, nor

have any of its two homologues in vertebrates, APLP1 and

APLP2 (Sprecher et al., 1993; Wasco et al., 1992), or any other

family member in invertebrates, which means that there is no

atomic resolution structure for the full-length, transmembrane

form of APP. This lack of a structure is actually not too

surprising, as the number of atomic resolution structures

available for transmembrane proteins is still much lower than

for soluble proteins. NMR studies have shown that the C-

terminal cytoplasmic tail of APP has no rigid structure

(Kroenke et al., 1997), while an a-helical structure is assumed

for the transmembrane segment (Kang et al., 1987) (see Fig. 1

for the overall structure of APP).

A variety of signaling adaptor proteins bind to the

cytoplasmic domain of APP, including G0 (Brouillet et al.,

1999), APP-BP1 (Chow et al., 1996), Fe65 (Borg et al., 1996),

X11 (Borg et al., 1996) and JIP-1 (Matsuda et al., 2001). The

structural basis of the interaction of the cytoplasmic domain

with some of these proteins has been inferred from solution

NMR studies on the isolated C-terminal tail (Ramelot et al.,

2000; Pastorino et al., 2006), while the interaction of the

transmembrane domain of APP with the g-secretase complex

has been inferred from biochemical experiments (Berezovska

et al., 2003) but not yet structurally elucidated. Investigating the

complexes of these proteins with APP fragments is an

important experimental challenge. However, definite knowl-

edge about the activation or the processing of APP will likely

depend on the availability of a structure of the entire

transmembrane APP, as the cytoplasmic and transmembrane

domains are both very small in relation to the full-length protein

and unable to fold independently into stable tertiary structures.

On the other hand, elucidation of the structure of the large

extracellular domain of APP (sAPP) would be a great step

forward in understanding the mechanisms mediated by the

intact transmembrane protein. Moreover, sAPP is produced in

vivo (Van Nostrand et al., 1990), is capable of independent

folding (Araki et al., 1991) and has potent biological activities

(as described in Section 2.3). For these reasons, obtaining the

structure of the extracellular portion of APP would also be of

significant value in itself.

The APP gene has 19 exons (Yoshikai et al., 1991). Several

isoforms of APP are generated by alternative splicing of exons

7, 8 and 15, all of them coding for domains localized in the

extracellular portion of the molecule; APLP2 is also

alternatively spliced in a very similar way (Sandbrink et al.,

1994; Yoshikai et al., 1990). In non-neuronal cells, the longer

isoforms containing exon 7 or exons 7 and 8 predominate, while

neurons express principally the isoform lacking exons 7 and 8

(Kang and Muller-Hill, 1990). APP isoforms lacking exon 15

serve as core proteins for a proteoglycan called appican

(Pangalos et al., 1995). Expression of these latter isoforms is

important in many cell types, but neurons express very little

APP- or APLP2-lacking exon 15 (Sandbrink et al., 1994).

After loss of the signal sequence and cleavage by a-

secretase, the extracellular portion of the neuronal isoform of

APP (sAPPa695) has 594 amino acid residues and an expected

molecular mass of 68 kDa, without accounting for eventual

post-translational modifications. Crystallization of the full-

length extracellular domain of APP has been attempted by

several groups, including our own, and to our knowledge has

not yet yielded satisfactory crystals. Moreover, the large

molecular mass of sAPPa still is a huge challenge for

successful three-dimensional structural determination by

NMR. In the following, we review structural information on

smaller fragments of APP in the context of available

biochemical knowledge on the full-length protein, before

proceeding to recent structural data on the full-length protein.

5.2. Protease inhibitory domain

The first APP fragment to be crystallized was the protease

inhibitory domain, present only in the non-neuronal isoforms

(sAPPa751 and sAPPa770; Hynes et al., 1990). As this domain is

coded for by a single exon and is inserted in the middle of the

APP sequence (see Fig. 1), and considering that it is quite

homologous to other Kunitz-type protease inhibitors (KPI;

Hynes et al., 1990), it seems likely that it constitutes an

independent folding unit and that the structure obtained for the

isolated domain represents well the structure of the domain in

the context of full-length APP. The protease inhibitory

properties of this isolated domain in vitro are very similar to

the inhibitory properties of full-length sAPPa, except for the

potentiation of inhibition by heparin in the case of the intact

protein (Wagner et al., 1992). APP and sAPP isoforms

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–32 19

containing the KPI domain are ligands of the LDL-receptor-

related protein (LRP) and can be internalized through

interaction with this receptor (Kounnas et al., 1995). Therefore,

the biological effects of those isoforms of APP and sAPP that

contain the KPI domain might be regulated by LRP. In the

longest isoform, APP770, the KPI domain is immediately

followed by a short stretch of 19 amino acids without significant

homology to other known proteins and without known structure

(Richards et al., 1995).

5.3. N-Terminal domain

One of the regions most conserved among APP and its

homologues in humans and other species is the N-terminal region

of 172 residues localized immediately after the signal sequence

(Daigle and Li, 1993). This region of APP contains 12 cysteine

residues, which suggests the presence of disulfide bonds. In 1999,

a 96-residue N-terminal subfragment was crystallized (HBD1 in

Fig. 1) and in fact showed three disulfide bonds that constrain a

very rigid tertiary structure rich in b-sheets (Rossjohn et al.,

1999). This isolated domain is capable of binding to heparin

(Mok et al., 1997), to the extracellular matrix protein fibulin

(Ohsawa et al., 2001) and also to Ab (Van Nostrand et al., 2002).

On the other hand, this subfragment has a rather hydrophobic

exposed surface that is prone to aggregation or interaction with

other protein surfaces, suggesting that it does not function as an

independent folding unit in full-length APP.

The sequence immediately following the N-terminal

subfragment contains a domain that binds Cu2+ in vitro (Hesse

et al., 1994). This domain is easily degraded in vitro (Rossjohn

et al., 1999), possibly as a consequence of free radicals

generated by Fenton reaction mediated by bound copper ions

(Multhaup et al., 1998). However, its structure was determined

by NMR and crystallization (Barnham et al., 2003; Kong et al.,

2007), localizing another three disulfide bonds and the copper-

binding site. A peptide sequence that binds Zn2+ in vitro is also

present in this amino acid sequence of APP, and Zn2+ binding

increases the affinity of APP for heparin (Bush et al., 1993).

However, recent evidence indicates that this sequence does not

contain all the amino acid residues necessary to fully chelate

Zn2+ (Ciuculescu et al., 2005). A possible solution to this

discrepancy would be the contribution of an additional amino

acid residue from another APP domain to Zn2+ chelation. These

observations suggest that the N-terminal fragments of APP, i.e.

the heparin, Cu2+- and Zn2+-binding regions, do not seem to be

independent structural or functional units in the APP molecule.

The stretch of APP contained between the Zn2+-binding

sequence and the KPI-domain insertion point [(DE)n in Fig. 1]

is also easily degraded in vitro (Rossjohn et al., 1999). It is very

rich in negatively charged residues and consists almost entirely

of a low complexity region (Wootton and Federhen, 1996).1

Because of the predominance of only a few types of amino

acids, a naıve use of alignment and structural prediction

1 Determined using the FASTA server on the site http://zeldia.cap.ed.ac.uk/

ncbi_blast.html.

algorithms would indicate high degrees of helicity and

sequence conservation among APP family members in this

stretch. However, low complexity domains do not usually

display permanent secondary structures and, therefore, only the

predominance of acidic amino acids may be conserved, not the

sequence itself (Wootton and Federhen, 1996). It has not yet

been possible to determine the structure of this part of APP in

isolated form or together with other domains, and it has also not

been determined if it interacts with other domains of the

protein, for example via electrostatic interactions. A possible

functional role of this region has recently been proposed in

regulating mitochondrial membrane translocation of APP

(Anandatheerthavarada et al., 2003).

5.4. Central domains

The second half of the extracellular domain of APP,

following the exons that are inserted in the non-neuronal

isoforms, consists of a 179-residue segment that is well

conserved in evolution (HBD2 in Fig. 1) followed by a 166-

residue segment that is specific for vertebrate APP (Daigle and

Li, 1993). The latter segment is rapidly degraded by proteases,

suggesting that it may exhibit a less compact conformation,

while the well-conserved region was predicted to have a high a-

helical content (Gralle et al., 2002; Sandbrink et al., 1994).

Indeed, the solution NMR structure of part of this region

(Dulubova et al., 2004) agrees perfectly with the crystal-

lographic structure of the entire conserved region (Wang and

Ha, 2004), which features a very long a-helix with two shorter

helix bundles at each end.

The helical domain contains sequences that bind heparin in

vitro (Mok et al., 1997), and the capacity of APP to bind

collagen and laminin also depends on sequences contained in

this domain (Beher et al., 1996; Narindrasorasak et al., 1992).

Binding of the morphoregulatory protein F-spondin to the

isolated helical domain is of special interest (Ho and Sudhof,

2004). With recent access to the crystallographic structure of

this domain, it may be expected that binding of these ligands

will soon be explored structurally. However, so far, there can be

no certainty as to whether the isolated highly elongated helical

domain behaves in the same way as within full-length APP or

sAPP. It is important to note that both heparin, as a substitute for

the side chains of proteoglycans such as glypican that might be

expected to constitute the real physiological ligands of APP

(Williamson et al., 1996), and laminin are large, extended

molecules and may, therefore, simultaneously interact with

more than one region of APP. In this regard, it is noteworthy

that binding of F-spondin inhibits cleavage of transmembrane

APP by b-secretase, and possibly by a-secretase as well (Ho

and Sudhof, 2004), which implies an interaction of the helical

domain with other regions of APP. Furthermore, the central

domain of APP contains an N-glycosylation site (Pahlsson and

Spitalnik, 1996), the effect of which could not be investigated in

the recombinant isolated domain.

All structural prediction algorithms employed so far indicate

that the region of extracellular APP nearest to the membrane

(indicated as RC in Fig. 1) does not possess standard secondary

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–3220

structure (Gralle et al., 2002; Sandbrink et al., 1994). As

mentioned above, this segment of the protein is specific for

vertebrates and is easily degraded by proteases in vitro. No

physiological ligands of this region of APP have been

characterized. However, as the b- and a-secretase cleavage

sites are localized within this sequence, there must necessarily

be contact between it and the secretases. Indeed, the structural

coupling of APP and b-secretase has been investigated through

mutations in this region that influence cleavage of APP by b-

secretase (Citron et al., 1995; Qahwash et al., 2004). It is

possible that this natively unstructured (Wright and Dyson,

1999) region of APP acquires secondary and/or tertiary

structure in the presence of the secretases or of other

macromolecules.

5.5. Interactions between APP domains

In Section 2, several functions attributed to APP in nervous

tissue were discussed. Their underlying molecular bases are the

interactions that APP establishes with the various types of

ligands described in the preceding sections. An important

question that arises, then, is what are the mechanisms that

connect the biochemical binding interactions with the functions

of APP at the cell and tissue levels.

Homophilic interactions between APP and its homologues

have recently been shown to promote cell–cell adhesion (Soba

et al., 2005). This clearly indicates that APP functions as a cell

adhesion molecule. On the other hand, as described above, the

extracellular portion of APP binds to many components of the

extracellular matrix. APP binding to, e.g., heparan sulfate

moieties might be involved in its cell-adhesive function (see

below), and also in interactions with the extracellular matrix,

which would make APP a member of the family of substrate

adhesion molecules that are equally important for brain

development and plasticity (Edelman and Cunningham,

1990). Still, while for some adhesion proteins (e.g., NCAM)

signaling pathways beginning with interation with an extra-

cellular ligand and ending in specific intracellular effects have

been unraveled (Dityatev et al., 2004), in the case of APP there

is as yet no clear connection between its binding to specific

extracellular ligands and subsequent intracellular events.

At first glance, the large number of possible biological

ligands of APP may appear somewhat puzzling, as each ligand

is expected to induce a structural transition of APP that leads to

a given biological effect. However, even without knowledge of

the complete three-dimensional structure of transmembrane

APP or full-length sAPP, it was possible to show that some of

those interactions are not independent: For example, binding of

Zn2+ increases the affinity of APP for heparin in vitro

(Multhaup et al., 1994) and potentiates the inhibitory activity

of APP in the coagulation cascade, which also depends on

heparan sulfate (Van Nostrand, 1995). The reduction of Cu2+ by

APP, when combined with the binding of APP to proteoglycan

heparan sulfate chains, causes controled and regulated

degradation of these proteoglycans (Cappai et al., 2005).

These observations indicate spatial proximity between the

copper reduction site, where the free radicals are produced, and

the heparan sulfate binding site(s), where the free radicals act

on the proteoglycan chains. As concerns the cell biology of

APP, on the other hand, it becomes clear that the multiple in

vitro activities of APP, which had been originally regarded as

independent from one another, may, in live cells, take part in

common biological events.

These examples show that the many binding partners of APP

may not act in isolation from one another. Instead, each piece of

information obtained on the structure of full-length APP or

sAPP will further our understanding of the relations between

the many binding partners of APP and of how binding of one

ligand in a given domain may impact interactions that take

place in other APP domains. The glycosylation of APP provides

one further example of such interdomain interactions. During

its passage through the Golgi complex, APP is N-glycosylated,

O-glycosylated, tyrosine sulfated and phosphorylated on

several residues on both sides of the membrane (for a review,

see Turner et al., 2003). While in an ovary-derived cell line no

role of APP glycosylation could be demonstrated (Pahlsson and

Spitalnik, 1996), N- and O-glycosylations of the extracellular

portion of APP are a precondition for phosphorylation of a

threonine residue in its cytoplasmic domain during neuronal

differentiation (Ando et al., 1999). This is a good example of

how human neurons use one type of post-translational

modification to regulate another type of post-translational

modification. This regulatory mechanism also indicates the

existence of a still unknown mechanism to transfer information

from the extracellular to the cytoplasmic domains of APP.

Phosphorylation of Thr668 (in APP695 numbering), in turn,

seems to be a signal for APP to be transported to the axon and to

participate in neuronal differentiation (Ando et al., 1999). At

the same time, Thr668 phosphorylation inhibits the stabiliza-

tion of the cytoplasmic domain of APP by Fe65 and, therefore,

abruptly decreases the nuclear concentration of the cytoplasmic

domain in embryonic neurons, possibly terminating a transient

signaling mediated by this domain (Kimberly et al., 2005).

Homo-oligomerization of APP has been reported (Chen

et al., 2006a; Hashimoto et al., 2003; Lu et al., 2003;

Scheuermann et al., 2001; Soba et al., 2005). Dimerization (or

monomerization) of transmembrane APP might be part of a

mechanism to transfer information about ligand binding or

environmental changes from one side of the membrane to the

other. Regarding the pathogenesis of Alzheimer’s disease, it is

especially interesting that Ab binds to the most N-terminal

domain of APP (Van Nostrand et al., 2002) and that Ab binding

may cause dimerization of transmembrane APP (Lu et al.,

2003) and neurotoxicity (Lorenzo et al., 2000). From a

structural point of view, it would be important to clarify how a

binding event involving the N-terminal domain translates into

an approximation of the transmembrane domains of APP

(Scheuermann et al., 2001).

Based on the arguments presented above, it is clear that

while a structure for full-length transmembrane APP does not

become available, a major current goal is obtaining high-

resolution structural information on the full-length extracellular

domain of the protein. This would provide insight into the

relations between different domains within sAPP as well as into

Fig. 2. Structural models of sAPPa695 (A) and sAPPa770 (B). Grey envelope:

Most probable SAXS model. Space-filling structures: The structures of HCZ

(yellow), of the C-terminal fragment (blue) and of the KPI domain (magenta, in

panel B) were positioned into the SAXS models. The putative heparin-binding

site in HCZ is indicated in orange, and the heparin-binding site in the C-

terminal fragment is shown in red. The glycosylation site is indicated in green.

Amino acid residues that participate in the proposed dimer interface are shown

in cyan. The two amino acid residues that form the protease inhibitory site are

shown in dark green within the magenta KPI domain. Model rotations are

indicated in the figures. Figure reprinted with permission from Gralle et al.

(2006).

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–32 21

the signal transduction mechanisms mediated by APP, with

special emphasis on its possible dimerization. This type of

information can be obtained by a combination of spectroscopic,

hydrodynamic and structural methods, including electronic

absorption, fluorescence and circular dichroism spectroscopies,

size-exclusion chromatography and analytical ultracentrifuga-

tion, and solution SAXS measurements. Just a few years ago,

the only published biophysical information on transmembrane

APP or full-length sAPP were secondary structure estimates

obtained from circular dichroism and infrared absorption

measurements (de La Fourniere-Besseuille et al., 1997). Much

work had already been published on the cellular effects of

sAPPa (Turner et al., 2003), but it was impossible to establish

reliable correlations between biological activities and bio-

chemical and structural characteristics of sAPPa. Fortunately,

considerable advances in our knowledge of the structure of full-

length sAPP have taken place in recent years, so that structure–

function correlations for this protein are now beginning to

emerge.

6. Structure of full-length sAPPa

6.1. Direct measurements on full-length sAPPa

We have recently investigated the structures of the full-

length, soluble extracellular domains of the two main isoforms

of APP, sAPPa695 and sAPPa770. sAPPa695 is the predominant

isoform in neurons (see Section 5.1), while sAPPa770 is

expressed in most non-neuronal cell types and contains an

insert of 75 amino acid residues, corresponding largely to a KPI

domain, in the middle of its sequence (Fig. 1). High-resolution

synchrotron radiation SAXS data were collected for sAPPa695

and sAPPa770 and permitted a model-free calculation of several

structural parameters, including maximum length, radius of

gyration and molecular weight (Botelho et al., 2003). These

results indicated that both isoforms were strictly monomeric in

solution and had elongated molecular shapes. The very high

quality of the scattering data also permitted the use of a

procedure capable of retrieving relatively detailed structural

information on the proteins. To that end, we represented the

proteins as chains of dummy residues and carried out simulated

annealing optimization to search for the configuration of the

backbone that minimizes the discrepancy between the ab initio

model and the experimental scattering data (Svergun et al.,

2001). Due to the need to incorporate a large number of dummy

residues in the model calculation, as well as due to the presence

of flexible domains in the structure of APP (Gralle et al., 2002),

the results of independent simulation runs led to the recovery of

slightly different independent structural models even using our

high-resolution data. Consequently, in order to retrieve the most

probable model for each isoform, an averaging procedure was

carried out. The final three-dimensional configuration is

represented as a space-filling model with a close packing of

spheres (Fig. 2, solid spheres), along with the superposition of

all independent solutions (semitransparent spheres) for each

isoform (Gralle et al., 2006). Importantly, the volumes of the

average structures recovered for the two isoforms of sAPPa

have the ratio expected from their molecular weights, i.e. 0.9,

indicating the internal consistency of the structural modelling

procedure.

Sedimentation velocity ultracentrifugation is another tech-

nique that gives information on the size and shape of particles in

solution. Sedimentation coefficients calculated from experi-

ments on sAPPa695 and sAPPa770 were 2.9 S for both isoforms,

considerably lower than the maximum sedimentation coeffi-

cients calculated for spherical proteins of equivalent molecular

weights and corroborating the notion that both sAPP isoforms

exhibit highly elongated and irregular shapes (Gralle et al.,

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–3222

2006). In fact, the sedimentation coefficients measured by

ultracentrifugation were even somewhat lower than the values

calculated from the SAXS models (3.77 S and 3.93 S for

sAPPa695 and sAPPa770, respectively). Consistent with our

previous observations (Botelho et al., 2003; Gralle et al., 2002),

this indicates that both sAPPa isoforms exhibit considerable

conformational flexibility. Thus, the filtered structural models

shown in Fig. 2 (dark spheres) should be considered time and

ensemble averages over the actual distributions of possible

conformations of sAPPa695 and sAPPa770 in solution.

Interestingly, the longer isoform, sAPPa770, contains an

additional structural domain that emerges laterally from one of

the ends of the molecule (Fig. 2B). That domain likely

corresponds to the KPI domain, which is present only in the

longer isoforms of APP (Kitaguchi et al., 1988), in addition to

other linker regions of the protein. The model thus indicates

that the KPI domain is freely exposed on the surface of APP,

facilitating its interaction with target proteases. The surface-

exposed location of the KPI domain is also in agreement with

the fact that its replacement by a yellow fluorescent protein

insert gives rise to a correctly localized APP chimera (Ehehalt

et al., 2003).

6.2. Topology and positioning of APP fragments

As described in Section 5, all the evolutionarily conserved

domains of APP have had their structures solved in isolation,

but it has not yet been possible to obtain suitable crystals or

perform NMR experiments that would solve the structure of

full-length sAPPa. In an attempt to overcome these difficulties,

in addition to the ab initio models of full-length sAPPa, we

performed SAXS measurements on the N-terminal fragment

that binds heparin, copper and zinc (see above; henceforth

denoted as HCZ) and showed that this fragment can best be

modelled as two rigid domains connected by a flexible linker

(Gralle et al., 2006). The available published data on high-

resolution structures of isolated sAPP fragments were then used

to establish topological correlations in the context of ab initio

SAXS models, based on a combination of (a) biochemical data

on the interaction of APP with known ligands, (b) a comparison

between the dimensional and conformational data available for

the crystallographic structures of isolated domains, (c) SAXS

models of the isolated HCZ domain and of the full-length

sAPPa isoforms, and (d) rigid body domain rotations and

translations guided by SAXS data.

The structures of HCZ and of the C-terminal domain (Wang

and Ha, 2004) were optimally positioned into the sAPPa695

model (Fig. 2A). Next, a similar procedure of rigid body

optimization was applied to position the KPI domain into the

salient protuberance of the sAPPa770 model, fixing the

positions of the other two domains as in the case sAPPa695

(Fig. 2B). The fits were found to be quite good in the angular

range conveying information on shape and quaternary

structure, though the existence of large structural gaps in the

models (i.e., regions of APP without defined structural

information) led to some deviation, notably at higher angles

(Gralle et al., 2006).

Positioning the high-resolution structures of individual

domains into the overall structure of sAPPa allowed the

localization of several functionally important sites of the protein.

For example, the sole N-glycosylation site contained within the

crystallized fragments is shown in green in Fig. 2. As expected, it

is fully exposed to the solvent in both isoforms, and its position

provides insight into the location of the glycan side chains in fully

glycosylated human APP. Two proposed heparan sulfate-binding

sites of APP (Rossjohn et al., 1999; Wang and Ha, 2004) are

shown in orange and red. The N-terminal-binding site (orange) is

localized in a groove between the N-terminal and C-terminal

fragments and may participate, along with nearby basic amino

acid residues on the C-terminal fragment, in heparin binding. On

the other hand, the C-terminal heparin-binding site (in red) is

fully exposed to the solvent in both isoforms. Interestingly, it has

been proposed that the isolated C-terminal domain forms dimers.

The amino acid residues participating in the suggested

hydrophobic dimerization interface (Wang and Ha, 2004) are

shown in cyan in Fig. 2. However, all the hydrodynamic data we

collected using analytical ultracentifugation, size-exclusion

chromatography and SAXS suggest that both sAPPa isoforms

are strictly monomeric in solution, in line with the fact that the

models indicate that part of the putative dimerization interface is

occluded in full-length sAPPa. This may be best appreciated in

the central panel of Fig. 2 for each isoform.

All the above-mentioned sites are present in the major

neuronal isoform, sAPPa695. An additional site of interest in the

structure of sAPPa770 is the protease inhibitory site in the KPI

domain (dark green in Fig. 2B), which corresponds to the

scissile bond in protease substrates and is expected to interact

most strongly with the proteases that are inhibited by sAPPa770

in vivo (Hynes et al., 1990). The structure shows that this

inhibitory site is fully exposed at one end of the sAPPa770

structure and, therefore, is poised for insertion at the active

center of a target protease.

7. Dimerization of sAPPa and APP

While it has recently been shown that a small fraction of

transmembrane APP may dimerize in intact cells (Soba et al.,

2005), the results described above indicate beyond doubt that the

extracellular domain of APP is monomeric in solution (Gralle

et al., 2002, 2006). Therefore, it is important to examine in more

depth the circumstances under which sAPP and APP might

dimerize and the possible consequences of this for the functions

of APP. Thewell-known binding of APP to heparan sulfate and of

APP-derived peptides to heparin (see Section 5.3) prompted us to

investigate the interaction of full-length sAPPa with heparin.

Combined size-exclusion chromatography, fluorescence spec-

troscopy, analytical ultracentrifugation and SAXS data sug-

gested that high-molecular-weight heparin chains induce the

formation of sAPP–heparin complexes at a sAPPa:heparin ratio

of 2:1 (Gralle et al., 2006). Structural models were also

calculated for heparin–APP dimers. From ab initio calculations

(imposing a two-fold symmetry axis), we obtained information

on the dimer shape. Several independent calculations of the

dimer model suggested that two configurations were possible,

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–32 23

with the two-fold axis parallel or perpendicular to the longer

molecular dimension. In order to investigate these possible

conformations, we used the structures of sAPPa695 monomers

previously calculated to generate the dimers and fit the models to

the experimental curve using rigid body modelling procedures to

obtain the best solutions. Parallel and anti-parallel dimer

configurations, both with approximately the same fit qualities,

were obtained. These results allowed the proposal of schematic

representations for the possible configurations of the heparin

(heparan sulfate)–APP complex at the cell membrane (Fig. 3A

and B; see below). The anti-parallel dimers connecting the

cellular membranes of neighboring cells may correspond to the

dimers discovered by cell aggregation assays, while co-

immunoprecipitation assays may have shown the presence of

both cis and trans dimers (Soba et al., 2005). Only biophysical

experiments such as those described above are capable of

indicating the presence of non-protein biomolecules, such as

heparan sulfate, in APP complexes.

Fig. 3. Alternative hypotheses for the conformation of transmembrane APP695 dimer

the sAPPa695–heparin complex, show the relations of scale between sAPPa695 m

intracellular and transmembrane domains of APP (yellow). As the SAXS data on the

sAPPa isoforms, the conformational changes that probably occur within each APP m

conformation of sAPPa and of heparin within the complex. Panel A: Parallel orient

molecular-weight heparin/heparin sulfate chain. The juxtaposition of the two trans

example by tyrosine phosphorylation of the cytoplasmic domain of APP. Note that, d

visible (yellow). Panel B: Anti-parallel orientation of two APP monomers present i

molecules, joined by a high-molecular-weight heparin/heparin sulfate chain, migh

reprinted with permission from Gralle et al. (2006).

8. Functional implications from structural studies with

sAPPa

Previous studies have tried to localize certain biological

activities of APP to smaller fragments of the protein (Jin et al.,

1994; Morimoto et al., 1998). However, as discussed in Section

4, proteins often function as coherent ‘‘machines’’, and their

workings cannot be completely understood by considering only

the activities of their constituent parts separately. The

importance of the recent structural results for full-length

sAPPa lies in that they allow the formulation of hypotheses on

the topological relations between the different domains of

sAPPa and APP and on the functions of these domains in the

context of the full-length protein.

The different types of receptors localized at the plasma

membrane can be classified as ion channel-coupled receptors,

G protein-coupled receptors, receptors with cytoplasmic

enzymatic activity (kinases, phosphatases and cyclases),

s. Both hypothetical conformations, derived from modeling of the SAXS data on

onomers (grey and red), heparin (green), the cellular membrane(s), and the

sAPPa695–heparin complex were of lower resolution than those on the isolated

onomer upon dimerization cannot be modeled. These changes may modify the

ation of two APP monomers in the same cellular membrane, joined by a high-

membrane and cytoplasmic domains might trigger intracellular signaling, for

ue to the tilt of the model, the cytoplasmic domain of only one APP monomer is

n the plasma membranes of different cells. The bridge formed by the two APP

t contribute to cell adhesion events as well as intercellular signaling. Figure

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–3224

receptors associated to cytoplasmic enzymes (tyrosine kinases)

and receptors that signal by regulated intramembrane

proteolysis (Alberts et al., 2002). Transmembrane APP does

not seem to be directly coupled to channels, and no enzymatic

activity of its cytoplasmic domain is known either. Thus, in

what other ways might APP participate in signal transduction

pathways?

Regulated intramembrane proteolysis is the most recently

discovered basic mechanism of signal transduction, and the best

studied example is the proteolysis of the Notch receptor by the

g-secretase complex (De Strooper and Annaert, 2000). Results

obtained in immortalized cell lines indicated that cleavage of

APP by g-secretase releases a cytoplasmic domain (known as

AICD) that may regulate gene transcription in the nucleus (Cao

and Sudhof, 2001; Pardossi-Piquard et al., 2005). While these

are interesting observations, another recent study suggests that

the presence of AICD may be restricted to a short period in

neuronal development (Kimberly et al., 2005). Moreover, other

recent studies in mitotic cells have denied the importance of

APP processing by g-secretase for gene regulation (Cao and

Sudhof, 2004; Hass and Yankner, 2005; Hebert et al., 2006).

Indeed, expression of all target genes reported to be affected by

AICD signaling was shown to be scarcely affected by over- or

underexpression of APP, the absence of presenilin or the

inhibition of g-secretase (Hebert et al., 2006). Whether or not

AICD has a signal transduction function in vivo, its production

has been shown not to be affected by cross-linking of APP

(Kimberly et al., 2005) and, therefore, is probably not

physiologically regulated by dimerization of APP.

A marked feature of the cytoplasmic domain of APP is the

presence of three tyrosine residues that can be partially

phosphorylated (Russo et al., 2001). Therefore, it is possible

that APP is part of the family of tyrosine kinase-associated

receptors (Alberts et al., 2002). These receptors do not have an

enzymatic activity of their own in their cytoplasmic domain.

Instead, they contain a domain which, after activation of the

receptor, becomes capable of binding an intracellular tyrosine

kinase. The interaction between APP and the tyrosine kinase Abl

was proposed based on models of neural development and was

then experimentally demonstrated: activated Abl phosphorylates

the most C-terminal tyrosine residue of APP (Zambrano et al.,

2001). It is also clear that the intracellular domain of APP can

only bind to the intracellular signaling protein Shc when

phosphorylated on this tyrosine residue (Russo et al., 2002).

Recent results suggest that APP dimerization leading to signaling

via tyrosine phosphorylation may be typical of immune cells but

not of neurons (Sondag and Combs, 2006).

The recent report that APP causes the phosphorylation of the

transcription factor Tip60 in a membrane-bound complex that

also includes Fe65 and cyclin-dependent kinase (Hass and

Yankner, 2005) suggests a similar mode of action, where APP,

though not enzymatically active itself, facilitates post-transla-

tional modifications on associated proteins. The stimuli and

mechanism for APP signaling have not yet been described for

this system.

Finally, APP may signal by activation of G0, the most

abundant G protein in neurons. APP has been shown to bind and

modulate G0 activity in vitro (Nishimoto et al., 1993; Okamoto

et al., 1995; Brouillet et al., 1999). Moreover, in insects,

physiological G0 signaling has been suggested to involve an

APP homologue (Swanson et al., 2005). While the majority of

G protein-coupled receptors are seven-transmembrane helix

proteins, APP is not the only single transmembrane domain

protein coupled to G proteins, as there are several other such

unconventional G protein-coupled receptors (Patel, 2004). The

mechanism used by APP to transmit an extracellular signal to

the protein G0 has not yet been completely elucidated, but there

are reasons to believe it might involve dimerization of APP

(Brouillet et al., 1999; Hashimoto et al., 2003), similar to other

G protein-coupled receptors regulated by dimerization (Her-

rick-Davis et al., 2005). In this case, the results mentioned in

Section 7 raise the question whether binding of heparan sulfate

(present in the extracellular matrix or on the surface of a cell) to

APP might induce APP dimerization and therefore influence

the activation of G0. The proposed consequences of APP-

mediated G0 activation vary strongly according to the cellular

model used, and include Ca2+-mediated reduction of motility in

migrating enteric neurons in vivo (Swanson et al., 2005), gene

transcription changes and later apoptotic changes in COS-NK1

cells (Ikezu et al., 1996; Yamatsuji et al., 1996a), and apoptotic

cell death in immortalized neurohybrid cells (Yamatsuji et al.,

1996b; Hashimoto et al., 2003).

9. Future perspectives

9.1. Possible questions to be addressed

From the perspective of establishing the signaling path-

way(s) in which it is involved, it would now be quite useful to

express full-length APP harboring mutations at strategic

residues and to investigate the cellular effects of such APP

mutants, similar to what is being done for the paradigmatic

neuronal adhesion molecule, NCAM (Dityatev et al., 2004).

Indeed, part of an intracellular pathway triggered by

dimerization of APP has already been teased apart in

immortalized cells, but the exact mechanism of dimerization

or the physiological ligand of APP that induces dimerization

has not been determined (Hashimoto et al., 2003). Now, based

on our recent data, heparin (as a surrogate for naturally

occurring heparin sulfate glycosaminoglycans) could be used

as model physiological extracellular ligand for such studies.

The availability of the structure of a large fragment of sAPPa

(Wang and Ha, 2004) and the known topological relations

between the fragments of sAPPa (Gralle et al., 2006) should

allow a better analysis of the amino acid residues possibly

involved in dimerization.

In the blood coagulation cascade (Zhang et al., 1997) and in

other tissues, especially in the brain, the ligand glycosami-

noglycan is probably present as part of a proteoglycan of the

heparan sulfate family. APP might suffer dimerization by the

action of heparan sulfate moieties present in the extracellular

matrix or associated to the membrane of neighboring cells. It is

quite probable that there are other ligands, beyond heparin or

heparin sulfate, which influence the dimerization of sAPPa and

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–32 25

APP, notably Cu2+, collagen and Ab (Beher et al., 1996; Hesse

et al., 1994; Lu et al., 2003; Scheuermann et al., 2001). Maybe

the inhibition of the production of Ab by the binding of F-

spondin to APP (Ho and Sudhof, 2004) is also mediated by

dimerization of the latter. If dimerization of transmembrane

APP by any given ligand can be demonstrated, the subcellular

localization of this interaction must be determined, if possible

in neurons. A strong candidate localization is the growth cone

(Sabo et al., 2003).

9.2. APP and cholinergic transmission

The best available model for the role of APP (and its

homologue APLP2) in synaptogenesis is the formation of

cholinergic synapses at the neuromuscular junction (Wang

et al., 2005) and in the submandibular ganglion (Yang et al.,

2005). Today, in addition to the general roles of cell adhesion

molecules in synaptogenesis, the specific roles of different cell

adhesion molecules in the formation of different types of

synapses are being investigated (Washbourne et al., 2004). It

has been shown that APP is important for the formation of

distinct subtypes of synapses (Salbaum and Ruddle, 1994). An

interesting hypothesis would be a specific role of APP/APLP2

in the formation and/or maintenance of cholinergic synapses in

the central nervous system as well, as dysfunctions of the

cholinergic system have been implied in Alzheimer’s disease.

In this case, APP signaling might be mediated by members of

the thrombospondin family, recently discovered synaptogenesis

inductors, which bind to some ligands known to be involved in

APP metabolism, such as LRP and heparan sulfate (Wash-

bourne et al., 2004).

Lack of NGF induces degeneration of cholinergic forebrain

neurons, which is typically observed at early stages of

Alzheimer’s disease. Indeed, one transgenic mouse model

for the disease produces antibodies against NGF exclusively in

adult neurons, which leads to depletion of NGF during aging

and to many neuropathological and behavioral symptoms of

Alzheimer’s disease (Capsoni et al., 2002). If APP is indeed

important for the establishment and maintenance of cholinergic

synapses (Wang et al., 2005; Yang et al., 2005), then

overproduction of APP might be a compensatory mechanism

to re-establish cholinergic transmission (Isacson et al., 2002).

The overproduction and deposition of Ab in neuritic plaques

observed in these mice could, thus, be secondary toxic effects

arising from the overproduction of APP; it remains to be seen if

such a mechanism is operative in AD, too.

9.3. Implications for APLP2

All of the structural work described above was designed to

investigate the structure and function of APP, one of three very

similar and functionally redundant proteins present in humans

(Heber et al., 2000). Although there are no data on the structure

of APLP2, it is quite probable that the structural model of

sAPPa obtained from SAXS measurements is also largely

applicable to the extracellular domain of APLP2, the nearest

homologue of APP (APLP1 is a smaller protein). The cellular

metabolism of APLP2 follows the same general lines as that of

APP: APLP2 is transported from the endoplasmic reticulum to

the plasma membrane or, in neurons, to the synaptic membrane

(Lyckman et al., 1998), and can be cleaved by the same

secretases that cleave APP (Walsh et al., 2003; Eggert et al.,

2004). Only double knock-out mice for APP and APLP2 have a

phenotype of synaptic dysfunctions, while each of the single

knock-outs has an essentially normal phenotype (Wang et al.,

2005; Yang et al., 2005). The only clear functional difference

between APP and APLP2 is the impossibility to generate Ab

from APLP2, and APP single knock-out mice have some subtle

neurological defects (Dawson et al., 1999; Zheng et al., 1995).

The high-homology regions conserved between the two

proteins are the same regions that have already been crystal-

lized and which we localized within our models of full-length

sAPPa (Fig. 2). Taking into account both the sequence

homology and the redundancy of function, it is probable that the

extracellular domain of APLP2 is also monomeric in isolation.

Furthermore, the possibility of formation of homo- and

heterodimers of APP, APLP1 and APLP2 (Soba et al., 2005)

suggests that APLP2 may also dimerize in the presence of

heparin or other biomolecules.

9.4. Unexpected functions of APLP2 and APP

Because of the high structural similarity and functional

redundance between APP and APLP2, it would be surprising if

APLP2 had functions fundamentally different from those

described above for APP. However, in addition to those

functions of APP described in Section 2, which agree perfectly

with those expected for a protein inserted in the plasma

membrane, and which seem to be largely common to both APP

and APLP2, other functions for the two proteins have been

described that have not yet been integrated into this general

view.

For example, it has been reported that, in mice, APLP2

interacts with the protein Kd, which has the function of

facilitating peptide presentation by the histocompatibility

complex MHC2 (Rassoulzadegan et al., 1998). This interaction

was reported to begin in the endoplasmic reticulum and the

Golgi complex, and the APLP2–Kd complex would be

transported to the cell surface. It is clear that the majority of

both APLP2 and APP is localized in the endoplasmic reticulum

and the Golgi complex (Wasco et al., 1993). The usual

explanation for this fact is the short half-life of APP (and

possibly APLP2) at the cell surface (Koo and Squazzo, 1994).

If, on the other hand, APLP2 interacts with Kd well before it

arrives at the cell surface, the possibility cannot be discarded

that APP, too, may have functionally important interactions that

take place in the initial compartments of the secretory pathway.

For example, part of the production of Ab42 from APP occurs in

the endoplasmic reticulum, which suggests an interaction

between APP and b- and g-secretase activities already in this

compartment (Wilson et al., 2002).

It has also been reported that APLP2 interacts with a specific

DNA sequence and localizes to specific spots in the nucleus

during interphase (Blangy et al., 1995). These observations

M. Gralle, S.T. Ferreira / Progress in Neurobiology 82 (2007) 11–3226

imply that APLP2 might be imported into the nucleus instead of

being inserted into the endoplasmic reticulum, as the majority

of reports suggest. Which structural elements of APLP2 would

permit its nuclear import and whether APP could also be

imported into the nucleus are questions that have still not been

addressed. The conclusions obtained from microinjection

experiments indicating that APLP2 plays an essential role in

the first cellular divisions of the embryo (Blangy et al., 1995),

because it is strictly necessary for chromatide segregation

(Sester et al., 2000), are in conflict with transgenic animal

results which show largely redundant functions for APP,

APLP1 and APLP2 (Heber et al., 2000; Herms et al., 2004;

Wang et al., 2005; Yang et al., 2005). There may be technical

reasons for this difference between the different transgenic

models, but the results on nuclear localization of APLP2 are not

disqualified by this conflict.

Surprisingly, recent reports suggest that APP can be inserted

into the inner mitochondrial membrane import channels

because of an ambiguous signaling sequence. High levels of

mitochondrial APP impair mitochondrial energy metabolism

and also appear to generate oxidative stress via enhanced

production of H2O2 (Anandatheerthavarada et al., 2003; Devi

et al., 2006). APP and APLP2 are relatively large proteins and

their intracellular trafficking in differentiated cells has only

been investigated successfully in the last few years. It is quite

possible that a cell may put APP and APLP2 to good use in

different functions, in the same way as it does so with other

proteins. As mentioned in Section 5.5, glycosylation of APP

allows the phosphorylation of a specific threonine residue in

neuron-derived cells, and this phosphorylation guides APP to

the axon (Ando et al., 1999). Other modifications can be

imagined which target APP or APLP2 to other cellular fates and

explain the great variety of functions described for the APP

family. The recent results discussed here may contribute to our

further understanding of the structural bases of those functions.

10. Conclusions

In the past 20 years, a vast amount of research has focused on

the structure and metabolism of Ab. By comparison, much less

attention has been given to investigating the structure and

metabolism of APP, and the complexity of the interactions

between APP and other biomolecules has only recently begun

to be illuminated. This review has focused on the role of APP as

an adhesion protein/contact receptor, without excluding other

possible functions. At this stage of APP research, there is a

cogent need to combine the great number of single observations

on cellular aspects of the biology of APP into more

comprehensive pictures while avoiding oversimplification or

one-sidedness. We have approached this from two different

directions: (1) considering the functions of APP in the intact

nervous tissue as the result of complex interactions between

cells and (2) analyzing the structure of the intact protein as the

result of complex interactions between domains. It is hoped that

a more coherent picture will emerge which may help

researchers in designing relevant experimental approaches

for investigating the roles of APP in the healthy and in the

demented brain.

Acknowledgements

We wish to thank Luiz Henrique Guerreiro and Drs.

Michelle Gralle Botelho, Cristiano L.P. de Oliveira, Iris

Torriani and Carlos H.I. Ramos who contributed to the

experimental work on sAPP described in this review. During

the preparation of this review, M.G. was supported by a Bert

Sakmann Nobel Laureate Stipend from the Max Planck

Society. Research in S.T.F.’s laboratory was supported by grants

from Howard Hughes Medical Institute and by the Brazilian

agencies Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico (CNPq), Programa de Nucleos de Excelencia

(Pronex) and Fundacao de Amparo a Pesquisa do Estado do Rio

de Janeiro (FAPERJ). S.T.F. is a Howard Hughes Medical

Institute International Scholar.

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