APP family member dimeric complexes are formed predominantly in synaptic compartments.

BACKGROUND: The amyloid precursor protein (APP), a key player in Alzheimer's disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions. RESULTS: We investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls. CONCLUSIONS: Together, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

Differential effects of familial Alzheimer's disease-causing mutations on amyloid precursor protein (APP) trafficking, proteolytic conversion, and synaptogenic activity.

The amyloid precursor protein (APP) is a key player in Alzheimer`s disease (AD) and the precursor of the Aß peptide, which is generated by consecutive cleavages of ß- and γ-secretases. Familial Alzheimer's disease (FAD) describes a hereditary subgroup of AD that represents a low percentage of AD cases with an early onset of the disease. Different APP FAD mutations are thought to have qualitatively different effects on its proteolytic conversion. However, few studies have explored the pathogenic and putative physiological differences in more detail. Here, we compared different FAD mutations, located at the ß- (Swedish), α- (Flemish, Arctic, Iowa) or γ-secretase (Iberian) cleavage sites. We examined heterologous expression of APP WT and FAD mutants in non-neuronal cells and their impact on presynaptic differentiation in contacting axons of co-cultured neurons. To decipher the underlying molecular mechanism, we tested the subcellular localization, the endocytosis rate and the proteolytic processing in detail by immunoprecipitation-mass spectrometry. Interestingly, we found that only the Iberian mutation showed altered synaptogenic function. Furthermore, the APP Iowa mutant shows significantly decreased α-secretase processing which is in line with our results that APP carrying the Iowa mutation was significantly increased in early endosomes. However, most interestingly, immunoprecipitation-mass spectrometry analysis revealed that the amino acid substitutions of APP FAD mutants have a decisive impact on their processing reflected in altered Aß profiles. Importantly, N-terminally truncated Aß peptides starting at position 5 were detected preferentially for APP Flemish, Arctic, and Iowa mutants containing amino acid substitutions around the α-secretase cleavage site. The strongest change in the ratio of Aß40/Aß42 was observed for the Iberian mutation while APP Swedish showed a substantial increase in Aß1-17 peptides. Together, our data indicate that familial AD mutations located at the α-, ß-, and γ-secretase cleavage sites show considerable differences in the underlying pathogenic mechanisms.

The Hidden Role of Non-Canonical Amyloid ß Isoforms in Alzheimer's Disease.

Recent advances have placed the pro-inflammatory activity of amyloid ß (Aß) on microglia cells as the focus of research on Alzheimer's Disease (AD). Researchers are confronted with an astonishing spectrum of over 100 different Aß variants with variable length and chemical modifications. With the exception of Aß1-42 and Aß1-40, the biological significance of most peptides for AD is as yet insufficiently understood. We therefore aim to provide a comprehensive overview of the contributions of these neglected Aß variants to microglia activation. First, the impact of Aß receptors, signaling cascades, scavenger mechanisms, and genetic variations on the physiological responses towards various Aß species is described. Furthermore, we discuss the importance of different types of amyloid precursor protein processing for the generation of these Aß variants in microglia, astrocytes, oligodendrocytes, and neurons, and highlight how alterations in secondary structures and oligomerization affect Aß neurotoxicity. In sum, the data indicate that gene polymorphisms in Aß-driven signaling pathways in combination with the production and activity of different Aß variants might be crucial factors for the initiation and progression of different forms of AD. A deeper assessment of their interplay with glial cells may pave the way towards novel therapeutic strategies for individualized medicine.

Brothers in arms: proBDNF/BDNF and sAPPα/Aß-signaling and their common interplay with ADAM10, TrkB, p75NTR, sortilin, and sorLA in the progression of Alzheimer's disease.

Brain-derived neurotrophic factor (BDNF) is an important modulator for a variety of functions in the central nervous system (CNS). A wealth of evidence, such as reduced mRNA and protein level in the brain, cerebrospinal fluid (CSF), and blood samples of Alzheimer's disease (AD) patients implicates a crucial role of BDNF in the progression of this disease. Especially, processing and subcellular localization of BDNF and its receptors TrkB and p75 are critical determinants for survival and death in neuronal cells. Similarly, the amyloid precursor protein (APP), a key player in Alzheimer's disease, and its cleavage fragments sAPPα and Aß are known for their respective roles in neuroprotection and neuronal death. Common features of APP- and BDNF-signaling indicate a causal relationship in their mode of action. However, the interconnections of APP- and BDNF-signaling are not well understood. Therefore, we here discuss dimerization properties, localization, processing by α- and γ-secretase, relevance of the common interaction partners TrkB, p75, sorLA, and sortilin as well as shared signaling pathways of BDNF and sAPPα.

The role of mycotoxins in neurodegenerative diseases: current state of the art and future perspectives of research.

Mycotoxins are fungal metabolites that can cause various diseases in humans and animals. The adverse health effects of mycotoxins such as liver failure, immune deficiency, and cancer are well-described. However, growing evidence suggests an additional link between these fungal metabolites and neurodegenerative diseases. Despite the wealth of these initial reports, reliable conclusions are still constrained by limited access to human patients and availability of suitable cell or animal model systems. This review summarizes knowledge on mycotoxins associated with neurodegenerative diseases and the assumed underlying pathophysiological mechanisms. The limitations of the common in vivo and in vitro experiments to identify the role of mycotoxins in neurotoxicity and thereby in neurodegenerative diseases are elucidated and possible future perspectives to further evolve this research field are presented.

The Rab5 activator RME-6 is required for amyloid precursor protein endocytosis depending on the YTSI motif.

Endocytosis of the amyloid precursor protein (APP) is critical for generation of ß-amyloid, aggregating in Alzheimer's disease. APP endocytosis depending on the intracellular NPTY motif is well investigated, whereas involvement of the YTSI (also termed BaSS) motif remains controversial. Here, we show that APP lacking the YTSI motif (ΔYTSI) displays reduced localization to early endosomes and decreased internalization rates, similar to APP ΔNPTY. Additionally, we show that the YTSI-binding protein, PAT1a interacts with the Rab5 activator RME-6, as shown by several independent assays. Interestingly, knockdown of RME-6 decreased APP endocytosis, whereas overexpression increased the same. Similarly, APP ΔNPTY endocytosis was affected by PAT1a and RME-6 overexpression, whereas APP ΔYTSI internalization remained unchanged. Moreover, we could show that RME-6 mediated increase of APP endocytosis can be diminished upon knocking down PAT1a. Together, our data identify RME-6 as a novel player in APP endocytosis, involving the YTSI-binding protein PAT1a.

Copper and zinc ions govern the trans-directed dimerization of APP family members in multiple ways.

The amyloid precursor protein (APP) and its homologs amyloid precursor-like protein 1 (APLP1) and APLP2 have central physiological functions in transcellular adhesion that depend on copper and zinc mediated trans-directed dimerization of the extracellular domains E1 and E2. Copper binds to three distinct sites in APP, one in the copper binding (CuBD) and growth factor-like (GFLD) domains each within E1, and one in the E2 domain. For APLP1 and APLP2, metal binding has so far only been shown for the E2 domain. Zinc binding has been reported for all APP family members to a unique site in the E2 domain and an additional site essential for APLP1 E2 domain trans-dimerization. Using isothermal titration calorimetry, co-immunoprecipitation, and in vitro bead aggregation assays, we show that copper promotes cis- as well as trans-directed dimerization of APLP1 and APLP2, similar as reported previously for APP. Furthermore, we report a APP-specific zinc binding site with nanomolar affinity located in the E1 domain, whereas no binding of zinc to the individual subdomains GFLD or CuBD was detected. Zinc binding did not affect the cis- but trans-dimerization of APP and APLP1. Furthermore, zinc binding inhibited copper-induced trans-directed dimerization of APP. Together, we identified a high-affinity APP-specific zinc binding site in the E1 domain and revealed contrasting cis- and trans-directed dimerization properties of APP, APLP1, and APLP2 in dependence on zinc and copper ions. Consequently, changes in metal ion homeostasis, as reported in the context of synaptic activity and neurodegenerative diseases, appear as key modulators of homo- and heterotypic trans-cellular APP/APLPs complexes.

Trafficking in Alzheimer's Disease: Modulation of APP Transport and Processing by the Transmembrane Proteins LRP1, SorLA, SorCS1c, Sortilin, and Calsyntenin.

The amyloid precursor protein (APP), one key player in Alzheimer's disease (AD), is extensively processed by different proteases. This leads to the generation of diverging fragments including the amyloid ß (Aß) peptide, which accumulates in brains of AD patients. Subcellular trafficking of APP is an important aspect for its proteolytic conversion, since the various secretases which cleave APP are located in different cellular compartments. As a consequence, altered subcellular targeting of APP is thought to directly affect the degree to which Aß is generated. The mechanisms underlying intracellular APP transport are critical to understand AD pathogenesis and can serve as a target for future pharmacological interventions. In the recent years, a number of APP interacting proteins were identified which are implicated in sorting of APP, thereby influencing APP processing at different angles of the secretory or endocytic pathway. This review provides an update on the proteolytic processing of APP and the interplay of the transmembrane proteins low-density lipoprotein receptor-related protein 1, sortilin-receptor with A-type repeats, SorCS1c, sortilin, and calsyntenin. We discuss the specific interactions with APP, the capacity to modulate the intracellular itinerary and the proteolytic conversion of APP, a possible involvement in the clearance of Aß, and the implications of these transmembrane proteins in AD and other neurodegenerative diseases.

Dimerization leads to changes in APP (amyloid precursor protein) trafficking mediated by LRP1 and SorLA.

Proteolytic cleavage of the amyloid precursor protein (APP) by α-, ß- and γ-secretases is a determining factor in Alzheimer's disease (AD). Imbalances in the activity of all three enzymes can result in alterations towards pathogenic Aß production. Proteolysis of APP is strongly linked to its subcellular localization as the secretases involved are distributed in different cellular compartments. APP has been shown to dimerize in cis-orientation, affecting Aß production. This might be explained by different substrate properties defined by the APP oligomerization state or alternatively by altered APP monomer/dimer localization. We investigated the latter hypothesis using two different APP dimerization systems in HeLa cells. Dimerization caused a decreased localization of APP to the Golgi and at the plasma membrane, whereas the levels in the ER and in endosomes were increased. Furthermore, we observed via live cell imaging and biochemical analyses that APP dimerization affects its interaction with LRP1 and SorLA, suggesting that APP dimerization modulates its interplay with sorting molecules and in turn its localization and processing. Thus, pharmacological approaches targeting APP oligomerization properties might open novel strategies for treatment of AD.

LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State.

The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aß secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third) but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin ß and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin ß, with the latter exhibiting a preference for APP in its dimeric state.

Fe65-PTB2 Dimerization Mimics Fe65-APP Interaction.

Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

APLP1 Is a Synaptic Cell Adhesion Molecule, Supporting Maintenance of Dendritic Spines and Basal Synaptic Transmission.

The amyloid precursor protein (APP), a key player in Alzheimer's disease, belongs to the family of synaptic adhesion molecules (SAMs) due to its impact on synapse formation and synaptic plasticity. These functions are mediated by both the secreted APP ectodomain that acts as a neurotrophic factor and full-length APP forming trans-cellular dimers. Two homologs of APP exist in mammals: the APP like proteins APLP1 and APLP2, exhibiting functions that partly overlap with those of APP. Here we tested whether APLP1 and APLP2 also show features of SAMs. We found that all three family members were upregulated during postnatal development coinciding with synaptogenesis. We observed presynaptic and postsynaptic localization of all APP family members and could show that heterologous expression of APLP1 or APLP2 in non-neuronal cells induces presynaptic differentiation in contacting axons of cocultured neurons, similar to APP and other SAMs. Moreover, APP/APLPs all bind to synaptic-signaling molecules, such as MINT/X11. Furthermore, we report that aged APLP1 knock-out mice show impaired basal transmission and a reduced mEPSC frequency, likely resulting from reduced spine density. This demonstrates an essential nonredundant function of APLP1 at the synapse. Compared to APP, APLP1 exhibits increased trans-cellular binding and elevated cell-surface levels due to reduced endocytosis. In conclusion, our results establish that APLPs show typical features of SAMs and indicate that increased surface expression, as observed for APLP1, is essential for proper synapse formation in vitro and synapse maintenance in vivoSIGNIFICANCE STATEMENT According to the amyloid-cascade hypothesis, Alzheimer's disease is caused by the accumulation of Aß peptides derived from sequential cleavage of the amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Here we show that all mammalian APP family members (APP, APLP1, and APLP2) exhibit synaptogenic activity, involving trans-synaptic dimerization, similar to other synaptic cell adhesion molecules, such as Neuroligin/Neurexin. Importantly, our study revealed that the loss of APLP1, which is one of the major substrates of BACE1, causes reduced spine density in aged mice. Because some therapeutic interventions target APP processing (e.g., BACE inhibitors), those strategies may alter APP/APLP physiological function. This should be taken into account for the development of pharmaceutical treatments of Alzheimer's disease.

Shedding of APP limits its synaptogenic activity and cell adhesion properties.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD) and has essential synapse promoting functions. Synaptogenic activity as well as cell adhesion properties of APP presumably depend on trans-cellular dimerization via its extracellular domain. Since neuronal APP is extensively processed by secretases, it raises the question if APP shedding affects its cell adhesion and synaptogenic properties. We show that inhibition of APP shedding using cleavage deficient forms of APP or a dominant negative α-secretase strongly enhanced its cell adhesion and synaptogenic activity suggesting that synapse promoting function of APP is tightly regulated by α-secretase mediated processing, similar to other trans-cellular synaptic adhesion molecules.

Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.

Early BDNF treatment ameliorates cell loss in the entorhinal cortex of APP transgenic mice.

Brain-derived neurotrophic factor (BDNF) improves molecular, cellular, and behavioral measures of neural dysfunction in genetic models of Alzheimer's disease (Blurton-Jones et al., 2009; Nagahara et al., 2009). However, BDNF treatment after disease onset has not been reported to improve neuronal survival in these models. We now report prevention of neuronal loss with early life BDNF treatment in mutant mice expressing two amyloid precursor protein (APP) mutations associated with early-onset familial Alzheimer's disease. APP transgenic mice underwent lentiviral BDNF gene delivery into the entorhinal cortices at age 2 months and were examined 5 months later. BDNF-treated mice exhibited significant improvements in hippocampal-dependent contextual fear conditioning compared with control-treated APP mice (p < 0.05). Stereological analysis of entorhinal cortical cell number demonstrated ∼20% reductions in neuronal number in layers II-VI of the entorhinal cortex in untreated APP mutant mice compared with wild-type mice (p < 0.0001), and significant amelioration of cell loss by BDNF (p < 0.001). Moreover, BDNF gene delivery improved synaptophysin immunoreactivity in the entorhinal cortex and, through anterograde BDNF transport, in the hippocampus (p < 0.01). Notably, BDNF did not affect amyloid plaque numbers, indicating that direct amyloid reduction is not necessary to achieve significant neuroprotective benefits in mutant amyloid models of Alzheimer's disease.

Age-dependent increases in tau phosphorylation in the brains of type 2 diabetic rats correlate with a reduced expression of p62.

Aging increases the co-incidence of Alzheimer's disease (AD) and type 2 diabetes (T2DM). However, the critical factors that contribute to the age-related increase in AD-T2DM comorbidity have yet to be clarified. In this study, aging effects and their relationship to AD-related pathology and T2DM as well as the underlying mechanisms of this process were investigated using obese rats with chronic T2DM. Tau pathology and its associated signaling pathways in the brain were compared between Otsuka Long-Evans Tokushima Fatty (OLETF) rats and corresponding non-diabetic controls at various ages. Tau phosphorylation at AD-related epitopes, including Thr212, Thr231, Ser262, and Ser396, increased with age in the soluble brain extracts of chronic OLETF rats and were accompanied by synaptic protein loss. There was also a marked age-dependent accumulation of polyubiquitinated substances in diabetic rats. Accordingly, tau proteins were highly polyubiquitinated in aged OLETF rats and a strong degree of co-localization existed between p-tau and ubiquitin in these neurons. In addition, the mRNA and protein levels of p62, a known cargo molecule that transports polyubiquitinated tau to proteasomal and autophagic degradation systems, decreased robustly with age in OLETF rats and there was an inverse correlation between protein levels of p62 and p-tau. The impaired degradation of polyubiquitinated p-tau due to age- and T2DM-dependent decreases in p62 transcription is a primary mechanism underlying increased AD-like pathology in a T2DM rat model as age increases. These results provide novel insight into the mechanisms supporting the age-related increase in AD-T2DM comorbidity.

A new Mint1 isoform, but not the conventional Mint1, interacts with the small GTPase Rab6.

Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer's Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to ß-amyloid in brains of patients suffering from Alzheimer's Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495-505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.

Amyloid precursor protein (APP) regulates synaptic structure and function.

The amyloid precursor protein (APP) plays a critical role in Alzheimer's disease (AD) pathogenesis. APP is proteolytically cleaved by ß- and γ-secretases to generate the amyloid ß-protein (Aß), the core protein component of senile plaques in AD. It is also cleaved by α-secretase to release the large soluble APP (sAPP) luminal domain that has been shown to exhibit trophic properties. Increasing evidence points to the development of synaptic deficits and dendritic spine loss prior to deposition of amyloid in transgenic mouse models that overexpress APP and Aß peptides. The consequence of loss of APP, however, is unsettled. In this study, we investigated whether APP itself plays a role in regulating synaptic structure and function using an APP knock-out (APP-/-) mouse model. We examined dendritic spines in primary cultures of hippocampal neurons and CA1 neurons of hippocampus from APP-/- mice. In the cultured neurons, there was a significant decrease (~35%) in spine density in neurons derived from APP-/- mice compared to littermate control neurons that were partially restored with sAPPα-conditioned medium. In APP-/- mice in vivo, spine numbers were also significantly reduced but by a smaller magnitude (~15%). Furthermore, apical dendritic length and dendritic arborization were markedly diminished in hippocampal neurons. These abnormalities in neuronal morphology were accompanied by reduction in long-term potentiation. Strikingly, all these changes in vivo were only seen in mice that were 12-15 months in age but not in younger animals. We propose that APP, specifically sAPP, is necessary for the maintenance of dendritic integrity in the hippocampus in an age-associated manner. Finally, these age-related changes may contribute to AD pathology independent of Aß-mediated synaptic toxicity.

Deletion of the amyloid precursor-like protein 2 (APLP2) does not affect hippocampal neuron morphology or function.

Amyloid precursor protein (APP), the parent molecule to amyloid ß peptide, is part of a larger gene family with two mammalian homologues, amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2). Initial knock-out studies demonstrated that while single APP family gene deletions produced relatively mild phenotypes, deficiency of APLP2 and one other member of the gene family resulted in perinatal lethality, suggesting vital roles masked by functional redundancy of the other homologues. Because of the importance of APP in Alzheimer's disease, the vast majority of studies to date have concentrated on the neuronal functions of APP, leaving limited data on its homologues. APLP2 is of particular interest as it contains high sequence homology with APP, is processed similarly, is expressed in overlapping spatial and temporal patterns, and is obligatory for lethality when combined with deficiency of either APLP1 or APP but does not contain the toxic amyloid ß sequence. Here we sought to test the role of APLP2 on neuronal structure and function using a combined approach involving in vitro and in vivo techniques in young and aged animals. Surprisingly, we found that unlike APP, APLP2 appears not to be essential for maintenance of dendritic structure, spine density, or synaptic function. Thus, there is clear divergence in the functional redundancy between APP and APLP2.

APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms.

The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.