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PubMed2006

Occurrence and co-localization of amyloid beta-protein and apolipoprotein E in perivascular drainage channels of wild-type and APP-transgenic mice.

The deposition of the amyloid beta-protein (Abeta) is a hallmark of Alzheimer's disease (AD). One reason for Abeta-accumulation and deposition in the brain may be an altered drainage along perivascular channels. Extracellular fluid is drained from the brain towards the cervical lymph nodes via perivascular channels. The perivascular space around cerebral arteries is the morphological correlative of these drainage channels. Here, we show that Abeta is immunohistochemically detectable within the perivascular space of 25 months old wild-type and amyloid precursor protein (APP)-transgenic mice harboring the Swedish double mutation driven by a neuron specific promoter. Only small amounts of Abeta can be detected immunohistochemically in the perivascular space of wild-type mice. Cerebrovascular and parenchymal Abeta-deposits were absent. In APP-transgenic mice, large amounts of Abeta were found in the perivascular drainage channels accompanied with cerebrovascular and parenchymal Abeta-deposition. The apolipoprotein E (apoE) immunostaining within the perivascular channels did not vary between wild-type and APP-transgenic mice. Almost 100% of the area that represents the perivascular space was stained with an antibody directed against apoE. Here, Abeta co-localized with apoE indicating an involvement of apoE in the perivascular clearance of Abeta. Fibrillar congophilic amyloid was not seen in wild-type mice. In APP-transgenic animals, congophilic fibrillar amyloid material was seen in the wall of cerebral blood vessels but not in the perivascular space. In conclusion, our results suggest that non-fibrillar forms of Abeta are drained along perivascular channels and that apoE is presumably involved in this clearance mechanism. Overloading such a clearance mechanism in APP-transgenic mice appears to result in insufficient Abeta-clearance, increased Abeta-levels in the brain and the perivascular drainage channels, and finally in Abeta-deposition. In so doing, our results strengthen the hypothesis that an alteration of perivascular drainage supports Abeta-deposition and the development of AD.

Aging↗
PubMed2006

Promoter polymorphisms which modulate APP expression may increase susceptibility to Alzheimer's disease.

Increasing evidence indicates that variants in promoter of the beta-amyloid precursor protein (APP) gene could up-regulate the APP gene expression and aggravate the amyloid beta protein (A beta) accumulation, thus contributing to the development of Alzheimer's disease (AD). In Chinese Han populations we found three polymorphisms in APP promoter: -877T/C(rs466433), -955A/G(rs364048) and -9G/C. The -877T and -955A alleles were over-represented in 209 sporadic AD (SAD) patients when compared to those in 437 healthy individuals. Furthermore, -877T/C and -955A/G were in strong linkage disequilibrium and they constructed a relatively risky -877T/-955A and a relatively protective -877C/-955G. Luciferase reporter assay indicated -877T/-955A had four times higher transcriptional activity than -877C/-955G. A more marked increase in -877T/-955A transcriptional activity was seen when under A beta(25-35) treatment. As for the -9G/C polymorphism, significant differences between the two alleles were not observed either in genetic evaluation or in functional assay. The present study provides strong evidence that APP promoter polymorphisms that significantly increase APP expression levels are associated with development of SAD.

Aged↗
PubMed2006

Intraneuronal amyloid beta and reduced brain volume in a novel APP T714I mouse model for Alzheimer's disease.

Transgenic mouse models of Alzheimer's disease (AD) expressing high levels of amyloid precursor protein (APP) with familial AD (FAD) mutations have proven to be extremely useful in understanding pathogenic processes of AD especially those that involve amyloidogenesis. We earlier described Austrian APP T714I pathology that leads to one of the earliest AD age-at-onsets with abundant intracellular and extracellular amyloid deposits in brain. The latter strikingly was non-fibrillar diffuse amyloid, composed of N-truncated A beta 42 in absence of A beta 40. In vitro, this mutation leads to one of the highest A beta 42/A beta 40 ratios among all FAD mutations. We generated an APP T714I transgenic mouse model that despite having 10 times lower transgene than endogenous murine APP deposited intraneuronal A beta in brain by 6 months of age. Accumulations increased with age, and this was paralleled by decreased brain sizes on volumetric MRI, compared to age-matched and similar transgene-expressing APP wild-type mice, although, with these levels of transgenic expression we did not detect neuronal loss or significant memory impairment. Immunohistochemical studies revealed that the majority of the intraneuronal A beta deposits colocalized with late endosomal markers, although some A beta inclusions were also positive for lysosomal and Golgi markers. These data support earlier observations of A beta accumulation in the endosomal-lysosomal pathway and the hypothesis that intraneuronal accumulation of A beta could be an important factor in the AD pathogenesis.

Alzheimer Disease↗
PubMed2006

Increased App expression in a mouse model of Down's syndrome disrupts NGF transport and causes cholinergic neuron degeneration.

Degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive dysfunction in Alzheimer's disease (AD) and Down's syndrome (DS). We used Ts65Dn and Ts1Cje mouse models of DS to show that the increased dose of the amyloid precursor protein gene, App, acts to markedly decrease NGF retrograde transport and cause degeneration of BFCNs. NGF transport was also decreased in mice expressing wild-type human APP or a familial AD-linked mutant APP; while significant, the decreases were less marked and there was no evident degeneration of BFCNs. Because of evidence suggesting that the NGF transport defect was intra-axonal, we explored within cholinergic axons the status of early endosomes (EEs). NGF-containing EEs were enlarged in Ts65Dn mice and their App content was increased. Our study thus provides evidence for a pathogenic mechanism for DS in which increased expression of App, in the context of trisomy, causes abnormal transport of NGF and cholinergic neurodegeneration.

Alzheimer Disease↗
PubMed2006

Critical role of methionine-722 in the stimulation of human brain G-proteins and neurotoxicity induced by London familial Alzheimer's disease (FAD) mutated V717G-APP(714-723).

We have demonstrated earlier that V717G-APP(714-723), the membrane fragment of the V717G ("London") familial Alzheimer's disease (FAD) mutant of amyloid precursor protein (APP), is a potent stimulator of G-proteins in human brain membranes. In this study, we tested the hypothesis that Met-722 in the V717G-APP(714-723) peptide (P2) plays a critical role in the P2-induced oxidative stimulation of G-proteins in the human temporal cortex membranes and in the neurotoxicity of the peptide in differentiated PC12 and cerebellar granular cells. We found that 10 microM P3, the Met-722 sulfoxide analog of P2, produced a twofold lower stimulation of G-proteins ([(35)S]-GTPgammaS binding) in control temporal cortex membranes compared with 10 microM P2. The stimulatory effect of 10 microM P4, the Met-722 sulfone analog of P2, was 2.5-fold lower than the effect of P2. In Alzheimer's disease (AD) temporal cortex, the P3 and P4 stimulation of G-proteins was slightly weaker than the P2 stimulation. Substitution of the Met-722 S-atom in P2 by -CH(2)- group (P5) led to the disappearance of P2 stimulatory effect on G-proteins. Glutathione (GSH), melatonin (Mel), desferrioxamine (DFO) and 17-beta-estradiol (17betaE) significantly reduced P2 stimulatory effect on G-proteins in human brain. Only DFO and Mel were able to reduce the moderate stimulation of G-proteins by P3, whereas none of the tested antioxidants influenced the weak stimulation by P4. P2 at 100 microM induced a 40% decrease in PC12 cell viability as revealed by MTT assay, the effect being significantly higher than that of P3 or P4, whereas P1 (wild-type APP(714-723)) did not affect cell viability. Trypan Blue exclusion assay demonstrated that 10 microM P2 and P3 induced 3.8- and 3.5-fold death in the cerebellar granular cells as compared with the respective control values. P1 and P4 at 10 microM induced 1.7- and 2.3-fold increase in cell death, respectively. Treatment of the cerebellar granular cells with pertussis toxin decreased the high neurotoxicity of P2 and P3, whereas the low toxicity of P1 and P4 was not influenced. These results support the hypothesis that the G-protein stimulatory effect and neurotoxicity of "London"-mutated V717G-APP(714-723) (P2) and its Met-722 oxidized analogs involve oxidative-dependent and oxidative-independent mechanisms and the oxidation state of Met-722 plays a critical role in determining the mechanism.

Aged↗
PubMed1990

Intact Alzheimer amyloid precursor protein (APP) is present in platelet membranes and is encoded by platelet mRNA.

Using antibodies directed against N-terminal and C-terminal epitopes we have immunologically detected APP species in the membrane and saline-soluble fractions of unstimulated platelets, and in the conditioned medium of thrombin-stimulated platelets. These studies demonstrate an intact 140 kD membrane-associated form of APP that is released on degranulation. Evidence that platelets synthesize at least one form of APP (APP751) was obtained by enzymatic amplification of specific mRNA using Polymerase Chain Reaction (PCR) and direct sequence analysis of PCR product. Processing of APP for release may occur via successive C-terminal truncations, and/or by the release and proteolysis of an intact membrane associated form. An intact form of APP in platelets provides a circulating substrate upon which proteases from many tissues may act to produce beta protein (AB) during pathologic conditions.

Alzheimer Disease↗
PubMed2002

Alteration of cortical EEG in mice carrying mutated human APP transgene.

Transgenic mice expressing human APPswe and PS1-A264E mutations mimic certain neuropathological features of Alzheimer's disease (AD). These mice have elevated levels of the highly fibrillogenic amyloid beta1-42 peptide (Abeta42) and develop amyloid plaques around the age of 9 months. Our aim was to find whether these transgenic mice differ electrophysiologically from non-transgenic mice and whether the alteration in EEG activity progresses with the accumulation of Abeta. The APP/PS1 mice had reduced cortical theta activity and enhanced beta and gamma activity, but these changes were not age-dependent. APP single mutant mice had similar EEG alterations in theta, beta and gamma bands as APP/PS1 double mutant mice while PS1 single mutant mice did not differ from non-transgenic controls. Insoluble Abeta40 and Abeta42 levels were robustly increased in APP/PS1 double mutant mice and insoluble Abeta40 moderately increased also in APP single mutant mice. Soluble Abeta42 was found in all APP mutant mice but also in lower concentrations in PS1 single mutant mice. Plaques were deposited in 13-month-old APP/PS1 double mutant mice but not in 8-month-old double mutant or 13-month-old single mutant mice. We conclude that the alteration of EEG activity in APP/PS1 double mutant and APP single mutant mice is related to their APP genotype rather than to deposition of beta-amyloid in the brain.

Action Potentials↗
PubMed1997

Cellular signaling roles of TGF beta, TNF alpha and beta APP in brain injury responses and Alzheimer's disease.

beta-Amyloid precursor protein (beta APP), transforming growth factor beta (TGF beta), and tumor necrosis factor-alpha (TNF alpha) are remarkably pleiotropic neural cytokines/neurotrophic factors that orchestrate intricate injury-related cellular and molecular interactions. The links between these three factors include: their responses to injury; their interactive effects on astrocytes, microglia and neurons; their ability to induce cytoprotective responses in neurons; and their association with cytopathological alterations in Alzheimer's disease. Astrocytes and microglia each produce and respond to TGF beta and TNF alpha in characteristic ways when the brain is injured. TGF beta, TNF alpha and secreted forms of beta APP (sAPP) can protect neurons against excitotoxic, metabolic and oxidative insults and may thereby serve neuroprotective roles. On the other hand, under certain conditions TNF alpha and the fibrillogenic amyloid beta-peptide (A beta) derivative of beta APP can promote damage of neuronal and glial cells, and may play roles in neurodegenerative disorders. Studies of genetically manipulated mice in which TGF beta, TNF alpha or beta APP ligand or receptor levels are altered suggest important roles for each factor in cellular responses to brain injury and indicate that mediators of neural injury responses also have the potential to enhance amyloidogenesis and/or to interfere with neuroregeneration if expressed at abnormal levels or modified by strategic point mutations. Recent studies have elucidated signal transduction pathways of TGF beta (serine/threonine kinase cascades), TNF alpha (p55 receptor linked to a sphingomyelin-ceramide-NF kappa B pathway), and secreted forms of beta APP (sAPP; receptor guanylate cyclase-cGMP-cGMP-dependent kinase-K+ channel activation). Knowledge of these signaling pathways is revealing novel molecular targets on which to focus neuroprotective therapeutic strategies in disorders ranging from stroke to Alzheimer's disease.

Alzheimer Disease↗
PubMed2002

Behavioral impairment of APP(V717F) mice in fear conditioning: is it only cognition?

Alzheimer's Disease (AD) is a devastating human neurodegenerative disorder associated with progressive deterioration of cognitive abilities. The APP(V717F) mouse, an animal model of AD showing robust overexpression of the human amyloid precursor protein (APP) carrying the mutation 717 V --> F, was also shown to exhibit learning and memory performance deficits. However, AD patients suffer from other abnormalities including altered emotionality. Emotionality has not been analyzed in AD mouse models. Here, motor and posture patterns exhibited by APP(V717F) mice are described in a detailed manner in fear conditioning, a paradigm that allows one to test both mnemonic and emotional characteristics of mice. Our results revealed a complex set of behavioral alterations in APP(V717F) mice in measures of exploratory behavior and fear suggesting that the effects of APP(V717F) overexpression in this mouse model are not limited to cognition and may need to be thoroughly examined in the future in a broad range of behavioral tests.

Alzheimer Disease↗
PubMed2001

Effects of apolipoprotein E (apoE) isoforms, beta-amyloid (Abeta) and apoE/Abeta complexes on protein kinase C-alpha (PKC-alpha) translocation and amyloid precursor protein (APP) processing in human SH-SY5Y neuroblastoma cells and fibroblasts.

We investigated the effects of different apolipoprotein E (apoE) isoforms, Abeta (1-42), and apoE/Abeta complexes on PKC-alpha translocation and APP processing in human SH-SY5Y neuroblastoma cells and fibroblasts. Treatment of cells with either 10 nM apoE3 or apoE4, 10 microM Abeta (1-42), or apoE/Abeta complexes induced significant translocation of PKC-alpha in both cell types. Effects were seen using both human recombinant apoE and apoE loaded into beta-very low density lipoprotein (beta-VLDL) particles. Time course (5-24 h) studies of APP processing revealed that some conditions induced transient or moderate increases in the secretion of proteins detected by 22C11. In contrast, the secretion of alpha-secretase cleaved APP was either not modified or transiently decreased, as determined by immunoblotting with the antibody 6E10. These results suggest that apoE, Abeta (1-42) and apoE/Abeta complexes can modulate PKC activity but do not have major consequences for APP processing. These effects could contribute to the reported PKC alterations seen in AD. However, it is unlikely that the contribution of different apoE isoforms to AD pathology occurs via effects on APP processing.

Amyloid beta-Peptides↗
PubMed2003

Normal cognitive behavior in two distinct congenic lines of transgenic mice hyperexpressing mutant APP SWE.

Amyloid deposition appears to be an early and crucial event in Alzheimer's disease (AD). To generate animal models of AD, mice expressing full-length amyloid precursor protein (APP), with mutations linked to FAD, have been created. These animals exhibit abnormalities characteristic of AD, including deposits of beta-amyloid (Abeta), neuritic plaques, and glial responses. In studies of cognition in these animals, there have been several reports of memory disturbances well before the appearance of amyloid deposits. We have developed two distinct lines of transgenic mice (C3-3 and E1-2) that express the "Swedish" variant of APP (APP(SWE)) at levels that are approximately three-fold higher than endogenous mouse APP. Both lines have been backcrossed to C57BL/6J mice for 10 generations. Here, we use longitudinal and cross-sectional studies to evaluate the cognitive performance of our animals, where the concentration of Abeta1-42 in brain increases with aging from low levels (2-10 pmol/g) at 6-14 months of age to relatively high levels (60-100 pmol/g) at 24-26 months, when deposits of Abeta were beginning to form. When 12-month-old mice were tested in tasks that assess reference and working memory, transgenic mice from both lines could not be distinguished from nontransgenic littermates. Further study of 24- to 26-month-old transgenic mice (C3-3 line) found no evidence of memory impairment despite the presence of high levels of human Abeta (60-100 pmol/g). Thus, the expression of APP(SWE) at approximately three-fold over endogenous levels, which is sufficient to induce amyloid deposition at advanced ages, does not significantly erode cognitive performance in aged mice.

Age Factors↗
PubMed2005

Altered APP processing in PDAPP (Val717 --> Phe) transgenic mice yields extended-length Abeta peptides.

Central to the pathology of Alzheimer's disease (AD) is the profuse accumulation of amyloid-beta (Abeta) peptides in the brain of affected individuals, and several amyloid precursor protein (APP) transgenic (Tg) mice models have been created to mimic Abeta deposition. Among these, the PDAPP Tg mice carrying the familial AD APP 717 Val --> Phe mutation have been widely used to test potential AD therapeutic interventions including active and passive anti-Abeta immunizations. The structure and biochemistry of the PDAPP Tg mice Abeta-related peptides were investigated using acid and detergent lysis of brain tissue, ultracentrifugation, FPLC, HPLC, enzymatic and chemical cleavage of peptides, Western blot, immunoprecipitation, and MALDI-TOF and SELDI-TOF mass spectrometry. Our experiments reveal that PDAPP mice produce a variety of C-terminally elongated Abeta peptides in addition to Abeta n-40 and Abeta n-42, as well as N-terminally truncated peptides, suggesting anomalous proteolysis of both APP and Abeta. Important alterations in the overall APP degradation also occur in this model, resulting in a striking comparative lack of CT83 and CT99 fragments, which may be inherent to the strain of mice, a generalized gamma-secretase failure, or the ultimate manifestation of the overwhelming amount of expressed human transgene; these alterations are not observed in other strains of APP Tg mice or in sporadic AD. Understanding at the molecular level the nature of these important animal models will permit a better understanding of therapeutic interventions directed to prevent, delay, or reverse the ravages of sporadic AD.

Amino Acid Sequence↗
PubMed2003

Protein phosphorylation and APP metabolism.

Numerous lines of evidence place signal transduction cascades at the core of many processes having a direct role in neurodegeneration and associated disorders. Key players include neurotransmitters, growth factors, cytokines, hormones, and even binding and targeting proteins. Indeed, abnormal phosphorylation of key control proteins has been detected in many cases and is thought to underlie the associated cellular dysfunctions. Several signaling cascades have been implicated, affecting processes as varied as protein processing, protein expression, and subcellular protein localization, among others. The Alzheimer's amyloid precursor protein (APP) is a phosphoprotein, with well-defined phosphorylation sites but whose function is not clearly understood. The factors and pathways regulating the processing of APP have been particularly elusive, both in normal ageing and the Alzheimer's disease (AD) condition. Not surprisingly, the physiological function(s) of the protein remain(s) to be elucidated, although many hypotheses have been advanced. Nonetheless, considerable data has accumulated over the last decade, placing APP in key positions to be modulated both directly and indirectly by phosphorylation and phosphorylation-dependent events. The pathological end product of APP processing is the main proteinaceous component of the hallmark senile plaques found in the brains of AD patients, that is, a toxic peptide termed Abeta. In this minireview we address the importance of phosphorylation and signal transduction cascades in relation to APP processing and Abeta production. The possible use of the identified molecular alterations as therapeutic targets is also addressed.

Alzheimer Disease↗
PubMed2005

Homo- and heterodimerization of APP family members promotes intercellular adhesion.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo- and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell-cell adhesion in a homo- and heterotypic fashion and that endogenous APLP2 is required for cell-cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo- and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo.

Amyloid beta-Protein Precursor↗
PubMed2003

Neisseria meningitidis App, a new adhesin with autocatalytic serine protease activity.

Neisseria meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract. Occasionally, it translocates to the bloodstream causing sepsis and from there it can cross the blood-brain barrier and cause meningitis. Many of the molecules, which mediate the interaction of N. meningitidis to host cells, are still unknown. Recently, App (Adhesion and penetration protein) was described as a member of the autotransporter family and a homologue to the Hap (Haemophilus adhesion and penetration) protein of Haemophilus influenzae, a molecule that plays a role in the interaction with human epithelial cells. In this study we expressed app in Escherichia coli in order to analyse the functional properties of the protein. We show that the protein is exported to the E. coli surface, processed by an endogenous serine-protease activity and released in the culture supernatant. Escherichia coli expressing app adhere to Chang epithelial cells, showing that App is able to mediate bacterial adhesion to host cells. The serine protease activity is localized at the amino-terminal domain, whereas the binding domain is in the carboxy-terminal region. The role of App in adhesion was confirmed also in N. meningitidis.

Amino Acid Sequence↗
PubMed2003

Employing a superior BACE1 cleavage sequence to probe cellular APP processing.

The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.

Alzheimer Disease↗
PubMed2006

Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664.

The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of beta-amyloid peptides, a set of 39-43 amino acid fragments derived proteolytically from beta-amyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp-664 to generate a second cytotoxic peptide, APP-C31, but whether this C-terminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp-664 caspase cleavage site. Surprisingly, whereas beta-amyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp-664. These results suggest that Asp-664 plays a critical role in the generation of Alzheimer-related pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via protein-protein interactions.

Alzheimer Disease↗
PubMed2004

Human BACE forms dimers and colocalizes with APP.

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.

Amyloid Precursor Protein Secretases↗