Degradation of amyloid β peptide by neprilysin expressed from Borna disease virus vector

Accumulation of amyloid β (Aβ40 and Aβ42) in the brain is a characteristic of Alzheimer disease (AD). Because neprilysin (NEP) is a major Aβ‐degrading enzyme, NEP delivery in the brain is a promising gene therapy for AD. Borna disease virus (BoDV) vector enables long‐term transduction of foreign genes in the central nerve system. Here, the proteolytic ability of NEP transduced by the BoDV vector was evaluated and it was found that the amounts of Aβ40 and Aβ42 decreased significantly, suggesting that NEP expressed from the BoDV vector is functional in that it degrades Aβ.

     

Mitochondrial accumulation of APP and Aβ: significance for Alzheimer disease pathogenesis

Accumulating evidence suggest that alterations in energy metabolism are among the earliest events that occur in the Alzheimer disease (AD) affected brain. Energy consumption is drastically decreased in the AD-affected regions of cerebral cortex and hippocampus pointing towards compromised mitochondrial function of neurons within specific brain regions. This is accompanied by an elevated production of reactive oxygen species contributing to increased rates of neuronal loss in the AD-affected brain regions. In this review, we will discuss the role of mitochondrial function and dysfunction in AD. We will focus on the consequences of amyloid precursor protein and amyloid-β peptide accumulation in mitochondria and their involvement in AD pathogenesis.     

Autosomal‐dominant Alzheimer's disease mutations at the same codon of amyloid precursor protein differentially alter Aβ production

Autosomal‐dominant Alzheimer's disease (ADAD) is a genetic disorder caused by mutations in Amyloid Precursor Protein (APP) or Presenilin (PSEN) genes. Studying the mechanisms underlying these mutations can provide insight into the pathways that lead to AD pathology. The majority of biochemical studies on APP mutations to‐date have focused on comparing mechanisms between mutations at different codons. It has been assumed that amino acid position is a major determinant of protein dysfunction and clinical phenotype. However, the differential effect of mutations at the same codon has not been sufficiently addressed. In the present study we compared the effects of the aggressive ADAD‐associated APP I716F mutation with I716V and I716T on APP processing in human neuroglioma and CHO‐K1 cells. All APP I716 mutations increased the ratio of Aβ42/40 and changed the product line preference of γ‐secretase towards Aβ38 production. In addition, the APP I716F mutation impaired the ε‐cleavage and the fourth cleavage of γ‐secretase and led to abnormal APP β‐CTF accumulation at the plasma membrane. Taken together, these data indicate that APP mutations at the same codon can induce diverse abnormalities in APP processing, some resembling PSEN1 mutations. These differential effects could explain the clinical differences observed among ADAD patients bearing different APP mutations at the same position.

     

Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770⁺ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770⁺ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (App^NL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770⁺:App^NL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A mutation protective against Alzheimer's disease renders amyloid β precursor protein incapable of mediating neurotoxicity

Expression of a familial Alzheimer's disease (AD)‐linked mutant of amyloid β precursor protein (APP) or the binding of transforming growth factor β2 to wild‐type (wt)‐APP causes neuronal death by activating an intracellular death signal (a APP‐mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro. These neuronal death models may therefore be regarded as Aβ‐independent neuronal death models related to AD. A recent study has shown that the A673T mutation in the APP isoform APP₇₇₀, corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP₆₉₅ (an AD‐protective mutant of APP), is linked to a reduction in the incidence rate of AD. Consistent with this, cells expressing the AD‐protective mutant of APP produce less Aβ than cells expressing wt‐APP. In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt‐APP, but not in those expressing the AD‐protective mutant of APP. This result suggests that the AD‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP‐mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis‐Dutch type also attenuated the APP‐mediated intracellular death signal.

     

Calmodulin‐like skin protein protects against spatial learning impairment in a mouse model of Alzheimer disease

Humanin and calmodulin‐like skin protein (CLSP) inhibits Alzheimer disease (AD)‐related neuronal cell death via the heterotrimeric humanin receptor in vitro . It has been suggested that CLSP is a central agonist of the heterotrimeric humanin receptor in vivo . To investigate the role of CLSP in the AD pathogenesis in vivo , we generated mouse CLSP‐1 transgenic mice, crossed them with the APPswe/PSEN1dE9 mice, a model mouse of AD, and examined the effect of CLSP over‐expression on the pathological phenotype of the AD mouse model. We found that over‐expression of the mouse CLSP‐1 gene attenuated spatial learning impairment, the loss of a presynaptic marker synaptophysin, and the inactivation of STAT3 in the APPswe/PSEN1dE9 mice. On the other hand, CLSP over‐expression did not affect levels of Aβ, soluble Aβ oligomers, or gliosis. These results suggest that the CLSP‐mediated attenuation of memory impairment and synaptic loss occurs in an Aβ‐independent manner. The results of this study may serve as a hint to the better understanding of the AD pathogenesis and the development of AD therapy.

     

Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2

Cleavage of the amyloid precursor protein (APP) is a crucial event in Alzheimer disease pathogenesis that creates the amyloid-beta peptide (Abeta) and liberates the carboxy-terminal APP intracellular domain (AICD) into the cytosol. The interaction of the APP C terminus with the adaptor protein Fe65 mediates APP trafficking and signalling, and is thought to regulate APP processing and Abeta generation. We determined the crystal structure of the AICD in complex with the C-terminal phosphotyrosine-binding (PTB) domain of Fe65. The unique interface involves the NPxY PTB-binding motif and two alpha helices. The amino-terminal helix of the AICD is capped by threonine T(668), an Alzheimer disease-relevant phosphorylation site involved in Fe65-binding regulation. The structure together with mutational studies, isothermal titration calorimetry and nuclear magnetic resonance experiments sets the stage for understanding T(668) phosphorylation-dependent complex regulation at a molecular level. A molecular switch model is proposed.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Differential regulation of the high‐affinity choline transporter by wild‐type and Swedish mutant amyloid precursor protein

The high‐affinity choline transporter (CHT) is responsible for choline uptake into cholinergic neurons, with this being the rate‐limiting step for acetylcholine production. Altering CHT protein disposition directly impacts choline uptake activity and cholinergic neurotransmission. Amyloid precursor protein (APP) interacts with CHT proteins and increases their endocytosis from the cell surface. The goal of this study was to examine regulation of CHT trafficking and activity by wild‐type APP (APP_wt) and determine if this differs with Swedish mutant APP (APP_Swe) in SH‐SY5Y human neuroblastoma cells. APP_Swe differs from APP_wt in its trafficking from the cell surface through endosomes. We report for the first time that CHT interacts significantly less with APP_Swe than with APP_wt. Surprisingly, however, CHT cell surface levels and choline uptake activity are decreased to the same extent and CHT co‐localization to early endosomes increased similarly in cells expressing either APP_wt or APP_Swe. A critical observation is that CHT co‐immunoprecipitates with βCTF from APP_Swe‐expressing cells. We propose that decreased CHT function is mediated differently by APP_wt and APP_Swe; APP_wt interaction with CHT facilitates its endocytosis from the cell surface, whereas the effect of APP_Swe on CHT is mediated indirectly potentially by binding to the βCTF fragment or by Aβ released from cells.

     

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-β polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a K_D of 15–47 μM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC₅₀ values in the range of 15–164 μM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.     

The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase

Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Calmodulin regulates the non-amyloidogenic metabolism of amyloid precursor protein in platelets

A balance between the proteolytic processing of amyloid precursor protein APP through the amyloidogenic and the non-amyloidogenic pathways controls the production and release of amyloid β-protein, whose accumulation in the brain is associated to the onset of Alzheimer Disease. APP is also expressed on circulating platelets. The regulation of APP processing in these cells is poorly understood. In this work we show that platelets store considerable amounts of APP fragments, including sAPPα, that can be released upon stimulation of platelets. Moreover, platelet stimulation also promotes the proteolysis of intact APP expressed on the cell surface. This process is supported by an ADAM metalloproteinase, and causes the release of sAPPα. Processing of intact platelet APP is promoted also by treatment with calmodulin antagonist W7. W7-induced APP proteolysis occurs through the non-amyloidogenic pathway, is mediated by a metalloproteinase, and causes the release of sAPPα. Co-immunoprecipitation and pull-down experiments revealed a physical association between calmodulin and APP. These results document a novel role of calmodulin in the regulation of non-amyloidogenic processing of APP.     

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.