The transmembrane domain of the amyloid precursor protein is required for antiamyloidogenic processing by α-secretase ADAM10.

Neurotoxic amyloid ß-peptides are thought to be a causative agent of Alzheimer's disease in humans. The production of amyloid ß-peptides from amyloid precursor protein (APP) could be diminished by enhancing α-processing; however, the physical interactions between APP and α-secretases are not well understood. In this study, we employed super-resolution light microscopy to examine in cell-free plasma membranes the abundance and association of APP and α-secretases ADAM10 (a disintegrin and metalloproteinase) and ADAM17. We found that both secretase molecules localize similarly closely to APP (within ≤50 nm). However, when cross-linking APP with antibodies directed against the GFP tag of APP, in confocal microscopy, we observed that only ADAM10 coaggregated with APP. Furthermore, we mapped the involved protein domain by using APP variants with an exchanged transmembrane segment or lacking cytoplasmic/extracellular domains. We identified that the transmembrane domain of APP is required for association with α-secretases and, as analyzed by Western blot, for α-processing. We propose that the transmembrane domain of APP interacts either directly or indirectly with ADAM10, but not with ADAM17, explaining the dominant role of ADAM10 in α-processing of APP. Further understanding of this interaction may facilitate the development of a therapeutic strategy based on promoting APP cleavage by α-secretases.

Revisiting APP secretases: an overview on the holistic effects of retinoic acid receptor stimulation in APP processing.

Alzheimer's disease (AD) is the leading cause of dementia worldwide and is characterized by the accumulation of the ß-amyloid peptide (Aß) in the brain, along with profound alterations in phosphorylation-related events and regulatory pathways. The production of the neurotoxic Aß peptide via amyloid precursor protein (APP) proteolysis is a crucial step in AD development. APP is highly expressed in the brain and is complexly metabolized by a series of sequential secretases, commonly denoted the α-, ß-, and γ-cleavages. The toxicity of resulting fragments is a direct consequence of the first cleaving event. ß-secretase (BACE1) induces amyloidogenic cleavages, while α-secretases (ADAM10 and ADAM17) result in less pathological peptides. Hence this first cleavage event is a prime therapeutic target for preventing or reverting initial biochemical events involved in AD. The subsequent cleavage by γ-secretase has a reduced impact on Aß formation but affects the peptides' aggregating capacity. An array of therapeutic strategies are being explored, among them targeting Retinoic Acid (RA) signalling, which has long been associated with neuronal health. Additionally, several studies have described altered RA levels in AD patients, reinforcing RA Receptor (RAR) signalling as a promising therapeutic strategy. In this review we provide a holistic approach focussing on the effects of isoform-specific RAR modulation with respect to APP secretases and discuss its advantages and drawbacks in subcellular AD related events.

Amyloid-Beta Mediates Homeostatic Synaptic Plasticity.

The physiological role of the amyloid-precursor protein (APP) is insufficiently understood. Recent work has implicated APP in the regulation of synaptic plasticity. Substantial evidence exists for a role of APP and its secreted ectodomain APPsα in Hebbian plasticity. Here, we addressed the relevance of APP in homeostatic synaptic plasticity using organotypic tissue cultures prepared from APP-/- mice of both sexes. In the absence of APP, dentate granule cells failed to strengthen their excitatory synapses homeostatically. Homeostatic plasticity is rescued by amyloid-ß and not by APPsα, and it is neither observed in APP+/+ tissue treated with ß- or γ-secretase inhibitors nor in synaptopodin-deficient cultures lacking the Ca2+-dependent molecular machinery of the spine apparatus. Together, these results suggest a role of APP processing via the amyloidogenic pathway in homeostatic synaptic plasticity, representing a function of relevance for brain physiology as well as for brain states associated with increased amyloid-ß levels.

Participation of Amyloid and Tau Protein in Post-Ischemic Neurodegeneration of the Hippocampus of a Nature Identical to Alzheimer's Disease.

Recent evidence suggests that amyloid and tau protein are of vital importance in post-ischemic death of CA1 pyramidal neurons of the hippocampus. In this review, we summarize protein alterations associated with Alzheimer's disease and their gene expression (amyloid protein precursor and tau protein) after cerebral ischemia, as well as their roles in post-ischemic hippocampus neurodegeneration. In recent years, multiple studies aimed to elucidate the post-ischemic processes in the development of hippocampus neurodegeneration. Their findings have revealed the dysregulation of genes for amyloid protein precursor, ß-secretase, presenilin 1 and 2, tau protein, autophagy, mitophagy, and apoptosis identical in nature to Alzheimer's disease. Herein, we present the latest data showing that amyloid and tau protein associated with Alzheimer's disease and their genes play a key role in post-ischemic neurodegeneration of the hippocampus with subsequent development of dementia. Therefore, understanding the underlying process for the development of post-ischemic CA1 area neurodegeneration in the hippocampus in conjunction with Alzheimer's disease-related proteins and genes will provide the most important therapeutic development goals to date.

Tuning of Glutamate, But Not GABA, Release by an Intrasynaptic Vesicle APP Domain Whose Function Can Be Modulated by ß- or α-Secretase Cleavage.

APP, whose mutations cause familial Alzheimer's disease (FAD), modulates neurotransmission via interaction of its cytoplasmic tail with the synaptic release machinery. Here we identified an intravesicular domain of APP, called intraluminal SV-APP interacting domain (ISVAID), which interacts with glutamatergic, but not GABAergic, synaptic vesicle proteins. ISVAID contains the ß- and α-secretase cleavage sites of APP: proteomic analysis of the interactome of ISVAID suggests that ß- and α-secretase cleavage of APP cuts inside the interaction domain of ISVAID and destabilizes protein-protein interactions. We have tested the functional significance of the ISVAID and of ß-/α-secretase-processing of APP using various ISVAID-derived peptides in competition experiments on both female and male mouse and rats hippocampal slices. A peptide encompassing the entire ISVAID facilitated glutamate, but not GABA, release acting as dominant negative inhibitor of the functions of this APP domain in acute hippocampal slices. In contrast, peptides representing the product of ß-/α-secretase-processing of ISVAID did not alter excitatory neurotransmitter release. These findings suggest that cleavage of APP by either ß- or α-secretase may inactivate the ISVAID, thereby enhancing glutamate release. Our present data support the notion that APP tunes glutamate release, likely through intravesicular and extravesicular interactions with synaptic vesicle proteins and the neurotransmitter release machinery, and that ß-/α cleavage of APP facilitates the release of excitatory neurotransmitter.SIGNIFICANCE STATEMENT Alzheimer's disease has been linked to mutations in APP. However, the biological function of APP is poorly understood. Here we show that an intravesicular APP domain interacts with the proteins that control the release of glutamate, but not GABA. Interfering with the function of this domain promotes glutamate release. This APP domain contains the sites cleaved by ß- and α-secretases: our data suggest that ß-/α cleavage of APP inactivates this functional APP domain promoting excitatory neurotransmitter release.

Ablation of neuronal ADAM17 impairs oligodendrocyte differentiation and myelination.

Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient propagation of the electric impulse in the nervous system and to maintain neuronal integrity. In the central nervous system (CNS), the development of oligodendrocytes and the process of myelination are regulated by the coordinated action of several positive and negative cell-extrinsic factors. We and others previously showed that secretases regulate the activity of proteins essential for myelination. We now report that the neuronal α-secretase ADAM17 controls oligodendrocyte differentiation and myelin formation in the CNS. Ablation of Adam17 in neurons impairs in vivo and in vitro oligodendrocyte differentiation, delays myelin formation throughout development and results in hypomyelination. Furthermore, we show that this developmental defect is, in part, the result of altered Notch/Jagged 1 signaling. Surprisingly, in vivo conditional loss of Adam17 in immature oligodendrocytes has no effect on myelin formation. Collectively, our data indicate that the neuronal α-secretase ADAM17 is required for proper CNS myelination. Further, our studies confirm that secretases are important post-translational regulators of myelination although the mechanisms controlling CNS and peripheral nervous system (PNS) myelination are distinct.

Proximity ligation assay reveals both pre- and postsynaptic localization of the APP-processing enzymes ADAM10 and BACE1 in rat and human adult brain.

BACKGROUND: Synaptic degeneration and accumulation of amyloid ß-peptides (Aß) are hallmarks of the Alzheimer diseased brain. Aß is synaptotoxic and produced by sequential cleavage of the amyloid precursor protein (APP) by the ß-secretase BACE1 and by γ-secretase. If APP is instead cleaved by the α-secretase ADAM10, Aß will not be generated. Although BACE1 is considered to be a presynaptic protein and ADAM10 has been reported to mainly localize to the postsynaptic density, we have previously shown that both ADAM10 and BACE1 are highly enriched in synaptic vesicles of rat brain and mouse primary hippocampal neurons. RESULTS: Here, using brightfield proximity ligation assay, we expanded our previous result in primary neurons and investigated the in situ synaptic localization of ADAM10 and BACE1 in rat and human adult brain using both pre- and postsynaptic markers. We found that ADAM10 and BACE1 were in close proximity with both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. The substrate APP was also detected both pre- and postsynaptically. Subcellular fractionation confirmed that ADAM10 and BACE1 are enriched to a similar degree in synaptic vesicles and as well as in the postsynaptic density. CONCLUSIONS: We show that the α-secretase ADAM10 and the ß-secretase BACE1 are located in both the pre- and postsynaptic compartments in intact brain sections. These findings increase our understanding of the regulation of APP processing, thereby facilitating development of more specific treatment strategies.

Participation of Amyloid and Tau Protein in Neuronal Death and Neurodegeneration after Brain Ischemia.

Current evidence indicates that postischemic brain injury is associated with the accumulation of folding proteins, such as amyloid and tau protein, in the intra- and extracellular spaces of neuronal cells. In this review, we summarize protein changes associated with Alzheimer's disease and their gene expression (amyloid protein precursor and tau protein) after brain ischemia, and their roles in the postischemic period. Recent advances in understanding the postischemic mechanisms in development of neurodegeneration have revealed dysregulation of amyloid protein precursor, α-, ß- and γ-secretase and tau protein genes. Reduced expression of the α-secretase gene after brain ischemia with recirculation causes neuronal cells to be less resistant to injury. We present the latest data that Alzheimer's disease-related proteins and their genes play a crucial role in postischemic neurodegeneration. Understanding the underlying processes of linking Alzheimer's disease-related proteins and their genes in development of postischemic neurodegeneration will provide the most significant goals to date for therapeutic development.

[Did Alzheimer research fail entirely? : Failure of amyloid-based clinical studies]. / Hat die Alzheimer-Forschung versagt? : Das Scheitern amyloidbasierter klinischer Studien.

Numerous amyloid-based clinical studies have recently failed. Does this mean that the mechanisms of Alzheimer's disease have to be reinvestigated and that amyloid is not the trigger of the disease? Strong genetic evidence from familial Alzheimer's disease contradicts this fatalistic opinion. Mutations in all genes associated with familial Alzheimer's disease affect amyloid metabolism and aggregation. Moreover, a protective mutation reduces amyloid production by 20-30% throughout the lifetime. Clinical studies rather failed because secretase inhibitors block cleavage of numerous other physiologically important substrates of secretases. Moreover, the disease is initiated decades before symptoms occur. Successful treatment attempts with anti-amyloid medication based on other prototype amyloidoses are described. Finally, new therapeutic target molecules expressed in microglia cells are discussed.

Modulation of BACE1 Activity by Chemically Modified Aptamers.

A modified DNA aptamer that binds BACE1, a therapeutic target involved in Alzheimer's disease has been developed. This ssXNA not only tightly binds to BACE1 but also inhibits its protease activity in vitro in the same range as a previously described unmodified aptamer. We report the in vitro selection of functional oligonucleotides incorporating two nucleobase modifications: 5-chlorouracil and 7-deazaadenine. The nucleoside analogue 5-chloro-2'-deoxyuridine has already been explored as a replacement for thymidine in a chemically modified genome of a bacterium. Thus, 5-chlorouracil modification is a good candidate to support genetic transfer in vivo as well as functional activity.

Diallyl trisulfide, a chemopreventive agent from Allium vegetables, inhibits alpha-secretases in breast cancer cells.

Breast cancer affects one in eight women throughout the course of their lifetime creating a demand for novel prevention strategies against this disease. The Notch signaling pathway is often aberrantly activated in human malignancies including breast cancer. Alpha secretases, including ADAM (A Disintegrin and Metalloprotease) -10 and -17, are proteases that play a key role in the cleavage of cell surface molecules and subsequent ligand-mediated activation of Notch signaling pathway. High expression levels of ADAM10 and 17 have been clinically associated with a lower disease-free survival in breast cancer patients. This study was undertaken to determine the effect of diallyl trisulfide (DATS), a bioactive organosulfide found in garlic and other Allium vegetables, on alpha secretases in breast cancer cells. Here we report for the first time that DATS inhibits the expression of ADAM10 and ADAM17 in estrogen-independent MDA-MB-231 and estrogen-dependent MCF-7 breast cancer cells, and in Harvey-ras (H-Ras) transformed MCF10A-H-Ras breast epithelial cells. We also show that DATS induces a dose-dependent reduction in colony formation ability of MDA-MB-231 and MCF-7 cells, suggesting a long-term effect of DATS on growth inhibition of breast cancer cells. Furthermore, we show that DATS inhibits the Notch ligands Jagged-1 and Jagged-2 involved in activation of Notch signaling pathway. Collectively, these findings show that DATS targets Notch pathway components overexpressed in breast cancer tumors and may serve as a functionally relevant bioactive for breast cancer prevention.

Brain ischaemia induces shedding of a BDNF-scavenger ectodomain from TrkB receptors by excitotoxicity activation of metalloproteinases and γ-secretases.

Stroke remains a leading cause of death and disability in the world with limited therapies available to restrict brain damage or improve functional recovery after cerebral ischaemia. A promising strategy currently under investigation is the promotion of brain-derived neurotrophic factor (BDNF) signalling through tropomyosin-related kinase B (TrkB) receptors, a pathway essential for neuronal survival and function. However, TrkB and BDNF-signalling are impaired by excitotoxicity, a primary pathological process in stroke also associated with neurodegenerative diseases. Pathological imbalance of TrkB isoforms is critical in neurodegeneration and is caused by calpain processing of BDNF high affinity full-length receptor (TrkB-FL) and an inversion of the transcriptional pattern of the Ntrk2 gene, to favour expression of the truncated isoform TrkB-T1 over TrkB-FL. We report here that both TrkB-FL and neuronal TrkB-T1 also undergo ectodomain shedding by metalloproteinases activated after ischaemic injury or excitotoxic damage of cortical neurons. Subsequently, the remaining membrane-bound C-terminal fragments (CTFs) are cleaved by γ-secretases within the transmembrane region, releasing their intracellular domains (ICDs) into the cytosol. Therefore, we identify TrkB-FL and TrkB-T1 as new substrates of regulated intramembrane proteolysis (RIP), a mechanism that highly contributes to TrkB-T1 regulation in ischaemia but is minor for TrkB-FL which is mainly processed by calpain. However, since the secreted TrkB ectodomain acts as a BDNF scavenger and significantly alters BDNF/TrkB signalling, the mechanism of RIP could contribute to neuronal death in excitotoxicity. These results are highly relevant since they reveal new targets for the rational design of therapies to treat stroke and other pathologies with an excitotoxic component.

Vitamin D and Its Analogues Decrease Amyloid-ß (Aß) Formation and Increase Aß-Degradation.

Alzheimer's disease (AD) is characterized by extracellular plaques in the brain, mainly consisting of amyloid-ß (Aß), as derived from sequential cleavage of the amyloid precursor protein. Epidemiological studies suggest a tight link between hypovitaminosis of the secosteroid vitamin D and AD. Besides decreased vitamin D level in AD patients, an effect of vitamin D on Aß-homeostasis is discussed. However, the exact underlying mechanisms remain to be elucidated and nothing is known about the potential effect of vitamin D analogues. Here we systematically investigate the effect of vitamin D and therapeutically used analogues (maxacalcitol, calcipotriol, alfacalcidol, paricalcitol, doxercalciferol) on AD-relevant mechanisms. D2 and D3 analogues decreased Aß-production and increased Aß-degradation in neuroblastoma cells or vitamin D deficient mouse brains. Effects were mediated by affecting the Aß-producing enzymes BACE1 and γ-secretase. A reduced secretase activity was accompanied by a decreased BACE1 protein level and nicastrin expression, an essential component of the γ-secretase. Vitamin D and analogues decreased ß-secretase activity, not only in mouse brains with mild vitamin D hypovitaminosis, but also in non-deficient mouse brains. Our results further strengthen the link between AD and vitamin D, suggesting that supplementation of vitamin D or vitamin D analogues might have beneficial effects in AD prevention.

PMEL Amyloid Fibril Formation: The Bright Steps of Pigmentation.

In pigment cells, melanin synthesis takes place in specialized organelles, called melanosomes. The biogenesis and maturation of melanosomes is initiated by an unpigmented step that takes place prior to the initiation of melanin synthesis and leads to the formation of luminal fibrils deriving from the pigment cell-specific pre-melanosomal protein (PMEL). In the lumen of melanosomes, PMEL fibrils optimize sequestration and condensation of the pigment melanin. Interestingly, PMEL fibrils have been described to adopt a typical amyloid-like structure. In contrast to pathological amyloids often associated with neurodegenerative diseases, PMEL fibrils represent an emergent category of physiological amyloids due to their beneficial cellular functions. The formation of PMEL fibrils within melanosomes is tightly regulated by diverse mechanisms, such as PMEL traffic, cleavage and sorting. These mechanisms revealed increasing analogies between the formation of physiological PMEL fibrils and pathological amyloid fibrils. In this review we summarize the known mechanisms of PMEL fibrillation and discuss how the recent understanding of physiological PMEL amyloid formation may help to shed light on processes involved in pathological amyloid formation.

Rheb GTPase regulates ß-secretase levels and amyloid ß generation.

The ß-site amyloid precursor protein (APP)-cleaving enzyme 1 (ß-secretase, BACE1) initiates amyloidogenic processing of APP to generate amyloid ß (Aß), which is a hallmark of Alzheimer disease (AD) pathology. Cerebral levels of BACE1 are elevated in individuals with AD, but the molecular mechanisms are not completely understood. We demonstrate that Rheb GTPase (Ras homolog enriched in brain), which induces mammalian target of rapamycin (mTOR) activity, is a physiological regulator of BACE1 stability and activity. Rheb overexpression depletes BACE1 protein levels and reduces Aß generation, whereas the RNAi knockdown of endogenous Rheb promotes BACE1 accumulation, and this effect by Rheb is independent of its mTOR signaling. Moreover, GTP-bound Rheb interacts with BACE1 and degrades it through proteasomal and lysosomal pathways. Finally, we demonstrate that Rheb levels are down-regulated in the AD brain, which is consistent with an increased BACE1 expression. Altogether, our study defines Rheb as a novel physiological regulator of BACE1 levels and Aß generation, and the Rheb-BACE1 circuitry may have a role in brain biology and disease.

Characterization of intermediate steps in amyloid beta (Aß) production under near-native conditions.

Processing of the amyloid precursor protein (APP) by γ-secretase results in generation of Aß peptides of different lengths ranging from 51 to 30 residues. Accumulation of Aß and in particular Aß42 is enhanced by familial Alzheimer disease (FAD) causing mutations in APP and is believed to play a pivotal role. The molecular mechanism underlying normal Aß production, the impact of FAD mutations on this process and how anti-amyloidogenic γ-secretase modulators (GSMs) cause a selective decrease in Aß40 and Aß42 and an increase in shorter Aß peptides, however, is poorly understood. By using a combined immuno- and LC-MS-based assay we identify several major intermediates, i.e. 3- and 4-peptides that line up head to head across the entire APP transmembrane sequence from Aß51 to Aß31/Aß30 and from Aß49 to Aß30/31. FAD APP mutations displayed a relative increase in 3- and 4-peptides from Aß48 to Aß38 compared with Aß49 to Aß37. These findings correlate with an increase in the Aß42/40 ratio. GSMs caused a decrease in Aß40 and Aß42 and an increase in Aß37 and Aß38 paralleled by an increase of the intermediates Aß40-38 and Aß42-39. Collectively, these data provide a thorough characterization of all intermediate steps in Aß production in native cell membranes and provide key mechanistic insights to genetic and pharmacological modulation of Aß generation.

Signature amyloid ß profiles are produced by different γ-secretase complexes.

γ-Secretase complexes are involved in the generation of amyloid-ß (Aß) in the brain. Therefore, γ-secretase has been proposed as a potential therapeutic target in Alzheimer disease (AD). Targeting γ-secretase activity in AD requires the pharmacological dissociation of the processing of physiological relevant substrates and the generation of "toxic" Aß. Previous reports suggest the differential targeting of γ-secretase complexes, based on their subunit composition, as a valid strategy. However, little is known about the biochemical properties of the different complexes, and key questions regarding their Aß product profiles should be first addressed. Here, we expressed, purified, and analyzed, under the same conditions, the endopeptidase and carboxypeptidase-like activities of the four γ-secretase complexes present in humans. We find that the nature of the catalytic subunit in the complex affects both activities. Interestingly, PSEN2 complexes discriminate between the Aß40 and Aß38 production lines, indicating that Aß generation in one or the other pathway can be dissociated. In contrast, the APH1 subunit mainly affects the carboxypeptidase-like activity, with APH1B complexes favoring the generation of longer Aß peptides. In addition, we determined that expression of a single human γ-secretase complex in cell lines retains the intrinsic attributes of the protease while present in the membrane, providing validation for the in vitro studies. In conclusion, our data show that each γ-secretase complex produces a characteristic Aß signature. The qualitative and quantitative differences between different γ-secretase complexes could be used to advance drug development in AD and other disorders.

γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of ß-carboxyl-terminal fragment.

γ-Secretase generates amyloid ß-protein (Aß), a pathogenic molecule in Alzheimer disease, through the intramembrane cleavage of the ß-carboxyl-terminal fragment (ßCTF) of ß-amyloid precursor protein. We previously showed the framework of the γ-secretase cleavage, i.e. the stepwise successive processing of ßCTF at every three (or four) amino acids. However, the membrane integrity of γ-secretase was not taken into consideration because of the use of the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized reconstituted γ-secretase system. Here, we sought to address how the membrane-integrated γ-secretase cleaves ßCTF by using γ-secretase associated with lipid rafts. Quantitative analyses using liquid chromatography-tandem mass spectrometry of the ßCTF transmembrane domain-derived peptides released along with Aß generation revealed that the raft-associated γ-secretase cleaves ßCTF in a stepwise sequential manner, but novel penta- and hexapeptides as well as tri- and tetrapeptides are released. The cropping of these peptides links the two major tripeptide-cleaving pathways generating Aß40 and Aß42 at several points, implying that there are multiple interactive pathways for the stepwise cleavages of ßCTF. It should be noted that Aß38 and Aß43 are generated through three routes, and γ-secretase modulator 1 enhances all the three routes generating Aß38, which results in decreases in Aß42 and Aß43 and an increase in Aß38. These observations indicate that multiple interactive pathways for stepwise successive processing by γ-secretase define the species and quantity of Aß produced.

MicroRNA-339-5p down-regulates protein expression of ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) in human primary brain cultures and is reduced in brain tissue specimens of Alzheimer disease subjects.

Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-ß (Aß) peptide as neuritic plaques in the brain. The short Aß peptide is derived from the large transmembrane Aß precursor protein (APP). The rate-limiting step in the production of Aß from APP is mediated by the ß-site APP-cleaving enzyme 1 (BACE1). Dysregulation of BACE1 levels leading to excess Aß deposition is implicated in sporadic AD. Thus, elucidating the full complement of regulatory pathways that control BACE1 expression is key to identifying novel drug targets central to the Aß-generating process. MicroRNAs (miRNAs) are expected to participate in this molecular network. Here, we identified a known miRNA, miR-339-5p, as a key contributor to this regulatory network. Two distinct miR-339-5p target sites were predicted in the BACE1 3'-UTR by in silico analyses. Co-transfection of miR-339-5p with a BACE1 3'-UTR reporter construct resulted in significant reduction in reporter expression. Mutation of both target sites eliminated this effect. Delivery of the miR-339-5p mimic also significantly inhibited expression of BACE1 protein in human glioblastoma cells and human primary brain cultures. Delivery of target protectors designed against the miR-339-5p BACE1 3'-UTR target sites in primary human brain cultures significantly elevated BACE1 expression. Finally, miR-339-5p levels were found to be significantly reduced in brain specimens isolated from AD patients as compared with age-matched controls. Therefore, miR-339-5p regulates BACE1 expression in human brain cells and is most likely dysregulated in at least a subset of AD patients making this miRNA a novel drug target.

Perturbations of the straight transmembrane α-helical structure of the amyloid precursor protein affect its processing by γ-secretase.

The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase of its C-terminal fragment produces amyloid-ß (Aß) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight α-helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the γ-secretase enzyme, as suggested by a series of Gly(700) mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of Aß38 compared with Aß40 peptides, which is reminiscent of the effect of γ-secretase modulators inhibitors.