Cleavage Of Β-amyloid Precursor Protein Research Articles

BACE Inhibitor Clinical Trials for Alzheimer's Disease.

The amyloid hypothesis posits that the amyloid-β aggregates in the brain initiate a cascade of events that eventually lead to neuron loss and Alzheimer's disease. Recent clinical trials of passive immunotherapy with anti-amyloid-β antibodies support this hypothesis, because clearing plaques led to better cognitive outcomes. Orally available small molecule BACE1 inhibitors are another approach to slowing the buildup of plaques and thereby cognitive worsening by preventing the cleavage of amyloid-β protein precursor (AβPP) into amyloid-β peptide, the major component of plaques. This approach is particularly attractive because of their ease of use, low cost, and advanced clinical stage. However, although effective in preventing amyloid-β production in late-stage clinical trials, BACE inhibitors have been associated with early, non-progressive, likely reversible, cognitive decline. The clinical trials tested high levels of BACE inhibition, greater than 50%, whereas genetics suggest that even a 30% inhibition may be sufficient to protect from Alzheimer's disease. Aside from AβPP, BACE1 cleaves many other substrates in the brain that may be contributing to the cognitive worsening. It is important to know what the cause of cognitive worsening is, and if a lower level of inhibition would sufficiently slow the progress of pathology while preventing these unwanted side effects. Should these side effects be mitigated, BACE inhibitors could rapidly move forward in clinical trials either as a primary prevention strategy in individuals that are at risk or biomarker positive, or as a maintenance therapy following amyloid clearance with an anti-amyloid antibody.

Alpha-lipoic acid alleviates cognitive deficits in transgenic APP23/PS45 mice through a mitophagy-mediated increase in ADAM10 α-secretase cleavage of APP

BackgroundAlpha-lipoic acid (ALA) has a neuroprotective effect on neurodegenerative diseases. In the clinic, ALA can improve cognitive impairments in patients with Alzheimer’s disease (AD) and other dementias. Animal studies have confirmed the anti-amyloidosis effect of ALA, but its underlying mechanism remains unclear. In particular, the role of ALA in amyloid-β precursor protein (APP) metabolism has not been fully elucidated.ObjectiveTo investigate whether ALA can reduce the amyloidogenic effect of APP in a transgenic mouse model of AD, and to study the mechanism underlying this effect.MethodsALA was infused into 2-month-old APP23/PS45 transgenic mice for 4 consecutive months and their cognitive function and AD-like pathology were then evaluated. An ALA drug concentration gradient was applied to 20E2 cells in vitro to evaluate its effect on the expression of APP proteolytic enzymes and metabolites. The mechanism by which ALA affects APP processing was studied using GI254023X, an inhibitor of A Disintegrin and Metalloproteinase 10 (ADAM10), as well as the mitochondrial toxic drug carbonyl cyanide m-chlorophenylhydrazone (CCCP).ResultsAdministration of ALA ameliorated amyloid plaque neuropathology in the brain tissue of APP23/PS45 mice and reduced learning and memory impairment. ALA also increased the expression of ADAM10 in 20E2 cells and the non-amyloidogenic processing of APP to produce the 83 amino acid C-terminal fragment (C83). In addition to activating autophagy, ALA also significantly promoted mitophagy. BNIP3L-knockdown reduced the mat/pro ratio of ADAM10. By using CCCP, ALA was found to regulate BNIP3L-mediated mitophagy, thereby promoting the α-cleavage of APP.ConclusionsThe enhanced α-secretase cleavage of APP by ADAM10 is the primary mechanism through which ALA ameliorates the cognitive deficits in APP23/PS45 transgenic mice. BNIP3L-mediated mitophagy contributes to the anti-amyloid properties of ALA by facilitating the maturation of ADAM10. This study provides novel experimental evidence for the treatment of AD with ALA.Graphical abstract

Quercetin-3-O-glc-1-3-rham-1-6-glucoside decreases Aβ production, inhibits Aβ aggregation and neurotoxicity, and prohibits the production of inflammatory cytokines

Alzheimer's disease (AD) is a progressive neurodegenerative disease with the hallmark of aggregation of beta-amyloid (Aβ) into extracellular fibrillar deposition. Accumulating evidence suggests that soluble toxic Aβ oligomers exert diverse roles in neuronal cell death, oxidative stress, neuroinflammation, and the eventual pathogenesis of AD. Aβ is derived from the sequential cleavage of amyloid-β precursor protein (APP) by β-secretase (BACE1) and γ-secretase. The current effect of single targeting is not ideal for the treatment of AD. Therefore, developing multipotent agents with multiple properties, including anti-Aβ generation and anti-Aβ aggregation, is attracting more attention for AD treatment. Previous studies indicated that Quercetin was able to attenuate the effects of several pathogenetic factors in AD. Here, we showed that naturally synthesized Quercetin-3-O-glc-1-3-rham-1-6-glucoside (YCC31) could inhibit Aβ production by reducing β-secretase activity. Further investigations indicated that YCC31 could suppress toxic Aβ oligomer formation by directly binding to Aβ. Moreover, YCC31 could attenuate Aβ-mediated neuronal death, ROS and NO production, and pro-inflammatory cytokines release. Taken together, YCC31 targeting multiple pathogenetic factors deserves further investigation for drug development of AD.

A Bioengineering Strategy to Control ADAM10 Activity in Living Cells.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer's disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-β precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.

PS1 Affects the Pathology of Alzheimer's Disease by Regulating BACE1 Distribution in the ER and BACE1 Maturation in the Golgi Apparatus.

Neuritic plaques are one of the major pathological hallmarks of Alzheimer's disease. They are formed by the aggregation of extracellular amyloid-β protein (Aβ), which is derived from the sequential cleavage of amyloid-β precursor protein (APP) by β- and γ-secretase. BACE1 is the main β-secretase in the pathogenic process of Alzheimer's disease, which is believed to be a rate-limiting step of Aβ production. Presenilin 1 (PS1) is the active center of the γ-secretase that participates in the APP hydrolysis process. Mutations in the PS1 gene (PSEN1) are the most common cause of early onset familial Alzheimer's disease (FAD). The PSEN1 mutations can alter the activity of γ-secretase on the cleavage of APP. Previous studies have shown that PSEN1 mutations increase the expression and activity of BACE1 and that BACE1 expression and activity are elevated in the brains of PSEN1 mutant knock-in mice, compared with wild-type mice, as well as in the cerebral cortex of FAD patients carrying PSEN1 mutations, compared with sporadic AD patients and controls. Here, we used a Psen1 knockout cell line and a PS1 inhibitor to show that PS1 affects the expression of BACE1 in vitro. Furthermore, we used sucrose gradient fractionation combined with western blotting to analyze the distribution of BACE1, combined with a time-lapse technique to show that PS1 upregulates the distribution and trafficking of BACE1 in the endoplasmic reticulum, Golgi, and endosomes. More importantly, we found that the PSEN1 mutant S170F increases the distribution of BACE1 in the endoplasmic reticulum and changes the ratio of mature BACE1 in the trans-Golgi network. The effect of PSEN1 mutations on BACE1 may contribute to determining the phenotype of early onset FAD.

Circulating micro-RNAs Differentially Expressed in Korean Alzheimer's Patients With Brain Aβ Accumulation Activate Amyloidogenesis.

Roles for extracellular vesicles (EVs) enriched with micro-RNAs (miRNAs) have been proposed in Alzheimer's disease (AD) pathogenesis, leading to the discovery of blood miRNAs as AD biomarkers. However, the diagnostic utility of specific miRNAs is not consistent. This study aimed to discover blood miRNAs that are differentially expressed in Korean AD patients, evaluate their clinical performance, and investigate their role in amyloidogenesis. We discovered miRNAs differentially expressed in AD (N = 8) from cognitively normal participants (CN, N = 7) or Parkinson's disease (PD) patients (N = 8). We evaluated the clinical performance of these miRNAs in plasma of subgroup (N = 99) and in plasma EVs isolated from the total cohort (N = 251). The effects of miRNAs on amyloidogenesis and on the regulation of their target genes were investigated in vitro. Among 17 upregulated and one downregulated miRNAs in AD (>twofold), miR-122-5p, miR-210-3p, and miR-590-5p were differentially expressed compared with CN or PD. However, the diagnostic performance of the selected plasma or EV miRNAs in total participants were limited (area under the curve < 0.8). Nevertheless, levels of 3 miRNAs in plasma or plasma EVs of participants who were amyloid positron emission tomography (Aβ-PET) positive were significantly higher than those from the Aβ-PET negative participants (p < .05). The selected miRNAs induced Aβ production (p < .05) through activation of β-cleavage of amyloid precursor protein (CTF-β; p < .01), and downregulated their target genes (ADAM metallopeptidase domain 10, Brain-derived neurotrophic factor, and Jagged canonical notch ligand 1; p < .05), which was further supported by pathway enrichment analysis of target genes of the miRNAs. In conclusion, despite of the limited diagnostic utility of selected miRNAs as plasma or plasma EV biomarkers, the discovered miRNAs may play a role in amyloidogenesis during AD onset and progression.

γ-Secretase structure and activity are modified by alterations in its membrane localization and ambient environment.

γ-Secretase cleaves type I transmembrane proteins in a hydrophobic membrane environment following ectodomain shedding. Mutations in PSEN genes, encoding the catalytic subunits of γ-secretase, presenilins, are the most common cause of familial Alzheimer's disease (ad). Pathogenic mutations in PSEN genes increase production of longer and neurotoxic amyloid-β (Aβ) by intramembrane cleavage of membrane-associated amyloid-β protein precursor (APP) carboxyl-terminal fragment β, which is generated via primary cleavage of APP by β-site APP cleaving enzyme 1. The longer Aβ is prone to aggregate and accumulate in the brain; however, the accumulation of Aβ in brain is also a pathological feature of sporadic ad. Increased pathogenic Aβ generation, even in the absence of pathogenic PSEN gene mutations, is one of proposed mechanisms for sporadic ad pathogenesis. γ-Secretase digests substrates in the transmembrane region, generating Aβ peptide intermediates of various lengths. The end products, shorter Aβ40 and Aβ38 peptides, are less neurotoxic, whereas PSEN gene mutations increase the production ratio of longer, neurotoxic Aβ species such as Aβ42, an intermediate in Aβ38 production. γ-Secretase activity or structures is altered because of its aberrant membrane localization or changes in the ambient environment such as luminal acidification. Interestingly, γ-secretase has a pH sensor in presenilins.

Lemur tail kinase 1 (LMTK1) regulates the endosomal localization of β-secretase BACE1.

Lemur tail kinase 1 (LMTK1), previously called apoptosis-associated tyrosine kinase (AATYK), is an endosomal Ser/Thr kinase. We recently reported that LMTK1 regulates axon outgrowth, dendrite arborization and spine formation via Rab11-mediated vesicle transport. Rab11, a small GTPase regulating recycling endosome trafficking, is shown to be associated with late-onset Alzheimer's disease (LOAD). In fact, genome-wide association studies identified many proteins regulating vesicle transport as risk factors for LOAD. Furthermore, LMTK1 has been reported to be a risk factor for frontotemporal dementia. Then, we hypothesized that LMTK1 contributes to AD development through vesicle transport and examined the effect of LMTK1 on the cellular localization of AD-related proteins, amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 (BACE1). The β-cleavage of APP by BACE1 is the initial and rate-limiting step in Aβ generation. We found that LMTK1 accumulated BACE1, but not APP, to the perinuclear endosomal compartment, whereas the kinase-negative(kn) mutant of LMTK1A did not. The β-C-terminal fragment was prone to increase under overexpression of LMTK1A kn. Moreover, the expression level of LMTK1A was reduced in AD brains. These results suggest the possibility that LMTK1 is involved in AD development through the regulation of the proper endosomal localization of BACE1.

Flexible and Accurate Substrate Processing with Distinct Presenilin/γ-Secretases in Human Cortical Neurons.

Mutations in the presenilin genes (PS1, PS2) have been linked to the majority of familial Alzheimer's disease (AD). Although great efforts have been made to investigate pathogenic PS mutations, which ultimately cause an increase in the toxic form of β-amyloid (Aβ), the intrinsic physiological functions of PS in human neurons remain to be determined. In this study, to investigate the physiological roles of PS in human neurons, we generated PS1 conditional knock-out (KO) induced pluripotent stem cells (iPSCs), in which PS1 can be selectively abrogated under Cre transduction with or without additional PS2 KO. We showed that iPSC-derived neural progenitor cells (NPCs) do not confer a maintenance ability in the absence of both PS1 and PS2, showing the essential role of PS in Notch signaling. We then generated PS-null human cortical neurons, where PS1 was intact until full neuronal differentiation occurred. Aβ40 production was reduced exclusively in human PS1/PS2-null neurons along with a concomitant accumulation of amyloid β precursor protein (APP)-C-terminal fragments CTFs, whereas Aβ42 was decreased in neurons devoid of PS2 Unlike previous studies in mice, in which APP cleavage is largely attributable to PS1, γ-secretase activity seemed to be comparable between PS1 and PS2. In contrast, cleavage of another substrate, N-cadherin, was impaired only in neurons devoid of PS1 Moreover, PS2/γ-secretase exists largely in late endosomes/lysosomes, as measured by specific antibody against the γ-secretase complex, in which Aβ42 species are supposedly produced. Using this novel stem cell-based platform, we assessed important physiological PS1/PS2 functions in mature human neurons, the dysfunction of which could underlie AD pathogenesis.

Amyloidogenic processing of amyloid β protein precursor (APP) is enhanced in the brains of alcadein α–deficient mice

Alzheimer's disease (AD) is a very common neurodegenerative disorder, chiefly caused by increased production of neurotoxic β-amyloid (Aβ) peptide generated from proteolytic cleavage of β-amyloid protein precursor (APP). Except for familial AD arising from mutations in the APP and presenilin (PSEN) genes, the molecular mechanisms regulating the amyloidogenic processing of APP are largely unclear. Alcadein α/calsyntenin1 (ALCα/CLSTN1) is a neuronal type I transmembrane protein that forms a complex with APP, mediated by the neuronal adaptor protein X11-like (X11L or MINT2). Formation of the ALCα-X11L-APP tripartite complex suppresses Aβ generation in vitro, and X11L-deficient mice exhibit enhanced amyloidogenic processing of endogenous APP. However, the role of ALCα in APP metabolism in vivo remains unclear. Here, by generating ALCα-deficient mice and using immunohistochemistry, immunoblotting, and co-immunoprecipitation analyses, we verified the role of ALCα in the suppression of amyloidogenic processing of endogenous APP in vivo We observed that ALCα deficiency attenuates the association of X11L with APP, significantly enhances amyloidogenic β-site cleavage of APP, especially in endosomes, and increases the generation of endogenous Aβ in the brain. Furthermore, we noted amyloid plaque formation in the brains of human APP-transgenic mice in an ALCα-deficient background. These results unveil a potential role of ALCα in protecting cerebral neurons from Aβ-dependent pathogenicity in AD.

Caspase Activation and Caspase-Mediated Cleavage of APP Is Associated with Amyloid β-Protein-Induced Synapse Loss in Alzheimer’s Disease

Amyloid β-protein (Aβ) toxicity is hypothesized to play a seminal role in Alzheimer's disease (AD) pathogenesis. However, it remains unclear how Aβ causes synaptic dysfunction and synapse loss. We hypothesize that one mechanism of Aβ-induced synaptic injury is related to the cleavage of amyloid β precursor protein (APP) at position D664 by caspases that release the putatively cytotoxic C31 peptide. In organotypic slice cultures derived from mice with a knock-in mutation in the APP gene (APP D664A) to inhibit caspase cleavage, Aβ-induced synaptic injury is markedly reduced in two models of Aβ toxicity. Loss of dendritic spines is also attenuated in mice treated with caspase inhibitors. Importantly, the time-dependent dendritic spine loss is correlated with localized activation of caspase-3 but is absent in APP D664A cultures. We propose that the APP cytosolic domain plays an essential role in Aβ-induced synaptic damage in the injury pathway mediated by localized caspase activation.

Amyloidogenic processing of Alzheimer's disease β-amyloid precursor protein induces cellular iron retention.

The proteolytic cleavage of β-amyloid precursor protein (APP) to form the amyloid beta (Aβ) peptide is related to the pathogenesis of Alzheimer's disease (AD) because APP mutations that influence this processing either induce familial AD or mitigate the risk of AD. Yet Aβ formation itself may not be pathogenic. APP promotes neuronal iron efflux by stabilizing the cell-surface presentation of ferroportin, the only iron export channel of cells. Mislocalization of APP can promote iron retention, thus we hypothesized that changes in endocytotic trafficking associated with altered APP processing could contribute to the neuronal iron elevation and oxidative burden that feature in AD pathology. Here, we demonstrate, using genetic and pharmacological approaches, that endocytotic amyloidogenic processing of APP impairs iron export by destabilizing ferroportin on the cell surface. Conversely, preferential non-amyloidogenic processing of APP at the cell surface promotes ferroportin stabilization to decrease intraneuronal iron. A new Aβ-independent hypothesis emerges where the amyloidogenic processing of APP, combined with age-dependent iron elevation in the tissue, increases pro-oxidant iron burden in AD.

Galectin 3 binding protein suppresses Aβ production by modulating β-cleavage of amyloid precursor protein (1117)

Galectin 3 binding protein suppresses Aβ production by modulating β-cleavage of amyloid precursor protein (1117)

Galectin 3–binding protein suppresses amyloid-β production by modulating β-cleavage of amyloid precursor protein

Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of β-amyloid (Aβ) peptides. Aβ is produced from amyloid precursor protein (APP) that is sequentially cleaved by β- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aβ production, we performed a gene microarray–based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aβ production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aβ production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3–binding protein (GAL3BP), which suppressed Aβ production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aβ production by directly interacting with APP and thereby inhibiting APP processing by β-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.

Inhibitory effects of curcumin on H2O2-induced cell damage and APP expression and processing in SH-SY5Y cells transfected with APP gene with Swedish mutation.

Alzheimer's disease (AD) is a neurodegenerative disorder, and the pathological mechanism of the disease is still far to understand. According to the amyloid cascade hypothesis in AD, Amyloid-β (Aβ) is considered as a key substance that contributes AD development. Aβ is a β-cleaving product from Amyloid-β protein precursor (APP). Mutations of APP including APPKM670/671670NL (Swedish mutation) result in Aβ overproduction and the development of early-onset familial AD. Increase of oxidative stress and damage also occurs in early stage of AD. In this study, we used a SH-SY5Y cell line that stably expresses APP gene with Swedish mutation (SH-SY5Y-APPswe), and the inhibitory effects of curcumin on H2O2-induced cell damage and APP processing were investigated. Cells were treated with curcumin (0 ~ 5μM) for 4h before hydrogen peroxide (H2O2). Cell growth was detected with CCK-8 assay, and cell damage was determined through the evaluation of release of lactate dehydrogenase (LDH) from the cytosol to the culture medium and the morphological change of nucleus. The ability of mitochondrial stress and the depolarization of mitochondrial membrane potential were assayed through the measuring the oxygen consumption rate (OCR) and the green/red fluorescence ratio of JC-1 dye respectively. The protein levels of APP, sAPPα, sAPPβ, and BACE1 were analyzed with Western blot assay. Aβ production was measured with enzyme-linked immunosorbent assay (ELISA). The results indicated that curcumin inhibits H2O2-induced decrease of cell growth and cell damage. Curcumin attenuates H2O2-induced damage on the ability to mitochondrial oxidative phosphorylation and membrane potential. Curcumin inhibits H2O2-induced increase of APP cleavage through β-cleavage pathway and of intracellular Aβ production. These results imply that curcumin can be used to treat AD through inhibiting oxidative damage-induced APP β-cleavage and intracellular Aβ generation.

Graphene oxide improves postoperative cognitive dysfunction by maximally alleviating amyloid beta burden in mice.

Rationale: Graphene oxide (GO) based nanomaterials have shown potential for the diagnosis and treatment of amyloid-β (Aβ)-related diseases, mainly on Alzheimer's disease (AD). However, these nanomaterials have limitations. How GO is beneficial to eliminate Aβ burden, and its physiological function in Aβ-related diseases, still needs to be investigated. Moreover, postoperative cognitive dysfunction (POCD) is an Aβ-related common central nervous system complication, however, nanomedicine treatment is lacking.Methods: To evaluate the effects of GO on Aβ levels, HEK293T-APP-GFP and SHSY5Y-APP-GFP cells are established. Intramedullary fixation surgery for tibial fractures under inhalation anesthesia is used to induce dysfunction of fear memory in mice. The fear memory of mice is assessed by fear conditioning test.Results: GO treatment maximally alleviated Aβ levels by simultaneously reducing Aβ generation and enhancing its degradation through inhibiting β-cleavage of amyloid precursor protein (APP) and improving endosomal Aβ delivery to lysosomes, respectively. In postoperative mice, the hippocampal Aβ levels were significantly increased and hippocampal-dependent fear memory was impaired. However, GO administration significantly reduced hippocampal Aβ levels and improved the cognitive function of the postoperative mice.Conclusion: GO improves fear memory of postoperative mice by maximally alleviating Aβ accumulation, providing new evidence for the application of GO-based nanomedicines in Aβ-related diseases.

Enhanced amyloid-β generation by γ-secretase complex in DRM microdomains with reduced cholesterol levels.

A neuropathologic hallmark of Alzheimer's disease (AD) is the presence of senile plaques that contain neurotoxic amyloid-β protein (Aβ) species, which are generated by the cleavage of amyloid β-protein precursor by secretases such as the γ-secretase complex, preferentially located in detergent-resistant membrane (DRM) regions and comprising endoproteolysed amino- and carboxy-terminal fragments of presenilin, nicastrin, anterior pharynx defective 1 and presenilin enhancer 2. Whereas some of familial AD patients harbor causative PSEN mutations that lead to more generation of neurotoxic Aβ42, the contribution of Aβ generation to sporadic/late-onset AD remains unclear. We found that the carboxy-terminal fragment of presenilin 1 was redistributed from DRM regions to detergent-soluble membrane (non-DRM) regions in brain tissue samples from individuals with sporadic AD. DRM fractions from AD brain sample had the ability to generate significantly more Aβ and had a lower cholesterol content than DRM fractions from non-demented control subjects. We further demonstrated that lowering the cholesterol content of DRM regions from cultured cells contributed to the redistribution of γ-secretase components and Aβ production. Taken together, the present analyses suggest that the lowered cholesterol content in DRM regions may be a cause of sporadic/late-onset AD by enhancing overall Aβ generation.

Familial L723P Mutation Can Shift the Distribution between the Alternative APP Transmembrane Domain Cleavage Cascades by Local Unfolding of the Ε-Cleavage Site Suggesting a Straightforward Mechanism of Alzheimer's Disease Pathogenesis.

Alzheimer's disease is an age-related pathology associated with accumulation of amyloid-β peptides, products of enzymatic cleavage of amyloid-β precursor protein (APP) by secretases. Several familial mutations causing early onset of the disease have been identified in the APP transmembrane (TM) domain. The mutations influence production of amyloid-β, but the molecular mechanisms of this effect are unclear. The "Australian" (L723P) mutation located in the C-termini of APP TM domain is associated with autosomal-dominant, early onset Alzheimer's disease. Herein, we describe the impact of familial L723P mutation on the structural-dynamic behavior of APP TM domain studied by high-resolution NMR in membrane-mimicking micelles and augmented by molecular dynamics simulations in explicit lipid bilayer. We found L723P mutation to cause local unfolding of the C-terminal turn of the APP TM domain helix and increase its accessibility to water required for cleavage of the protein backbone by γ-secretase in the ε-site, thus switching between alternative ("pathogenic" and "non-pathogenic") cleavage cascades. These findings suggest a straightforward mechanism of the pathogenesis associated with this mutation, and are of generic import for understanding the molecular-level events associated with APP sequential proteolysis resulting in accumulation of the pathogenic forms of amyloid-β. Moreover, age-related onset of Alzheimer's disease can be explained by a similar mechanism, where the effect of mutation is emulated by the impact of local environmental factors, such as oxidative stress and/or membrane lipid composition. Knowledge of the mechanisms regulating generation of amyloidogenic peptides of different lengths is essential for development of novel treatment strategies of the Alzheimer's disease.

Sez6 levels are elevated in cerebrospinal fluid of patients with inflammatory pain-associated conditions.

Seizure-related protein 6 (Sez6) contributes to chronic pain development as sez6 knockout mice show attenuated pain behaviours after peripheral nerve injury, compared with control mice. The type I transmembrane isoform of Sez6 is cleaved by the β-amyloid precursor protein cleavage enzyme 1 (BACE1), resulting in Sez6 extracellular domain shedding from the neuron surface. To determine whether this BACE1-shed form of Sez6 can be detected in the cerebrospinal fluid (CSF) and whether Sez6 levels in the CSF are altered in neuropathic pain or chronic inflammatory pain (IP). We analysed the CSF samples collected during surgery from patients with chronic neuropathic pain (n = 8) or IP (n = 33), comparing them to the CSF samples from patients with suspected subarachnoid haemorrhage that was subsequently excluded (nonsurgical group, n = 5). Western blots were used to determine the relative Sez6 levels in the CSF from the different patient and nonsurgical comparison groups. The results show that BACE1-shed Sez6 can be readily detected in the CSF by Western blot and that the levels of Sez6 are significantly higher in the IP group than in the nonsurgical comparison group. The association between elevated Sez6 levels in the CSF and IP is further evidence for persistent alterations in central nervous system activity in chronic IP conditions.

Reduced Aβ secretion by human neurons underconditions of strongly increased BACE activity.

The initial step in the amyloidogenic cascade of amyloid precursor protein (APP) processing is catalyzed by beta-site APP-cleaving enzyme (BACE), and this protease has increased activities in affected areas of Alzheimer's disease brains. We hypothesized that altered APP processing, because of augmented BACE activity, would affect the actions of direct and indirect BACE inhibitors. We therefore compared post-mitotic human neurons (LUHMES) with their BACE-overexpressing counterparts (BLUHMES). Although β-cleavage of APP was strongly increased in BLUHMES, they produced less full-length and truncated amyloid beta (Aβ) than LUHMES. Moreover, low concentrations of BACE inhibitors decreased cellular BACE activity as expected, but increased Aβ1-40 levels. Several other approaches to modulate BACE activity led to a similar, apparently paradoxical, behavior. For instance, reduction in intracellular acidification by bepridil increased Aβ production in parallel with decreased BACE activity. In contrast to BLUHMES, the respective control cells (LUHMES or BLUHMES with catalytically inactive BACE) showed conventional pharmacological responses. Other non-canonical neurochemical responses (so-called 'rebound effects') are well-documented for the Aβ pathway, especially for γ-secretase: a partial block of its activity leads to an increased Aβ secretion by some cell types. We therefore compared LUHMES and BLUHMES regarding rebound effects of γ-secretase inhibitors and found an Aβ rise in LUHMES but not in BLUHMES. Thus, different cellular factors are responsible for the γ-secretase- versus BACE-related Aβ rebound. We conclude that increased BACE activity, possibly accompanied by an altered cellular localization pattern, can dramatically influence Aβ generation in human neurons and affect pharmacological responses to secretase inhibitors. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.