APP Protein Research Articles

Systemic Exposure to Air Pollution Induces Oxidative Stress and Inflammation in Mouse Brain, Contributing to Neurodegeneration Onset.

In northern Italy, biomass burning-derived (BB) particles and diesel exhaust particles (DEP) are considered the most significant contributors to ultrafine particle (UFP) emission. However, a comparison between their impact on different brain regions was not investigated until now. Therefore, male BALB/c mice were treated with a single or three consecutive intratracheal instillations using 50 µg of UFPs in 100 µL of isotonic saline solution or 100 µL of isotonic saline solution alone, and brains were collected and analyzed. Proteins related to oxidative stress and inflammation, as well as Alzheimer’s disease markers, were examined in the hippocampus, cerebellum, and the rest of the brain (RoB). Histopathological examination of the brain was also performed. Moreover, correlations among different brain, pulmonary, and cardiovascular markers were performed, allowing us to identify the potentially most stressful UFP source. Although both acute exposures induced inflammatory pathways in mouse brain, only DEP showed strong oxidative stress. The sub-acute exposure also induced the modulation of APP and BACE1 protein levels for both UFPs. We observed that DEP exposure is more harmful than BB, and this different response could be explained by this UFP’s different chemical composition and reactivity.

Alzheimer\u2019s disease progression characterized by alterations in the molecular profiles and biogenesis of brain extracellular vesicles

BackgroundThe contributions of brain intercellular communication mechanisms, specifically extracellular vesicles (EV), to the progression of Alzheimer’s disease (AD) remain poorly understood.MethodsHere, we investigated the role(s) of brain EV in the progressive course of AD through unbiased proteome-wide analyses of temporal lobe-derived EV and proteome-label quantitation of complementary remaining brain portions. Furthermore, relevant proteins identified were further screened by multiple reaction monitoring.ResultsOur data indicate that EV biogenesis was altered during preclinical AD with the genesis of a specific population of EV containing MHC class-type markers. The significant presence of the prion protein PrP was also manifested in these brain vesicles during preclinical AD. Similarly, sequestration of amyloid protein APP in brain EV coincided with the observed PrP patterns. In contrast, active incorporation of the mitophagy protein GABARAP in these brain vesicles was disrupted as AD progressed. Likewise, disrupted incorporation of LAMP1 in brain EV was evident from the initial manifestation of AD clinical symptoms, although the levels of the protein remained significantly upregulated in the temporal lobe of diseased brains.ConclusionsOur findings indicate that impaired autophagy in preclinical AD coincides with the appearance of proinflammatory and neuropathological features in brain extracellular vesicles, facts that moderately remain throughout the entire AD progression. Thus, these data highlight the significance of brain EV in the establishment of AD neuropathology and represent a further leap toward therapeutic interventions with these vesicles in human dementias.

Alterations in seminal plasma proteomic profile in men with primary and secondary infertility

Proteome of seminal plasma provides profound information related to the male reproductive health. This pilot study was conducted to characterize proteomic profile of seminal plasma from men with primary, or secondary infertility and compare it with proven fertile men. Study participants (n = 59) were recruited at the Cleveland Clinic and divided according to their fertility status: proven fertile (n = 39); primary infertility (n = 11) and secondary infertility (n = 9). Proteomic shotgun analysis revealed a total of 515 peptides common to primary infertility and control group; whereas 523 peptides were common to secondary infertility and control group. Bioinformatic analysis revealed dysregulation of biological processes such as cell secretion and vesicle mediated transport in primary infertility, whereas immune system response, regulation of proteolysis and iron homeostasis were dysregulated in secondary infertility. Western blot validation showed overexpression of ANXA2 and CDC42, and underexpression of SEMG2 proteins in primary infertility; and overexpression of ANXA2 and APP proteins in secondary infertility. This study elucidates the potential role of differentially expressed proteins in the seminal plasma as diagnostic biomarker for primary and secondary infertility. Furthermore, our results suggest maturation failure and immune reaction response as the main cause of infertility in men with primary and secondary infertility, respectively. Additional validation of the proteins involved in the above pathways is warranted.

Mitigating Effect of Korea Red Ginseng Against Multi‐Tissue Iipotoxicity Caused by High‐Fat Diet Feeding

BackgroundsExcessive consumption of fat and its aberrant deposit in the body has been linked to the low‐grade inflammation in various susceptible organs and eventually causes tissue toxicity. This study examines the multifaceted suppressive effects of Korean Red ginseng extract (KRG) against high‐fat diet (HFD)‐induced lipotoxicity and inflammatory responses occurring in the liver, aorta, and brain.MethodsThe standardized KRG powder was prepared by Korea Ginseng Corporation and was dissolved in phosphate buffered saline as a concentration of 30 mg/mL. Male C57BL/6 mice were fed HFD with or without KRG (10 or 50 mg/kg body weight; orally) for 12 weeks (n = 10 per group). The anti‐inflammatory potential of KRG was determined in the blood and the aorta, liver, and brain.ResultsKRG significantly inhibited HMG‐CoA reductase activity in vitro. In vivo, KRG supplementation suppressed HFD‐associated body weight gain, lipid profile changes, excessive fat deposition in the liver, and increases of leptin, insulin, and ALT levels in the blood. Inflammatory markers of the aorta (ICAM‐1 protein level and wall thickening of the aorta), liver (COX‐2 expression and liver function tests), and brain (APP, BACE1, and Tau protein levels) were also significantly reduced by KRG. In microvascular endothelial cells, 15% cyclic stretch‐mediated upregulation of ICAM‐1 and VCAM‐1 expression was significantly attenuated in the presence of KRG.ConclusionKRG supplementation attenuates HFD‐mediated body weight gain, lipid profile changes, and multi‐tissue inflammatory responses including the liver, aorta, and brain.

Characterization and Neuroprotection Potential of Seleno-Polymannuronate.

Seleno-polymannuronate (Se-PM) was prepared from alginate-derived polymannuronate (PM) through a sulfation followed by a selenylation replacement reaction. The organic selenium content of Se-PM was 437.25 μg/g and its average molecular weight was 2.36 kDa. The neuroprotection effect of Se-PM and corresponding molecular mechanisms were investigated. Our results showed that, comparing to both sulfated PM (S-PM) and PM, Se-PM remarkably inhibited the aggregation of Aβ1–42 oligomer in vitro and significantly reduced the APP and BACE1 protein expression in N2a-sw cells, highlighting the critical function of the selenium presented in Se-PM. Moreover, Se-PM decreased the expression of cytochrome c and the ratio of Bax to Bcl-2, and enhanced the mitochondrial membrane potential in N2a-sw cells. These results suggested that Se-PM treatment can markedly inhibit N2a-sw cell apoptosis and promote N2a-sw cell survival and that Se-PM might be a potential therapeutic agent for the prevention of neurodegeneration owing to its remarkable neuroprotection effect.

Modulatory Effects of Ginkgo biloba Against Amyloid Aggregation Through Induction of Heat Shock Proteins in Aluminium Induced Neurotoxicity.

Protein misfolding and aggregation of amyloid beta (Aβ) peptide, as well as formation of neurofibrillary tangles (NFTs) are the signature hallmarks of Alzheimer's disease (AD) pathology. To prevent this, molecular chaperones come into play as they facilitate the refolding of the misfolded proteins and cell protection under stress. Here, we have evaluated the possible effects of Ginkgo biloba (GBE) against aggregation of the Aβ through activation of heat shock proteins (HSPs) in the Aluminium (Al) induced AD based model. GBE (100mg/kg body weight) was administered per oral to the female SD rats in conjunction with intraperitoneal (i.p.) injection of Al lactate (10mg/kg body weight) for six weeks. Pretreated animals were administered GBE for additional two weeks prior to any exposure of Al. GBE administration resulted in decrease in Aβ aggregation, ubiquitin deposition, accompanying a significant decline in APP & Tau protein hyperphosphorylation which can be attributed to activation of Heat shock factor (HSF-1) and upregulation in the protein expression of HSPs. Histopathological investigation studies have also shown the decrease in aggregation of Aβ peptide by GBE administration. Additionally, the decrease in ROS levels and Aβ aggregation by GBE administration prohibited the decline in the neurotransmitter levels and monoamine oxidase levels in hippocampus and cortex. This further caused improvement in learning and memory of the animals. In conclusion, our results indicate that GBE prevents the symptoms of Al induced AD like pathophysiology by upregulating the HSPs levels and decreasing the aggregation load.

Purification of Full-Length <i>β</i>-Secretase Involved in Alzheimer’s Disease, and Proteomic Identification of Binding Partners

β-Secretase (BACE1 or β-site APP cleaving enzyme) is an acid protease that releases the neurotoxic 40 - 42 residue peptides (β-amyloid or A-β) from its glycoprotein precursor, (APP or amyloid precursor protein) which when released in brain is thought to give rise to cognitive decline in patients with Alzheimer’s Disease. Most structural studies on β-secretase have previously been performed with recombinant forms of the protease, in which the transmembrane coding region has been deleted. However, interactions with proteins of the same species are best studied using the full-length β-secretase as interactions are likely to be influenced by the hydrophobic nature and localization of its transmembrane regions. Here we develop a multi-step purification procedure that isolates a complex containing BACE1 from recombinant human cells using mild detergents in a procedure that retains other proteins within the complex and remains active in its β-site APP cleaving activity. Some of these proteins, eg reticulon 4, are identified by proteomics, and are known by previous studies performed by others to regulate the activity of BACE1 against APP. These interactions may aid the development of small proteins and peptides that could inhibit the release of aggregated forms of β-amyloid, and thus be useful therapeutically.

Icaritin Improves Memory and Learning Ability by Decreasing BACE-1 Expression and the Bax/Bcl-2 Ratio in Senescence-Accelerated Mouse Prone 8 (SAMP8) Mice.

Icaritin (ICT) is the main component in the traditional Chinese herb Epimedium, and it has been shown to have anti-Alzheimer's disease (AD) effects, but its neuroprotective effects and the pharmacological mechanisms are unclear. In the present study, senescence-accelerated mouse prone 8 (SAMP8) mice were randomly divided into a model group and an ICT-treated group. Learning and memory abilities were detected by the Morris water maze assay, and the expression of amyloid beta protein (Aβ) and β-site APP cleavage enzyme 1 (BACE1) was determined by Western blotting and polymerase chain reaction (PCR). Histological changes in CA1 and CA3 were detected by hematoxylin-eosin staining (H&E staining), and the immunohistochemical analysis was used to detect the expression and localization of Bax and Bcl-2. The results showed that compared with the SAMP8 mice, the ICT-treated SAMP8 mice showed improvements in spatial learning and memory retention. In addition, the number of necrotic cells and the morphological changes in CA1 and CA3 areas were significantly alleviated in the group of ICT-treated SAMP8 mice, and the expression of BACE1, Aβ1-42 levels, and the Bax/Bcl-2 ratio in the hippocampus was obviously decreased in the ICT-treated group compared with the control group. The results demonstrated that ICT reduced BACE-1 levels, the contents of Aβ1-42, and the Bax/Bcl-2 ratio, suggesting that ICT might have potential therapeutic benefits by delaying or modifying the progression of AD.

Edaravone Exerts Brain Protective Function by Reducing the Expression of AQP4, APP and Aβ Proteins.

This study aims to investigate the changes of aquaporin-4 (AQP4), β-amyloid precursor proteins (APP) and β-amyloid (Aβ) in brain tissues after cerebral ischemiareperfusion injury (CIRI), and evaluate the effect of edaravone. The Middle Cerebral Artery Occlusion was used to establish CIRI in rats. Rats were divided into control, model and edaravone groups. The neurological deficits in the model group were obvious and the neurological score increased compared to the control group, while the neurological deficits of the edaravone group were improved as the neurological score decreased compared to the model group. The number of pyramidel cells in the hippocampus of the model group was significantly decreased whereas edaravone could reverse this decrease. The model group had significantly higher levels of Aβ, APP and AQP4 than the control group and edaravone group, suggesting that they might be involved in the neuronal cell damage. Meanwhile, the increased AQP4 might enhance the permeability of cells, and thus cause cell damage and neurological deficit. Conclusively, edaravone could reduce brain edema, protect neuronal cells and improve the neurological impairment of rats possibly by decreasing the expression of Aβ, APP and AQP4. Therefore, edaravone may have the potential to treat neurodegenerative diseases (such as Alzheimer's disease).

P2‐091: SV40T‐MEDIATED NEURONAL CELL CYCLE DYSREGULATION IN MICE INDUCES Aβ AND TAU PATHOLOGIES AND NEUROINFLAMMATION

The majority of the Alzheimer's disease (AD) cases are sporadic and generate Aβ plaques and neurofibrillary tangles in the absence of mutations in the APP or tau proteins. The presence of cell cycle markers in postmortem AD brains has led a number of researchers to suggest a potential role of cell cycle activation in AD. My previously published findings demonstrated that cell cycle induction in postmitotic neurons in mice produces Aβ and tau pathologies in the absence of APP or tau mutations (Park et al., 2007, J Neurosci). Our recent work examines the microglial and astrocytic responses in these mice. SV40T expression was restricted to postmitotic neurons in our mice using the tet-off system. Our inducible transgenic mice express SV40T under the regulation of TRE promoter (Tag mice). Tag mice were paired with Camk2a-tTA transgenic mice (Off mice) and maintained on doxycycline (dox) diet. The offspring were maintained on dox diet until 30 days of age, at which point they were continuously maintained on standard diet. Tag mice were used as SV40T transgene controls. Animals were examined at 2, 4, 6, and 12 months of age. Vibratome sectioned brains were immunofluorescent labeled for Iba1, CD45, CD68, MHCII and GFAP. Cellular senescence was assessed using anti-p16 antibody. Microglia and astrocyte activations are observed as early as 2 months of age after neuronal cell cycle activation. Tag/Off mice demonstrated increase in GFAP immunolabeling compared to Tag mice, indicative of astrocyte activation. Tag/Off mice also showed increased Iba1 immunostaining compared to Tag mice. A subset of Iba1-positive microglia showed co-labeling with CD45, CD68, or MHCII antibodies, demonstrating activated microglia. At 12-months of age, microglia demonstrates blebbing and p16 immunopositivity, both hallmarks of senescent phenotype. Our Tag/Off mice demonstrate early and persistent activation of microglia and astrocytes. Additionally, these mice develop senescent microglia phenotype along with Aβ and tau pathologies following chronic and persistent neuronal cell cycle activation. Our mice simultaneously display the major pathological features of AD in the absence of APP or tau mutations, and thereby may represent a sporadic AD mouse model.

Ganglioside GQ1b ameliorates cognitive impairments in an Alzheimer\u2019s disease mouse model, and causes reduction of amyloid precursor protein

Brain-derived neurotrophic factor (BDNF) plays crucial roles in memory impairments including Alzheimer’s disease (AD). Previous studies have reported that tetrasialoganglioside GQ1b is involved in long-term potentiation and cognitive functions as well as BDNF expression. However, in vitro and in vivo functions of GQ1b against AD has not investigated yet. Consequently, treatment of oligomeric Aβ followed by GQ1b significantly restores Aβ1–42-induced cell death through BDNF up-regulation in primary cortical neurons. Bilateral infusion of GQ1b into the hippocampus ameliorates cognitive deficits in the triple-transgenic AD mouse model (3xTg-AD). GQ1b-infused 3xTg-AD mice had substantially increased BDNF levels compared with artificial cerebrospinal fluid (aCSF)-treated 3xTg-AD mice. Interestingly, we also found that GQ1b administration into hippocampus of 3xTg-AD mice reduces Aβ plaque deposition and tau phosphorylation, which correlate with APP protein reduction and phospho-GSK3β level increase, respectively. These findings demonstrate that the tetrasialoganglioside GQ1b may contribute to a potential strategy of AD treatment.

Mitochondrial methionine sulfoxide reductase B2 links oxidative stress to Alzheimer's disease-like pathology

Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial protein that protects cell from oxidative stress. The antioxidant activity suggests that MSRB2 may play a role in the pathophysiology of Alzheimer's disease (AD). Here, we report that in APP/PS1 mice, an animal model of AD, MSRB2 protein levels were decreased in the hippocampus at both young (6 mon) and old (18 mon) age, and in the cortex only at an old age, respectively. In HEK293 cells that stably express human full-length β-amyloid precursor protein (APP, HEK/APP), MSRB2 reduced the protein and mRNA levels of APP and β-amyloid converting enzyme 1 (BACE1), and the consequent amyloid beta peptide (Aβ) 1–40 and Aβ1-42 levels. MSRB2 overexpression or knockdown also oppositely affected Tau phosphorylation at selective sites, with the concomitant alteration of the phosphorylated extracellular signal regulated kinase (p-ERK) and AMP-activated protein kinase (p-AMPK) levels. Moreover, in cells treated with long-term (24 h) hydrogen peroxide, the alterations of APP processing and Tau phosphorylation were reversed by MSRB2 overexpression. We further found that MSRB2-mediated regulation of APP transcription involved JNK and ERK signaling, as MSRB2 also reduced the levels of phosphorylated JNK (p-JNK), and JNK or ERK inhibitor attenuated the effect of MSRB2 on APP proteins and transcripts. Finally, MSRB2 reduced apoptosis-related proteins Bax and caspase3 and enhanced the anti-apoptotic protein Bcl2. These results indicated that the role for MSRB2 in AD-like pathology was closely associated with its antioxidant activity. By attenuating both amyloidogenesis and Tau phosphorylation, MSRB2 may serve as a potential therapeutic target for AD.

Microvesicles from cerebrospinal fluid of patients with Alzheimer\u2019s disease display reduced concentrations of tau and APP protein

Microvesicles are small membranous particles generated during cellular activation or stress. The analysis of the content and the surface of microvesicles allow conclusions about the cells they are originating from and the underlying pathology. Therefore, CSF microvesicles have been suggested to be promising targets to monitor the (etio)pathology of neurodegenerative diseases. Microvesicles in the CSF of 15 patients with Alzheimer’s disease and 15 controls were analyzed by flow cytometry regarding the levels of CD3, CD4, CD45, CD64, BACE1, Aβ, APP and tau. The results were replicated in a second cohort comprising 14 patients with Alzheimer’s disease and 9 controls. The levels of tau and APP were reduced in microvesicles of Alzheimer’s disease patients. A significant change was neither observed in the number of microvesicles nor in the expression of the other antigens. Tau and APP in microvesicles separated patients with Alzheimer’s disease from controls with an AUC of 0.84 and 0.89 respectively. We conclude that tau and APP in CSF microvesicles are promising biomarkers which could directly provide information about the Alzheimer pathology on a cellular level.

Effect of Aluminum-Maltolate on the Content of Aβ Protein and the Expression of ApoER2, VLDLRs, and LRP1 in PC12-ApoE4 Cells.

To investigate the effect of aluminum-maltolate [Al(mal)3] on the expression of ApoER2, VLDLRs, and LRP1 in PC12-ApoE4 cells. The lentiviral vector carrying human ApoE4 gene was transfected into PC12 cells; after screening with puromycin, PC12 cells carrying ApoE4 gene (PC12-ApoE4 cells) were established. After 24-h treatment with Al(mal)3, the cell survival rate was measured by CCK-8 assay. The expression of Aβ40 and Aβ42 was detected by ELISA assay; the expression of the APP, ApoER2, LRP1, and VLDLRs genes was detected by RT-PCR, and Western blot assay was used to detect the expression of the APP, ApoER2, LRP1, and VLDLRs proteins. Factorial experiment design was performed to analyze interaction between cell type and Al dose. Al(mal)3 treatment induced dose-dependent decreases of survival rate in the two cell groups and dose-dependent increases of Aβ42 content(P< 0.05). The expressions of ApoER2, LRP1, and VLDLR proteins and their mRNA transcription decreased gradually with the increase of Al(mal)3 doses (P < 0.05), while the expression of APP protein and mRNA transcription gradually increased with the increase of Al(mal)3 doses (P < 0.05). As regard to the interaction of cell type and Al dose, the decrease of cell survival rate and the increase of the Aβ42 were both statistically significant (P< 0.05). And the decrease of ApoER2 and LRP1 proteins was both statistically significant too (P< 0.05). The effect of Al(mal)3 and ApoE4 gene on the survival rate and the increase of Aβ content in PC12 cells. That is to say, there is interaction between ApoE4 gene and aluminum on the Aβ content, especially the change of the Aβ42 content, which may be related to the down-regulation of the expression of ApoER2 and LRP1 proteins.

Partial loss of endothelial nitric oxide leads to increased cerebrovascular beta amyloid

Cerebral amyloid angiopathy (CAA) is present in over half of the elderly population and in 80–90% of Alzheimer’s disease (AD) patients. CAA is defined by the deposition of beta amyloid (Aβ) in small cerebral arteries and capillaries. Cardiovascular risk factors are associated with an increased incidence of CAA. We utilized 18-month-old endothelial nitric oxide synthase (eNOS) heterozygous knockout (+/−) mice, a clinically relevant model of endothelial dysfunction, to examine the role of endothelial nitric oxide (NO) in vascular Aβ accumulation. eNOS+/− mice had significantly higher vascular levels of Aβ40 (P < 0.05). Aβ42 was not detected. There was no difference in Aβ in brain tissue. Amyloid precursor protein and β-site APP cleavage enzyme 1 protein levels were unaltered, while levels of the α-secretase enzyme, a disintegrin and metalloproteinase 10, were significantly lower in eNOS + /− microvascular tissue (P < 0.05). Insulin degrading enzyme and low-density lipoprotein receptor-related protein 1 were significantly increased in eNOS+/− microvascular tissue, most likely an adaptive response to locally higher Aβ concentrations. Lastly, catalase and CuZn superoxide dismutase were significantly elevated in eNOS+/− microvascular tissue (P < 0.05). These data demonstrate decreased availability of endothelial NO leads to increased cerebrovascular concentration of Aβ along with compensatory mechanisms to protect the vasculature.

Novel upregulation of amyloid-\u03b2 precursor protein (APP) by microRNA-346 via targeting of APP mRNA 5\u2032-untranslated region: Implications in Alzheimer\u2019s disease

In addition to the devastating symptoms of dementia, Alzheimer’s disease (AD) is characterized by accumulation of the processing products of the amyloid-β (Aβ) peptide precursor protein (APP). APP’s non-pathogenic functions include regulating intracellular iron (Fe) homeostasis. MicroRNAs are small (~ 20 nucleotides) RNA species that instill specificity to the RNA-induced silencing complex (RISC). In most cases, RISC inhibits mRNA translation through the 3′-untranslated region (UTR) sequence. By contrast, we report a novel activity of miR-346: specifically, that it targets the APP mRNA 5′-UTR to upregulate APP translation and Aβ production. This upregulation is reduced but not eliminated by knockdown of argonaute 2. The target site for miR-346 overlaps with active sites for an iron-responsive element (IRE) and an interleukin-1 (IL-1) acute box element. IREs interact with iron response protein1 (IRP1), an iron-dependent translational repressor. In primary human brain cultures, miR-346 activity required chelation of Fe. In addition, miR-346 levels are altered in late-Braak stage AD. Thus, miR-346 plays a role in upregulation of APP in the CNS and participates in maintaining APP regulation of Fe, which is disrupted in late stages of AD. Further work will be necessary to integrate other metals, and IL-1 into the Fe-miR-346 activity network. We, thus, propose a “FeAR” (Fe, APP, RNA) nexus in the APP 5′-UTR that includes an overlapping miR-346-binding site and the APP IRE. When a “healthy FeAR” exists, activities of miR-346 and IRP/Fe interact to maintain APP homeostasis. Disruption of an element that targets the FeAR nexus would lead to pathogenic disruption of APP translation and protein production.

Methamphetamine exposure induces neuropathic protein β-Amyloid expression

Methamphetamine (METH) abusing contributes to dopaminergic neurons degeneration, resulting inParkinson's disease (PD)-like changes. More recently, the association between METH exposure and the Alzheimer's disease (AD)-like changes gained more attention, however, the underlying mechanisms remain poorly understood. In the present study, we aimed to investigate whether METH exposure promotes the formation of Aβ42, one of the key AD-like pathological proteins. With the cell model PC-12 cell line, it showed that METH treatment significantly increased the level of the precursor protein APP and its hydrolysates CTFs expression in a dose-dependent manner. In parallel, with the ELISA assay, we found that METH exposure contributed to an obvious elevation of the Aβ1–42 excretion in the cell culture supernatant. Therefore, we examined the expression of p-GSK3α and BACE-1, which were responsible for APP and Aβ1–42 generation respectively, it suggested in that METH obviously activated the p-GSK3α and increased the level of BACE-1, and the expression of BACE-1 was also detected by the immunofluorescence, with the significant elevation of the BACE-1 fluorescence intensity. In conclusion, METH treatment promotes the expression of Aβ precursor protein APP and its hydrolysis product CTFs and Aβ1–42, and p-GSK3α as well as BACE-1 may be involved in this process.

Functional amyloidogenesis in immunocytes from the colonial ascidian Botryllus schlosseri: Evolutionary perspective

Cytotoxic morula cells (MCs) and phagocytes are the circulating immunocytes of the colonial ascidian Botryllus schlosseri: Both these cells can synthesise amyloid fibrils, supporting the idea that physiological amyloidogenesis is involved in inflammation and modulation of immune responses. Intriguingly, amyloid of B. schlosseri immunocytes is made of two different proteins. MCs, the first cells to sense non-self and involved in the allorejection reaction between contacting genetically incompatible colonies, use melanin encapsulation as the principal method to fight non-self. They release amyloid fibrils formed by p102 protein that allow the packaging and deposit of melanin and other toxic molecules nearby the invader or in the contact region of incompatible colonies. Phagocytes release amyloid-based extracellular traps when challenged with microbes: their amyloid fibrils harbour BsAPP, an orthologue of the vertebrate amyloidogeneic protein APP. This strategy of immune response, present also in human neutrophils, allows phagocytes to block and engulf bacteria and fungi.

Lipid bilayer position and orientation of novel carprofens, modulators of γ-secretase in Alzheimer's disease

γ-Secretase is an integral membrane protein complex and is involved in the cleavage of the amyloid precursor protein APP to produce amyloid-β peptides. Amyloid-β peptides are considered causative agents for Alzheimer's disease and drugs targeted at γ-secretase are investigated as therapeutic treatments. We synthesized new carprofen derivatives, which showed γ-secretase modulating activity and determined their precise position, orientation, and dynamics in lipid membranes by combining neutron diffraction, solid-state NMR spectroscopy, and molecular dynamics simulations. Our data indicate that the carprofen derivatives are inserted into the membrane interface, where the exact position and orientation depends on the lipid phase. This knowledge will help to understand the docking of carprofen derivatives to γ-secretase and in the design of new potent drugs. The approach presented here promises to serve as a general guideline how drug/target interactions in membranes can be analyzed in a comprehensive manner.

Quantifying the Proteolytic Cleavage of Plasma Membrane Proteins in Living Cells.

The standard approach to study the activity of proteases consists of lysing cells and measuring the changes in the fluorescence properties of a synthetic substrate after cleavage in vitro. Here, a general protocol that uses a bi-fluorescent chimeric construct of a known substrate protein that follows the proteolytic processing in living cells is described. This approach is useful, in particular, to search for pharmacological conditions altering the cleavage rate of a certain protease, or to investigate the biological factors influencing a certain proteolytic mechanism. Three different methods (microscopy, flow cytometry, and spectroscopy) to detect fluorescence changes due to alteration in the processing are described. This approach was originally developed for studying conditions affecting the proteolytic activity of the β-secretase Bace1 on the amyloid precursor protein APP, but can in principle be applied to investigate any membrane protein undergoing ectodomain shedding by proteolytic cleavage. © 2018 by John Wiley & Sons, Inc.