Alzheimer's Amyloid Precursor Research Articles

Iron response elements (IREs)-mRNA of Alzheimer's amyloid precursor protein binding to iron regulatory protein (IRP1): a combined molecular docking and spectroscopic approach

The interaction between the stem-loop structure of the Alzheimer's amyloid precursor protein IRE mRNA and iron regulatory protein was examined by employing molecular docking and multi-spectroscopic techniques. A detailed molecular docking analysis of APP IRE mRNA∙IRP1 reveals that 11 residues are involved in hydrogen bonding as the main driving force for the interaction. Fluorescence binding results revealed a strong interaction between APP IRE mRNA and IRP1 with a binding affinity and an average binding sites of 31.3 × 106 M−1 and 1.0, respectively. Addition of Fe2+(anaerobic) showed a decreased (3.3-fold) binding affinity of APP mRNA∙IRP1. Further, thermodynamic parameters of APP mRNA∙IRP1 interactions were an enthalpy-driven and entropy-favored event, with a large negative ΔH (–25.7 ± 2.5 kJ/mol) and a positive ΔS (65.0 ± 3.7 J/mol·K). A negative ΔH value for the complex formation suggested the contribution of hydrogen bonds and van der Waals forces. The addition of iron increased the enthalpic contribution by 38% and decreased the entropic influence by 97%. Furthermore, the stopped-flow kinetics of APP IRE mRNA∙IRP1 also confirmed the complex formation, having the rate of association (kon) and the rate of dissociation (koff) as 341 μM−1 s−1, and 11 s−1, respectively. The addition of Fe2+ has decreased the rate of association (kon) by ~ three-fold, whereas the rate of dissociation (koff) has increased by ~ two-fold. The activation energy for APP mRNA∙IRP1 complex was 52.5 ± 2.1 kJ/mol. The addition of Fe2+ changed appreciably the activation energy for the binding of APP mRNA with IRP1. Moreover, circular dichroism spectroscopy has confirmed further the APP mRNA∙IRP1 complex formation and IRP1 secondary structure change with the addition of APP mRNA. In the interaction between APP mRNA and IRP1, iron promotes structural changes in the APP IRE mRNA∙IRP1 complexes by changing the number of hydrogen bonds and promoting a conformational change in the IRP1 structure when it is bound to the APP IRE mRNA. It further illustrates how IRE stem-loop structure influences selectively the thermodynamics and kinetics of these protein-RNA interactions.

Abstract 1397: Targeting the Iron Response Element (IRE)-mRNA of Alzheimer's Amyloid Precursor Protein for Binding to Iron Response Protein (IRP1)

Abstract 1397: Targeting the Iron Response Element (IRE)-mRNA of Alzheimer's Amyloid Precursor Protein for Binding to Iron Response Protein (IRP1)

NeuronRead, an open source semi-automated tool for morphometric analysis of phase contrast and fluorescence neuronal images.

Neurons are specialized cells of the Central Nervous System whose function is intricately related to the neuritic network they develop to transmit information. Morphological evaluation of this network and other neuronal structures is required to establish relationships between neuronal morphology and function, and may allow monitoring physiological and pathophysiologic alterations. Fluorescence-based microphotographs are the most widely used in cellular bioimaging, but phase contrast (PhC) microphotographs are easier to obtain, more affordable, and do not require invasive, complicated and disruptive techniques. Despite the various freeware tools available for fluorescence-based images analysis, few exist that can tackle the more elusive and harder-to-analyze PhC images. To surpass this, an interactive semi-automated image processing workflow was developed to easily extract relevant information (e.g. total neuritic length, average cell body area) from both PhC and fluorescence neuronal images. This workflow, named 'NeuronRead', was developed in the form of an ImageJ macro. Its robustness and adaptability were tested and validated on rat cortical primary neurons under control and differentiation inhibitory conditions. Validation included a comparison to manual determinations and to a golden standard freeware tool for fluorescence image analysis. NeuronRead was subsequently applied to PhC images of neurons at distinct differentiation days and exposed or not to DAPT, a pharmacological inhibitor of the γ-secretase enzyme, which cleaves the well-known Alzheimer's amyloid precursor protein (APP) and the Notch receptor. Data obtained confirms a neuritogenic regulatory role for γ-secretase products and validates NeuronRead as a time- and cost-effective useful monitoring tool.

Alzheimer's disease against peptides products of enzymatic cleavage APP protein: Biological, pathobiological and physico-chemical properties of fibrillating peptides.

Various peptides products of enzymatic cleavage of key for Alzheimer's disease Amyloid Precursor Protein (APP) are well known, but still are matter of scientific debate. The Aβ type products are especially challenging for experimental and medical research. This paper outlines several, still poorly known, biological and medical processes such as peptides biology, i.e., formation, biodistribution, translocation, transport and finally removal from brain compartments and body fluids like Intracellular Fluid (ICF), Cerebrospinal Fluid (CSF), Interstitial Fluid (ISF), blood serum or urine. In addition, the following studies concerning AD patients might prove challenging and simultaneously promising: peptides translocation through Blood-Brain - Barrier (BBB) and Blood-Cerebrospinal Fluid Barrier (BCSFB) and their removal from the brain according to a new concept of glymphatic system; - diagnostic difficulties that stem from physico-chemical properties and the nature of proteins or fibrillating peptides itself like low concentration, short half-live and from experimental-technical problems as well like high adsorption or low solubility of Aβ, tau or amylin. The study of diagnostic parameters is very important, as it may better reflect early changes before the disease develops; one such parameter is the Aβ42/Aβ40 ratio, or the ratio with the total tau concentration combination and other new biomarkers like Aβ1-38; other factors include oxidative stress and inflammation process proteins, complement factor H, alpha-2-macroglobulin, or clusterin. The study of various forms of pathological amyloid deposits that emerge in different but specific brain regions AD patients seems to be crucial as well. The composition of the first initial pathological, pre-fibrillating monomers of fibrillating peptides and their role in AD development and disease progression have been described as well. They are even more challenging for science and simultaneously might be more promising in early diagnosis for AD patients. As always in science, research leads to endless discoveries and further inquiry. Fundamental problems in this field most probably are still far from being definitively comprehended, and multiple crucial questions await better answers. What we really need is to study more and deeper into this matter.

Crosstalk between angiotensin and the nonamyloidogenic pathway of Alzheimer's amyloid precursor protein

The association between hypertension and an increased risk for Alzheimer's disease (AD) and dementia is well established. Many data suggest that modulation of the renin-angiotensin system may be meaningful for the prevention and therapy of neurodegenerative disorders, in particular AD. Proteolytic cleavage of the amyloid precursor protein (APP) by α-secretase precludes formation of neurotoxic Aβ peptides and is expected to counteract the development of AD. An established approach for the up-regulation of α-secretase cleavage is the activation of G protein-coupled receptors (GPCRs). Therefore, our study aimed to analyze whether stimulation of angiotensin AT1 or AT2 receptors stably expressed in HEK cells influence the nonamyloidogenic pathway of APP processing. Treatment of both receptors with angiotensin II clearly showed that only activation of the AT1 receptor increased several fold the α-secretase-mediated shedding of APP. This effect was completely abolished by treatment with the AT1 receptor-specific antagonist telmisartan. Using the BIM-46187 inhibitor, we demonstrate that the Gαq protein-mediated pathway is involved in this stimulation process. Stimulation of AT1 receptors with the β-arrestin-biased agonist SII was ineffective regarding α-secretase-mediated APP shedding. This result discloses that only the G protein-dependent pathway is involved in the Ang II-induced APP shedding. Blocking of Gβγ subunits by the inhibitor gallein completely prevented constitutive and Ang II-induced APP shedding. Our findings provide evidence that induction of APP shedding via Ang II/AT1 receptor stimulation is effected by G protein activation with Gβγ subunits playing important roles.

Alteration of Alzheimer Amyloid Precursor Protein after Treatment with Carbamazepine in Colon Cancer

Background: Amyloid precursor protein (APP), which is involved in cell proliferation, is observed to be over expressed in human cancer. Thus, it is crucial to investigate its role in cancer and suggest treatment strategies to alter its effects on cell growth. Objectives: The main purpose of this study was to determine APP concentrations in colon cancer SW480 cell line after treatment with histone deacetylase inhibitors (HDACIs), namely valproic acid (VPA) and carbamazepine (CBZ). Methods: In the present experimental study, carried out during the period 2014 - 2016 in Iran, 1000000 cells were seeded and incubated in a six-well plate for each sample preparation. The cells were then treated with drugs and APP was evaluated. The concentration of this protein was detected using the enzyme-linked immunosorbent assay (ELISA) method. Results: We found that treatment with CBZ (P < 0.001) and VPA (P < 0.001) reduced the APP levels significantly compared to controls. The APP level reduction by CBZ was 41.6% more than that by VPA. Conclusions: The results of this study suggest that APP reduction by HDACIs can apparently play an important role in the treatment of colon cancer.

A role for amyloid precursor protein translation to restore iron homeostasis and ameliorate lead (Pb) neurotoxicity.

Iron supplementation ameliorates the neurotoxicity of the environmental contaminant lead (Pb); however, the mechanism remains undefined. Iron is an essential nutrient but high levels are toxic due to the catalytic generation of destructive hydroxyl radicals. Using human neuroblastoma SH-SY5Y cells to model human neurons, we investigated the effect of Pb on proteins of iron homeostasis: the Alzheimer's amyloid precursor protein (APP), which stabilizes the iron exporter ferroportin 1; and, the heavy subunit of the iron-storage protein, ferritin (FTH). Lead (Pb(II) and Pb(IV) inhibited APP translation and raised cytosolic iron(II). Lead also increased iron regulatory protein-1 binding to the cognate 5'untranslated region-specific iron-responsive element (IRE) of APP and FTH mRNAs. Concurrent iron treatment rescued cells from Pb toxicity by specifically restoring APP synthesis, i.e. levels of the APP-related protein, APLP-2, were unchanged. Significantly, iron/IRE-independent over-expression of APP695 protected SH-SY5Y cells from Pb toxicity, demonstrating that APP plays a key role in maintaining safe levels of intracellular iron. Overall, our data support a model of neurotoxicity where Pb enhances iron regulatory protein/IRE-mediated repression of APP and FTH translation. We propose novel treatment options for Pb poisoning to include chelators and the use of small molecules to maintain APP and FTH translation. We propose the following cascade for Lead (Pb) toxicity to neurons; by targeting the interaction between Iron regulatory protein-1 and Iron-responsive elements, Pb caused translational repression of proteins that control intracellular iron homeostasis, including the Alzheimer's amyloid precursor protein (APP) that stabilizes the iron exporter ferroportin, and the ferroxidase heavy subunit of the iron-storage protein, ferritin. When unregulated, IRE-independent over-expression of APP695 protected SH-SY5Y neurons from Pb toxicity. There is a novel and key role for APP in maintaining safe levels of intracellular iron pertinent to lead toxicity.

Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function.

Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer's disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain.

Deficiency of Neuronal p38α MAPK Attenuates Amyloid Pathology in Alzheimer Disease Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of the p38α MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.

BRI2 and BRI3 are functionally distinct phosphoproteins

Three BRI protein family members have been identified. Among these are BRI3 and BRI2, the latter is associated with Familial Danish and Familial British dementias. ‘In silico’ sequence analysis identified putative PP1 binding sites in BRI2 and BRI3. This is singularly important, given that protein phosphorylation is a major mechanism regulating intracellular processes. Protein phosphatase 1 (PP1) interacting proteins (PIPs) are fundamental in determining substrate specificity and subcellular localization of this phosphatase. More than 200 PIPs have thus far been reported.Both BRI2 and BRI3 are type II transmembrane glycoproteins relevant in neuronal systems. Using Myc-BRI2 and Myc-BRI3, wild type and PP1 binding mutant constructs, it was possible to show, for the first time, that in fact BRI2 and BRI3 bind PP1. The complexes BRI2:PP1 and BRI3:PP1 were validated in vitro and in vivo. The subcellular distribution of BRI2 and BRI3 is similar; both localize to the perinuclear area and Golgi apparatus in non-neuronal cells. However, in SH-SY5Y cells, BRI2 and BRI3 could also be detected in elongated cellular projections (‘processes’) and in rat cortical neurons both are broadly distributed throughout the cell body, neuritis and the nucleus. Consistently, co-localization of BRI2 and BRI3 with PP1 was evident. The functional significance of these complexes is apparent given that both BRI proteins are substrates of PP1, thus simultaneously this is the first report of BRI2 and BRI3 as phosphoproteins. Moreover, we show that when BRI2 is phosphorylated a significant increase in neuronal outgrowth and differentiation is evident.Interestingly, the Alzheimer's amyloid precursor protein (APP), forms a trimeric complex composed of PP1 and Fe65, with PP1 having the capacity to dephosphorylate APP at Thr668 residue. The emerging consensus appears to be that PP1 containing complexes are crucial in regulating signaling events underlying neuropathological conditions.

Phosphorylation of FE65 Ser610 by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer's disease amyloid precursor protein processing.

Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting 36 million people worldwide. Genetic and biochemical research indicate that the excessive generation of amyloid-β peptide (Aβ) from amyloid precursor protein (APP), is a major part of AD pathogenesis. FE65 is a brain-enriched adaptor protein that binds to APP. However, the role of FE65 in APP processing and the mechanisms that regulate binding of FE65 to APP are not fully understood. In the present study, we show that serum- and glucocorticoid-induced kinase 1 (SGK1) phosphorylates FE65 on Ser(610) and that this phosphorylation attenuates FE65 binding to APP. We also show that FE65 promotes amyloidogenic processing of APP and that FE65 Ser(610) phosphorylation inhibits this effect. Furthermore, we found that the effect of FE65 Ser(610) phosphorylation on APP processing is linked to a role of FE65in metabolic turnover of APP via the proteasome. Thus FE65 influences APP degradation via the proteasome and phosphorylation of FE65 Ser(610) by SGK1 regulates binding of FE65 to APP, APP turnover and processing.

Long-term treatment with Ginkgo biloba extract EGb 761 improves symptoms and pathology in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid β peptide (Aβ) and microglia-dominated neuroinflammation. The therapeutic options for AD are currently limited. In this study, we investigated the antiinflammatory effects and the underlying molecular mechanisms of Ginkgo biloba extract EGb 761 when administered to TgCRND8 AD mice, which overexpress human Alzheimer's amyloid precursor protein (APP) specifically in neurons. We gave APP-transgenic mice EGb 761 as a dietary supplement for 2 or 5months. Plasma concentrations of EGb 761 components in mice were in the same range as such concentrations in humans taking EGb 761 at the recommended dose (240mg daily). Treatment with EGb 761 for 5months significantly improved the cognitive function of the mice as measured by the Barnes Maze test. It also attenuated the loss of synaptic structure proteins, such as PSD-95, Munc18-1, and SNAP25. Treatment with EGb 761 for 5months inhibited microglial inflammatory activation in the brain. The effects of treatment with EGb 761 for 2months were weak and not statistically significant. Moreover, EGb 761 activated autophagy in microglia. Treatment with EGb 761 decreased Aβ-induced microglial secretion of TNF-α and IL-1β and activation of caspase-1, both of which were abolished by the inhibition of autophagy. Treatment with EGb 761 also reduced the concentrations of NLRP3 protein that colocalized with LC3-positive autophagosomes or autolysosomes in microglia. Additionally, long-term treatment with EGb 761 may reduce cerebral Aβ pathology by inhibiting β-secretase activity and Aβ aggregation. Therefore, long-term treatment with G. biloba extract EGb 761, a clinically available and well-tolerated herbal medication, ameliorates AD pathology by antiinflammatory and Aβ-directed mechanisms.

Proneurogenic Group II mGluR antagonist improves learning and reduces anxiety in Alzheimer Aβ oligomer mouse.

Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer's pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger γ-secretase-inhibitor-sensitive biogenesis of Aβ42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer's amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-β (Aβ) oligomers but never develop Aβ plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch Aβ-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain Aβ monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer's disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair.

SAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Flotillins bind to the dileucine sorting motif of β‐site amyloid precursor protein‐cleaving enzyme 1 and influence its endosomal sorting

The β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a protease that participates in the amyloidogenic cleavage of the Alzheimer amyloid precursor protein. Trafficking of BACE1 has been shown to be largely mediated by an acidic cluster dileucine motif in its cytoplasmic tail. This sorting signal functions both in endocytosis and endosomal sorting/recycling of BACE1 by providing a binding site for various sorting factors, such as the Golgi-localizing γ-ear containing ADP ribosylation factor binding (GGA) proteins that mediate BACE1 sorting within endosomes. Because flotillin-1 has been suggested to bind to BACE1 cytoplasmic tail, we analyzed the role of flotillins in BACE1 sorting. We show that flotillin-1 directly binds to the dileucine motif in the cytoplasmic tail of BACE1, whereas flotillin-2 binding is mainly mediated by its interaction with flotillin-1. Depletion of flotillins results in altered subcellular localization of BACE1 in endosomes and stabilization of BACE1 protein. Furthermore, amyloidogenic processing of Alzheimer amyloid precursor protein is increased. Flotillins compete with GGA proteins for binding to the dileucine motif in the BACE1 tail, suggesting that they play an important role in endosomal sorting of BACE1. The present study shows for the first time that flotillins are involved in endosomal sorting of BACE1. Because the endosomal localization of BACE1 affects its function as the β-secretase by increasing amyloidogenic processing of the amyloid precursor protein, flotillins may play a novel role in Alzheimer's disease. The present study is the first to show that flotillins bind to a canonical sorting signal and influence the binding of endosomal sorting factors onto cargo tails.

F-box Only Protein 2 (Fbxo2) Regulates Amyloid Precursor Protein Levels and Processing

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (μ1-μ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in μ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the μ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of μ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type μ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered μ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of μ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non-canonical site of μ4.

83-Year-Old Man With Nausea, Constipation, and Peripheral Neuropathy

83-Year-Old Man With Nausea, Constipation, and Peripheral Neuropathy

Major Carboxyl Terminal Fragments Generated by γ-Secretase Processing of the Alzheimer Amyloid Precursor Are 50 and 51 Amino Acids Long

To understand the cleavage of the amyloid β protein (Aβ) precursor (APP) by γ-secretase and to determine its changes in a representative familial Alzheimer disease (FAD) mutation.Transfected cells expressing wild-type and FAD mutant APP were analyzed for changes in the levels of the major secreted Aβ species and of the corresponding intracellular C-terminal APP fragments (APP intracellular domain, AICD) generated by γ-secretase, whereas radio-sequencing was used to precisely identify the resulting cleavage site(s).The AICD fragment(s) generated by γ-secretase cleavage comigrated in gels with a 50-residue synthetic peptide used as control, which is smaller than the 59 and 57 residues predicted from Aβ ending at positions 40 (Aβ40) and 42 (Aβ42), respectively. In agreement with previous findings, an FAD mutant form of presenilin 1 (PS1-M139V) significantly increased the longer Aβ42 while showing trends toward reducing Aβ40. AICD levels were reduced by the mutation, suggesting that γ-secretase activity may be actually impaired by the mutation. Radiosequence analysis in cells expressing wild-type PS1 detected γ-secretase cleavage sites at the Aβ peptide bond L(49)-V(50) to generate a 50-amino acid (aa) AICD fragment (AICD50) and the Aβ peptide bond T(48)-L(49), generating an AICD of 51 aa (AICD51). No other cleavage sites were reliably detected.Based on findings that the FAD mutation that increases Aβ42 also reduces AICD, we propose that γ-secretase activity is impaired by FAD mutations and predict that physiologic and environmental agents that inhibit γ-secretase will actually induce AD pathogenesis rather that prevent it. Furthermore, we propose that the cleavage site to generate AICD is naturally ragged and occurs predominantly at two sites 48 and 49 aa from the start of the Aβ sequence. Thus, end specific antibodies to these two sites will need to be generated to study the quantitative relationships between these two cleavages in sporadic AD and FAD.

Mild oxidative stress induces redistribution of BACE1 in non-apoptotic conditions and promotes the amyloidogenic processing of Alzheimer's disease amyloid precursor protein.

BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP), which represents the first step in the production of amyloid β (Aβ) peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer’s disease (AD). The association between oxidative stress (OS) and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10–40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS), as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H2O2 concentration (100 µM) caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.