Endogenous Aβ Research Articles

Rho-associated protein kinase 1 (ROCK1) is increased in Alzheimer's disease and ROCK1 depletion reduces amyloid-β levels in brain.

Alzheimer's disease (AD) is the leading cause of dementia and mitigating amyloid-β (Aβ) levels may serve as a rational therapeutic avenue to slow AD progression. Pharmacologic inhibition of the Rho-associated protein kinases (ROCK1 and ROCK2) is proposed to curb Aβ levels, and mechanisms that underlie ROCK2's effects on Aβ production are defined. How ROCK1 affects Aβ generation remains a critical barrier. Here, we report that ROCK1 protein levels were elevated in mild cognitive impairment due to AD (MCI) and AD brains compared to controls. Aβ42 oligomers marginally increased ROCK1 and ROCK2 protein levels in neurons but strongly induced phosphorylation of Lim kinase 1 (LIMK1), suggesting that Aβ42 activates ROCKs. RNAi depletion of ROCK1 or ROCK2 suppressed endogenous Aβ40 production in neurons, and Aβ40 levels were reduced in brains of ROCK1 heterozygous knock-out mice compared to wild-type littermate controls. ROCK1 knockdown decreased amyloid precursor protein (APP), and treatment with bafilomycin accumulated APP levels in neurons depleted of ROCK1. These observations suggest that reduction of ROCK1 diminishes Aβ levels by enhancing APP protein degradation. Collectively, these findings support the hypothesis that both ROCK1 and ROCK2 are therapeutic targets to combat Aβ production in AD. Mitigating amyloid-β (Aβ) levels is a rational strategy for Alzheimer's disease (AD) treatment, however, therapeutic targets with clinically available drugs are lacking. We hypothesize that Aβ accumulation in mild cognitive impairment because of AD (MCI) and AD activates the RhoA/ROCK pathway which in turn fuels production of Aβ. Escalation of this cycle over the course of many years may contribute to the buildup of amyloid pathology in MCI and/or AD.

Effect of curcumin on accumulation in mononuclear cells and secretion in incubation medium of Аβ(40) and cytokines under local excess of Аβ(42)-homoaggregates.

The aim of the work was to investigate accumulation of endogenous Aβ40 and cytokines (IL-1β, TNFα, IL-6, IL-10) in mononuclear cells and their secretion into incubation medium under Aβ42-aggregates’ toxicity and anti-inflammatory effects of curcumin. Mononuclear cells were isolated in Ficoll-Urografin density gradient from venous blood of healthy donors, resuspended and used for testing of homoaggregates of Aβ42 (15 nM), curcumin (54 pM) and their combinations on various timescales (0, 1, 2, 3, 6 and 24 hours). Endogenous Aβ40 and cytokines were detected in mononuclear cells and (separately) in incubation medium by ELISA. We demonstrated for the first time that homoaggregates of Aβ42 cause rapid accumulation of endogenous Aβ40 in mononuclear cells and accelerate its secretion into incubation medium. We found increased concentration of TNFα after 3 hours of incubation, and no changes in IL-1β concentration due to secretion of these pro-inflammatory factors into incubation medium. The concentrations of IL-6 in mononuclear cells were increased under effects of Aβ42 homoaggregates, and it was being secreted profoundly into incubation medium. Aβ42 did not affect IL-10 secretion, yet caused an increase in its intracellular concentration after 1 hour of incubation, which was subsequently suppressed. Curcumin prevented the increase in Aβ40 concentration in mononuclear cells and significantly decreased its secretion resulting from Aβ42 toxicity. Curcumin negated the activating effect of Aβ42 on pro-inflammatory cytokines, starting immediately for IL-1β and on 3-6 hours for TNFα, which resulted in decreased extracellular concentrations of these cytokines. The polyphenol also potentiated repleni­shing of intracellular IL-6 and IL-10 concentrations and their secretion into incubation medium.

Characterization of a sensitive mouse Aβ40 PD biomarker assay for Alzheimer's disease drug development in wild-type mice.

Transgenic mice that overexpress human amyloid precursor protein with Swedish or London (APPswe or APPlon) mutations have been widely used for preclinical Alzheimer's disease (AD) drug development. AD patients, however, rarely possess these mutations or overexpress APP. We developed a sensitive ELISA that specifically and accurately measures low levels of endogenous Aβ40 in mouse plasma, brain and CSF. In wild-type mice treated with a bispecific anti-TfR/BACE1 antibody, significant Aβ reductions were observed in the periphery and the brain. APPlon transgenic mice showed a slightly less reduction, whereas APPswe mice did not have any decrease. This sensitive and well-characterized mouse Aβ40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.

Zinc-induced heterodimer formation between metal-binding domains of intact and naturally modified amyloid-beta species: implication to amyloid seeding in Alzheimer’s disease?

Zinc ions and modified amyloid-beta peptides (Aβ) play a critical role in the pathological aggregation of endogenous Aβ in Alzheimer’s disease (AD). Zinc-induced Aβ oligomerization is mediated by the metal-binding domain (MBD) which includes N-terminal residues 1–16 (Aβ1–16). Earlier, it has been shown that Aβ1–16 as well as some of its naturally occurring variants undergoes zinc-induced homodimerization via the interface in which zinc ion is coordinated by Glu11 and His14 of the interacting subunits. In this study using surface plasmon resonance technique, we have found that in the presence of zinc ions Aβ1–16 forms heterodimers with MBDs of two Aβ species linked to AD: Aβ containing isoAsp7 (isoAβ) and Aβ containing phosphorylated Ser8 (pS8-Aβ). The heterodimers appear to possess the same interface as the homodimers. Simulation of 200 ns molecular dynamic trajectories in two constructed models of dimers ([Aβ1–16/Zn/Aβ1–16] and [isoAβ1–16/Zn/Aβ1–16]), has shown that conformational flexibility of the N-terminal fragments of the dimer subunits is controlled by the structure of corresponding sites 6–8. The data suggest that isoAβ and pS8-Aβ can be involved in the AD pathogenesis by means of their zinc-dependent interactions with endogenous Aβ resulting in the formation of heterodimeric seeds for amyloid aggregation.

Effect of β-amyloid peptide 42 on the dynamics of expression and formation of Аβ40, IL -1β, TNF α, IL -6, IL -10 by peripheral blood mononuclear cells in vitro and its correction by curcumin.

The toxic effect of Аβ-oligomers accompanies chronic inflammation, with cytokines as main mediators. Therefore, the cytokine link of inflammation becomes a new target on the way to restrain amyloidosis. The aim of the study was the effect of aggregated Аβ42 on the dynamics of expression and formation of endogenous Аβ40 and cytokines (IL-1β, TNFα, IL-6, IL-10) by peripheral blood mononuclear cells in vitro and its correction by curcumin. A suspension of mononuclear cells isolated ex tempore using ficoll-urografin gradient from venous blood samples of healthy volunteers were used to study the effects of Аβ42 (15 nM), curcumin (54 pM), and their combined action (at similar concentrations) in time dynamics: 0, 1, 3, 6 and 24 h incubation at 37 °C. Polymerase chain reaction with appropriate primers was used to determine the relative expression of mRNA for AβPP, TNFα, IL-1β, IL-6, IL-10 and enzyme-linked immunosorbent assay – to determine the content of Аβ40 and cytokines in mononuclear suspension during all periods of incubation. The individual dynamics AβPP and cytokine expression was shown under the action of the Aβ42, which had influence on the content of Aβ40, TNFα, IL-1β, IL-6 and IL-10 in mononuclear suspension. Curcumin displayed the inhibitory effect on gene expression of AβPP, TNFα and IL6, which resulted in the decrease of the level of these two cytokines and Aβ40. Thus, the dynamics of anti-inflammatory effect of curcumin in vitro for transcriptional and translational levels of cytokine’s formation by mononuclear cells was shown in the work. Direct inhibitory effect of curcumin on the concentration of endogenous Aβ40 during the 24 h incubation in conditions of toxic action of Aβ42 aggregates was established.

Amyloid-β(1-42)Oligomer Models Developed using Combined Solid State NMR and Sequence Specific Hydroxyl Radical Footprinting Data

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) are the pathogenic species in Alzheimer's disease (AD). However, detailed structural information on these species remains scarce due to low levels of endogenous Aβ oligomers and uncertainties surrounding current in vitro model systems. Herein, we describe a hydroxyl radical footprinting (HRF) study of Aβ42 monomers, dimers, and specially prepared stable and homogeneous oligomers. Specific side chain solvent accessibilities of individual residues in the folded and aggregated forms of Aβ42 are measured with respect to the same residues of Aβ42 in a fully exposed reference state. These data provide residue specific side chain solvent accessibility protection factors and are used in complement with biophysical characterizations and ss-NMR analyses of the same systems. Results are discussed in the context of proposed NMR models of Aβ oligomers with implications towards further development of therapeutic and diagnostic strategies.

Commentary: Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy.

Commentary: Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy.

Isobaric Quantification of Cerebrospinal Fluid Amyloid-β Peptides in Alzheimer's Disease: C-Terminal Truncation Relates to Early Measures of Neurodegeneration.

The amyloid beta (Aβ) peptide is the main constituent of the plaques characteristic of Alzheimer's disease (AD). Measurement of Aβ1-42 in cerebrospinal fluid (CSF) is a valuable marker in AD research, where low levels indicate AD. Although the use of immunoassays measuring Aβ1-38 and Aβ1-40 in addition to Aβ1-42 has increased, quantitative assays of other Aβ peptides remain rarely explored. We recently discovered novel Aβ peptides in CSF using antibodies recognizing the Aβ mid-domain region. Here we have developed a method using both Aβ N-terminal and mid-domain antibodies for immunoprecipitation in combination with isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of endogenous Aβ peptides in CSF. The developed method was used in a pilot study to produce Aβ peptide profiles from 38 CSF samples. Statistical comparison between CSF samples from 19 AD patients and 19 cognitively healthy controls revealed no significant differences at group level. A significant correlation was found between several larger C-terminally truncated Aβ peptides and protein biomarkers for neuronal damage, particularly prominent in the control group. Comparison of the isobaric quantification with immunoassays measuring Aβ1-38 or Aβ1-40 showed good correlation (r(2) = 0.84 and 0.85, respectively) between the two analysis methods. The developed method could be used to assess disease-modifying therapies directed at Aβ production or degradation.

Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ) and the aggregated proteins that cause Alzheimer’s disease (AD). We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ) by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1–42, protein levels of beclin-1, autophagy-related protein5 (Atg5), and SIRT1 decreased significantly. Pretreatment with cilostazol (10–30 μM) or resveratrol (20 μM) prevented these Aβ1–42 evoked suppressions. LC3-II (a marker of mammalian autophagy) levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation) were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition). Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion). After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein), cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

Fasting activates macroautophagy in neurons of Alzheimer's disease mouse model but is insufficient to degrade amyloid-beta.

We developed a new technique to observe macroautophagy in the brain in vivo, and examined whether fasting induced macroautophagy in neurons and how the induction was different between Alzheimer’s disease (AD) model and control mice. Lentivirus for EGFP-LC3 injected into the brain successfully visualized autophagosome in living neurons by two-photon microscopy. The time-lapse imaging revealed that fasting increased the number, size and signal intensity of autophagosome in neurons. In AD model mice, these parameters of autophagosome were higher at the basal levels before starvation, and increased more rapidly by fasting than in control mice. However, metabolism of exogenous labeled Aβ evaluated by the new technique suggested that the activated macroautophagy was insufficient to degrade the intracellular Aβ increased by enhanced uptake from extracellular space after fasting. Ordinary immunohistochemistry also revealed that fasting increased intracellular accumulation of endogenous Aβ, triggered cell dysfunction but did not mostly decrease extracellular Aβ accumulation. Moreover, we unexpectedly discovered a circadian rhythm of basal level of macroautophagy. These results revealed new aspects of neuronal autophagy in normal/AD states and indicated usefulness of our method for evaluating autophagy functions in vivo.

Inhibition of JAK2 attenuates the increase in inflammatory markers in microglia from APP/PS1 mice

There is a wealth of evidence indicating that macrophages adopt distinct phenotypes when exposed to specific stimuli and, in the past few years, accumulating data suggest that microglia behave somewhat similarly. Therefore, microglia can adopt the so-called M1 or M2 phenotypes in response to interferon-γ (IFNγ) and interleukin-4, respectively. Although it has yet to be unequivocally proven in the context of microglia, acutely activated M1 cells are probably protective, although a persistent M1 state is likely to be damaging, whereas M2 cells may be reparative and restorative. In this case, particularly because the current evidence suggests the development of a predominantly M1 state with age and in neurodegenerative diseases, it is important to identify mechanisms by which polarization of microglia can be modulated. The present findings indicate that exposure of cultured microglia to IFNγ increased expressions of the archetypal markers of the M1 phenotype, tumour necrosis factor-α, and inducible nitric oxide synthase, and preexposure of cells to amyloid-β (Aβ) sensitized microglia to subsequent stimulation with IFNγ. Importantly, this synergy was also evident in microglia prepared from the brains of transgenic mice that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1, APP/PS1 mice) and are exposed to a combination of increasing concentrations of endogenous Aβ from 4 or 5 months of age and an age-related increase in IFNγ. Significantly, the JAK2 inhibitor, TG101209, attenuated the IFNγ-induced changes in cultured microglia and in isolated microglia prepared from APP/PS1 mice. These findings suggest that targeting JAK2 may be a potential strategy for reducing neuroinflammation in Alzheimer's disease.

Calcium flux-independent NMDA receptor activity is required for Aβ oligomer-induced synaptic loss.

Synaptic loss is one of the major features of Alzheimer's disease (AD) and correlates with the degree of dementia. N-methyl-d-aspartate receptors (NMDARs) have been shown to mediate downstream effects of the β-amyloid peptide (Aβ) in AD models. NMDARs can trigger intracellular cascades via Ca2+ entry, however, also Ca2+-independent (metabotropic) functions of NMDARs have been described. We aimed to determine whether ionotropic or metabotropic NMDAR signaling is required for the induction of synaptic loss by Aβ. We show that endogenous Aβ as well as exogenously added synthetic Aβ oligomers induced dendritic spine loss and reductions in pre- and postsynaptic protein levels in hippocampal slice cultures. Synaptic alterations were mitigated by blocking glutamate binding to NMDARs using NMDAR antagonist APV, but not by preventing ion flux with Ca2+ chelator BAPTA or open-channel blockers MK-801 or memantine. Aβ increased the activity of p38 MAPK, a kinase involved in long-term depression and inhibition of p38 MAPK abolished the loss of dendritic spines. Aβ-induced increase of p38 MAPK activity was prevented by APV but not by BAPTA, MK-801 or memantine treatment highlighting the role of glutamate binding to NMDARs but not Ca2+ flux for synaptic degeneration by Aβ. We further show that treatment with the G protein inhibitor pertussis toxin (PTX) did not prevent dendritic spine loss in the presence of Aβ oligomers. Our data suggest that Aβ induces the activation of p38 MAPK and subsequent synaptic loss through Ca2+ flux- and G protein-independent mechanisms.

Calcineurin and Sorting-Related Receptor with A-Type Repeats Interact to Regulate the Renal Na⁺-K⁺-2Cl⁻ Cotransporter.

The furosemide-sensitive Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin Aβ isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with A-type repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin Aβ in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin Aβ. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Aβ and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Aβ and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors.

Interaction between therapeutic interventions for Alzheimer's disease and physiological Aβ clearance mechanisms.

Most therapeutic agents are designed to target a molecule or pathway without consideration of the mechanisms involved in the physiological turnover or removal of that target. In light of this and in particular for Alzheimer’s disease, a number of therapeutic interventions are presently being developed/investigated which target the amyloid-β peptide (Aβ). However, the literature has not adequately considered which Aβ physiological clearance pathways are necessary and sufficient for the effective action of these therapeutics. In this review, we evaluate the therapeutic strategies targeting Aβ presently in clinical development, discuss the possible interaction of these treatments with pathways that under normal physiological conditions are responsible for the turnover of Aβ and highlight possible caveats. We consider immunization strategies primarily reliant on a peripheral sink mechanism of action, small molecules that are reliant on entry into the CNS and thus degradation pathways within the brain, as well as lifestyle interventions that affect vascular, parenchymal and peripheral degradation pathways. We propose that effective development of Alzheimer’s disease therapeutic strategies targeting Aβ peptide will require consideration of the age- and disease-specific changes to endogenous Aβ clearance mechanisms in order to elicit maximal efficacy.

γ-Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation.

Soluble γ-secretase modulators (SGSMs) selectively decrease toxic amyloid β (Aβ) peptides (Aβ42). However, their effect on the physiologic functions of γ-secretase has not been tested in human model systems. γ-Secretase regulates fate determination of neural progenitor cells. Thus, we studied the impact of SGSMs on the neuronal differentiation of ReNcell VM (ReN) human neural progenitor cells (hNPCs). Quantitative PCR analysis showed that treatment of neurosphere-like ReN cell aggregate cultures with γ-secretase inhibitors (GSIs), but not SGSMs, induced a 2- to 4-fold increase in the expression of the neuronal markers Tuj1 and doublecortin. GSI treatment also induced neuronal marker protein expression, as shown by Western blot analysis. In the same conditions, SGSM treatment selectively reduced endogenous Aβ42 levels by ∼80%. Mechanistically, we found that Notch target gene expressions were selectively inhibited by a GSI, not by SGSM treatment. We can assert, for the first time, that SGSMs do not affect the neuronal differentiation of hNPCs while selectively decreasing endogenous Aβ42 levels in the same conditions. Our results suggest that our hNPC differentiation system can serve as a useful model to test the impact of GSIs and SGSMs on both endogenous Aβ levels and γ-secretase physiologic functions including endogenous Notch signaling.

Endogenous murine Aβ increases amyloid deposition in APP23 but not in APPPS1 transgenic mice

Endogenous murine amyloid-β peptide (Aβ) is expressed in most Aβ precursor protein (APP) transgenic mouse models of Alzheimer's disease but its contribution to β-amyloidosis remains unclear. We demonstrate ∼35% increased cerebral Aβ load in APP23 transgenic mice compared with age-matched APP23 mice on an App-null background. No such difference was found for the much faster Aβ-depositing APPPS1 transgenic mouse model between animals with or without the murine App gene. Nevertheless, both APP23 and APPPS1 mice codeposited murine Aβ, and immunoelectron microscopy revealed a tight association of murine Aβ with human Aβ fibrils. Deposition of murine Aβ was considerably less efficient compared with the deposition of human Aβ indicating a lower amyloidogenic potential of murine Aβ in vivo. The amyloid dyes Pittsburgh Compound-B and pentamer formyl thiophene acetic acid did not differentiate between amyloid deposits consisting of human Aβ and deposits of mixed human-murine Aβ. Our data demonstrate a differential effect of murine Aβ on human Aβ deposition in different APP transgenic mice. The mechanistically complex interaction of human and mouse Aβ may affect pathogenesis of the models and should be considered when models are used for translational preclinical studies.

Opposing effects of viral mediated brain expression of apolipoprotein E2 (apoE2) and apoE4 on apoE lipidation and Aβ metabolism in apoE4-targeted replacement mice.

BackgroundHuman apolipoprotein E (apoE) exists in three major isoforms: apoE2, apoE3 and apoE4. In the brain, apoE is produced mostly by astrocytes and transports cholesterol to neurons via apoE receptors. Among the gene alleles encoding the three isoforms, the APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD), whereas APOE2 is protective. ApoE4 confers a gain of toxic function, a loss of neuroprotective function or a combination of both in AD pathogenesis. Given that therapeutic impacts of modulating apoE expression may be isoform-dependent, we sought to investigate the relationship between overexpressing apoE isoform and apoE-related functions in apoE-targeted replacement (TR) mice. Specifically, apoE isoform expression driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was built into an adeno-associated virus serotype 8 (AAV8) vector and injected into the ventricles of postnatal day 2 (P2) apoE3-TR or apoE4-TR mice. Upon confirmation of apoE isoform expression, effects on apoE lipidation and the levels of amyloid-β (Aβ) in the brain were assessed.ResultsAAV8-GFAP-apoE isoforms were specifically expressed in astrocytes throughout all brain regions, which led to overall increased apoE levels in the brain. Viral mediated overexpression of apoE4 in the apoE4-TR background increased poorly-lipidated apoE lipoprotein particles and decreased apoE-associated cholesterol in apoE4-TR mice. Conversely, apoE2 overexpression in apoE4-TR mice enhanced apoE lipidation and associated cholesterol. Furthermore, overexpression of apoE4 elevated the levels of endogenous Aβ, whereas apoE2 overexpression trended to lower endogenous Aβ.ConclusionsOverexpression of apoE isoforms induces differential effects in the apoE4-TR background: apoE4 decreases apoE lipidation and enhances Aβ accumulation, whereas apoE2 has the opposite effects. Our findings suggest that increasing apoE2 in APOE4 carriers is a beneficial strategy to treat AD, whereas increasing apoE4 in APOE4 carriers is likely harmful. We have also established novel methods to express apoE isoforms in mouse brain to study apoE-related pathways in AD and related dementia.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0001-3) contains supplementary material, which is available to authorized users.

Sequence Specific Radiolytic Footprinting Study of Monomer, Oligomeric and Fibrillar Amyloid Beta (1-42)

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) are the pathogenic species in Alzheimer's disease (AD). However, detailed structural information on these species remains scarce due to low levels of endogenous Aβ oligomers and uncertainties surrounding current in vitro model systems. Herein, we describe a hydroxyl radical footprinting (HRF) study of Aβ42 fibrils and a stable and homogeneous preparation of Aβ42 oligomers. Specific side chain solvent accessibilities of individual residues in the aggregated and fibril forms of Aβ42 are measured with respect to the same residues of Aβ42 in a fully exposed reference state. These data provide residue specific side chain solvent accessibility protection factors and are used in complement with biophysical characterizations and ss-NMR analyses of these systems. Results are discussed in the context of proposed NMR models of Aβ oligomers and fibrils with implications towards further development of therapeutic and diagnostic strategies.

CALHM1 ion channel elicits amyloid-β clearance by insulin-degrading enzyme in cell lines and in vivo in the mouse brain.

Alzheimer's disease is characterized by amyloid-β (Aβ) peptide accumulation in the brain. CALHM1, a cell-surface Ca(2+) channel expressed in brain neurons, has anti-amyloidogenic properties in cell cultures. Here, we show that CALHM1 controls Aβ levels in vivo in the mouse brain through a previously unrecognized mechanism of regulation of Aβ clearance. Using pharmacological and genetic approaches in cell lines, we found that CALHM1 ion permeability and extracellular Ca(2+) were required for the Aβ-lowering effect of CALHM1. Aβ level reduction by CALHM1 could be explained by an increase in extracellular Aβ degradation by insulin-degrading enzyme (IDE), extracellular secretion of which was strongly potentiated by CALHM1 activation. Importantly, Calhm1 knockout in mice reduced IDE enzymatic activity in the brain, and increased endogenous Aβ concentrations by up to ∼50% in both the whole brain and primary neurons. Thus, CALHM1 controls Aβ levels in cell lines and in vivo by facilitating neuronal and Ca(2+)-dependent degradation of extracellular Aβ by IDE. This work identifies CALHM1 ion channel as a potential target for promoting amyloid clearance in Alzheimer's disease.

Toxic oligomer species of amyloid-β in Alzheimer's disease, a timing issue.

A decade following the paradigm-shifting concept that endogenous forms of soluble, non-fibrillar amyloid-β (Aβ) might constitute the major bioactive entity causing synaptic loss and cognitive decline in Alzheimer's disease (AD), our understanding of these oligomeric species still remains conspicuously superficial. The current lack of direct evaluation tools for each endogenous Aβ oligomer hampers our ability to readily address crucial question such as: (i) where they form and accumulate?; (ii) when they first appear in human brains and body fluids?; (iii) what is the longitudinal expression of these putative toxins during the course of the disease?; (iv) and how do these soluble Aβ assemblies alter synaptic and neuronal function in the brain? Despite these limitations, indirect ex vivo measurement and isolation from biological specimens has been possible and have allowed parsing out intrinsic differences between putative endogenous Aβ oligomers. In this review, I integrated recent findings and extrapolated emerging hypotheses derived from these studies with the hope to provide a clarified view on the putative role of endogenous Aβ oligomers in AD, with a particular emphasis on the timing at which these soluble species might act in the aging and diseased brain.