Affect Aβ Production Research Articles

The role and mechanism of Aβ clearance dysfunction in the glymphatic system in Alzheimer’s disease comorbidity

Alzheimer’s disease (AD) is the leading type of dementia globally, characterized by a complex pathogenesis that involves various comorbidities. An imbalance in the production and clearance of amyloid β-protein (Aβ) peptides in the brain is a key pathological mechanism of AD, with the glymphatic system playing a crucial role in Aβ clearance. Comorbidities associated with AD, such as diabetes, depression, and hypertension, not only affect Aβ production but also impair the brain’s lymphatic system. Abnormalities in the structure and function of this system further weaken Aβ clearance capabilities, and the presence of comorbidities may exacerbate this process. This paper aims to review the role and specific mechanisms of impaired Aβ clearance via the glymphatic system in the context of AD comorbidities, providing new insights for the prevention and treatment of AD. Overall, the damage to the glymphatic system primarily focuses on aquaporin-4 (AQP4) and perivascular spaces (PVS), suggesting that maintaining the health of the glymphatic system may help slow the progression of AD and its comorbidities. Additionally, given the ongoing controversies regarding the structure of the glymphatic system, this paper revisits this structure and discusses the principles and characteristics of current detection methods for the glymphatic system.

MiR-3940-5p reduces amyloid β production via selectively targeting PSEN1.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid beta (Aβ) in brain. Mounting evidence has revealed critical roles of microRNAs (miRNAs) in AD pathogenesis; however, the miRNAs directly targeting presenilin1 (PSEN1), which encodes the catalytic core subunit of γ-secretase that limits the production of Aβ from amyloid precursor protein (APP), are extremely understudied. The present study aimed to identify miRNAs targeting PSEN1 and its effect on Aβ production. This study first predicted 5 candidate miRNAs that may target PSEN1,through websites such as TargetScan, miRDB, and miRwalk. Subsequently, the targeting specificity of the candidate miRNAs towards PS1 was validated using dual-luciferase reporter assays. To investigate the regulatory effect of miR-3940-5p on gene expression based on its targeting of PS1, miR-3940-5p mimics or inhibitors were transiently transfected into SH-SY5Y cells. Changes in PSEN1 transcription and translation in the tested cells were detected using RT-qPCR and Western Blot, respectively. Finally, to explore whether miR-3940-5p affects Aβ production, SH-SY5Y APPswe cells overexpressing the Swedish mutant type of APP were transiently transfected with miR-3940-5p mimics, and the expression level of Aβ was detected using ELISA. The results are as follows: The dual-luciferase reporter assays validated the targeting specificity of miR-3940-5p for PSEN1. Overexpression of miR-3940-5p significantly reduced the mRNA and protein levels of PSEN1 in SH-SY5Y cells. Conversely, inhibition of miR-3940-5p led to an increase in PSEN1 mRNA levels. Transfection of miR-3940-5p mimics into SH-SY5Y-APPswe cells resulted in a significant reduction in Aβ42 and Aβ40. Lentiviral-mediated overexpression of miR-3940-5p significantly decreased the expression of PSEN1 and did not significantly affect the expression of other predicted target genes. Furthermore, stable overexpression of miR-3940-5p in SH-SY5Y-APPswe cells mediated by lentivirus significantly reduced the expression of PSEN1 and the production of Aβ42 and Aβ40. Therefore, our study demonstrates for the first time the functional importance of miR-3940-5p in antagonizing Aβ production through specific and direct targeting of PSEN1.

The role of ATP-binding cassette transporter G1 (ABCG1) in Alzheimer's disease: A review of the mechanisms.

The maintenance of cholesterol homeostasis is essential for central nervous system function. Consequently, factors that affect cholesterol homeostasis are linked to neurological disorders and pathologies. Among them, ATP-binding cassette transporter G1 (ABCG1) plays a significant role in atherosclerosis. However, its role in Alzheimer's disease (AD) is unclear. There is inconsistent information regarding ABCG1's role in AD. It can increase or decrease amyloid β (Aβ) levels in animals' brains. Clinical studies show that ABCG1 is involved in AD patients' impairment of cholesterol efflux capacity (CEC) in the cerebrospinal fluid (CSF). Lower Aβ levels in the CSF are correlated with ABCG1-mediated CEC dysfunction. ABCG1 modulates α-, β-, and γ-secretase activities in the plasma membrane and may affect Aβ production in the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) cell compartment. Despite contradictory findings regarding ABCG1's role in AD, this review shows that ABCG1 has a role in Aβ generation via modulation of membrane secretases. It is, however, necessary to investigate the underlying mechanism(s). ABCG1 may also contribute to AD pathology through its role in apoptosis and oxidative stress. As a result, ABCG1 plays a role in AD and is a candidate for drug development.

POTENTIAL MECHANISMS THROUGH WHICH INDUCING HEART RATE OSCILLATIONS AFFECTS PLASMA AMYLOID BETA LEVELS

Abstract Alzheimer’s disease-related protein amyloid beta (Aβ) plasma levels increase with age, and Aβ40 levels are associated with greater mortality. Most plasma Aβ40 is produced via platelets. Platelet activity is regulated by endothelial cells, which are stimulated by blood flow. Slow paced breathing with a 10-second cycle maximizes heart rate oscillations, which may stimulate endothelial-platelet interactions that affect Aβ production. We investigated this idea by randomizing healthy adults (54 younger and 54 older adults) into two groups with the opposite goals: increasing (Osc+) or decreasing (Osc-) heart rate oscillations. Four weeks of daily practice (20-40 min) resulted in large changes in plasma Aβ40 and Aβ42 levels. We also found that changes in Aβ were not associated with changes in resting-state physiology including heart rate variability. But the Aβ changes were associated with a frequency measure of heart rate oscillations induced during practice, suggesting that the Aβ changes were more driven by blood flow changes during practice. P-selectin is an indicator of endothelial activity. We found a significant intervention effect where the Osc+ intervention increased p-selectin and the Osc- intervention decreased p-selectin in older adults. Changes in p-selectin also showed strong negative correlations with changes in Aβ40 and Aβ42 in older adults. The results suggest that blood flow oscillations caused by high amplitude heart rate oscillations stimulated endothelial cells, which suppressed platelet activity and consequently reduced the production of Aβ in older adults. In follow-up research, we are investigating relationships between plasma Ab changes and changes in brain and cognition.

Apolipoprotein E4 inhibits γ-secretase activity via binding to the γ-secretase complex.

The mechanisms of amyloid accumulation in familial Alzheimer's disease (FAD) and sporadic AD (SAD) are controversial. In FAD, mutations in presenilin (PSEN) impair γ-secretase activity and lead to abnormal amyloid β-protein (Aβ) production, thereby increasing the Aβ42/40 ratio. SAD is postulated to be caused by decreased Aβ clearance of apolipoprotein E4 (APOE4), the strongest risk factor for SAD. However, whether intracellular APOE4 affects Aβ production is unclear. Using APOE3 and APOE4 knock-in (KI) mouse brain and primary cultured fibroblasts from these mice, in this study, we demonstrated that APOE3 and APOE4 bind to the γ-secretase complex and isoform-dependently regulate its activity and Aβ production. We found that Aβ40 levels and γ-secretase activity were higher in APOE knockout mouse brain than in wild-type mouse brain. APOE4-KI fibroblasts had significant lower Aβ levels and γ-secretase activity but higher Aβ42/40 ratio compared with APOE3-KI cells, indicating that APOE4-KI reduces Aβ production by inhibiting γ-secretase activity. Interestingly, the levels of γ-secretase complex bound to APOE4 are higher than those bound to APOE3, and the levels of γ-secretase complex in the brain and fibroblasts of APOE4-KI mice were higher than those of APOE3-KI mice. Taken together, our findings demonstrate that intracellular APOE4 inhibits Aβ production, more preferentially inhibits Aβ40 production, and thereby induces an increase in the Aβ42/40 ratio via binding to the γ-secretase complex. These results suggest a novel mechanism in which intracellular APOE4 contributes to the pathogenesis of SAD by inhibiting γ-secretase activity.

Structural Studies Providing Insights into Production and Conformational Behavior of Amyloid-β Peptide Associated with Alzheimer's Disease Development.

Alzheimer’s disease is the most common type of neurodegenerative disease in the world. Genetic evidence strongly suggests that aberrant generation, aggregation, and/or clearance of neurotoxic amyloid-β peptides (Aβ) triggers the disease. Aβ accumulates at the points of contact of neurons in ordered cords and fibrils, forming the so-called senile plaques. Aβ isoforms of different lengths are found in healthy human brains regardless of age and appear to play a role in signaling pathways in the brain and to have neuroprotective properties at low concentrations. In recent years, different substances have been developed targeting Aβ production, aggregation, interaction with other molecules, and clearance, including peptide-based drugs. Aβ is a product of sequential cleavage of the membrane glycoprotein APP (amyloid precursor protein) by β- and γ-secretases. A number of familial mutations causing an early onset of the disease have been identified in the APP, especially in its transmembrane domain. The mutations are reported to influence the production, oligomerization, and conformational behavior of Aβ peptides. This review highlights the results of structural studies of the main proteins involved in Alzheimer’s disease pathogenesis and the molecular mechanisms by which perspective therapeutic substances can affect Aβ production and nucleation.

Membrane dynamics of γ-secretase with the anterior pharynx-defective 1B subunit.

The four-subunit protease complex γ-secretase cleaves many single-pass transmembrane (TM) substrates, including Notch and β-amyloid precursor protein to generate amyloid-β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx-defective 1 (APH-1) and presenilin (PS) exist in two homologous forms APH1-A and APH1-B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ-secretase medicine. Here, we developed the first complete structural model of the APH-1B subunit using the published cryo-electron microscopy (cryo-EM) structures of APH1-A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all-atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH-1B alone and in γ-secretase without and with substrate C83-bound. We show that APH-1B adopts a 7TM topology with a water channel topology similar to APH-1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo-EM structures with APH-1A, however with subtle differences: The substrate-bound APH-1B γ-secretase was quite stable, but some TM helices of PS1 and APH-1B rearranged in the membrane consistent with the disorder seen in the cryo-EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH-1B, that is, it represents a more closed state, due to interactions with the C-terminal fragment of PS1. Our structural-dynamic model of APH-1B alone and in γ-secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.

Pathophysiological Mechanisms of Cognitive Impairment and Neurodegeneration by Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular parasite considered one of the most successful pathogens in the world, owing to its ability to produce long-lasting infections and to persist in the central nervous system (CNS) in most warm-blooded animals, including humans. This parasite has a preference to invade neurons and affect the functioning of glial cells. This could lead to neurological and behavioral changes associated with cognitive impairment. Although several studies in humans and animal models have reported controversial results about the relationship between toxoplasmosis and the onset of dementia as a causal factor, two recent meta-analyses have shown a relative association with Alzheimer’s disease (AD). AD is characterized by amyloid-β (Aβ) peptide accumulation, neurofibrillary tangles, and neuroinflammation. Different authors have found that toxoplasmosis may affect Aβ production in brain areas linked with memory functioning, and can induce a central immune response and neurotransmitter imbalance, which in turn, affect the nervous system microenvironment. In contrast, other studies have revealed a reduction of Aβ plaques and hyperphosphorylated tau protein formation in animal models, which might cause some protective effects. The aim of this article is to summarize and review the newest data in regard to different pathophysiological mechanisms of cerebral toxoplasmosis and their relationship with the development of AD and cognitive impairment. All these associations should be investigated further through clinical and experimental studies.

O-GlcNAcylation of Amyloid-β Protein Precursor by Insulin Signaling Reduces Amyloid-β Production.

Alzheimer's disease (AD) is caused by the accumulation of neurotoxic amyloid-β (Aβ) peptides. Aβ is derived from amyloid-β protein precursor (AβPP). In the non-amyloidogenic pathway, AβPP is cleaved by α-secretase and γ-secretase at the plasma membrane, excluding Aβ production. Alternatively, AβPP in the plasma membrane is internalized via endocytosis, and delivered to early endosomes and lysosomes, where it is cleaved by β-secretase and γ-secretase. Recent studies have shown that insulin in the periphery crosses the blood-brain barrier, and plays important roles in the brain. Furthermore, impaired insulin signaling has been linked to the progression of AD, and intranasal insulin administration improves memory impairments and cognition. However, the underlying molecular mechanisms of insulin treatment remain largely unknown. To investigate the effects of insulin on AβPP processing, we tested the effects of insulin on neuroblastoma SH-SY5Y cells overexpressing AβPP, and cultured rat cortical neurons. We found that insulin increased the level of cell surface AβPP, decreasing the endocytosis rate of AβPP. Insulin reduced Aβ generation through upregulation of AβPP O-GlcNAcylation via Akt insulin signaling. Our present data suggest that insulin affects Aβ production by regulating AβPP processing through AβPP O-GlcNAcylation. These results provide mechanistic insight into the beneficial effects of insulin, and a possible link between insulin deficient diabetes and cerebral amyloidosis in the pathogenesis of AD.

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) delays Alzheimer's progression in vivo.

Deposition of amyloid β protein (Aβ) to form neuritic plaques in the brain is the pathological hallmark of Alzheimer's disease (AD). Aβ is produced by β- and γ-cleavages of amyloid β precursor protein (APP). Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme that cleaves ubiquitin at its carboxyl terminal. Dysfunction of UCHL1 has been reported in neurodegenerative diseases. However, whether UCHL1 affects Aβ production and AD progression remains unknown. Here we report that UCHL1 interacts with APP and regulates Aβ production. UCHL1 increases free ubiquitin level and accelerates the lysosomal degradation of APP by promoting its ubiquitination. Furthermore, we demonstrate that overexpression of UCHL1 by intracranial injection of UCHL1-expressing rAAV reduces Aβ production, inhibits neuritic plaque formation and improves memory deficits in AD transgenic model mice. Our study suggests that UCHL1 may delay Alzheimer's progression by regulating APP degradation in a long-term fashion, and that overexpression of UCHL1 may be a safe and effective disease-modifying strategy to treat AD.

IKKβ deficiency in myeloid cells ameliorates Alzheimer's disease-related symptoms and pathology.

Alzheimer's disease (AD) is characterized by extracellular amyloid-β (Aβ) deposits and microglia-dominated inflammatory activation. Innate immune signaling controls microglial inflammatory activities and Aβ clearance. However, studies examining innate immunity in Aβ pathology and neuronal degeneration have produced conflicting results. In this study, we investigated the pathogenic role of innate immunity in AD by ablating a key signaling molecule, IKKβ, specifically in the myeloid cells of TgCRND8 APP-transgenic mice. Deficiency of IKKβ in myeloid cells, especially microglia, simultaneously reduced inflammatory activation and Aβ load in the brain and these effects were associated with reduction of cognitive deficits and preservation of synaptic structure proteins. IKKβ deficiency enhanced microglial recruitment to Aβ deposits and facilitated Aβ internalization, perhaps by inhibiting TGF-β-SMAD2/3 signaling, but did not affect Aβ production and efflux. Therefore, inhibition of IKKβ signaling in myeloid cells improves cognitive functions in AD mice by reducing inflammatory activation and enhancing Aβ clearance. These results contribute to a better understanding of AD pathogenesis and could offer a new therapeutic option for delaying AD progression.

A critique of the drug discovery and phase 3 clinical programs targeting the amyloid hypothesis for Alzheimer disease.

In 1906, Alois Alzheimer described the neuropathology of the disease that was to bear his name. Subsequently, our understanding of Alzheimer disease (AD) has grown significantly. The autosomal dominant mutations to the amyloid precursor protein (APP), presenilin (PS) 1, and PS2 genes that cause early onset AD have been very informative, leading to the articulation of the amyloid cascade hypothesis. This hypothesis has been the basis for several disease-modifying therapeutic approaches for AD. This has been partly because it provided a coherent framework for understanding AD pathogenesis but also because several pharmacological approaches that targeted the amyloid peptide (amyloidocentric) were sufficiently well-founded scientifically to enter clinical development. In the past 5 years, there have been 6 amyloidocentric programs that completed phase 3 clinical testing. None met their primary outcome measures (Table 1), although 1, solanezumab, showed encouraging results in a prespecified secondary outcome measure. This disappointing track record has brought into question the amyloidocentric therapeutic approach. This review will consider these programs from the following perspectives:

Erlin-2 is associated with active γ-secretase in brain and affects amyloid β-peptide production

The transmembrane protease complex γ-secretase is responsible for the generation of the neurotoxic amyloid β-peptide (Aβ) from its precursor (APP). Aβ has a causative role in Alzheimer disease, and thus, γ-secretase is a therapeutic target. However, since there are more than 70 γ-secretase substrates besides APP, selective inhibition of APP processing is required. Recent data indicates the existence of several γ-secretase associated proteins (GSAPs) that affect the selection and processing of substrates. Here, we use a γ-secretase inhibitor for affinity purification of γ-secretase and associated proteins from microsomes and detergent resistant membranes (DRMs) prepared from rat or human brain. By tandem mass spectrometry we identified a novel brain GSAP; erlin-2. This protein was recently reported to reside in DRMs in the ER. A proximity ligation assay, as well as co-immunoprecipitation, confirmed the association of erlin-2 with γ-secretase. We found that a higher proportion of erlin-2 was associated with γ-secretase in DRMs than in soluble membranes. siRNA experiments indicated that reduced levels of erlin-2 resulted in a decreased Aβ production, whereas the effect on Notch processing was limited. In summary, we have found a novel brain GSAP, erlin-2, that resides in DRMs and affects Aβ production.

Elevated amyloid β production in senescent retinal pigment epithelium, a possible mechanism of subretinal deposition of amyloid β in age-related macular degeneration

Age-related macular degeneration (AMD) is the most common cause of legal blindness in the elderly individuals in developed countries. Subretinally-deposited amyloid β (Aβ) is a main contributor of developing AMD. However, the mechanism causing Aβ deposition in AMD eyes is unknown. Aging is the most significant risk of AMD, thus, we examined the effect of aging on subretinal Aβ deposition. mRNAs and cell lysates were isolated from retinal pigment epithelial (RPE) cells derived from 24-month-old (24M RPE) and 2-month-old (2M RPE) C57BL/6 mice. Aβ concentration in culture supernatants was measured by ELISA. Activity and expression of proteins that regulate Aβ level were examined by activity assay and real time PCR. Effect of β-secretase (BACE) on Aβ production was examined by siRNA silencing. Aβ amounts in supernatants of 24M RPE were significantly higher than 2M RPE. Activity and mRNA levels of neprilysin, an Aβ degrading enzyme, were significantly decreased in 24M RPE compared to 2M RPE. PCR analysis found that BACE2 was significantly more abundantly expressed than BACE1 in RPE cells, however, inactivation of BACE2 gene did not affect Aβ production. BACE1 protein amounts did not differ between 24M and 2M RPE, however, BACE1 activity was significantly higher in 24M RPE compared to 2M RPE. There were no significant changes in the activities of α- or γ-secretase between 2M and 24M RPE. In conclusion, RPE cells produce more amounts of Aβ when they are senescent, and this is probably caused by a decrease in Aβ degradation due to a reduction in the expression and activity of neprilysin and an increase in Aβ synthesis due to increased activity of BACE1.

Neuronal β‐amyloid generation is independent of lipid raft association of β‐secretase BACE1: analysis with a palmitoylation‐deficient mutant

β-Secretase, BACE1 is a neuron-specific membrane-associated protease that cleaves amyloid precursor protein (APP) to generate β-amyloid protein (Aβ). BACE1 is partially localized in lipid rafts. We investigated whether lipid raft localization of BACE1 affects Aβ production in neurons using a palmitoylation-deficient mutant and further analyzed the relationship between palmitoylation of BACE1 and its shedding and dimerization. We initially confirmed that BACE1 is mainly palmitoylated at four C-terminal cysteine residues in stably transfected neuroblastoma cells. We found that raft localization of mutant BACE1 lacking the palmitoylation modification was markedly reduced in comparison to wild-type BACE1 in neuroblastoma cells as well as rat primary cortical neurons expressing BACE1 via recombinant adenoviruses. In primary neurons, expression of wild-type and mutant BACE1 enhanced production of Aβ from endogenous or overexpressed APP to similar extents with the β-C-terminal fragment (β-CTF) of APP mainly distributed in nonraft fractions. Similarly, β-CTF was recovered mainly in nonraft fractions of neurons expressing Swedish mutant APP only. These results show that raft association of BACE1 does not influence β-cleavage of APP and Aβ production in neurons, and support the view that BACE1 cleaves APP mainly in nonraft domains. Thus, we propose a model of neuronal Aβ generation involving mobilization of β-CTF from nonraft to raft domains. Additionally, we obtained data indicating that palmitoylation plays a role in BACE1 shedding but not dimerization.

Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified γ-Secretase Complexes

BackgroundMutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1–42/Aβ1–40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1–42 production, decreasing Aβ1–40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q.Methodology/Principal FindingsWe took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1–40, Aβ1–42 and APP intracellular domain productions in vitro.Conclusion/SignificanceOur data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1–42/Aβ1–40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease.

Identification of Novel γ-Secretase-associated Proteins in Detergent-resistant Membranes from Brain

In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.

Prenatal Lead Levels, Plasma Amyloid β Levels, and Gene Expression in Young Adulthood

Background: Animal studies suggest that early-life lead exposure influences gene expression and production of proteins associated with Alzheimer’s disease (AD).Objectives: We attempted to assess the relationship between early-life lead exposure and potential biomarkers for AD among young men and women. We also attempted to assess whether early-life lead exposure was associated with changes in expression of AD-related genes.Methods: We used sandwich enzyme-linked immunosorbent assays (ELISA) to measure plasma concentrations of amyloid β proteins Aβ40 and Aβ42 among 55 adults who had participated as newborns and young children in a prospective cohort study of the effects of lead exposure on development. We used RNA microarray techniques to analyze gene expression.Results: Mean plasma Aβ42 concentrations were lower among 13 participants with high umbilical cord blood lead concentrations (≥ 10 μg/dL) than in 42 participants with lower cord blood lead concentrations (p = 0.08). Among 10 participants with high prenatal lead exposure, we found evidence of an inverse relationship between umbilical cord lead concentration and expression of ADAM metallopeptidase domain 9 (ADAM9), reticulon 4 (RTN4), and low-density lipoprotein receptor-related protein associated protein 1 (LRPAP1) genes, whose products are believed to affect Aβ production and deposition. Gene network analysis suggested enrichment in gene sets involved in nerve growth and general cell development.Conclusions: Data from our exploratory study suggest that prenatal lead exposure may influence Aβ-related biological pathways that have been implicated in AD onset. Gene network analysis identified further candidates to study the mechanisms of developmental lead neurotoxicity.

Crystal Structure of the E2 Domain of Amyloid Precursor Protein-like Protein 1 in Complex with Sucrose Octasulfate

Missense mutations in the amyloid precursor protein (APP) gene can cause familial Alzheimer disease. It is thought that APP and APP-like proteins (APLPs) may play a role in adhesion and signal transduction because their ectodomains interact with components of the extracellular matrix. Heparin binding induces dimerization of APP and APLPs. To help explain how these proteins interact with heparin, we have determined the crystal structure of the E2 domain of APLP1 in complex with sucrose octasulfate (SOS). A total of three SOS molecules are bound to the E2 dimer. Two SOSs are bound inside a narrow intersubdomain groove, and the third SOS is bound near the two-fold axis of the protein. Mutational analyses show that most residues interacting with SOS also contribute to heparin binding, although in varying degrees; a deep pocket, defined by His-376, Lys-422, and Arg-429, and an interfacial site between Lys-314 and its symmetry mate are most important in the binding of the negatively charged polysaccharide. Comparison with a lower resolution APP structure shows that all key heparin binding residues are conserved and identically positioned, suggesting that APLP1 and APP may bind heparin similarly. In transfected HEK-293 cells, mutating residues responsible for heparin binding causes little change in the proteolysis of APP by the secretases. However, mutating a pair of conserved basic residues (equivalent to Arg-414 and Arg-415 of APLP1) immediately adjacent to the heparin binding site affects both the maturation and the processing of APP.

Flavonoids lower Alzheimer's Aβ production via an NFκB dependent mechanism.

Alzheimer's disease (AD) is characterized by the brain accumulation of Aβ peptides and by the presence of neurofibrillary tangles. Aβ is believed to play an important role in AD and it has been shown that certain flavonoids can affect Aβ production. Recently, it was suggested that the Aβ lowering properties of flavonoids are mediated by a direct inhibition the β-secretase (BACE-1) activity, the rate limiting enzyme responsible for the production of Aβ peptides. Westernblots and ELISAs were employed to monitor the impact of flavonoids on amyloid precursor protein processing and Aβ production. A cell free chemoluminescent assay using human recombinant BACE-1 was used to assess the effect of flavonoids on BACE-1 activity. The effect of flavonoids on NFκB activation was determined by using a stable NFκB luciferase reporter cell line. Molecular docking simulations were performed to predict the binding of flavonoids to the BACE-1 catalytic site. Real time quantitative PCR was used to determine the effect of flavonoids on BACE-1 transcription. We show in a cell free assay that flavonoids are only weak inhibitors of BACE-1 activity. Docking simulation studies with different BACE-1 structures also suggest that flavonoids are poor BACE-1 inhibitors as they appear to adopt various docking poses in the active site pocket and have weak docking scores that differ as a function of the BACE-1 structures studied. Moreover, a weak correlation was observed between the effect of flavonoids on Aβ production in vitro and their ability to lower BACE-1 activity suggesting that the Aβ lowering properties of flavonoids in whole cells are not mediated via direct inhibition of BACE-1 activity. We found however a strong correlation between the inhibition of NFκB activation by flavonoids and their Aβ lowering properties suggesting that flavonoids inhibit Aβ production in whole cells via NFκB related mechanisms. As NFκB has been shown to regulate BACE-1 expression, we show that NFκB lowering flavonoids inhibit BACE-1 transcription in human neuronal SH-SY5Y cells. Altogether, our data suggest that flavonoids inhibit Aβ and sAPPβ production by regulating BACE-1 expression and not by directly inhibiting BACE-1 activity.