Increased γ-Secretase Activity in Idiopathic Normal Pressure Hydrocephalus Patients with β-Amyloid Pathology

The potential similarity between the brain pathology of idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer disease (AD) is intriguing and thus further studies focusing on the underlying molecular mechanisms may offer valuable information for differential diagnostics and the development of treatments for iNPH. Here, we investigated β- and γ-secretase activities in relation to amyloid-β (Aβ) pathology in the brain tissue samples collected from iNPH and AD patients. β- and γ-secretase activities were measured from the frontal cortical biopsies of 26 patients with suspected iNPH as well as post-mortem tissue samples from the inferior temporal cortex of 74 AD patients and eight subjects without neurofibrillary pathology. In iNPH samples with detectable Aβ plaques, γ-secretase activity was significantly increased (∼1.6-fold) when compared to iNPH samples without Aβ plaques (p = 0.009). In the AD samples, statistically significant differences in the γ-secretase activity were not observed with respect to disease severity (mild, moderate and severe AD according to neurofibrillary pathology). Conversely, β-secretase activity was unaltered in iNPH samples with or without Aβ plaques, while it was significantly increased in relation to disease severity in the AD patients. These results show for the first time increased γ-secretase but not β-secretase activity in the biopsy samples from the frontal cortex of iNPH patients with AD-like Aβ pathology. Conversely, the opposite was observed in these secretase activities in AD patients with respect to neurofibrillary pathology. Despite the resemblances in the Aβ pathology, iNPH and AD patients appear to have marked differences in the cellular mechanisms responsible for the production of Aβ.     

Longitudinal Study of CSF Biomarkers in Patients with Alzheimer's Disease

Background

The CSF biomarkers tau and Aβ42 can identify patients with AD, even during the preclinical stages. However, previous studies on longitudinal changes of tau and Aβ42 in individual patients with AD and elderly controls report somewhat inconsistent results.

Methodology/Principal Findings

We investigated the levels of tau and Aβ42 at baseline and after 1 year in 100 patients with AD. In a second cohort of 45 AD patients we measured the CSF biomarkers at baseline and after 2 years. Moreover, in 34 healthy elderly controls the CSF biomarkers were followed for 4 years. The baseline levels of tau were increased with >60% in AD patients compared to controls (p<0.001), while baseline Aβ42 levels were decreased with >50% (p<0.001). In the AD group followed for 2 years, tau increased with 16% compared to the baseline levels (p<0.05). However, the levels of tau were stable over 4 years in the controls. The levels of Aβ42 did not change significantly over time in any of the groups. In the patients with AD, tau was moderately associated with worse cognitive performance already at baseline (p<0.05).

Conclusions/Significance

Tau and Aβ42 in CSF seem to reflect the underlying disease state in both early and late stages of AD. The slight increase in tau over time observed in the patients with AD is modest when compared to the relatively large difference in absolute tau levels between AD patients and controls. Therefore, these markers maintain their usefulness as state markers over time and might serve as surrogate markers for treatment efficacy in clinical trials.     

Pathway-Based Analysis of Genome-Wide siRNA Screens Reveals the Regulatory Landscape of App Processing

The progressive aggregation of Amyloid-β (Aβ) in the brain is a major trait of Alzheimer''s Disease (AD). Aβ is produced as a result of proteolytic processing of the β-amyloid precursor protein (APP). Processing of APP is mediated by multiple enzymes, resulting in the production of distinct peptide products: the non-amyloidogenic peptide sAPPα and the amyloidogenic peptides sAPPβ, Aβ40, and Aβ42. Using a pathway-based approach, we analyzed a large-scale siRNA screen that measured the production of different APP proteolytic products. Our analysis identified many of the biological processes/pathways that are known to regulate APP processing and have been implicated in AD pathogenesis, as well as revealing novel regulatory mechanisms. Furthermore, we also demonstrate that some of these processes differentially regulate APP processing, with some mechanisms favouring production of certain peptide species over others. For example, synaptic transmission having a bias towards regulating Aβ40 production over Aβ42 as well as processes involved in insulin and pancreatic biology having a bias for sAPPβ production over sAPPα. In addition, some of the pathways identified as regulators of APP processing contain genes (CLU, BIN1, CR1, PICALM, TREM2, SORL1, MEF2C, DSG2, EPH1A) recently implicated with AD through genome wide association studies (GWAS) and associated meta-analysis. In addition, we provide supporting evidence and a deeper mechanistic understanding of the role of diabetes in AD. The identification of these processes/pathways, their differential impact on APP processing, and their relationships to each other, provide a comprehensive systems biology view of the “regulatory landscape” of APP.     

Cerebrospinal Fluid (CSF) Neuronal Biomarkers across the Spectrum of HIV Infection: Hierarchy of Injury and Detection

The character of central nervous system (CNS) HIV infection and its effects on neuronal integrity vary with evolving systemic infection. Using a cross-sectional design and archived samples, we compared concentrations of cerebrospinal fluid (CSF) neuronal biomarkers in 143 samples from 8 HIV-infected subject groups representing a spectrum of untreated systemic HIV progression and viral suppression: primary infection; four groups of chronic HIV infection neuroasymptomatic (NA) subjects defined by blood CD4+ T cells of >350, 200–349, 50–199, and <50 cells/µL; HAD; treatment-induced viral suppression; and ‘elite’ controllers. Samples from 20 HIV-uninfected controls were also examined. The neuronal biomarkers included neurofilament light chain protein (NFL), total and phosphorylated tau (t-tau, p-tau), soluble amyloid precursor proteins alpha and beta (sAPPα, sAPPβ) and amyloid beta (Aβ) fragments 1–42, 1–40 and 1–38. Comparison of the biomarker changes showed a hierarchy of sensitivity in detection and suggested evolving mechanisms with progressive injury. NFL was the most sensitive neuronal biomarker. Its CSF concentration exceeded age-adjusted norms in all HAD patients, 75% of NA CD4<50, 40% of NA CD4 50–199, and 42% of primary infection, indicating common neuronal injury with untreated systemic HIV disease progression as well as transiently during early infection. By contrast, only 75% of HAD subjects had abnormal CSF t-tau levels, and there were no significant differences in t-tau levels among the remaining groups. sAPPα and β were also abnormal (decreased) in HAD, showed less marked change than NFL with CD4 decline in the absence of HAD, and were not decreased in PHI. The CSF Aβ peptides and p-tau concentrations did not differ among the groups, distinguishing the HIV CNS injury profile from Alzheimer''s disease. These CSF biomarkers can serve as useful tools in selected research and clinical settings for patient classification, pathogenetic analysis, diagnosis and management.     

A Luminex Assay Detects Amyloid β Oligomers in Alzheimer’s Disease Cerebrospinal Fluid

Amyloid beta (aβ) protein assembles into larger protein aggregates during the pathogenesis of Alzheimer’s disease (AD) and there is increasing evidence that soluble aβ oligomers are a critical pathologic species. Diagnostic evaluations rely on the measurement of increased tau and decreased aβ42 in the cerebrospinal fluid (CSF) from AD patients and evidence for oligomeric aβ in patient CSF is conflicting. In this study, we have adapted a monoclonal single antibody sandwich ELISA assay to a Luminex platform and found that this assay can detect oligomerized aβ42 and sAPPα fragments. We evaluated oligomeric aβ reactivity in 20 patients with AD relative to 19 age matched controls and compared these values with a commercially available Alzbio3 kit that detects tau, phosphorylated tau and aβ42 on the same diagnostic platform. We found that CSF samples of patients with AD had elevated aβ oligomers compared to control subjects (p < 0.05) and the ratio of aβ oligomers to aβ42 was also significantly elevated (p < 0.0001). Further research to develop high sensitivity analytical platforms and rigorous methods of developing stable assay standards will be needed before the analysis of oligomeric aβ becomes a routine diagnostic assay for the evaluation of late onset AD patients.     

Cognitive Performance and Cerebrospinal Fluid Biomarkers of Neurodegeneration: A Study of Patients with Bipolar Disorder and Healthy Controls

The purpose of the present study was to investigate if cerebrospinal fluid (CSF) biomarkers of neurodegeneration are associated with cognition in bipolar disorder and healthy controls, respectively. CSF concentrations of total and phosphorylated tau, amyloid beta (Aβ)1-42, ratios of Aβ42/40 and Aβ42/38, soluble amyloid precursor protein α and β, and neurofilament light chain protein were analyzed in relation to neuropsychological performance in 82 euthymic bipolar disorder patients and 71 healthy controls. Linear regression models were applied to account for performance in five cognitive domains using the CSF biomarkers. In patients, the CSF biomarkers explained a significant proportion of the variance (15–36%, p=.002 - <.0005) in all cognitive domains independently of age, medication, disease status, and bipolar subtype I or II. However, the CSF biomarkers specifically mirroring Alzheimer-type brain changes, i.e., P-tau and Aβ1-42, did not contribute significantly. In healthy controls, CSF biomarkers did not explain the variance in cognitive performance. Selected CSF biomarkers of neurodegenerative processes accounted for cognitive performance in persons with bipolar disorder, but not for healthy controls. Specifically, the ratios of Aβ42/40 and Aβ42/38 were consistently associated with altered cognitive performance.     

Cerebrospinal Fluid PKR Level Predicts Cognitive Decline in Alzheimer’s Disease

The cerebrospinal fluid (CSF) levels of the proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR) are increased in Alzheimer’s disease (AD), but whether CSF PKR concentrations are associated with cognitive decline in AD patients remain unknown. In this study, 41 consecutive patients with AD and 11 patients with amnestic mild cognitive impairment (aMCI) from our Memory Clinic were included. A lumbar puncture was performed during the following month of the clinical diagnosis and Mini-Mental State Examination (MMSE) evaluations were repeated every 6 months during a mean follow-up of 2 years. In AD patients, linear mixed models adjusted for age and sex were used to assess the cross-sectional and longitudinal associations between MMSE scores and baseline CSF levels of Aβ peptide (Aβ 1-42), Tau, phosphorylated Tau (p-Tau 181), PKR and pPKR. The mean (SD) MMSE at baseline was 20.5 (6.1) and MMSE scores declined over the follow-up (-0.12 point/month, standard error [SE] = 0.03). A lower MMSE at baseline was associated with lower levels of CSF Aβ 1–42 and p-Tau 181/Tau ratio. pPKR level was associated with longitudinal MMSE changes over the follow-up, higher pPKR levels being related with an exacerbated cognitive deterioration. Other CSF biomarkers were not associated with MMSE changes over time. In aMCI patients, mean CSF biomarker levels were not different in patients who converted to AD from those who did not convert.These results suggest that at the time of AD diagnosis, a higher level of CSF pPKR can predict a faster rate of cognitive decline.     

No correlation between time-linked plasma and CSF Aβ levels

Plasma β-amyloid protein (Aβ) isoforms are considered potential biomarkers for Alzheimer's disease (AD) and dementia. The relation between plasma and cerebrospinal fluid (CSF) levels of Aβ isoforms remains unclear. In order to identify possible correlations between Aβ levels in plasma and CSF we determined Aβ levels in time-linked plasma and CSF samples. Aβ concentrations in plasma (Aβ_1–42 and Aβ_N–42) and CSF (Aβ_1–42) samples from 49 AD patients, 47 non-Alzheimer's disease dementia (NONAD) patients, 39 MCI patients and 29 controls were determined using a multi-parameter fluorimetric bead-based immunoassay using xMAP^® technology (for plasma) and a conventional single-parameter ELISA (for CSF). Plasma Aβ_1–42 concentrations did not correlate with CSF Aβ_1–42 concentrations in the total study population, or in the different diagnostic groups. No correlations between plasma Aβ_N–42 and CSF Aβ_1–42 levels were found either. The CSF/serum albumin index did not show any significant differences between AD, NONAD, MCI and controls.These results suggest that the Aβ levels in plasma are independent of the Aβ levels in CSF both in dementia and controls. The fact that CSF and plasma Aβ do not correlate in patients as well as controls and no significant differences in plasma Aβ_1–42 or Aβ_N–42 between patients and controls can be detected hampers the diagnostic utility of the plasma Aβ levels as biomarkers for dementia.     

Protection of TGF-β1 against Neuroinflammation and Neurodegeneration in Aβ1–42-Induced Alzheimer’s Disease Model Rats

Neuroinflammation has been reported to be associated with Alzheimer’s disease (AD) pathogenesis. Neuroinflammation is generally considered as an outcome of glial activation; however, we recently demonstrated that T helper (Th)17 cells, a subpopulation of proinflammatory CD4+ T cells, are also involved in AD pathogenesis. Transforming growth factor (TGF)-β1, a cytokine that can be expressed in the brain, can be immunosuppressive, but its effects on lymphocyte-mediated neuroinflammation in AD pathogenesis have not been well addressed. In the current study we administered TGF-β1 via intracerebroventricle (ICV) and intranasal (IN) routes in AD model rats to investigate its antiinflammatory and neuroprotective effects. The AD rat model was prepared by bilateral hippocampal injection of amyloid-β (Aβ)_1–42. TGF-β1 was administered via ICV one hour prior to Aβ_1–42 injection or via both nares seven days after Aβ_1–42 injection. ICV administration of TGF-β1 before Aβ_1–42 injection remarkably ameliorated Aβ_1–42-induced neurodegeneration and prevented Aβ_1–42-induced increases in glia-derived proinflammatory mediators (TNF-α, IL-1β and iNOS), as well as T cell-derived proinflammatory cytokines (IFN-γ, IL-2, IL-17 and IL-22), in the hypothalamus, serum or cerebrospinal fluid (CSF) in a concentration-dependent manner. TGF-β1 pretreatment also prevented Aβ_1–42-induced decreases in the neurotrophic factors, IGF-1, GDNF and BDNF, and in the antiinflammatory cytokine, IL-10. Similarly, IN administration of TGF-β1 after Aβ_1–42 injection reduced neurodegeneration, elevation of proinflammatory mediators and cytokines, and reduction of neurotrophic and antiinflammatory factors, in the hypothalamus, serum or CSF. These findings suggest that TGF-β1 suppresses glial and T cell-mediated neuroinflammation and thereby alleviates AD-related neurodegeneration. The effectiveness of IN administered TGF-β1 in reducing Aβ_1–42 neurotoxicity suggests a possible therapeutic approach in patients with AD.     

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Alzheimer''s disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of β-amyloid peptide (Aβ) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of Aβ (Aβ42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in Aβ or tau levels, making it possible to detect subtle alterations over time. In combination with Aβ and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF Aβ42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF Aβ or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.Open in a separate windowClick here to view.^{(49M, flv)}     

In Vivo Turnover of Tau and APP Metabolites in the Brains of Wild-Type and Tg2576 Mice: Greater Stability of sAPP in the β-Amyloid Depositing Mice

The metabolism of the amyloid precursor protein (APP) and tau are central to the pathobiology of Alzheimer''s disease (AD). We have examined the in vivo turnover of APP, secreted APP (sAPP), Aβ and tau in the wild-type and Tg2576 mouse brain using cycloheximide to block protein synthesis. In spite of overexpression of APP in the Tg2576 mouse, APP is rapidly degraded, similar to the rapid turnover of the endogenous protein in the wild-type mouse. sAPP is cleared from the brain more slowly, particularly in the Tg2576 model where the half-life of both the endogenous murine and transgene-derived human sAPP is nearly doubled compared to wild-type mice. The important Aβ degrading enzymes neprilysin and IDE were found to be highly stable in the brain, and soluble Aβ40 and Aβ42 levels in both wild-type and Tg2576 mice rapidly declined following the depletion of APP. The cytoskeletal-associated protein tau was found to be highly stable in both wild-type and Tg2576 mice. Our findings unexpectedly show that of these various AD-relevant protein metabolites, sAPP turnover in the brain is the most different when comparing a wild-type mouse and a β-amyloid depositing, APP overexpressing transgenic model. Given the neurotrophic roles attributed to sAPP, the enhanced stability of sAPP in the β-amyloid depositing Tg2576 mice may represent a neuroprotective response.     

COPS5 Protein Overexpression Increases Amyloid Plaque Burden,Decreases Spinophilin-immunoreactive Puncta,and Exacerbates Learning and Memory Deficits in the Mouse Brain

Brain accumulation of neurotoxic amyloid β (Aβ) peptide because of increased processing of amyloid precursor protein (APP), resulting in loss of synapses and neurodegeneration, is central to the pathogenesis of Alzheimer disease (AD). Therefore, the identification of molecules that regulate Aβ generation and those that cause synaptic damage is crucial for future therapeutic approaches for AD. We demonstrated previously that COPS5 regulates Aβ generation in neuronal cell lines in a RanBP9-dependent manner. Consistent with the data from cell lines, even by 6 months, COPS5 overexpression in APΔE9 mice (APΔE9/COPS5-Tg) significantly increased Aβ40 levels by 32% (p < 0.01) in the cortex and by 28% (p < 0.01) in the hippocampus, whereas the increases for Aβ42 were 37% (p < 0.05) and 34% (p < 0.05), respectively. By 12 months, the increase was even more robust. Aβ40 levels increased by 63% (p < 0.001) in the cortex and by 65% (p < 0.001) in the hippocampus. Similarly, Aβ42 levels were increased by 69% (p < 0.001) in the cortex and by 71% (p < 0.011) in the hippocampus. Increased Aβ levels were translated into an increased amyloid plaque burden both in the cortex (54%, p < 0.01) and hippocampus (64%, p < 0.01). Interestingly, COPS5 overexpression increased RanBP9 levels in the brain, which, in turn, led to increased amyloidogenic processing of APP, as reflected by increased levels of sAPPβ and decreased levels of sAPPα. Furthermore, COPS5 overexpression reduced spinophilin in both the cortex (19%, p < 0.05) and the hippocampus (20%, p < 0.05), leading to significant deficits in learning and memory skills. Therefore, like RanBP9, COPS5 also plays a pivotal role in amyloid pathology in vivo.     

Evaluating Amyloid-β Oligomers in Cerebrospinal Fluid as a Biomarker for Alzheimer’s Disease

The current study evaluated amyloid-β oligomers (Aβo) in cerebrospinal fluid as a clinical biomarker for Alzheimer’s disease (AD). We developed a highly sensitive Aβo ELISA using the same N-terminal monoclonal antibody (82E1) for capture and detection. CSF samples from patients with AD, mild cognitive impairment (MCI), and healthy controls were examined. The assay was specific for oligomerized Aβ with a lower limit of quantification of 200 fg/ml, and the assay signal showed a tight correlation with synthetic Aβo levels. Three clinical materials of well characterized AD patients (n = 199) and cognitively healthy controls (n = 148) from different clinical centers were included, together with a clinical material of patients with MCI (n = 165). Aβo levels were elevated in the all three AD-control comparisons although with a large overlap and a separation from controls that was far from complete. Patients with MCI who later converted to AD had increased Aβo levels on a group level but several samples had undetectable levels. These results indicate that presence of high or measurable Aβo levels in CSF is clearly associated with AD, but the overlap is too large for the test to have any diagnostic potential on its own.     

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1)

Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer’s disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.     

Alzheimer Presenilin-1 Mutations Dramatically Reduce Trimming of Long Amyloid β-Peptides (Aβ) by γ-Secretase to Increase 42-to-40-Residue Aβ

The presenilin-containing γ-secretase complex produces the amyloid β-peptide (Aβ) through intramembrane proteolysis, and >100 presenilin mutations are associated with familial early-onset Alzheimer disease (AD). The question of whether these mutations result in AD through a gain or a loss of function remains highly controversial. Mutations in presenilins increase ratios of 42- to 40-residue Aβ critical to pathogenesis, but other Aβs of 38–49 residues are also formed by γ-secretase. Evidence in cells suggests the protease first cleaves substrate within the transmembrane domain at the ϵ site to form 48- or 49-residue Aβ. Subsequent cleavage almost every three residues from the C terminus is thought to occur along two pathways toward shorter secreted forms of Aβ: Aβ49 → Aβ46 → Aβ43 → Aβ40 and Aβ48 → Aβ45 → Aβ42 → Aβ38. Here we show that the addition of synthetic long Aβ peptides (Aβ45–49) directly into purified preparations of γ-secretase leads to the formation of Aβ40 and Aβ42 whether the protease complex is detergent-solubilized or reconstituted into lipid vesicles, and the ratios of products Aβ42 to Aβ40 follow a pattern consistent with the dual-pathway hypothesis. Kinetic analysis of five different AD-causing mutations in presenilin-1 revealed that all result in drastic reduction of normal carboxypeptidase function. Altered trimming of long Aβ peptides to Aβ40 and Aβ42 by mutant proteases occurs at multiple levels, independent of the effects on initial endoproteolysis at the ϵ site, all conspiring to increase the critical Aβ42/Aβ40 ratio implicated in AD pathogenesis. Taken together, these results suggest that specific reduction of carboxypeptidase function of γ-secretase leads to the gain of toxic Aβ42/Aβ40.     

Levels of Soluble Apolipoprotein E/Amyloid-β (Aβ) Complex Are Reduced and Oligomeric Aβ Increased with APOE4 and Alzheimer Disease in a Transgenic Mouse Model and Human Samples

Human apolipoprotein E (apoE) isoforms may differentially modulate amyloid-β (Aβ) levels. Evidence suggests physical interactions between apoE and Aβ are partially responsible for these functional effects. However, the apoE/Aβ complex is not a single static structure; rather, it is defined by detection methods. Thus, literature results are inconsistent and difficult to interpret. An ELISA was developed to measure soluble apoE/Aβ in a single, quantitative method and was used to address the hypothesis that reduced levels of soluble apoE/Aβ and an increase in soluble Aβ, specifically oligomeric Aβ (oAβ), are associated with APOE4 and AD. Previously, soluble Aβ42 and oAβ levels were greater with APOE4 compared with APOE2/APOE3 in hippocampal homogenates from EFAD transgenic mice (expressing five familial AD mutations and human apoE isoforms). In this study, soluble apoE/Aβ levels were lower in E4FAD mice compared with E2FAD and E3FAD mice, thus providing evidence that apoE/Aβ levels isoform-specifically modulate soluble oAβ clearance. Similar results were observed in soluble preparations of human cortical synaptosomes; apoE/Aβ levels were lower in AD patients compared with controls and lower with APOE4 in the AD cohort. In human CSF, apoE/Aβ levels were also lower in AD patients and with APOE4 in the AD cohort. Importantly, although total Aβ42 levels decreased in AD patients compared with controls, oAβ levels increased and were greater with APOE4 in the AD cohort. Overall, apoE isoform-specific formation of soluble apoE/Aβ modulates oAβ levels, suggesting a basis for APOE4-induced AD risk and a mechanistic approach to AD biomarkers.     

Brain Endothelial Cells Produce Amyloid β from Amyloid Precursor Protein 770 and Preferentially Secrete the O-Glycosylated Form

Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.     

Octyl Gallate Markedly Promotes Anti-Amyloidogenic Processing of APP through Estrogen Receptor-Mediated ADAM10 Activation

Our previous studies showed that the green tea-derived polyphenolic compound (−)-epigallocatechin-3 gallate (EGCG) reduces amyloid-β (Aβ) production in both neuronal and mouse Alzheimer’s disease (AD) models in concert with activation of estrogen receptor-α/phosphatidylinositide 3-kinase/protein kinase B (ERα/PI3K/Akt) signaling and anti-amyloidogenic amyloid precursor protein (APP) α-secretase (a disintegrin and metallopeptidase domain-10, ADAM10) processing. Since the gallate moiety in EGCG may correspond to the 7α position of estrogen, thereby facilitating ER binding, we extensively screened the effect of other gallate containing phenolic compounds on APP anti-amyloidogenic processing. Octyl gallate (OG; 10 µM), drastically decreased Aβ generation, in concert with increased APP α-proteolysis, in murine neuron-like cells transfected with human wild-type APP or “Swedish” mutant APP. OG markedly increased production of the neuroprotective amino-terminal APP cleavage product, soluble APP-α (sAPPα). In accord with our previous study, these cleavage events were associated with increased ADAM10 maturation and reduced by blockade of ERα/PI3k/Akt signaling. To validate these findings in vivo, we treated Aβ-overproducing Tg2576 mice with OG daily for one week by intracerebroventricular injection and found decreased Aβ levels associated with increased sAPPα. These data indicate that OG increases anti-amyloidogenic APP α-secretase processing by activation of ERα/PI3k/Akt signaling and ADAM10, suggesting that this compound may be an effective treatment for AD.     

Dynamin 1 Regulates Amyloid Generation through Modulation of BACE-1

Background

Several lines of investigation support the notion that endocytosis is crucial for Alzheimer’s disease (AD) pathogenesis. Substantial evidence have already been reported regarding the mechanisms underlying amyloid precursor protein (APP) traffic, but the regulation of beta-site APP-Cleaving Enzyme 1 (BACE-1) distribution among endosomes, TGN and plasma membrane remains unclear. Dynamin, an important adaptor protein that controls sorting of many molecules, has recently been associated with AD but its functions remain controversial. Here we studied possible roles for dynamin 1 (dyn1) in Aβ biogenesis.

Principal Findings

We found that genetic perturbation of dyn1 reduces both secreted and intracellular Aβ levels in cell culture. There is a dramatic reduction in BACE-1 cleavage products of APP (sAPPβ and βCTF). Moreover, dyn1 knockdown (KD) leads to BACE-1 redistribution from the Golgi-TGN/endosome to the cell surface. There is an increase in the amount of surface holoAPP upon dyn1 KD, with resultant elevation of α–secretase cleavage products sAPPα and αCTF. But no changes are seen in the amount of nicastrin (NCT) or PS1 N-terminal fragment (NTF) at cell surface with dyn1 KD. Furthermore, treatment with a selective dynamin inhibitor Dynasore leads to similar reduction in βCTF and Aβ levels, comparable to changes with BACE inhibitor treatment. But combined inhibition of BACE-1 and dyn1 does not lead to further reduction in Aβ, suggesting that the Aβ-lowering effects of dynamin inhibition are mainly mediated through regulation of BACE-1 internalization. Aβ levels in dyn1^−/− primary neurons, as well as in 3-month old dyn1 haploinsufficient animals with AD transgenic background are consistently reduced when compared to their wildtype counterparts.

Conclusions

In summary, these data suggest a previously unknown mechanism by which dyn1 affects amyloid generation through regulation of BACE-1 subcellular localization and therefore its enzymatic activities.