Retinoic Acid-Elicited RARα/RXRα Signaling Attenuates Aβ Production by Directly Inhibiting γ-Secretase-Mediated Cleavage of Amyloid Precursor Protein

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Residues at P2-P1 positions of - and ζ-cleavage sites are important in formation of β-amyloid peptide

Most of the Alzheimer's disease (AD)-linked mutations in amyloid precursor protein (APP), which cause abnormal production of β-amyloid (Aβ), are localized at the major β-secretase-and γ-secretase cleavage sites. In this study, using an APP-knockout mouse neuronal cell line, our data demonstrated that at the P2-P1 positions of the -cleavage site at Aβ49 and the ζ-cleavage site at Aβ46, aromatic amino acids caused a strong reduction in total Aβ. On the other hand, residues with a long side chain caused a decrease in Aβ₄₀ and a concomitant increase in Aβ₄₂ and Aβ₃₈. These findings indicate that the structures of the substituting residues at these key positions strongly determine the efficiency and preference of γ-secretase-mediated APP processing, which determines the ratio of different secreted Aβ species, a crucial factor in the disease development. Our findings provide new insight into the mechanisms of γ-secretase-mediated APP processing and, specifically, into why most AD-linked APP mutations are localized at major γ-secretase cleavage sites. This information may contribute to the development of methods of prevention and treatment of Alzheimer's disease aimed at modulating γ-secretase activity.     

Quantitative Analysis of Axonal Transport by Using Compartmentalized and Surface Micropatterned Culture of Neurons

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New Mouse Model of Alzheimer’s

Amyloid β-peptide (Aβ) accumulation is a key characteristic of Alzheimer’s disease (AD); therefore, mouse models of AD exhibiting Aβ pathology are valuable tools for unraveling disease mechanisms. However, the overexpression of Aβ precursor protein (APP) used in previous mouse models may cause Aβ-independent artifacts that influence data interpretation. To circumvent these problems, we used an APP knock-in (KI) strategy to introduce mutations to the mouse APP gene to develop a new generation of AD mouse models. These new models, termed APP^NL-F and APP^NL-G-F, have endogenous APP levels and develop robust Aβ amyloidosis, which induce synaptic degeneration and memory impairments. Thus, we suggest that these novel APP KI mice will serve as important tools to elucidate molecular mechanisms of AD.     

Olive-Oil-Derived Oleocanthal Enhances β-Amyloid Clearance as a Potential Neuroprotective Mechanism against Alzheimer’s Disease: In Vitro and in Vivo Studies

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Regional Tau Effects on Prospective Cognitive Change in Cognitively Normal Older Adults

Studies suggest that tau deposition starts in the anterolateral entorhinal cortex (EC) with normal aging, and that the presence of β-amyloid (Aβ) facilitates its spread to neocortex, which may reflect the beginning of Alzheimer''s disease (AD). Functional connectivity between the anterolateral EC and the anterior-temporal (AT) memory network appears to drive higher tau deposition in AT than in the posterior-medial (PM) memory network. Here, we investigated whether this differential vulnerability to tau deposition may predict different cognitive consequences of EC, AT, and PM tau. Using ¹⁸F-flortaucipir (FTP) and ¹¹C-Pittsburgh compound-B (PiB) positron emission tomography (PET) imaging, we measured tau and Aβ in 124 cognitively normal human older adults (74 females, 50 males) followed for an average of 2.8 years for prospective cognition. We found that higher FTP in all three regions was individually related to faster memory decline, and that the effects of AT and PM FTP, but not EC, were driven by Aβ+ individuals. Moreover, when we included all three FTP measures competitively in the same model, only AT FTP significantly predicted memory decline. Our data support a model whereby tau, facilitated by Aβ, transits from EC to cortical regions that are most closely associated with the anterolateral EC, which specifically affects memory in the initial stage of AD. Memory also appears to be affected by EC tau in the absence of Aβ, which may be less clinically consequential. These findings may provide clarification of differences between normal aging and AD, and elucidate the transition between the two stages.SIGNIFICANCE STATEMENT Tau and β-amyloid (Aβ) are hallmarks of Alzheimer''s disease (AD) but are also found in cognitively normal people. It is unclear whether, and how, this early deposition of tau and Aβ may affect cognition in normal aging and the asymptomatic stage of AD. We show that tau deposition in the entorhinal cortex (EC), which is common in advanced age, predicts memory decline in older adults independent of Aβ, likely reflecting normal, age-related memory loss. In contrast, tau in anterior-temporal (AT) regions is most predictive of memory decline in Aβ+ individuals. These data support the idea that tau preferentially spreads to specific cortical regions, likely through functional connections, which plays a primary role in memory decline in the early stage of AD.     

Vascular endothelial growth factor (VEGF) affects processing of amyloid precursor protein and β-amyloidogenesis in brain slice cultures derived from transgenic Tg2576 mouse brain

The up-regulation of the angiogenic vascular endothelial growth factor (VEGF) in brains of Alzheimer patients in close relationship to β-amyloid (Aβ) plaques, suggests a link of VEGF action and processing of the amyloid precursor protein (APP). To reveal whether VEGF may affect APP processing, brain slices derived from 17-month-old transgenic Tg2576 mice were exposed with 1 ng/ml VEGF for 6, 24, and 72 h, followed by assessing cytosolic and membrane-bound APP expression, level of both soluble and fibrillar Aβ-peptides, as well as activities of α- and β-secretases in brain slice tissue preparations.Treatment of brain slices with VEGF did not significantly affect the expression level of APP, regardless of the exposure time studied. In contrast, VEGF exposure of brain slices for 6 h reduced the formation of soluble, SDS extractable Aβ(1–40) and Aβ(1–42) as compared to brain slice cultures incubated in the absence of any drug, while the fibrillar Aβ peptides did not change significantly. This effect was less pronounced 24 h after VEGF exposure, but was no longer detectable when brain slices were exposed by VEGF for 72 h, which indicates an adaptive response to chronic VEGF exposure. The VEGF-mediated reduction in Aβ formation was accompanied by a transient decrease in β-secretase activity peaking 6 h after VEGF exposure. To reveal whether the VEGF-induced changes in soluble Aβ-level may be due to actions of VEGF on Aβ fibrillogenesis, the fibrillar status of Aβ was examined using the thioflavin-T binding assay. Incubation of Aβ preparations obtained from Tg2576 mouse brain cortex, in the presence of VEGF slightly decreased the fibrillar content with increasing incubation time up to 72 h. The data demonstrate that VEGF may affect APP processing, at least in vitro, suggesting a role of VEGF in the pathogenesis of Alzheimer's disease.     

Application of quantitative LC–MS surrogate peptide methodology in the analysis of the amyloid beta peptide (Aβ) biosynthetic intermediate protein APP–βCTF

An area of current research in Alzheimer's disease (AD) is the biosynthetic pathway of amyloid beta peptides (Aβ) via consecutive proteolytic cleavages of the amyloid beta precursor protein (APP) by BACE and γ-secretase enzymes. APP is first cleaved by BACE to form a C-terminal fragment APP–βCTF, or also called C99, which then undergoes further cleavage by γ-secretase to form Aβ. Inhibitors of γ-secretase have been observed to yield a so-called ‘Aβ rise’ phenomenon whereby low inhibitor concentrations result in an increase in Aβ levels while high inhibitor concentrations result in lower Aβ levels. A previous report from our labs indicated that this phenomenon was related to ratios of APP–βCTF substrate relative to γ-secretase enzyme. A quantitative Western blot analysis was used with a recombinant C100 protein as calibration standards to assess the relationship of APP–βCTF, γ-secretase enzyme and various inhibitors resulting in the ‘Aβ rise’. An on-line liquid chromatography mass spectrometry (LC–MS) method employing the ‘surrogate peptide’ methodology was developed to accurately quantify the recombinant C100 used in the Western blot analyses. The surrogate peptide approach utilizes tryptic digestion of the protein to stoichiometrically yield a unique peptide fragment, in this case ^C100Aβ17–28 (LVFFAEDVGSNK) that can be readily detected by LC–MS. The absolute quantitative assessment of C100 was accomplished using synthetic Aβ17–28 to generate calibration curves over a 0.001–1 μM range and 15N isotopically labeled Aβ1–40 as the internal standard for enzymatic digestion and its proteolytic peptide [15N]-Aβ17–28 for the analysis.     

Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase

The Alzheimer's disease β-amyloid peptide (Aβ) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of β- and then γ-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of β-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal β-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the β-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Aβ. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.     

Hypertension enhances Aβ-induced neurovascular dysfunction,promotes β-secretase activity,and leads to amyloidogenic processing of APP

Hypertension (HTN) doubles the risk of Alzheimer’s disease (AD), but the mechanisms remain unclear. Amyloid-β (Aβ), a key pathogenic factor in AD, induces cerebrovascular dysfunction. We hypothesized that HTN acts in concert with Aβ to amplify its deleterious cerebrovascular effects and to increase Aβ production. Infusion of angiotensin II (ANGII; intravenously) elevated blood pressure and attenuated the cerebral blood flow (CBF) response to whisker stimulation or the endothelium-dependent vasodilator acetylcholine (ACh) (P < 0.05). Neocortical application of Aβ in mice receiving ANGII worsened the responses to ACh (P < 0.05). The cerebrovascular dysfunction observed in Tg2576 mice, in which Aβ is elevated both in blood and in brain due to expression of mutated amyloid precursor protein (APP), was not aggravated by neocortical application of ANGII or by a 2-week administration of ‘slow pressor’ of ANGII (600 ng/kg per minute; subcutaneously). In contrast, ANGII aggravated the dysfunction in TgSwDI mice, in which Aβ is increased only in brain. Slow-pressor ANGII induced microvascular amyloid deposition in Tg2576 mice and enhanced β-secretase APP cleavage. In Chinese hamster ovary (CHO) cells producing Aβ, ANGII increased β-secretase activity, Aβ1–42, and the Aβ42/40 ratio. We conclude that HTN enhances amyloidogenic APP processing, effects that may contribute to the pathogenic interaction between HTN and AD.     

HIF-1alpha inhibition ameliorates neonatal brain injury in a rat pup hypoxic-ischemic model

In this study, we tested if caspase-3 inhibition decreased ischemia-induced Aβ elevation by reducing β-secretase (BACE1) activity. Changes in caspase-3, Aβ and BACE1 levels were detected in rat striatum on different days after middle cerebral artery occlusion using immunostaining. We found that the positive labeled cells of activated caspase-3, Aβ, and BACE1 were significantly and time-dependently increased in the ipsilateral striatum. The results of Western blotting and RT-PCR showed that caspase-3 inhibitor Z-DEVD-FMK reduced BACE1 mRNA and protein levels, and inhibited its protease activity, thereby decreasing the amount of APP C99 and Aβ in ischemic brains. Moreover, Z-DEVD-FMK reduced BACE1 and GFAP double-labeled cells, but not GFAP protein levels or GFAP-labeled cells, in the ipsilateral striatum. Thus, we demonstrated that caspase-3 inhibition attenuated ischemia-induced Aβ formation by reducing BACE1 production and activity. This finding provides a therapeutic strategy for preventing Aβ accumulation and reducing the risk of neurodegeneration after stroke.     

Unbalanced Regulation of α7 nAChRs by Ly6h and NACHO Contributes to Neurotoxicity in Alzheimer's Disease

α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain where they promote fast cholinergic synaptic transmission and serve important neuromodulatory functions. However, their high permeability to Ca²⁺ also predisposes them to contribute to disease states. Here, using transfected HEK-tsa cells and primary cultured hippocampal neurons from male and female rats, we demonstrate that two proteins called Ly6h and NACHO compete for access to α7 subunits, operating together but in opposition to maintain α7 assembly and activity within a narrow range that is optimal for neuronal function and viability. Using mixed gender human temporal cortex and cultured hippocampal neurons from rats we further show that this balance is perturbed during Alzheimer''s disease (AD) because of amyloid β (Aβ)-driven reduction in Ly6h, with severe reduction leading to increased phosphorylated tau and α7-mediated neurotoxicity. Ly6h release into human CSF is also correlated with AD severity. Thus, Ly6h links cholinergic signaling, Aβ and phosphorylated tau and may serve as a novel marker for AD progression.SIGNIFICANCE STATEMENT One of the earliest and most persistent hypotheses regarding Alzheimer''s disease (AD) attributes cognitive impairment to loss of cholinergic signaling. More recently, interest has focused on crucial roles for amyloid β (Aβ) and phosphorylated tau in Alzheimer''s pathogenesis. Here, we demonstrate that these elements are linked by Ly6h and its counterpart, NACHO, functioning in opposition to maintain assembly of nicotinic acetylcholine receptors (nAChRs) within the physiological range. Our data suggests that Aβ shifts the balance away from Ly6h and toward NACHO, resulting in increased assembly of Ca²⁺-permeable nAChRs and thus a conversion of basal cholinergic to neurotoxic signaling.     

Parkinson's Disease with Fatigue: Clinical Characteristics and Potential Mechanisms Relevant to α-Synuclein Oligomer

MethodsPD patients (n=102) were evaluated using a fatigue severity scale and scales for motor and nonmotor symptoms. The levels of three pathological proteins—α-synuclein oligomer, β-amyloid (Aβ)_1-42, and tau—were measured in 102 cerebrospinal fluid (CSF) samples from these PD patients. Linear regression analyses were performed between fatigue score and the CSF levels of the above-listed pathological proteins in PD patients.ResultsThe frequency of fatigue in the PD patients was 62.75%. The fatigue group had worse motor symptoms and anxiety, depression, and autonomic dysfunction. The CSF level of α-synuclein oligomer was higher and that of Aβ_1-42 was lower in the fatigue group than in the non-fatigue group. In multiple linear regression analyses, fatigue severity was significantly and positively correlated with the α-synuclein oligomer level in the CSF of PD patients, after adjusting for confounders.ConclusionsPD patients experience a high frequency of fatigue. PD patients with fatigue have worse motor and part nonmotor symptoms. Fatigue in PD patients is associated with an increased α-synuclein oligomer level in the CSF.     

Distribution of β-amyloid and amyloid precursor protein in the brain of spawning (senescent) salmon: a natural, brain-aging model

Brain amyloid precursor protein (APP), a normal constituent of neurons, glial cells and cerebrospinal fluid, has several proposed functions (e.g., in neuronal growth and survival). It appears, however, that altered processing of APP is an initial or downstream step in the neuropathology of brain aging, Alzheimer's disease (AD), and Down's syndrome (DS). Some studies suggest that proteolytic cleavage of APP, producing β-amyloid (Aβ_1–42), could have neurotoxic or neuroprotective effects. In this study, we utilized antibodies to human APP₆₉₅ and Aβ_1–42, and Congo red staining, to search for amyloid deposition in the brain of semelparous spawning kokanee salmon (Oncorhynchus nerka kennerlyi). Intracellular APP₆₉₅ immunoreactivity (APP-ir) was observed in brain regions involved in gustation (glomerulosus complex), olfaction (putative hippocampus, olfactory bulb), vision (optic tectum), the stress response (nucleus preopticus and nucleus lateralis tuberis), reproductive behavior (nucleus preopticus magnocellularis, nucleus preopticus periventricularis, ventral telencephalon), and coordination (cerebellum). Intra- and extra-neuronal Aβ_1–42 immunoreactivity (Aβ-ir) were present in all APP-ir regions except the nucleus lateralis tuberis and Purkinje cells of the cerebellum (coordination). Thus, the relationship between APP and Aβ deposition during brain aging could shed light on the processing of APP into Aβ, neurodegeneration, and possible protection of neurons that are functioning in spawning but senescent salmon. Pacific salmon, with their predictable and synchronized life history, could provide research options not available with the existing models for studies of brain aging and amyloidosis.     

Mechanisms that lessen benefits of β-secretase reduction in a mouse model of Alzheimer's disease

The β-secretase enzyme BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), which initiates amyloid-β (Aβ) production, is an excellent therapeutic target for Alzheimer''s disease (AD). However, recent evidence raises concern that BACE1-inhibiting approaches may encounter dramatic declines in their abilities to ameliorate AD-like pathology and memory deficits during disease progression. Here, we used BACE1 haploinsufficiency as a therapeutic relevant model to evaluate the efficacy of partial inhibition of this enzyme. Specifically, we crossed BACE1^+/− mice with 5XFAD transgenic mice and investigated the mechanisms by which Aβ accumulation and related memory impairments become less sensitive to rescue by BACE1^+/− reduction. Haploinsufficiency lowered BACE1 expression by ∼50% in 5XFAD mice regardless of age in concordance with reduction in gene copy number. However, profound Aβ plaque pathology and memory deficits concomitant with BACE1 equivalent to wild-type control levels remained in BACE1^+/−·5XFAD mice with advanced age (15–18 months old). Therefore, BACE1 haploinsufficiency is not sufficient to block the elevation of BACE1 expression (approximately twofold), which is also reported to occur during human AD progression, in 5XFAD mice. Our investigation revealed that PERK (PKR-endoplasmic reticulum-related kinase)-dependent activation of eIF2α (eukaryotic translation initiation factor-2α) accounts for the persistent BACE1 upregulation in BACE1^+/−·5XFAD mouse brains at 15–18 months of age. Moreover, BACE1 haploinsufficiency was also no longer able to prevent reduction in the expression of neprilysin, a crucial Aβ-degrading enzyme, in 5XFAD mice with advanced age. These findings demonstrate that partial BACE1 suppression cannot attenuate deleterious BACE1-elevating or neprilysin-reducing mechanisms, limiting its capabilities to reduce cerebral Aβ accumulation and rescue memory defects during the course of AD development.     

Reduction of β-amyloid deposits by γ-secretase inhibitor is associated with the attenuation of secondary damage in the ipsilateral thalamus and sensory functional improvement after focal cortical infarction in hypertensive rats

Abnormal β-amyloid (Aβ) deposits in the thalamus have been reported after cerebral cortical infarction. In this study, we investigated the association of Aβ deposits, with the secondary thalamic damage after focal cortical infarction in rats. Thirty-six stroke-prone renovascular hypertensive rats were subjected to distal middle cerebral artery occlusion (MCAO) and then randomly divided into MCAO, vehicle, and N-[N-(3,5-difluorophenacetyl)--alanyl]-S-phenylglycine t-butyl ester (DAPT) groups and 12 sham-operated rats as control. The DAPT was administered orally at 72 hours after MCAO. Seven days after MCAO, sensory function, neuron loss, and glial activation and proliferation were evaluated using adhesive removal test, Nissl staining, and immunostaining, respectively. Thalamic Aβ accumulation was evaluated using immunostaining and enzyme-linked immunosorbent assay (ELISA). Compared with vehicle group, the ipsilateral thalamic Aβ, neuronal loss, glial activation and proliferation, and the mean time to remove the stimulus from right forepaw significantly decreased in DAPT group. The mean time to remove the stimulus from the right forepaw and thalamic Aβ burden were both negatively correlated with the number of thalamic neurons. These findings suggest that Aβ deposits are associated with the secondary thalamic damage. Reduction of thalamic Aβ by γ-secretase inhibitor may attenuate the secondary damage and improve sensory function after cerebral cortical infarction.     

Amyloid β-Peptide 25–35 Self-Assembly and Its Inhibition: A Model Undecapeptide System to Gain Atomistic and Secondary Structure Details of the Alzheimer’s Disease Process and Treatment

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