Processing Of Precursor Protein Research Articles

The complex link between amyloid and neuronal dysfunction in Alzheimer's disease.

This scientific commentary refers to ‘Regional brain hypometabolism is unrelated to regional amyloid plaque burden’ by Altmann et al. (doi:10.1093/awv278). The amyloid cascade hypothesis of Alzheimer’s disease states that aberrant amyloid-β precursor protein (APP) processing is the starting point of a series of pathogenic events that ends in neuronal dysfunction and neurodegeneration, and results in cognitive decline (Hardy and Allsop, 1991) (Fig. 1A). While the general link between amyloid pathology and Alzheimer’s disease is indisputable, the detailed link between amyloid and neurodegenerative dysfunction is less clear. Early neuropathological studies suggested both spatially and temporally dissociating trajectories for patterns of regional amyloid plaque deposition and neurodegenerative changes such as neurofibrillary tangle (tau) pathology or cell loss (Braak and Braak, 1991). In particular, the spatial dissociation between amyloid deposition and neurodegenerative changes challenged the idea of a direct, i.e. regional, link between amyloid pathology and neurodegeneration. The use of PET-based imaging techniques for measuring amyloid pathology and neurodegenerative dysfunction allows the associations between these factors to be examined in living human subjects. However, attempts to establish a quantitative link between regional amyloid pathology and neuronal hypometabolism have yielded inconsistent results so far. In this issue of Brain , Altmann et al. (2015) report that global cerebral amyloid load is robustly associated with regionally-specific reductions in cortical glucose metabolism; however, this does not translate into a consistent and generalizable association between amyloid load and neuronal dysfunction at the regional level. Figure 1 Models of amyloid and tau-pathology interactions. ( A ) Canonical amyloid cascade hypothesis (Hardy and Allsop, 1991). ( B ) Amyloid facilitates MTL tau pathology and its cortical spread (Sperling et al. , 2014). MTL tau pathology is age-related (Crary et al. , 2014). MTL = medial temporal lobe. Previous in vivo imaging studies using amyloid-sensitive PET have revealed a highly reproducible neocortical-predominant deposition …

Genetic Variation in δ-Opioid Receptor Associates with Increased β- and γ-Secretase Activity in the Late Stages of Alzheimer’s Disease

The agonist-induced activation of human δ-opioid receptor (δOR) has been shown to increase β- (BACE1) and γ-secretase activities leading to increased production of amyloid-β (Aβ) peptide. We have recently shown that phenylalanine to cysteine substitution at amino acid 27 in δOR (δOR-Phe27Cys) increases amyloid-β protein precursor processing through altered endocytic trafficking. Also, a genetic meta-analysis of the δOR-Phe27Cys variation (rs1042114) in two independent Alzheimer's disease (AD) patient cohorts indicated that the heterozygosity of δOR-Phe27Cys increases the risk of AD. Here, we investigated α-, β-, and γ-secretase activities in human brain with respect to δOR-Phe27Cys variation in the temporal cortex of 71 subjects with varying degree of AD-related neurofibrillary pathology (Braak stages I-VI). As a result, a significant increase in β- (p = 0.03) and γ- (p = 0.01), but not α-secretase, activities was observed in late stage AD samples (Braak stages V-VI), which were heterozygous for δOR-Phe27Cys as compared to the δOR-Phe27 and δOR-Cys27 homozygotes. The augmented β-secretase activity was not associated with increased mRNA expression or protein levels of BACE1 in the late stage AD patients, who were heterozygous for the δOR-Phe27Cys variation. These findings suggest that δOR-Phe27Cys variation modulates β- and γ-secretase activity in the late stages of AD likely via post-translational mechanisms other than alterations in the mRNA or protein levels of BACE1, or, in the expression of γ-secretase complex components.

Acute Effects of Muscarinic M1 Receptor Modulation on AβPP Metabolism and Amyloid-β Levels in vivo: A Microdialysis Study.

Indirect modulation of cholinergic activity by cholinesterase inhibition is currently a widely established symptomatic treatment for Alzheimer's disease (AD). Selective activation of certain muscarinic receptor subtypes has emerged as an alternative cholinergic-based amyloid-lowering strategy for AD, as selective muscarinic M1 receptor agonists can reduce amyloid-β (Aβ) production by shifting endoproteolytic amyloid-β protein precursor (AβPP) processing toward non-amyloidogenic pathways. In this study, we addressed the hypothesis that acute stimulation of muscarinic M1 receptors can inhibit Aβ production in awake and freely moving AβPP transgenic mice. By combining intracerebral microdialysis with retrodialysis, we determined hippocampal Aβ concentrations during simultaneous pharmacological modulation of brain M1 receptor function. Infusion with a M1 receptor agonist AF102B resulted in a rapid reduction of interstitial fluid (ISF) Aβ levels while treatment with the M1 antagonist dicyclomine increased ISF Aβ levels reaching significance within 120 minutes of treatment. The reduction in Aβ levels was associated with PKCα and ERK activation resulting in increased levels of the α-secretase ADAM17 and a shift in AβPP processing toward the non-amyloidogenic processing pathway. In contrast, treatment with the M1 receptor antagonist dicyclomine caused a decrease in levels of phosphorylated ERK that was independent of PKCα, and led to an elevation of β-secretase levels associated with increased amyloidogenic AβPP processing. The results of this study demonstrate rapid effects of in vivo M1 receptor modulation on the ISF pool of Aβ and suggest that intracerebral microdialysis with retrodialysis is a useful technical approach for monitoring acute treatment effects of muscarinic receptor modulators on AβPP/Aβ metabolism.

Abstract 71: Endothelium-specific Deficiency of PPARδ Selectively Decreases Level of sAPPα in Hippocampus

We tested the hypothesis that endothelial PPARδ plays an important role in the protection of neurovascular unit. Cerebral microvessels were isolated from mice with endothelium-specific deficiency of PPARδ (ePPARδ-/-) and littermate wild type (WT) mice. After ex vivo incubation of cerebral microvessels for 24 h, supernatant was collected and levels of soluble amyloid precursor protein α [sAPPα, a product of non-amyloidogenic processing of amyloid precursor protein (APP)] were measured using an ELISA kit. The production of sAPPα in cerebral microvessels of ePPARδ-/- mice was reduced by 54%, as compared to WT mice (n=6-7, P<0.05). Western blot analysis demonstrated that microvascular expression of ADAM10 was reduced in ePPARδ-/- mice (80%) (n=18, P<0.05), while ADAM17 protein level was increased in by 132% (n=4, P<0.05), as compared to WT mice. Protein expressions of APP, ADAM9, and BACE1 were not significantly different between cerebral microvessels derived from ePPARδ-/- mice and WT mice. Real-time RT-PCR demonstrated that in brain microvessels of ePPARδ-/- mice mRNA levels of ADAM10 were also decreased by 56% (n=5, P<0.05). Furthermore, Western blot analysis of the brain regions showed that sAPPα content was selectively reduced in hippocampus of ePPARδ-/- mice by 80% (n=11, P<0.05). This was associated with significantly reduced expression of ADAM10 in the hippocampus (n=11, P<0.05). In contrast, protein levels of ADAM10, and sAPPα were not altered in cortex and cerebellum of ePPARδ-/- mice. In addition, we did not detect any changes in Aβ40 and Aβ42 levels (measured using ELISA assay) in all three examined brain regions. Since sAPPα is a potent neurotrophic and neuro-protective molecule, our results suggest that loss of endothelial PPARδ may selectively increase vulnerability of hippocampus. This mechanism may contribute to susceptibility of hippocampus to injury during initiation and progression of neuro-degenerative diseases.

The Expression of Transthyretin and Amyloid-β Protein Precursor is Altered in the Brain of Idiopathic Normal Pressure Hydrocephalus Patients.

Idiopathic normal pressure hydrocephalus (iNPH) is a dementing condition in which Alzheimer's disease (AD)-related amyloid-β (Aβ) plaques are frequently observed in the neocortex. iNPH patients with prominent Aβ pathology show AD-related alterations in amyloid-β protein precursor (AβPP) processing resulting from increased γ-secretase activity. Our goal was to assess potential alterations in the global gene expression profile in the brain of iNPH patients as compared to non-demented controls and to evaluate the levels of the identified targets in the cerebrospinal fluid (CSF) of iNPH patients. The genome-wide expression profile of ~35,000 probes was assessed in the RNA samples obtained from 22 iNPH patients and eight non-demented control subjects using a microarray chip. The soluble levels of sAβPPα, sAβPPβ, and transthyretin (TTR) were measured from the CSF of 102 iNPH patients using ELISA. After correcting the results for multiple testing, significant differences in the expression of TTR and A βPP were observed between iNPH and control subjects. The mRNA levels of TTR were on average 17-fold lower in iNPH samples compared to control samples. Conversely, the expression level of A βPP was on average three times higher in iNPH samples as compared to control samples. Interestingly, the expression of α-secretase (ADAM10) was also increased in iNPH patients. In the lumbar CSF samples, soluble TTR levels showed a significant positive correlation with sAβPPα and sAβPPβ, but TTR levels did not predict the brain pathology or the shunt response. These findings suggest differences in the expression profile of key factors involved in AD-related cellular events in the brain of iNPH patients.

ABCA5 regulates amyloid-β peptide production and is associated with Alzheimer's disease neuropathology.

Brain cholesterol homeostasis is regulated by a group of proteins called ATP-binding cassette subfamily A (ABCA) transporters. Certain ABCA transporters regulate amyloid-β protein precursor (AβPP) processing to generate amyloid-β peptides (Aβ) and are associated with an increased risk for late-onset Alzheimer's disease (AD). ABCA5 is a little-known member of the ABCA subfamily with no known function. In this study we undertook a comprehensive analysis of ABCA5 expression in the human and mouse brains. We explored the potential role of ABCA5 in AβPP processing associated with AD pathology. ABCA5 was differentially expressed in multiple regions of both human and mouse brains. It was strongly expressed in neurons with only weak expression in microglia, astrocytes, and oligodendrocytes. ABCA5 was able to stimulate cholesterol efflux in neurons. ABCA5 expression was specifically elevated in the hippocampus of AD brains. Using two in vitro cell systems we demonstrated that ABCA5 reduces Aβ production, both Aβ40 and Aβ42, without altering AβPP mRNA and protein levels, indicating that the decrease in the Aβ levels was due to changes in AβPP processing and not AβPP expression. This report represents the first extensive expression and functional study of ABCA5 in the human brain and our data suggest a plausible function of ABCA5 in the brain as a cholesterol transporter associated with Aβ generation, information that may offer a potential new target for controlling Aβ levels in the brain.

Self-assembling nanofibers alter the processing of amyloid precursor protein in a transgenic mouse model of Alzheimer’s disease

Background:Alzheimer’s disease (AD) is one of the most common dementia, which is not effectively cured to date. Amyloid-beta (Abeta) deposition cascade and disintegrity of brain extracellular matrix (ECM) scaffold attribute to the progress of AD. Thus, it maybe an effective way to treat AD by altering the processing of amyloid precursor protein (APP) and regaining the integrity of ECM. The peptide amphiphile (PA) with a laminin epitope isoleucine–lysine–valine–alanine–valine (IKVAV) (IKVAV-PA) can be trigged into ECM in vivo. In addition, IKVAV-PA could significantly improve cognitive impairment with remarkable increase of endoneurogensis in the hippocampus, as well as reduction of burden of amyloid plaque in the brain.Methods:We used heterozygous AbetaPPswe/PS1dE9 double transgenic mice as the animal model of AD. After 1 week of initial stereotaxic administration into bilateral hippocampus, the mice were subjected to the Morris Water Maze (MWM) test. At the end of MWM test, immunohistochemical staining, Western blot and real-time polymerase chain reaction (PCR) were performed in mice.Results:Here we showed that IKVAV-PA significantly improved cognitive impairment accompanying with reducing the burden of Abeta plaques, as well as the levels of soluble Abeta1-40 and Abeta1-42 in the cortex and hippocampus after 2 weeks of initial administration into bilateral hippocampus. Further examination demonstrated that IKVAV-PA also altered the processing of APP via inhibiting the gene expression of beta-secretase (BACE1), as well as improving the gene expression of insulin-degrading enzyme (IDE) and neprilysin (NEP).Conclusion:Our data suggest that IKVAV-PA may serve as an alternative therapeutic intervention for treating the learning and memory losses in AD.

ADAM10: α-secretase in Alzheimer’s disease and regulator of neurobiology

Proteolytic and amyloidogenic processing of amyloid precursor protein (APP) by β- and γ-secretases are pathological hallmarks of Alzheimer’s disease (AD). These proteolytic activities lead to release of the amyloid-β peptides believed to cause neurological pathology and be linked to pathological progression in AD. Due to its capability to cleave APP within the toxic peptide sequence, the metalloproteinase ADAM10 (“a disintegrin and metalloprotease”) is a known antagonist of the disease-causing pathway. ADAM10 also plays a major role in the ectodomain shedding of a number of important cell surface proteins. In addition, ADAM10 is involved in the proteolytic activation cascade of the Notch receptor, which is of crucial function in developmental processes. The study of ADAM10-deficient mice also revealed that ADAM10 regulates synaptic function and synaptogenesis. Pharmacological activation of ADAM10 is postulated to represent a valuable strategy for prevention of AD. However, due to the multiple roles of ADAM10 in the brain, it will be challenging to find a suitable therapeutic window.

Increased Levels of Plasma p3-Alcα35, a Major Fragment of Alcadeinα by γ-Secretase Cleavage, in Alzheimer's Disease

p3-Alcα is a metabolic fragment of Alcadeinα (Alcα). Similar to the generation of the p3 fragment from amyloid-β protein precursor (AβPP) processing, Alcα is cleaved by α- and γ-secretases, leading to the secretion of p3-Alcα peptides into cerebrospinal fluid (CSF). p3-Alcα is also detected in the plasma, similar to amyloid-β (Aβ), which is a metabolic fragment of AβPP cleaved by amyloidogenic β- and γ-secretases. Because p3-Alcα is a non-aggregatable and stable peptide, unlike aggregatable Aβ and metabolically labile p3 of AβPP, the changes of p3-Alcα in quality and/or quantity in CSF and plasma are expected to be a marker for assessing alteration of substrate cleavage by γ-secretase, such as Aβ generation from AβPP. The present study describes a sandwich enzyme-linked immunosorbent assay for quantifying levels of p3-Alcα35, the major form of the p3-Alcα species, and examines levels of p3-Alcα35 in the plasma of three independent Japanese cohorts. In two of the three cohorts, the p3-Alcα35 levels were significantly increased with a concomitant decrease in the Mini-Mental State Examination score, or in clinically diagnosed Alzheimer's disease (AD) patients, when compared with age-matched non-demented subjects. The values were significantly lower in AD subjects who were administered donepezil, when compared to AD subjects without donepezil treatment. The increase in plasma p3-Alcα35 levels may indicate an endophenotype in subjects in whom AD is due to a progressing cognitive impairment in subjects with a γ-secretase malfunction, or a disorder of the clearance of peptides.

An intronic rare mutation in Presenilin-1 (PSEN-1) gene may be involved in the developement of Alzheimer’s disease

Background The Presenilin-1 gene (PSEN-1) encodes a protein component of gamma-secretase complex which is involved in processing of amyloid precursor protein (APP). The PSEN-1 is involved in many cardinal mechanisms in several molecular pathway which when impaired leads to the manifestation of Alzheimer’s disease (AD). The aim of the study was to investigate the role of PSEN-1 gene in the developement of AD in Indian Bengali population.

Homage to Rita Levi-Montalcini. Molecular mechanisms of Alzheimer's disease: NGF modulation of APP processing

The therapeutic focus of the scientific community in Alzheimer's disease (AD) has been moving in the last years from attempting late rescue against cholinergic degeneration and amyloid beta plaque clearance to (i) the discovery of blood or cerebrospinal fluid markers for early diagnosis, and (ii) early therapeutic intervention with modulators of amyloid precursor protein (APP) processing. It is currently accepted idea that subtle synaptic alterations determine the first neuronal dysfunctions and cognitive deterioration, progressing overtime into neuronal degeneration in the sporadic and late onset form AD (LOAD), the most diffuse one (90% of AD cases). Synaptic loss occurs long before the appearance of a frank neuronal degeneration in LOAD. The perturbation of the nerve growth factor (NGF) signaling system in brain neurodegenerative disease like AD and Down's syndrome was for long time considered to be a pathological event, subsequent to amyloid-driven disruption of NGF retrograde transport from the cortex and hippocampus to the cell bodies of basal forebrain cholinergic neurons. Nowadays, an increasing amount of data indicate that the observed dysregulation of NGF-TrkA (tyrosine kinase A) and proNGF-p75NTR signaling systems is a good candidate for being the primus movens in the neuropathology of sporadic AD. Accordingly, an amyloid-independent strong correlation of cognitive deficits with reduction of NGF and TrkA, and accumulation of proNGF has been recently observed in mild cognitive impairment and its progression to AD. This review highlights the current knowledge about NGF and early events occurring in LOAD. The involvement of muscarinic acetylcholine receptors, cholesterol, sirtuin1, insulin-like growth factor-1, and Sunday driver protein in APP processing are also discussed. The studies reported here confirm a multitasking ability of NGF in slowing down, through both distinct and overlapping mechanisms, the amyloidogenic processing of APP in neurons of the central nervous system. Adipobiology 2013; 5: 7-18.

Optimization of Furin Inhibitors To Protect against the Activation of Influenza Hemagglutinin H5 and Shiga Toxin

Proprotein convertases (PCs) are crucial in the processing and entry of viral or bacterial protein precursors and confer increased infectivity of pathogens bearing a PC activation site, which results in increased symptom severity and lethality. Previously, we developed a nanomolar peptide inhibitor of PCs to prevent PC activation of infectious agents. Herein, we describe a peptidomimetic approach that increases the stability of this inhibitor for use in vivo to prevent systemic infections and cellular damage, such as that caused by influenza H5N1 and Shiga toxin. The addition of azaβ(3)-amino acids to both termini of the peptide successfully prevented influenza hemagglutinin 5 fusogenicity and Shiga toxin Vero toxicity in cell-based assays. The results from a cell-based model using stable shRNA-induced proprotein convertase knockdown indicate that only furin is the major proprotein convertase required for HA5 cleavage.

Difluorophenylglycinols as New Modulators of Proteolytic Processing of Amyloid Precursor Proteins

Synthesis and evaluation of difluorophenylglycinols as new modulators of proteolytic processing of the amyloid-β precursor proteins for Alzheimer's therapies were described. A range of N-substituted (R)- and (S)-difluorophenylglycinols, structured on the amino alcohol framework, were explored by incorporating the arylsulfonyl moieties and various N-substituents. Evans' chiral auxiliary strategy was employed for the asymmetric synthesis of these enantiomeric difluorophenylglycinols. Compounds with effects on the γ-secretase inhibition and ERK-mediated signaling pathways were evaluated on cell-based assays. Among them, N-cyclopropylmethyl derivatives R-12c and R-13c showed modest γ-secretase inhibition as well as ERK-dependent activation.

Hypoxic stress, brain vascular system, and β-amyloid: A primary cell culture study

This study stresses the hypothesis whether hypoxic events contribute to formation and deposition of β-amyloid (Aβ) in cerebral blood vessels by affecting the processing of endothelial amyloid precursor protein (APP). Therefore, cerebral endothelial cells (ECs) derived from transgenic Tg2576 mouse brain, were subjected to short periods of hypoxic stress, followed by assessment of formation and secretion of APP cleavage products sAPPα, sAPPβ, and Aβ as well as the expression of endothelial APP. Hypoxic stress of EC leads to enhanced secretion of sAPPβ into the culture medium as compared to normoxic controls, which is accompanied by increased APP expression, induction of vascular endothelial growth factor (VEGF) synthesis, nitric oxide production, and differential changes in endothelial p42/44 (ERK1/2) expression. The hypoxia-mediated up-regulation of p42/44 at a particular time of incubation was accompanied by a corresponding down-regulation of the phosphorylated form of p42/44. To reveal any role of hypoxia-induced VEGF in endothelial APP processing, ECs were exposed by VEGF. VEGF hardly affected the amount of sAPPβ and Aβ(1–40) secreted into the culture medium, whereas the suppression of the VEGF receptor action by SU-5416 resulted in decreased release of sAPPβ and Aβ(1–40) in comparison to control incubations, suggesting a role of VEGF in controlling the activity of γ-secretase, presumably via the VEGF receptor-associated tyrosine kinase. The data suggest that hypoxic stress represents a mayor risk factor in causing Aβ deposition in the brain vascular system by favoring the amyloidogenic route of endothelial APP processing. The hypoxic-stress-induced changes in β-secretase activity are presumably mediated by altering the phosphorylation status of p42/44, whereas the stress-induced up-regulation of VEGF appears to play a counteracting role by maintaining the balance of physiological APP processing.

Regulation of dynamic BACE1 trafficking in neurons

Proteolytic processing of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase generates Aβ peptides. APP is constitutively trafficked through the secretory and endocytic pathways in cultured cells and neurons. The identities of cellular organelles and sorting pathways involved in amyloidogenic processing of APP have been extensively investigated. Although a consensus has not yet emerged, there is a general agreement from biochemical and genetic studies on the importance of endocytic APP trafficking for Aβ production. In neurons, APP is trafficked anterogradely along peripheral and central axons, and proteolytically processed during transit. Recent in vivo studies estimated that ~70% of Aβ released in the brain requires ongoing endocytosis, and synaptic activity regulates the vast majority of this endocytosis-dependent Aβ. BACE1 cleavage is thought to be the rate-limiting step in amyloidogenic processing of APP. Little, however, is known about endocytic BACE1 sorting and dynamic transport in neurons. We investigated BACE1 trafficking in cultured hippocampal neurons using live-cell imaging and selective labeling. This approach revealed dynamic neuronal transport of internalized BACE1 in dendrites and axons. BACE1 was transported in vesicles that were positive for markers of recycling endosomes. Dominant-negative expression and siRNA knock-down of proteins involved in endocytic transit revealed that BACE1 is dynamically transported in recycling endosomes and this process significantly contributes to amyloidogenic APP processing. Our results suggest that BACE1 trafficking in neuronal recycling endosomes is likely relevant for presynaptic Aβ production and contributes to Alzheimer’s disease pathogenesis.

Regulation of key proteins in Alzheimer’s disease molecular pathogenesis by ubiquilin-1

Background Ubiquilin-1 is a ubiquitin-like protein involved in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative disorders via different mechanisms. UBQLN1 UBQ-8i genetic variation associates with increased AD risk. Ubiquilin-1 protein functions as a molecular chaperone controlling amyloid precursor protein (APP) trafficking and processing and presenilin (PS) levels and subcellular targeting. Ubiquilin-1 harbors a ubiquitin-like (UBL) domain interacting with the proteasome and a ubiquitinassociated (UBA) domain binding polyubiquitinated proteins. Thus, it may target polyubiquitinated proteins to degradation. Alternative splicing of UBQLN1 generates four transcript variants (TV) with varying domain structures, suggesting that diverse variants may specifically interact with and regulate different proteins and cellular functions.

Effects of rivastigmine on secreted amyloid precursor protein and beta-amyloid secretion in neuroblastoma SK-N-SH cells

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically by progressive impairment of memory and cognition. The currently available pharmacological treatment of AD consists mainly of cholinesterase inhibitors. Rivastigmine is one of the cholinesterase inhibitors clinically used to treat this disease, and many clinical trials have indicated that it did alleviate some AD symptoms without causing apparent side effects. Since the amyloid precursor protein (APP) processing imbalance plays a crucial role in AD pathogenesis, the effects of rivastigmine on APP processing were investigated. In neuroblastoma SK-N-SH cells, rivastigmine significantly increased the secretion of sAPPα and decreased the release of Aβ40 and Aβ42 as compared with control group, but it has no effect on cellular full length APP expression. Rivastigmine significantly increased α-secretase activity and decreased β-secretase activity as compared with control group. The increased sAPPα can be partially blocked by muscarinic receptor inhibitor scopolamine but not by nicotinic receptor antagonist α-Bungarotoxin. The effect of rivastigmine on sAPPα can be partially reversed by PKC inhibitor GF109203X, ERK inhibitor PD98059 and JNK inhibitor SP600125. The data present here indicated that rivastigmine can regulate APP processing in vitro by increasing sAPPα secretion and decreasing Aβ release and this pharmacological property may underlie the clinical effect of the drug in the treatment of AD patients.

Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein.

The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1-interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.

Abstract WMP120: Long-Term Estrogen Deprivation Enhances Hippocampal Amyloidogenic Processing and Cognitive Decline after Global Cerebral Ischemia

Women who enter menopause prematurely have a doubled lifetime risk of cognitive decline and dementia. The mechanism underlying this phenomenon is unclear, but it is postulated to be associated with dramatic and prolonged deprivation of the neuroprotective ovarian hormone 17beta-estradiol (E2). To shed light on this issue, 3-month-old female Sprague Dawley rats were bilaterally ovariectomized and had subcutaneous, osmotic mini-pumps containing a low, Diestrus I dose of E2 implanted either immediately (short-term E2 deprivation - STED) or 10 weeks later (long-term E2 deprivation - LTED). One week following continuous E2 replacement, all animals were subjected to 10 minutes of global cerebral ischemia (GCI) and sacrificed for tissue harvest. Intriguingly, we observed a profound induction of Alzheimer’s disease-related proteins (beta-amyloid and hyperphosphorylated-tau) throughout the hippocampus of LTED females who experienced GCI. In the same animals, we also noted a switch to amyloidogenic processing of amyloid precursor protein (APP), such that hippocampal expression of beta-amyloid cleaving enzyme 1 (BACE1) was enhanced and hippocampal expression of A D isintegrin A nd M etalloprotease 17 (ADAM17) was diminished. Hippocampal expression of an alternate alpha secretase, ADAM10, remained unchanged. Furthermore, LTED females demonstrated enhanced post-ischemic cognitive decline, as they performed worse than STED females on the Morris Water Maze after GCI. Importantly, while immediate replacement of ovarian E2 was capable of preventing these events in STED females, delayed treatment with low-dose E2 was ineffective. Finally, c-Jun N-terminal Kinase (JNK) was identified as a critical mediator of post-ischemic amyloidogenesis, cognitive decline, and apoptosis in LTED females, as delayed treatment with a JNK inhibitor was able to prevent these events. These data could help explain why surgically menopausal women have an increased risk of cognitive decline and dementia and why postmenopausal women fare worse than age-matched men following a stroke. Furthermore, they lend support to the critical period hypothesis, suggesting that only perimenopausal replacement of ovarian E2 will provide neurological benefit for post-menopausal women.

Investigation of Nebivolol as a Novel Therapeutic Agent for the Treatment of Alzheimer's Disease

Nebivolol is a selective β1 adrenergic receptor antagonist with nitric oxide-mediated vasodilatory properties utilized in the treatment of hypertension. Previously, nebivolol was shown to modulate amyloid-β protein precursor processing in vitro. In this study, we investigated the in vivo effects of nebivolol on the modulation of amyloid neuropathology in the Tg2576 mouse model of Alzheimer's disease (AD). We found that nebivolol is brain bioavailable and can be readily detected in the brain following three weeks of treatment at a dose of 1 mg/kg/day. Moreover, this treatment regime resulted in a significant reduction of amyloid-β neuropathology in the brain, and this reduction was inversely correlated with plasma levels of amyloid-β. Chronic nebivolol treatment of Tg2576 mice with established amyloid neuropathology and cognitive impairments significantly reduced brain amyloid content but failed to improve cognitive function. Our study demonstrates that nebivolol is highly tolerable and safe and can significantly reduce amyloid neuropathology in the brain, which could be one of the most important parameters for primary prevention of AD. Our studies support the continued investigation of nebivolol for the treatment of AD at very early stages of the disease.