Targeting apolipoprotein E and N-terminal amyloid ß-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer's mice.

Apolipoprotein E (apoE) interaction with amyloid ß-protein precursor (APP) has garnered attention as the therapeutic target for Alzheimer's disease (AD). Having discovered the apoE antagonist (6KApoEp) that blocks apoE binding to N-terminal APP, we tested the therapeutic potential of 6KApoEp on AD-relevant phenotypes in amyloid ß-protein precursor/presenilin 1 (APP/PS1) mice that express each human apoE isoform of apoE2, apoE3, or apoE4 (designated APP/PS1/E2, APP/PS1/E3, or APP/PS1/E4 mice). At 12 months of age, we intraperitoneally administered 6KApoEp (250 µg/kg) or vehicle once daily for 3 months. At 15 months of age, blockage of apoE and N-terminal APP interaction by 6KApoEp treatment improved cognitive impairment in most tests of learning and memory, including novel object recognition and maze tasks in APP/PS1/E2, APP/PS1/E3, and APP/PS1/E4 mice versus each vehicle-treated mouse line and did not alter behavior in nontransgenic littermates. Moreover, 6KApoEp therapy ameliorated brain parenchymal and cerebral vascular ß-amyloid deposits and decreased abundance of amyloid ß-protein (Aß) in APP/PS1/E2, APP/PS1/E3, and APP/PS1/E4 mice versus each vehicle-treated mouse group. Notably, the highest effect in Aß-lowering by 6KApoEp treatment was observed in APP/PS1/E4 mice versus APP/PS1/E2 or APP/PS1/E3 mice. These effects occured through shifting toward lessened amyloidogenic APP processing due to decreasing APP abundance at the plasma membrane, reducing APP transcription, and inhibiting p44/42 mitogen-activated protein kinase phosphorylation. Our findings provide the preclinical evidence that 6KApoEp therapy aimed at targeting apoE and N-terminal APP interaction is a promising strategy and may be suitable for patients with AD carrying the apoE4 isoform.

Familial Alzheimer's disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid ß-peptides of ≥45 residues.

Production of amyloid ß-protein (Aß) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid ß-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aß (Aß42) to 40-residue Aß (Aß40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aß49→Aß46→Aß43→Aß40 and Aß48→Aß45→Aß42→Aß38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aß peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aß48 and Aß49 was determined by mass spectrometry. Levels of individual Aß peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aß40 and Aß42 by specific ELISAs and production of equimolar levels of Aß and AICD. Not all mutant substrates led to increased Aß42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aß ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aß peptides apparently remain bound for subsequent trimming and are not released and reassociated.

Gallic acid is a dual α/ß-secretase modulator that reverses cognitive impairment and remediates pathology in Alzheimer mice.

Several plant-derived compounds have demonstrated efficacy in pre-clinical Alzheimer's disease (AD) rodent models. Each of these compounds share a gallic acid (GA) moiety, and initial assays on this isolated molecule indicated that it might be responsible for the therapeutic benefits observed. To test this hypothesis in a more physiologically relevant setting, we investigated the effect of GA in the mutant human amyloid ß-protein precursor/presenilin 1 (APP/PS1) transgenic AD mouse model. Beginning at 12 months, we orally administered GA (20 mg/kg) or vehicle once daily for 6 months to APP/PS1 mice that have accelerated Alzheimer-like pathology. At 18 months of age, GA therapy reversed impaired learning and memory as compared with vehicle, and did not alter behavior in nontransgenic littermates. GA-treated APP/PS1 mice had mitigated cerebral amyloidosis, including brain parenchymal and cerebral vascular ß-amyloid deposits, and decreased cerebral amyloid ß-proteins. Beneficial effects co-occurred with reduced amyloidogenic and elevated nonamyloidogenic APP processing. Furthermore, brain inflammation, gliosis, and oxidative stress were alleviated. We show that GA simultaneously elevates α- and reduces ß-secretase activity, inhibits neuroinflammation, and stabilizes brain oxidative stress in a pre-clinical mouse model of AD. We further demonstrate that GA increases abundance of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10, Adam10) proprotein convertase furin and activates ADAM10, directly inhibits ß-site APP cleaving enzyme 1 (BACE1, Bace1) activity but does not alter Adam10 or Bace1 transcription. Thus, our data reveal novel post-translational mechanisms for GA. We suggest further examination of GA supplementation in humans will shed light on the exciting therapeutic potential of this molecule.

The influence of the amyloid ß-protein and its precursor in modulating cerebral hemostasis.

Ischemic and hemorrhagic strokes are a significant cause of brain injury leading to vascular cognitive impairment and dementia (VCID). These deleterious events largely result from disruption of cerebral hemostasis, a well-controlled and delicate balance between thrombotic and fibrinolytic pathways in cerebral blood vessels and surrounding brain tissue. Ischemia and hemorrhage are both commonly associated with cerebrovascular deposition of amyloid ß-protein (Aß). In this regard, Aß directly and indirectly modulates cerebral thrombosis and fibrinolysis. Further, major isoforms of the Aß precursor protein (AßPP) function as a potent inhibitor of pro-thrombotic proteinases. The purpose of this review article is to summarize recent research on how cerebral vascular Aß and AßPP influence cerebral hemostasis. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia, edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Toward the structure of presenilin/γ-secretase and presenilin homologs.

Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer's disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembrane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein-protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases.

rTg-D: A novel transgenic rat model of cerebral amyloid angiopathy Type-2.

Background: Cerebral amyloid angiopathy (CAA) is common disorder of the elderly, a prominent comorbidity of Alzheimer's disease, and causes vascular cognitive impairment and dementia. Previously, we generated a transgenic rat model of capillary CAA type-1 that develops many pathological features of human disease. However, a complementary rat model of larger vessel CAA type-2 disease has been lacking. Methods: A novel transgenic rat model (rTg-D) was generated that produces human familial CAA Dutch E22Q mutant amyloid ß-protein (Aß) in brain and develops larger vessel CAA type-2. Quantitative biochemical and pathological analyses were performed to characterize the progression of CAA and associated pathologies in aging rTg-D rats. Results: rTg-D rats begin to accumulate Aß in brain and develop varying levels of larger vessel CAA type-2, in the absence of capillary CAA type-1, starting around 18 months of age. Larger vessel CAA was mainly composed of the Aß40 peptide and most prominent in surface leptomeningeal/pial vessels and arterioles of the cortex and thalamus. Cerebral microbleeds and small vessel occlusions were present mostly in the thalamic region of affected rTg-D rats. In contrast to capillary CAA type-1 the amyloid deposited within the walls of larger vessels of rTg-D rats did not promote perivascular astrocyte and microglial responses or accumulate the Aß chaperone apolipoprotein E. Conclusion: Although variable in severity, the rTg-D rats specifically develop larger vessel CAA type-2 that reflects many of the pathological features of human disease and provide a new model to investigate the pathogenesis of this condition.

Role of γ-Secretase Inhibitors for the Treatment of Diverse Disease Conditions through Inhibition of Notch Signaling Pathway.

γ-secretase is an intramembrane protease sub-assembly that sunders transmembrane proteins. It is involved in intramembrane proteolysis and also contributes to the regeneration of transmembrane protein. The amyloid precursor proteins (APPs) are typical γ-secretase substrates. These proteins are cleaved to produce 36-43 amyloid-beta (Aß) amino acid peptides. Abnormal folding of these proteins fragments leads to amyloid plaques, which are frequently encountered in Alzheimer's disease. Some Type I class of integral membrane proteins is processed under the influence of γ-secretase, such as receptor tyrosine-protein kinase erbB4 and CD44 glycoprotein. γ-Secretase is being explored in several diseases as a clinical goal. Both γ-secretase inhibitors (GSIs) and γ-secretase modulators (GSMs) are being evaluated for this purpose. A large amount of γ-secretase inhibitors (GSIs) from peptide to non-peptide have been disclosed, offering several lead compounds for the design and optimization of γ-secretase targets, but most GSIs lack sufficient potency, exhibit low penetration in the brain, and manifest low selectiveness. γ-secretase inhibitors are obliquely a regulator of a γ-secretase substrate Notch, and valuable in the development of ß-amyloid peptide (Aß). These γ-secretase inhibitors block the Notch signaling pathway in autoimmune and lymphoproliferative disorders, like autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosus (SLE), and perhaps even in cancerous cell proliferation, angiogenesis, and cellular differentiation of human-induced pluripotent stem cells (hiPSC). The current review portrays the mechanism, regulation, and inhibition of γ-secretase in the management of a wide assortment of diseases.

Mutation of the Kunitz-type proteinase inhibitor domain in the amyloid ß-protein precursor abolishes its anti-thrombotic properties in vivo.

INTRODUCTION: Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid ß-protein precursor (AßPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AßPP are abundant in brain. Regions of AßPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AßPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo. METHODS: To determine the extent of KPI function of AßPP in regulating cerebral thrombosis we generated a recombinant reactive center KPIR13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AßPP knock out and AßPP/KPIR13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage. RESULTS: Recombinant mutant KPIR13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AßPP/KPIR13I mutant mice were similarly deficient as AßPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage. CONCLUSIONS: We demonstrate that the anti-thrombotic function of AßPP primarily resides in the KPI activity of the protein.

Down Syndrome, Partial Trisomy 21, and Absence of Alzheimer's Disease: The Role of APP.

Overexpression of the amyloid precursor protein (APP) gene on chromosome 21 in Down syndrome (DS) has been linked to increased brain amyloid levels and early-onset Alzheimer's disease (AD). An elderly man with phenotypic DS and partial trisomy of chromosome 21 (PT21) lacked triplication of APP affording an opportunity to study the role of this gene in the pathogenesis of dementia. Multidisciplinary studies between ages 66-72 years comprised neuropsychological testing, independent neurological exams, amyloid PET imaging with 11C-Pittsburgh compound-B (PiB), plasma amyloid-ß (Aß) measurements, and a brain autopsy examination. The clinical phenotype was typical for DS and his intellectual disability was mild in severity. His serial neuropsychological test scores showed less than a 3% decline as compared to high functioning individuals with DS who developed dementia wherein the scores declined 17-28% per year. No dementia was detected on neurological examinations. On PiB-PET scans, the patient with PT21 had lower PiB standard uptake values than controls with typical DS or sporadic AD. Plasma Aß42 was lower than values for demented or non-demented adults with DS. Neuropathological findings showed only a single neuritic plaque and neurofibrillary degeneration consistent with normal aging but not AD. Taken together the findings in this rare patient with PT21 confirm the obligatory role of APP in the clinical, biochemical, and neuropathological findings of AD in DS.

Stabilization of intracellular trafficking and metabolism of amyloid ß-protein precursor and Alcadein ß by apolipoprotein E.

Intracellular metabolism of amyloid ß-protein precursor (APP) is important for the pathogenesis of Alzheimer's disease (AD). Alcadeins (Alcα, Alcß, and Alcγ) are neural membrane proteins similar to APP in their localization, metabolism, and cellular function. Isoform ε4 of apolipoprotein E (ApoE) is a major risk factor for AD. We found that ApoE expression attenuated intracellular trafficking of APP and Alcß, resulting in metabolic stabilization of both proteins. By contrast, Alcα intracellular proteolysis was facilitated by ApoE expression, which was not due to an increase in the primary cleavage of Alcα. This difference may result from binding of ApoE to membrane proteins.

Arachidonic or Docosahexaenoic Acid Diet Prevents Memory Impairment in Tg2576 Mice.

It is believed that the amyloid ß-protein (Aß) plays a causative role in the development of Alzheimer's disease (AD). The amyloid-ß protein precursor (AßPP), a substrate of Aß, and ß-secretase and γ-secretase complex proteins, which process AßPP to generate Aß, are all membrane proteins. Thus, it is reasonable to assume that alterations in brain lipid metabolism modulate AßPP and/or Aß metabolism. However, the role of cellular polyunsaturated fatty acids in AßPP processing has not been completely understood yet. We report here that 4 months of treatment of Tg2576 mice with an arachidonic acid (ARA)- or a docosahexaenoic acid (DHA)-containing (ARA+ or DHA+) diet prevented memory impairment at 13 months of age. Although, AßPP processing to generate soluble AßPP and induce Aß synthesis was enhanced, Aß(1- 42)/Aß(1- 40) ratio decreased in 14-month-old Tg2576 mice fed with the ARA+ or DHA+ diet. The ARA+ or DHA+ diet did not alter the AßPP levels and the expression levels of Aß-degrading enzymes. In cortical primary neuron cultures, ARA or DHA treatment also increased soluble AßPP and Aß(1- 40) levels, and decreased Aß(1- 42)/Aß(1- 40) ratio, which are similar to what were observed in Tg2576 mice fed with ARA+ or DHA+ diet. These findings suggest that not only the DHA+ diet, but also the ARA+ diet could prevent cognitive dysfunction in Tg2576 mice through the alteration of AßPP processing.

Lessons from a Rare Familial Dementia: Amyloid and Beyond.

Here we review the similarities between a rare inherited disorder, familial British dementia (FBD), and the most common of all late-life neurological conditions, Alzheimer's diseases (AD). We describe the symptoms, pathology and genetics of FBD, the biology of the BRI2 protein and mouse models of FBD and familial Danish dementia. In particular, we focus on the evolving recognition of the importance of protein oligomers and aberrant processing of the amyloid ß-protein precursor (APP) - themes that are common to both FBD and AD. The initial discovery that FBD is phenotypically similar to AD, but associated with the deposition of an amyloid peptide (ABri) distinct from the amyloid ß-protein (Aß) led many to assume that amyloid production alone is sufficient to initiate disease and that ABri is the molecular equivalent of Aß. Parallel with work on Aß, studies of ABri producing animal models and in vitro ABri toxicity experiments caused a revision of the amyloid hypothesis and a focus on soluble oligomers of Aß and ABri. Contemporaneous other studies suggested that loss of the ABri precursor protein (BRI2) may underlie the cognitive deficits in FBD. In this regard it is important to note that BRI2 has been shown to interact with and regulate the processing of APP, and that mutant BRI2 leads to altered cleavage of APP. A synthesis of these results suggests that a "two-hit mechanism" better explains FBD than earlier toxic gain of function and toxic loss of function models. The lessons learned from the study of FBD imply that the molecular pathology of AD is also likely to involve both aberrant aggregation (in AD, Aß) and altered APP processing. With regard to FBD, we propose that the C-terminal 11 amino acid of FBD-BRI2 interfere with both the normal function of BRI2 and promotes the production of cystine cross-linked toxic ABri oligomers. In this scenario, loss of BRI2 function leads to altered APP processing in as yet underappreciated ways. Given the similarities between FBD and AD it seems likely that study of the structure of ABri oligomers and FBD-induced changes in APP metabolites will further our understanding of AD.

Effect of Anticoagulants on Amyloid ß-Protein Precursor and Amyloid Beta Levels in Plasma.

Altered levels of amyloid ß-protein precursor (AßPP) and/or amyloid beta (Aß) are characteristic of several neurological disorders including Alzheimer's disease (AD), Down syndrome (DS), Fragile X syndrome (FXS), Parkinson's disease (PD), autism and epilepsy. Thus, these proteins could serve as valuable blood-based biomarkers for assessing disease severity and pharmacological efficacy. We have observed significant differences in Aß1-42 levels in human plasma dependent on the anticoagulant utilized during blood collection. Our data suggests that anticoagulants alter AßPP processing and that care needs to be used in comparing published studies that have not utilized the same blood collection methodology.