The inhibition of cellular toxicity of amyloid-ß by dissociated transthyretin.

The protective effect of transthyretin (TTR) on cellular toxicity of ß-amyloid (Aß) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and cerebrospinal fluid, the pathogenic aggregation of which causes systemic amyloidosis. However, studies have documented a protective effect of TTR against cellular toxicity of pathogenic Aß, a protein associated with Alzheimer's disease. TTR binds Aß, alters its aggregation, and inhibits its toxicity both in vitro and in vivo In this study, we investigate whether the amyloidogenic ability of TTR and its antiamyloid inhibitory effect are associated. Using protein aggregation and cytotoxicity assays, we found that the dissociation of the TTR tetramer, required for its amyloid pathogenesis, is also necessary to prevent cellular toxicity from Aß oligomers. These findings suggest that the Aß-binding site of TTR may be hidden in its tetrameric form. Aided by computational docking and peptide screening, we identified a TTR segment that is capable of altering Aß aggregation and toxicity, mimicking TTR cellular protection. EM, immune detection analysis, and assessment of aggregation and cytotoxicity revealed that the TTR segment inhibits Aß oligomer formation and also promotes the formation of nontoxic, nonamyloid amorphous aggregates, which are more sensitive to protease digestion. Finally, this segment also inhibits seeding of Aß catalyzed by Aß fibrils extracted from the brain of an Alzheimer's patient. Together, these findings suggest that mimicking the inhibitory effect of TTR with peptide-based therapeutics represents an additional avenue to explore for the treatment of Alzheimer's disease.

An RNA aptamer with potent affinity for a toxic dimer of amyloid ß42 has potential utility for histochemical studies of Alzheimer's disease.

Oligomers of ß-amyloid 42 (Aß42), rather than fibrils, drive the pathogenesis of Alzheimer's disease (AD). In particular, toxic oligomeric species called protofibrils (PFs) have attracted significant attention. Herein, we report RNA aptamers with higher affinity toward PFs derived from a toxic Aß42 dimer than toward fibrils produced from WT Aß42 or from a toxic, conformationally constrained Aß42 variant, E22P-Aß42. We obtained these RNA aptamers by using the preincubated dimer model of E22P-Aß42, which dimerized via a linker located at Val-40, as the target of in vitro selection. This dimer formed PFs during incubation. Several physicochemical characteristics of an identified aptamer, E22P-AbD43, suggested that preferential affinity of this aptamer toward PFs is due to its higher affinity for the toxic dimer unit (KD = 20 ± 6.0 nm) of Aß42 than for less-toxic Aß40 aggregates. Comparison of CD data from the full-length and random regions of E22P-AbD43 suggested that the preferential binding of E22P-AbD43 toward the dimer might be related to the formation of a G-quadruplex structure. E22P-AbD43 significantly inhibited the nucleation phase of the dimer and its associated neurotoxicity in SH-SY5Y human neuroblastoma cells. Of note, E22P-AbD43 also significantly protected against the neurotoxicity of WT Aß42 and E22P-Aß42. Furthermore, in an AD mouse model, E22P-AbD43 preferentially recognized diffuse aggregates, which likely originated from PFs or higher-order oligomers with curvilinear structures, compared with senile plaques formed from fibrils. We conclude that the E22P-AbD43 aptamer is a promising research and diagnostic tool for further studies of AD etiology.

High temperature promotes amyloid ß-protein production and γ-secretase complex formation via Hsp90.

Alzheimer's disease (AD) is characterized by neuronal loss and accumulation of ß-amyloid-protein (Aß) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25-40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aß production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aß production. Here, we report temperature-dependent regulation of Aß production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aß production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aß production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aß production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.

Metal ion coordination delays amyloid-ß peptide self-assembly by forming an aggregation-inert complex.

A detailed understanding of the molecular pathways for amyloid-ß (Aß) peptide aggregation from monomers into amyloid fibrils, a hallmark of Alzheimer's disease, is crucial for the development of diagnostic and therapeutic strategies. We investigate the molecular details of peptide fibrillization in vitro by perturbing this process through addition of differently charged metal ions. Here, we used a monovalent probe, the silver ion, that, similarly to divalent metal ions, binds to monomeric Aß peptide and efficiently modulates Aß fibrillization. On the basis of our findings, combined with our previous results on divalent zinc ions, we propose a model that links the microscopic metal-ion binding to Aß monomers to its macroscopic impact on the peptide self-assembly observed in bulk experiments. We found that substoichiometric concentrations of the investigated metal ions bind specifically to the N-terminal region of Aß, forming a dynamic, partially compact complex. The metal-ion bound state appears to be incapable of aggregation, effectively reducing the available monomeric Aß pool for incorporation into fibrils. This is especially reflected in a decreased fibril-end elongation rate. However, because the bound state is significantly less stable than the amyloid state, Aß peptides are only transiently redirected from fibril formation, and eventually almost all Aß monomers are integrated into fibrils. Taken together, these findings unravel the mechanistic consequences of delaying Aß aggregation via weak metal-ion binding, quantitatively linking the contributions of specific interactions of metal ions with monomeric Aß to their effects on bulk aggregation.

NMR-based site-resolved profiling of ß-amyloid misfolding reveals structural transitions from pathologically relevant spherical oligomer to fibril.

Increasing evidence highlights the central role of neurotoxic oligomers of the 42-residue-long ß-amyloid (Aß42) in Alzheimer's disease (AD). However, very limited information is available on the structural transition from oligomer to fibril, particularly for pathologically relevant amyloids. To the best of our knowledge, we present here the first site-specific structural characterization of Aß42 misfolding, from toxic oligomeric assembly yielding a similar conformation to an AD-associated Aß42 oligomer, into a fibril. Transmission EM (TEM) analysis revealed that a spherical amyloid assembly (SPA) of Aß42 with a 15.6 ± 2.1-nm diameter forms in a ∼30-µm Aß42 solution after a ∼10-h incubation at 4 °C, followed by a slow conversion into fibril at ∼180 h. Immunological analysis suggested that the SPA has a surface structure similar to that of amylospheroid (ASPD), a patient-derived toxic Aß oligomer, which had a diameter of 10-15 nm in negative-stain TEM. Solid-state NMR analyses indicated that the SPA structure involves a ß-loop-ß motif, which significantly differed from the triple-ß motif observed for the Aß42 fibril. The comparison of the 13C chemical shifts of SPA with those of the fibril prepared in the above conditions and interstrand distance measurements suggested a large conformational change involving rearrangements of intermolecular ß-sheet into in-register parallel ß-sheet during the misfolding. A comparison of the SPA and ASPD 13C chemical shifts indicated that SPA is structurally similar to the ASPD relevant to AD. These observations provide insights into the architecture and key structural transitions of amyloid oligomers relevant for AD pathology.

Copper stabilizes antiparallel ß-sheet fibrils of the amyloid ß40 (Aß40)-Iowa variant.

Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue-long ß-amyloid peptide (Aß40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aß42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel ß-sheet, whereas previous studies by other researchers have reported parallel ß-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.

Garcinoic acid prevents ß-amyloid (Aß) deposition in the mouse brain.

Garcinoic acid (GA or δ-T3-13'COOH), is a natural vitamin E metabolite that has preliminarily been identified as a modulator of nuclear receptors involved in ß-amyloid (Aß) metabolism and progression of Alzheimer's disease (AD). In this study, we investigated GA's effects on Aß oligomer formation and deposition. Specifically, we compared them with those of other vitamin E analogs and the soy isoflavone genistein, a natural agonist of peroxisome proliferator-activated receptor γ (PPARγ) that has therapeutic potential for managing AD. GA significantly reduced Aß aggregation and accumulation in mouse cortical astrocytes. Similarly to genistein, GA up-regulated PPARγ expression and apolipoprotein E (ApoE) efflux in these cells with an efficacy that was comparable with that of its metabolic precursor δ-tocotrienol and higher than those of α-tocopherol metabolites. Unlike for genistein and the other vitamin E compounds, the GA-induced restoration of ApoE efflux was not affected by pharmacological inhibition of PPARγ activity, and specific activation of pregnane X receptor (PXR) was observed together with ApoE and multidrug resistance protein 1 (MDR1) membrane transporter up-regulation in both the mouse astrocytes and brain tissue. These effects of GA were associated with reduced Aß deposition in the brain of TgCRND8 mice, a transgenic AD model. In conclusion, GA holds potential for preventing Aß oligomerization and deposition in the brain. The mechanistic aspects of GA's properties appear to be distinct from those of other vitamin E metabolites and of genistein.

Galectin 3-binding protein suppresses amyloid-ß production by modulating ß-cleavage of amyloid precursor protein.

Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of ß-amyloid (Aß) peptides. Aß is produced from amyloid precursor protein (APP) that is sequentially cleaved by ß- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aß production, we performed a gene microarray-based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aß production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aß production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3-binding protein (GAL3BP), which suppressed Aß production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aß production by directly interacting with APP and thereby inhibiting APP processing by ß-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.

Coagulation factor XIIIa cross-links amyloid ß into dimers and oligomers and to blood proteins.

In cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), the amyloid ß (Aß) peptide deposits along the vascular lumen, leading to degeneration and dysfunction of surrounding tissues. Activated coagulation factor XIIIa (FXIIIa) covalently cross-links proteins in blood and vasculature, such as in blood clots and on the extracellular matrix. Although FXIIIa co-localizes with Aß in CAA, the ability of FXIIIa to cross-link Aß has not been demonstrated. Using Western blotting, kinetic assays, and microfluidic analyses, we show that FXIIIa covalently cross-links Aß40 into dimers and oligomers (kcat/Km = 1.5 × 105 m-1s-1), as well as to fibrin, platelet proteins, and blood clots under flow in vitro Aß40 also increased the stiffness of platelet-rich plasma clots in the presence of FXIIIa. These results suggest that FXIIIa-mediated cross-linking may contribute to the formation of Aß deposits in CAA and Alzheimer's disease.

A clinical dose of angiotensin-converting enzyme (ACE) inhibitor and heterozygous ACE deletion exacerbate Alzheimer's disease pathology in mice.

Inhibition of angiotensin-converting enzyme (ACE) is a strategy used worldwide for managing hypertension. In addition to converting angiotensin I to angiotensin II, ACE also converts neurotoxic ß-amyloid protein 42 (Aß42) to Aß40. Because of its neurotoxicity, Aß42 is believed to play a causative role in the development of Alzheimer's disease (AD), whereas Aß40 has neuroprotective effects against Aß42 aggregation and also against metal-induced oxidative damage. Whether ACE inhibition enhances Aß42 aggregation or impairs human cognitive ability are very important issues for preventing AD onset and for optimal hypertension management. In an 8-year longitudinal study, we found here that the mean intelligence quotient of male, but not female, hypertensive patients taking ACE inhibitors declined more rapidly than that of others taking no ACE inhibitors. Moreover, the sera of all AD patients exhibited a decrease in Aß42-to-Aß40-converting activity compared with sera from age-matched healthy individuals. Using human amyloid precursor protein transgenic mice, we found that a clinical dose of an ACE inhibitor was sufficient to increase brain amyloid deposition. We also generated human amyloid precursor protein/ACE+/- mice and found that a decrease in ACE levels promoted Aß42 deposition and increased the number of apoptotic neurons. These results suggest that inhibition of ACE activity is a risk factor for impaired human cognition and for triggering AD onset.

In vivo localization of human acetylcholinesterase-derived species in a ß-sheet conformation at the core of senile plaques in Alzheimer's disease.

Many neurodegenerative diseases are characterized by amyloid deposition. In Alzheimer's disease (AD), ß-amyloid (Aß) peptides accumulate extracellularly in senile plaques. The AD amyloid cascade hypothesis proposes that Aß production or reduced clearance leads to toxicity. In contrast, the cholinergic hypothesis argues for a specific pathology of brain cholinergic pathways. However, neither hypothesis in isolation explains the pattern of AD pathogenesis. Evidence suggests that a connection exists between these two scenarios: the synaptic form of human acetylcholinesterase (hAChE-S) associates with plaques in AD brains; among hAChE variants, only hAChE-S enhances Aß fibrillization in vitro and Aß deposition and toxicity in vivo Only hAChE-S contains an amphiphilic C-terminal domain (T40, AChE575-614), with AChE586-599 homologous to Aß and forming amyloid fibrils, which implicates T40 in AD pathology. We previously showed that the amyloid scavenger, insulin-degrading enzyme (IDE), generates T40-derived amyloidogenic species that, as a peptide mixture, seed Aß fibrillization. Here, we characterized 11 peptides from a T40-IDE digest for ß-sheet conformation, surfactant activity, fibrillization, and seeding capability. We identified residues important for amyloidogenicity and raised polyclonal antibodies against the most amyloidogenic peptide. These new antisera, alongside other specific antibodies, labeled sections from control, hAChE-S, hAPPswe, and hAChE-S/hAPPswe transgenic mice. We observed that hAChE-S ß-sheet species co-localized with Aß in mature plaque cores, surrounded by hAChE-S α-helical species. This observation provides the first in vivo evidence of the conformation of hAChE-S species within plaques. Our results may explain the role of hAChE-S in Aß deposition and aggregation, as amyloidogenic hAChE-S ß-sheet species might seed Aß aggregation.

Combined treatment with the phenolics (-)-epigallocatechin-3-gallate and ferulic acid improves cognition and reduces Alzheimer-like pathology in mice.

"Nutraceuticals" are well-tolerated natural dietary compounds with drug-like properties that make them attractive as Alzheimer's disease (AD) therapeutics. Combination therapy for AD has garnered attention following a recent National Institute on Aging mandate, but this approach has not yet been fully validated. In this report, we combined the two most promising nutraceuticals with complementary anti-amyloidogenic properties: the plant-derived phenolics (-)-epigallocatechin-3-gallate (EGCG, an α-secretase activator) and ferulic acid (FA, a ß-secretase modulator). We used transgenic mice expressing mutant human amyloid ß-protein precursor and presenilin 1 (APP/PS1) to model cerebral amyloidosis. At 12 months of age, we orally administered EGCG and/or FA (30 mg/kg each) or vehicle once daily for 3 months. At 15 months, combined EGCG-FA treatment reversed cognitive impairment in most tests of learning and memory, including novel object recognition and maze tasks. Moreover, EGCG- and FA-treated APP/PS1 mice exhibited amelioration of brain parenchymal and cerebral vascular ß-amyloid deposits and decreased abundance of amyloid ß-proteins compared with either EGCG or FA single treatment. Combined treatment elevated nonamyloidogenic soluble APP-α and α-secretase candidate and down-regulated amyloidogenic soluble APP-ß, ß-C-terminal APP fragment, and ß-secretase protein expression, providing evidence for a shift toward nonamyloidogenic APP processing. Additional beneficial co-treatment effects included amelioration of neuroinflammation, oxidative stress, and synaptotoxicity. Our findings offer preclinical evidence that combined treatment with EGCG and FA is a promising AD therapeutic approach.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells.

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular ß-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

Amyloid-ß oligomers have a profound detergent-like effect on lipid membrane bilayers, imaged by atomic force and electron microscopy.

The ability of amyloid-ß peptide (Aß) to disrupt membrane integrity and cellular homeostasis is believed to be central to Alzheimer's disease pathology. Aß is reported to have various impacts on the lipid bilayer, but a clearer picture of Aß influence on membranes is required. Here, we use atomic force and transmission electron microscopies to image the impact of different isolated Aß assembly types on lipid bilayers. We show that only oligomeric Aß can profoundly disrupt the bilayer, visualized as widespread lipid extraction and subsequent deposition, which can be likened to an effect expected from the action of a detergent. We further show that Aß oligomers cause widespread curvature and discontinuities within lipid vesicle membranes. In contrast, this detergent-like effect was not observed for Aß monomers and fibers, although Aß fibers did laterally associate and embed into the upper leaflet of the bilayer. The marked impact of Aß oligomers on membrane integrity identified here reveals a mechanism by which these oligomers may be cytotoxic.

Proteolytic shedding of the prion protein via activation of metallopeptidase ADAM10 reduces cellular binding and toxicity of amyloid-ß oligomers.

The cellular prion protein (PrPC) is a key neuronal receptor for ß-amyloid oligomers (AßO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer's disease (AD). Similarly to the amyloid precursor protein (APP), PrPC is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrPC shedding would alter the cellular binding and cytotoxicity of AßO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrPC shedding and reduces the binding of AßO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrPC shedding and increased AßO binding, which was blocked by the PrPC-specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrPC shedding and decreased AßO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AßO binding to PrPC Besides blocking AßO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aß and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrPC in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AßO, revealing a potential therapeutic benefit of ADAM10 activation in AD.

Humanization of the entire murine Mapt gene provides a murine model of pathological human tau propagation.

In cortical regions of brains from individuals with preclinical or clinical Alzheimer's disease (AD), extracellular ß-amyloid (Aß) deposition precedes the aggregation of pathological intracellular tau (the product of the gene microtubule-associated protein tau (MAPT)). To our knowledge, current mouse models of tauopathy reconstitute tau pathology by overexpressing mutant human tau protein. Here, through a homologous recombination approach that replaced the entire murine Mapt gene with the human ortholog, we developed knock-in mice with humanized Mapt to create an in vivo platform for studying human tauopathy. Of note, the humanized Mapt expressed all six tau isoforms present in humans. We next cross-bred the MAPT knock-in mice with single amyloid precursor protein (App) knock-in mice to investigate the Aß-tau axis in AD etiology. The double-knock-in mice exhibited higher tau phosphorylation than did single MAPT knock-in mice but initially lacked apparent tauopathy and neurodegeneration, as observed in the single App knock-in mice. We further observed that tau humanization significantly accelerates cell-to-cell propagation of AD brain-derived pathological tau both in the absence and presence of Aß-amyloidosis. In the presence of Aß-amyloidosis, tau accumulation was intensified and closely associated with dystrophic neurites, consistently showing that Aß-amyloidosis affects tau pathology. Our results also indicated that the pathological human tau interacts better with human tau than with murine tau, suggesting species-specific differences between these orthologous pathogenic proteins. We propose that the MAPT knock-in mice will make it feasible to investigate the behaviors and characteristics of human tau in an animal model.

Heme and hemoglobin suppress amyloid ß-mediated inflammatory activation of mouse astrocytes.

Glial immune activity is a key feature of Alzheimer's disease (AD). Given that the blood factors heme and hemoglobin (Hb) are both elevated in AD tissues and have immunomodulatory roles, here we sought to interrogate their roles in modulating ß-amyloid (Aß)-mediated inflammatory activation of astrocytes. We discovered that heme and Hb suppress immune activity of primary mouse astrocytes by reducing expression of several proinflammatory cytokines (e.g. RANTES (regulated on activation normal T cell expressed and secreted)) and the scavenger receptor CD36 and reducing internalization of Aß(1-42) by astrocytes. Moreover, we found that certain soluble (>75-kDa) Aß(1-42) oligomers are primarily responsible for astrocyte activation and that heme or Hb association with these oligomers reverses inflammation. We further found that heme up-regulates phosphoprotein signaling in the phosphoinositide 3-kinase (PI3K)/Akt pathway, which regulates a number of immune functions, including cytokine expression and phagocytosis. The findings in this work suggest that dysregulation of Hb and heme levels in AD brains may contribute to impaired amyloid clearance and that targeting heme homeostasis may reduce amyloid pathogenesis. Altogether, we propose heme as a critical molecular link between amyloid pathology and AD risk factors, such as aging, brain injury, and stroke, which increase Hb and heme levels in the brain.

Mammalian amyloidogenic proteins promote prion nucleation in yeast.

Fibrous cross-ß aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-ß (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes.

Prion protein stabilizes amyloid-ß (Aß) oligomers and enhances Aß neurotoxicity in a Drosophila model of Alzheimer's disease.

The cellular prion protein (PrPC) can act as a cell-surface receptor for ß-amyloid (Aß) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aß isoforms and PrPC in the Drosophila brain. We found that co-expression of Aß and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aß deposition in the fly brain. In contrast, under the same conditions, expression of Aß or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aß as oligomeric assemblies and fragment-preformed Aß fibers. The ability of membrane-anchored PrPC to trap Aß as cytotoxic oligomers at the membrane surface and fragment inert Aß fibers suggests a mechanism by which PrPC exacerbates Aß deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aß toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aß and PrPC represents a potential therapeutic strategy.

Histone deacetylase-mediated regulation of endolysosomal pH.

The pH of the endolysosomal system is tightly regulated by a balance of proton pump and leak mechanisms that are critical for storage, recycling, turnover, and signaling functions in the cell. Dysregulation of endolysosomal pH has been linked to aging, amyloidogenesis, synaptic dysfunction, and various neurodegenerative disorders, including Alzheimer's disease. Therefore, understanding the mechanisms that regulate luminal pH may be key to identifying new targets for managing these disorders. Meta-analysis of yeast microarray databases revealed that nutrient-limiting conditions inhibited the histone deacetylase (HDAC) Rpd3 and thereby up-regulated transcription of the endosomal Na+/H+ exchanger Nhx1, resulting in vacuolar alkalinization. Consistent with these findings, Rpd3 inhibition by the HDAC inhibitor and antifungal drug trichostatin A induced Nhx1 expression and vacuolar alkalinization. Bioinformatics analysis of Drosophila and mouse databases revealed that caloric control of the Nhx1 orthologs DmNHE3 and NHE6, respectively, is also mediated by HDACs. We show that NHE6 is a target of the transcription factor cAMP-response element-binding protein (CREB), a known regulator of cellular responses to low-nutrient conditions, providing a molecular mechanism for nutrient- and HDAC-dependent regulation of endosomal pH. Of note, pharmacological targeting of the CREB pathway to increase NHE6 expression helped regulate endosomal pH and correct defective clearance of amyloid Aß in an apoE4 astrocyte model of Alzheimer's disease. These observations from yeast, fly, mouse, and cell culture models point to an evolutionarily conserved mechanism for HDAC-mediated regulation of endosomal NHE expression. Our insights offer new therapeutic strategies for modulation of endolysosomal pH in fungal infection and human disease.