Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease

Near-infrared fluorescence (NIRF) molecular imaging has been widely applied to monitoring therapy of cancer and other diseases in preclinical studies; however, this technology has not been applied successfully to monitoring therapy for Alzheimer’s disease (AD). Although several NIRF probes for detecting amyloid beta (Aβ) species of AD have been reported, none of these probes has been used to monitor changes of Aβs during therapy. In this article, we demonstrated that CRANAD-3, a curcumin analog, is capable of detecting both soluble and insoluble Aβ species. In vivo imaging showed that the NIRF signal of CRANAD-3 from 4-mo-old transgenic AD (APP/PS1) mice was 2.29-fold higher than that from age-matched wild-type mice, indicating that CRANAD-3 is capable of detecting early molecular pathology. To verify the feasibility of CRANAD-3 for monitoring therapy, we first used the fast Aβ-lowering drug LY2811376, a well-characterized beta-amyloid cleaving enzyme-1 inhibitor, to treat APP/PS1 mice. Imaging data suggested that CRANAD-3 could monitor the decrease in Aβs after drug treatment. To validate the imaging capacity of CRANAD-3 further, we used it to monitor the therapeutic effect of CRANAD-17, a curcumin analog for inhibition of Aβ cross-linking. The imaging data indicated that the fluorescence signal in the CRANAD-17–treated group was significantly lower than that in the control group, and the result correlated with ELISA analysis of brain extraction and Aβ plaque counting. It was the first time, to our knowledge, that NIRF was used to monitor AD therapy, and we believe that our imaging technology has the potential to have a high impact on AD drug development.Alzheimer’s disease (AD) has been considered incurable, because none of the clinically tested drugs have shown significant effectiveness (1–4). Therefore, seeking effective therapeutics and imaging probes capable of assisting drug development is highly desirable. The amyloid hypothesis, in which various Aβ species are believed to be neurotoxic and one of the leading causes of AD, has been considered controversial in recent years because of the failures of amyloid beta (Aβ)-based drug development (1, 3, 5–8). However, no compelling data can prove that this hypothesis is wrong (2, 5), and no other theories indicate a clear path for AD drug development (4). Thus the amyloid hypothesis is still an important framework for AD drug development (1–5, 9–14). Additionally, Kim and colleagues (15) recently reported that a 3D cell-culture model of human neural cells could recapture AD pathology. In this study, their finding that the accumulation of Aβs could drive tau pathology provided strong support for the amyloid hypothesis (15).It is well known that Aβ species, including soluble monomers, dimers, oligomers, and insoluble fibrils/aggregates and plaques, play a central role in the neuropathology of AD (2, 5). Initially, it was thought that insoluble deposits/plaques formed by the Aβ peptides in an AD brain cause neurodegeneration. However, studies have shown that soluble dimeric and oligomeric Aβ species are more neurotoxic than insoluble deposits (16–21). Furthermore, it has been shown that soluble and insoluble species coexist during disease progression. The initial stage of pathology is represented by an excessive accumulation of Aβ monomers resulting from imbalanced Aβ clearance (22, 23). The early predominance of soluble species gradually shifts with the progression of AD to a majority of insoluble species (24, 25). Therefore imaging probes capable of detecting both soluble and insoluble Aβs are needed to monitor the full spectrum of amyloidosis pathology in AD.Thus far, three Aβ PET tracers have been approved by the Food and Drug Administration (FDA) for clinical applications. However, they are not approved for positive diagnosis of AD; rather, they are recommended for excluding the likelihood of AD. The fundamental limitation of these three tracers and others under development is that they bind primarily to insoluble Aβs, not the more toxic soluble Aβs (26–32). Clearly, work remains to be done in developing imaging probes based on Aβs, and imaging probes capable of diagnosing AD positively are undeniably needed.Numerous agents reportedly are capable of inhibiting the generation and aggregation of Aβs in vitro; however, only few have been tested in vivo. Partially, this lack of testing arises from the lack of reliable imaging methods that can monitor the agents’ therapeutic effectiveness in vivo. PET tracers, such as ¹¹C-Pittsburgh compound B (¹¹C-PiB) and ¹⁸F-AV-45, recently have been adapted to evaluate the efficacy of experimental AD drugs in clinical trials (33). However, they are rarely used to monitor drug treatment in small animals (30–32), likely because of the insensitivity of the tracers for Aβ species [particularly for soluble species (34–36)], complicated experimental procedures and data analysis in small animals, the high cost of PET probe synthesis and scanning, and the use of radioactive material. Therefore, a great demand for imaging agents that could be used in preclinical drug development to monitor therapeutic effectiveness in small animals remains unmet.Because of its low cost, simple operation, and easy data analysis, near infrared fluorescence (NIRF) imaging is generally more suitable than PET imaging for animal studies. Several NIRF probes for insoluble Aβs have been reported (37–45). It has been almost 10 y since the first report of NIRF imaging of Aβs by Hintersteiner et al. in 2005 (41). However, to the best of our knowledge, successful application of NIRF probes for monitoring therapeutic efficacy has not yet been reported. Our group recently has designed asymmetrical CRANAD-58 to match the hydrophobic (LVFF) and hydrophilic (HHQK) segments of Aβ peptides and demonstrated its applicability for the detection of both insoluble and soluble Aβs in vitro and in vivo (46). In this report, CRANAD-3 was designed to enhance the interaction with Aβs by replacing the phenyl rings of curcumin with pyridyls to introduce potential hydrogen bonds. Additionally, we demonstrated, for the first time to our knowledge, that the curcumin analog CRANAD-3 could be used as an NIRF imaging probe to monitor the Aβ-lowering effectiveness of therapeutics.     

PP2A methylation controls sensitivity and resistance to β-amyloid–induced cognitive and electrophysiological impairments

Elevated levels of the β-amyloid peptide (Aβ) are thought to contribute to cognitive and behavioral impairments observed in Alzheimer’s disease (AD). Protein phosphatase 2A (PP2A) participates in multiple molecular pathways implicated in AD, and its expression and activity are reduced in postmortem brains of AD patients. PP2A is regulated by protein methylation, and impaired PP2A methylation is thought to contribute to increased AD risk in hyperhomocysteinemic individuals. To examine further the link between PP2A and AD, we generated transgenic mice that overexpress the PP2A methylesterase, protein phosphatase methylesterase-1 (PME-1), or the PP2A methyltransferase, leucine carboxyl methyltransferase-1 (LCMT-1), and examined the sensitivity of these animals to behavioral and electrophysiological impairments caused by exogenous Aβ exposure. We found that PME-1 overexpression enhanced these impairments, whereas LCMT-1 overexpression protected against Aβ-induced impairments. Neither transgene affected Aβ production or the electrophysiological response to low concentrations of Aβ, suggesting that these manipulations selectively affect the pathological response to elevated Aβ levels. Together these data identify a molecular mechanism linking PP2A to the development of AD-related cognitive impairments that might be therapeutically exploited to target selectively the pathological effects caused by elevated Aβ levels in AD patients.Multiple observations suggest a role for the serine/threonine protein phosphatase 2A (PP2A) in the molecular pathways that underlie Alzheimer’s disease (AD). Analyses conducted on postmortem AD brains have found reduced PP2A expression and activity, and studies conducted in animal models have found that inhibiting PP2A produces AD-like tau pathology and cognitive impairment (1–3). One of the ways in which PP2A may affect AD is through its role as the principal tau phosphatase (4–7). PP2A also interacts with a number of kinases implicated in AD including glycogen synthase kinase 3β (GSK3β), cyclin-dependent kinase 5 (CDK5), and ERK and JNK as well as amyloid precursor protein and the NMDA and metabotropic glutamate receptors (reviewed in ref. 2).PP2A is a heterotrimeric protein composed of a catalytic, scaffolding, and regulatory subunit. Each subunit is encoded by multiple genes and splice isoforms, and the subunit composition of a particular PP2A molecule determines its subcellular distribution and substrate specificity (reviewed in ref. 2). One of the ways in which PP2A activity is regulated is through C-terminal methylation of the catalytic subunit (reviewed in refs. 8 and 9). Impaired methyl-donor metabolism is a risk factor for AD (10, 11), and PP2A dysregulation caused by impaired methylation is thought to be one of the molecular mechanisms contributing to this increased risk (12–14). Methylation promotes the formation of PP2A holoenzymes that contain Bα regulatory subunits (7, 13, 15–19), and these forms of PP2A exhibit the greatest tau phosphatase activity (6, 7).PP2A methylation is catalyzed in vivo by the methyl transferase, leucine carboxyl methyltransferase 1 (LCMT-1) (20–22), and its demethylation is catalyzed by the methylesterase, protein phosphatase methylesterase 1 (PME-1) (23–25). To explore the role of PP2A in AD further, we generated lines of transgenic mice that overexpress these enzymes and tested their effect on the sensitivity of animals to electrophysiogical and behavioral impairments caused by β-amyloid (Aβ). We found that LCMT-1 overexpression protected animals from Aβ-induced impairments, whereas overexpression of PME-1 worsened Aβ neurotoxicity. Neither transgene affected endogenous Aβ levels, suggesting that they acted by altering the response to Aβ rather than Aβ production. We also found that PME-1 and LCMT-1 overexpression were without effect on the electrophysiological response to picomolar Aβ application, suggesting that they selectively affected the response to pathological Aβ concentrations. Together these data indicate that this pathway has potential as a therapeutic avenue for AD that acts not by targeting Aβ production but by selectively altering the response to pathological levels of Aβ.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

High-speed atomic force microscopy reveals structural dynamics of amyloid β1–42 aggregates

Aggregation of amyloidogenic proteins into insoluble amyloid fibrils is implicated in various neurodegenerative diseases. This process involves protein assembly into oligomeric intermediates and fibrils with highly polymorphic molecular structures. These structural differences may be responsible for different disease presentations. For this reason, elucidation of the structural features and assembly kinetics of amyloidogenic proteins has been an area of intense study. We report here the results of high-speed atomic force microscopy (HS-AFM) studies of fibril formation and elongation by the 42-residue form of the amyloid β-protein (Aβ_1–42), a key pathogenetic agent of Alzheimer''s disease. Our data demonstrate two different growth modes of Aβ_1–42, one producing straight fibrils and the other producing spiral fibrils. Each mode depends on initial fibril nucleus structure, but switching from one growth mode to another was occasionally observed, suggesting that fibril end structure fluctuated between the two growth modes. This switching phenomenon was affected by buffer salt composition. Our findings indicate that polymorphism in fibril structure can occur after fibril nucleation and is affected by relatively modest changes in environmental conditions.Amyloid fibril accumulation is associated with numerous neurodegenerative diseases, including Alzheimer’s (AD) (1–3), prionoses (4–7), Parkinson’s (8–11), and Huntington''s (12). Nonhomologous genes encode the proteins involved in each disease, namely the amyloid β-protein (Aβ), prions (e.g., PrP, Sup35, Het-s), α-synuclein, and huntingtin, respectively. Each of these proteins assembles from a monomer state through a variety of intermediates to form insoluble amyloid fibrils that accumulate in brain tissues. The suggestion that brain Aβ accumulation and neurodegeneration are correlated remains an area of contention. Studies of human brain extracts and transgenic mice suggest such a correlation does not exist (13, 14), whereas other studies support this relationship (15). Amyloid deposition in the brain does correlate with progress from mild cognitive impairment to AD (16). The amount of brain amyloid in asymptomatic elderly people generally is less than in AD patients (17). Historically, amyloid fibrils have been regarded as the key pathologic agents in AD. This idea has been supplanted by theories in which oligomers are central (18, 19). A variety of studies support this view (20–23). In addition, oligomers appear to be more toxic to cultured cells than are fibrils (24, 25). Nevertheless, Aβ40 fibrils are neurotoxic (24, 26–28) and fibrillar Aβ appears to be associated with inflammation (29, 30) and oxidative damage (31, 32) in the brain. We believe that an unbiased assessment of working theories of disease causation does not allow one to conclude that the two theories are mutually exclusive. It is more likely that both types of assemblies are involved in AD pathogenesis. In fact, Lu et al. have provided support for this more inclusive theory by arguing that oligomers and fibrils exist in equilibrium (33). Such an equilibrium is thought to include, in addition, monomers and protofibrils (34).Although specific intermediates and fibrils have been studied in isolation at particular stages of Aβ assembly, it has been more difficult to observe structural transitions that may occur among assembly types. Techniques such as thioflavin-T (ThT) fluorescence, circular dichroism spectroscopy, and Fourier transform infrared spectroscopy are widely used to monitor development of β-sheet structure, but these methods do not provide information on aggregate tertiary or quaternary structure. Structural studies using X-ray crystallography or solid-state NMR have provided useful information on protein structure at the atomic level, but this information is static in nature and does not reveal aspects of the gross structural transitions among assembly states. Electron microscopy also is a static method.The process of fibril formation itself has been shown to be more complicated than originally thought. One reason is the huge conformational space of the intrinsically disordered Aβ monomer, which gives rise to many different oligomer structures, some of which are on-pathway for fibril formation and some of which are not. This diversity of prefibrillar structures is reflected in the structures of the fibrils that then form. Fibrils are polymorphic—Aβ forms fibrils with distinct structures depending on experimental conditions (35, 36). Additionally, different fibril types have different impacts on neurodegeneration (26, 33, 37–39). Thus, characterization of the structural dynamics of the fibril formation process is an important endeavor. Real-time visualization of monomer aggregation and fibril formation offers the possibility of understanding the dynamics of the system, developing hypotheses about assembly mechanisms, and elucidating aggregation mechanisms.In the present study, we used high-speed atomic force microscopy (HS-AFM) (40, 41) to study the dynamics of Aβ_1–42 assembly. We were able to visualize initial fibril nucleation and subsequent fibril elongation. We observed two distinct growth modes for Aβ_1–42 fibrils—one producing straight fibrils and one producing spiral fibrils—and, unexpectedly, morphological switching between these two modes.     

Peptide dimer structure in an Aβ(1–42) fibril visualized with cryo-EM

Alzheimer’s disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of β-amyloid (Aβ) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and Aβ fibrillation is extensively documented, much is still unknown about the formation of these Aβ aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by Aβ(1–42), a particularly pathogenic variant of Aβ peptide. Our model reveals that the individual layers of the Aβ fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal β-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of Aβ peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length Aβ peptide.Amyloid fibrils are the terminal assembly states of the β-amyloid (Aβ) fibrillogenic pathway. They are responsible for the neuronal damage in cerebral amyloid angiopathy and form the core of Alzheimer’s disease (AD)-specific amyloid plaques (1, 2). These plaques can locally accumulate toxic Aβ oligomers and may be surrounded by halos of altered neuronal activity (2). Our understanding of Aβ fibril structures is limited because it is difficult to explain biochemical and biological properties of Aβ and its aggregates from current fibril models. First, why does the AD-specific extension of the Aβ C terminus from Aβ(1–40) to Aβ(1–42) yield a peptide variant that is more favorable for the aggregated state and, thus, more pathogenic (3)? Second, why do fibrils formed from these two peptides exhibit a limited capacity to form mixed fibrils in vitro (4)? Third, why do the charged residues Glu22 and Asp23 disturb the fibril state such that their genetic mutation accelerates fibril formation in vitro and leads to early onset familial AD in patients (5)? Lastly, why are fibrillation inhibitors particularly effective if they target the Aβ C terminus (6, 7), and how is oligomeric assembly of toxic intermediates reflected by the structure of the fibril (1, 3)?To address these questions, we determined the structure of an Aβ(1–42) fibril morphology by electron cryomicroscopy (cryo-EM). Cryo-EM is an established technique for visualizing the 3D structure of macromolecular assemblies at near-atomic resolution (8). The technique does not require crystals and is therefore particularly well suited for the study of polymorphic amyloid structures in solution. Cryo-EM has been applied to fibrils formed from SH3 domains (9), transthyretin fragments (10), β2-microglobulin (11), and Alzheimer’s Aβ peptide (12–14). Furthermore, recent cryo-EM reconstructions have identified a common protofilament substructure in Aβ(1–40) and Aβ(1–42) fibrils in which the cross-β repeats were formed by peptide dimers (12, 15).     

Edaravone alleviates Alzheimer’s disease-type pathologies and cognitive deficits

Alzheimer’s disease (AD) is one of most devastating diseases affecting elderly people. Amyloid-β (Aβ) accumulation and the downstream pathological events such as oxidative stress play critical roles in pathogenesis of AD. Lessons from failures of current clinical trials suggest that targeting multiple key pathways of the AD pathogenesis is necessary to halt the disease progression. Here we show that Edaravone, a free radical scavenger that is marketed for acute ischemic stroke, has a potent capacity of inhibiting Aβ aggregation and attenuating Aβ-induced oxidation in vitro. When given before or after the onset of Aβ deposition via i.p. injection, Edaravone substantially reduces Aβ deposition, alleviates oxidative stress, attenuates the downstream pathologies including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, synaptic dysfunction, and rescues the behavioral deficits of APPswe/PS1 mice. Oral administration of Edaravone also ameliorates the AD-like pathologies and memory deficits of the mice. These findings suggest that Edaravone holds a promise as a therapeutic agent for AD by targeting multiple key pathways of the disease pathogenesis.Alzheimer’s disease (AD) is the most common form of dementia among the elderly, and the incidence increases with the aging population worldwide, causing a huge social and economic burden for families and societies (1, 2). Accumulating evidence indicates that amyloid-β (Aβ) and its oligomers play central roles in the pathogenesis of AD (3). Despite significant progress that has been made toward understanding the pathogenesis of AD in recent years, no efficient disease-modifying therapeutics are available for the management of AD (4). In recent years, a number of drug candidates targeting Aβ through immunotherapy or using secretase inhibitors have proceeded to clinical trials but all failed to improve cognitive functions in patients (5). Clearly, lessons have been learned through failed clinical trials, indicating that a drug targeting a single target or pathway does not work on this complex disease (6). Aβ, overproduced and accumulated in AD brains, triggers subsequent pathological events such as synaptic degeneration, Tau-hyperphosphorylation, oxidative stress, neuroinflammation, neurite degeneration, and neuronal loss (7, 8). These secondary pathological events can form vicious cycles themselves and accelerate the disease progression (9–11). Therefore, we proposed that it is critical to discover novel drugs, which target multiple key pathways in the pathogenesis of AD, to improve or halt the progression of the disease (6).As a series of new drugs for AD failed in clinical trials, it is necessary to choose drugs with both an established safety profile and a mechanism-based rationale for future clinical trials. One approach is to screen current drugs approved by regulatory bodies for other indications and reposition them for AD (12). In the present study, we took such an approach and investigated the potential therapeutic effect of Edaravone, an oxygen radical scavenger that is currently used for the treatment of acute ischemic stroke (13, 14). Oxidative imbalance is a manifestation of AD even preceding Aβ deposition and neurofibrillary tangle (NFT) (15). Aβ is a highly redox active peptide that generates reactive oxygen species (ROS) (16, 17). ROS is one of the key factors, which promote several Aβ-driven vicious cycles and propagate the pathogenesis of AD (9–11). Previous study found that Edaravone was able to attenuate Aβ-induced oxidative stress and neurotoxicity (18, 19). Aβ accumulation and aggregation into amyloid plaques in the brain are considered to trigger the AD pathogenesis. In the present study, we found that Edaravone can interact with Aβ and is competent in inhibiting Aβ aggregation and disaggregating preformed Aβ fibrils, suggesting that Edaravone is a scavenger for both ROS and Aβ. In animal models, we found that Edaravone, given before or after the onset of Aβ deposition, reduced Aβ burden in the brain and cerebral arterioles by inhibiting Aβ deposition and reducing BACE1 processing of the amyloid-β precursor protein (APP), attenuated oxidative stress and neuroinflammation, inhibited Tau hyperphosphorylation, protected brain neurons from loss and synaptic degeneration, and finally rescued the cognitive deficits of aged APPswe/PS1dE9 (APP/PS1) mice.     

Na,K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly

Neurodegeneration correlates with Alzheimer’s disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na⁺/K⁺-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ–derived “thorns” responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn⁸⁷⁹ and Trp⁸⁸⁰ is essential for ASPD–NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD–NAKα3 interaction.Alzheimer’s disease (AD) brains characteristically display fibrillar and nonfibrillar (oligomeric) protein assemblies composed of the amyloid β-protein (Aβ) (1–6). Aβ has been shown to bind to postsynaptic receptors, such as α7-nicotinic acetylcholine receptor (α7nAChR) (7), receptor for advanced glycation end products (RAGE) (8), receptor tyrosine kinase EPHB2 (9), and cellular prion protein PrP^C (10). These “Aβ receptors,” except for RAGE, have been reported to mediate toxicity of Aβ oligomers through modulating NMDA receptors (NMDAR) (11). Aβ oligomers, including dimers from AD brains (12, 13), dodecamers (Aβ*56) from AD model mice (14), and in vitro-generated Aβ-derived diffusible ligands (ADDLs) (15, 16), induce synaptic impairment by affecting NMDAR (11). Thus, NMDAR are a common target for synaptic impairment in AD. However, these oligomers do not cause neuronal death (12, 14). The atomic resolution structures of neurotoxic Aβ oligomers and their in vivo targets leading to neuronal death in AD remain unclear (6), even though neuronal death is the central mechanism responsible for symptomatic onset in AD (17).We previously isolated neurotoxic Aβ oligomers, termed amylospheroids (ASPD), from the brains of AD patient (18–20). ASPD appear in transmission electron microscopic (TEM) images as spheres of diameter ∼11.9 ± 1.7 nm (19). ASPD appear to be unique Aβ assemblies, as determined immunochemically. These structures are recognized strongly by ASPD-specific antibodies (K_d ∼ pM range), but not with the oligomer-specific polyclonal antiserum A11 (19). ASPD are distinct from Aβ dimers, ADDLs, dodecamers, and other A11-reactive entities (19).ASPD cause severe degeneration of mature human neurons (19). ASPD levels in the cortices of AD patients correlate well with disease severity (19). In contrast, ASPD-like oligomers were minimally detectable in the brains of transgenic mice expressing human amyloid precursor protein (APP), in which no significant neuronal loss is observed (19). These findings suggest that ASPD are an important effector of neuronal death in AD patients. We sought to elucidate mechanisms of ASPD-induced neurotoxicity. We report here that ASPD interact with the α-subunit of neuron-specific Na⁺/K⁺-ATPase (NAKα3), resulting in presynaptic calcium overload and neuronal death.     

From the Cover: Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation

Metal ions have emerged to play a key role in the aggregation process of amyloid β (Aβ) peptide that is closely related to the pathogenesis of Alzheimer’s disease. A detailed understanding of the underlying mechanistic process of peptide–metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic Aβ–Zn²⁺ binding features as well as how Zn²⁺ modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn²⁺ effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc Aβ₄₀ aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn²⁺, the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn²⁺ transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn²⁺ ion, forming a marginally stable, short-lived folded Aβ₄₀ species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded Aβ₄₀–Zn²⁺ complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for Aβ₄₀.Neurodegenerative disorders, such as Alzheimer’s disease (AD), have their origin in protein misfolding and generation of amyloid aggregates that have been shown to mediate toxic effects on neurons (1, 2). The aggregation of the amyloid β (Aβ) peptide is closely linked to the pathogenesis of AD (3) and is strongly dependent on environmental conditions. Metal ions have been suggested to play a key role in AD pathogenesis (4, 5), and they have been suggested to be involved in generation of amyloid and modulation of cytotoxicity (6, 7). It seems that Zn²⁺ ions have a protective effect on Aβ’s toxicity, at low Zn²⁺ concentrations, whereas higher concentrations may enhance toxicity (8, 9). To this date, it is unclear how the Zn²⁺ levels are altered in the AD brain, and seemingly contradictory studies have reported both elevated and decreased zinc levels (ref. 5 and references therein). In particular, zinc and copper ions have, however, been observed to be enriched in the amyloid plaques from brain tissues of AD patients (i.e., metal ions seem to coaggregate with Aβ) (4, 10, 11).In in vitro studies, Zn²⁺ has been reported to inhibit formation of amyloid fibrils at a metal:peptide ratio of 2:1 (7). However, at high Zn²⁺ concentration it is suggested that amorphous aggregates are formed (12–14). Structural NMR studies on Aβ–Zn²⁺ interactions showed that Zn²⁺ binds to the N terminus of the 40-residue variant of Aβ (Aβ₄₀) (15–17) where the first 16 residues are the minimal peptide sequence for Zn²⁺ binding (16). In Aβ₄₀ the Zn²⁺ ion is coordinated by four ligands, the histidines H6, H13, and H14 and the N-terminal D1 (15). Interaction between Zn²⁺ and Aβ₄₀ causes NMR signal loss of the N-terminal residues (15, 17), and these data suggest that NMR signal loss may be attributed to a chemical exchange process on an NMR intermediate time scale (18) as reported for similar exchanging systems (19–21).In general, modulation of amyloid aggregation by potential inhibitors may occur through interactions with monomers, oligomeric and/or fibrillar species that influence primary and secondary nucleation reactions and/or fibril-end elongation (22, 23). With a kinetic analysis of aggregation profiles the dominating microscopic aggregation mechanism can be determined (24–26). This approach can be applied to characterize which microscopic event(s) during primary and/or secondary pathway is(are) prevented by an inhibitor (23).Despite the huge numbers of studies of the interaction between Aβ₄₀ and metals, and the subsequent effect on self-assembly and aggregation, no detailed model for the molecular mechanism of the modulation of fibril formation has been proposed. Here, we analyze Aβ₄₀ aggregation kinetics in the absence and presence of substoichiometric concentrations of Zn²⁺ ions to elucidate at which level and which microscopic rate constant(s) is(are) modulated by Zn²⁺. In addition, we use NMR spectroscopy to follow the details of the zinc binding and folding of Aβ₄₀ around the zinc ion.     

Serial propagation of distinct strains of Aβ prions from Alzheimer’s disease patients

An increasing number of studies argues that self-propagating protein conformations (i.e., prions) feature in the pathogenesis of several common neurodegenerative diseases. Mounting evidence contends that aggregates of the amyloid-β (Aβ) peptide become self-propagating in Alzheimer’s disease (AD) patients. An important characteristic of prions is their ability to replicate distinct strains, the biological information for which is enciphered within different conformations of protein aggregates. To investigate whether distinct strains of Aβ prions can be discerned in AD patients, we performed transmission studies in susceptible transgenic mice using brain homogenates from sporadic or heritable (Arctic and Swedish) AD cases. Mice inoculated with the Arctic AD sample exhibited a pathology that could be distinguished from mice inoculated with the Swedish or sporadic AD samples, which was judged by differential accumulation of Aβ isoforms and the morphology of cerebrovascular Aβ deposition. Unlike Swedish AD- or sporadic AD-inoculated animals, Arctic AD-inoculated mice, like Arctic AD patients, displayed a prominent Aβ38-containing cerebral amyloid angiopathy. The divergent transmission behavior of the Arctic AD sample compared with the Swedish and sporadic AD samples was maintained during second passage in mice, showing that Aβ strains are serially transmissible. We conclude that at least two distinct strains of Aβ prions can be discerned in the brains of AD patients and that strain fidelity was preserved on serial passage in mice. Our results provide a potential explanation for the clinical and pathological heterogeneity observed in AD patients.Alzheimer’s disease (AD) is the most common human neurodegenerative disease, and it is characterized by the accumulation of extracellular amyloid plaques composed of aggregated amyloid-β (Aβ) peptide as well as intracellular neurofibrillary tangles composed of aggregated and hyperphosphorylated tau protein in the brain. The Aβ peptide is generated by the sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase enzymes. The amyloid cascade hypothesis posits that the accumulation and subsequent deposition of Aβ in the brain are the initiating pathological events in AD that lead to the downstream aggregation of tau (1). Although most AD cases are sporadic, a minority results from mutations in the genes encoding APP or γ-secretase components.Mounting evidence argues that the progressive nature of AD as well as many other neurodegenerative illnesses may stem from the formation and subsequent spread of self-propagating, β-sheet–rich protein conformations (i.e., prions) in the brain (2–4). The term prion, derived from the words “protein” and “infectious,” was introduced to define a novel pathogen lacking nucleic acids (5). Because the mechanism of prion propagation was shown to involve template-directed conformational change, the prion paradigm was recognized to apply more broadly in biology, including non-Mendelian phenotypic inheritance in yeast (6) and maintenance of synapse-specific changes in neurons (7). Diverse studies have converged to argue that prions, formed from normal proteins, cause many, if not most, neurodegenerative diseases (8). Some investigators prefer to use other terms for these self-propagating protein aggregates, including prion-like protein aggregates, prionoids, and proteopathic seeds (9, 10).A wealth of studies argues that the formation of Aβ prions is involved in the pathogenesis of AD. In AD, cerebral Aβ deposition follows a stereotypical progression, in which the neocortex is targeted first, followed by spreading to subcortical regions of the brain (11). Moreover, inoculation of susceptible transgenic (Tg) mice or rats expressing mutant or WT human APP with brain homogenate containing Aβ aggregates, purified Aβ amyloid fibrils, or synthetic Aβ aggregates induced widespread cerebral Aβ deposition, revealing that Aβ aggregates are self-propagating and hence, prions (12–17).An important characteristic of prions is their ability to replicate distinct strains, which can be distinguished by their transmission behavior as well as their biochemical and pathogenic properties. Prion strain-specific biological information is enciphered within different conformations of protein aggregates (18–20). Distinct conformations of Aβ aggregates have been described as formed either spontaneously from synthetic Aβ (21–23) or after seeding of synthetic Aβ by Aβ aggregates from AD brains (24, 25). Furthermore, AD is a clinically heterogeneous disease (26), which could potentially be explained by the existence of multiple Aβ strains.Based on the observation that different mutations in the human prion protein (PrP) encipher distinct strains of PrP prions (19), we hypothesized that heritable AD caused by different APP mutations (particularly mutations that result in the production of mutant Aβ) might result in the formation of distinct Aβ prion strains. The Arctic mutation in APP (E693G) occurs within the sequence of Aβ (E22G), causes enhanced Aβ protofibril formation, produces distinct fibril morphology, and results in a distinct AD pathology (27–30). In contrast, the Swedish mutation (K670M/N671L) occurs outside the Aβ sequence but results in the overproduction of WT Aβ and typical AD pathology (31–33). Here, we report that brain homogenates from Arctic and Swedish AD patients induced distinct disease phenotypes after multiple passages in susceptible Tg mice, suggesting that distinct Aβ strains form in the brains of AD patients.     

Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques

Through a comprehensive analysis of organellar markers in mouse models of Alzheimer’s disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer’s disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer’s disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology.Alzheimer’s disease (AD) is the most common form of dementia associated with aging. Nonetheless, more than a century after the original definition of the disease, the identification of the fundamental cell biological processes that cause AD remains a major challenge. Major defining features of AD brain pathology, as elucidated by molecular and genetic studies in humans and mice, are as follows: the proteolytic processing of the amyloid precursor protein (APP) by the successive action of β- and γ-secretases to generate the toxic Aβ peptides, the accumulation of extracellular aggregates of Aβ, synapse dysfunction, and death of specific subpopulations of neurons (1–6). However, although mutations that result in enhanced APP expression and/or altered processing of APP into Aβ peptides drive the development of a subset of early onset familial forms of AD, the causes of the vastly more common late-onset AD are much less well understood.One major aspect of AD pathology that is observed in both humans and mouse models of the disease is the formation of amyloid plaques. These structures contain a core of aggregated extracellular Aβ that is surrounded by swollen, dystrophic neurites and microglial processes (7–13). Multiple studies in humans and mice have additionally reported an elevated abundance of putative lysosomes and/or lysosomal proteins around amyloid plaques (9–11, 14–16). These observations indicate an influence of extracellular β-amyloid deposits on the physiology of surrounding cells and raise questions about the underlying cell biological mechanisms and the contributions of such pathological changes to AD.The goal of this study was to investigate the cell biology of AD amyloid plaques to advance understanding of how the interactions between extracellular Aβ aggregates and surrounding brain tissues might contribute to disease pathology. Through studies of mouse models of AD, we found a robust relationship between extracellular Aβ aggregates and the massive accumulation of lysosomes (but not other organelles) within swollen axons adjacent to such aggregates. A striking new feature of the lysosomes that accumulate within these dystrophic axons is their relatively low levels of multiple lysosomal proteases. Because we also identified a subpopulation of lysosomes with similar properties in the distal neuronal compartments of normal brain tissue and primary neuron cultures, the distinct composition of the axonal lysosomes that accumulate at amyloid plaques most likely reflects a blockade in their retrograde transport and maturation. More broadly, our characterization of a distinct population of axonal lysosomes that is selectively accumulated at amyloid plaques provides a foundation for the future elucidation of the mechanisms that underlie their biogenesis, function, and contributions to neuronal physiology and pathology.     

Aβ induces astrocytic glutamate release,extrasynaptic NMDA receptor activation,and synaptic loss

Synaptic loss is the cardinal feature linking neuropathology to cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here, using FRET-based glutamate sensor imaging, we show that amyloid-β peptide (Aβ) engages α7 nicotinic acetylcholine receptors to induce release of astrocytic glutamate, which in turn activates extrasynaptic NMDA receptors (eNMDARs) on neurons. In hippocampal autapses, this eNMDAR activity is followed by reduction in evoked and miniature excitatory postsynaptic currents (mEPSCs). Decreased mEPSC frequency may reflect early synaptic injury because of concurrent eNMDAR-mediated NO production, tau phosphorylation, and caspase-3 activation, each of which is implicated in spine loss. In hippocampal slices, oligomeric Aβ induces eNMDAR-mediated synaptic depression. In AD-transgenic mice compared with wild type, whole-cell recordings revealed excessive tonic eNMDAR activity accompanied by eNMDAR-sensitive loss of mEPSCs. Importantly, the improved NMDAR antagonist NitroMemantine, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from Aβ-induced damage both in vitro and in vivo.Emerging evidence suggests that the injurious effects of amyloid β peptide (Aβ) in Alzheimer’s disease (AD) may be mediated, at least in part, by excessive activation of extrasynaptic or perisynaptic NMDARs (eNMDARs) containing predominantly NR2B subunits (1, 2). In contrast, in several neurodegenerative paradigms, physiological synaptic NMDAR (sNMDAR) activity can be neuroprotective (refs. 3–8, but see ref. 9). Soluble oligomers of Aβ_1–42 are thought to underlie dementia, mimic extracellular glutamate stimulation of eNMDARs, and disrupt synaptic plasticity and long-term potentiation, eventually leading to synaptic loss (1, 6, 10, 11). However, mechanistic insight into the action of Aβ that causes excessive eNMDAR stimulation and the potential link between eNMDARs and synaptic damage remain to be elucidated. Here, we examine the cascade involved in eNMDAR activation by oligomeric Aβ and its consequences on miniature excitatory postsynaptic currents (mEPSCs). We found that eNMDAR activation is triggered by extrasynaptic glutamate released from astrocytes in response to Aβ peptide. In turn, eNMDAR stimulation is followed rapidly by a decrease in mEPSC frequency with accompanying generation of nitric oxide (NO), hyperphosphorylation of tau, and activation of caspase-3. Pharmacological blockade of eNMDARs with relative sparing of sNMDARs abrogated NO production, tau phosphorylation, caspase activation, and subsequent synaptic loss. These results suggest a glutamate-mediated cascade triggered by Aβ in which early eNMDAR activation may contribute to subsequent synaptic damage and consequent cognitive decline in AD.     

Structural basis of human γ-secretase assembly

The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer’s disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Å resolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase.Alzheimer’s disease (AD), characterized by formation of β-amyloid plaque in the brain of a patient, is closely associated with γ-secretase (1, 2). Amyloid precursor protein (APP) is processed by β-secretase in the extracellular space to produce a membrane-tethered fragment known as C99 (3). APP C99 then undergoes sequential cleavages by γ-secretase, generating a series of β-amyloid peptides (Aβ) exemplified by Aβ42 and Aβ40 (4, 5). Among all Aβs, Aβ42 is particularly prone to aggregation, resulting in formation of β-amyloid plaque and presumably contributing to the development of AD (6).Mature γ-secretase contains four components: presenilin, Pen-2, nicastrin, and Aph-1. The catalytic subunit presenilin is predicted to contain nine transmembrane segments (TMs), with two catalytic aspartate residues on TM6 and TM7. During assembly of γ-secretase, presenilin undergoes an autocatalytic cleavage to yield two polypeptide fragments, NTF (comprising TMs 1–6) and CTF (comprising TMs 7–9) (7, 8). PS1 is the target of most mutations derived from early onset familial Alzheimer’s disease patients (1). The largest component nicastrin has only one TM but contains a highly glycosylated extracellular domain (ECD), which presumably recognizes the amino terminus of substrate protein (9–11). The smallest component Pen-2 is thought to be required for the autocatalytic maturation of presenilin and γ-secretase activity (12, 13). Aph-1, required for assembly of γ-secretase (14), appears to have a previously unidentified fold with seven predicted TMs.The assembly and intersubunit interactions of γ-secretase constitute an important basis for its mechanistic understanding and have been extensively investigated during the past decade. As the central component of γ-secretase, PS1 was shown to interact with both Pen-2 and Aph-1 and form distinct subcomplexes (15–21). The only TM of nicastrin was thought to bind Aph-1 and contribute to interactions with PS1. Rationalization of these biochemical findings and other functional observations requires detailed 3D structural information on γ-secretase.In contrast to rapid accumulation of biochemical and functional data on γ-secretase, structural determination has been slow to emerge, largely due to the technical challenges associated with expression and manipulation of the intact γ-secretase. Several EM analyses have yielded low-resolution images of γ-secretase (22–27), with the overall shapes diverging from each other. Investigation of γ-secretase by other biophysical methods produced an NMR structure of the presenilin CTF (28) and X-ray structures of an archaeal homolog of presenilin (29) and a eukaryotic homolog of nicastrin (30).The high-resolution cryo-electron microscopy (cryo-EM) structure of human γ-secretase, determined at 4.5-Å resolution and in the presence of amphipols, revealed an overall architecture that is qualitatively different from all previous structures (31). The EM densities allowed identification of 19 TMs and construction of an atomic model for the ECD (31). However, these densities lacked connectivity between TMs and exhibited few side-chain features in the TMs, disallowing specific TM assignment to the four components. The use of amphipols also raises the question of whether the structure of human γ-secretase is dependent upon the choice of detergent used. In this study, we address these concerns and report, to our knowledge, the first structure of an intact γ-secretase with all TMs assigned.     

Low levels of copper disrupt brain amyloid-β homeostasis by altering its production and clearance

Whereas amyloid-β (Aβ) accumulates in the brain of normal animals dosed with low levels of copper (Cu), the mechanism is not completely known. Cu could contribute to Aβ accumulation by altering its clearance and/or its production. Because Cu homeostasis is altered in transgenic mice overexpressing Aβ precursor protein (APP), the objective of this study was to elucidate the mechanism of Cu-induced Aβ accumulation in brains of normal mice and then to explore Cu’s effects in a mouse model of Alzheimer’s disease. In aging mice, accumulation of Cu in brain capillaries was associated with its reduction in low-density lipoprotein receptor-related protein 1 (LRP1), an Aβ transporter, and higher brain Aβ levels. These effects were reproduced by chronic dosing with low levels of Cu via drinking water without changes in Aβ synthesis or degradation. In human brain endothelial cells, Cu, at its normal labile levels, caused LRP1-specific down-regulation by inducing its nitrotyrosination and subsequent proteosomal-dependent degradation due in part to Cu/cellular prion protein/LRP1 interaction. In APP^sw/0 mice, Cu not only down-regulated LRP1 in brain capillaries but also increased Aβ production and neuroinflammation because Cu accumulated in brain capillaries and, unlike in control mice, in the parenchyma. Thus, we have demonstrated that Cu’s effect on brain Aβ homeostasis depends on whether it is accumulated in the capillaries or in the parenchyma. These findings should provide unique insights into preventative and/or therapeutic approaches to control neurotoxic Aβ levels in the aging brain.Copper (Cu), an essential trace element, is an integral component of cuproproteins required for many physiological functions, such as energy production, scavenging of free radicals, connective tissue production, iron mobilization, and neurotransmission (1, 2). Almost all of plasma Cu (16–20 μM) is bound to ceruloplasmin (Cp), and the remainder, non-Cp bound Cu, (labile Cu) is bound to albumin, transcuprein, peptides, and amino acids (3, 4). Excess free Cu is toxic (5, 6). Imbalance in cerebral Cu homeostasis plays a role in the pathogenesis of Alzheimer’s disease (AD), and possibly other neurodegenerative diseases (7, 8). Increased Cu levels in plasma and/or brain have been associated with AD (9–12). In phase II clinical trials, PBT2, a modified 8-hydroxyquinoline analogue, and Cu chaperone, are showing promise (13). However, further studies are needed on effects of Cu on the CNS to fully understand the benefits of this potential therapy.Amyloid-β (Aβ) accumulates around cerebral blood vessels and in brain parenchyma in rabbits dosed with low levels of Cu (0.12 mg/L) via their drinking water and cholesterol in their chow for 10 wk (14). Similar data were obtained in beagles (15), but the mechanism is unclear. However, in transgenic (Tg) mice overexpressing amyloid-β precursor protein (APP), soluble Aβ levels in brain were reduced in APP23 mice dosed with higher Cu levels (16). In TgCRND8 mice crossed with mice that had Cu toxicities (toxic-milk mice), brain Aβ levels were also reduced (17). In Tg2576 mice, Cu levels in brain were reduced (18, 19). High levels of Cu inhibited in vitro Aβ production (20). Thus, Tg mice overexpressing APP may be unsuitable to study Cu’s normal role in Aβ homeostasis. APP expression is normal in the sporadic form of AD (21). The objective of this study was to elucidate the mechanisms of Cu-induced brain Aβ accumulation in normal young mice, so as to provide a better understanding of Cu’s role in Aβ homeostasis before exploring its role in a mouse model of AD.Brain Aβ levels are regulated by its rate of production from APP and by its rate of clearance (22). Clearance of Aβ from brain involves enzymatic degradation by proteases, including insulin-degrading enzyme (IDE) and neprilysin (NEP) (23), bulk flow of interstitial fluid (24), and transport across the blood–brain barrier (BBB) via low-density lipoprotein receptor-related protein 1 (LRP1) (25). LRP1 is expressed in brain endothelium, and reduced expression was observed in aging rodents and in patients with AD (25, 26), but the mechanism is still unclear. Herein, we uniquely show that Cu accumulated in brain capillaries but not in the parenchyma and that this was associated with reduced levels of LRP1 and increased brain Aβ levels in the aging mouse brain. Long-term exposure to low levels of Cu reproduced these effects and specifically caused LRP1 down-regulation, due, in part, to Cu/cellular prion/LRP1 interaction, LRP1 nitrotyrosination, and its proteosomal degradation. In a mouse model of AD, unlike normal mice, Cu levels were also increased in the parenchyma and caused increased Aβ production and neuroinflammation.     

From the Cover: KTKEGV repeat motifs are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers and neurotoxicity

α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into β-sheet–rich inclusions in Parkinson’s disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix–rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs β- and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescent-protein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in buffer-insoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics.Alpha-synuclein (αS) is an abundant neuronal protein that may function in synaptic vesicle trafficking (1–3). A portion of αS forms insoluble neuronal aggregates (Lewy bodies and neurites) during aging and in some neurodegenerative diseases, such as PD, dementia with Lewy bodies, multiple system atrophy, and Alzheimer’s disease (4). Genetic evidence increasingly implicates αS dyshomeostasis as a cause of PD, via missense mutations, copy number variants, or up-regulated expression (5–8). Two decades of research suggest that αS occurs principally as a natively unfolded monomer in neurons, and this assumption is commonly stated in articles and reviews of αS. In the last 4 y, unexpected findings from our (9–11) and other (3, 12–14) laboratories have provided evidence that αS forms physiological multimers, principally tetramers, that have α-helical conformation. Several criteria indicate that the tetramers/multimers present in intact, healthy neurons are physiological and we thus refer to these species as “multimers” to emphasize their distinction from pathological, β-sheet–rich aggregates traditionally called oligomers.We performed chemical cross-linking of endogenous αS in intact cells, including primary neurons, that support the existence of soluble, low-n multimers (10). We trapped abundant αS in ∼60-kDa species, the size of four monomers (4 × 14,502 Da = 58,010 Da). Known monomeric and multimeric proteins were trapped by cross-linking in their expected monomeric or multimeric states. Multiple controls ruled out artifactual induction of multimers, abnormally migrating monomers or hetero-multimers (10). If cells were lysed before cross-linking, multimers appeared depolymerized unless cross-linking was done at high protein concentration, suggesting that molecular crowding may stabilize native tetramers/multimers. Several endogenous multimeric proteins did not show this cell lysis sensitivity, except αS homolog β-synuclein (βS). These findings suggested to us that a dynamic intracellular population of metastable αS multimers and monomers coexists normally, and others have proposed similar models of dynamic/metastable tetramers (12), multimers (15), or “conformers” that may represent multimers (16). Analogous dynamic equilibria have been proposed for well-known tetrameric proteins such as hemoglobin (17) and p53 (18). An older study, from when αS was assumed to be solely monomeric, showed cross-linked low-n αS multimers in intact cells, and the authors discussed the possibility that synuclein normally exists in cells as low molecular mass oligomers, primarily dimers and trimers, that are in equilibrium with monomeric synuclein and are stabilized by experimentally induced covalent association (19).After our initial report (9), two laboratories published data supporting the earlier model of αS existing as natively unfolded monomers. These studies either did not use cross-linking of intact cells (20) or considered any cross-linked multimeric αS to be nonspecific (21). One of the reports suggested that a small and variable portion of brain αS was helically folded (20) and, subsequently, this group reported multimers (including abundant tetramers) on membranes/vesicles (14). The unresolved but central debate about the existence and biological relevance of native αS tetramers/multimers led us to perform systematic mutagenesis of αS in intact neural cells to learn whether there are discrete sequence requirements that could mediate tetramer/multimer formation and, if so, what the cellular consequences of fully depolymerizing αS might be. To quantify multimers in these experiments, we used two independent intact-cell methods: cell-penetrant covalent cross-linking and fluorescent protein complementation. We hypothesized that if mutating αS residues abolished multimers and led to detection of only monomers in cells, then αS multimers are not induced by the two intact-cell detection methods we use. Moreover, mutants that make αS solely monomeric might be toxic to cells and lead to αS-rich inclusions. Finally, solely monomeric αS mutants could be useful tools for future studies to identify whether tetramers or monomers (or both) are the functional form of αS in neurons.     

Crystal structure of the γ-secretase component nicastrin

γ-Secretase is an intramembrane protease responsible for the generation of amyloid-β (Aβ) peptides. Aberrant accumulation of Aβ leads to the formation of amyloid plaques in the brain of patients with Alzheimer''s disease. Nicastrin is the putative substrate-recruiting component of the γ-secretase complex. No atomic-resolution structure had been identified on γ-secretase or any of its four components, hindering mechanistic understanding of γ-secretase function. Here we report the crystal structure of nicastrin from Dictyostelium purpureum at 1.95-Å resolution. The extracellular domain of nicastrin contains a large lobe and a small lobe. The large lobe of nicastrin, thought to be responsible for substrate recognition, associates with the small lobe through a hydrophobic pivot at the center. The putative substrate-binding pocket is shielded from the small lobe by a lid, which blocks substrate entry. These structural features suggest a working model of nicastrin function. Analysis of nicastrin structure provides insights into the assembly and architecture of the γ-secretase complex.An intramembrane protease, γ-secretase, cleaves the type I integral membrane proteins within their transmembrane domains (1, 2). One of the most prominent substrates is the amyloid precursor protein (APP). Sequential cleavage of APP by β-secretase and γ-secretase gives rise to the amyloid-β (Aβ) peptides, particularly those containing 40 and 42 amino acids (Aβ40 and Aβ42). The Aβ peptides are the main constituent of the amyloid plaques found in the brains of patients who have Alzheimer''s disease (AD). Modulation of the activity and specificity of γ-secretase represents a potential therapeutic strategy for the treatment of Alzheimer''s disease (3–6).γ-Secretase consists of four components: presenilin (PS), presenilin enhancer 2 (Pen-2), anterior pharynx-defective 1 (Aph-1), and nicastrin (7–9). PS is an aspartyl protease and functions as the catalytic component of γ-secretase (10, 11). PS contains nine transmembrane helices (TMs); the two catalytic aspartate residues are located in the sixth and seventh TMs (12). Pen-2, bearing two TMs, is thought to facilitate the maturation of PS and enhance the γ-secretase activity (13). Aph-1 is a seven-transmembrane protein known to stabilize the γ-secretase complex (13, 14). Nicastrin is a type I transmembrane glycoprotein with a large extracellular domain (ECD) and a single TM at the C terminus. As the largest component of γ-secretase with 709 amino acids and 30- to 70-kDa glycosylation (15), nicastrin accounts for approximately two-thirds of the 230-kDa apparent molecular mass of the intact human γ-secretase. The nicastrin ECD is thought to play a critical role in the recruitment of γ-secretase substrate (16–19).At present, there is no atomic-resolution structure for the intact γ-secretase or any of its four components. The limited structural information comes from low-resolution electron microscopic (EM) analysis of γ-secretase (20–24), an NMR structure of the C-terminal three TMs of PS1 (25), and a crystal structure of a PS homolog from archaea (12). Consequently, mechanistic understanding of γ-secretase has been slow to emerge. Our recent cryo-EM structure of human γ-secretase, at 4.5-Å resolution, revealed its overall 3D architecture and most secondary structural elements, including 19 TMs (26). In this study, we present the high-resolution crystal structure of the nicastrin ECD from the eukaryote Dictyostelium purpureum and discuss its functional implications.     

α2A adrenergic receptor promotes amyloidogenesis through disrupting APP-SorLA interaction

Accumulation of amyloid β (Aβ) peptides in the brain is the key pathogenic factor driving Alzheimer’s disease (AD). Endocytic sorting of amyloid precursor protein (APP) mediated by the vacuolar protein sorting (Vps10) family of receptors plays a decisive role in controlling the outcome of APP proteolytic processing and Aβ generation. Here we report for the first time to our knowledge that this process is regulated by a G protein-coupled receptor, the α_2A adrenergic receptor (α_2AAR). Genetic deficiency of the α_2AAR significantly reduces, whereas stimulation of this receptor enhances, Aβ generation and AD-related pathology. Activation of α_2AAR signaling disrupts APP interaction with a Vps10 family receptor, sorting-related receptor with A repeat (SorLA), in cells and in the mouse brain. As a consequence, activation of α_2AAR reduces Golgi localization of APP and concurrently promotes APP distribution in endosomes and cleavage by β secretase. The α_2AAR is a key component of the brain noradrenergic system. Profound noradrenergic dysfunction occurs consistently in patients at the early stages of AD. α_2AAR-promoted Aβ generation provides a novel mechanism underlying the connection between noradrenergic dysfunction and AD. Our study also suggests α_2AAR as a previously unappreciated therapeutic target for AD. Significantly, pharmacological blockade of the α_2AAR by a clinically used antagonist reduces AD-related pathology and ameliorates cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α₂AR antagonists would be an effective therapeutic strategy for AD.Excess amyloid β (Aβ) peptides in the brain are a neuropathological hallmark of Alzheimer’s disease (AD) and are generally accepted as the key pathogenic factor of the disease (1). Aβ is generated by two sequential cleavages of amyloid precursor protein (APP) by β and γ secretase, whereas cleavage by α secretase within the Aβ domain precludes Aβ generation (2, 3). APP and the secretases undergo endocytic sorting into various organelles, such as the trans-Golgi network, the plasma membrane, and endosomes (2–6). The initial step of APP processing by α versus β secretase preferentially occurs in distinct compartments of the cell. Although α secretase-mediated cleavage of APP occurs on the plasma membrane, β secretase primarily interacts with and cleaves APP in endosomes (2–6). Therefore, endocytic sorting of APP into different membranous compartments, causing it to coreside or avoid a particular secretase, plays a decisive role in APP proteolytic processing. Consistent with this notion, abnormalities of the endocytic pathway have been found to precede Aβ deposition in late-onset AD (7).Retrograde sorting of APP from endosomes to trans-Golgi network mediated by the vacuolar protein sorting-10 (Vps10) family proteins and the retromer complex represents a critical mechanism to prevent amyloidogenic processing of APP (8–10) and has recently emerged as a potential target for therapeutic intervention (11). In particular, the sorting-related receptor with A repeat (SorLA) directs retrograde transport of APP to trans-Golgi network by binding to both APP and the retromer complex (12, 13) and retains APP in the Golgi (14), thus preventing its proteolytic processing. A connection between SorLA and AD was first revealed in patients with late-onset AD, in whom the levels of SorLA at the steady state are markedly reduced (15). Further human genetic studies identified variations of SORL1 (the gene encoding SorLA) resulting in a lower level of expression that are associated with late-onset sporadic AD (12, 16, 17). Moreover, nonsense and missense mutations of SORL1 cause autosomal dominant early-onset AD (18), supporting an etiological role of SorLA in AD. The function of SorLA in inhibiting Aβ production is confirmed by mouse genetic studies showing that loss of SorLA significantly increases Aβ levels in the brain (14) and enhances AD-related early pathology (19). Despite the importance of SorLA-dependent APP sorting in controlling Aβ metabolism and AD pathogenesis, how this process may be targeted by extracellular stimuli, such as neurotransmitters and hormones, to modulate amyloidogenesis remains largely unstudied.The α_2A adrenergic receptor (AR) belongs to the G protein-coupled receptor (GPCR) superfamily and is a crucial component of the brain noradrenergic (NA) system, controlling both NA input to the cerebral cortex and the resulting response in this brain region (20). Profound dysfunction of the NA system consistently occurs at the early stage of AD (21), raising the possibility of involvement of the α_2AAR in AD pathogenesis. Here we report for the first time to our knowledge that α_2AAR signaling regulates SorLA-dependent APP sorting and promotes amyloidogenic processing of APP by beta-site amyloid precursor protein cleaving enzyme (BACE) 1. The initial cleavage of APP by BACE1 is the rate-limiting factor of Aβ generation (22, 23). Furthermore, blockade of α_2AAR by a clinical antagonist reduces AD-related pathology and rescues cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α₂AR antagonists would be a novel effective strategy for AD treatment.     

Activation of the factor XII-driven contact system in Alzheimer’s disease patient and mouse model plasma

Alzheimer’s disease (AD) is characterized by accumulation of the β-amyloid peptide (Aβ), which likely contributes to disease via multiple mechanisms. Increasing evidence implicates inflammation in AD, the origins of which are not completely understood. We investigated whether circulating Aβ could initiate inflammation in AD via the plasma contact activation system. This proteolytic cascade is triggered by the activation of the plasma protein factor XII (FXII) and leads to kallikrein-mediated cleavage of high molecular-weight kininogen (HK) and release of proinflammatory bradykinin. Aβ has been shown to promote FXII-dependent cleavage of HK in vitro. In addition, increased cleavage of HK has been found in the cerebrospinal fluid of patients with AD. Here, we show increased activation of FXII, kallikrein activity, and HK cleavage in AD patient plasma. Increased contact system activation is also observed in AD mouse model plasma and in plasma from wild-type mice i.v. injected with Aβ42. Our results demonstrate that Aβ42-mediated contact system activation can occur in the AD circulation and suggest new pathogenic mechanisms, diagnostic tests, and therapies for AD.Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with a complex and still poorly defined etiology. Although multiple factors are likely involved in AD onset and development, a growing body of evidence implicates both neuroinflammation and peripheral inflammation in the disease (1–3). Pathways capable of triggering inflammatory processes are therefore of particular interest to AD etiology and pathogenesis. One such pathway is the contact activation system, which is initiated when the plasma protein factor XII (FXII) is exposed to negatively charged surfaces (contact activation). Contact-activated FXII (FXIIa) triggers plasma kallikrein-mediated cleavage of high molecular-weight kininogen (HK) to release bradykinin, which promotes inflammatory processes including increased blood–brain barrier permeability, edema, and cytokine expression (4) via interaction with receptors B₁ and B₂ (5). In AD, a possible surface for FXII activation could be the AD-associated peptide beta-amyloid (Aβ), which has been shown to stimulate FXII-dependent plasma kallikrein activity (6, 7) and kallikrein-mediated HK cleavage (6, 8) in vitro.Although the contact activation system is primarily thought to function in the circulation, there is evidence for its dysregulation in AD brain tissue: FXII is found in Aβ plaques (9), increased plasma kallikrein activity is observed in the AD brain parenchyma (10), and elevated levels of cleaved HK are found in cerebrospinal fluid (CSF) of patients with AD (11). To our knowledge, FXII activation and HK cleavage in the periphery of AD patients have not been demonstrated.Here, we show increased levels of FXIIa, HK cleavage, and kallikrein activity in the plasma of AD patients compared with nondemented (ND) control plasma. Furthermore, plasma HK cleavage is increased in a mouse model of AD and in wild-type mice i.v. injected with Aβ42, supporting a role for Aβ42 in AD-associated activation of the contact system. Activation of the contact system and associated bradykinin release in the AD circulation could contribute to the inflammatory and vascular dysfunction observed in the disease (3, 12). Plasma HK cleavage may also be a useful, minimally invasive biomarker for identifying AD patients who could benefit from therapeutic strategies directed against FXII.     

Allosteric regulation of γ-secretase activity by a phenylimidazole-type γ-secretase modulator

γ-Secretase is an intramembrane-cleaving protease responsible for the generation of amyloid-β (Aβ) peptides. Recently, a series of compounds called γ-secretase modulators (GSMs) has been shown to decrease the levels of long toxic Aβ species (i.e., Aβ42), with a concomitant elevation of the production of shorter Aβ species. In this study, we show that a phenylimidazole-type GSM allosterically induces conformational changes in the catalytic site of γ-secretase to augment the proteolytic activity. Analyses using the photoaffinity labeling technique and systematic mutational studies revealed that the phenylimidazole-type GSM targets a previously unidentified extracellular binding pocket within the N-terminal fragment of presenilin (PS). Collectively, we provide a model for the mechanism of action of the phenylimidazole-type GSM in which binding at the luminal side of PS induces a conformational change in the catalytic center of γ-secretase to modulate Aβ production.Gamma-secretase is responsible for the production of amyloid-β (Aβ) peptide, which is thought to play a key role in the pathogenesis of Alzheimer’s disease (AD) (1, 2). γ-Secretase is a membrane protein complex comprising a catalytic subunit, presenilin (PS), and the other three membrane protein subunits: nicastrin, anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2) (2, 3). PS is endoproteolyzed into N- and C-terminal fragments (NTF and CTF, respectively) through the maturation process of the γ-secretase complex. In the Aβ production pathway, amyloid-β precursor protein (APP) is first cleaved by β-secretase to generate a CTF of APP, C99. This stub is then cleaved by γ-secretase to release the APP intracellular domain at the cytoplasmic border of the membrane, which is called ε-cleavage. γ-Secretase then processively trims every three to four residues from the ε-site as γ-cleavage to generate Aβ fragments heterogeneous in their C termini, resulting in Aβ species ranging from 46 to 38 residues in length (4, 5). Previous immunohistological, genetic, and biochemical studies indicate that Aβ of 42 residues in length (Aβ42) is the most aggregation-prone and pathogenic species among the various Aβ peptides (6). To date, clinical trials of γ-secretase inhibitors (GSIs) have failed due to severe adverse effects, presumably attributable to the simultaneous inhibition of the cleavage of the other γ-secretase substrates, including the notch receptor (7). Recently, a series of compounds called γ-secretase modulators (GSMs) has emerged as promising therapeutic candidates, because GSMs selectively decrease Aβ42 production without affecting notch cleavage (8, 9). GSMs are chemically classified into two groups (10): acidic GSMs with carboxylic acid groups, which are derived from NSAIDs, and nonacidic or phenylimidazole-type GSMs, which are generally more potent than acidic GSMs. Phenylimidazole-type GSMs reduce the production of Aβ40 and Aβ42 while increasing the production of Aβ37, Aβ38, and Aβ39. Photoaffinity labeling experiments revealed that the phenylimidazole-type GSMs directly target the NTF of PS (11, 12). However, the precise mode of binding of phenylimidazole-type GSMs, as well as the molecular mechanism underlying the modulation of the proteolytic reaction of γ-secretase, still remains unclear. Here, we report that phenylimidazole-type GSMs facilitate the formation of the transition-state structure and augment the catalytic activity of γ-secretase by directly targeting the extracellular pocket formed by hydrophilic loop 1 (HL1) of PS. Together with the homology model derived from the crystal structure of presenilin homolog (PSH) from the archaea Methanoculleus marisnigri (13), we propose a mode of action of phenylimidazole-type GSMs, namely, the activation of the processive cleaving activity of γ-secretase, which reduces the number of long Aβ species.