Dynamic PET and SPECT imaging with radioiodinated,amyloid-reactive peptide p5 in mice: A positive role for peptide dehalogenation

Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72 h post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients.     

AL Amyloid Imaging and Therapy with a Monoclonal Antibody to a Cryptic Epitope on Amyloid Fibrils

The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA) containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL) amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K_D of ∼10 nM. Binding was inhibited in the presence of the –Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas) as evidenced by single photon emission (SPECT) imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.     

Validation and Characterization of a Novel Peptide That Binds Monomeric and Aggregated β-Amyloid and Inhibits the Formation of Neurotoxic Oligomers

Although the formation of β-amyloid (Aβ) deposits in the brain is a hallmark of Alzheimer disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to Aβ toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble Aβ oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15-amino acid peptide (15-mer), isolated via phage display screening, targeted Aβ and attenuated its neurotoxicity (Taddei, K., Laws, S. M., Verdile, G., Munns, S., D''Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Neurobiol. Aging 31, 203–214). The aim of the current study was to generate and biochemically characterize analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15-amino acid peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t = 300 min, c.f. t = 30 min for the parent peptide). This candidate reduced the formation of soluble Aβ42 oligomers, with the concurrent generation of non-toxic, insoluble aggregates measuring up to 25–30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric Aβ42, as shown by coimmunoprecipitation and surface plasmon resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar Aβ42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated Aβ42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies.     

Early-onset Formation of Parenchymal Plaque Amyloid Abrogates Cerebral Microvascular Amyloid Accumulation in Transgenic Mice

The fibrillar assembly and deposition of amyloid β (Aβ) protein, a key pathology of Alzheimer disease, can occur in the form of parenchymal amyloid plaques and cerebral amyloid angiopathy (CAA). Familial forms of CAA exist in the absence of appreciable parenchymal amyloid pathology. The molecular interplay between parenchymal amyloid plaques and CAA is unclear. Here we investigated how early-onset parenchymal amyloid plaques impact the development of microvascular amyloid in transgenic mice. Tg-5xFAD mice, which produce non-mutated human Aβ and develop early-onset parenchymal amyloid plaques, were bred to Tg-SwDI mice, which produce familial CAA mutant human Aβ and develop cerebral microvascular amyloid. The bigenic mice presented with an elevated accumulation of Aβ and fibrillar amyloid in the brain compared with either single transgenic line. Tg-SwDI/Tg-5xFAD mice were devoid of microvascular amyloid, the prominent pathology of Tg-SwDI mice, but exhibited larger parenchymal amyloid plaques compared with Tg-5xFAD mice. The larger parenchymal amyloid deposits were associated with a higher loss of cortical neurons and elevated activated microglia in the bigenic Tg-SwDI/Tg-5xFAD mice. The periphery of parenchymal amyloid plaques was largely composed of CAA mutant Aβ. Non-mutated Aβ fibril seeds promoted CAA mutant Aβ fibril formation in vitro. Further, intrahippocampal administration of biotin-labeled CAA mutant Aβ peptide accumulated on and adjacent to pre-existing parenchymal amyloid plaques in Tg-5xFAD mice. These findings indicate that early-onset parenchymal amyloid plaques can serve as a scaffold to capture CAA mutant Aβ peptides and prevent their accumulation in cerebral microvessels.     

Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling

Proteolytic processing of the amyloid precursor protein (APP) by the β- and γ-secretases releases the amyloid-β peptide (Aβ), which deposits in senile plaques and contributes to the etiology of Alzheimer''s disease (AD). The α-secretase cleaves APP in the Aβ peptide sequence to generate soluble APPα (sAPPα). Upregulation of α-secretase activity through the 5-hydroxytryptamine 4 (5-HT₄) receptor has been shown to reduce Aβ production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT₄ receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT₄ receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT_4d receptor-stimulated α-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for α-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for α-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT₄ receptor-induced α-secretase activation.     

Dipeptidyl peptidase 4 contributes to Alzheimer’s disease–like defects in a mouse model and is increased in sporadic Alzheimer’s disease brains

The amyloid cascade hypothesis, which proposes a prominent role for full-length amyloid β peptides in Alzheimer’s disease, is currently being questioned. In addition to full-length amyloid β peptide, several N-terminally truncated fragments of amyloid β peptide could well contribute to Alzheimer’s disease setting and/or progression. Among them, pyroGlu3–amyloid β peptide appears to be one of the main components of early anatomical lesions in Alzheimer’s disease–affected brains. Little is known about the proteolytic activities that could account for the N-terminal truncations of full-length amyloid β, but they appear as the rate-limiting enzymes yielding the Glu3–amyloid β peptide sequence that undergoes subsequent cyclization by glutaminyl cyclase, thereby yielding pyroGlu3–amyloid β. Here, we investigated the contribution of dipeptidyl peptidase 4 in Glu3–amyloid β peptide formation and the functional influence of its genetic depletion or pharmacological blockade on spine maturation as well as on pyroGlu3–amyloid β peptide and amyloid β 42–positive plaques and amyloid β 42 load in the triple transgenic Alzheimer’s disease mouse model. Furthermore, we examined whether reduction of dipeptidyl peptidase 4 could rescue learning and memory deficits displayed by these mice. Our data establish that dipeptidyl peptidase 4 reduction alleviates anatomical, biochemical, and behavioral Alzheimer’s disease–related defects. Furthermore, we demonstrate that dipeptidyl peptidase 4 activity is increased early in sporadic Alzheimer’s disease brains. Thus, our data demonstrate that dipeptidyl peptidase 4 participates in pyroGlu3–amyloid β peptide formation and that targeting this peptidase could be considered as an alternative strategy to interfere with Alzheimer’s disease progression.     

Deciphering the Glycolipid Code of Alzheimer's and Parkinson's Amyloid Proteins Allowed the Creation of a Universal Ganglioside-Binding Peptide

A broad range of microbial and amyloid proteins interact with cell surface glycolipids which behave as infectivity and/or toxicity cofactors in human pathologies. Here we have deciphered the biochemical code that determines the glycolipid-binding specificity of two major amyloid proteins, Alzheimer''s β-amyloid peptide (Aβ) and Parkinson''s disease associated protein α-synuclein. We showed that both proteins interact with selected glycolipids through a common loop-shaped motif exhibiting little sequence homology. This 12-residue domain corresponded to fragments 34-45 of α-synuclein and 5-16 of Aβ. By modulating the amino acid sequence of α-synuclein at only two positions in which we introduced a pair of histidine residues found in Aβ, we created a chimeric α-synuclein/Aβ peptide with extended ganglioside-binding properties. This chimeric peptide retained the property of α-synuclein to recognize GM3, and acquired the capacity to recognize GM1 (an Aβ-inherited characteristic). Free histidine (but not tryptophan or asparagine) and Zn²⁺ (but not Na⁺) prevented this interaction, confirming the key role of His-13 and His-14 in ganglioside binding. Molecular dynamics studies suggested that the chimeric peptide recognized cholesterol-constrained conformers of GM1, including typical chalice-shaped dimers, that are representative of the condensed cholesterol-ganglioside complexes found in lipid raft domains of the plasma membrane of neural cells. Correspondingly, the peptide had a particular affinity for raft-like membranes containing both GM1 and cholesterol. The chimeric peptide also interacted with several other gangliosides, including major brain gangliosides (GM4, GD1a, GD1b, and GT1b) but not with neutral glycolipids such as GlcCer, LacCer or asialo-GM1. It could inhibit the binding of Aβ1-42 onto neural SH-SY5Y cells and did not induce toxicity in these cells. In conclusion, deciphering the glycolipid code of amyloid proteins allowed us to create a universal ganglioside-binding peptide of only 12-residues with potential therapeutic applications in infectious and neurodegenerative diseases that involve cell surface gangliosides as receptors.     

The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species

The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase.     

Variations in the Electrostatic Landscape of Class II Human Leukocyte Antigen Molecule Induced by Modifications in the Myelin Basic Protein Peptide: A Theoretical Approach

The receptor-ligand interactions involved in the formation of the complex between Class II Major Histocompatibility Complex molecules and antigenic peptides, which are essential for establishing an adaptive immunological response, were analyzed in the Class II Human Leukocyte Antigen (HLA) - Myelin Basic Protein (MBP) peptide complex (HLA-DRβ1*1501-MBP) using a multipolar molecular electrostatic potential approach. The Human Leukocyte Antigen - peptide complex system was divided into four pockets together with their respective peptide fragment and the corresponding occupying amino acid was replaced by each of the remaining 19 amino acids. Partial atomic charges were calculated by a quantum chemistry approach at the Hatree Fock/3-21*G level, to study the behavior of monopole, dipole and quadrupole electrostatic multipolar moments. Two types of electrostatic behavior were distinguished in the pockets'' amino acids: “anchoring” located in Pocket 1 and 4, and “recognition” located in Pocket 4 and 7. According to variations in the electrostatic landscape, pockets were ordered as: Pocket 1>Pocket 9≫Pocket 4≈Pocket 7 which is in agreement with the binding ability reported for Class II Major Histocompatibility Complex pockets. In the same way, amino acids occupying the polymorphic positions β13R, β26F, β28D, β9W, β74A, β47F and β57D were shown to be key for this Receptor-Ligand interaction. The results show that the multipolar molecular electrostatic potential approach is appropriate for characterizing receptor-ligand interactions in the MHC–antigenic peptide complex, which could have potential implications for synthetic vaccine design.     

Proteomic Analysis of Highly Prevalent Amyloid A Amyloidosis Endemic to Endangered Island Foxes

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.     

Amyloid-β Peptide Exacerbates the Memory Deficit Caused by Amyloid Precursor Protein Loss-of-Function in Drosophila

The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD). APP can undergo two exclusive proteolytic pathways: cleavage by the α-secretase initiates the non-amyloidogenic pathway while cleavage by the β-secretase initiates the amyloidogenic pathway that leads, after a second cleavage by the γ-secretase, to amyloid-β (Aβ) peptides that can form toxic extracellular deposits, a hallmark of AD. The initial events leading to AD are still unknown. Importantly, aside from Aβ toxicity whose molecular mechanisms remain elusive, several studies have shown that APP plays a positive role in memory, raising the possibility that APP loss-of-function may participate to AD. We previously showed that APPL, the Drosophila APP ortholog, is required for associative memory in young flies. In the present report, we provide the first analysis of the amyloidogenic pathway’s influence on memory in the adult. We show that transient overexpression of the β-secretase in the mushroom bodies, the center for olfactory memory, did not alter memory. In sharp contrast, β-secretase overexpression affected memory when associated with APPL partial loss-of-function. Interestingly, similar results were observed with Drosophila Aβ peptide. Because Aβ overexpression impaired memory only when combined to APPL partial loss-of-function, the data suggest that Aβ affects memory through the APPL pathway. Thus, memory is altered by two connected mechanisms—APPL loss-of-function and amyloid peptide toxicity—revealing in Drosophila a functional interaction between APPL and amyloid peptide.     

β-Amyloid Fibrils in Alzheimer Disease Are Not Inert When Bound to Copper Ions but Can Degrade Hydrogen Peroxide and Generate Reactive Oxygen Species

According to the “amyloid cascade” hypothesis of Alzheimer disease, the formation of Aβ fibrils and senile plaques in the brain initiates a cascade of events leading to the formation of neurofibrillary tangles, neurodegeneration, and the symptom of dementia. Recently, however, emphasis has shifted away from amyloid fibrils as the predominant toxic form of Aβ toward smaller aggregates, referred to as “soluble oligomers.” These oligomers have become one of the prime suspects for involvement in the early oxidative damage that is evident in this disease. This raises the question whether or not Aβ fibrils are actually “inert tombstones” present at the end of the aggregation process. Here we show that, when Aβ(1–42) aggregates, including fibrils, are bound to Cu(II) ions, they retain their redox activity and are able to degrade hydrogen peroxide (H₂O₂) with the formation of hydroxyl radicals and the consequent oxidation of the peptide (detected by formation of carbonyl groups). We find that this ability increases as the Cu(II):peptide ratio increases and is accompanied by changes in aggregate morphology, as determined by atomic force microscopy. When aggregates are prepared in the copresence of Cu(II) and Zn(II) ions, the ratio of Cu(II):Zn(II) becomes an important factor in the degeneration of H₂O₂, the formation of carbonyl groups in the peptide, and in aggregate morphology. We believe, therefore, that Aβ fibrils can destroy H₂O₂ and generate damaging hydroxyl radicals and, so, are not necessarily inert end points.     

Effects of the English (H6R) and Tottori (D7N) Familial Alzheimer Disease Mutations on Amyloid β-Protein Assembly and Toxicity

Mutations in the amyloid β-protein (Aβ) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid β-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the Aβ40 and Aβ42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil → α/β → β and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide oligomers using in situ chemical cross-linking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial β-strand (H6R) or α/β (D7N) structure, in contrast to the predominately statistical coil structure of wild type Aβ oligomers. Mutant oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter Aβ assembly at its earliest stages, monomer folding and oligomerization, and produce oligomers that are more toxic to cultured neuronal cells than are wild type oligomers.     

Pharmacological removal of serum amyloid P component from intracerebral plaques and cerebrovascular Aβ amyloid deposits in vivo

Human amyloid deposits always contain the normal plasma protein serum amyloid P component (SAP), owing to its avid but reversible binding to all amyloid fibrils, including the amyloid β (Aβ) fibrils in the cerebral parenchyma plaques and cerebrovascular amyloid deposits of Alzheimer''s disease (AD) and cerebral amyloid angiopathy (CAA). SAP promotes amyloid fibril formation in vitro, contributes to persistence of amyloid in vivo and is also itself directly toxic to cerebral neurons. We therefore developed (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a drug that removes SAP from the blood, and thereby also from the cerebrospinal fluid (CSF), in patients with AD. Here we report that, after introduction of transgenic human SAP expression in the TASTPM double transgenic mouse model of AD, all the amyloid deposits contained human SAP. Depletion of circulating human SAP by CPHPC administration in these mice removed all detectable human SAP from both the intracerebral and cerebrovascular amyloid. The demonstration that removal of SAP from the blood and CSF also removes it from these amyloid deposits crucially validates the strategy of the forthcoming ‘Depletion of serum amyloid P component in Alzheimer''s disease (DESPIAD)’ clinical trial of CPHPC. The results also strongly support clinical testing of CPHPC in patients with CAA.     

Effect of Copper and Zinc on the Single Molecule Self-Affinity of Alzheimer’s Amyloid-β Peptides

The presence of trace concentrations of metallic ions, such as copper and zinc, has previously been shown to drastically increase the aggregation rate and neurotoxicity of amyloid-β (Aβ), the peptide implicated in Alzheimer’s disease (AD). The mechanism of why copper and zinc accelerate Aβ aggregation is poorly understood. In this work, we use single molecule force spectroscopy (SMFS) to probe the kinetic and thermodynamic parameters (dissociation constant, K_d, kinetic dissociation rate, k_off, and free energy, ΔG) of the dissociation of an Aβ dimer, the amyloid species which initiates the amyloid cascade. Our results show that nanomolar concentrations of copper do not change the single molecule affinity of Aβ to another Aβ peptide in a statistically significant way, while nanomolar concentrations of zinc decrease the affinity of Aβ-Aβ by an order of magnitude. This suggests that the binding of zinc ion to Aβ may interfere with the binding of Aβ-Aβ, leading to a lower self-affinity.     

“Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don''t-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.     

Long-Term Oral Administration of Hop Flower Extracts Mitigates Alzheimer Phenotypes in Mice

Coincident with the expanding population of aged people, the incidence of Alzheimer disease (AD) is rapidly increasing in most advanced countries. At present, no effective prophylactics are available. Among several pathological mechanisms proposed for AD, the “amyloid hypothesis” has been most widely accepted, in which accumulation or deposition of Aβ is considered to be the initial event. Thus, prevention of Aβ production would be an ideal strategy for the treatment or prevention of AD. Aβ is produced via the proteolytic cleavage of its precursor protein, APP (amyloid precursor protein), by two different enzymes, β and γ-secretases. Indeed, inhibitors against either or both enzymes have been developed and tested for clinical efficacy. Based on the “amyloid hypothesis”, we developed a luciferase-based screening method to monitor γ-secretase activity, screened more than 1,600 plant extracts, most of which have long been used in Chinese medicine, and observed that Hop extracts significantly inhibit Aβ production in cultured cells. A major component of the inhibitory activity was purified, and its chemical identity was determined by NMR to be Garcinielliptone HC. In vivo, oral administration of Hop extracts to AD model mice decreased Aβ depositions in the cerebral cortex of the parietal lobe, hippocampus, and artery walls (amyloid angiopathy) in the brains. In a Morris water maze test, AD model mice that had daily consumed Hop extracts in their drinking water showed significant mitigation of memory impairment at ages of 9 and 12 months. Moreover, in the open field test oral administration of Hop extracts also prevented an emotional disturbance that appeared in the AD mice at 18 months. Despite lifelong consumption of Hop extracts, no deleterious side effects were observed at any age. These results support the “amyloid hypothesis”, and indicate that Hop extract is a promising candidate for an effective prophylactic for AD.     

Detection of Amyloid Plaques Targeted by Bifunctional USPIO in Alzheimer’s Disease Transgenic Mice Using Magnetic Resonance Microimaging

Amyloid plaques are a key pathological hallmark of Alzheimer’s disease (AD). The detection of amyloid plaques in the brain is important for the diagnosis of AD, as well as for following potential amyloid targeting therapeutic interventions. Our group has developed several contrast agents to detect amyloid plaques in vivo using magnetic resonance microimaging (µMRI) in AD transgenic mice, where we used mannitol to enhance blood brain barrier (BBB) permeability. In the present study, we used bifunctional ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, chemically coupled with Aβ1-42 peptide to image amyloid plaque deposition in the mouse brain. We coupled the nanoparticles to polyethylene glycol (PEG) in order to improve BBB permeability. These USPIO-PEG-Aβ1-42 nanoparticles were injected intravenously in AD model transgenic mice followed by initial in vivo and subsequent ex vivo μMRI. A 3D gradient multi-echo sequence was used for imaging with a 100 µm isotropic resolution. The amyloid plaques detected by T2*-weighted μMRI were confirmed with matched histological sections. The region of interest-based quantitative measurement of T2* values obtained from the in vivo μMRI showed contrast injected AD Tg mice had significantly reduced T2* values compared to wild-type mice. In addition, the ex vivo scans were examined with voxel-based analysis (VBA) using statistical parametric mapping (SPM) for comparison of USPIO-PEG-Aβ1-42 injected AD transgenic and USPIO alone injected AD transgenic mice. The regional differences seen by VBA in the USPIO-PEG-Aβ1-42 injected AD transgenic correlated with the amyloid plaque distribution histologically. Our results indicate that USPIO-PEG-Aβ1-42 can be used for amyloid plaque detection in vivo by intravenous injection without the need to co-inject an agent which increases permeability of the BBB. This technique could aid the development of novel amyloid targeting drugs by allowing therapeutic effects to be followed longitudinally in model AD mice.     

Matrix Metalloproteinase-9 Reduces Islet Amyloid Formation by Degrading Islet Amyloid Polypeptide

Deposition of islet amyloid polypeptide (IAPP) as amyloid is a pathological hallmark of the islet in type 2 diabetes, which is toxic to β-cells. We previously showed that the enzyme neprilysin reduces islet amyloid deposition and thereby reduces β-cell apoptosis, by inhibiting fibril formation. Two other enzymes, matrix metalloproteinase (MMP)-2 and MMP-9, are extracellular gelatinases capable of degrading another amyloidogenic peptide, Aβ, the constituent of amyloid deposits in Alzheimer disease. We therefore investigated whether MMP-2 and MMP-9 play a role in reducing islet amyloid deposition. MMP-2 and MMP-9 mRNA were present in mouse islets but only MMP-9 activity was detectable. In an islet culture model where human IAPP (hIAPP) transgenic mouse islets develop amyloid but nontransgenic islets do not, a broad spectrum MMP inhibitor (GM6001) and an MMP-2/9 inhibitor increased amyloid formation and the resultant β-cell apoptosis. In contrast, a specific MMP-2 inhibitor had no effect on either amyloid deposition or β-cell apoptosis. Mass spectrometry demonstrated that MMP-9 degraded amyloidogenic hIAPP but not nonamyloidogenic mouse IAPP. Thus, MMP-9 constitutes an endogenous islet protease that limits islet amyloid deposition and its toxic effects via degradation of hIAPP. Because islet MMP-9 mRNA levels are decreased in type 2 diabetic subjects, islet MMP-9 activity may also be decreased in human type 2 diabetes, thereby contributing to increased islet amyloid deposition and β-cell loss. Approaches to increase islet MMP-9 activity could reduce or prevent amyloid deposition and its toxic effects in type 2 diabetes.     

MALDI-Mass Spectrometry Imaging Identifies Vitronectin as a Common Constituent of Amyloid Deposits

Amyloids are pathological intra- and extracellular fibrillar aggregates of polypeptides with a cross-β-sheet structure and characteristic tinctorial properties. The amyloid deposits commonly enclose several non-fibrillar components of the extracellular matrix. Their potential to regulate the formation and aggregation process of amyloid fibrils is still poorly understood. For a better understanding of the role of the extracellular matrix in amyloidosis, it is essential to gain deeper insights into the composition of amyloid deposits. Here, we utilized matrix-assisted laser desorption and ionization mass spectrometry imaging to identify extracellular matrix compounds in amyloid deposits. Using this technique, we identified and determined the spatial distribution of vitronectin within AApoAI-, ALλ-, ATTR- and AIns amyloid deposits and, using immunohistochemistry, validated the spatial overlap of vitronectin with amyloids in 175 cases with diverse types of amyloid in several different tissues.