Alzheimer's Disease Amyloid Plaques Research Articles

MR Microimaging of amyloid plaques in Alzheimer’s disease transgenic mice

Alzheimer's disease (AD) is the most prevalent neurological condition affecting industrialized nations and will rapidly become a healthcare crisis as the population ages. Currently, the post-mortem histological observation of amyloid plaques and neurofibrillary tangles is the only definitive diagnosis available for AD. A pre-mortem biological or physiological marker specific for AD used in conjunction with current neurological and memory testing could add a great deal of confidence to the diagnosis of AD and potentially allow therapeutic intervention much earlier in the disease process. Our group has developed MRI techniques to detect individual amyloid plaques in AD transgenic mouse brain in vivo. We are also developing contrast-enhancing agents to increase the specificity of detection of amyloid plaques. Such in vivo imaging of amyloid plaques will also allow the evaluation of anti-amyloid therapies being developed by the pharmaceutical industry in pre-clinical trials of AD transgenic mice. This short review briefly discusses our progress in these areas.

Promotion of Amyloid β Protein Misfolding and Fibrillogenesis by a Lipid Oxidation Product

Oxidatively damaged lipid membranes are known to promote the aggregation of amyloid β proteins and fibril formation. Oxidative damage typically produces 4-hydroxy-2-nonenal when lipid membranes contain ω-6 polyunsaturated fatty acyl chains, and this compound is known to modify the three His residues in Aβ proteins by Michael addition. In this report, the ability of 4-hydroxy-2-nonenal to reproduce the previously observed amyloidogenic effects of oxidative lipid damage on amyloid β proteins is demonstrated and the mechanism by which it exerts these effects is examined. Results indicate that 4-hydroxy-2-nonenal modifies the three His residues in amyloid beta proteins, which increases their membrane affinity and causes them to adopt a conformation on membranes that is similar to their conformation in a mature amyloid fibril. As a consequence, fibril formation is accelerated at relatively low protein concentrations, and the ability to seed the formation of fibrils by unmodified amyloid beta proteins is enhanced. These in vitro findings linking oxidative stress to amyloid fibril formation may be significant to the in vivo mechanism by which oxidative stress is linked to the formation of amyloid plaques in Alzheimer's disease.

Activity of γ -Secretase on Substrates Other than APP

Gamma-secretase is an intramembranous protein complex that cleaves many type-I membrane proteins, including the Notch receptor and the beta-amyloid precursor protein (APP). Interest in gamma-secretase comes, in part, from the fact that this multiprotein complex is responsible for the cleavage of APP that generates the amyloid-beta peptide (Abeta), one of the primary components of amyloid plaques in Alzheimer's disease (AD). Over the last years, molecular identification of the complex has shown that gamma-secretase is an aspartyl protease composed of four different members that are essential for the enzymatic activity: presenilin 1, aph1, pen-2 and nicastrin. In recent years, an increasing number of type-I membrane proteins have been shown to be cleaved by gamma-secretase. How the enzyme cleaves such a set of substrates with diverse functions and subcellular localizations is not well understood. In overexpression assays, the gamma-secretase cleavage of some substrates releases intracellular domains with signaling properties. On the other hand, the loose specificity required for intramembrane cleavage has raised the possibility of gamma-secretase as the membrane proteasome. The impact of gamma-secretase on other substrates has clear implications for the development of new therapies for AD, and in particular for the search of gamma-secretase inhibitors or modulators. Interference with the cleavage of some of the gamma-secretase substrates has been shown to be associated with serious adverse effects in animal models. The understanding of the mechanism by which gamma-secretase recognizes and cleaves all these proteins is of great importance to clarify the function of gamma-secretase and its role as a therapeutic target in AD, and possibly in other diseases in which gamma-secretase is involved.

Vulnerability of the Orbitofrontal Cortex to Age‐Associated Structural and Functional Brain Changes

Cross-sectional and longitudinal findings from the Baltimore Longitudinal Study of Aging (BLSA) neuroimaging study indicate that the orbitofrontal cortex (OFC) is among those regions vulnerable to age-associated tissue loss in older adults without dementia. Neuropathologic and recent in vivo amyloid imaging studies indicate that the OFC is also among the earliest neocortical regions to show deposition of amyloid plaques in aging and Alzheimer's disease. We performed behavioral and imaging studies to investigate age effects on specific aspects of OFC function. We compared performance in young (age 20-40) and old (age 60 and older) adults on cognitive tasks selected for differential sensitivity to OFC versus dorsolateral prefrontal cortex (DLPFC). Overall, greater age differences were seen in the OFC tasks compared to DLPFC tasks, with Delayed Match and Non-Match to Sample tasks showing the greatest effect size among OFC tasks and Self-Ordered Pointing Task showing the greatest effect size among DLPFC tasks. A functional magnetic resonance imaging study was conducted in parallel to probe the neural underpinnings of age differences in OFC function using the Delayed Match and Non-Match to Sample paradigm. Young but not old adults showed the expected OFC activation. Older compared with young adults showed greater activation in association with successful performance for several posterior regions, perhaps indicating compensation in the face of OFC deficits. Together, these findings indicate a vulnerability of the OFC to age-related decline in brain structure and function. Future studies using new in vivo imaging probes will help determine whether neuropathologic changes underlie the structural and functional changes.

A natural antisense transcript against Rad18, specifically expressed in neurons and upregulated during β‐amyloid‐induced apoptosis

Apoptosis, the main form of programmed cell death, is associated to a complex and dynamic transcriptional and post-transcriptional programme. By microarray analysis, we have previously implicated 241 genes differentially expressed in rat cortical neurons exposed to beta-amyloid (Abeta) protein, the major constituent of amyloid plaques in Alzheimer's disease. A large number of identified genes have no name or known function. In the present study, we have investigated one of these genes that encodes for a natural antisense transcript against Rad18 (NAT-Rad18). Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) confirmed differential expression of this transcript in cortical neurons exposed to Abeta. In situ hybridization, qRT-PCR and immunohistochemistry were used to assess the regional and cellular distribution of NAT-Rad18 in adult rat brain. These experiments showed a widespread distribution of NAT-Rad18, with the highest levels in the cerebellum, brainstem and cortex, where it was specifically expressed by neurons. NAT-Rad18 was also strongly expressed in epithelial cells of choroid plexus and cerebral vessels. At the cellular level, expression of Rad18 was counterbalanced by that of its natural antisense transcript, as shown by both in situ hybridization and immunohistochemistry. These experiments suggest the existence of a NAT that exerts a post-transcriptional control over Rad18.

Pharmacokinetics and Amyloid Plaque Targeting Ability of a Novel Peptide-Based Magnetic Resonance Contrast Agent in Wild-Type and Alzheimer's Disease Transgenic Mice

A novel magnetic resonance (MR) imaging contrast agent based on a derivative of human amyloid beta (Abeta) peptide, Gd[N-4ab/Q-4ab]Abeta 30, was previously shown to cross the blood-brain barrier (BBB) and bind to amyloid plaques in Alzheimer's disease (AD) transgenic mouse (APP/PS1) brain. We now report extensive plasma and brain pharmacokinetics of this contrast agent in wild-type (WT) and in APP/PS1 mice along with a quantitative summary of various physiological factors that govern its efficacy. Upon i.v. bolus administration, (125)I-Gd[N-4ab/Q-4ab]Abeta 30 was rapidly eliminated from the plasma following a three-exponential disposition, which is saturable at higher concentrations. Nevertheless, the contrast agent exhibited rapid and nonsaturable absorption at the BBB. The brain pharmacokinetic profile of (125)I-Gd[N-4ab/Q-4ab]Abeta 30 showed a rapid absorption phase followed by a slower elimination phase. No significant differences were observed in the plasma or brain kinetics of WT and APP/PS1 animals. Emulsion autoradiography studies conducted on WT and APP/PS1 mouse brain after an i.v. bolus administration of (125)I-Gd[N-4ab/Q-4ab]Abeta 30 in vivo confirmed the brain pharmacokinetic data and also demonstrated the preferential localization of the contrast agent on the plaques for an extended period of time. These attributes of the contrast agent are extremely useful in providing an excellent signal/noise ratio during longer MR scans, which may be essential for obtaining a high resolution image. In conclusion, this study documents the successful plaque targeting of Gd[N-4ab/Q-4ab]Abeta 30 and provides crucial pharmacokinetic information to determine the dose, mode of administration, and scan times for future in vivo MR imaging of amyloid plaques in AD transgenic mice.

Involvement of β-site APP cleaving enzyme 1 (BACE1) in amyloid precursor protein-mediated enhancement of memory and activity-dependent synaptic plasticity

The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid-beta protein (Abeta), which forms amyloid plaques in Alzheimer's disease (AD), secreted APPalpha (sAPPalpha) which enhances memory, and the APP intracellular domain (AICD), which has been implicated in the regulation of gene transcription and calcium signaling. The beta-site APP cleaving enzyme 1 (BACE1) cleaves APP in an activity-dependent manner to form Abeta, AICD, and secreted APPbeta. Because this neural activity was shown to diminish synaptic transmission in vitro [Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R (2003) Neuron 37:925-937], the prevailing notion has been that this pathway diminishes synaptic function. Here we investigated the role of this pathway in vivo. We studied transgenic mice overproducing APP that do not develop AD pathology or memory deficits but instead exhibit enhanced spatial memory. We showed enhanced synaptic plasticity in the hippocampus that depends on prior synaptic activity. We found that the enhanced memory and synaptic plasticity are abolished by the ablation of one or both copies of the BACE1 gene, leading to a significant decrease in AICD but not of any other APP cleavage products. In contrast to the previously described negative effect of BACE1-mediated cleavage of APP on synaptic function in vitro, our in vivo work indicates that BACE1-mediated cleavage of APP can facilitate learning, memory, and synaptic plasticity.

Proteomics analysis of plasma for potential biomarkers in the diagnosis of Alzheimer's disease

The objective of this study was to search for biological markers associated with Alzheimer's disease (AD). Plasma specimens obtained from ten pathologically diagnosed AD patients and ten non-demented (ND) control subjects were analyzed by a combination of 2-DE and MS. This strategy allowed us to identify six plasma proteins (alpha-1-antitrypsin, vitamin D-binding protein, inter-alpha-trypsin inhibitor family heavy chain-related protein, apolipoprotein J precursor, cAMP-dependent protein kinase catalytic subunit alpha 1, and an orf) whose 2-DE spot densities were different between the AD and ND groups. Due to their involvements in AD amyloid plaque formation, the plasma concentrations of alpha-1-antitrypsin and apolipoprotein J were further validated using either ELISA or Western blot. The results revealed that the plasma levels of alpha-1-antitrypsin in AD were higher than those of controls, confirming the 2-DE findings. However, no difference in total apolipoprotein J concentration was observed between the AD and ND groups. Considering the difference in resolving power to differentially quantitate protein isoforms provided by 2-DE and Western blot, 2-DE analysis combined with MS protein identification offers distinctive advantages when a disease-related protein isoform-specific variance is investigated.

Metal Ions Differentially Influence the Aggregation and Deposition of Alzheimer's β-Amyloid on a Solid Template

The abnormal deposition and aggregation of beta-amyloid (Abeta) on brain tissues are considered to be one of the characteristic neuropathological features of Alzheimer's disease (AD). Environmental conditions such as metal ions, pH, and cell membranes are associated with Abeta deposition and plaque formation. According to the amyloid cascade hypothesis of AD, the deposition of Abeta42 oligomers as diffuse plaques in vivo is an important earliest event, leading to the formation of fibrillar amyloid plaques by the further accumulation of soluble Abeta under certain environmental conditions. In order to characterize the effect of metal ions on amyloid deposition and plaque growth on a solid surface, we prepared a synthetic template by immobilizing Abeta oligomers onto a N-hydroxysuccinimide ester-activated solid surface. According to our study using ex situ atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and thioflavin T (ThT) fluorescence spectroscopy, Cu2+ and Zn2+ ions accelerated both Abeta40 and Abeta42 deposition but resulted only in the formation of "amorphous" aggregates. In contrast, Fe3+ induced the deposition of "fibrillar" amyloid plaques at neutral pH. Under mildly acidic environments, the formation of fibrillar amyloid plaques was not induced by any metal ion tested in this work. Using secondary ion mass spectroscopy (SIMS) analysis, we found that binding Cu ions to Abeta deposits on a solid template occurred by the possible reduction of Cu ions during the interaction of Abeta with Cu2+. Our results may provide insights into the role of metal ions on the formation of fibrillar or amorphous amyloid plaques in AD.

In vivo targeting of antibody fragments to the nervous system for Alzheimer’s disease immunotherapy and molecular imaging of amyloid plaques

Targeting therapeutic or diagnostic proteins to the nervous system is limited by the presence of the blood-brain barrier. We report that a F(ab')(2) fragment of a monoclonal antibody against fibrillar human Abeta42 that is polyamine (p)-modified has increased permeability at the blood-brain barrier, comparable binding to the antigen, and comparable in vitro binding to amyloid plaques in Alzheimer's disease (AD) transgenic mouse brain sections. Intravenous injection of the pF(ab')(2)4.1 in the AD transgenic mouse demonstrated efficient targeting to amyloid plaques throughout the brain, whereas the unmodified fragment did not. Removal of the Fc portion of this antibody derivative will minimize the inflammatory response and cerebral hemorrhaging associated with passive immunization and provide increased therapeutic potential for treating AD. Coupling contrast agents/radioisotopes might facilitate the molecular imaging of amyloid plaques with magnetic resonance imaging/positron emission tomography. The efficient delivery of immunoglobulin G fragments may also have important applications to other neurodegenerative disorders or for the generalized targeting of nervous system antigens.

Identification of Multiple Amyloidogenic Sequences in Laminin-1

Amyloid fibril formation is associated with several pathologies, including Alzheimer's disease, Parkinson's disease, type II diabetes, and prion diseases. Recently, a relationship between basement membrane components and amyloid deposits has been reported. The basement membrane protein, laminin, may be involved in amyloid-related diseases, since laminin is present in amyloid plaques in Alzheimer's disease and binds to amyloid precursor protein. Recently, we showed that peptide A208 (AASIKVAVSADR), the IKVAV-containing peptide, formed amyloid-like fibrils. We previously identified 60 cell adhesive sequences in laminin-1 using a total of 673 12-mer synthetic peptides. Here, we screened for additional amyloidogenic sequences among 60 cell adhesive peptides derived from laminin-1. We first examined amyloid-like fibril formation by the 60 active peptides with Congo red, a histological dye binding to many amyloid-like proteins. Thirteen peptides were stained with Congo red. Four of the 13 peptides promoted cell attachment and neurite outgrowth like the IKVAV-containing peptide. The four peptides also showed amyloid-like fibril formation in both X-ray diffraction and electron microscopic analyses. The amyloidogenic peptides contain consensus amino acid components, including both basic and acidic amino acids and Ser and Ile residues. These results indicate that at least five laminin-derived peptides can form amyloid-like fibrils. We conclude that the laminin-derived amyloidogenic peptides have the potential to form amyloid-like fibrils in vivo, possibly when laminin-1 is degraded.

Finding diamonds in the rubble

A recent publication by Vogt and colleagues illustrates both the power and pitfalls of using gene expression profiling of neurodegenerative disease (24). ‘Neurodegeneration’ is a generic term which describes the death of neurons, and, as such, could be applied to acute injury such as brain trauma. In contrast, the term ‘neurodegenerative disease’ connotes those disorders in which an endogenous process produces dysfunction and death of neurons over the course of years. In the past decade, there has been increasing recognition of the remarkable similarities among the neurodegenerative diseases (2,13,17, 20). For many of these disorders, genetic forms of the disease have been identified (6,8,12, 19,23). For many of these disorders, there are characteristic proteinaceous aggregates: amyloid plaques in Alzheimer's disease, Lewy bodies in Parkinson's disease (PD), neuronal intranuclear inclusions in Huntington's disease, Bunina bodies in amyotrophic lateral sclerosis, and glial cytoplasmic inclusions in multiple system atrophy (MSA) and progressive supranuclear palsy. Interestingly, it is not known if these characteristic proteins aggregates are in themselves toxic, or if they represent a compensatory cellular response. Another recent realization is that abnormal symptoms may not result purely from neuronal death, but may reflect abnormal functioning of the remaining neurons. Thus, defining the scope of neuronal dysfunction is critical in unraveling these disorders. Brain cells – neurons – are unique in that these post-mitotic cells encode experience-dependent alterations in their activity, so-called synaptic plasticity. Whereas a decade ago, the prevailing theory was that synaptic plasticity was a phenomenon that occurred primarily at the synapse, there is increasing realization that transcriptional events are critical to the encoding and maintenance of synaptic plasticity. What neurons do is alter their activity in response to external signals; we now understand that this type of lifelong plasticity depends on a continual interrogation of the genome. Transcription is the process whereby cells read specified portions of the DNA. Thus, increasing evidence demonstrates that neurons depend on transcriptional events in order to function normally. Expression profiling is the technique that permits simultaneous assessment of expression levels of thousands of genes. Using DNA microarrays, one can obtain an instantaneous snapshot of cellular activity. These measurements can often lead to novel hypotheses regarding pathogenesis. The first application of DNA microarrays to human neurologic disease revealed gene expression abnormalities in a transgenic mouse model of Huntington's disease (HD) (15). Subsequently, gene expression abnormalities have been found in a number of neurodegenerative diseases, including AD (1), ALS (5), PD (9,10,16), prion diseases (25), frontotemporal dementia and PSP (10), and multiple sclerosis (22), suggesting that impacting the genome is another common feature of these disorders. These studies of human

Differential Distribution of Alzheimer's Amyloid Precursor Protein Family Variants in Human Sperm

The Alzheimer's amyloid precursor protein (APP) is a type I transmembrane glycoprotein with receptor-like characteristics that originates the Abeta peptide by proteolytic processing. Abeta is potentially cytotoxic and the major component of the cerebral amyloid plaques in Alzheimer's disease (AD) patients. APP is known to be ubiquitously expressed in mammalian cells, with a broad tissue distribution, and Abeta deposition has been reported to occur also in many cells outside the nervous system. Although many putative functions have been suggested for APP, its precise physiological role remains to be elucidated. As several results point to a role of chronic inflammation in AD pathogenesis and suggest that AD might be a systemic disorder, the importance of APP function in non-neuronal cells/tissues has gained increased relevance. Previous studies have shown that amyloid precursor-like protein 2 (APLP2) is highly expressed in testis and sperm, but failed to unambiguously prove the presence of APP itself in mammalian sperm. The use of a battery of available antibodies that detect APP-specific epitopes or epitopes shared with other APP family members, revealed quite distinct distributions in human sperm. Our results are consistent with previous observations of APLP2 in sperm and unequivocally demonstrate the presence of APP itself in human sperm, thus suggesting a putative role for this important protein in sperm function.

In Vivo Magnetic Resonance Microimaging of Individual Amyloid Plaques in Alzheimer's Transgenic Mice

In Vivo Magnetic Resonance Microimaging of Individual Amyloid Plaques in Alzheimer's Transgenic Mice

Dyrk1A Phosphorylates α-Synuclein and Enhances Intracellular Inclusion Formation

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.

Matrix Metalloproteinases Expressed by Astrocytes Mediate Extracellular Amyloid-β Peptide Catabolism

It has been postulated that the development of amyloid plaques in Alzheimer's disease (AD) may result from an imbalance between the generation and clearance of the amyloid-beta peptide (Abeta). Although familial AD appears to be caused by Abeta overproduction, sporadic AD (the most prevalent form) may result from impairment in clearance. Recent evidence suggests that several proteases may contribute to the degradation of Abeta. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of Abeta degradation. In this study, we examined the possibility that matrix metalloproteinases (MMPs), proteases known to be expressed and secreted by astrocytes, could play a role in extracellular Abeta degradation. We found that astrocytes surrounding amyloid plaques showed enhanced expression of MMP-2 and MMP-9 in aged amyloid precursor protein (APP)/presenilin 1 mice. Moreover, astrocyte-conditioned medium (ACM) degraded Abeta, lowering levels and producing several fragments after incubation with synthetic human Abeta(1-40) and Abeta(1-42). This activity was attenuated with specific inhibitors of MMP-2 and -9, as well as in ACM derived from mmp-2 or -9 knock-out (KO) mice. In vivo, significant increases in the steady-state levels of Abeta were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls. Furthermore, pharmacological inhibition of the MMPs with N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001) increased brain interstitial fluid Abeta levels and elimination of half-life in APPsw mice. These results suggest that MMP-2 and -9 may contribute to extracellular brain Abeta clearance by promoting Abeta catabolism.

Acceleration of Amyloid β-Peptide Aggregation by Physiological Concentrations of Calcium

Alzheimer disease is characterized by the accumulation of aggregated amyloid beta-peptide (Abeta) in the brain. The physiological mechanisms and factors that predispose to Abeta aggregation and deposition are not well understood. In this report, we show that calcium can predispose to Abeta aggregation and fibril formation. Calcium increased the aggregation of early forming protofibrillar structures and markedly increased conversion of protofibrils to mature amyloid fibrils. This occurred at levels 20-fold below the calcium concentration in the extracellular space of the brain, the site at which amyloid plaque deposition occurs. In the absence of calcium, protofibrils can remain stable in vitro for several days. Using this approach, we directly compared the neurotoxicity of protofibrils and mature amyloid fibrils and demonstrate that both species are inherently toxic to neurons in culture. Thus, calcium may be an important predisposing factor for Abeta aggregation and toxicity. The high extracellular concentration of calcium in the brain, together with impaired intraneuronal calcium regulation in the aging brain and Alzheimer disease, may play an important role in the onset of amyloid-related pathology.

Neuropathology of Alzheimer disease: pathognomonic but not pathogenic

Neuropathological changes in subjects with dementia are, by definition, end-stage phenomena. While such changes allow case characterization and lend themselves to disease classification and modeling, the lesions themselves are not etiological. This truth would appear to be self-evident, yet the medical and scientific literature suggests otherwise. Indeed it is now customary to view amyloid plaques in Alzheimer disease as primary etiological, neurotoxic lesions and, hence, removing them (e.g., by immunotherapy) is believed to lead to clinical improvement. The foundation for this line of thinking lies in the existence of rare kindreds with mutations in amyloid-beta, or mutations believed to be involved in the processing of amyloid-beta, and then the extrapolation of the inherited condition to sporadic disease. We believe that this overall construct ignores early events that are more critical to onset and progression of sporadic disease. Likewise, we have studied subjects with sporadic Alzheimer disease, as well as early onset familial Alzheimer disease and Down's syndrome, over a spectrum of ages, and have found that markers of oxidative stress precede amyloid deposits in all three conditions. Amyloid and neurofibrillary pathology in the Alzheimer brain show a decrease in oxidative stress relative to vulnerable but morphologically intact neurons, suggesting that neurodegenerative lesions are compensatory phenomena, and thus manifestations of cellular adaptation. The pathology of neurodegenerative diseases should be viewed as the end-stage consequence, as opposed to cause, of the disease processes, so that early disease processes that are amenable to intervention can be properly recognized and treated.

Novel Benzofuran Derivatives for PET Imaging of β-Amyloid Plaques in Alzheimer's Disease Brains

A novel series of benzofuran derivatives as potential positron emission tomography (PET) tracers targeting amyloid plaques in Alzheimer's disease (AD) were synthesized and evaluated. The syntheses of benzofurans were successfully achieved by an intramolecular Wittig reaction between triphenylphosphonium salt and 4-nitrobenzoyl chloride. When in vitro binding studies using AD brain gray matter homogenates were carried out with a series of benzofuran derivatives, all the derivatives examined displayed high binding affinities with K(i) values in the subnanomolar range. Among these benzofuran derivatives, compound 8, 5-hydroxy-2-(4-methyaminophenyl)benzofuran, showed the lowest K(i) value (0.7 nM). In vitro fluorescent labeling of AD sections with compound 8 intensely stained not only amyloid plaques, but also neurofibrillary tangles. The (11)C labeled compound 8, [(11)C]8, was prepared by reacting the normethyl precursor, 5-hydroxy-2-(4-aminophenyl)benzofuran, with [(11)C]methyl triflate. The [(11)C]8 displayed moderate lipophilicity (log P = 2.36), very good brain penetration (4.8%ID/g at 2 min after iv injection in mice), and rapid washout from normal brains (0.4 and 0.2%ID/g at 30 and 60 min, respectively). In addition, this PET tracer showed in vivo amyloid plaque labeling in APP transgenic mice. Taken together, the data suggest that a relatively simple benzofuran derivative, [(11)C]8, may be a useful candidate PET tracer for detecting amyloid plaques in the brains of patients with Alzheimer's disease.

Physiological and Biophysical Factors That Influence Alzheimer's Disease Amyloid Plaque Targeting of Native and Putrescine Modified Human Amyloid β40

Amyloid beta40 (Abeta40) and its derivatives are being developed as probes for the ante-mortem diagnosis of Alzheimer's disease. Putrescine-Abeta40 (PUT-Abeta40) showed better plaque targeting than the native Abeta40, which was not solely explained by the differences in their blood-brain-barrier (BBB) permeabilities. The objective of this study was to elucidate the physiological and biophysical factors influencing the differential targeting of Abeta40 and PUT-Abeta40. Despite better plaque-targeting ability 125I-PUT-Abeta40 was more rapidly cleared from the systemic circulation than amyloid beta40 labeled with 125I (125I-Abeta40) after i.v. administration in mice. The BBB permeability of both compounds was inhibited by circulating peripheral Abeta40 levels. 125I-Abeta40 but not 125I-PUT-Abeta40 was actively taken up by the mouse brain slices in vitro. Only fluorescein-Abeta40, not fluorescein-PUT-Abeta40, was localized in the brain parenchymal cells in vitro. The metabolism of 125I-Abeta40 in the brain slices was twice as great as 125I-PUT-Abeta40. 125I-Abeta40 efflux from the brain slices was saturable and found to be 5 times greater than that of 125I-PUT-Abeta40. Thioflavin-T fibrillogenesis assay demonstrated that PUT-Abeta40 has a greater propensity to form insoluble fibrils compared with Abeta40, most likely due to the ability of PUT-Abeta40 to form beta sheet structure more readily than Abeta40. These results demonstrate that the inadequate plaque targeting of Abeta40 is due to cellular uptake, metabolism, and efflux from the brain parenchyma. Despite better plaque targeting of PUTAbeta40, its propensity to form fibrils may render it less suitable for human use and thus allow increased focus on the development of novel derivatives of Abeta with improved characteristics.