In vivo imaging of amyloid plaques in the brain

The pathological hallmark of Alzheimer's disease (AD) is the deposition of senile plaques (SPs) and neurofibrillary tangles. The deposition of SPs, which initially occurs in the neocortex, is regarded as the critical event for the pathogenesis of AD. Previous pathological studies indicated that the earliest deposition of SPs is suspected to occur before any detectable cognitive decline. Therefore, noninvasive imaging of brain SPs is considered to be an ideal diagnostic method for presymptomatic detection of AD. For in vivo detection of SPs in the brain, a lipophilic probe that can selectively binds SPs is indispensable to the examination using positron emission tomography (PET) or single photon emission computed tomography (SPECT). For this purpose, many kinds of amyloid binding agents have been developed, such as FDDNP, BTA-1, IMPY. We have also developed a series of promising compounds for in vivo imaging of amyloid deposits. One of these compounds, compound-A, achieves high binding affinity for A beta fibrils. This agent also showed abundant initial brain uptake and short brain clearance half-life in normal mice. Furthermore, to investigate the in vivo binding property of compound-A to amyloid plaques, compound-A was intravenously administered to APP transgenic mice. Brain slices at 120 min post injection demonstrated specific binding of compound-A to amyloid plaques in the brain. These data emphasize the potential usefulness of compound-A as in vivo imaging probe for early diagnosis of AD.     

Nanoparticles enhance brain delivery of blood-brain barrier-impermeable probes for in vivo optical and magnetic resonance imaging

Several imaging modalities are suitable for in vivo molecular neuroimaging, but the blood-brain barrier (BBB) limits their utility by preventing brain delivery of most targeted molecular probes. We prepared biodegradable nanocarrier systems made up of poly(n-butyl cyanoacrylate) dextran polymers coated with polysorbate 80 (PBCA nanoparticles) to deliver BBB-impermeable molecular imaging probes into the brain for targeted molecular neuroimaging. We demonstrate that PBCA nanoparticles allow in vivo targeting of BBB-impermeable contrast agents and staining reagents for electron microscopy, optical imaging (multiphoton), and whole brain magnetic resonance imaging (MRI), facilitating molecular studies ranging from individual synapses to the entire brain. PBCA nanoparticles can deliver BBB-impermeable targeted fluorophores of a wide range of sizes: from 500-Da targeted polar molecules to 150,000-Da tagged immunoglobulins into the brain of living mice. The utility of this approach is demonstrated by (i) development of a "Nissl stain" contrast agent for cellular imaging, (ii) visualization of amyloid plaques in vivo in a mouse model of Alzheimer's disease using (traditionally) non-BBB-permeable reagents that detect plaques, and (iii) delivery of gadolinium-based contrast agents into the brain of mice for in vivo whole brain MRI. Four-dimensional real-time two-photon and MR imaging reveal that brain penetration of PBCA nanoparticles occurs rapidly with a time constant of ～18 min. PBCA nanoparticles do not induce nonspecific BBB disruption, but collaborate with plasma apolipoprotein E to facilitate BBB crossing. Collectively, these findings highlight the potential of using biodegradable nanocarrier systems to deliver BBB-impermeable targeted molecular probes into the brain for diagnostic neuroimaging.     

Immunohistochemical investigation of the brain of aged dogs. I. Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, an oxidative damage product, in senile plaques.

In the aging dog brain lesions develop spontaneously. They share some morphological characteristics with those of Alzheimer 's disease in man. Diffuse and primitive plaques are well known, whereas neuritic plaques rarely develop. Neurofibrillary tangles have not been seen in the canine. The aim of the present investigation was to study major age-related changes of the dog's brain using paraffin sections with respect to cross-immunoreactivity of tau, A beta protein and other immunoreactive components including hydroxynonenal protein, which is a marker for oxidative damage. The occurrence of neurofibrillary tangles and of the protein tau therein was studied in serial brain sections of two dogs with the Gallyas stain and by immunohistochemistry with three different antibodies against tau. Senile plaques were stained with a monoclonal anti-A beta (residues 8-17), polyclonal anti-apolipoprotein E and a monoclonal antibody against 4-hydroxynonenal (HNE). Amyloid deposits and controls were screened by Congo red staining viewed in fluorescent light, followed by polarized light for green birefringence. With the Gallyas stain and one of the antisera against tau, neurofibrillary tangles were revealed in a similar dispersed pattern, whereas the other antitau antisera gave negative results. With the anti-HNE a positive reaction was found in cerebral amyloid deposits and in vascular wall areas where amyloid deposition was confirmed by Congo-red staining, and in perivascular cells and in some neurons. These results indicate that the canine with his tangles and plaques which show oxidative changes, forms a spontaneous modelfor understanding the early changes and their interrelationships in Alzheimer's disease.     

Immunological approaches for amyloid-beta clearance toward treatment for Alzheimer's disease

The pathology of Alzheimer's disease is characterized primarily by extracellular plaques and intracellular neurofibrillary tangles. Plaques are composed mainly of the amyloid-beta peptide, whereas tangles are derived from the cytoskeletal protein tau. The most studied hypothesis of development of the disease is that of the amyloid cascade, which states that overproduction of amyloid-beta peptide, or failure to clear this peptide, leads to Alzheimer's disease primarily through amyloid deposition, which is presumed to be involved in neurofibrillary tangle formation; these lesions are then associated with cell death, which is reflected in memory impairment, the hallmarks of this dementia. We developed a new concept showing that site-directed antibodies against amyloid-beta peptide may modulate formation of amyloid filaments, which has become the theoretical basis of the immunological approach for treatment of Alzheimer's disease. The performance of anti-beta-amyloid antibodies in transgenic mouse models of Alzheimer's disease showed they are delivered to the central nervous system, clearing amyloid plaques and protecting the mice from learning and age-related memory deficits. Amyloid plaque clearance via specific anti-amyloid-beta peptide antibodies follows multiple mechanisms. As immunotherapy is at the crossroads of immunology and the nervous system, a deeper understanding of the amyloid-beta peptide clearance mechanism may lead to an optimized therapeutic approach to the treatment of Alzheimer's disease. Antibodies generated with the first-generation vaccine might not have the desired therapeutic properties to target the "correct" mechanism, however, new immunological approaches are now under consideration.     

Binding and safety profile of novel benzoxazole derivative for in vivo imaging of amyloid deposits in Alzheimer's disease

Background:   In vivo detection of amyloid deposits in the brain is potentially useful for early diagnosis of Alzheimer's disease (AD) and tracking the efficacy of anti-amyloid therapy.
Methods:   To develop an amyloid-binding agent for positron emission tomography, we screened over 2600 compounds.
Results:   We found benzoxazole derivatives as candidate compounds for in vivo amyloid imaging probes. One of these agents, 2-(2-[2-dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole (BF-227), displays high binding affinity to Aβ fibrils. BF-227 binding increased linearly with increasing Aβ fibril formation. In temporal and hippocampal AD brain sections, BF-227 selectively bound to amyloid plaques. In contrast, no staining was evident in the cerebellum. Compared with the previously reported compound BF-168, ¹⁸F-labeled BF-227 displayed selective in vivo labeling of amyloid fibrils and rapid washout from white matter areas in an Aβ-injected rat model. An acute and subacute toxicity study of BF-227 indicated sufficient safety for clinical use as a positron emission tomography probe.
Conclusions:   These findings suggest that BF-227 is feasible as an in vivo imaging probe of amyloid deposits in AD patients.     

An in vivo model for the neurodegenerative effects of beta amyloid and protection by substance P.

Deposition of the beta-amyloid protein in senile plaques is a pathologic hallmark of Alzheimer disease (AD). Focal deposition of beta amyloid in the adult rat cerebral cortex caused profound neurodegenerative changes, including neuronal loss and degenerating neurons and neurites. Chronic induction of the Alz-50 antigen appeared in neurons around focal cortical deposits of beta amyloid. Immunoblot analysis showed that beta amyloid induced Alz-50-immunoreactive proteins in rat cerebral cortex that were very similar to the proteins induced in human cerebral cortex from patients with AD. The neuropeptide substance P prevented beta-amyloid-induced neuronal loss and expression of Alz-50 proteins when coadministered into the cerebral cortex. Systemic administration of substance P also provided protection against the effects of intracerebral beta amyloid. Thus, beta amyloid is a potent neurotoxin in the adult brain in vivo, and its effects can be blocked by substance P.     

Reversible in vitro growth of Alzheimer disease beta-amyloid plaques by deposition of labeled amyloid peptide.

The salient pathological feature of Alzheimer disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of victims. The plaques are predominantly composed of human beta-amyloid peptide (beta A4), a 40-mer whose neurotoxicity is related to its aggregation. Radioiodinated human beta A4 is rapidly deposited in vitro from a dilute (less than 10 pM) solution onto neuritic and diffuse plaques and cerebrovascular amyloid in AD brain tissue, whereas no deposition is detectable in tissue without performed plaques. This growth of plaques by deposition of radiolabeled beta A4 to plaques is reversible, with a dissociation half-time of approximately 1 h. The fraction of grey matter occupied by plaques that bind radiolabeled beta A4 in vitro is dramatically larger in AD cortex (23 +/- 11%) than in age-matched normal controls (less than 2%). In contrast to the human peptide, rat/mouse beta A4 (differing at three positions from human beta A4) does not affect the deposition of radiolabeled human beta A4. beta A4 has no detectable interaction with tachykinin receptors in rat or human brain. The use of radioiodinated beta A4 provides an in vitro system for the quantitative evaluation of agents or conditions that may inhibit or enhance the growth or dissolution of AD plaques. This reagent also provides an extremely sensitive method for visualizing various types of amyloid deposits and a means for characterizing and locating sites of amyloid peptide binding to cells and tissues.     

Patients with Alzheimer's disease display a pro-inflammatory phenotype

BACKGROUND: Inflammation plays a pivotal role in amyloid plaque progression thereby contributing to Alzheimer's disease-related neurodegeneration. We hypothesized that patients with Alzheimer's disease have an innate pro-inflammatory phenotype, as compared to control subjects without dementia. METHODS: Patients with a diagnosis of probable Alzheimer's disease (n=12) and control subjects without signs of dementia (n=18) were enrolled. Whole blood samples were stimulated ex vivo with endotoxin under standard conditions. Cytokine levels were assessed by ELISA and compared by Mann-Whitneyll-test after log transformation. RESULTS: Patients with Alzheimer's disease had seven- to ten-fold higher IL-1beta production relative to the amount of IL-10 both at the low (p=0.006) and high concentration of endotoxin (p=0.007). Subjects who display a pro-inflammatory phenotype as defined by a high IL-1beta/IL-10 ratio had 13.0-fold higher odds (95% CI: 2.1-82) to have dementia. CONCLUSION: The data support the hypothesis that a pro-inflammatory phenotype contributes to the development of Alzheimer's disease.     

Vector-mediated delivery of 125I-labeled beta-amyloid peptide A beta 1-40 through the blood-brain barrier and binding to Alzheimer disease amyloid of the A beta 1-40/vector complex.

The brain amyloid of Alzheimer disease (AD) may potentially be imaged in patients with AD by using neuroimaging technology and a radiolabeled form of the 40-residue beta-amyloid peptide A beta 1-40 that is enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. Transport of 125I-labeled A beta 1-40 (125I-A beta 1-40) through the BBB was found to be negligible by experiments with both an intravenous injection technique and an internal carotid artery perfusion method in anesthetized rats. In addition, 125I-A beta 1-40 was rapidly metabolized after either intravenous injection or internal carotid artery perfusion. BBB transport was increased and peripheral metabolism was decreased by conjugation of monobiotinylated 125I-A beta 1-40 to a vector-mediated drug delivery system, which consisted of a conjugate of streptavidin (SA) and the OX26 monoclonal antibody to the rat transferrin receptor, which undergoes receptor-mediated transcytosis through the BBB. The brain uptake, expressed as percent of injected dose delivered per gram of brain, of the 125I,bio-A beta 1-40/SA-OX26 conjugate was 0.15 +/- 0.01, a level that is 2-fold greater than the brain uptake of morphine. The binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was demonstrated by both film and emulsion autoradiography performed on frozen sections of AD brain. Binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was completely inhibited by high concentrations of unlabeled A beta 1-40. In conclusion, these studies show that BBB transport and access to amyloid within brain may be achieved by conjugation of A beta 1-40 to a vector-mediated BBB drug delivery system.     

An antiserum to the N-terminal subsequence of the Alzheimer amyloid beta protein does not react with neurofibrillary tangles

Polyclonal antibodies were raised against a synthetic peptide corresponding to a subsequence for the first 10 residues of the beta amyloid protein A4 (1-10 beta PA4). In an immunoperoxidase study of Alzheimer brain tissue, these antibodies immunostained senile plaque cores, amyloid vessel walls, and amyloid fibrils surrounding senile plaques and angiopathic vessels. Neurofibrillary tangles stained with thioflavin S or immunostained with anti-Tau immune serum were never immunodetected with the anti 1-10 beta PA4. We confirm that the neurofibrillary tangles do not contain epitopes corresponding to the first 10 residues of the beta PA4.     

Neuritic plaques and cerebrovascular amyloid in Alzheimer disease are antigenically related.

A synthetic peptide (Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr), homologous to the amino terminus of a protein purified from cerebrovascular amyloid (beta protein), induced antibodies in BALB/c mice that were used immunohistochemically to stain not only amyloid-laden cerebral vessels but neuritic plaques as well. These findings suggest that the amyloid in neuritic plaques shares antigenic determinants with beta protein of cerebral vessels. Since the amino acid compositions of plaque amyloid and cerebrovascular amyloid are similar, it is likely that plaque amyloid also consists of beta protein. This possibility suggests a model for the pathogenesis of Alzheimer disease involving beta protein.     

Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models

Autosomal dominant forms of familial Alzheimer's disease (FAD) are associated with increased production of the amyloid beta peptide, Abeta42, which is derived from the amyloid protein precursor (APP). In FAD, as well as in sporadic forms of the illness, Abeta peptides accumulate abnormally in the brain in the form of amyloid plaques. Here, we show that overexpression of FAD(717V-->F)-mutant human APP in neurons of transgenic mice decreases the density of presynaptic terminals and neurons well before these mice develop amyloid plaques. Electrophysiological recordings from the hippocampus revealed prominent deficits in synaptic transmission, which also preceded amyloid deposition by several months. Although in young mice, functional and structural neuronal deficits were of similar magnitude, functional deficits became predominant with advancing age. Increased Abeta production in the context of decreased overall APP expression, achieved by addition of the Swedish FAD mutation to the APP transgene in a second line of mice, further increased synaptic transmission deficits in young APP mice without plaques. These results suggest a neurotoxic effect of Abeta that is independent of plaque formation.     

Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease.

A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major beta/A4-protein (A beta) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences of these peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-A beta component of AD amyloid) and its precursor NACP. NAC is the second component, after A beta, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form beta-structures consistent with its association with amyloid. NACP is detected as a M(r) 19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD.     

Amyloid precursor protein in aged nonhuman primates.

In individuals with Alzheimer disease and in aged nonhuman primates, deposits of amyloid occur in senile plaques in brain parenchyma and in the walls of some meningeal and cortical vessels. Amyloid is primarily composed of beta/A4, a 4-kDa peptide derived from the transmembrane form of an amyloid precursor protein (APP). We examined the distribution of beta/A4 and APP (outside the beta/A4 domain) in cerebral cortices of monkeys ranging in age from 4 to 41 years. In all animals, APP immunoreactivity was present in cell bodies, proximal dendrites, and axons of cortical neurons. In aged animals, all of which showed senile plaques, large APP-positive axons were conspicuous, and APP immunoreactivity was present in neurites around beta/A4-immunoreactive plaques. In some plaques, APP-immunoreactive elements were located in proximity to deposits of beta/A4. The presence of APP immunoreactivity in neuronal perikarya, dendrites, axons, and in neurites within beta/A4-containing plaques supports the hypothesis that neurons can serve as one source of amyloid deposited in brain parenchyma.     

Amyloidogenic and anti-amyloidogenic properties of recombinant transthyretin variants.

Most transthyretin (TTR) mutations lead to TTR amyloid depositions in patients with familial amyloidotic polyneuropathy and familial amyloidotic cardiomyopathy. However, though an amyloidogenic protein itself, TTR inhibits aggregation of Alzheimer's amyloid beta protein (A beta) in vitro and in vivo. The pathogenic relationship between two amyloidogenic processes remains unclear. To understand how TTR mutations influence the ability of TTR to inhibit A beta amyloidosis, forty-seven recombinant TTR variants were produced and analyzed. We showed that all recombinant proteins formed tetramers and were functional in thyroxine binding. Acid denaturation at pH 3.8 resulted in aggregation and fibril formation of all TTR variants. However, only TTR G42 and TTR P55 formed fibrils at pH 6.8. Most TTR variants bound to A beta and inhibited A beta aggregation in vitro. TTR variants S64, A71, Q89, V107, H114 and I122 revealed decreased binding to A beta and decreased inhibition of A beta aggregation. Only TTR G42 and TTR P55 completely failed to bind A beta and to inhibit A beta aggregation. We suggest that TTR variants characterized by decreased binding to A beta or by decreased inhibition of A beta aggregation in vitro may contribute to A beta amyloid formation in vivo. These TTR variants might be important targets for epidemiological studies in Alzheimer's disease.     

Mitochondrial dysfunction in sporadic and genetic Alzheimer's disease

Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of many common age-related neurodegenerative diseases, including Alzheimer's disease (AD). AD is the most common neurodegenerative disorder characterized by dementia, memory loss, neuronal apoptosis and eventually death of the affected individuals. AD is characterized by two pathologic hallmark lesions that consist of extracellular plaques of amyloid-beta peptides and intracellular neurofibrillary tangles composed of hyperphosphorylated microtubular protein tau. Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimer's disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein and tau alterations in this type of dementia remains controversial.     

Amyloid deposition in the hippocampus and entorhinal cortex: quantitative analysis of a transgenic mouse model

Various transgenic mouse models of Alzheimer's disease (AD) have been developed that overexpress mutant forms of amyloid precursor protein in an effort to elucidate more fully the potential role of beta-amyloid (A beta) in the etiopathogenesis of the disease. The present study represents the first complete 3D reconstruction of A beta in the hippocampus and entorhinal cortex of PDAPP transgenic mice. A beta deposits were detected by immunostaining and thioflavin fluorescence, and quantified by using high-throughput digital image acquisition and analysis. Quantitative analysis of amyloid load in hippocampal subfields showed a dramatic increase between 12 and 15 months of age, with little or no earlier detectable deposition. Three-dimensional reconstruction in the oldest brains visualized previously unrecognized sheets of A beta coursing through the hippocampus and cerebral cortex. In contrast with previous hypotheses, compact plaques form before significant deposition of diffuse A beta, suggesting that different mechanisms are involved in the deposition of diffuse amyloid and the aggregation into plaques. The dentate gyrus was the hippocampal subfield with the greatest amyloid burden. Sublaminar distribution of A beta in the dentate gyrus correlated most closely with the termination of afferent projections from the lateral entorhinal cortex, mirroring the selective vulnerability of this circuit in human AD. This detailed temporal and spatial analysis of A beta and compact amyloid deposition suggests that specific corticocortical circuits express selective, but late, vulnerability to the pathognomonic markers of amyloid deposition, and can provide a basis for detecting prior vulnerability factors.     

Selection of peptides binding to the amyloid b-protein reveals potential inhibitors of amyloid formation.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by extracellular amyloid plaques, cerebrovascular amyloid deposits, intracellular neurofibrillary tangles, and neuronal loss. Amyloid deposits are composed of insoluble fibers of a 39-43 amino acid peptide named the amyloid beta-protein (A beta). Neuropathological and genetic studies provide strong evidence of a key role for A beta amyloidosis in the pathogenesis of AD. Therefore, an obvious pharmacological target for treatment of AD is the inhibition of amyloid growth and/or inhibition of amyloid function. We took an unbiased approach to generate new inhibitors of amyloid formation by screening a FliTrx combinatorial peptide library for A beta binding peptides and identified four groups of peptides with different A beta binding motifs. In addition, we designed and examined peptides mimicking the A beta binding domain of transthyretin (TTR). Our results showed that A beta binding peptides selected from FliTrx peptide library and from TTR-peptide analogs are capable of inhibiting A beta aggregation and A beta deposition in vitro. These properties demonstrate that binding of selected peptides to the amyloid beta-protein may provide potent therapeutic compounds for the treatment AD.     

Antiphospholipid antibodies may be a new prognostic parameter in aggressive non-Hodgkin's lymphoma

OBJECTIVES: Patients with malignancies have an increased prevalence of antiphospholipid antibodies (APA). The aim of this study was to determine the prevalence of IgG, IgM, and IgA anticardiolipin antibodies (aCL) and anti-beta-2 glycoprotein I antibodies (anti-beta2-GPI) in patients with non-Hodgkin's lymphoma (NHL), and to investigate their clinical and prognostic significance. METHODS: The study group included 86 patients with NHL. Enzyme-linked immunosorbent assay kits were used to measure the concentrations of aCL and anti-beta2-GPI, and coagulation tests, to measure lupus anticoagulant (LAC) activity. Blood was collected at diagnosis in all patients and at follow-up in 15. Median follow-up time was 1.9 yr. RESULTS: Elevated APA levels were found in 35 patients (41%) at diagnosis: one patient aCL IgG, five patients aCL IgM, five aCL IgA, one anti-beta2-GPI IgG, 14 anti-beta2-GPI IgM, and 19 anti-beta2-GPI IgA; LAC activity was found in three of 67 patients (4.5%). There was no significant correlation between elevated APA levels and patient's age or sex, disease stage or grade, bone marrow involvement, B symptoms, serum lactate dehydrogenase levels, serum beta2 microglobulin levels, International Prognostic Index (IPI) score, performance status, type of treatment, or response to treatment. There was a correlation between elevated APA and absence of extranodal disease (P = 0.045). A strong negative correlation was found between elevated APA at diagnosis and survival time. Two-year survival was 90 +/- 5% for patients without APA at diagnosis compared with 63 +/- 11% for patients with an elevated APA levels (P = 0.0025). APA added to the predictive value of IPI for event-free and overall survival. CONCLUSIONS: APA are elevated in 41% of NHL patients at diagnosis and are correlated with shortened survival. Their level may serve as an independent prognostic variable in aggressive NHL.