Anti-α-synuclein immunotherapy reduces α-synuclein propagation in the axon and degeneration in a combined viral vector and transgenic model of synucleinopathy.

Neurodegenerative disorders such as Parkinson's Disease (PD), PD dementia (PDD) and Dementia with Lewy bodies (DLB) are characterized by progressive accumulation of α-synuclein (α-syn) in neurons. Recent studies have proposed that neuron-to-neuron propagation of α-syn plays a role in the pathogenesis of these disorders. We have previously shown that antibodies against the C-terminus of α-syn reduce the intra-neuronal accumulation of α-syn and related deficits in transgenic models of synucleinopathy, probably by abrogating the axonal transport and accumulation of α-syn in in vivo models. Here, we assessed the effect of passive immunization against α-syn in a new mouse model of axonal transport and accumulation of α-syn. For these purpose, non-transgenic, α-syn knock-out and mThy1-α-syn tg (line 61) mice received unilateral intra-cerebral injections with a lentiviral (LV)-α-syn vector construct followed by systemic administration of the monoclonal antibody 1H7 (recognizes amino acids 91-99) or control IgG for 3 months. Cerebral α-syn accumulation and axonopathy was assessed by immunohistochemistry and effects on behavior were assessed by Morris water maze. Unilateral LV-α-syn injection resulted in axonal propagation of α-syn in the contra-lateral site with subsequent behavioral deficits and axonal degeneration. Passive immunization with 1H7 antibody reduced the axonal accumulation of α-syn in the contra-lateral side and ameliorated the behavioral deficits. Together this study supports the notion that immunotherapy might improve the deficits in models of synucleinopathy by reducing the axonal propagation and accumulation of α-syn. This represents a potential new mode of action through which α-syn immunization might work.

Reducing C-terminal-truncated alpha-synuclein by immunotherapy attenuates neurodegeneration and propagation in Parkinson's disease-like models.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are common neurodegenerative disorders of the aging population, characterized by progressive and abnormal accumulation of α-synuclein (α-syn). Recent studies have shown that C-terminus (CT) truncation and propagation of α-syn play a role in the pathogenesis of PD/DLB. Therefore, we explored the effect of passive immunization against the CT of α-syn in the mThy1-α-syn transgenic (tg) mouse model, which resembles the striato-nigral and motor deficits of PD. Mice were immunized with the new monoclonal antibodies 1H7, 5C1, or 5D12, all directed against the CT of α-syn. CT α-syn antibodies attenuated synaptic and axonal pathology, reduced the accumulation of CT-truncated α-syn (CT-α-syn) in axons, rescued the loss of tyrosine hydroxylase fibers in striatum, and improved motor and memory deficits. Among them, 1H7 and 5C1 were most effective at decreasing levels of CT-α-syn and higher-molecular-weight aggregates. Furthermore, in vitro studies showed that preincubation of recombinant α-syn with 1H7 and 5C1 prevented CT cleavage of α-syn. In a cell-based system, CT antibodies reduced cell-to-cell propagation of full-length α-syn, but not of the CT-α-syn that lacked the 118-126 aa recognition site needed for antibody binding. Furthermore, the results obtained after lentiviral expression of α-syn suggest that antibodies might be blocking the extracellular truncation of α-syn by calpain-1. Together, these results demonstrate that antibodies against the CT of α-syn reduce levels of CT-truncated fragments of the protein and its propagation, thus ameliorating PD-like pathology and improving behavioral and motor functions in a mouse model of this disease.

ß-Amyloid inhibits E-S potentiation through suppression of cannabinoid receptor 1-dependent synaptic disinhibition.

It has been widely reported that ß-amyloid peptide (Aß) blocks long-term potentiation (LTP) of hippocampal synapses. Here, we show evidence that Aß more potently blocks the potentiation of excitatory postsynaptic potential (EPSP)-spike coupling (E-S potentiation). This occurs, not by direct effect on excitatory synapses or postsynaptic neurons, but rather through an indirect mechanism: reduction of endocannabinoid-mediated peritetanic disinhibition. During high-frequency (tetanic) stimulation, somatic synaptic inhibition is suppressed by endocannabinoids. We find that Aß prevents this endocannabinoid-mediated disinhibition, thus leaving synaptic inhibition more intact during tetanic stimulation. This intact inhibition opposes the normal depolarization of hippocampal pyramidal neurons that occurs during tetanus, thus opposing the induction of synaptic plasticity. Thus, a pathway through which Aß can act to modulate neural activity is identified, relevant to learning and memory and how it may mediate aspects of the cognitive decline seen in Alzheimer's disease.

Secreted amyloid ß-proteins in a cell culture model include N-terminally extended peptides that impair synaptic plasticity.

Evidence for a central role of amyloid ß-protein (Aß) in the genesis of Alzheimer's disease (AD) has led to advanced human trials of Aß-lowering agents. The "amyloid hypothesis" of AD postulates deleterious effects of small, soluble forms of Aß on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aß could be therapeutically advantageous, it is important to understand the full range of soluble Aß derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aß species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aß40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aß-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aß. These N-terminally extended Aß-containing monomeric fragments are distinct from soluble Aß oligomers formed from Aß1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing ß-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aß-containing species can arise from APP processing events N-terminal to the classical ß-secretase cleavage site.

Vascular alterations in PDAPP mice after anti-Aß immunotherapy: Implications for amyloid-related imaging abnormalities.

BACKGROUND: Clinical studies of ß-amyloid (Aß) immunotherapy in Alzheimer's disease (AD) patients have demonstrated reduction of central Aß plaque by positron emission tomography (PET) imaging and the appearance of amyloid-related imaging abnormalities (ARIA). To better understand the relationship between ARIA and the pathophysiology of AD, we undertook a series of studies in PDAPP mice evaluating vascular alterations in the context of central Aß pathology and after anti-Aß immunotherapy. METHODS: We analyzed PDAPP mice treated with either 3 mg/kg/week of 3D6, the murine form of bapineuzumab, or isotype control antibodies for periods ranging from 1 to 36 weeks and evaluated the vascular alterations in the context of Aß pathology and after anti-Aß immunotherapy. The number of mice in each treatment group ranged from 26 to 39 and a total of 345 animals were analyzed. RESULTS: The central vasculature displayed morphological abnormalities associated with vascular Aß deposits. Treatment with 3D6 antibody induced clearance of vascular Aß that was spatially and temporally associated with a transient increase in microhemorrhage and in capillary Aß deposition. Microhemorrhage resolved over a time period that was associated with a recovery of vascular morphology and a decrease in capillary Aß accumulation. CONCLUSIONS: These data suggest that vascular leakage events, such as microhemorrhage, may be related to the removal of vascular Aß. With continued treatment, this initial susceptibility period is followed by restoration of vascular morphology and reduced vulnerability to further vascular leakage events. The data collectively suggested a vascular amyloid clearance model of ARIA, which accounts for the currently known risk factors for the incidence of ARIA in clinical studies.

Axonopathy in an α-synuclein transgenic model of Lewy body disease is associated with extensive accumulation of C-terminal-truncated α-synuclein.

Progressive accumulation of α-synuclein (α-syn) in limbic and striatonigral systems is associated with the neurodegenerative processes in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). The murine Thy-1 (mThy1)-α-syn transgenic (tg) model recapitulates aspects of degenerative processes associated with α-syn accumulation in these disorders. Given that axonal and synaptic pathologies are important features of DLB and PD, we sought to investigate the extent and characteristics of these alterations in mThy1-α-syn tg mice and to determine the contribution of α-syn c-terminally cleaved at amino acid 122 (CT α-syn) to these abnormalities. We generated a novel polyclonal antibody (SYN105) against the c-terminally truncated sequence (amino acids 121 to 123) of α-syn (CT α-syn) and performed immunocytochemical and ultrastructural analyses in mThy1-α-syn tg mice. We found abundant clusters of dystrophic neurites in layers 2 to 3 of the neocortex, the stratum lacunosum, the dentate gyrus, and cornu ammonis 3 of the hippocampus, striatum, thalamus, midbrain, and pons. Dystrophic neurites displayed intense immunoreactivity detected with the SYN105 antibody. Double-labeling studies with antibodies to phosphorylated neurofilaments confirmed the axonal location of full-length and CT α-syn. α-Syn immunoreactive dystrophic neurites contained numerous electrodense laminated structures. These results show that neuritic dystrophy is a prominent pathologic feature of the mThy1-α-syn tg model and suggest that CT α-syn might play an important role in the process of axonal damage in these mice as well as in DLB and PD.

Sustained levels of antibodies against Aß in amyloid-rich regions of the CNS following intravenous dosing in human APP transgenic mice.

Passive immunization with anti-Aß antibodies leads to the reduction of AD-like neuropathology in transgenic mice. Previously we showed that anti-Aß antibodies enter the brain and bind to amyloid plaques. Now using (125)I-labeled 3D6, the mouse parent antibody of the clinical candidate bapineuzumab, we further characterized the pharmacokinetic profile of this antibody in the brain and serum. Our studies demonstrated that following a single intravenous injection, the labeled antibody accumulates and persists in plaque rich regions of the brain in transgenic PDAPP mice. Accumulation was specific to amyloid since it did not occur in non-transgenic animals lacking human APP, could not be measured in transgenic animals prior to plaque deposition, and correlated with the level of plaque burden in aging transgenic mice. After a single intravenous injection, CNS levels of (125)I-labeled 3D6 continued to increase for 14 days even as serum levels of the antibody declined. The calculated half-life of antibody in the circulation was 6 days, while antibody levels in the CNS remained stable for nearly a month. When given at supra-therapeutic levels, unlabeled antibody did not compete with tracer levels of labeled antibody for accumulation in the CNS, indicating that the binding capacity of plaques was very high. Our results demonstrate that even when administered in the periphery at very low (tracer) doses, 3D6 and bapineuzumab cross the blood brain barrier to accumulate in plaque rich regions of the brain. CNS clearance is markedly slower than in the serum and correlates with binding to deposited amyloid in a transgenic model of Alzheimer's disease.

Neutralization of soluble, synaptotoxic amyloid ß species by antibodies is epitope specific.

Several anti-amyloid ß (Aß) antibodies are under evaluation for the treatment of Alzheimer's disease (AD). Clinical studies using the N-terminal-directed anti-Aß antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of Aß plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of Aß plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble Aß species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of Aß(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble Aß moieties. Importantly C-terminal anti-Aß antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-Aß antibodies effectively interact with both soluble and insoluble forms of Aß and therefore appear particularly well suited for testing the Aß hypothesis of AD.

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.

Passive immunization reduces behavioral and neuropathological deficits in an alpha-synuclein transgenic model of Lewy body disease.

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB.

Generation and characterization of anti-AA amyloid-specific monoclonal antibodies.

AA amyloidosis results from the pathologic deposition in the kidneys and other organs of fibrils composed of N-terminal fragments of serum amyloid A protein (SAA). Given that there are only limited means to visualize these deposits, we have developed a series of mAbs, 2A4, 7D8, and 8G9, that bind specifically with nanomolar affinity to a carboxy-terminal epitope generated following proteolysis of SAA that yields the predominant component of AA amyloid deposits. Notably, these antibodies do not recognize native SAA, they retain their immunoreactivity when radiolabeled with I-125 and, after injection into AA amyloidotic mice, localize, as evidenced by autoradiography and micro-single photon emission computed tomography imaging, to histologically confirmed areas of amyloid deposition; namely, spleen, liver, and pancreas. The results of our in vitro and in vivo studies demonstrate the AA fibril-selectivity of mAbs 2A4, 7D8, and 8G9 and warrant further investigation into their role as novel diagnostic agents for patients with AA amyloidosis.

The role of animal models in advancing amyloid-beta immunotherapy to the clinic.

The amyloid-beta (Aß) hypothesis of Alzheimer's disease (AD) causality is now well into its third decade and is finally entering a phase of rigorous clinical testing in numerous late stage clinical trials. The use of Aß-based animal models of AD has been essential to the discovery and/or preclinical validation of many of these therapeutic approaches. While several neuropathologically based results from preclinical studies have translated nicely into AD patients, the full clinical value of Aß-directed therapies awaits results from trials now in progress.

Structural correlates of antibodies associated with acute reversal of amyloid beta-related behavioral deficits in a mouse model of Alzheimer disease.

Immunotherapy targeting of amyloid beta (Abeta) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Abeta-targeted immunotherapy has also been demonstrated to reverse Abeta-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Abeta, Abeta(3-7), differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Abeta in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Abeta. The conformation of the Abeta peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Abeta:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Abeta species postulated to underlie cognitive deficits in AD.

Abeta immunotherapy: intracerebral sequestration of Abeta by an anti-Abeta monoclonal antibody 266 with high affinity to soluble Abeta.

Amyloid beta (Abeta) immunotherapy is emerging as a promising disease-modifying therapy for Alzheimer's disease, although the precise mechanisms whereby anti-Abeta antibodies act against amyloid deposition and cognitive deficits remain elusive. To test the "peripheral sink" theory, which postulates that the effects of anti-Abeta antibodies in the systemic circulation are to promote the Abeta efflux from brain to blood, we studied the clearance of (125)I-Abeta(1-40) microinjected into mouse brains after intraperitoneal administration of an anti-Abeta monoclonal antibody 266. (125)I-Abeta(1-40) was rapidly eliminated from brains with a half-life of approximately 30 min in control mice, whereas 266 significantly retarded the elimination of Abeta, presumably due to formation of Abeta-antibody complex in brains. Administration of 266 to APP transgenic mice increased the levels of monomer Abeta species in an antibody-bound form, without affecting that of total Abeta. We propose a novel mechanism of Abeta immunotherapy by the class of anti-Abeta antibodies that preferentially bind soluble Abeta, i.e., intracerebral, rather than peripheral, sequestration of soluble, monomer form of Abeta, thereby preventing the accumulation of multimeric toxic Abeta species in brains.

Immunotherapy reduces vascular amyloid-beta in PDAPP mice.

In addition to parenchymal amyloid-beta (Abeta) plaques, Alzheimer's disease (AD) is characterized by Abeta in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in the majority of patients. Recent studies investigating vascular Abeta (VAbeta) in amyloid precursor protein transgenic mice have suggested that passive immunization with anti-Abeta antibodies may clear parenchymal amyloid but increase VAbeta and the incidence of microhemorrhage. However, the influences of antibody specificity and exposure levels on VAbeta and microhemorrhage rates have not been well established, nor has any clear causal relationship been identified. This report examines the effects of chronic, passive immunization on VAbeta and microhemorrhage in PDAPP mice by comparing antibodies with different Abeta epitopes (3D6, Abeta(1-5); 266, Abeta(16-23)) and performing a 3D6 dose-response study. VAbeta and microhemorrhage were assessed using concomitant Abeta immunohistochemistry and hemosiderin detection. 3D6 prevented or cleared VAbeta in a dose-dependent manner, whereas 266 was without effect. Essentially complete absence of VAbeta was observed at the highest 3D6 dose, whereas altered morphology suggestive of ongoing clearance was seen at lower doses. The incidence of microhemorrhage was increased in the high-dose 3D6 group and limited to focal, perivascular sites. These colocalized with Abeta deposits having altered morphology and apparent clearance in the lower-dose 3D6 group. Our results suggest that passive immunization can reduce VAbeta levels, and modulating antibody dose can significantly mitigate the incidence of microhemorrhage while still preventing or reducing VAbeta. These observations raise the possibility that Abeta immunotherapy can potentially slow or halt the course of CAA development in AD that is implicated in vascular dysfunction.

Progress in the active immunotherapeutic approach to Alzheimer's disease: clinical investigations into AN1792-associated meningoencephalitis.

BACKGROUND: In a phase 2a clinical trial of AN1792 (Study 201), a potential immunotherapeutic agent for use in Alzheimer's disease (AD), approximately 6% of the treated AD patients (18/300) developed meningoencephalitis (ME). OBJECTIVE: To elucidate potential immune mechanisms of treatment-induced ME. METHODS: Peripheral blood mononuclear cells obtained from patients who received AN1792 were stimulated in vitro either with beta-amyloid (Abeta) or various overlapping peptides of Abeta(1-42), followed by quantification of cytokine-secreting cells by enzyme-linked immunosorbent spot assay. RESULTS: A significant difference in the quality of the T-cell responses between patients in Study 201 and those in earlier studies of AN1792 was noted. T-cell responses specific to the carboxy terminus of Abeta elicited from patients' peripheral blood mononuclear cells in an earlier multiple dose study (Study 102) were Th2 biased, while those from Study 201 were biased toward a proinflammatory Th1 response. Antibody responses in both studies were quantitatively and qualitatively similar (as determined by epitope mapping). The addition of polysorbate 80 to the formulation used in Study 201 is the most likely explanation for the difference in the T-cell response. CONCLUSION: ME following injection of AN1792 may be related to immune response differences driven by a formulation change. To address this, a novel peptide-carrier protein conjugate using an amino-terminal fragment of Abeta (ACC-001) has been developed to avoid potentially harmful T-cell responses, while maintaining a similar antibody response to that of AN1792. Immunotherapeutic trials using this treatment approach in AD patients are in progress.

Red blood cells are the major source of alpha-synuclein in blood.

BACKGROUND: Alpha-synuclein has been directly linked to Parkinson's disease etiology by mutations in and multiplication of its gene that result in a familial form of Parkinson's disease. Alpha-synuclein has been detected in blood, and was found to be elevated in the blood of those individuals with the alpha-synuclein gene multiplication. OBJECTIVE: A complete analysis of the level of alpha-synuclein in blood has not been performed. In this report, we determine the quantitative distribution of alpha-synuclein in the plasma and different cellular fractions of human blood. The levels of alpha-synuclein in human and mouse blood are compared. METHODS: Alpha-synuclein levels in the different fractions of blood were quantified by a sandwich ELISA with purified recombinant alpha-synuclein as an assay standard. Samples were further characterized by Western immunoblot analysis. RESULTS: More than 99% of the alpha-synuclein resides in the red blood cells (RBCs) with less than 1% of the total detected in the plasma, platelets and peripheral blood mononuclear cells. CONCLUSIONS: More than 99% of the alpha-synuclein in human blood is present in the peripheral blood cells, with the remainder in plasma. Fractionation of peripheral blood cells from human blood and quantification of alpha-synuclein revealed that only a very small amount of the total alpha-synuclein is present in peripheral blood mononuclear cells, and platelets, with the majority of alpha-synuclein in blood being present in RBCs. Considering the abundance and fragility of RBCs, alpha-synuclein levels in these other blood fractions or other bodily fluids such as cerebrospinal fluid may be artificially elevated by contamination with intact or lysed RBCs.

Antibody capture of soluble Abeta does not reduce cortical Abeta amyloidosis in the PDAPP mouse.

BACKGROUND: In vivo administration of antibodies against the amyloid-beta (Abeta) peptide has been shown to reduce and reverse the progressive amyloidosis that develops in a variety of mouse models of Alzheimer's disease (AD). This work has been extended to clinical trials where subsequent autopsy cases of AD subjects immunized against Abeta showed similar reductions in parenchymal amyloid plaques, suggesting this approach to reduce neuropathology in man is feasible. OBJECTIVE: Multiple hypotheses have been advanced to explain how anti-Abeta antibodies may lower amyloid burden. In this report, we compare approaches utilizing either plaque-binding or peptide-capturing anti-Abeta antibodies for effectiveness in reducing amyloidosis in a mouse model of AD. METHODS: A plaque-binding monoclonal antibody (3D6) and an Abeta peptide-capturing monoclonal antibody (266) were compared in chronic treatment and prevention paradigms using a transgenic mouse model of AD. The effects of antibody therapy on plaque burden and plasma clearance of Abeta were investigated by quantitative imaging and clearance studies of intravenously injected (125)I-Abeta. RESULTS: The plaque-binding antibody 3D6 was highly effective in either treatment or prevention of amyloidosis. In these studies, the peptide-capture antibody 266 showed no reduction in amyloidosis in either paradigm and showed trends towards increasing amyloidosis. Antibody 266 was also found to greatly prolong (>180-fold) the normally rapid peripheral clearance of Abeta, in contrast to that found with 3D6 (>24-fold). CONCLUSION: Reversing and preventing Alzheimer's type amyloidosis is most effectively accomplished with anti-amyloid antibodies that avidly bind plaque.

Distribution of Abeta peptide in whole blood.

The measurement of amyloid beta peptides (Abeta) in blood and plasma is expected to be a useful biomarker as potential therapeutics designed to lower Abeta peptide enter clinical trials. Many reports have suggested that Abeta could bind to substances in blood that may influence the recovery of Abeta peptide in plasma, its detection by conventional ELISAs or the actual turnover and half-life of the peptide in blood. In this study we describe a process for analyzing total Abeta in whole blood and plasma using denaturing solid-phase extraction followed by reverse-phase HPLC linked to ELISA. Comparison of total Abeta peptide levels in whole blood and plasma from the same bleed showed that most of the Abeta peptide is captured in the plasma if the samples are first denatured. In contrast, plasma that was assayed without denaturation could show greater than 70% reduction in apparent total Abeta peptide. This suggested that there was a pool of Abeta peptide in non-denatured plasma that is occluded from detection by ELISA, perhaps by binding to plasma proteins.

Effect of statins on Alzheimer's disease biomarkers in cerebrospinal fluid.

BACKGROUND: Treatment with HMG-CoA reductase inhibitors ("statins") has been variably associated with a reduced risk of Alzheimer's disease (AD) in epidemiologic studies and reduced amyloid-beta (Abeta) deposition in animal models of AD. Putative neuroprotective effects of statins may vary in relation to their ability to penetrate into the central nervous system (CNS). METHODS: We measured levels of cerebrospinal fluid (CSF) AD biomarkers following 14 weeks of treatment with simvastatin (a CNS permeant statin; n=10) at 40 mg/day or pravastatin (a CNS impermeant statin; n=13) at 80 mg/day in hypercholesterolemic subjects without dementia. RESULTS: Simvastatin, but not pravastatin, reduced CSF levels of phospho-tau-181 (p-tau181) in all subjects. There were no differences in CSF levels of total tau, Abeta42, Abeta40, soluble amyloid beta protein precursor (sAbetaPP) alpha or beta, or F2-isoprostanes. CONCLUSIONS: Statins may modulate the phosphorylation of tau in humans and this effect may depend on the CNS availability of the statin. These results suggest another mechanism by which statins may act to reduce the risk of AD.