Intracerebroventricular passive immunization with anti-Abeta antibody in Tg2576

Current Alzheimer's disease (AD) research has established the fact that excessive genesis of Abeta derived from amyloidogenic processing of beta-amyloid (Abeta) precursor protein is fundamental to AD pathogenesis. There has been considerable interest in using immunization strategies for clearing excessive Abeta. Studies in animal models of AD have shown that active immunizations or systemic passive immunizations reduced cerebral plaque load and improved behavioral deficits. However, clinical translation of an active immunization strategy was interrupted because of evidence for meningoencephalitis produced in some patients who received Abeta vaccine. Studies in animal models have shown perimicrovascular hemorrhages and inflammation after sustained systemic immunizations in animals with vascular amyloid. In this light, our data showing the effects of a single intracerebroventricular (ICV) injection of anti-Abeta in the Alzheimer's Swedish mutant model Tg2576 are intriguing. We have previously demonstrated that a single ICV injection of anti-Abeta into the third ventricle of 10-month-old Tg2576 mice reduced cerebral plaques, reversed Abeta-induced depletion of presynaptic SNAP-25, and abolished astroglial activation as seen 1 month post-injection (Chauhan and Siegel [2002] J. Neurosci. Res. 69:10-23). The present report demonstrates that a single ICV injection of 10 microg anti-Abeta in 10-month-old Tg2576 mice reduced cerebral plaques, with decreased inflammation at this stage as evidenced by a reduced number of interleukin-1beta-positive microglia surrounding Congophilic plaques. Moreover, at this particular age, no microhemorrhage was discernible, as evidenced by the absence of hemosiderin deposition after a single ICV injection of anti-Abeta. This is the first report demonstrating absence of microhemorrhage and reduced inflammation after the ICV introduction of anti-Abeta in Tg2576 mice at 10 months of age. These facts indicate that, although invasive, ICV injection of anti-Abeta may be a safer method of vaccination in AD, possibly through reducing the vascular exposure to antibody. Further studies are warranted to determine the lasting effects of a single ICV anti-Abeta injection in animals with and without abundant plaque burden and at older ages.     

Intracerebroventricular passive immunization with anti-oligoAbeta antibody in TgCRND8

Based on the central dogma of beta-amyloid (Abeta) as a key seeding event in the pathogenesis of Alzheimer disease (AD), immunoneutralization strategies have been actively pursued both in AD and in models of AD as a potential means for treating AD. Both active and passive immunizations targeted at fibrillar Abeta successfully remove cerebral plaque load and attenuate Abeta-induced toxicity. Consistently with this, intracerebroventricular (ICV) passive immunization established in our laboratory using antibody against fibrillar Abeta (anti-fAbeta) reduced cerebral plaque load and reversed early synaptic deficits at pre/early plaque stage when there is an abundance of soluble dimeric/oligomeric Abeta but sparse fibrillar Abeta, indicating that anti-fAbeta-mediated partial neutralization of toxic oligomeric Abeta species might have reduced early synaptotoxicity. In the previous investigation, we found that immunoneutralization with anti-fAbeta transiently reduced cerebral Abeta and associated toxicity. The current investigation tested whether ICV im munization using antibody to conformationally changed oligomeric Abeta (anti-oligoAbeta) will overcome the transient restorative nature of anti-fAbeta and produce persistent, long-lasting preventive effects. Because oligomeric Abeta is strongly correlated with synaptotoxicity, we investigated whether immunoneutralization of oligomeric Abeta will reverse synaptic deficits by analyzing presynaptic molecular marker (SNAP-25) profile within hippocampal dendritic fields, where SNAP-25 is abundantly expressed. Results show that, in contrast to ICV anti-fAbeta antibody, ICV anti-oligoAbeta antibody significantly prevented cerebral Abeta build and almost completely restored SNAP-25 immunoreaction up to 8 weeks postinjection in TgCRND8 brain. Results show that ICV passive immunization with anti-oligoAbeta antibody might be an improved ICV immunization strategy for preventing permanent structural damage in AD.     

Microglial activation facilitates Abeta plaque removal following intracranial anti-Abeta antibody administration

The mechanisms by which anti-Abeta antibodies clear amyloid plaques in Abeta depositing transgenic mice are unclear. In the current study, we demonstrate that inhibition of anti-Abeta antibody-induced microglial activation with anti-inflammatory drugs, such as dexamethasone, inhibits removal of fibrillar amyloid deposits. We also show that anti-Abeta F(ab')(2) fragments fail to activate microglia and are less efficient in removing fibrillar amyloid than the corresponding complete IgG. Diffuse Abeta deposits are cleared by antibodies under all circumstances. These data suggest that microglial activation is necessary for efficient removal of compact amyloid deposits with immunotherapy. Inhibition of this activation may result in an impaired clinical response to vaccination against Abeta.     

Viral-induced inflammation is accompanied by beta-amyloid plaque reduction in brains of amyloid precursor protein transgenic Tg2576 mice

Amyloid plaques, one of the neuropathological hallmarks of Alzheimer's disease, and their main constituent, the amyloid beta-peptide (Abeta), are triggers of the activation of innate inflammatory mechanisms involving the activation of microglia. To dissect the effects of a non-Abeta-specific microglial activation on the Abeta metabolism, we employed a viral infection-based model. Transgenic mice expressing a mutated form of the human amyloid precursor protein (Tg2576) were used. In preceding experiments, 2-week-old transgenic mice and non-transgenic littermates were infected intracerebrally with the neurotropic Borna disease virus and investigated at 2, 4 and 14 weeks post-infection. The Borna disease virus-inoculated mice showed a persisting, subclinical infection of cortical and limbic brain areas characterized by slight T-cell infiltrates, expression of cytokines and a massive microglial activation in the hippocampus and neocortex. Viral-induced effects reached their peak at 4 weeks post-infection. In 14-month-old Tg2576 mice, characterized by the deposition of diffuse and dense-core amyloid plaques in cortical brain regions, Borna disease virus-induced microglial activation in the vicinity of Abeta deposits was used to investigate the influence of a local inflammatory response on these deposits. At 4 weeks post-infection, histometric analyses employing Abeta immunohistochemistry revealed a decrease of the cortical and hippocampal Abeta-immunopositive area. This overall decrease was accompanied by a decrease of parenchymal thioflavin-S-positive amyloid deposits and an increase of such deposits in the walls of cerebral vessels, which indicates that the elicitation of a non-Abeta-specific microglial activation may contribute to a reduction of Abeta in the brain parenchyma.     

A limited role for microglia in antibody mediated plaque clearance in APP mice

Amyloid-beta (Abeta) accumulation in senile plaques is a hallmark of Alzheimer's disease (AD). Immunotherapy is a leading approach for amyloid clearance, despite the early termination of the Elan clinical trial with active immunization due to a few cases of meningoencephalitis. The mechanisms of immunotherapy-mediated amyloid clearance and this deleterious side effect are largely unknown. While clearance of Abeta probably results in part from microglia-mediated inflammation, it can be microglia independent. Therefore, establishing the role of microglia in Abeta clearance is important for the treatment of AD. We analyzed the effects of direct microglia activation and inhibition on antibody-mediated Abeta clearance. Robust microglia activation with interferon-gamma led to modest Abeta clearance alone but did not potentiate antibody-mediated clearance. Microglia elimination/inactivation with immunotoxin or minocycline only partially limited antibody-induced Abeta clearance suggesting that although there is a role for microglia in Abeta clearance, it does not account for the majority of the effect observed after anti-Abeta antibody treatment.     

Anti-Abeta single-chain antibody delivery via adeno-associated virus for treatment of Alzheimer's disease

Immunization of mouse models of Alzheimer disease (AD) with amyloid-peptide (Abeta) reduces Abeta deposits and attenuates their memory and learning deficits. Recent clinical trials were halted due to meningoencephalitis, presumably induced by T cell mediated and/or Fc-mediated immune responses. Because injection of anti-Abeta F(ab')(2) antibodies also induces clearance of amyloid plaques in AD mouse models, we have tested a novel gene therapy modality where an adeno-associated virus (AAV) encoding anti-Abeta single-chain antibody (scFv) is injected into the corticohippocampal regions of AD mouse models. One year after injection, expression of scFv was readily detectable in the neurons of the hippocampus without discernible neurotoxicity. AD mouse models subjected to AAV injection had much less amyloid deposits at the injection sites than the mouse models subjected to PBS injection. Because the scFv lacks the Fc portion of the immunoglobulin molecule, this modality may be a feasible solution for AD without eliciting inflammation.     

Antibodies against beta-amyloid reduce Abeta oligomers, glycogen synthase kinase-3beta activation and tau phosphorylation in vivo and in vitro

Although active and passive immunization against the beta-amyloid peptide (Abeta) of amyloid plaque-bearing transgenic mice markedly reduces amyloid plaque deposition and improves cognition, the mechanisms of neuroprotection and impact on toxic oligomer species are not understood. We demonstrate that compared to control IgG2b, passive immunization with intracerebroventricular (icv) anti-Abeta (1-15) antibody into the AD HuAPPsw (Tg2576) transgenic mouse model reduced specific oligomeric forms of Abeta, including the dodecamers that correlate with cognitive decline. Interestingly, the reduction of soluble Abeta oligomers, but not insoluble Abeta, significantly correlated with reduced tau phosphorylation by glycogen synthase kinase-3beta (GSK-3beta), a major tau kinase implicated previously in mediating Abeta toxicity. A conformationally-directed antibody against amyloid oligomers (larger than tetramer) also reduced Abeta oligomer-induced activation of GSK3beta and protected human neuronal SH-SY5Y cells from Abeta oligomer-induced neurotoxicity, supporting a role for Abeta oligomers in human tau kinase activation. These data suggest that antibodies that are highly specific for toxic oligomer subspecies may reduce toxicity via reduction of GSK-3beta, which could be an important strategy for Alzheimer's disease (AD) therapeutics.     

Minocycline reduces engraftment and activation of bone marrow-derived cells but sustains their phagocytic activity in a mouse model of Alzheimer's disease

Bone marrow (BM)-derived monocytes contribute to the development of microglial reaction around beta-amyloid (Abeta) plaques in Alzheimer's disease (AD) and possibly clear Abeta. Therefore, it is of great importance to separate the proinflammatory actions of monocytic cells from Abeta phagocytic effects. We used minocycline (mino) to systemically downregulate microglial activation and studied proliferation, expression of markers for activated microglia, and Abeta removal in vitro and in vivo. Mino did not affect proliferation or phagocytic activity of BM-derived cells toward Abeta in vitro. Intrahippocampal LPS injection used to induce inflammation and increase recruitment of BM cells from periphery, reduced Abeta burden in BM-transplanted AD transgenic mice. All engrafted cells expressed CD45, approximately 50% expressed Iba-1, and <0.5% of these cells expressed CD3e. About 40% of the engrafted cells were mitotically active. LPS increased immunoreactivity for Iba-1, MHC II, a marker of antigen presenting cells, and CD68, a marker of lysosomal activity. The endogenous microglia largely contributed to these LPS-induced immunoreactivities. Mino reduced the engraftment of BM-derived cells and blocked the LPS-induced MHC II and Iba-1 immunoreactivities, but did not prevent the increased CD68-immunoreactivity or the reduced Abeta burden. Importantly, mino did not block the association of eGFP-positive cells with Abeta deposits and the percentage of mitotically active BM-derived cells. In conclusion, mino reduces overall inflammatory potential of BM-derived monocytic cells without preventing their phagocytic activity. The separation of harmful activation of microglia/monocytic cells from their Abeta clearing mechanism may hold important therapeutic potential.     

Minocycline affects microglia activation, Abeta deposition, and behavior in APP-tg mice

Activated microglia and reactive astrocytes invade and surround cerebral beta amyloid (Abeta) plaques in Alzheimer's disease (AD), but the role of microglia in plaque development is still unclear. In this study, minocycline was administered for 3 months, prior to and early in Abeta plaque formation in amyloid precursor protein transgenic mice (APP-tg). When minocycline was given to younger mice, there was a small but significant increase in Abeta deposition in the hippocampus, concurrent with improved cognitive performance relative to vehicle treated mice. If APP-tg mice received minocycline after Abeta deposition had begun, microglial activation was suppressed but this did not affect Abeta deposition or improve cognitive performance. In vitro studies demonstrated that minocycline suppressed microglial production of IL-1beta, IL-6, TNF, and NGF. Thus, minocycline has different effects on Abeta plaque deposition and microglia activation depending on the age of administration. Our data suggest that this may be due to the effects of minocycline on microglial function. Therefore, anti-inflammatory therapies to suppress microglial activation or function may reduce cytokine production but enhance Abeta plaque formation early in AD.     

Abeta42 gene vaccination reduces brain amyloid plaque burden in transgenic mice

OBJECTIVE: To demonstrate that in APPswe/PS1DeltaE9 transgenic mice, gene gun mediated Abeta42 gene vaccination elicits a high titer of anti-Abeta42 antibodies causal of a significant reduction of Abeta42 deposition in brain. METHODS: Gene gun immunization is conducted with transgenic mice using the Abeta42 gene in a bacterial plasmid with the pSP72-E3L-Abeta42 construct. Enzyme-linked immunoabsorbent assays (ELISA) and Western blots are used to monitor anti-Abeta42 antibody levels in serum and Abeta42 levels in brain tissues. Enzyme-linked immunospot (ELISPOT) assays are used for detection of peripheral blood T cells to release gamma-interferon. Immunofluorescence detection of Abeta42 plaques and quantification of amyloid burden of brain tissue were measured and sections were analyzed with Image J (NIH) software. RESULTS: Gene gun vaccination with the Abeta42 gene resulted in high titers of anti-Abeta42 antibody production of the Th2-type. Levels of Abeta42 in treated transgenic mouse brain were reduced by 60-77.5%. The Mann-Whitney U-test P=0.0286. INTERPRETATION: We have developed a gene gun mediated Abeta42 gene vaccination method that is efficient to break host Abeta42 tolerance without using adjuvant and induces a Th2 immune response. Abeta42 gene vaccination significantly reduces the Abeta42 burden of the brain in treated APPswe/PS1DeltaE9 transgenic mice with no overlap between treated and control mice.     

Deglycosylation of anti-beta amyloid antibodies inhibits microglia activation in BV-2 cellular model

Immunotherapy has become a strategy for treatment of Alzheimer's disease, by inducing antibody response to amyloid-beta peptide (AbetaP) or by passive administration of anti-AbetaP antibodies. Clearance of amyloid plaques involves interaction of immunoglobulin Fc receptor (FcR)-expressing microglia and antibodyopsonized Abeta deposits, stimulating phagocytosis but may promote neuroinflammation. Carbohydrate moiety of Fc of the immunoglobulin G molecule plays a significant role in modulating binding to FcR and its effector functions. Here, we enzymatically removed Fc glycan from monoclonal antibody 196 raised against AbetaP Antigen binding ability and in vitro stability of deglycosylated antibody were unaffected by deglycosylation. Moreover, the deglycosylated antibody exhibits low affinity to FcR on microglial BV-2 cells and has limited ability to mediate microglial chemotaxis and antibodydependent cytotoxicity compared to native antibody. These data suggest that deglycosylation of anti-Abeta antibodies before in vivo administration might prevent microglial overactivation, thus reducing the risk of neuroinflammatory response during passive immunization.     

beta-amyloid (Abeta)42(43), abeta42, abeta40 and apoE immunostaining of plaques in fatal head injury

beta-Amyloid (Abeta) deposits are found in the brains of approximately one-third of patients who die within days after a severe head injury; their presence correlating strongly with possession of an apolipoprotein E (apoE)-epsilon4 allele. The aim of the study was to investigate the relationship between Abeta42, Abeta40 and apoE immunostaining of Abeta plaques in the cerebral cortex and the relevance of apoE genotype in 23 fatally head-injured patients. These cases were known to have Abeta deposits from a previous study in which they were examined and semiquantified and related to apoE genotype. In the present study, the temporal cortex was probed using four different antibodies that recognize Abeta42(43), Abeta40 and an antibody to apoE. Abeta42(43)-positive plaques were observed in all of the 23 cases and Abeta40 immunoreactivity in only 11 of the 23 cases. In addition, semiquantitative analysis showed that relatively fewer plaques were detected with anti-Abeta40 than anti-Abeta42(43). ApoE-immunoreactive plaques were identified in 18 of the 23 cases. The number of plaques stained for apoE was relatively less than for Abeta42(43) but greater than for Abeta40. Furthermore, the density of Abeta plaques detected using either Abeta42(43), Abeta40 or apoE antibodies was associated with possession of apoE-epsilon4 in an allele dose-dependent manner. The results are consistent with Abeta42(43) as the initially deposited species in brain parenchyma and provide evidence that apoE is involved in the early stages of amyloid deposition. Further, the findings may be of relevance to the role of apoE genotype in influencing outcome after acute brain injury.     

Distribution of intraventricularly administered antiamyloid-beta peptide (Abeta) antibody in the mouse brain

There is considerable interest in utilizing the intracerebroventricular (icv) route of administration of antibodies in the brain for various studies and for the therapy of malignancies, but very little is known about the anatomic extent of distribution of the antibody in brain after injection into the third ventricle. To explore the potential for icv administration of antiamyloid-beta peptide (Abeta) in reducing Abeta toxicity in brain in Alzheimer's disease, we first mapped the time course and path of transit of horseradish peroxidase (HRP)-labeled antibody. The results show that, after a single injection into the mouse third venticle, the HRP-labeled antibody is localized within the microvasculature, first that of the corticohippocampal region close to the site of injection at 3 hr. By 24 hr, the antibody is distributed throughout the hippocampus and frontoparietal cortex close to the injection site, as well as in the deep and outer cerebral cortex and cerebellar cortex remote from the injection site. The injected antibody is almost entirely removed by 4 days. Therefore, the antibody had diffused throughout all the brain by 24 hr, showing the feasibility of small quantities of anti-Abeta antibody infused into the third ventricle to reach extracellular epitopes throughout the brain parenchyma rapidly.     

UV light-induced autofluorescence of full-length Abeta-protein deposits in the human brain

The formation of amyloid plaques is a hallmark of Alzheimer's disease (AD). Amyloid plaques and vascular amyloid deposits in cerebral amyloid angiopathy (CAA) consist of the beta-amyloid protein (Abeta) in association with other proteins. These Abeta-deposits can be visualized by thioflavin S, Congo red staining, silver staining methods and immunohistochemistry. Senile plaques also have been shown to exhibit blue autofluorescence. Here we report that UV light-induced autofluorescence is restricted to full-length Abeta-containing amyloid plaques and is also seen in blood vessels affected by CAA. Different types of samples from AD and control cortices were examined: native samples, formalin-fixed paraffin and polyethylene glycol-embedded tissue sections. These samples were viewed with a fluorescence microscope under UV light excitation (360 - 370 nm). By emitting blue fluorescence (>420 nm), amyloid plaques and blood vessels affected by CAA were detected in AD and CAA samples. Combination with immunofluorescence against anti-Abeta1-42, anti-Abeta17-24, and anti-Abeta8-17 demonstrated co-localization of the autofluorescent deposits with full-length Abeta containing Abeta-deposits. N-terminal truncated Abeta-deposits, such as the fleecy amyloid, do not exhibit autofluorescence. In doing so, Abeta-autofluorescence is a suitable method for screening native tissue samples for full-length Abeta-deposits. In contradistinction to conventional and immunohistochemical procedures, detection of plaques and CAA by autofluorescence enables the recognition of full-length Abeta-deposits in the human brain without any chemical interaction whatsoever on the part of Abeta.     

Reversal of amyloid beta toxicity in Alzheimer's disease model Tg2576 by intraventricular antiamyloid beta antibody

There are considerable data on synaptic dysfunction in Alzheimer's disease (AD). However, the precise molecular basis for synaptotoxicity in AD is not known. We tested the hypothesis that amyloid beta (Abeta), as produced in Tg2576 mice overexpressing a mutant form of amyloid precursor protein, leads to changes in SNAP-25, a molecule required for Ca-sensitive neurotransmitter vesicle exocytosis. Anti-Abeta antibody was injected into the third ventricle (icv) of 10-month-old Tg2576 mice, preceding formation of plaques. Immunodensity of glial fibrillary acidic protein (GFAP) and SNAP-25 were quantitated in the hippocampus 1 month later. SNAP-25 was reduced by 96% in the inner molecular layer (SMi) of dentate gyrus, by 95% in the hilum, and by 75-76% in stratum lucidum (SL), stratum oriens (SO), and stratum radiatum (SR) of CA1-CA3 of the Tg2576 mice. GFAP was increased by more than 50-fold, specifically within the neuropil of CA1-CA3, and by twofold in portions of fimbria. One injection of 10 microg of anti-Abeta antibody into the third ventricle at 10 months completely prevented or restored changes in GFAP at 11 months of age. The restoration of SNAP-25 by anti-Abeta antibody compared with wild type was 69% in CA1-SO, 93% in CA1-SR, 85% in CA3-SL, 77% in SMi, and 60-73% in hilum. In addition, whereas control injections of saline or IgG produced greatly increased GFAP diffusely in the hippocampus of Tg2576 animals, there was no increase in GFAP after anti-Abeta injection, suggesting a synergistic interaction of nonspecific trauma with Abeta in the transgenic mice. This is the first report of depleted SNAP-25 immunoreactivity in Tg models and the first report of icv injection of anti-Abeta antibody in this model of AD. The largest reductions of the SNAP-25 are in hilum and SMi, so either reduction in the septal-hilum-SMi path is primary or reduction in this path begins at an earlier age than in CA3-CA1 fields. A single icv injection of anti-Abeta antibody is potent in reversing Abeta effects and, therefore, represents a suitable model for investigating early Abeta toxicity. In addition, intrathecal or icv antibody may be an efficient means of treating or preventing toxicity in AD, particularly under conditions of immune hyporesponsivity.     

Deglycosylated anti-amyloid beta antibodies reduce microglial phagocytosis and cytokine production while retaining the capacity to induce amyloid beta sequestration

Accumulation of amyloid beta (Abeta) is a pathological hallmark of Alzheimer's disease, and lowering Abeta is a promising therapeutic approach. Intact anti-Abeta antibodies reduce brain Abeta through two pathways: enhanced microglial phagocytosis and Abeta transfer from the brain to the periphery (Abeta sequestration). While activation of microglia, which is essential for microglial phagocytosis, is necessarily accompanied by undesired neuroinflammatory events, the capacity for sequestration does not seem to be linked to such effects. We and other groups have found that simple Abeta binding agents are sufficient to reduce brain Abeta through the sequestration pathway. In this study, we aimed to eliminate potentially deleterious immune activation from antibodies without affecting the ability to induce sequestration. The glycan portion of immunoglobulin is critically involved in interactions with immune effectors including the Fc receptor and complement c1q; deglycosylation eliminates these interactions, while antigen (Abeta)-binding affinity is maintained. In this study, we investigated whether deglycosylated anti-Abeta antibodies reduce microglial phagocytosis and neuroinflammation without altering the capacity to induce Abeta sequestration. Deglycosylated antibodies maintained Abeta binding affinity. Deglycosylated antibodies did not enhance Abeta phagocytosis or cytokine release in primary cultured microglia, whereas intact antibodies did so significantly. Intravenous injection of deglycosylated antibodies elevated plasma Abeta levels and induced Abeta sequestration to a similar or greater degree compared with intact antibodies in an Alzheimer's transgenic mouse model without or with Abeta plaque pathology. We conclude that deglycosylated antibodies effectively induced Abeta sequestration without provoking neuroinflammation; thus, these deglycosylated antibodies may be optimal for sequestration therapy for Alzheimer's disease.     

Learning deficits and dysfunctional synaptic plasticity induced by aggregated amyloid deposits in the dentate gyrus are rescued by chronic treatment with indomethacin

The amyloid pathology in Alzheimer's disease is accompanied by a chronic inflammatory response characterized by gliosis and activated microglial cells surrounding senile plaques. Epidemiological studies have shown nonsteroidal anti-inflammatory drug treatment reduces the risk of Alzheimer's disease. We have previously shown that injection of a combination of Abeta40 and Abeta43 in the dentate gyrus of the rat induces aggregated amyloid deposits and inflammation associated with dysfunctional synaptic plasticity and learning deficits. Here we characterize the effectiveness of nonsteroidal anti-inflammatory treatment in this model and show that this treatment restores the working memory deficit and decremental long-term potentiation in the dentate gyrus. Importantly, we observe no qualitative difference in the presence of aggregated material but a substantial reduction in microglial-induced inflammation, suggesting that mature aggregated plaques may not be directly responsible for the deficits but may trigger an inflammatory response which has a detrimental effect on synaptic function and memory.     

CD40L disruption enhances Abeta vaccine-mediated reduction of cerebral amyloidosis while minimizing cerebral amyloid angiopathy and inflammation

Amyloid-beta (Abeta) immunization efficiently reduces amyloid plaque load and memory impairment in transgenic mouse models of Alzheimer's disease (AD). Active Abeta immunization has also yielded favorable results in a subset of AD patients. However, a small percentage of patients developed severe aseptic meningoencephalitis associated with brain inflammation and infiltration of T-cells. We have shown that blocking the CD40-CD40 ligand (L) interaction mitigates Abeta-induced inflammatory responses and enhances Abeta clearance. Here, we utilized genetic and pharmacologic approaches to test whether CD40-CD40L blockade could enhance the efficacy of Abeta(1-42) immunization, while limiting potentially damaging inflammatory responses. We show that genetic or pharmacologic interruption of the CD40-CD40L interaction enhanced Abeta(1-42) immunization efficacy to reduce cerebral amyloidosis in the PSAPP and Tg2576 mouse models of AD. Potentially deleterious pro-inflammatory immune responses, cerebral amyloid angiopathy (CAA) and cerebral microhemorrhage were reduced or absent in these combined approaches. Pharmacologic blockade of CD40L decreased T-cell neurotoxicity to Abeta-producing neurons. Further reduction of cerebral amyloidosis in Abeta-immunized PSAPP mice completely deficient for CD40 occurred in the absence of Abeta immunoglobulin G (IgG) antibodies or efflux of Abeta from brain to blood, but was rather correlated with anti-inflammatory cytokine profiles and reduced plasma soluble CD40L. These results suggest CD40-CD40L blockade promotes anti-inflammatory cellular immune responses, likely resulting in promotion of microglial phagocytic activity and Abeta clearance without generation of neurotoxic Abeta-reactive T-cells. Thus, combined approaches of Abeta immunotherapy and CD40-CD40L blockade may provide for a safer and more effective Abeta vaccine.     

Active immunization of mice with an Abeta-Hsp70 vaccine

Heat-shock proteins are highly immunogenic. Complexed with an antigen, they act as adjuvants, inducing a humoral and cellular immune response against both the antigen and the chaperone. In this study, we produced an Hsp70-supported vaccine to induce the generation of antibodies against amyloid-beta (Abeta) peptides, the major constituent of beta-amyloid plaques in Alzheimer's disease. The vaccine consisted of synthetic human Abeta42 covalently cross-linked with DnaK, an Hsp70 homolog of Escherichia coli. Active immunization of mice with this vaccine resulted in the generation of antibodies against Abeta, that were detectable in sera after the first booster immunization. Antibody titers varied markedly with the genetic background of the mice. Prophylactic short-term immunization of transgenic mice (APP tg2576) before the onset of plaques, however, did not prevent amyloid plaque deposition. There were no differences in the plaque load and in the level of Triton X-100-soluble Abeta peptides in the brains of immunized and control-treated transgenic mice. Unexpectedly, the level of formic-acid soluble Abeta peptides tended to be higher in immunized mice. The reason for the increase may be an enhanced deposition of Abeta in the small cerebral blood vessels. These data emphasize the need for anti-Abeta antibodies that remove Abeta peptides from the central nervous system without negative side effects.     

Anti-Abeta: The good, the bad, and the unforeseen

Alzheimer's disease (AD) is characterized in part by the deposition of amyloid beta protein (Abeta) in compact fibrillar plaques. These structures can induce an innate immune response in the brain, which triggers progressive inflammation, neuronal loss, and further acceleration of Abeta plaque formation. Compared with the case in normal individuals, the T and B lymphocytes in AD patients and murine models are hyporesponsive to Abeta. However, depending on the route of delivery, tolerance can be overcome by vaccination, with the induction of an anti-Abeta-mediated immune response. Through mechanisms that are incompletely understood, immunized APP transgenic animals show markedly reduced Abeta deposition, preservation of normal neuronal architecture, and improved performance in memory and spatial learning tasks. In human trials, Abeta vaccination stabilized cognition and slowed the progression of dementia. Neuropathologic examination of a vaccinated subject showed reduced cortical Abeta without changes in other AD-associated pathology. However, in some patients, vaccination induced severe meningoencephalitis, causing the trial to be terminated. Thus, vaccination appears to activate both beneficial and deleterious anti-Abeta immunity, suggesting that the vaccine can have potent clinical utility if an appropriate immunologic response can be generated.