Antibodies from a DNA peptide vaccination decrease the brain amyloid burden in a mouse model of Alzheimer’s disease

The neuropathology of Alzheimer's disease(AD) is characterized by the accumulation of amyloid peptide Abeta in the brain derived from proteolytic cleavage of the amyloid precursor protein (APP). Vaccination of mice with plasmid DNA coding for the human Abeta42 peptide together with low doses of preaggregated peptide induced antibodies with detectable titers after only 2 weeks. One serum was directed against the four aminoterminal amino acids DAEF and differs from previously described ones. Both immune sera and monoclonal antibodies solubilized preformed aggregates of Abeta42 in vitro and recognized amyloid plaques in brain sections of mice transgenic for human APP. Passive immunization of transgenic AD mice caused a significant and rapid reduction in brain amyloid plaques within 24 h. The combined DNA peptide vaccine may prove useful for active immunization with few inoculations and low peptide dose which may prevent the recently described inflammatory reactions inpatients. The monoclonal antibodies are applicable for passive immunization studies and may lead to a therapy of AD.     

Amyloid-beta vaccination, but not nitro-nonsteroidal anti-inflammatory drug treatment, increases vascular amyloid and microhemorrhage while both reduce parenchymal amyloid

Vaccination with Abeta(1-42) and treatment with NCX-2216, a novel nitric oxide releasing flurbiprofen derivative, have each been shown separately to reduce amyloid deposition in transgenic mice and have been suggested as potential therapies for Alzheimer's disease. In the current study we treated doubly transgenic amyloid precursor protein and presenilin-1 (APP+PS1) mice with Abeta(1-42) vaccination, NCX-2216 or both drugs simultaneously for 9 months. We found that all treatments reduced amyloid deposition, both compact and diffuse, to the same extent while only vaccinated animals, with or without nonsteroidal anti-inflammatory drug (NSAID) treatment, showed increased microglial activation associated with the remaining amyloid deposits. We also found that active Abeta vaccination resulted in significantly increased cerebral amyloid angiopathy and associated microhemorrhages, while NCX-2216 did not, in spite of similar reductions in parenchymal amyloid. Co-administration of NCX-2216 did not attenuate this effect of the vaccine. This is the first report showing that active immunization can result in increased vascular amyloid and microhemorrhage, as has been observed with passive immunization. Co-administration of an NSAID agent with Abeta vaccination does not substantially modify the effects of Abeta immunotherapy. The difference between these treatments with respect to vascular amyloid development may reflect the clearance-promoting actions of the vaccine as opposed to the production-modifying effects proposed for flurbiprofen.     

Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies

The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.     

Reduced effectiveness of Abeta1-42 immunization in APP transgenic mice with significant amyloid deposition.

Vaccinations with Abeta1-42 have been shown to reduce amyloid burden in transgenic models of Alzheimer's disease (AD). We have further tested the efficacy of Abeta1-42 immunization in the Tg2576 mouse model of AD by immunizing one group of mice with minimal Abeta deposition, one group of mice with modest Abeta deposition, and one group with significant Abeta deposition. The effects of immunization on Abeta deposition were examined using biochemical and immunohistochemical methods. In Tg2576 mice immunized prior to significant amyloid deposition, Abeta1-42 immunization was highly effective. Biochemically extracted Abeta40 and Abeta42 levels were significantly reduced and immunohistochemical plaque load was also reduced. Immunization of mice with modest amounts of pre-existing Abeta deposits selectively reduced Abeta42 without altering Abeta40, although plaque load was reduced. In contrast, in Tg2576 mice with significant pre-existing Abeta loads, Abeta1-42 immunization only minimally decreased Abeta42 levels, whereas no alteration in Abeta40 levels or in plaque load was observed. These results indicate that in Tg2576 mice, Abeta1-42 immunization is more effective at preventing additional Abeta accumulation and does not result in significant clearance of pre-existing Abeta deposits.     

Intracerebroventricular passive immunization in transgenic mouse models of Alzheimer's disease

Since uncontrolled production of beta-amyloid (Abeta) is considered a key seeding event underlying progression of Alzheimer's disease (AD), elimination of excessive Abeta and preventing its reaccumulation constitute the primary therapeutic goal in preventing and treating AD. To date, immunoneutralization has been the most effective strategy in removing pre-existing cerebral Abeta. Both active and systemic passive immunizations are known to reduce cerebral Abeta and improve memory in transgenic murine models of AD. However, active immunization is associated with adverse effects such as encephalitis with perivascular inflammation and hemorrhage, while passive immunization has the potential to disrupt cerebral vasculature that is laden with amyloid and exposed to high levels of antibody in the blood. Intriguingly, intracerebroventricular passive immunization established in the authors' laboratory circumvented these problems. The authors demonstrated that a single intracerebroventricular injection of anti-Abeta antibody reduced the cerebral Abeta burden and Abeta-related astrocytosis, retarded reaccumulation of Abeta and restored Abeta-induced depletion of presynaptic SNAP-25, for at least 1 month and reduced inflammatory reactions for 1 week in AD murine models without producing inflammation, microhemorrhage or systemic histotoxicity. These facts suggest that intracerebroventricular anti-Abeta may be a safe method for the rapid clearance of pre-existing Abeta and retarding reaccumulation of Abeta in AD. Intracerebroventricular administration via a catheter and reservoir, may be combined with the development of humanized monoclonal antibody against Abeta. Intraventricular shunts and ventriculostomy are frequently employed with acceptable risk-to-benefit ratios in the treatment of various brain disorders, while humanized antibodies are currently used in clinical trials of brain diseases such as multiple sclerosis and lymphoma.     

Generation and characterization of the humoral immune response to DNA immunization with a chimeric beta-amyloid-interleukin-4 minigene

Active immunization with fibrillar beta-amyloid peptide (Abeta(42)) as well as passive transfer of anti-Abeta antibodies significantly reduces Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brain of mutant amyloid precursor protein (APP)-transgenic mice. Although the mechanism(s) of clearance of Abeta from the brain following active or passive immunization remains to be determined, it is clear that anti-Abeta antibodies are critical for clearance. DNA immunization provides an attractive alternative to direct peptide and adjuvant approaches for inducing a humoral response to Abeta. We constructed a DNA minigene with Abeta fused to mouse interleukin-4 (pAbeta(42)-IL-4) as a molecular adjuvant to generate anti-Abeta antibodies and enhance the Th2-type of immune responses. Gene gun immunizations induced primarily IgG1 and IgG2b anti-Abeta antibodies. Fine epitope analysis with overlapping peptides of the Abeta(42) sequence identified the 1-15 region as a dominant B cell epitope. The DNA minigene-induced anti-Abeta antibodies bound to Abeta plaques in brain tissue from an Alzheimer's disease patient demonstrating functional activity of the antibodies and the potential for therapeutic efficacy.     

Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model

We have previously shown that chronic treatment with the monoclonal antibody m266, which is specific for amyloid beta-peptide (Abeta), increases plasma concentrations of Abeta and reduces Abeta burden in the PDAPP transgenic mouse model of Alzheimer's disease (AD). We now report that administration of m266 to PDAPP mice can rapidly reverse memory deficits in both an object recognition task and a holeboard learning and memory task, but without altering brain Abeta burden. We also found that an Abeta/antibody complex was present in both the plasma and the cerebrospinal fluid of m266-treated mice. Our data indicate that passive immunization with this anti-Abeta monoclonal antibody can very rapidly reverse memory impairment in certain learning and memory tasks in the PDAPP mouse model of AD, owing perhaps to enhanced peripheral clearance and (or) sequestration of a soluble brain Abeta species.     

Immunological and anti-chaperone therapeutic approaches for Alzheimer disease

Alzheimer disease (AD) is the most common cause of dementia. Currently available therapies only provide symptomatic relief. A number of therapeutic approaches are under development that aim to increase the clearance of brain Abeta peptides. These include immune mediated clearance of Abeta and the inhibition of the interaction between Abeta and its pathological chaperones. Both active and passive immunization has been shown to have robust effects in transgenic mouse models of AD on amyloid reduction and behavioral improvements. However, a human trial of active immunization has been associated with significant toxicity in a minority of patients. New generation vaccines are being developed which likely will reduce the potential for cell-mediated toxicity. In addition, the recent development of anti-chaperone therapy opens a new therapeutic avenue which is unlikely to be associated with toxicity.     

Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzheimer's disease-associated pathology in transgenic mice

Transgenic mice with brain amyloid-beta (Abeta) plaques immunized with aggregated Abeta1-42 have reduced cerebral amyloid burden. However, the use of Abeta1-42 in humans may not be appropriate because it crosses the blood brain barrier, forms toxic fibrils, and can seed fibril formation. We report that immunization in transgenic APP mice (Tg2576) for 7 months with a soluble nonamyloidogenic, nontoxic Abeta homologous peptide reduced cortical and hippocampal brain amyloid burden by 89% (P = 0.0002) and 81% (P = 0.0001), respectively. Concurrently, brain levels of soluble Abeta1-42 were reduced by 57% (P = 0.0019). Ramified microglia expressing interleukin-1beta associated with the Abeta plaques were absent in the immunized mice indicating reduced inflammation in these animals. These promising findings suggest that immunization with nonamyloidogenic Abeta derivatives represents a potentially safer therapeutic approach to reduce amyloid burden in Alzheimer's disease, instead of using toxic Abeta fibrils.     

Amyloid-beta peptide remnants in AN-1792-immunized Alzheimer's disease patients: a biochemical analysis

Experiments with amyloid-beta (Abeta)-42-immunized transgenic mouse models of Alzheimer's disease have revealed amyloid plaque disruption and apparent cognitive function recovery. Neuropathological examination of patients vaccinated against purified Abeta-42 (AN-1792) has demonstrated that senile plaque disruption occurred in immunized humans as well. Here, we examined tissue histology and quantified and biochemically characterized the remnant amyloid peptides in the gray and white matter and leptomeningeal/cortical vessels of two AN-1792-vaccinated patients, one of whom developed meningoencephalitis. Compact core and diffuse amyloid deposits in both vaccinated individuals were focally absent in some regions. Although parenchymal amyloid was focally disaggregated, vascular deposits were relatively preserved or even increased. Immunoassay revealed that total soluble amyloid levels were sharply elevated in vaccinated patient gray and white matter compared with Alzheimer's disease cases. Our experiments suggest that although immunization disrupted amyloid deposits, vascular capture prevented large-scale egress of Abeta peptides. Trapped, solubilized amyloid peptides may ultimately have cascading toxic effects on cerebrovascular, gray and white matter tissues. Anti-amyloid immunization may be most effective not as therapeutic or mitigating measures but as a prophylactic measure when Abeta deposition is still minimal. This may allow Abeta mobilization under conditions in which drainage and degradation of these toxic peptides is efficient.     

Primate-like amyloid-beta sequence but no cerebral amyloidosis in aged tree shrews.

A central pathological feature of Alzheimer's disease is the profuse deposition of amyloid-beta protein (Abeta) in the brain parenchyma and vessel walls. Abeta also forms deposits in the brains of a variety of mammals, including all aged non-human primates studied to date. The sequence of Abeta in these animals is identical to that in humans. No Abeta deposits have been found in the brains of wild-type rats and mice, suggesting that the three amino acid differences between their Abeta and that of amyloid-bearing mammals impedes the fibrillogenicity of Abeta. Analysis of the primary sequence of the beta-amyloid precursor protein in tree shrews revealed a 98% similarity and 97% identity with the human protein. Furthermore, the predicted amino acid sequence of Abeta in tree shrews is identical to that in humans. However, immunohistochemical analysis failed to reveal beta-amyloid deposits in the neural parenchyma or vasculature of eight aged (7-8 years) tree shrews (Tupaia belangeri). The lack of correlation between the Abeta sequence and amyloid formation suggests that other factors contribute to cerebral amyloid deposition in aged animals.     

Passive immunotherapy against Abeta in aged APP-transgenic mice reverses cognitive deficits and depletes parenchymal amyloid deposits in spite of increased vascular amyloid and microhemorrhage

BACKGROUND: Anti-Abeta immunotherapy in transgenic mice reduces both diffuse and compact amyloid deposits, improves memory function and clears early-stage phospho-tau aggregates. As most Alzheimer disease cases occur well past midlife, the current study examined adoptive transfer of anti-Abeta antibodies to 19- and 23-month old APP-transgenic mice. METHODS: We investigated the effects of weekly anti-Abeta antibody treatment on radial-arm water-maze performance, parenchymal and vascular amyloid loads, and the presence of microhemorrhage in the brain. 19-month-old mice were treated for 1, 2 or 3 months while 23-month-old mice were treated for 5 months. Only the 23-month-old mice were subject to radial-arm water-maze testing. RESULTS: After 3 months of weekly injections, this passive immunization protocol completely reversed learning and memory deficits in these mice, a benefit that was undiminished after 5 months of treatment. Dramatic reductions of diffuse Abeta immunostaining and parenchymal Congophilic amyloid deposits were observed after five months, indicating that even well-established amyloid deposits are susceptible to immunotherapy. However, cerebral amyloid angiopathy increased substantially with immunotherapy, and some deposits were associated with microhemorrhage. Reanalysis of results collected from an earlier time-course study demonstrated that these increases in vascular deposits were dependent on the duration of immunotherapy. CONCLUSIONS: The cognitive benefits of passive immunotherapy persist in spite of the presence of vascular amyloid and small hemorrhages. These data suggest that clinical trials evaluating such treatments will require precautions to minimize potential adverse events associated with microhemorrhage.     

Inflammatory responses to amyloidosis in a transgenic mouse model of Alzheimer's disease

Mutations in the amyloid precursor protein (APP) and presenilin-1 and -2 genes (PS-1, -2) cause Alzheimer's disease (AD). Mice carrying both mutant genes (PS/APP) develop AD-like deposits composed of beta-amyloid (Abeta) at an early age. In this study, we have examined how Abeta deposition is associated with immune responses. Both fibrillar and nonfibrillar Abeta (diffuse) deposits were visible in the frontal cortex by 3 months, and the amyloid load increased dramatically with age. The number of fibrillar Abeta deposits increased up to the oldest age studied (2.5 years old), whereas there were less marked changes in the number of diffuse deposits in mice over 1 year old. Activated microglia and astrocytes increased synchronously with amyloid burden and were, in general, closely associated with deposits. Cyclooxygenase-2, an inflammatory response molecule involved in the prostaglandin pathway, was up-regulated in astrocytes associated with some fibrillar deposits. Complement component 1q, an immune response component, strongly colocalized with fibrillar Abeta, but was also up-regulated in some plaque-associated microglia. These results show: i) an increasing proportion of amyloid is composed of fibrillar Abeta in the aging PS/APP mouse brain; ii) microglia and astrocytes are activated by both fibrillar and diffuse Abeta; and iii) cyclooxygenase-2 and complement component 1q levels increase in response to the formation of fibrillar Abeta in PS/APP mice.     

Mostly separate distributions of CLAC- versus Abeta40- or thioflavin S-reactivities in senile plaques reveal two distinct subpopulations of beta-amyloid deposits

Collagenous Alzheimer amyloid plaque component (CLAC) is a unique non-Abeta amyloid component of senile plaques (SP) derived from a transmembrane collagen termed CLAC-precursor. Here we characterize the chronological and spatial relationship of CLAC with other features of SP amyloid in the brains of patients with Alzheimer's disease (AD), Down syndrome (DS), and of PSAPP transgenic mice. In AD and DS cerebral cortex, CLAC invariably colocalized with Abeta42 but often lacked Abeta40- or thioflavin S (thioS)-reactivities. Immunoelectron microscopy of CLAC-positive SP showed labeling of fibrils that are more loosely dispersed compared to typical amyloid fibrils in CLAC-negative SP. In DS cerebral cortex, diffuse plaques in young patients were negative for CLAC, whereas a subset of SP became CLAC-positive in patients aged 35 to 50 years, before the appearance of Abeta40. In DS cases over 50 years of age, Abeta40-positive SP dramatically increased, whereas CLAC burden remained at a constant level. In PSAPP transgenic mice, CLAC was positive in the diffuse Abeta deposits surrounding huge-cored plaques. Thus, CLAC and Abeta40 or thioS exhibit mostly separate distribution patterns in SP, suggesting that CLAC is a relatively early component of SP in human brains that may have inhibitory effects against the maturation of SP into beta-sheet-rich amyloid deposits.     

Chlamydia pneumoniae induces Alzheimer-like amyloid plaques in brains of BALB/c mice

Amyloid deposits resembling plaques found in Alzheimer's disease (AD) brains were formed in the brains of non-transgenic BALB/c mice following intranasal infection with Chlamydia pneumoniae. The mice were infected at 3 months of age with C. pneumoniae isolated from an AD brain. Infection was confirmed by light and electron microscopy in olfactory tissues of the mice. C. pneumoniae was still evident in these tissues 3 months after the initial infection indicating that a persistent infection had been established. Amyloid beta (Abeta) 1-42 immunoreactive deposits were identified in the brains of infected BALB/c mice up to 3 months post-infection with the density, size, and number of deposits increasing as the infection progressed. A subset of deposits exhibited thioflavin-s labeling. Intracellular Abeta1-42 labeling was observed in neuronal cells. Experimental induction of amyloid deposition in brains of non-transgenic BALB/c mice following infection with C. pneumoniae may be a useful model for furthering our understanding of mechanisms, linked to infection, involved in the initiation of the pathogenesis of sporadic AD.     

Extracellular amyloid formation and associated pathology in neural grafts

Amyloid precursor protein (APP) processing and the generation of beta-amyloid peptide (Abeta) are important in the pathogenesis of Alzheimer's disease. Although this has been studied extensively at the molecular and cellular levels, much less is known about the mechanisms of amyloid accumulation in vivo. We transplanted transgenic APP23 and wild-type B6 embryonic neural cells into the neocortex and hippocampus of both B6 and APP23 mice. APP23 grafts into wild-type hosts did not develop amyloid deposits up to 20 months after grafting. In contrast, both transgenic and wild-type grafts into young transgenic hosts developed amyloid plaques as early as 3 months after grafting. Although largely diffuse in nature, some of the amyloid deposits in wild-type grafts were congophilic and were surrounded by neuritic changes and gliosis, similar to the amyloid-associated pathology previously described in APP23 mice. Our results indicate that diffusion of soluble Abeta in the extracellular space is involved in the spread of Abeta pathology, and that extracellular amyloid formation can lead to neurodegeneration.     

Augmented senile plaque load in aged female beta-amyloid precursor protein-transgenic mice

Transgenic mice (Tg2576) overexpressing human beta-amyloid precursor protein with the Swedish mutation (APP695SWE) develop Alzheimer's disease-like amyloid beta protein (Abeta) deposits by 8 to 10 months of age. These mice show elevated levels of Abeta40 and Abeta42, as well as an age-related increase in diffuse and compact senile plaques in the brain. Senile plaque load was quantitated in the hippocampus and neocortex of 8- to 19-month-old male and female Tg2576 mice. In all mice, plaque burden increased markedly after the age of 12 months. At 15 and 19 months of age, senile plaque load was significantly greater in females than in males; in 91 mice studied at 15 months of age, the area occupied by plaques in female Tg2576 mice was nearly three times that of males. By enzyme-linked immunosorbent assay, female mice also had more Abeta40 and Abeta42 in the brain than did males, although this difference was less pronounced than the difference in histological plaque load. These data show that senescent female Tg2576 mice deposit more amyloid in the brain than do male mice, and may provide an animal model in which the influence of sex differences on cerebral amyloid pathology can be evaluated.     

Lifelong immunization with human beta-amyloid (1-42) protects Alzheimer's transgenic mice against cognitive impairment throughout aging

Although both active and passive beta-amyloid (Abeta) immunotherapy have been shown to protect against or lessen cognitive impairment in various Alzheimer's transgenic mouse lines, these studies have focused on a single task and involved standard statistical analysis. Because Alzheimer's disease impacts multiple cognitive domains, the current study employed an extensive behavioral battery and multimetric analysis therein to determine the impact of Abeta immunization given throughout most of adult life (from 2-16 1/2 months of age) to APP+PS1 transgenic mice. At both adult (4 1/2-6 month) and aged (15-16 1/2 month) test points, the same 6-week behavioral battery was administered. Results indicate that Abeta immunotherapy partially or completely protected APP+PS1 mice at both test points from otherwise impaired performance in a variety of tasks spanning multiple cognitive domains (reference learning/memory, working memory, search/recognition). At both adult and aged test points, the cognitive benefits of Abeta immunotherapy were evident even when behavioral measures were analyzed collectively (as "overall" performance) through discriminant function analysis. Since behavioral protection at the 15-16 1/2 month test point occurred without a decrease in (or correlation to) Abeta deposition, the mechanism of Abeta immunotherapy's action most likely involves neutralization/removal of small Abeta oligomers from the brain. However, in factor analysis performed at this aged test point, brain Abeta deposition measures loaded heavily with key cognitive measures. Collectively, our results suggest that the entire process of Abeta deposition deleteriously impacts cognitive performance and that Abeta-based preventative strategies can provide long-term cognitive benefits extending well into older age.