Chica Mori

Nasal administration of amyloid‐β peptide decreases cerebral amyloid burden in a mouse model of Alzheimer's disease

Progressive cerebral deposition of amyloid‐β (Aβ) peptide, an early and essential feature of Alzheimers disease (AD), is accompanied by an inflammatory reaction marked by microgliosis, astrocytosis, and the release of proinflammatory cytokines. Mucosal administration of disease‐implicated proteins can induce antigen‐specific anti‐inflammatory immune responses in mucosal lymphoid tissue which then act systemically. We hypothesized that chronic mucosal administration of Aβ peptide might induce an anti‐inflammatory process in AD brain tissue that could beneficially affect the neuropathological findings. To test this hypothesis, we treated PDAPP mice, a transgenic line displaying numerous neuropathological features of AD, between the ages of ∼5 and ∼12 months with human Aβ synthetic peptide mucosally each week. We found significant decreases in the cerebral Aβ plaque burden and Aβ42 levels in mice treated intranasally with Aβ peptide versus controls treated with myelin basic protein or left untreated. This lower Aβ burden was associated with decreased local microglial and astrocytic activation, decreased neuritic dystrophy, serum anti‐Aβ antibodies of the IgG1 and IgG2b classes, and mononuclear cells in the brain expressing the anti‐inflammatory cytokines interleukin‐4, interleukin‐10, and tumor growth factor‐β. Our results demonstrate that chronic nasal administration of Aβ peptide can induce an immune response to Aβ that decreases cerebral Aβ deposition, suggesting a novel mucosal immunological approach for the treatment and prevention of AD. Ann Neurol 2000;48:567–579

Intraneuronal Aβ42 accumulation in Down syndrome brain

Alzheimers disease (AD) brains display A beta (Aβ) plaques, inflammatory changes and neurofibrillary tangles (NFTs). Converging evidence suggests a neuronal origin of Aβ. We performed a temporal study of intraneuronal Aβ accumulation in Down syndrome (DS) brains. Sections from temporal cortex of 70 DS cases aged 3 to 73 years were examined immunohistochemically for immunoreactivity (IR) for the Aβ N-terminal, the Aβ40 C-terminus and the Aβ42 C-terminus. N-terminal antibodies did not detect intracellular Aβ Aβ40 antibodies did not detect significant intracellular Aβ, but older cases showed Aβ40 IR in mature plaques. in contrast, Aβ42 antibodies revealed clear-cut intraneuronal IR. All Aβ42 antibodies tested showed strong intraneuronal Aβ42 IR in very young DS patients, especially in the youngest cases studied (e.g., 3 or 4 yr. old), but this IR declined as extracellular Applaques gradually accumulated and matured. No inflammatory changes were associated with intraneuronal Aβ. We also studied the temporal development of gliosis and NFT formation, revealing that in DS temporal cortex, inflammation and NFT follow A β deposition. We conclude that Aβ42 accumulates intracellulary prior to extracellular Aβ deposition in Down syndrome, and that subsequent maturation of extracellular Aβ deposits elicits inflammatory responses and precedes NFTs.

Evidence for peripheral clearance of cerebral Aβ protein following chronic, active Aβ immunization in PSAPP mice

Immunization with amyloid-β (Aβ) peptide in mouse models of Alzheimer’s disease has been reported to decrease cerebral Aβ levels and improve behavioral deficits. Several mechanisms have been proposed, including antibody-induced phagocytosis of Aβ by cerebral microglia and increased efflux of Aβ from the brain to the periphery. The latter mechanism was suggested in mice undergoing acute, passive transfer of an Aβ monoclonal antibody. Here, PSAPP transgenic mice were actively immunized by a single intraperitoneal injection of synthetic Aβ followed by chronic intranasal administration of Aβ with the mucosal adjuvant, Escherichia coli heat-labile enterotoxin, LT, twice weekly for 8 weeks. Serum from Aβ-immunized mice had an average of 240 μg/ml of anti-Aβ-specific antibodies; these antibodies had epitope(s) within Aβ1-15 and were of immunoglobulin (Ig) isotypes IgG2b, IgG2a, and IgG1. Immunization led to a 75% decrease in plaque number (P < 0.0001) and a 58% decrease in Aβx–42 levels (P < 0.026) in brain, and gliosis and neuritic dystrophy were diminished. No pathological effects of the immunization were observed in kidney, spleen, or snout. Serum Aβ levels increased 28-fold in immunized mice (53.06 ng/ml) compared to controls (1.87 ng/ml). Most of the Aβ in the serum of the immunized mice was bound to antibodies. We conclude that following active immunization, anti-Aβ antibodies sequester serum Aβ and may increase central nervous system to serum Aβ clearance.

Nasal Aβ Treatment Induces Anti‐Aβ Antibody Production and Decreases Cerebral Amyloid Burden in PD‐APP Mice

Amyloid accumulation1 and accompanying inflammation—including both the activation of glial cells2 and the accrual of inflammatory proteins, such as complement,3,4 cytokines,5 and acute phase proteins6–8—play key roles in the pathogenesis of Alzheimer’s disease (AD).9 Mucosal administration of proteins implicated in a disease can decrease organ-specific inflammatory processes in a number of animal models of autoimmune disorders, including those affecting the nervous system, principally by inducing antiinflammatory IL-4/IL-10 (Th2) and TGFβ immune responses in mucosal lymphoid tissue that then act systemically.10 For example, oral or nasal administration of myelin basic protein (MBP)11–13 or the acetylcholine receptor14,15 can suppress experimental autoimmune encephalomyelitis (EAE) and experimental myasthenia gravis, respectively. In an effort to reduce the inflammation associated with Aβ deposition via mucosal tolerance, we tested the effects of nasal or oral administration of Aβ1–40 peptide and a control protein, myelin basic protein (MBP), by treating 52 PD-APP transgenic mice, an animal model with certain key features of AD,16–18 on a weekly basis for seven months (ages 5 to 12 months). Doses were chosen based on preliminary nasal and oral studies in nontransgenic mice. Treatment groups included (1) untreated (n = 7); (2) MBP oral, 500 μg (n = 5); (3) MBP nasal, 50 μg (n = 6); (4) Aβ oral, 10 μg (n = 9); (5) Aβ oral, 100 μg (n = 9); (6) Aβ nasal, 5 μg (n = 7); and (7) Aβ nasal, 25 μg (n = 9). During the first week, mice were fed five times or nasally treated three times on consecutive days. Thereafter, mice were fed or nasally treated each week for seven months and then sacrificed. The brain from each mouse was removed and divided in half along the sagittal midline. One hemisphere was formalin fixed and embedded in paraffin for immunohistochemical analysis. Of the contralateral hemispheres, half were snap frozen for biochemical analysis; the other half were embedded sagittally in OCT and snap frozen for cryosectioning and immunohistochemistry.

Temporal Accrual of Complement Proteins in Amyloid Plaques in Down’s Syndrome with Alzheimer’s Disease

The complement system constitutes a series of enzymatic steps involved in the inflammatory response and is activated in Alzheimers disease (AD). Using Downs syndrome (DS) brains as a temporal model for the progression of AD, we examined components of the complement cascade and their relationship to other principal events in AD pathology: Abeta42 deposition, neuritic changes, neurofibrillary tangles (NFTs), and gliosis (reactive astrocytes, activated microglia). Adjacent sections of frontal cortex from 24 DS subjects ranging in age from 12 to 73 years were immunohistochemically examined for immunoreactivity (IR) of classical complement proteins (Clq and C3), markers indicating activation of complement (C4d and C5b-9), the complement inhibitor apolipoprotein J (apo J), and markers of AD neuropathology. Abeta42-labeled diffuse plaques were first detected in a 12-year-old DS subject and were not labeled by any of the complement antibodies. Colocalization of Abeta42 with Clq, C3, C4d, and/or apo J was first detected in compacted plaques in the brain of a 15-year-old DS patient with features of mature AD pathology, such as reactive astrocytes, activated microglia, dystrophic neurites, and a few NFTs. IR for C4d and C5b-9 (membrane attack complex, MAC) was observed in small numbers of plaque-associated dystrophic neurites and in focal regions of pyramidal neurons in this 15-year-old. The only other young (</=30 years) DS brain to show extensive complement IR was that of a 29-year-old DS subject who also displayed the full range of AD neuropathological features. All middle-aged and old DS brains showed IR for Clq and C3, primarily in compacted plaques. In these cases, C4d IR was found in a subset of Abeta42 plaques and, along with C5b-9 IR, was localized to dystrophic neurites in a subset of neuritic plaques, neurons, and some NFTs. Our data suggest that in AD and DS, the classical complement cascade is activated after compaction of Abeta42 deposits and, in some instances, can progress to the local neuronal expression of the MAC as a response to Abeta plaque maturation.

Intranasal immunotherapy for the treatment of Alzheimer’s disease: Escherichia coli LT and LT(R192G) as mucosal adjuvants

Alzheimers disease (AD) is the most common form of dementia worldwide, yet there is currently no effective treatment or cure. Extracellular deposition of amyloid-beta protein (Abeta) in brain is a key neuropathological characteristic of AD. In 1999, Schenk et al. first reported that an injected Abeta vaccine given to PDAPP mice, an AD mouse model displaying Abeta deposition in brain, led to the lowering of Abeta levels in brain. In 2000, we demonstrated that intranasal (i.n.) immunization with human synthetic Abeta1-40 peptide for 7 months led to a 50-60% reduction in cerebral Abeta burden in PDAPP mice; serum Abeta antibody titers were low (approximately 26 microg/ml). More recently, we have optimized our i.n. Abeta immunization protocol in wild-type (WT) mice. When low doses Escherichia coli heat-labile enterotoxin (LT) were given as a mucosal adjuvant with Abeta i.n., there was a dramatic 12-fold increase in Abeta antibody titers in WT B6D2F1 mice treated two times per week for 8 weeks compared to those of mice receiving i.n. Abeta without adjuvant. A non-toxic form of LT, designated LT(R192G), showed even better adjuvanticity; anti-Abeta antibody titers were 16-fold higher than those seen in mice given i.n. Abeta without adjuvant. In both cases, the serum Abeta antibodies recognized epitopes within Abeta1-15 and were of the immunoglobulin (Ig) isotypes IgG2b, IgG1, IgG2a and low levels of IgA. This new and improved Abeta vaccine protocol is now being tested in AD mouse models with the expectation that higher Abeta antibody titers may be more effective in reducing cerebral Abeta levels.

Amyloid-Beta Immunization in Alzheimer's Disease Transgenic Mouse Models and Wildtype Mice

Alzheimers disease is the most prevalent form of dementia worldwide. Therapies are desperately needed to prevent and cure the disease. Mouse models of amyloid-β deposition [APP and PSAPP transgenic (tg) mice] have been useful in determining the role of amyloid-β (Aβ) in both the pathogenesis and cognitive changes in AD. In addition, they have allowed scientists to investigate potential AD therapies in living animals. Active and passive Aβ immunizations have been employed successfully in APP and PSAPP tg mice to lower cerebral Aβ levels and improve cognition. Optimization of immunization protocols and characterization of immune responses in wildtype mice have been reported. Based on the promising results of Aβ immunization studies in mice, a clinical trial was initiated for Aβ vaccination in humans with AD. Although no adverse effects were reported in the Phase I safety trials, about 5% of AD patients in the phase II clinical trial developed meningoencephalitis, ending the trial prematurely in March 2002. Studies in AD mouse models and wildtype mice may help elucidate the mechanism for these unwanted side effects and will be useful for testing newer, safer vaccines for future use in human clinical trials.