Scott D. Webster

Morphology and Toxicity of Aβ-(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of Alzheimer's Disease

In the course of analyzing the chemical composition of Alzheimers disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Aβ peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 M guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic Aβ-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Aβ-(1-40/42) were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal killing. It is possible that these potentially pathogenic Aβ-(1-40/42) dimers and trimers from Alzheimers disease amyloid represent the soluble oligomers of Aβ recently described in Alzheimers disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).

Molecular and Cellular Characterization of the Membrane Attack Complex, C5b-9, in Alzheimer’s Disease

The membrane attack complex, C5b-9, is of considerable importance in many inflammatory reactions. It is the terminal, cytolytic component of both classical and alternative pathway activation, and its presence presupposes other potentially destructive complement constituents, including anaphylotoxins and opsonins. We have characterized C5b-9 and its C9 constituent in the Alzheimers disease (AD) and nondemented elderly (ND) brain using immunohistochemistry at the light and electron microscopic levels, Western blot analysis, and the reverse transcriptase polymerase chain reaction. We have also conducted in vitro ELISA assays of amyloid beta-peptide-stimulated SC5b-9 production. C5b-9 is abundantly present in Alzheimers disease cortex, associated with neurofibrillary tangle containing neurons, dystrophic neurites within neuritic plaques, and neuropil threads, but is weakly detected, if at all, in nondemented elderly cortex under the same conditions. Staining of Alzheimers disease sections is abolished both by deletion of primary antibody or preabsorption with purified SC5b-9.

Complement activation by neurofibrillary tangles in Alzheimer's disease

Brain inflammation is widely documented to occur in Alzheimers disease (AD), but its sources are still incompletely understood. Here, we present in vitro and in situ evidence that, like amyloid beta peptide (Abeta), tau, the major protein constituent of the neurofibrillary tangle, is a potent, antibody-independent activator of the classical complement pathway. Complement activation, in turn, is known to drive numerous inflammatory responses, including scavenger cell activation and cytokine production. Because Abeta deposits and extracellular tangles are present from early preclinical to terminal stages of AD, their ability to activate complement provides a ready mechanism for initiating and sustaining chronic, low-level inflammatory responses that may cumulate over the disease course.

Complement Component C1q Modulates the Phagocytosis of Aβ by Microglia

Recent studies showing that microglia internalize the amyloid beta-peptide (Abeta) suggest that these cells have the potential for clearing Abeta deposits in Alzheimers disease, and mechanisms that regulate the removal of Abeta may therefore be of clinical interest. Previous studies from this laboratory showing that C1q enhances phagocytosis of cellular targets by rat microglia prompted the current investigations characterizing the effects of C1q on microglial phagocytosis of Abeta. Microglia were shown to phagocytose Abeta1-42, in agreement with observations of other investigators. Uptake of Abeta1-42 was observed for concentrations of 5-50 microM, and phagocytosis of peptides containing (14)C or fluorescein (FM) labels was not affected by the interaction of microglia with C1q-coated surfaces. However, inclusion of C1q (125 nM-1.4 microM) in solutions of 50 microM Abeta1-42 inhibited the uptake of (14)C-Abeta1-42 and FM-Abeta1-42, suggesting that C1q blocks the interaction of Abeta with microglia. Uptake of Abeta was partially blocked by the scavenger receptor ligands polyinosinic acid and maleylated BSA. Inhibition of Abeta uptake by C1q may contribute to the accumulation of fibrillar, C1q-containing plaques that occurs in parallel with disease progression. These data suggest that mechanisms which interfere with the binding of C1q to Abeta may be of therapeutic value both through inhibition of the inflammatory events resulting from complement activation and via altered access of Abeta sites necessary for ingestion by microglia.

Irreversible dimerization/tetramerization and post-translational modifications inhibit proteolytic degradation of Aβ peptides of Alzheimer's disease

Experimental evidence increasingly implicates the beta-amyloid peptide in the pathogenesis of Alzheimers disease. Beta-amyloid filaments dramatically accumulate in the neuritic plaques and vascular deposits as the result of the brains inability to clear these structures. In this paper, we demonstrate that in addition to the intrinsic stability of A beta N-42, the time dependent generation of irreversibly associated A beta dimers and tetramers incorporated into A beta filaments are themselves resistant to proteolytic degradation. The presence of post-translational modifications such as isomerization of aspartyls 1 and 7, cyclization of glutamyl 3 to pyroglutamyl and oxidation of methionyl 35, further contribute to the insolubility and stability of A beta. All these factors promote the accumulation of neurotoxic amyloid in the brains of patients with Alzheimers disease, and should be considered in therapeutic strategies directed towards the dissociation of the brains A beta filaments.

Antibody-Mediated Phagocytosis of the Amyloid β-Peptide in Microglia Is Differentially Modulated by C1q

Microglial ingestion of the amyloid β-peptide (Aβ) has been viewed as a therapeutic target in Alzheimer’s disease, in that approaches that enhance clearance of Aβ relative to its production are predicted to result in decreased senile plaque formation, a proposed contributor to neuropathology. In vitro, scavenger receptors mediate ingestion of fibrillar Aβ (fAβ) by microglia. However, the finding that cerebral amyloid deposition in a transgenic mouse model of Alzheimer’s disease was diminished by inoculation with synthetic Aβ has suggested a possible therapeutic role for anti-Aβ Ab-mediated phagocytosis. Microglia also express C1qRP, a receptor for complement protein C1q, ligation of which in vitro enhances phagocytosis of immune complexes formed with IgG levels below that required for optimal FcR-mediated phagocytosis. The data presented here demonstrate FcR-dependent ingestion of Aβ-anti-Aβ complexes (IgG-fAβ) by microglia that is a function of the amount of Ab used to form immune complexes. In addition, C1q incorporated into IgG-fAβ enhanced microglial uptake of these complexes when they contained suboptimal levels of anti-Aβ Ab. Mannose binding lectin and lung surfactant protein A, other ligands of C1qRP, also enhanced ingestion of suboptimally opsonized IgG-fAβ, whereas control proteins did not. Our data suggest that C1qRP-mediated events may promote efficient ingestion of Aβ at low Ab titers, and this may be beneficial in paradigms that seek to clear amyloid via FcR-mediated mechanisms by minimizing the potential for destructive Ab-induced complement-mediated processes.

Structural and functional evidence for microglial expression of C1qR(P), the C1q receptor that enhances phagocytosis.

Microglial activation has been associated with several degenerative diseases of the central nervous system (CNS). One consequence of activation is the induction of a more efficient phagocytic response, and it is therefore important to determine what factors regulate microglial phagocytosis and whether this capacity influences the progression of neurodegenerative changes. Previous studies have demonstrated that complement component C1q enhances Fc receptor‐ and CR1‐mediated phagocytosis in cells of the myeloid lineage via a cell surface receptor, C1qRP. Because C1q has been found in the area of lesions in several degenerative CNS diseases, the current investigations were carried out to characterize the effects of C1q on microglial phagocytosis. Neonatal rat microglia were shown to express C1qRP as assessed by flow cytometry and immuno‐cytochemistry. Interaction of these cells with substrate‐bound C1q was shown to enhance both FcR‐and CR1‐mediated phagocytosis two‐ to fourfold. In addition, introduction of an antibody raised against the carboxy‐terminal, cytoplasmic domain of C1qRP into microglia by electroporation markedly diminished the ability of C1q to enhance uptake of IgG‐coated targets, whereas nonspecific IgG had no such effect. These results suggest that C1q in areas of active degeneration may promote the phagocytic capacity of microglia via interaction with microglial C1qRP. J. Leukoc. Biol. 67: 109–116; 2000.

Cholinergic deafferentation of the rabbit cortex: a new animal model of Aβ deposition

Brain deposition of the amyloid beta-peptide (Abeta) is a critical step in the pathogenesis of Alzheimers disease (AD) and human cerebral amyloid angiopathy (CAA). A small fraction of AD and CAA cases are caused by gene mutations leading to increased production and deposition of Abeta, but for the majority, there is no known direct genetic cause. We have hypothesized that Abeta deposition in these sporadic cases occurs as a result of cortical cholinergic deafferentation. Here we show that cortical cholinergic deafferentation, induced in rabbits by a selective immunotoxin, leads to Abeta deposition in cerebral blood vessels and perivascular neuropil. Biochemical measurements confirmed that lesioned animals had 2.5- and 8-fold elevations of cortical Abeta40 and Abeta42, respectively. Cholinergic deafferentation may be one factor that can contribute to Abeta deposition.

Novel Aβ peptide immunogens modulate plaque pathology and inflammation in a murine model of Alzheimer's disease

BackgroundAlzheimers disease, a common dementia of the elder, is characterized by accumulation of protein amyloid deposits in the brain. Immunization to prevent this accumulation has been proposed as a therapeutic possibility, although adverse inflammatory reactions in human trials indicate the need for novel vaccination strategies.MethodHere vaccination with novel amyloid peptide immunogens was assessed in a transgenic mouse model displaying age-related accumulation of fibrillar plaques.ResultsImmunization with any conformation of the amyloid peptide initiated at 12 months of age (at which time fibrillar amyloid has just begun to accumulate) showed significant decrease in total and fibrillar amyloid deposits and in glial reactivity relative to control transgenic animals. In contrast, there was no significant decrease in amyloid deposition or glial activation in mice in which vaccination was initiated at 16 months of age, despite the presence of similar levels anti-Aβ antibodies in young and old animals vaccinated with a given immunogen. Interestingly, immunization with an oligomeric conformation of Aβ was equally as effective as other amyloid peptides at reducing plaque accumulation. However, the antibodies generated by immunization with the oligomeric conformation of Aβ have more limited epitope reactivity than those generated by fAβ, and the microglial response was significantly less robust.ConclusionThese results suggest that a more specific immunogen such as oligomeric Aβ can be designed that achieves the goal of depleting amyloid while reducing potential detrimental inflammatory reactions. In addition, the data show that active immunization of older Tg2576 mice with any amyloid conformation is not as efficient at reducing amyloid accumulation and related pathology as immunization of younger mice, and that serum anti-amyloid antibody levels are not quantitatively related to reduced amyloid-associated pathology.

Inflammatory Pathology in Alzheimer’s Disease

A large body of evidence suggests that there is an inflammatory component to Alzheimer’s disease (AD) pathogenesis. The reactive microglial cells of neuritic plaques are characterized by the presence of a variety of inflammatory markers, including HLA-DR, HLA-DP, HLA-DQ, β-2 integrins and Fc receptors (reviewed by Rogers et al., 1992a). Also evident within plaques are the As associated proteins (Selkoe, 1991), many of which are related to inflammatory processes. Some of the most studied are α 1-antichymotrypsin (ACT) (Abraham et al., 1988), complement proteins (McGeer and McGeer, 1992; McGeer and Rogers, 1992), serum amyloid P (SAP) (Coria et al., 1988) and heparin sulfate proteoglycans (HSPG) (Snow et al., 1988). Furthermore, altered levels of the cytokine interleukin 1 (IL-1) and IL-6 have been demonstrated in AD (reviewed in Wood et al., 1993).