Human Alzheimer's Disease Patients Research Articles

Single chain variable fragments against beta-amyloid (Abeta) can inhibit Abeta aggregation and prevent abeta-induced neurotoxicity.

Beta-amyloid (Abeta) is a major pathological determinant of Alzheimer's disease (AD). Both active and passive immunization studies have shown that antibodies against Abeta are effective in decreasing cerebral Abeta levels, reducing Abeta accumulation, and attenuating cognitive deficits in animal models of AD. However, the therapeutic potential of these antibodies in human AD patients is limited because of adverse inflammatory reactions and cerebral hemorrhaging associated with the treatments. Here we show that single chain variable fragments (scFv's) represent an attractive alternative to more conventional antibody-based therapeutics to reduce Abeta toxicity. The binding affinities and binding epitopes of two different scFv's to Abeta were characterized using a surface plasmon resonance (SPR) biosensor. An scFv binding the 17-28 region of Abeta effectively inhibited in vitro aggregation of Abeta as determined by thioflavin T (ThT) fluorescence staining and atomic force microscopy (AFM) analysis, while an scFv binding the carboxyl-terminal region of Abeta (residues 29-40) did not inhibit aggregation. The scFv to the 17-28 region when co-incubated with Abeta not only decreased aggregation but also eliminated any toxic effects of aggregated Abeta on the human neuroblastoma cell line, SH-SY5Y. The ability of scFv's to inhibit both aggregation and cytotoxicity of Abeta indicates that scFv's have potential therapeutic value for treating AD.

Synthesis and evaluation of two 18F-labeled 6-iodo-2-(4'-N,N-dimethylamino)phenylimidazo[1,2-a]pyridine derivatives as prospective radioligands for beta-amyloid in Alzheimer's disease.

This study evaluated (18)F-labeled IMPY [6-iodo-2-(4'-N,N-dimethylamino)phenylimidazo[1,2-a]pyridine] derivatives as agents for imaging beta-amyloid plaque with positron emission tomography (PET). The precursor for radiolabeling and reference compounds was synthesized in up to five steps from commercially accessible starting materials. One of the two N-methyl groups of IMPY was substituted with either a 3-fluoropropyl (FPM-IMPY) or a 2-fluoroethyl (FEM-IMPY) group. FPM-IMPY and FEM-IMPY were found to have moderate affinity for Abeta-aggregates with K(i) = 27 +/- 8 and 40 +/- 5 nM, respectively. A "one-pot" method for (18)F-2-fluoroethylation and (18)F-3-fluoropropylation of the precursor was developed. The overall decay-corrected radiochemical yields were 26-51%. In PET experiments with normal mouse, high uptake of activity was obtained in the brain after iv injection of each probe: 6.4% ID/g for [(18)F]FEM-IMPY at 1.2 min, and 5.7% ID/g for [(18)F]FPM-IMPY at 0.8 min. These values were similar to those of [(123)I/(125)I]IMPY (7.2% ID/g at 2 min). Polar and nonpolar radioactive metabolites were observed in both plasma and brain homogenates after injection of [(18)F]FEM or [(18)F]FPM-IMPY. In contrast to the single-exponential washout of [(123)I/(125)I]IMPY, the washouts of brain activity for the two fluorinated analogues were biphasic, with an initial rapid phase over 20 min and a subsequent much slower phase. Residual brain activity at 2 h, which may represent polar metabolites trapped in the brain, was 4.5% ID/g for [(18)F]FEM-IMPY and 2.1% ID/g for [(18)F]FPM-IMPY. Substantial skull uptake of [(18)F]fluoride was also clearly observed. With a view to slow the metabolism of [(18)F]FEM-IMPY, an analogue was prepared with deuteriums substituted for the four ethyl hydrogens. However, D(4)-[(18)F]FEM-IMPY showed the same brain uptake and clearance as the protio analogue. Metabolism of the [(18)F]FEM-IMPY was appreciably slower in rhesus monkey than in mouse. Autoradiography of postmortem brain sections of human Alzheimer's disease patients with [(18)F]FEM-IMPY showed high displaceable uptake in gray matter and low nonspecific binding in the white matter. This study demonstrates that the IMPY derivatives have favorable in vivo brain pharmacokinetics and a moderate affinity for imaging beta-amyloid plaques; however, further improvements are needed to reduce radioactive metabolites, increase binding affinity, and reduce lipophilicity.

Advanced glycation endproducts and pro-inflammatory cytokines in transgenic Tg2576 mice with amyloid plaque pathology.

Increased expression and altered processing of the amyloid precursor protein (APP) and generation of beta-amyloid peptides is important in the pathogenesis of amyloid plaques in Alzheimer's disease (AD). Transgenic Tg2576 mice overexpressing the Swedish mutation of human APP exhibit beta-amyloid deposition in the neocortex and limbic areas, accompanied by gliosis and dystrophic neurites. However, murine plaques appear to be less cross-linked and the mice show a lower degree of inflammation and neurodegeneration than AD patients. 'Advanced glycation endproducts (AGEs)', formed by reaction of proteins with reactive sugars or dicarbonyl compounds, are able to cross-link proteins and to activate glial cells, and are thus contributing to plaque stability and plaque-induced inflammation in AD. In this study, we analyze the tissue distribution of AGEs and the pro-inflammatory cytokines IL-1beta and TNF-alpha in 24-month-old Tg2576 mice, and compare the AGE distribution in these mice with a younger age group (13 months old) and a typical Alzheimer's disease patient. Around 70% of the amyloid plaque cores in the 24-month-old mice are devoid of AGEs, which might explain their solubility in physiological buffers. Plaque associated glia, which express IL-1beta and TNF-alpha, contain a significant amount of AGEs, suggesting that plaques, i.e. Abeta as its major component, can induce intracellular AGE formation and the expression of the cytokines on its own. In the 13-month-old transgenic mice, AGEs staining can neither be detected in plaques nor in glial cells. In contrast, AGEs are present in high amounts in both plaques and glia in the human AD patient. The data obtained in this show interesting differences between the transgenic mouse model and AD patients, which should be considered using the transgenic approach to test therapeutical strategies to eliminate plaques or to attenuate the inflammatory response in AD.

Modeling Alzheimer's disease immune therapy mechanisms: interactions of human postmortem microglia with antibody-opsonized amyloid beta peptide.

The induction of an antibody response to amyloid beta (Abeta) peptide has become a strategy for the treatment of Alzheimer's disease (AD). This has proven effective in reducing the plaque burden in transgenic mice that develop Abeta plaques similar to human AD patients. The mechanism for enhanced clearance of Abeta is partly due to the interaction of immunoglobulin Fcgamma receptor-expressing microglia and specific antibody-opsonized Abeta deposits. This interaction can stimulate Fcgamma receptor-mediated phagocytosis, but also results in inflammatory activation of these cells. Consequently, interaction of microglia with antibody-antigen complexes could exacerbate the existing inflammation in the brains of AD patients. In this study, we used substrate-bound Abeta and cultured human microglia from AD and non-demented cases to model interaction of microglia and antibody-opsonized plaques in AD brains. Enhanced production of tumor necrosis factor-alpha, macrophage colony stimulating factor, interleukin-10, and superoxide ions was detected. We also demonstrated enhanced uptake of opsonized Abeta by microglia, which was reduced significantly in the presence of excess IgG, indicative of the involvement of Fcgamma receptor-mediated mechanisms. Human microglia were shown in this study to express mRNA for Fcgamma receptors I, IIa, IIb, and III. The expression of Fcgamma receptor II was augmented by proinflammatory stimulation. These results suggest that initial interactions of human microglia with antibody-opsonized amyloid could result in increased inflammation. The consequence of this on inflammatory pathology in AD brains needs to be considered before immunization is used as a strategy for treating AD.

Preclinical experiments on cognition enhancement in Alzheimer's disease: Drugs affecting nicotinic acetylcholine receptors

AbstractNicotinic acetylcholine receptors (nAChRs) play a role in a variety of diseases of the central nervous system including Alzheimer's disease (AD). There is currently great interest in evaluating AD‐related nAChR changes, and pharmacological treatment of nAChR deficits is a promising therapy. In AD, α7 nAChRs remain relatively stable in contrast to α4β2 nAChRs that are lost in substantial numbers. However, α7 nAChRs may be functionally impaired in AD because β‐amyloid, a major neuropathology in AD, blocks α4β2 and α7 nAChRs. Agonists selective to α7 or α4β2 nAChRs are neuroprotective against β‐amyloid. A preclinical test of cognition‐enhancing drugs affecting nAChRs is eyeblink classical conditioning. This task is severely impaired in human probable AD patients and is impaired by antagonists to nAChRs. Three drugs with different mechanisms of action on nAChRs (partial α7 nAChR agonism [GTS‐21], acetylcholinesterase inhibition and allosteric modulation [galantamine], nootropic activation [nefiracetam]) were tested in young and older rabbits using eyeblink classical conditioning. All three drugs ameliorated learning and memory impairments in older rabbits and reversed an antagonist to nAChRs in young rabbits. Galantamine, with its allosteric modulatory action, was the only drug that facilitated learning in young rabbits. On the basis of efficacy of these drugs that affect nAChRs in preclinical studies and in Phase I (GTS‐21), Phase II (nefiracetam), or Phase III (galantamine) clinical trials, exploration of nAChRs as targets for therapeutic intervention via a number of different pathways seems warranted. Drug Dev. Res. 56:335–346, 2002. © 2002 Wiley‐Liss, Inc.