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Previous studies have reported immunoglobulin-positive neurons in Alzheimer's disease (AD) brains, an observation indicative of blood-brain barrier (BBB) breakdown. Recently, we demonstrated the nearly ubiquitous presence of brain-reactive autoantibodies in human sera. The significance of these observations to AD pathology is unknown. Here, we show that IgG-immunopositive neurons are abundant in brain regions exhibiting AD pathology, including intraneuronal amyloid-β(42) (Aβ(42)) and amyloid plaques, and confirm by western analysis that brain-reactive autoantibodies are nearly ubiquitous in human serum. To investigate a possible interrelationship between neuronal antibody binding and Aβ pathology, we tested the effects of human serum autoantibodies on the intraneuronal deposition of soluble Aβ(42) peptide in adult mouse neurons in vitro (organotypic brain slice cultures). Binding of human autoantibodies to mouse neurons dramatically increased the rate and extent of intraneuronal Aβ(42) accumulation in the mouse cerebral cortex and hippocampus. Additionally, individual sera exhibited variable potency related to their capacity to enhance intraneuronal Aβ(42) peptide accumulation and immunolabel neurons in AD brain sections. Replacement of human sera with antibodies targeting abundant neuronal surface proteins resulted in a comparable enhancement of Aβ(42) accumulation in mouse neurons. Overall, results suggest that brain-reactive autoantibodies are ubiquitous in the blood and that a defective BBB allows these antibodies to access the brain interstitium, bind to neuronal surfaces and enhance intraneuronal deposition of Aβ(42) in AD brains. Thus, in the context of BBB compromise, brain-reactive autoantibodies may be an important risk factor for the initiation and/or progression of AD as well as other neurodegenerative diseases.

SUMMARYHuman salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: 1) hydrolysis of starch; 2) binding to hydroxyapatite; and 3) binding to bacteria (e.g. viridans streptococci). Although the active site of HSAmy for starch hydrolysis is well characterized, the regions responsible for the bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharidebinding sites in which aromatic residues are prominently located, we hypothesized that one or more of the secondary saccharide binding sites harboring the aromatic residues may play an important role in bacterial binding. To test this hypothesis, the aromatic residues at five secondary binding sites were mutated to alanine to generate six mutants representing either single (W203A, Y276A and W284A), double (Y276A/W284A and W316A/W388A) or multiple (HSAmy-ar; W134A/W203A/ Y276A/W284A/W316A/W388A) mutations. The crystal structure of HSAmy-ar was determined at a resolution of 1.5 Å as an acarbose complex and compared with the existing wild type acarbose complex. The wild type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch binding, hydroxyapatite and bacterial binding activities. Our results clearly showed that 1) mutation of aromatic residues does not alter the overall conformation of the molecule; 2) the single or double mutants showed either moderate or minimal changes in both starch and bacterial binding activities activity whereas the HSAmy-ar showed significant reduction in these activities; 3) the starch hydrolytic activity was reduced 10-fold in HSAmy-ar; 4) oligosaccharide hydrolytic activity was reduced in all the mutants but the action pattern was similar to that of the wild type enzyme; and 5) the hydroxyaptite binding was unaffected in HSAmy-ar. These results clearly show that the aromatic residues at the secondary saccharide binding sites in HSAmy play a critical role in bacterial binding and starch hydrolytic functions of HSAmy.

Using a porcine model of diabetes mellitus and hypercholesterolaemia, we previously showed that diabetes mellitus and hypercholesterolaemia is associated with a chronic increase in blood-brain barrier permeability in the cerebral cortex, leading to selective binding of immunoglobulin G and deposition of amyloid-beta 1-42 peptide in pyramidal neurons. Treatment with Darapladib (GlaxoSmithKline, SB480848), an inhibitor of lipoprotein-associated phospholipase-A2, alleviated these effects. Here, investigation of the effects of chronic diabetes mellitus and hypercholesterolaemia on the pig retina revealed a corresponding increased permeability of the blood-retina barrier coupled with a leak of plasma components into the retina, alterations in retinal architecture, selective IgG binding to neurons in the ganglion cell layer, thinning of retinal layers due to cell loss and increased glial fibrillary acidic protein expression in Müller cells, all of which were curtailed by treatment with Darapladib. These findings suggest that chronic diabetes mellitus and hypercholesterolaemia induces increased blood-retina barrier permeability that may be linked to altered expression of blood-retina barrier-associated tight junction proteins, claudin and occludin, leading to structural changes in the retina consistent with diabetic retinopathy. Additionally, results suggest that drugs with vascular anti-inflammatory properties, such as Darapladib, may have beneficial effects on eye diseases strongly linked to vascular abnormalities such as diabetic retinopathy and age-related macular degeneration.

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