Specific amyloid ß clearance by a catalytic antibody construct.

Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid ß (Aß)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aß C terminus noncovalently and hydrolyzed Aß rapidly, with no reactivity to the Aß precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aß dipeptide unit. The catabody dissolved preformed Aß aggregates and inhibited Aß aggregation more potently than an Aß-binding IgG. Intravenous catabody treatment reduced brain Aß deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aß hydrolysis appears to be an innate immune function that could be applied for therapeutic Aß removal.

Antibody uptake into neurons occurs primarily via clathrin-dependent Fcγ receptor endocytosis and is a prerequisite for acute tau protein clearance.

Tau immunotherapy is effective in transgenic mice, but the mechanisms of Tau clearance are not well known. To this end, Tau antibody uptake was analyzed in brain slice cultures and primary neurons. Internalization was rapid (<1 h), saturable, and substantial compared with control mouse IgG. Furthermore, temperature reduction to 4 °C, an excess of unlabeled mouse IgG, or an excess of Tau antibodies reduced uptake in slices by 63, 41, and 62%, respectively (p = 0.002, 0.04, and 0.005). Uptake strongly correlated with total and insoluble Tau levels (r(2) = 0.77 and 0.87 and p = 0.002 and 0.0002), suggesting that Tau aggregates influence antibody internalization and/or retention within neurons. Inhibiting phagocytosis did not reduce uptake in slices or neuronal cultures, indicating limited microglial involvement. In contrast, clathrin-specific inhibitors reduced uptake in neurons (≤ 78%, p < 0.0001) and slices (≤ 35%, p = 0.03), demonstrating receptor-mediated endocytosis as the primary uptake pathway. Fluid phase endocytosis accounted for the remainder of antibody uptake in primary neurons, based on co-staining with internalized dextran. The receptor-mediated uptake is to a large extent via low affinity FcγII/III receptors and can be blocked in slices (43%, p = 0.04) and neurons (53%, p = 0.008) with an antibody against these receptors. Importantly, antibody internalization appears to be necessary for Tau reduction in primary neurons. Overall, these findings clarify that Tau antibody uptake is primarily receptor-mediated, that these antibodies are mainly found in neurons with Tau aggregates, and that their intracellular interaction leads to clearance of Tau pathology, all of which have major implications for therapeutic development of this approach.

Postsynaptic degeneration as revealed by PSD-95 reduction occurs after advanced Aß and tau pathology in transgenic mouse models of Alzheimer's disease.

Impairment of synaptic plasticity underlies memory dysfunction in Alzheimer's disease (AD). Molecules involved in this plasticity such as PSD-95, a major postsynaptic scaffold protein at excitatory synapses, may play an important role in AD pathogenesis. We examined the distribution of PSD-95 in transgenic mice of amyloidopathy (5XFAD) and tauopathy (JNPL3) as well as in AD brains using double-labeling immunofluorescence and confocal microscopy. In wild type control mice, PSD-95 primarily labeled neuropil with distinct distribution in hippocampal apical dendrites. In 3-month-old 5XFAD mice, PSD-95 distribution was similar to that of wild type mice despite significant Aß deposition. However, in 6-month-old 5XFAD mice, PSD-95 immunoreactivity in apical dendrites markedly decreased and prominent immunoreactivity was noted in neuronal soma in CA1 neurons. Similarly, PSD-95 immunoreactivity disappeared from apical dendrites and accumulated in neuronal soma in 14-month-old, but not in 3-month-old, JNPL3 mice. In AD brains, PSD-95 accumulated in Hirano bodies in hippocampal neurons. Our findings support the notion that either Aß or tau can induce reduction of PSD-95 in excitatory synapses in hippocampus. Furthermore, this PSD-95 reduction is not an early event but occurs as the pathologies advance. Thus, the time-dependent PSD-95 reduction from synapses and accumulation in neuronal soma in transgenic mice and Hirano bodies in AD may mark postsynaptic degeneration that underlies long-term functional deficits.

Tau antibody chimerization alters its charge and binding, thereby reducing its cellular uptake and efficacy.

BACKGROUND: Bringing antibodies from pre-clinical studies to human trials requires humanization, but this process may alter properties that are crucial for efficacy. Since pathological tau protein is primarily intraneuronal in Alzheimer's disease, the most efficacious antibodies should work both intra- and extracellularly. Thus, changes which impact uptake or antibody binding will affect antibody efficacy. METHODS: Initially, we examined four tau mouse monoclonal antibodies with naturally differing charges. We quantified their neuronal uptake, and efficacy in preventing toxicity and pathological seeding induced by human-derived pathological tau. Later, we generated a human chimeric 4E6 (h4E6), an antibody with well documented efficacy in multiple tauopathy models. We compared the uptake and efficacy of unmodified and chimeric antibodies in neuronal and differentiated neuroblastoma cultures. Further, we analyzed tau binding using ELISA assays. FINDINGS: Neuronal uptake of tau antibodies and their efficacy strongly depends on antibody charge. Additionally, their ability to prevent tau toxicity and seeding of tau pathology does not necessarily go together. Particularly, chimerization of 4E6 increased its charge from 6.5 to 9.6, which blocked its uptake into human and mouse cells. Furthermore, h4E6 had altered binding characteristics despite intact binding sites, compared to the mouse antibody. Importantly, these changes in uptake and binding substantially decreased its efficacy in preventing tau toxicity, although under certain conditions it did prevent pathological seeding of tau. CONCLUSIONS: These results indicate that efficacy of chimeric/humanized tau antibodies should be thoroughly characterized prior to clinical trials, which may require further engineering to maintain or improve their therapeutic potential. FUND: National Institutes of Health (NS077239, AG032611, R24OD18340, R24OD018339 and RR027990, Alzheimer's Association (2016-NIRG-397228) and Blas Frangione Foundation.

Role of active-site residues of dispersin B, a biofilm-releasing beta-hexosaminidase from a periodontal pathogen, in substrate hydrolysis.

Dispersin B (DspB), a family 20 beta-hexosaminidase from the oral pathogen Aggregatibacter actinomycetemcomitans, cleaves beta(1,6)-linked N-acetylglucosamine polymer. In order to understand the substrate specificity of DspB, we have undertaken to characterize several conserved and nonconserved residues in the vicinity of the active site. The active sites of DspB and other family 20 hexosaminidases possess three highly conserved acidic residues, several aromatic residues and an arginine at subsite -1. These residues were mutated using site-directed mutagenesis and characterized for their enzyme activity. Our results show that a highly conserved acid pair in beta-hexosaminidases D183 and E184, and E332 play a critical role in the hydrolysis of the substrates. pH activity profile analysis showed a shift to a higher pH (6.8) in the optimal activity for the E184Q mutant, suggesting that this residue might act as the acid/base catalyst. The reduction in k(cat) observed for Y187A and Y278A mutants suggests that the Y187 residue (unique to DspB) located on a loop might play a role in substrate specificity and be a part of subsite +1, whereas the hydrogen-bond interaction between Y278A and the N-acetyl group might help to stabilize the transition state. Mutation of W237 and W330 residues abolished hydrolytic activity completely suggesting that alteration at these positions might collapse the binding pocket for the N-acetyl group. Mutation of the conserved R27 residue (to R27A or R27K) also caused significant reduction in k(cat) suggesting that R27 might be involved in stabilization of the transition state. From these results, we conclude that in DspB, and possibly in other structurally similar family 20 hydrolases, some residues at the active site assist in orienting the N-acetyl group to participate in the substrate-assisted mechanism, whereas other residues such as R27 and E332 assist in holding the terminal N-acetylglucosamine during the hydrolysis.

Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-amylase in substrate hydrolysis and bacterial binding.

Human salivary alpha-amylase (HSAmy) has three distinct functions relevant to oral health: (1) hydrolysis of starch, (2) binding to hydroxyapatite (HA), 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 bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding 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 [W134A/W203A/Y276A/W284A/W316A/W388A; human salivary alpha-amylase aromatic residue multiple mutant (HSAmy-ar)] mutations. The crystal structure of HSAmy-ar as an acarbose complex was determined at a resolution of 1.5 A 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 activity, HA-binding activity, and bacterial binding activity. Our results clearly showed that (1) mutation of aromatic residues does not alter the overall conformation of the molecule; (2) single or double mutants showed either moderate or minimal changes in both starch-binding activity and bacterial binding activity, whereas HSAmy-ar showed significant reduction in these activities; (3) starch-hydrolytic activity was reduced by 10-fold in HSAmy-ar; (4) oligosaccharide-hydrolytic activity was reduced in all mutants, but the action pattern was similar to that of the wild-type enzyme; and (5) HA 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 in starch-hydrolytic functions of HSAmy.