Transthyretin Mimetics as Anti-ß-Amyloid Agents: A Comparison of Peptide and Protein Approaches.

ß-Amyloid (Aß) aggregation is causally linked to neuronal pathology in Alzheimer's disease; therefore, several small molecules, antibodies, and peptides have been tested as anti-Aß agents. We developed two compounds based on the Aß-binding domain of transthyretin (TTR): a cyclic peptide cG8 and an engineered protein mTTR, and compared them for therapeutically relevant properties. Both mTTR and cG8 inhibit fibrillogenesis of Aß, with mTTR inhibiting at a lower concentration than cG8. Both inhibit aggregation of amylin but not of α-synuclein. They both bind more Aß aggregates than monomer, and neither disaggregates preformed fibrils. cG8 retained more of its activity in the presence of biological materials and was more resistant to proteolysis than mTTR. We examined the effect of mTTR or cG8 on Aß binding to human neurons. When mTTR was co-incubated with Aß under oligomer-forming conditions, Aß morphology was drastically changed and Aß-cell deposition significantly decreased. In contrast, cG8 did not affect morphology but decreased the amount of Aß deposited. These results provide guidance for further evolution of TTR-mimetic anti-amyloid agents.

Cerebrospinal Fluid Proteins as Regulators of Beta-amyloid Aggregation and Toxicity.

Amyloid disorders, such as Alzheimer's, are almost invariably late-onset diseases. One defining diagnostic feature of Alzheimer's disease is the deposition of beta-amyloid as extracellular plaques, primarily in the hippocampus. This raises the question: are there natural protective agents that prevent beta-amyloid from depositing, and is it loss of this protection that leads to onset of disease? Proteins in cerebrospinal fluid (CSF) have been suggested to act as just such natural protective agents. Here, we describe some of the early evidence that led to this suggestion, and we discuss, in greater detail, two CSF proteins that have garnered the bulk of the attention.

Influence of gold nanoparticle surface chemistry and diameter upon Alzheimer's disease amyloid-ß protein aggregation.

BACKGROUND: Deposits of aggregated amyloid-ß protein (Aß) are a pathological hallmark of Alzheimer's disease (AD). Thus, one therapeutic strategy is to eliminate these deposits by halting Aß aggregation. While a variety of possible aggregation inhibitors have been explored, only nanoparticles (NPs) exhibit promise at low substoichiometric ratios. With tunable size, shape, and surface properties, NPs present an ideal platform for rationally designed Aß aggregation inhibitors. In this study, we characterized the inhibitory capabilities of gold nanospheres exhibiting different surface coatings and diameters. RESULTS: Both NP diameter and surface chemistry were found to modulate the extent of aggregation, while NP electric charge influenced aggregate morphology. Notably, 8 nm and 18 nm poly(acrylic acid)-coated NPs abrogated Aß aggregation at a substoichiometric ratio of 1:2,000,000. Theoretical calculations suggest that this low stoichiometry could arise from altered solution conditions near the NP surface. Specifically, local solution pH and charge density are congruent with conditions that influence aggregation. CONCLUSIONS: These findings demonstrate the potential of surface-coated gold nanospheres to serve as tunable therapeutic agents for the inhibition of Aß aggregation. Insights gained into the physiochemical properties of effective NP inhibitors will inform future rational design of effective NP-based therapeutics for AD.

Anthoxanthin Polyphenols Attenuate Aß Oligomer-induced Neuronal Responses Associated with Alzheimer's Disease.

AIMS: Epidemiological evidence implicates polyphenols as potential natural therapeutics for Alzheimer's disease (AD). To investigate this prospect, five anthoxanthin polyphenols were characterized for their ability to reduce amyloid-ß (Aß) oligomer-induced neuronal responses by two mechanisms of action, modulation of oligomerization and antioxidant activity, as well as the synergy between these two mechanisms. METHODS: Anthoxanthin oligomerization modulation and antioxidant capabilities were evaluated and correlated with anthoxanthin attenuation of oligomer-induced intracellular reactive oxygen species (ROS) and caspase activation using human neuroblastoma cell treatments designed to isolate these mechanisms of action and to achieve dual-action. RESULTS: While modulation of oligomerization resulted in only minor reductions to neuronal responses, anthoxanthin antioxidant action significantly attenuated oligomer-induced intracellular ROS and caspase activation. Kaempferol uniquely exhibited synergism when the two mechanisms functioned in concert, leading to a pronounced reduction in both ROS and caspase activation. CONCLUSIONS: Together, these findings identify the dominant mechanism by which these anthoxanthins attenuate Aß oligomer-induced neuronal responses, elucidate their prospective synergy, and demonstrate the potential of anthoxanthin polyphenols as natural AD therapeutics.

Conformational Dynamics of Specific Aß Oligomers Govern Their Ability To Replicate and Induce Neuronal Apoptosis.

Oligomers of amyloid-ß (Aß) have emerged as the primary toxic agents responsible for early synaptic dysfunction and neuronal death in Alzheimer's disease (AD). Characterization of oligomers is an important step in the progress toward delineating the complex molecular mechanisms involved in AD pathogenesis. In our previous reports, we established that a distinct 12-24mer neurotoxic oligomer of Aß42, called Large Fatty Acid derived Oligomers (LFAOs), exhibits a unique property of replication in which LFAOs directly duplicate to quantitatively larger amounts upon interacting with monomers. This self-propagative process of replication is somewhat reminiscent of prion propagation. In this report, we sought to investigate the concentration-dependent conformational dynamics LFAOs undergo and how such transitions manifest in their ability to replicate and induce neuronal apoptosis. The results indicate that LFAOs undergo a concentration-dependent transition between 12mers and disperse 12-24mers with a dissociation constant (Kd) of 0.1 µM. The two species differ in their respective tertiary/quaternary structures but not their secondary structures. This conformational dynamics of LFAOs correlates with their ability to replicate and to induce apoptosis in SH-SY5Y human neuroblastoma cells, with 12mers being more neurotoxic and prone to replication than 12-24mers. The latter result implicates the replication process dominates at low physiological concentrations. The observations made in this report may have profound significance in deciphering the elusive roles of Aß oligomer phenotypes and in determining their prion-type behavior in AD pathology.

A methacrylate-based polymeric imidazole ligand yields quantum dots with low cytotoxicity and low nonspecific binding.

This paper assesses the biocompatibility for fluorescence imaging of colloidal nanocrystal quantum dots (QDs) coated with a recently-developed multiply-binding methacrylate-based polymeric imidazole ligand. The QD samples were purified prior to ligand exchange via a highly repeatable gel permeation chromatography (GPC) method. A multi-well plate based protocol was used to characterize nonspecific binding and toxicity of the QDs toward human endothelial cells. Nonspecific binding in 1% fetal bovine serum was negligible compared to anionically-stabilized QD controls, and no significant toxicity was detected on 24h exposure. The nonspecific binding results were confirmed by fluorescence microscopy. This study is the first evaluation of biocompatibility in QDs initially purified by GPC and represents a scalable approach to comparison among nanocrystal-based bioimaging scaffolds.

Self-propagative replication of Aß oligomers suggests potential transmissibility in Alzheimer disease.

The aggregation of amyloid-ß (Aß) peptide and its deposition in parts of the brain form the central processes in the etiology of Alzheimer disease (AD). The low-molecular weight oligomers of Aß aggregates (2 to 30 mers) are known to be the primary neurotoxic agents whose mechanisms of cellular toxicity and synaptic dysfunction have received substantial attention in the recent years. However, how these toxic agents proliferate and induce widespread amyloid deposition throughout the brain, and what mechanism is involved in the amplification and propagation of toxic oligomer species, are far from clear. Emerging evidence based on transgenic mice models indicates a transmissible nature of Aß aggregates and implicates a prion-like mechanism of oligomer propagation, which manifests as the dissemination and proliferation of Aß toxicity. Despite accumulating evidence in support of a transmissible nature of Aß aggregates, a clear, molecular-level understanding of this intriguing mechanism is lacking. Recently, we reported the characterization of unique replicating oligomers of Aß42 (12-24 mers) in vitro called Large Fatty Acid-derived Oligomers (LFAOs) (Kumar et al., 2012, J. Biol. Chem). In the current report, we establish that LFAOs possess physiological activity by activating NF-κB in human neuroblastoma cells, and determine the experimental parameters that control the efficiency of LFAO replication by self-propagation. These findings constitute the first detailed report on monomer - oligomer lateral propagation reactions that may constitute potential mechanism governing transmissibility among Aß oligomers. These data support the previous reports on transmissible mechanisms observed in transgenic animal models.

In vitro controlled release of an anti-inflammatory from daily disposable therapeutic contact lenses under physiological ocular tear flow.

Novel molecularly imprinted, therapeutic contact lenses capable of controlled release of the non-steroidal anti-inflammatory (NSAID) diclofenac sodium were synthesized, exploiting ionic non-covalent interactions. Poly(HEMA-co-DEAEM-co-PEG200DMA) soft contact lenses were prepared (105±5 µm thickness, diameter 15.0±0.2mm, base curve of 8.6±0.2mm) with different monomer to template ratios and dynamic release studies were conducted in artificial lacrimal solution using two different in vitro methods. Under infinite sink conditions, imprinted contact lenses demonstrated concentration dependent release kinetics. Under physiological flow rates, by increasing the M/T ratio from 1 to 10.5, the release rate decreased from 11.72 µg/h to 6.75 µg/h during the first 48 h. The release rate was more constant, moving toward zero-order release. To use these lenses as daily disposable lenses, the first 24h of release was studied and found to be linear with a rate of 17.27, 11.99, and 8.74 µg/h for M/T ratios of 1, 3.5, and 10.5, respectively. Furthermore, the lenses prepared with a M/T ratio of 10.5 released diclofenac at a rate close to the maximum dose delivered by commercial eye drops, making them ideal for use as daily disposable lenses, and potentially leading to better patient benefit with substantially increased efficacy.

Extended release of high molecular weight hydroxypropyl methylcellulose from molecularly imprinted, extended wear silicone hydrogel contact lenses.

Symptoms of contact lenses induced dry eye (CLIDE) are typically treated through application of macromolecular re-wetting agents via eye drops. Therapeutic soft contact lenses can be formulated to alleviate CLIDE symptoms by slowly releasing comfort agent from the lens. In this paper, we present an extended wear silicone hydrogel contact lens with extended, controllable release of 120 kDa hydroxypropyl methylcellulose (HPMC) using a molecular imprinting strategy. A commercial silicone hydrogel lens was tailored to release approximately 1000 µg of HPMC over a period of up to 60 days in a constant manner at a rate of 16 µg/day under physiological flowrates, releasing over the entire range of continuous wear. Release rates could be significantly varied by the imprinting effect and functional monomer to template ratio (M/T) with M/T values 0, 0.2, 2.8, 3.4 corresponding to HPMC release durations of 10, 13, 23, and 53 days, respectively. Lenses had high optical quality and adequate mechanical properties for contact lens use. This work highlights the potential of imprinting in the design and engineering of silicone hydrogel lenses to release macromolecules for the duration of wear, which may lead to decreased CLIDE symptoms and more comfortable contact lenses.