Spin Labeled Fluorene Compounds are a Versatile Sword in the Fight Against Amyloid Beta Peptide of Alzheimer's Disease

Amyloid-β (Aβ) peptide is generated after sequential cleavage of the constitutively expressed amyloid precursor protein (APP) by γ and β secretases, and is recognized as the primary causative agent underlying the neuropathogenesis of Alzheimer’sDisease (AD). Once generated, monomeric Aβ demonstrates a high propensity to aggregate into toxic Aβ oligomers (AβO) of various sizes, which eventually accumulate in the brain in the form of amyloid plaques. Mutations in either the gene for APP or one or both of its processing genes, presenilin-1 (PS1) and presenilin-2 (PS2) of the secretases complex leading to accumulation of Aβ and early-onset familial AD. Late onset AD is modulated by mutations in the gene for apolipoprotein E (apo-E), with the isoform apo-E4 leading to an approximate eight-fold increase in risk for AD, and by environmental and life style factors. The Alzheimer’s disease process develops over decades, with substantial neurological loss occurring before a clinical diagnosis of dementia can be rendered. A major roadblock to the management of AD is the inability to definitively diagnose AD until post-mortem examination. It is therefore imperative to develop methods that permit safe, early detection and monitoring of disease progression. Magnetic resonance imaging (MRI) is a non-invasive way to detect and monitor AD progression and therapy, but so far MRI contrast has been obtained only using Gd(III) based contrast agents. Fluorene compounds have garnered attention as amyloid imaging agents. Our lab has developed a spin labeled fluorene (SLF) compound that contains a fluorene moiety with known affinity for Aβ and a pyrroline nitroxyl spin-label moiety. We hypothesized that the SLF compound will specifically coat assemblies of amyloid beta in the brain and, by establishing a boundary of magnetic field inhomogeneity, produce MRI contrast in tissues with elevated levels of the Aβ peptide. I found that labeling of brain specimens with the SLF compound produces negative contrast in samples from AD model mice whereas no negative contrast is seen in specimens harvested from wild-type mice. Injection of SLF into live mice resulted in good brain penetration, with the compound able to generate contrast 24-hr post injection. (Abstract shortened by ProQuest.

Spectroscopic Characterization of Structural Changes in Membrane Scaffold Proteins Entrapped within Mesoporous Silica Gel Monoliths.

The changes in the orientation and conformation of three different membrane scaffold proteins (MSPs) upon entrapment in sol-gel-derived mesoporous silica monoliths were investigated. MSPs were examined in either a lipid-free or a lipid-bound conformation, where the proteins were associated with lipids to form nanolipoprotein particles (NLPs). NLPs are water-soluble, disk-shaped patches of a lipid bilayer that have amphiphilic MSPs shielding the hydrophobic lipid tails. The NLPs in this work had an average thickness of 5 nm and diameters of 9.2, 9.7, and 14.8 nm. We have previously demonstrated that NLPs are more suitable lipid-based structures for silica gel entrapment than liposomes because of their size compatibility with the mesoporous network (2-50 nm) and minimally altered structure after encapsulation. Here we further elaborate on that work by using a variety of spectroscopic techniques to elucidate whether or not different MSPs maintain their protein-lipid interactions after encapsulation. Fluorescence spectroscopy and quenching of the tryptophan residues with acrylamide, 5-DOXYL-stearic acid, and 16-DOXYL-stearic acid were used to determine the MSP orientation. We also utilized fluorescence anisotropy of tryptophans to measure the relative size of the NLPs and MSP aggregates after entrapment. Finally, circular dichroism spectroscopy was used to examine the secondary structure of the MSPs. Our results showed that, after entrapment, all of the lipid-bound MSPs maintained orientations that were minimally changed and indicative of association with lipids in NLPs. The tryptophan residues appeared to remain buried within the hydrophobic core of the lipid tails in the NLPs and appropriately spaced from the bilayer center. Also, after entrapment, lipid-bound MSPs maintained a high degree of α-helical content, a secondary structure associated with protein-lipid interactions. These findings demonstrate that NLPs are capable of serving as viable hosts for functional integral membrane proteins in the synthesis of sol-gel-derived bioinorganic hybrid nanomaterials

Spectroscopic Characterization of Structural Changes in Membrane Scaffold Proteins Entrapped within Mesoporous Silica Gel Monoliths

The changes in the orientation and conformation of three different membrane scaffold proteins (MSPs) upon entrapment in sol–gel-derived mesoporous silica monoliths were investigated. MSPs were examined in either a lipid-free or a lipid-bound conformation, where the proteins were associated with lipids to form nanolipoprotein particles (NLPs). NLPs are water-soluble, disk-shaped patches of a lipid bilayer that have amphiphilic MSPs shielding the hydrophobic lipid tails. The NLPs in this work had an average thickness of 5 nm and diameters of 9.2, 9.7, and 14.8 nm. We have previously demonstrated that NLPs are more suitable lipid-based structures for silica gel entrapment than liposomes because of their size compatibility with the mesoporous network (2–50 nm) and minimally altered structure after encapsulation. Here we further elaborate on that work by using a variety of spectroscopic techniques to elucidate whether or not different MSPs maintain their protein–lipid interactions after encapsulation. Fluorescence spectroscopy and quenching of the tryptophan residues with acrylamide, 5-DOXYL-stearic acid, and 16-DOXYL-stearic acid were used to determine the MSP orientation. We also utilized fluorescence anisotropy of tryptophans to measure the relative size of the NLPs and MSP aggregates after entrapment. Finally, circular dichroism spectroscopy was used to examine the secondary structure of the MSPs. Our results showed that, after entrapment, all of the lipid-bound MSPs maintained orientations that were minimally changed and indicative of association with lipids in NLPs. The tryptophan residues appeared to remain buried within the hydrophobic core of the lipid tails in the NLPs and appropriately spaced from the bilayer center. Also, after entrapment, lipid-bound MSPs maintained a high degree of α-helical content, a secondary structure associated with protein–lipid interactions. These findings demonstrate that NLPs are capable of serving as viable hosts for functional integral membrane proteins in the synthesis of sol–gel-derived bioinorganic hybrid nanomaterials

A Bifunctional Anti-Amyloid Blocks Oxidative Stress and the Accumulation of Intraneuronal Amyloid-Beta.

There is growing recognition regarding the role of intracellular amyloid beta (Aβ) in the Alzheimer's disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aβ likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aβ and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aβ accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aβ. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aβ. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aβ-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aβ targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aβ, its promotion of ROS, and Aβ metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aβ oligomer formation

A Bifunctional Anti-Amyloid Blocks Oxidative Stress and the Accumulation of Intraneuronal Amyloid-Beta

There is growing recognition regarding the role of intracellular amyloid beta (Aβ) in the Alzheimer’s disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aβ likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aβ and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aβ accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aβ. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aβ. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aβ-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aβ targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aβ, its promotion of ROS, and Aβ metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aβ oligomer formation

Analysis of lipid phase behavior and protein conformational changes in nanolipoprotein particles upon entrapment in sol-gel-derived silica.

The entrapment of nanolipoprotein particles (NLPs) and liposomes in transparent, nanoporous silica gel derived from the precursor tetramethylorthosilicate was investigated. NLPs are discoidal patches of lipid bilayer that are belted by amphiphilic scaffold proteins and have an average thickness of 5 nm. The NLPs in this work had a diameter of roughly 15 nm and utilized membrane scaffold protein (MSP), a genetically altered variant of apolipoprotein A-I. Liposomes have previously been examined inside of silica sol-gels and have been shown to exhibit instability. This is attributed to their size (∼150 nm) and altered structure and constrained lipid dynamics upon entrapment within the nanometer-scale pores (5-50 nm) of the silica gel. By contrast, the dimensional match of NLPs with the intrinsic pore sizes of silica gel opens the possibility for their entrapment without disruption. Here we demonstrate that NLPs are more compatible with the nanometer-scale size of the porous environment by analysis of lipid phase behavior via fluorescence anisotropy and analysis of scaffold protein secondary structure via circular dichroism spectroscopy. Our results showed that the lipid phase behavior of NLPs entrapped inside of silica gel display closer resemblance to its solution behavior, more so than liposomes, and that the MSP in the NLPs maintain the high degree of α-helix secondary structure associated with functional protein-lipid interactions after entrapment. We also examined the effects of residual methanol on lipid phase behavior and the size of NLPs and found that it exerts different influences in solution and in silica gel; unlike in free solution, silica entrapment may be inhibiting NLP size increase and/or aggregation. These findings set precedence for a bioinorganic hybrid nanomaterial that could incorporate functional integral membrane proteins

Novel Stilbene-Nitroxyl Hybrid Compounds Display Discrete Modulation of Amyloid Beta Toxicity and Structure.

Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These "toxic oligomers" have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AβO) and the oxidative stress elicited by AβO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer's disease in a powerful, synergistic manner. Examination of AβO structure by various biophysical tools shows that each stilbene candidate uniquely alters AβO conformation and toxicity, providing insight towards the future development of structural correctors for AβO. Correlations of AβO structural modulation and bioactivity displayed by each provides insights for future testing in vivo. The multi-target activity of these hybrid molecules represents a highly advantageous feature for disease modification in Alzheimer's, which displays a complex, multifactorial etiology. Importantly, these novel small molecules intervene with intraneuronal AβO, a necessary feature to counter the cycle of dysregulation, oxidative stress and inflammation triggered during the earliest stages of disease progression