Efficacy of Multifunctionalized Saccharide Constructs for the Attenuation of Amyloid-beta Toxicity

There is evidence that amyloid-beta (Aβ) toxicity is mediated through interactions and binding with neuronal surface sialic acids in Alzheimer’s disease (AD). The binding affinity is higher if the sialic acids are clustered and toxicity of Aβ was attenuated by removal of neuronal sialic acids. Thus, interfering with cell membrane-Aβ binding using biomimetics that could reproduce the clustered sialic acid structure could present us with a potential target for therapeutic intervention in AD. Based on this hypothesis, we developed several multifunctionalized sialic acid labeled chitosan compounds of different valency, or number of sialic acid per chitosan molecule, to attenuate Aβ toxicity. A cross-linker, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was used, which provided control over the degree of labeling of chitosan. After characterization, the ability of the complexes to attenuate toxicity of Aβ(1-40) was investigated in vitro. We found that all linear polysialylated complexes showed significant ability to attenuate Aβ toxicity, with optimum balance between intrinsic toxicity and protection around 37% labeling of chitosan. Moreover, unlabeled chitosan also showed some level of protective properties to the labeled compounds. Then, four biological sugars that are structural analogs of sialic acid (N-Acetylneuraminic acid) were used to decorate approximately 35% of the chitosan backbone using EDC chemistry. After characterization, the ability of these sugar complexes to attenuate toxicity of Aβ was investigated in vitro. We investigated whether sugars other than sialic acid provided better toxicity attenuation and attempted to understand the impact of sub-structures or unique –R groups of sialic acid and its analogs in Aβ toxicity attenuation. Our results show that oxygen substitution in the ring structure contributes to the intrinsic toxicity but also plays a role in Aβ toxicity attenuation. Similarly, the multi –OH tail present in sialic acid plays an important role in Aβ toxicity attenuation. This approach of designing effective biomimetics and of determining the structure-activity relationship has relevance with respect to the development of new intelligent class of therapeutic agents for AD. Although this work focuses on AD, this approach can be extended to other diseases involving misfolded proteins

Sialic Acid Conjugated Chitosan for the Attenuation of Amyloid-beta Toxicity

Amyloid-beta (Aβ), a 39 to 43 amino acid long peptide, is the primary species identified in senile plaques associated with Alzheimer’s disease (AD) and has been implicated in the neurotoxicity associated with AD. It is believed that Aβ toxicity is mediated through the interaction with neuronal membranes. A variety of evidence indicates that 1) Aβ may bind to the cell surface sialic acids, 2) the affinity of this interaction is higher if the gangliosides or sialic acids on the cell surface are clustered, 3) the removal of the surface sialic acids attenuate Aβ toxicity. Based on this data, we hypothesized that a biomimetic compound could be synthesized which would reproduce the clustered sialic acid structure of the cell surface, having antibody-like affinity towards Aβ, thus competing with the cell surface for Aβ binding. Our technique relies on attacking the theoretical “bottleneck” region in the Alzheimer’s process, i.e. the interaction of Aβ with neurons. This area can be considered as a bottleneck as there are several mechanisms that can transform the Aβ peptide into its toxic form. Also, the exact toxic form of Aβ peptide that attacks neurons is not agreed upon. However, it is agreed that preventing neuronal interaction prevents toxicity making the Aβ-cell interaction the “bottleneck” region. To explore this hypothesis further, we developed different sialic acid labeled compounds of different valency or number of sialic acids per molecule to attenuate Aβ toxicity. For this purpose, chitosan was used as a carrier molecule for sialic acids. EDC along with Sulfo-NHS was used as a cross-linker to couple the sialic acids with chitosan, with control over the degree of labeling. After verifying the presence of sialic acids on chitosan, the ability of this sialic acid-chitosan complex to attenuate the toxicity of aggregated Aβ was investigated in-vitro. Preliminary results indicate that the complex synthesized is biocompatible. Also, the results suggested that the compound has Aβ toxicity attenuating properties. Further studies will help elucidate the role of cell-surface sialic acids in Aβ toxicity. Drugs available today are merely symptoms alleviating and thus, these results can have implications in the design of intelligent compounds that can bind pathogenic Aβ for the treatment of Alzheimer’s disease

Synthesis, characterization, and preclinical validation of a PET radiopharmaceutical for interrogating Aβ (β-amyloid) plaques in Alzheimer’s disease

BACKGROUND: PET radiopharmaceuticals capable of imaging β-amyloid (Aβ) plaque burden in the brain could offer highly valuable diagnostic tools for clinical studies of Alzheimer’s disease. To further supplement existing armamentarium of FDA-approved agents as well as those under development, and to correlate multiphoton-imaging data reported earlier, herein, we describe preclinical validation of a PET tracer. METHODS: A novel PET radiopharmaceutical ((18)F-7B) was synthesized and characterized. To assess its affinity for Aβ, binding assays with Aβ(1-42) fibrils, Alzheimer’s disease (AD) homogenates, and autoradiography studies and their IHC correlations were performed. For assessing its overall pharmacokinetic profiles in general and its ability to cross the blood-brain barrier (BBB) in particular, biodistribution studies in normal mice were performed. Finally, for evaluating potential for (18)F-7B to serve as a targeted Aβ probe, the microPET/CT imaging was performed in age-matched amyloid precursor protein/presenilin-1 (APP/PS1) mice and wild-type (WT) counterparts. RESULTS: The radiotracer (18)F-7B shows saturable binding to autopsy-confirmed AD homogenates (K(d) = 17.7 nM) and Aβ(1-42) fibrils (K(d) = 61 nM). Preliminary autoradiography studies show binding of (18)F-7B to cortical Aβ plaques in autopsy-confirmed AD tissue sections, inhibition of that binding by unlabeled counterpart 7A-indicating specificity, and a good correlation of tracer binding with Aβ immunostaining. The agent indicates high initial penetration into brains (7.23 ± 0.47%ID/g; 5 min) of normal mice, thus indicating a 5-min/120-min brain uptake clearance ratio of 4.7, a benchmark value (>4) consistent with the ability of agents to traverse the BBB to enable PET brain imaging. Additionally, (18)F-7B demonstrates the presence of parental species in human serum. Preliminary microPET/CT imaging demonstrates significantly higher retention of (18)F-7B in brains of transgenic mice compared with their WT counterparts, consistent with expected binding of the radiotracer to Aβ plaques, present in APP/PS1 mice, compared with their age-matched WT counterparts lacking those Aβ aggregates. CONCLUSIONS: These data offer a platform scaffold conducive to further optimization for developing new PET tracers to study Aβ pathophysiology in vitro and in vivo

VCP suppresses proteopathic seeding in neurons

BACKGROUND: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43. METHODS: We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice. RESULTS: One hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons. CONCLUSION: Using an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP\u27s surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP

Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation

Aggregates of the RNA-binding protein TDP-43 (TAR DNAbinding protein) are a hallmark of the overlapping neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stage is primarily driven by TDP-43’s RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid–liquid phase separation and detected similar detergentresistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than forWTTDP-43 aggregates.We provide evidence for aninitial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbationsin TDP-43 homeostasisin protein aggregation and ALS-FTD pathogenesis

Fluselenamyl: A novel benzoselenazole derivative for PET detection if amyloid plaques (Aβ) in Alzheimer\u27s disease

Fluselenamyl (5), a novel planar benzoselenazole shows traits desirable of enabling noninvasive imaging of Aβ pathophysiology in vivo; labeling of both diffuse (an earlier manifestation of neuritic plaques) and fibrillar plaques in Alzheimer’s disease (AD) brain sections, and remarkable specificity for mapping Aβ compared with biomarker proteins of other neurodegenerative diseases. Employing AD homogenates, [(18)F]-9, a PET tracer demonstrates superior (2–10 fold higher) binding affinity than approved FDA tracers, while also indicating binding to high affinity site on Aβ plaques. Pharmacokinetic studies indicate high initial influx of [(18)F]-9 in normal mice brains accompanied by rapid clearance in the absence of targeted plaques. Following incubation in human serum, [(18)F]-9 indicates presence of parental compound up to 3h thus indicating its stability. Furthermore, in vitro autoradiography studies of [(18)F]-9 with AD brain tissue sections and ex vivo autoradiography studies in transgenic mouse brain sections show cortical Aβ binding, and a fair correlation with Aβ immunostaining. Finally, multiphoton- and microPET/CT imaging indicate its ability to penetrate brain and label parenchymal plaques in transgenic mice. Following further validation of its performance in other AD rodent models and nonhuman primates, Fluselenamyl could offer a platform technology for monitoring earliest stages of Aβ pathophysiology in vivo

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn

Quantifying regional α -synuclein, amyloid β, and tau accumulation in Lewy body dementia

OBJECTIVE: Parkinson disease (PD) is defined by the accumulation of misfolded α-synuclein (α-syn) in Lewy bodies and Lewy neurites. It affects multiple cortical and subcortical neuronal populations. The majority of people with PD develop dementia, which is associated with Lewy bodies in neocortex and referred to as Lewy body dementia (LBD). Other neuropathologic changes, including amyloid β (Aβ) and tau accumulation, occur in some LBD cases. We sought to quantify α-syn, Aβ, and tau accumulation in neocortical, limbic, and basal ganglia regions. METHODS: We isolated insoluble protein from fresh frozen postmortem brain tissue samples for eight brains regions from 15 LBD, seven Alzheimer disease (AD), and six control cases. We measured insoluble α-syn, Aβ, and tau with recently developed sandwich ELISAs. RESULTS: We detected a wide range of insoluble α-syn accumulation in LBD cases. The majority had substantial α-syn accumulation in most regions, and dementia severity correlated with neocortical α-syn. However, three cases had low neocortical levels that were indistinguishable from controls. Eight LBD cases had substantial Aβ accumulation, although the mean Aβ level in LBD was lower than in AD. The presence of Aβ was associated with greater α-syn accumulation. Tau accumulation accompanied Aβ in only one LBD case. INTERPRETATION: LBD is associated with insoluble α-syn accumulation in neocortical regions, but the relatively low neocortical levels in some cases suggest that other changes contribute to impaired function, such as loss of neocortical innervation from subcortical regions. The correlation between Aβ and α-syn accumulation suggests a pathophysiologic relationship between these two processes