A Novel Dual Fluorochrome Near-Infrared Imaging Probe for Potential Alzheimer’s Enzyme Biomarkers-BACE1 and Cathepsin D

Abstract

A molecular imaging probe to fluorescently image the β-site of the amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) and cathepsin D (CatD) enzymes associated with Alzheimer’s disease (AD) was designed and synthesized. This imaging probe was built upon iron oxide nanoparticles (cross-linked dextran iron oxide nanoparticles, or CLIO). Peptide substrates containing a terminal near-infrared fluorochrome (fluorophore emitting at 775 nm for CatD or fluorophore emitting at 669 nm for BACE1) were conjugated to the CLIO nanoparticles. The CatD substrate contained a phenylalanine-phenylalanine cleavage site more specific to CatD than BACE1. The BACE1 substrate contained the sequence surrounding the leucine-asparagine cleavage site of the BACE1 found in the Swedish mutation of APP, which is more specific to BACE1 than CatD. These fluorescently-labeled peptide substrates were then conjugated to the nanoparticle. The nanoparticle probes were purified by gel filtration, and their fluorescence intensities were determined using a fluorescence plate reader. The CatD peptide substrate demonstrated a 15.5-fold increase in fluorescence when incubated with purified CatD enzyme, and the BACE1 substrate exhibited a 31.5-fold increase in fluorescence when incubated with purified BACE1 enzyme. Probe specificity was also demonstrated in the human H4 neuroglioma cells and the H4 cells stably transfected with BACE1 in which the probe monitored enzymatic cleavage. In the H4 and H4-BACE1 cells, BACE1 and active CatD activity increased, an occurrence that was reflected in enzyme expression levels as determined by immunoblotting. These results demonstrate the applicability of this probe for detecting potential Alzheimer’s enzyme biomarkers.

Body Fluid Biomarkers for Alzheimer’s Disease—An Up-To-Date Overview

“…The generation of Aβ monomeric forms is dependent upon the activity of BACE1, this being directly related to synaptic functions, plasticity and homeostasis [ 69 , 72 ]. Studies have shown the significantly increased concentrations and activity rates of BACE1 in AD brains and CSF, which is thought to cause a vicious cycle by producing Aβ peptides near synapses [ 69 , 72 , 73 , 74 , 75 ]. Other synaptic dysfunction-associated biomarkers for AD include synaptotagmin, a calcium sensor protein, SNAP-25, a component of the soluble N-ethylmaleimide sensitive factor attachment protein receptor complex, GAP-43, a pre-synaptic membrane protein, and synaptophysin, which has exhibited increased levels in the CSF of AD patients [ 69 , 76 ].…”

Design and Synthesis of New Benzo[d]oxazole-Based Derivatives and Their Neuroprotective Effects on β-Amyloid-Induced PC12 Cells

“…BACE1 is considered as the major form of β-secretases. Therefore, reducing the expression of BACE1 has become one of the therapeutic targets for AD [ 34 ]. The results indicate that compound 5c inhibited expression of BACE1 in PC12 cells ( p < 0.05, p < 0.01 or p < 0.001, Figure 6 ).…”

Induced-volatolomics, a new research field in chemical biology

“…For instance, simultaneously probing several glycosidase activities through the conversion of the corresponding 4-methylumbelliferyl glycosides is not possible because all these probes release the same fluorescent tracer. While activatable dual and/or multimodal fluorescent probes [81][82][83][84] can be envisioned to highlight at least two catalytic activities, their development remains limited due to imaging interferences and/or high background noise. Moreover, differences in the sensitivity between certain imaging modalities (e.g.…”