A group from the Harvard Medical School has developed a molecular imaging probe that can fluorescently image three key proteins associated with the development of Alzheimer’s disease (AD) and related dementias (ADRD). These include the amyloid precursor protein (APP), cleaving enzyme 1 (BACE1) and cathepsin D (CatD) enzymes.
The team has developed and characterized a novel molecular imaging (MI) probe capable of simultaneously detecting AD enzymatic biomarkers, BACE1 and CatD. The detection of such proteins provides an encouraging prognosis for AD. The rationale behind this conclusion is based on knowledge of the enzymic cleavage activities in the pathophysiology of AD.
The socio-economic burden of AD as ADRDs have driven effective prevention measures including positron emission tomography (PET) radioligands for Aβ amyloid plaques, brain structural and vascular changes, tau tangles, and, Lewy bodies, etc. The detection and quantification of these biomarkers aid an understanding of the etiopathogenesis of AD and ADRD.
They also provide therapeutic endpoints in dementia treatment; at present, current biomarkers are invasive or expensive. As such it is imperative to develop imaging probes of biomarkers that can differentiate normal and abnormal brain function that can reliably indicate or correlate decline in cognition. This has motivated the team’s work in the development of molecular imaging probes that are capable of characterizing, diagnosing, and predicting outcomes in neurodegenerative diseases.
The production of Aβ peptide is thought to be a critical step in the progression of AD. This peptide can aggregate into plaques and is produced by 2 cleavage events by the protease enzymes β- and γ-secretase. These enzymes catalyze the endo proteolysis of the amyloid precursor protein (APP); it is this secretase step that represents the first committed event in Aβ amyloidogenesis.
BACE1 is regarded as the major class of β-secretase and has, therefore, become a therapeutic target. CatD is a cathepsin which forms part of the lysosomal system, is also instrumental in the progression of the disease. Cathepsins influence both the production and clearance of Aβ; they are upregulated, disrupting the lysosomal system, which culminates in an increase in intracellular Aβ concentrations.
This overproduction of Aβ Results in their secretion into the extracellular space, prompting aggregation. Previous studies have shown that APP degradation results from overexpression of several proteases which disrupts the cycle of generation and degradation in the lysosomal compartments. A key finding in these studies was the understanding that the protease function is segregated between cysteine and non-cysteine proteases; CatD is a non-cysteine protease.
These enzymatic processes provide biomarkers for AD disease progression. To be able to understand the action of the enzymes involved in the production and degradation of APP, the development of an imaging probe is necessary. Previous molecular imaging (MI) probes have focused on those which can be visualized using MRI, CT or single-photon emission computed tomography (SPECT).
Increasingly, however, fluorescent imaging is being utilized. The team concludes that BACE1 and CatD1 serve as effective biomarkers; by understanding their roles, the pathogenesis of AD can be understood. To achieve this, the team produced a multimodal, multi-wavelength, near-infrared fluorescence (NIRF) capable of detecting CatD and BACE1 in both cell culture and cell-free conditions, based on a previous probe that used a nanoparticle foundation to quench fluorescently labeled substrates.
The peptide substrates had distinct wavelengths with minimal spectral overlap; in addition, these fluorochromes had wavelengths in the near-infrared region of the spectrum, resulting in greater photo penetration. This probe represents a way to accelerate the preclinical and clinical evaluation of new therapeutic substances for AD.
The probe is based on iron oxide nanoparticles (cross-linked dextran iron oxide nanoparticles, called CLIO). Proteins that possess a terminal near-infrared fluorochrome can be conjugated to CLIO nanoparticles. The fluorescence is quenched until CatD or BACE cleaves this complex (comprised of an iron oxide nanoparticle and fluorescently labeled peptide substrates).
This cleavage of these fluorescently labeled peptide substrates results in a fluorescence signal increase., which was purified by gel filtration and subject to fluorescence intensity quantification using a fluorescence plate reader. The CatD substrate increased in fluorescence intensity by 15.5-fold following incubation with the CatD enzyme while the BACE1 substrate showed a 31.5-fold increase following cleavage by its corresponding enzyme.
Although both enzymes BACE1 and CatD, have distinct specificity, the team speculate that future experiments should include the Michalis-Menten kinetics to quantitatively compare enzyme specificity. The team determined that despite the fluorescence activation of the imaging probe indicating the expression in immunoblots, fluorescence activation was also caused by non-specific proteases. The inclusion of Michalis-Menten kinetics will, therefore, determine these non-specific activities.
The team concludes that the CLIO/NIRF-based molecular imaging probe offers a new means of detecting BACE1 and CatD enzymatic activities.
Future direction of study is hoped to include the testing the specificity of BACE1, together with the measurement of the Michaelis Menten enzyme parameters for both the substrate and the enzyme. the parameters for other pro teasers I hoped to build a better picture of the behavior of the CatD and BACE1 probes.
A further suggestion by Tam et al. is testing of the iron oxide cores as magnetic resonance contrast agents – alongside their role as an optical probe, which Is hoped to validate the role of the proteases in the etiopathogenesis of AD.
Tam, J.M. et al. (2020) A Novel Dual Fluorochrome Near-Infrared Imaging Probe for Potential Alzheimer’s Enzyme Biomarkers-BACE1 and Cathepsin D. Molecules. DOI: https://doi.org/10.3390/molecules25020274
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Last Updated: Mar 12, 2020
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