Developing amyloid-β chaperone lipocalin-type prostaglandin D synthase protein as a magnetic resonance active early diagnostic tool

• Main Author: Sharma, Bhargy
• Other Authors: Konstantin Pervushin
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences::Biophysics
• Online Access: https://hdl.handle.net/10356/136881
• Institution: Nanyang Technological University

Lipocalin-type prostaglandin D synthase (L-PGDS) is an endogenous brain protein, shown previously as a significant chaperone for amyloid β (Aβ) peptide. It binds to monomeric Aβ as well as mature fibrils and is capable of inhibiting their aggregation. We recently showed that L-PGDS could successfully breakdown mature Aβ fibrils (manuscript submitted), which opens avenues for its use as a therapeutic agent in cases of Alzheimer's disease (AD). This thesis is aimed at utilizing the theranostic (therapeutic + diagnostic) potential of L-PGDS in AD by early detection of Aβ using MRI. For this purpose, we covalently conjugated the recombinant L-PGDS protein with iron oxide-based nanoparticles with different outer coatings. Four different nanoparticles were compared for their T2 contrast enhancement. The functionality of the conjugated protein, its inhibition activity towards Aβ, the effect on cellular viability and the tendency to form aggregates were compared to choose the most efficient composition. For this purpose, we used a multi-disciplinary approach using chemical and biophysical assays, spectrophotometric techniques, structural and morphological studies, cellular assays and tissue histology, and pre-clinical mouse imaging to develop an L-PGDS-based theranostic molecular probe. Based on the results of in vitro assay, the biological probes designed by covalent conjugation of magnetic resonance active ferritin protein nanocages conjugated with L-PGDS were used for studies in AD mice. Injections in diseased mice showed hypointensity in mouse brain areas correlated with the presence of amyloid-rich structures compared to age-matched healthy mice. All MRI data were acquired using Bruker spectrometers, and Paravision 6.0 software was used for processing and analysis. T2 relaxation curves were prepared for nanoparticles to compare relaxivity differences in the biological buffer. Differences in T2-star weighted image intensities were identified in the brains of healthy mice after non-invasive intranasal administration of conjugated L-PGDS probes. L-PGDS conjugated with ferritin nanocages injected in the ventricular chambers were seen to disperse towards the amyloid-rich brain regions in AD mice. To this end, we have shown here that L-PGDS protein has great outlook as a diagnostic agent for early identification of AD hallmarks in disease-prone populations, and potential as a therapeutic intervention.