Mechanisms of aggregation and pathogenesis of the human aortic medial amyloid peptide medin

Abstract

Cardiovascular amyloidosis affects the health of a significant proportion of the global population. These diseases are characterised by the deposition of aggregates of misfolded protein called amyloid in the heart and vascular tissues. The commonly known conditions are cerebral amyloid angiopathy [CAA; a comorbidity in at least 40% of patients of Alzheimer’s disease (AD)], AL (deposition of immunoglobulin light chains), AA (deposition of serum amyloid A) and ATTR (transthyretin aggregation) amyloidoses. Over the past three decades, some more vascular diseases of the upper body, such as giant cell arteritis (GCA; the most common arterial inflammatory disease), thoracic aortic aneurysm (TAA) and dissection, and vascular dementia (VaD), have been added to the list due to the predominance of an amyloidogenic peptide called medin involved in their prognosis. Medin is one of the most common senile localised amyloids in humans, found in the aorta of almost all Europeans above the age of 60. It was first discovered in the tunica media of the aorta, the largest artery in humans and the main artery supplying oxygenated blood from the heart to the entire body. Medin is a 50-residue peptide formed by the enzymatic cleavage of the glycoprotein lactadherin which is involved in phagocytic clearance of apoptotic bodies. Medin aggregation affects vascular wall elasticity, vascular cell viability and inflammatory status. Medin colocalises with and enhances deposition of amyloid-β (Aβ) aggregates in the cerebral vasculature, which are linked with AD-type CAA. A correlation of cerebrovascular medin deposits with cognitive decline has suggested a potential role of medin aggregation in the disruption of the blood-brain barrier (BBB) in CAA. Despite its widespread occurrence in vascular pathology, medin biology suffers from a lack of a clear mechanistic description of medin aggregation, sparse information about how the human body regulates medin aggregation under physiological conditions, lack of in vitro and in vivo models of medin’s pathogenesis, irreproducibility in lab-based production protocols of medin for research purposes, and, hence, an absence of probes and clinical modulatory tools of medin pathogenesis. This thesis summarises a battery of approaches to address some of these gaps in the literature on medin. It reports:

the development of a protocol for the recombinant production of human medin to high purity and peptide integrity, as well as approaches for labelling medin for various applications in biochemistry and molecular biology (Chapter 3), microscopic characterisation of kinetics of medin aggregation (Chapter 4), comparison of the cytocidal effects of different microscopic steps in the aggregation pathway of medin on brain endothelial cells (Chapter 5), mechanistic insights into how different aggregation intermediates of medin influence the kinetics of aggregation of Aβ alloforms relevant to CAA (Chapter 6), the role of extracellular chaperone machinery, particularly clusterin, on modulating medin aggregation (Chapter 7), and the application of artificial intelligence to discover novel small molecule inhibitors of medin aggregation (Chapter 8).

Medin aggregation is dominated by extremely rapid fibril elongation and is sustained almost equally by primary and saturated secondary pathways of aggregate proliferation with very low nucleation energy barrier. Medin has a higher elongation rate constant compared to other pathological amyloids such as Aβ, ⍺-synuclein (⍺Syn) and tau. Different intermediates in the medin aggregation pathway interact differently with the physiological milieu. Medin self-assembly determines the relative efficiency of medin aggregation intermediates in catalysing the aggregation of Aβ40, the most abundant Aβ isoform and the one predominant in CAA, under physiological conditions in vitro. The strongest cytotoxic response in cultured brain endothelial cells was observed under monomer-plus-seed conditions, consistent with toxicity arising during secondary-pathway-driven aggregation. This may involve transient oligomeric intermediates. The extracellular chaperone clusterin inhibits medin aggregation in vitro by preventing the dominant microscopic step of medin aggregation viz. fibril elongation. Finally, using deep learning-based mining of purchasable small molecule libraries, we have identified evacetrapib as a repurposable drug acting as a pharmacological chaperone that inhibits medin aggregation potentially by sequestering medin in its native non-toxic monomeric state. Our results are a stepping stone in understanding why medin aggregation is widespread and aggressive in human vasculature and how the normal physiological environment in the human body tightly regulates medin deposition under healthy conditions. This study also helps identify target microscopic steps in medin aggregation for development of therapeutic strategies. In the second section of this thesis, we further report the development of single-domain antibodies, also known as nanobodies, for the detection of antigens involved in central nervous system (CNS) disorders and nanobody encapsulation strategies for a noninvasive approach of drug delivery to the CNS via the nose-to-brain route that essentially bypasses the blood-brain barrier (Chapter 10).