Date of Award
May 2020
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biomedical and Chemical Engineering
Advisor(s)
Shikha Nangia
Keywords
Blood-brain barrier, Claudin, Metadynamics, Molecular Dynamics, Paracellular, Tight Junction
Subject Categories
Engineering
Abstract
The molecular interface of the blood-brain barrier (BBB) is a highly selective physiological barrier. The BBB shields the central nervous system (CNS) for harmful agents while also preventing lifesaving drugs from entering the CNS. With the prevalence of neurogenerative disease on the rise, there is a growing interest to design therapeutic interventions that can surpass the BBB. Such efforts necessitate a thorough understanding of the BBB, requiring one to decipher: why the BBB is so selective? what governing molecular rules govern selectivity across the BBB? and how does it impact physiology. As a contribution towards this understanding the following dissertation discusses nuances of the BBB see from the perspective of its tight junctions (TJ). Tight junctions are a protein-protein adhesion structures that seal the paracellular space for small solutes. Tight junctions are a common feature in many epithelial and endothelial tissues and a crucial component of the BBB. The BBB tight junctions are shown to be regulate a size and charge selective barrier that permeates only molecules of 800 Da in size. In the following chapters a computational microscopy approach was utilized to probe different structural and biochemical features of the tight junction. Chapter 2 discusses the molecular assembly of tight junction proteins investigates for the first time under molecular dynamics simulations. The key findings included the discovery of dimeric interfaces that are seen to form tight structural contacts between conserved residues. An experimental investigation with formaldehyde as a cross-linker in HeLa cells validated the existence of such contacts. Chapter 3 investigated the tight junction assembly in the paracellular space of adjacent cells by mimicking this interface with two membranes. These simulations revealed the structural aspects of the pores that are feasible under claudin-5 tight junction assembly. We performed a mutation experiment that distinguished the dimeric interfaces between claudin-3 and claudin-5, further a biophysical investigation showed how the flexibility of the transmembrane domains affect the dimerization of claudins. Chapter 4 extends upon the discoveries from chapters 2 and 3 to other claudins that are relevant for the tight junction biology. There is an inherent need to compare different members of the claudin family of proteins to enhance the overall understanding about tight junction biology and consequently the BBB tight junctions. Major findings include the discovery of a putative trimeric receptor assembly for Clostridium perfringens enterotoxin. The pore assemblies of claudin-2 and the dynamics of ions across the pores. Chapter 5 investigates the ion selectivity of claudin-5 and claudin-2 in a greater detail. The key findings include that the barrier to charge selectivity in the claudin pores are due to charge repulsion from the pore lining residues. The electrostatic interaction dominates the pore selectivity while the steric interaction plays a role for divalent cations. These biophysical evidence reveal how the claudin-5 tight junction pores that line the BBB screen charged ions and water. These computational findings push the boundaries of current knowledge on the BBB and sets the stage for applications targeted towards drug delivery strategies. The computational methods and tools discussed herein sets precedent for its transferability to the investigation of other tight junction proteins and in wider scope other membrane proteins.
Access
Open Access
Recommended Citation
Irudayanathan, Flaviyan Jerome, "MOLECULAR PHYSIOLOGY OF BLOOD-BRAIN BARRIER TIGHT JUNCTIONS" (2020). Dissertations - ALL. 1155.
https://surface.syr.edu/etd/1155
