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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Laird, Dale W.

Abstract

The connexin family of gap junction-forming proteins are expressed in most cells of the human body. The critical roles of connexins in physiology and lifelong health are highlighted by the link between ~1000 mutations in 11 connexin-encoding genes and at least 30 developmental and early life disorders, including deafness, cataracts, and skin diseases. Collectively, these disorders are common and affect approximately 1 in 1500 live births. Among these, 33 gene mutations in the genes encoding Cx30.3, Cx31, and Cx43 are clinically associated with erythrokeratodermia variabilis et progressiva (EKVP), a rare incurable skin disorder characterized by painful erythematous and hyperkeratotic lesions that worsen with age. EKVP treatment involves symptom management that generally fails to achieve long-term clearance of disease. As such, there is a pressing need to understand the breadth of mechanisms that underpin this debilitating skin condition to guide future research into effective targeted therapeutics for EKVP. Here, we utilized rat epidermal keratinocytes (REKs) to investigate the consequences of connexin variant expression on connexin and keratinocyte physiology. In the first study, we discovered that the Cx43-P283L variant exhibited a modest increase in its lifespan, possibly due to impaired phosphorylation on nearby residues, while the Cx43-T290N variant appeared benign. In our second study, we discovered a trio of variants, Cx30.3-G12D, Cx30.3-T85P, and Cx30.3-F189Y, failed to form functional gap junctions due to a trafficking defect resulting in their entrapment within the secretory pathway. However, these Cx30.3 variants formed gap junctions when intermixed with co-expressed Cx30.3, Cx26, Cx30, or Cx43. Dye uptake studies also revealed these variants assembled into leaky hemichannels (HCs) and/or otherwise impaired cell membrane integrity. Finally, in our third study we showed the Cx30.3-R22H, Cx30.3-S26Y, Cx30.3-P61R, Cx30.3-C86S, Cx30.3-E99K, Cx30.3-T130M, and Cx30.3-M190L variants each altered one or more aspects of the connexin lifecycle, including protein stability and channel function. Additionally, the expression of Cx30.3-P61R and Cx30.3-S26Y impaired keratinocyte viability. Collectively, the range of molecular changes identified are complex and may all be sufficient to cause EKVP. Overall, this project enhanced our understanding of 12 EKVP-associated connexin variants and laid the foundation for the development of targeted treatments for this debilitating disorder.

Summary for Lay Audience

The connexin family of proteins are found in nearly all cells of the human body where they enable contacting cells to directly “talk” to each other by exchanging small signaling molecules and ions. This canonical role of connexins is critical for normal cellular functions and lifelong organ health as nearly 1 in 1500 live births will have disease due to variants in a connexin gene. Collectively, there are at least 30 developmental and early life disorders–encompassing hearing loss, cataracts, and skin disease–that can occur when variants disrupt the normal localization, function, or lifespan of these essential proteins. So far, 33 variants in the genes encoding three key connexins found in the skin have been clinically linked to a rare inherited skin disorder known as erythrokeratodermia variabilis et progressiva (EKVP). Patients with EKVP suffer from regions of thickened skin and painful patches of reddened, inflamed skin, both of which arise early in childhood and tend to worsen with age. Since there is currently no cure for EKVP and treatment options are limited, there is a pressing need to unravel how each of these variant connexins leads to EKVP as a better understanding of all the mechanisms involved will allow for the development of targeted therapeutics. In this project, we introduced the connexin variants into skin cells derived from the epidermis and assessed their localization, function and lifespan, as well as how these proteins altered the behaviour of skin cells. Overall, this project aimed to comprehensively examine how variants in two connexins (Cx30.3 and Cx43) cause EKVP. We also screened and assessed the potential of Health Canada approved drugs and experimental small molecule drugs that can improve variant connexin assembly, trafficking, and function in our pre-clinical model of the skin. This work helped to uncover the underlying mechanistic causes of EKVP and laid the groundwork for the development of effective treatments for patients that suffer from this debilitating skin disease.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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