The physiological and pathological aggregation of transforming growth factor-β-induced and amyloid precursor protein in drosophila secondary cells

Amyloid proteins abound in nature, share several familial commonalities, and more often than not, play important physiological roles. The same is true of human amyloids, which similarly serve crucial functions, and whose potential aberrancy only come to light with the manifestation of disease. Thus in humans, amyloid or pre-amyloid proteins function normally as peptide hormones, are required for nervous system development, are part of the coagulation cascade, provide a scaffolding for melanin in melanocytes, and are essential for the transparency of the cornea, to name but a few of their known roles.
Drosophila secondary cells located in the male fly’s prostate-like accessory glands are unique as a genetically tractable system in containing very large secretory compartments, which are easily visualized ex vivo, making them invaluable in the study of secretory processes in living cells. Secondary cells contain a single large dense-core granule (DCG) in each compartment, which I show is comprised of high levels of Midline Fasciclin (MFAS), an amyloid which is the homologue of the human structural and potentially amyloidogenic corneal protein Transforming Growth Factor Beta-Induced (TGFBI).
The biogenesis of DCGs is a robust physiological process, and their secretion has been found by the Wilson laboratory to be crucial in reproduction. This earlier work also presciently suggested links to neurodegeneration, cancer and diabetes. Here, I show that DCG formation is impacted by specific manipulations, chief among these being mfas knockdown, which completely blocks the aggregation of proteins into DCGs. Furthermore, knockdown of the Drosophila homologue of Amyloid Precursor Protein (APP) called APP-like (APPL), leads to the formation of multiple mini-cores in each compartment, which fail to fuse together. Intriguingly, DCG formation is also impacted by expression of mutations in related human and Drosophila amyloid proteins containing amyloidogenic mutations. Thus, amyloidogenic mutations in MFAS’ homologue TGFBI, as well as mutations in human APP or its cleaved amyloidogenic amyloid-β peptides, which cause Familial Alzheimer’s Disease, when expressed in secondary cells, affect the coalescence of MFAS. When different mutant forms of human TGFBI and APP were combined with mini-core-inducing mutations (knockdowns of Drosophila Appl and GAPDH2), a spectrum of outcomes was produced, ranging from intermediate phenotypes to near-complete rescues.
My work shows that amyloids interact with other amyloid proteins to influence each other’s aggregation both physiologically and pathologically, a long-suspected but experimentally undemonstrated conjecture, at least in amyloid pathology. The impossibility of providing proof lay in the fact that amyloidogenesis is an inscrutable, decades-long process, combined with the obvious unethicality of performing brain biopsies on living patients. My work proves this Grail-like surmise and demonstrates a physiological role for APP proteins that is inhibited by amyloidogenic versions of this molecule. It also showcases the depth and power of this system in modelling amyloid diseases of the brain and cornea, which is now leading to the development of a new model explaining the precise molecular function of APP in secretion and the likely route by which this role is compromised in disease.

Advisor: Wilson, C
Harris, A