Award details

Regulation and activities of amyloidogenic proteins APP and TGFBI in physiological and pathological protein aggregation

Reference	BB/W00707X/1
Principal Investigator / Supervisor	Professor Clive Wilson
Co-Investigators / Co-Supervisors
Institution	University of Oxford
Department	Physiology Anatomy and Genetics
Funding type	Research
Value (£)	567,179
Status	Current
Type	Research Grant
Start date	01/04/2022
End date	31/03/2025
Duration	36 months

Abstract

We have developed the adult Drosophila secondary cell (SC) as a new in vivo model to study protein aggregation into dense-core granules (DCGs) in real-time. To do this, we have generated an extensive genetic toolbox, producing SCs in which membrane-bound compartments, DCGs and vesicles are fluorescently marked and can be genetically manipulated to knock down or overexpress genes in an SC-specific way. Exploiting these tools and the SC's uniquely large DCG compartments, we found that the Drosophila homologues of APP and TGFBI co-operate together to trigger protein aggregation in DCGs, a novel cell biological role for these amyloidogenic molecules. Expressing defective forms of these proteins alters DCG assembly and/or disassembly, providing a new model to study pathological amyloidogenesis. We now propose to use these methodologies to: 1. Characterise how APP and TGFBI normally work together to control protein aggregation in DCGs, using gene knockdown and overexpression approaches, and how this process and disassembly of secreted DCGs are disrupted by amyloidogenic versions of these proteins; 2. Determine how physiological protein aggregation in DCGs is affected by intracellular trafficking and exosome biogenesis, and whether amyloidogenic versions of APP/A-beta and TGFBI alter this response; 3. Test whether knockdown of candidate genes implicated in Alzheimer's Disease affects DCG protein aggregation in the presence of normal and amyloidogenic forms of APP and TGFBI, and characterise the precise roles of genes that we identify. These studies will determine the normal cell biological functions of APP and TGFBI in triggering protein aggregation, and reveal how protein assembly and disassembly is disrupted by amyloidogenic forms of these proteins. By identifying genetic modifiers of this process, we will highlight candidate genes and mechanisms that can be tested in mammalian models and human cells as potential therapeutic targets in amyloid disease.

Summary

Amyloidogenesis is the aggregation of normally soluble proteins into insoluble fibres. It is commonly observed in neurodegenerative disorders. For example, in Alzheimer's Disease, processed forms of a protein called APP self-aggregate to produce amyloid plaques in patients, and these plaques affect the survival of neurons. Similarly, defective forms of the TGFBI protein produce amyloid in the cornea and blindness. However, amyloid formation is not just pathological. For example, some hormones normally form inert amyloid structures called dense cores, which can be stored in cells prior to secretion. Combatting amyloid disease requires a better understanding of how amyloid formation is triggered and controlled. But studies in patients and in animals with Alzheimer's-like disease primarily focus on the end products of this process, plaques and memory loss. In fact, for proteins like APP and TGFBI, it has been unclear whether they are normally involved in protein aggregation or whether this is a uniquely pathological process. Large protein aggregates are typically only seen in disease, while normal protein aggregation generally involves the assembly of much smaller structures, making it difficult to follow this process using microscopy techniques suitable for living cells. We have been studying dense core formation in the fruit fly. Flies have equivalents of about 70% of human disease genes, including APP and TGFBI. It is often much easier to identify genes involved in biological processes and disease in flies and to watch these processes take place in living tissues. In fact, these animals are extensively used to study degenerative processes in Alzheimer's. We have identified a specific prostate-like cell in flies, the secondary cell, which makes dense cores that are over one thousand times larger than cores in other cells. This allowed us to follow the rapid formation of dense cores and how this is controlled for the first time in living tissues. Remarkably, we found that the fly equivalents of APP and TGFBI have complementary roles in making these dense cores. TGFBI is required for protein aggregation to take place, while APP ensures that micro-core structures coalesce together to make a single giant core. If a pathological form of APP is made in secondary cells, it changes the organisation of TGFBI in dense cores and stabilises them, so that they fail to disperse when secreted. Using the genetic approaches available in flies, we have already shown parallels between the control of dense core formation in secondary cells and plaque formation in humans, which both seem to involve small membranous droplets called exosomes. We will now study how APP and TGFBI work together to control dense core formation in secondary cells and how this process goes wrong when pathological versions of these amyloid proteins are made in these cells. We will also work out how altering the ways in which secreted proteins are guided through the secretory pathway changes these protein aggregation events in the cell, particularly focusing on whether pathological forms of APP or TGFBI behave differently to the normal proteins. Finally, we will test which of the large number of genes and cellular processes that have been suggested to play a role in Alzheimer's and neurodegeneration through studies in patients and animals are involved in normal and pathological APP and TGFBI aggregation in secondary cells. We will then work out precisely how they affect these processes. Our work will allow us to define how APP and TGFBI, two important amyloid proteins in disease, normally drive protein aggregation into insoluble dense cores. We will identify the genes that control this process and how pathological forms of APP and TGFBI interfere with this control. We will then be ideally positioned to collaborate with other researchers to determine which of these mechanisms is involved in amyloid disease in humans, and to develop approaches to block them.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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