Investigating the effects of the insulin/PI3 kinase/mTORC1 signalling pathway on Alzheimer’s relevant secretory events using the Drosophila melanogaster secondary cell model

Thesis

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterised by two hallmark pathologies, extracellular amyloid plaques and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. In addition to these well-known pathologies, increasing evidence indicates that early defects in secretory and endolysosomal trafficking play a central role in disease progression. Insulin resistance and impaired insulin signalling are also frequently observed in brains with AD, leading to the proposal that metabolic dysregulation may contribute to neurodegeneration. However, the cellular mechanisms linking disrupted insulin signalling to pathological protein trafficking remain poorly understood.

This study investigated how modulation of the insulin/PI3K/mTORC1 signalling pathway regulates dense core granule (DCG) biogenesis and endolysosomal trafficking using Drosophila melanogaster male accessory gland secondary cells (SCs) as a specialised secretory model. Insulin/PI3K/mTORC1 signalling activity was altered through targeted genetic manipulation of key components, including InR, PI3K, PTEN, Akt, Tsc1/2, Rheb and mTOR. In parallel, human Tau 2N4R wild type was overexpressed in SCs to model tauopathy observed in AD. DCGs were visualised via GFP-tagged MFAS, while lysosomes and acidified compartments were labelled with LysoTracker and analysed by live cell imaging.

Modulation of insulin/PI3K/mTORC1 signalling in healthy SCs revealed that both pathway upregulation and downregulation disrupted normal DCG biogenesis. Reduced signalling, particularly following PI3K knockdown, produced pronounced mini-core phenotypes and increased DCG acidification and lysosomal area, consistent with enhanced endolysosomal trafficking. In contrast, increased signalling promoted DCG compartments formation but also led to structural abnormalities. hTau2N4R overexpression induced DCG compartments with cylindrical cores, increased compartment numbers and expanded lysosomal area, indicating substantial trafficking defects. Notably, both up- and down- regulation of insulin/PI3K/mTORC1 signalling partially rescued tau-induced DCG abnormalities, although normal endolysosomal trafficking was not fully restored.

Together, these findings demonstrate that insulin/PI3K/mTORC1 signalling showed bidirectional control over secretory and endolysosomal pathways and must be tightly balanced to maintain cellular homeostasis. Disruption of this equilibrium may predispose cells to early pathological changes associated with AD, supporting a mechanistic link between metabolic dysregulation and neurodegenerative progression.

Authors: Kong, L

• DOI: 10.5287/ora-vjroxypjr
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: dense core granules; endolysosomal trafficking; secondary cells; insulin/PI3K/mTORC1 signalling; Alzheimer’s disease; tauopathy; Drosophila melanogaster; diabetes
• Subjects: Nervous system--Degeneration; Genetics; Neurosciences; Neurobiology; Molecular biology