Mitochondrial and synaptic dysfunction in primary neuronal models of Alzheimer’s disease

Abstract

Alzheimer’s disease is the most common form of dementia, clinically diagnosed by the presence of amyloid-β and tau aggregates in the brain. However, difficulties in establishing accurate disease models to more clearly understand its pathogenesis has slowed the development of therapeutics. In this thesis, I explore the use of primary neuronal cell culture models of aspects of AD, using lentiviral transduction to overexpress pathogenic tau, and knockdown-replacement of amyloid precursor protein (APP).
I first confirmed that these disease-associated proteins were expressed and produced pathological hallmarks such as tau phosphorylation and increased Aβ production in neuronal cultures. I then focused on how tau overexpression impacted mitochondria and synapses, two subcellular components implicated in AD pathogenesis.
At mitochondria tau overexpression caused the loss of two proteins involved in mitochondria dynamics, Drp1 and Mfn2. This loss did not, however, translate to changes in mitochondria morphology, although I did observe a reduction in ATP levels following tauWT and pathogenic tauP301L overexpression, indicating disruption to mitochondrial function. To explore this dysfunction more broadly, I carried out a mass-spectrometry screen of proteins in isolated mitochondrial fractions from primary neurons, which identified a large number of proteins that were significantly altered following tauP301L overexpression. Proteomic ‘hits’ included proteins involved in RNA silencing, synaptic activation signalling and linked to low-density lipoprotein cholesterol.
I also investigated changes to the synapses following tau overexpression, carrying out automated quantification of synapses using the newly developed SynBot image analysis tool. I found a loss of both excitatory and inhibitory synapses, with a specific increase in excitatory/inhibitory ratios in cells overexpressing tauP301L, indicating a switch towards hyperexcitability in these cells.
Overall, this work has explored the use of primary neuronal cultures to model aspects of AD and provides starting points for subsequent investigation of these proteins effects in cells.

Date of Award	18 Mar 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jeremy M Henley (Supervisor)