Investigating Argonaute phosphorylation and miR-134 in Alzheimer's disease

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA-mediated silencing of synaptic
proteins via Argonaute (AGO) as part of the RNA-induced Silencing Complex (RISC). miRNAs are
known to be involved in regulation of synapses and dendritic spine morphology and specific
miRNAs have been implicated in the pathogenesis of Alzheimer’s Disease (AD). However, how
RISC protein machinery is affected by AD pathology is less well understood.
Limk kinase 1 (LIMK1) activity stabilises and maintains mature dendritic spine structure. miRNA134 (miR-134) plays a role in shrinkage of dendritic spines following Long Term Depression (LTD)
by targeting LIMK1 mRNA for translational silencing. Preliminary data from the Hanley lab
indicated that phosphorylation of AGO at S387 and the association of AGO to the RISC is
increased in the J20 mouse model of AD and in a primary neuronal culture model of AD.
Furthermore, LIMK1 expression levels were found to be decreased in late stage cortical AD
tissue. This thesis aimed to investigate the potential role of AGO2 phosphorylation and miR-134
in the synaptic dysfunction and dendritic spine loss seen in AD by determining at what point in
the disease course these changes occur, and to produce a model in which rescue strategies
targeting AGO2 phosphorylation could potentially be tested.
Technical challenges with the primary neuronal culture model used previously led me to use
and characterise an alternative model using lentiviral expression of Amyloid Precursor Protein
(APP) with AD-associated mutations and to design a method for quantifying miRNA activity in
this model. Changes in levels of AGO2-S387 phosphorylation or LIMK1 expression seen in the
previous model were not successfully reproduced, however, I demonstrated proof of concept
for this novel miRNA activity assay.
Surprisingly, when I quantified LIMK1 expression levels in cortical samples from AD patients at
Braak stages 1-6 by Western blotting I found no significant differences during the disease
course. However, Western blotting of cortical and hippocampal samples from the J20 AD mouse
model at 6months and 18months revealed that, despite no change in LIMK1 levels. pS387-AGO2
was significantly decreased. These results may indicate altered miRISC assembly and regulation
in the AD hippocampus and provide a potentially important foundation for future research into
the role of AGO2 phosphorylation in AD.

Date of Award	21 Mar 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jonathan G Hanley (Supervisor) & Michael Ashby (Supervisor)