Investigating Argonaute phosphorylation and miR-134 as novel therapeutic targets for Alzheimer's disease

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During the pre-clinical period of Alzheimer's Disease (AD) significant deficits in synaptic transmission and dendritic spine loss develop. This process underpins the circuit dysfunction and cognitive decline seen in later stages of the disease. 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 dendrite morphology and specific miRNAs have been implicated in the pathogenesis of AD. However, how RISC protein machinery is affected by AD pathology is less well understood. Preliminary data from this lab suggests that phosphorylation of Ago at S387 and binding of Ago to RISC scaffold protein GW182 is increased in the hAPP-J20 mouse model of AD. This suggests that miRNA activity may be dysregulated as a consquence of AD-related pathology and contributing to spine loss and synaptic dysfunction. Hypothesis: Blocking Ago phosphorylation at S387 in models of amyloidopathy will reduce synaptic deficits and spine shrinkage observed in these models via resetting of miRNA activity.Methods: Endogenous Argonaute will be replaced with phospho-null mutant in primary neuronal cultures overexpressing APP and miRNA activity will be quantified using dual luciferase assays using luciferase reporter constructs carrying the 3'UTR seed sequence of key synaptic proteins. In a mouse model of amyloidopathy the following three rescue strategies will be utilised by use of stereotactic injection into the CA1 region of the hippocampus: a) lentiviral replacement of endogenous Argonaute with a phospho-null mutant, b) lentiviral overexpression of key synaptic protein 3) Antagomirs to bind and repress activity of key synaptic miRNAs.Slices will be prepared from these mice for electrophysiological recording and spine analysis by confocal microscopy to assess synaptic function and dendrite morphology.