Mechanisms Underlying Memory Retrieval and the Effect of Amyloid Precursor Protein in a Transgenic Mouse Model

Abstract

Introduction
Research into Alzheimer’s disease (AD) would greatly benefit from a deeper understanding of the mechanisms underlying memory retrieval. Previous studies have found impairments in memory retrieval present in very young amyloid precursor transgenic (APPtg) mice, at a stage which would be considered preclinical in humans, however, the molecular and cellular mechanisms behind this impairment are unknown. Dysfunctional amyloid precursor protein (APP) processing is thought to be central to AD pathogenesis and mutations in the APP gene are one of the leading causes of familial Alzheimer’s disease (FAD).

Objective
This thesis aims to contribute to a better understanding of the mechanisms underlying memory loss in a genetically modified preclinical mouse model resembling aspects of AD. These mechanisms will be elucidated via the quantitative analysis of certain mitochondrial protein levels, identification of biochemical and cellular pathways involved in memory retrieval via proteomics analysis, analysis of the activity of various enzymes in the mouse brain in the context of memory retrieval and the quantification of key mitochondrial metabolites in the mouse brain tissue samples.

Methods
Brain tissue from a mouse model overexpressing APP with two mutations linked to FAD (APPtg), was used in this project. Four groups consisting of APPtg and wild-type (WT) mice at basal levels (with no behavioural task) and during memory retrieval (sacrificed 20 seconds after 7-day probe trial, following Morris Water Maze behavioural task) were used in this study. Purified synaptosome samples were used for western blotting, enzymatic activity assays, and proteomic analysis.

Results
Western blotting against several synaptic and mitochondrial markers revealed increased expression of VDAC1 and the mitochondrial fission and fusion proteins Drp1 and Mfn1 in the APPtg mice at both the basal level and during memory retrieval. Several complexes of the electron transport chain also showed synapse specific expression increases in the APPtg mice at both the basal levels and during the attempted retrieval of a memory.

Using a combination of tools including gene-ontology (GO), protein-protein interaction (PPI) networks and functional dependency analysis, proteomic analysis revealed that the insertion of the APP transgene causes upregulation of proteins implicated in mitochondrial dysfunction and disease pathology in both basal and memory retrieval groups . Based on the proteomics findings, failure to upregulate proteins involved in ATP production (Ndufa7, Ndufa6, Ndufb6, Ndufb4, Ndufb2) structural support (Nefl, Ina, Gfap), proteasome complex (Psma3, Psmb2, Psma7, Psmc1, Psmd4) response to oxidative stress (Prdx3, Prdx2, Gsr, Park7, Snca), and the negative regulation of apoptotic processes (Slc25a27, Nefl, Prdx3, Prdx2, Park7, Snca) are suggested to contribute to the memory deficits in this APPtg mouse model.

Whilst these methodologies have been previously used in Alzheimer’s research, performing them in the context of memory retrieval (specifically, mice sacrificed at the point of attempted memory retrieval) is novel and provides new insights into the mechanisms underlying memory loss in preclinical APPtg mice.

Future work
Further work investigating the metabolic changes in WT mice during memory retrieval, when compared to basal levels, and how these changes differ in the preclinical model of FAD, can help to identify metabolites involved in healthy memory retrieval and how they differ in FAD. Alongside this, further assessment of the activity of enzymes involved in the various stages of aerobic respiration will provide insight into the disease process, offering opportunities for testing of enzymatic response to targeted therapies which, if delivered at a preclinical stage, could provide a means to prevent of delay memory loss in AD.