Investigating the potential of stem cell based therapy in murine models of Alzheimer's disease

Abstract

Forebrain cholinergic neuronal loss is strongly correlated with the memory impairment of Alzheimer’s disease (AD). Current therapeutic options provide short term symptomatic relief and only minimally affect disease progression. Thus, any potential stem cell therapy with the ability to reverse the cell loss and halt the disease progression would be a valuable therapeutic option. The overarching aim of this thesis was to investigate the ability of transplanted neural progenitor cells to restore cognitive function in mouse models of Alzheimer’s disease. A dual reporter system was employed to identify specific populations of mouse embryonic stem (mES) cells, with mcherry (a red florescent protein) targeted to the ROSA26 (thumpd 3) promoter and β lactamase-T2A-β- galactosidase targeted to the Lhx8 promoter, designed to enrich cultured cells for cholinergic progenitors. Two different mouse models of AD, the acquired immunotoxin based model and the genetic triple transgenic model of AD (3xTg-AD), were validated using two behavioural paradigms measuring hippocampal (spatial memory) and cortical (declarative memory) function via a modified water maze and novel object recognition paradigm. Immunotoxin treated mice were later implanted with neural precursor cells and their cognitive function was then evaluated post-transplantation. In the first chapter (chapter3), the immunotoxin mouse model was investigated. The model uses mu-p75-saporin, a ribosomal inactivating toxin directed against the p75 nerve growth factor receptor, to cause the degeneration of forebrain cholinergic neurons as observed in AD. Intracerebroventricular injections of mu-p75- saporin caused cholinergic cell loss which was behaviourally correlated with spatial memory deficits in the water maze but intact recognition memory in novel object paradigm. Post mortem analyses revealed a reduction in the number of choline acetyltransferase (ChAT) positive cells in the principle regions providing cholinergic innervations to the hippocampus. Together, these data suggests that mu- p75-saporin treated mice represent a suitable model for the investigation of spatial memory deficits in future stem cell experiments. In chapter four, the 3xTg-AD mouse model was investigated. 3xTg-AD mice express three human transgenes, the amyloid precursor protein (APPswe), presenilin 1 (PS1M146V), and tau (tauP301L), and exhibit age related insoluble Aβ and neurofibrillary tangle (NFT) pathologies, as observed in human AD patients. Cognitive function in 3xTg-AD mice was assessed at 12 - 14 months and 16 - 18 months of age, again using the same modified water maze and novel object recognition paradigms. Deficits in recognition memory were observed in both male and female 3xTg-AD mice at both age points. However, in the water maze both WT and 3xTg-AD mice exhibited spatial memory deficits, making the data inconclusive. Tau based tangle pathology was detected in the hippocampus, amygdala and cortex of transgenic mice; however Aβ pathology was completely absent in the aforementioned regions. Due to the lack of Aβ pathology, the model was not used for future stem cell transplantation studies.In chapter five, the cholinergic differentiation potential of the Lhx8-AMP-T2A-lacZ‐FneoF reporter cell line was characterised under monolayer and neurosphere culture conditions. Lhx8 is a vital transcription factor involved in cholinergic differentiation. In the monolayer Lhx8+ cell culture a very low number of cholinergic neurons were detected at day 18 of differentiation. A moderately higher number of cholinergic neurons were detected in the neurosphere cultures therefore neural precursor cells obtained from neurosphere culture conditions were selected for use in future transplantation experiments. In the final experimental study, immunotoxin treated mice were implanted with neural precursor cells derived from neurosphere culture. The mice were first injected with mu-p75-saporin and their spatial memory was assessed using the water maze paradigm. After a month, treated and control mice were injected with 100,000 neural precursor cells (bilaterally administered into the hippocampus) and were behaviourally assessed again to detect any potential effects of cell transplantation on cognitive function. Whilst treatment with mu-p75-saporin induced a cognitive decline in spatial memory as measured using the water maze and altered nesting behaviour in treated mice, cell transplantation was only able to influence the nesting behaviour, with the behavioural deficits observed in the water maze remaining unchanged. Implanted cells were detected in the hippocampus and expressed proteins were indicative of a neuronal phenotype, demonstrating that the transplanted cells were able to survive in the brain over the experimental period. In conclusion, this thesis demonstrates that the implantation of neural precursor cells derived using a specific knock-in fluorescent reporter cell line has the potential to improve cognitive function in an acquired mouse model of AD. These preliminary studies indicate that further investigations are warranted and suggest that this approach could serve as an important model system in which to study brain development and neurodegenerative processes.