Modulation of the PI3K/Akt Pathway in Oligodendrogenesis

Abstract

Oligodendrocytes are highly specialised cells that ensure the rapid and
efficient conduction of impulses by producing myelin, a phospholipid substance that enwraps and insulates the axons. Myelin disruption and oligodendrocytes
impairment are hallmarks of many neurodegenerative diseases, such as multiple
sclerosis, stroke and Alzheimer’s disease. Identifying the multiple aspects that
regulate oligodendrogenesis is therefore relevant for new therapies to
neurodegenerative diseases. The PI3K/Akt signalling pathway is downstream of
multiple factors that regulate oligodendrocyte precursor cells (OPCs) proliferation, survival and differentiation into myelinating oligodendrocytes in the developing CNS. However, its role in the adult CNS is still poorly understood. In this thesis, I used the adult optic nerve and postnatal cerebellar slice ex vivo organotypic culture models to investigate the impact on oligodendrocyte lineage cells by targeting the PI3K/Akt pathway. Transgenic mice models in which expression of fluorescent reporters was driven by the Sox10 and proteolipid protein (PLP) genes, together with microarray, (RT)-qPCR and immunoassays were utilised to identify and evaluate the role of this pathway in driving oligodendrocyte generation. Inhibition of PI3K/Akt was achieved with LY294002, a well-known PI3K inhibitor, and the effects were compared with 3’-O-methylepicatechin, a flavonoid that has been shown to be neuroprotective acting via PI3K/Akt. The results showed that strong inhibition of PI3K/Akt by LY294002 resulted in a dramatic loss of oligodendrocyte lineage cells, whereas partial inhibition of PI3K/Akt by lower doses of LY294002 proved beneficial. Notably, 3’-O-methylepicatechin was also shown to inhibit PI3K/Akt and to be cytoprotective for mature oligodendrocytes and increased MBP mRNA expression. A key objective of this PhD was to synthesise 3,3’,4’,5,7- pentaacetyl epicatechin, a less hydrophilic derivative of the flavonoid (-)- epicatechin, in order to increase its membrane permeability and potentially facilitate its uptake across the blood-brain barrier. In silico molecular modelling demonstrated that 3,3’,4’,5,7-pentaacetyl epicatechin had a greater affinity than that of LY294002, epicatechin and 3’-O-methylepicatechin at the ATP-binding site of PI3K, the site of enzyme inhibitions. In addition, immunoassays determined that 3,3’,4’,5,7-pentaacetyl epicatechin was 200-fold more efficient than LY294002 inducing PI3K/Akt inhibition. Furthermore, 3,3’,4’,5,7-pentaacetyl epicatechin was shown to be cytoprotective and increase MBP mRNA at nanomolar concentrations, compared to micromolar concentrations for LY294002 and 3’-O-methylepicatechin.
This study identified the PI3K/Akt/mTOR pathway as the common mechanism by
which LY294002 and the derivatives of epicatechin might exert protective or
proliferative effects on oligodendrocytes. Altogether, these findings indicated a
potential new strategy to target oligodendrocyte and myelin via development of
new pharmacological agents aimed to modulate the PI3K/Akt/mTOR signalling
pathway.

Date of Award	22 Jun 2016
Original language	English
Awarding Institution	University of Portsmouth
Supervisor	Arthur Butt (Supervisor)