Background: Multiple sclerosis (MS) is an autoimmune disease characterized by the destruction of oligodendrocytes (OLGs) and myelin, as well as neuronal axonal loss. OLGs are the myelinating cells of the central nervous system (CNS). The function of myelin is to ensure rapid saltatory conduction along axons. People with MS often suffer from many symptoms including muscle spasms and weakness, pain, fatigue, and blurred vision. The prevalence of MS in North America ranges from one in 500 to one in 1000 people depending on the region. Current therapies include anti-inflammatory drugs with little pharmacological selectivity. Recently, the δ and γ isoforms of the phosphoinositide 3-kinase (PI3K) family have been suggested as potential targets to treat inflammatory diseases, such as MS. However, it is not known whether these PI3K subtypes are expressed in OLGs. Our laboratory has previously shown that the PI3K/Akt pathway is essential for OLG growth and differentiation. PI3K, a key regulator of cell survival and metabolic control, catalyzes phosphorylation of the inositol ring of PI at the 3' position. PI3Ks are divided into three classes based on their size, structure, function and substrate. Class I PI3Ks consist of four isoforms (α, β, δ, and γ) and their differential activation may control downstream cellular processes, depending on cell type and context. One key downstream effector of PI3K is the Ser/Thr kinase Akt. Upon its activation by PDK1, Akt can phosphorylate and, consequently, inactivate pro-apoptotic factors leading to cell growth and proliferation. Objective: The goal of my project is to functionally characterize the specific isoforms of PI3K in terms of their involvement in OLG development and microglial growth. Methods: Primary cultures of OLG progenitors (OLPs) and microglia were prepared from the brains of newborn Sprague-Dawley rats. The specific PI3K isoforms expressed in these glial cells were determined by Western blotting using antibodies against the α, β and γ isoforms. In order to determine whether specific PI3K isoforms affect microglial and OLG growth and development, OLGs and microglia were stimulated with IGF-1 and GM-CSF respectively, and then treated with PI3K isoform-specific inhibitors. These glial cultures were then tested for survival and proliferation using MTT and 3H-thymidine assays, respectively. As an index of PI3K activity, Akt phosphorylation was also assessed under similar conditions. Results: My results demonstrate that OLGs express PI3Kα throughout all stages of development, whereas PI3Kβ is only expressed in OLPs, while PI3Kγ was not detected. Furthermore, PI3Kα was expressed in all CNS glial cells, whereas PI3Kγ expression was restricted to microglia. Using selective inhibitors, the data also suggest that PI3Kα, but not β or γ, is required for IGF-1 stimulated OLG survival. PI3Kα and surprisingly, PI3Kγ were required for OLG proliferation. GM-CSF proved to be an effective stimulant for both microglia survival and proliferation. PI3Kα but, surprisingly, not PI3Kγ was required for GM-CSF stimulated microglial survival and proliferation. Conclusion: Our results indicate that PI3Kγ might be a good potential target to treat the inflammatory component in MS since this isoform is not expressed in OLGs but expressed at high levels in microglia. / N/A
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.104742 |
| Date | January 2011 |
| Creators | Kastner, Derek |
| Contributors | Guillermina Almazan (Supervisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Pharmacology & Therapeutics) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | Electronically-submitted theses. |
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