Moffat, Tia Corinne2013-11-082013-11-0820072007Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4565.http://hdl.handle.net/10393/29510http://dx.doi.org/10.20381/ruor-12992Apoptosis is known to underlie neuronal loss in many human neurodegenerative disorders, such as Alzheimer disease (AD), HIV-dementia and ischemic stroke. Phospholipid signalling is involved in each of these conditions; changes in phospholipid membrane composition and phospholipid synthesis enzyme activity occurs in AD, while in HIV-dementia and ischemia there is an increased production and accumulation of inflammatory bioactive phospholipid mediators. Pathophysiological exposure to the phospholipid neuromodulator platelet activating factor (PAF) can initiate apoptotic pathways, and has been hypothesized to be a key mediator of neuronal death in these neurodegenerative disorders. It has been assumed that PAF signals exclusively through its G-protein coupled receptor (PAFR). However, our laboratory has shown that PAF can also induce apoptosis independently of PAFR and that ectopic expression of PAFR renders cells resistant to PAF-induced toxicity. This is relevant to neurodegenerative disease since PAFR expression in the human brain is found predominantly in non-neuronal cells suggesting that PAFR-independent signalling is also involved in neurodegeneration. This thesis focused on elucidating the PAF-induced, PAFR-independent apoptotic pathways in human neurons with the overarching goal of identifying new therapeutic targets capable of inhibiting neuronal loss in neurodegenerative disease. Here, PAF is shown to induce endoplasmic reticulum (ER) stress resulting in caspase-2 activation, leading to the activation of caspase-7, and culminating in DNA fragmentation in human neurons (hNTs) lacking PAFR. Mitochondrial effects, specifically release of cytochrome c from the mitochondria, caspase-9 activation, reactive oxygen species (ROS) production, and mitochondrial uncoupling protein-2 (UCP2) upregulation were initiated downstream of this primary ER cascade. To target this pathway, a panel of natural and synthetic compounds was screened for their ability to protect hNTs from neurotoxicity initiated by PAF and by the AD-related neurotoxic peptide amyloid-beta 1-42 (Abeta). Several compounds were identified as PAF/Abeta inhibitors. One of these compounds, nelfinavir (NFV), was tested for in vivo efficiency using the middle cerebral artery occlusion (MCAO) model of focal ischemia. NFV reduced infarct size, prevented neuronal apoptotic-like death, and improved behavioural recovery, solidifying the relation between PAF, AD, HIV-dementia, and ischemia-induced neuronal apoptosis. Together, these studies demonstrate that PAF can trigger neuronal apoptosis independently of PAFR, that inhibiting PAF-mediated pathways of neurotoxicity can protect human neurons in vitro from PAF and Abeta, and that targeting secondary apoptotic pathways elicited by PAF can reduce neuronal death in vivo in mouse models of human disease. The identification of compounds capable of inhibiting the primary and secondary PAFR-independent apoptotic events triggered by PAF represents a new means of targeting phospholipid signalling in neurodegenerative disease.146 p.enBiology, Molecular.Thesis