Alzheimer’s disease (AD) is the most common form of dementia among the elderly. The disease is characterized by amyloid-β (Aβ) plaques, neurofibrillary tangles, loss of synapses and neurons, and chronic neuroinflammation. The significance of neuroinflammatory processes in the on-set and progression of AD have been debated, as activated and reactive glial cells demonstrated protective and damaging properties. However, patients with AD treated with anti-inflammatory drugs demonstrated a lesser than average extent of disease development, indicating an important role of neuroinflammation in AD. Current strategies for the treatment of Alzheimer’s disease are minimally effective, as we lack the methods of diagnosis at an early stage. Numerous cases present significant neuronal loss prior to diagnosis and, as restoration of function is unlikely, treatment options focus on limiting further neuronal loss. It is my hypothesis that deposition of Aβ peptide can activate the innate immune system via pattern recognition receptors (PRRs), including complement, and evoke Alzheimer's pathology. In the current study, we focused on the role of the innate immunity system of the brain in the initiation and the propagation of inflammatory process in AD and the interplay between TLRs/NLRs and the complement system. Silencing the expression of various receptors demonstrated the involvement of NLRP3 in Aβ recognition, and use of confocal microscopy confirmed the association of Aβ interactions with this inflammasome and complement receptors. These results were confirmed in human astrocytes stimulated with Aβ and in brain tissue slides of patients with AD. Using human astrocytes and fluorescence resonance energy transfer (FRET), the associations of cell surface TLRs (such as TLR2, TLR4)with CRs (C5aR1, C3aR) were investigated; TLR2 and TLR4 were 6 | P a g e found to interact with C5aR1. In addition, Aβ-induced responses were augmented in the presence of complement and Aβ appeared to induce the interactions of TLR4 and C5aR1 on the cell surface. The trafficking of PRRs and Aβ in human astrocytes was further investigated to determine whether internalization and trafficking of Aβ is crucial for triggering a proinflammatory response. The results demonstrated that, along with Aβ and TLR4, complement internalized in the Rab5+ endosomes, and recruited NF-kB-inducing kinase (NIK) to these endosomes in a complement-dependent manner. MAC complexes were previously demonstrated to activate an Akt+NIK+ signalosome in human endothelial cells. To the best of our knowledge, this is the first to identify such signalosomes in Rab5+ endosomes in response to complement and Aβ. These results suggest a novel possibility of a therapeutic target in the case of an Aβ-induced neuroinflammation. C5a is the most potent anaphylatoxin, thus upon the identification of the complement-inflammasome interactions, the role of C5a in the Aβ recognition was investigated in human astrocytes. The results demonstrated that C5a augmented the Aβ-induced IL-1β production, activated pro-IL-1β and was caspase-1-dependent. In addition, a C5a-induced IL-1β release was observed following Signal 2 stimulation by Ca2+ and lysosomal damage. Since the signal activation resulting in inflammation in AD appears to involve multiple receptors, the therapy should be combinational and not targeting single molecules. Thus, C5aR1 blocking antibodies and PMX53 were utilized to identify a combinatory therapeutic approach in human astrocytes. The results demonstrated that the secretion of IL-1β was further reduced in the combinatory treatment of PMX53+MCC950 7 | P a g e compared with their single treatments, suggesting a combinatory treatment to tackle the effect of Aβ in human cells. The results of the present study add on to the current knowledge of the molecular mechanisms involved in AD, regarding Aβ association, and may lead to the design of targeted therapeutic interventions for AD.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:715993 |
| Date | January 2016 |
| Creators | Loizou, Florentia |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/100532/ |
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