Proteomic analysis of molecular mechanisms in ageing associated neurodegenerative diseases
Alzheimer’s disease (AD), as one of the most common ageing-related neurodegenerative diseases, contributes to 60-70% of dementia cases and is the fourth-largest cause of death for people over 65 years old. Brain abnormalities and eye pathologies have been noted during AD progression, with the abnormal accumulation of amyloid β (Aβ) protein playing a vital role. However, the molecular mechanisms induced by Aβ toxicity, especially in the early stage of AD, have not been clearly demonstrated. Investigation of the molecular changes induced by Aβ in the brain and eye is an active area for the discovery of potential ocular biomarkers for AD diagnosis and treatment. This thesis aims to enhance our knowledge of ageing-related neurodegenerative diseases by characterizing disease activated proteins and analyzing associated biological pathways.
Initially, different protein and peptide fractionation approaches for label-free shotgun quantitative proteomic analysis were compared to find an optimal method to achieve more protein and peptide identifications. Subsequently, a strategy with the optimized method and TMT-labelling technique was applied to all studies included in this thesis. These generated proteomic data reveal the molecular responses and biochemical pathways induced by Aβ in brain and eye using different in vitro (SH-SY5Y human neuroblastoma cells and 661W mice photoreceptor cells) and in vivo animal models (APP/PS1 double transgenic mice), along with the molecular mechanisms underlying the retinal disorders induced by glaucoma in glaucoma model rats.
For the in vitro experiments, SH-SY5Y neuronal cells and 661W photoreceptor cells were separately exposed to two different concentrations of Aβ for two time-points, to mimic the neuronal damage in the brain and the retinal disorder in the eye which occur with AD progression. These studies revealed the impacts of Aβ toxicity on the two cell types are both dose- and timedependent, although the exposure time induced greater changes in cellular proteome compared to the impacts of concentration. In addition, the Aβ regulated proteins and associated biochemical pathways in both cells are also demonstrated.
For the in vivo experiments, brain and retina tissues from the animal model APP/PS1 mice were separately analyzed. Increased expression of APP protein was found in all brain areas and retinas with AD. Early-stage impacts of AD and different regulations were found in the proteomes of the hippocampus, frontal cortex, parietal cortex and cerebellum, with the most abundant protein expression changes discovered in hippocampus and cortices. In addition, more altered protein expression was seen in retinas from the older APP/PS1 mice than from the younger, including elevated levels of proteolytic enzymes and proteasomal proteins associated with the processing of noxious proteins, and decreased abundance of ribosomal subunits related to protein synthesis.
Since there are similarities between glaucoma and AD as ageing-related neurodegenerative diseases and eye disorders, the proteome remodeling underlying the retinal disorder caused by glaucoma was demonstrated in glaucoma model rats produced by chronic exposure to increased intraocular pressure. Compared with the findings from AD research, similar, yet different processes were identified in retinas with glaucoma. Greater knowledge of the molecular changes in AD and glaucoma may open up the possibility of early diagnosis of both diseases through a simple and non-invasive eye imaging or examination.
All these studies demonstrate the efficacy of the quantitative proteomic analysis in biomedical research, and illustrate the molecular mechanisms underlying ageing-related neurodegenerative diseases.