Using stem cells to identify regulatory genes in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders and it affects over 30 million people worldwide. It is categorised into sporadic (SAD) and familial (FAD) forms, and FAD is caused by the autosomal dominant mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) or presenilin 2 (PSEN2). The aetiology of SAD and exact mechanism of AD progression is not yet clearly understood, with pathological changes starting up to 20 years before the first symptoms appear. FAD accounts for less than 5% of the total AD cases; however, the hallmarks of AD such as amyloid plaques, neurofibrillary tangles, glial and inflammatory response, synaptic and neuronal loss are present in both FAD and SAD. In order to identify targets for early diagnosis and drug discovery, it is essential to understand the metabolic pathways involved in mediating the changes that results in the hallmarks of AD. Identification of master regulatory genes using transcriptomics can provide an insight into the progression of complex disease such as AD, considering that the master regulatory genes are involved in multiple complex signalling pathways. In this thesis, cortical neurons were differentiated from induced pluripotent stem cells of an FAD patient with a PSEN1 L286V mutation and 3 healthy controls and RNA-sequencing was performed using the Illumina sequencing platform. The aim of the thesis was to identify altered metabolic pathways and candidate master regulatory genes in neurons containing the FAD-causing PSEN1 L286V mutation. Pathways relevant to AD such as synaptogenesis signalling pathway, neuroinflammation signalling pathway, axonal guidance signalling, protein kinase A signalling, Wnt/β-catenin signalling and HIF1α signalling were overrepresented in neurons containing the FAD-causing PSEN1 L286V mutation as compared to the control neurons. PBX1 was identified as a novel master regulatory gene that affected the APP processing pathway at various points. The siRNA knockdown of PBX1 in neuroblastoma SH-SY5Y cells resulted in a reduction in both Aβ40 and Aβ42, with no alteration to the Aβ42/Aβ40 ratio. PBX1 knockdown also led to a reduction in APP, sAPPα, and mature ADAM10, while sAPPβ and immature ADAM10 remained unaltered. This indicates that sAPPα might be reduced owing to decreased APP and a reduction in the mature form of ADAM10. Also, the knockdown of PBX1 potentially reduced secreted Aβ40 and Aβ42 by affecting the endoproteolysis of PSEN1, as there was a reduction in the active C- and N- terminal fragments of PSEN1. These data illustrate the importance of identifying master regulatory genes to understand AD pathogenesis and identifies the novel role of PBX1 as a constitutive regulator of the APP processing pathway. Further investigation of the mechanism of action of PBX1 and its effect on other pathological hallmarks of AD may provide insights into its potential role in AD pathogenesis.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Doig (Supervisor), Stuart Pickering-Brown (Supervisor) & Nigel Hooper (Supervisor)