Familial Alzheimer’s disease (AD) and frontotemporal dementia (FTD) are characterised by an age of onset typically between 30 and 50 years of age and are often linked to autosomal dominant mutations. This dissertation focuses on APP and MAPT mutations causal to early onset AD or FTD, which affect the dosage or isoform ratio balance of the key proteins underlying AD/FTD: amyloid beta and tau. Using a combination of in vitro iPSC-derived neuronal cultures carrying MAPT E10+16 and APP duplication mutations and post-mortem human brain tissue from patients carrying MAPT E10+16, APP V717L and APP duplication mutations, I describe the generation of transcriptomics and proteomics datasets with the goal of uncovering transcriptional and translational pathways driving AD linked to APP and MAPT mutations.

The first half of this thesis focuses on the differentiation of patient-derived iPSCs carrying MAPT E10+16 and APP duplication mutations and isogenic control iPSCs into neuronal cultures using the directed differentiation protocol. I first describe the CRISPR/Cas9 strategy to correct APP copy number in iPSCs derived from patients carrying APP duplication mutation. Using MiSeq sequencing, I confirm a successful monoallelic APP knockout in iPSCs carrying APP duplication mutation, thus generating two isogenic APP duplication iPSC lines (APPDUPiso). Next, I characterise the neuronal cultures derived from iPSCs carrying MAPT E10+16 and APP duplication mutations and isogenic control iPSCs differentiated using the directed differentiation protocol. Last, using a single-cell RNA sequencing dataset of mature cultures derived from each iPSC line, I identify an upregulation of cholesterol biosynthesis genes in excitatory and inhibitory neurons and astrocytes carrying MAPT E10+16 mutation compared to the isogenic control cells. I corroborate cholesterol synthesis upregulation in VGLUT1+ excitatory neurons carrying MAPT E10+16 mutation using an independent organoid-derived single-cell RNA sequencing dataset (Bertucci et al., 2023). Small RNA sequencing from bulk cultures carrying MAPT E10+16 mutation points out miRNA-mediated repression of cholesterol efflux, suggesting an overall prioritisation of cholesterol internalisation in the cultures carrying MAPT E10+16 mutation.

The second half of this dissertation focuses on the generation of spatial transcriptomic and bulk proteomic datasets using frontal lobe human brain post-mortem tissue from patients carrying patients carrying MAPT E10+16, APPV717I, APP V717L and APP duplication mutations. Using spatial transcriptomics on FFPE slides from three patients carrying MAPT E10+16 mutation and five control patients, I identify dysregulation of transcripts potentially relevant to the MAPT E10+16 tauopathy in the prototype spatial transcriptomic dataset. Using bulk TMT proteomics design from a bigger cohort of patients, including patients carrying APP V717L and APP duplication mutations, I corroborate the results from the spatial transcriptomics dataset. In addition, I identify differential protein abundance in samples from patients carrying MAPT E10+16 mutation, including a potentially novel marker of MAPT E10+16 tauopathy: RNA-binding protein Nop58. Preliminary results from immunohistochemistry experiments staining for Nop58 in the FFPE slides from patients carrying MAPT E10+16 mutation suggests formation of Nop58+ inclusions in the grey matter of these patients, potentially related to stress granule formation. The role of Nop58 in the brain or in tauopathies has not yet been described.

The results from my dissertation provide a platform for future examination of cholesterol biosynthesis and Nop58 in tauopathy underlined by MAPT E10+16 mutation. Whilst these results may be specific to the experimental models deployed in this dissertation, cholesterol synthesis upregulation was previously described as potentially related to pathological hyperexcitability in neurons carrying MAPT mutations, and stress granule formation remains an intensive area of research in neurodegenerative diseases.