Abstract
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The hallmark pathological features of AD are intraneuronal aggregates of hyper-phosphorylated tau protein and extracellular plaques of amyloid-beta (Aβ) protein. Aggregated forms of Aβ, such as oligomers and protofibrils, induce a wide array of neuro- and synapto-toxic effects upon neurons and glial cells. In contrast, rodent models have shown that secreted amyloid precursor protein-alpha (sAPPα), which is generated from the parent protein that gives rise to Aβ, protects neurons against Aβ-induced neurotoxicity, induces neurogenesis, and enhances synaptic plasticity mechanisms including altered protein synthesis. This suggests that sAPPα may have disease-modifying potential, however, to date, investigation of sAPPα’s effects in other neural cell types, such as astrocytes, and in neural cells of human origin, has been extremely limited.
Therefore, the overarching hypothesis of this thesis is that the previously observed effects of sAPPα would extend to astrocytes and be observed in human neurons. Accordingly, I used primary murine cultures highly enriched in astrocytes to explore whether sAPPα (1 nM) could exert neuroprotective actions upon astrocytes by enhancing their ingestion of Aβ protofibrils (AβPFs). Immunocytochemical detection of AβPFs using the Aβ-specific antibody 4G8 revealed that administration of sAPPα significantly reduced the area and number, but not the integrated density, of AβPF deposits within astrocyte cultures following a 24 h AβPF insult. These findings suggest that either the sAPPα-treated astrocytes internalised less AβPF than astrocytes that were not exposed to sAPPα or, alternatively, that sAPPα administration altered astrocytic processing of ingested AβPFs, causing a similar amount of ingested material to be packaged more compactly inside the cell. The additional experiments required to tease apart these two possibilities are proposed.
As microRNA (miRNA) are important regulators of protein synthesis, I next assessed whether sAPPα and/or AβPFs altered the miRNA cargo of astrocyte-derived extracellular vesicles (ADEVs), using highly sensitive TaqMan Advanced miRNA arrays representing a panel of 168 neurodegeneration-associated miRNAs. Notably, exposure of primary murine astrocytes to AβPFs significantly upregulated the expression of let-7c-5p, miR-29a-3p and miR-34a-5p, while sAPPα enhanced the level of miR-99b-5p and concurrent exposure of astrocytes to sAPPα and AβPF led to increased expression of miR-181d-5p within ADEVs. Importantly, bioinformatic analysis of biological pathways likely targeted by these differentially expressed miRNAs revealed that AβPF-instigated changes were associated with pathways previously linked to AD. In contrast, the upregulation of ADEV-associated miR-99b-5p following sAPPα treatment is likely to promote neuroprotective outcomes in ADEV-ingesting neurons.
As sAPPα robustly upregulates protein synthesis in rodent hippocampal neurons, using the fluorescent non-canonical amino acid tagging with proximity ligation assay (FUNCAT-PLA), I investigated its effects on primary rodent hippocampal astrocytes, finding that sAPPα (1 nM; 2 h) significantly promoted de novo protein synthesis in a manner comparable to the neurotrophin brain-derived neurotrophic factor. Surprisingly, however, in induced glutamatergic neuron-like cells derived from directly reprogrammed adult human dermal fibroblasts, increasing concentrations of sAPPα (1, 10 and 20 nM for 30 min) significantly reduced de novo protein synthesis as detected by the surface sensing of translation (SUnSET) puromycin incorporation assay.
In summary, these results provide evidence that sAPPα exerts neuroprotective and protein synthesis-enhancing effects upon rodent astrocytes, but suggest that further investigation is required to establish the actions and most beneficial concentration of sAPPα in a human cell culture model.