Human cholinergic neurons from nucleus basalis of Meynert: a new promising tool to study pathogenetic mechanisms affecting neurogenesis

The degeneration of basal forebrain cholinergic neurons within the nucleus basalis of Meynert (NBM) is responsible for the cognitive decline in neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Indeed, the major therapeutic strategies have been directed toward the cholinergic system. However, no effective therapies exist to contrast NBM cholinergic neuron loss and investigations in the field are strongly restricted by the lack of human models. Thus, the work of this thesis firstly contributed to establish and characterise a novel primary culture of cholinergic neurons from the human foetal NBM (hfNBM). An extensive phenotypic and functional characterization confirmed the cholinergic identity of hfNBM cells, including positivity for specific markers (ChAT, VAChT and AChE) and acetylcholine release as well as the presence of functional cholinergic receptors. Treatment with Nerve Growth Factor (NGF), the main neurotrophin for NBM neurons, activated the specific NGF/TrkA signalling pathway and promoted differentiation of hfNBM cells. Moreover, when intravenously injected in a NBM-lesioned rat model, hfNBM cells led to a significant improvement in cognitive and memory functions, confirming the functional value of these cells. Given the key role of neuroinflammation in the onset and progression of neurodegenerative diseases, another objective of this work was to investigate the effects of the main pro-inflammatory cytokine, tumor necrosis factor α (TNF-α), on cholinergic neuron development and plasticity. TNF-α exposure reduced immature neuronal markers (nestin, β-tubulin III) and increased the mature marker MAP2 along with neurite elongation. Interestingly, TNF-α treatment significantly reduced the number of neurons expressing primary cilium, a non-motile sensory antenna required for neurogenesis. Furthermore, a significant reduction of TrkA along with an increase of p75NTR, the high- and low-affinity NGF receptors, essential for survival or apoptotic signals, respectively, were observed upon TNF-α stimulation. Lastly, based on the emerging evidences demonstrating inflammation-driven epigenetic modifications, a significant increase of DNMT1, one of the key enzymes regulating DNA methylation, was detected after TNF-α stimulation. Interestingly, the genome-wide methylome analysis of hfNBM cells revealed an alteration of methylation pattern after the inflammatory insult. In particular, TNF-α exposure for 48 hours led to promoter hypermethylation of genes involved in neuronal development, such as chordin like-1 (CHRDL1) and mesoderm specific transcript (MEST). Accordingly, the mRNA levels of both genes were significantly reduced by TNF-α treatment. Overall, these results suggest that TNF-α-mediated inflammation may affect hfNBM cell development and maturation most likely interfering with the DNA methylation status. In conclusion, our findings indicate that hfNBM cells may represent a proven powerful in vitro model for studying disorders of the human nervous system, such as those affecting the differentiation and maintenance of cholinergic NBM neurons.