Single-cell transcriptional patterns impairment in an adult neurogenesis model of Alzheimer´s disease

Alzheimer's disease (AD) disrupts neurogenesis and neuronal differentiation, processes critical for cognitive function. This study investigates transcriptional and regulatory changes during neuronal differentiation under the influence of amyloid-beta (Aß) peptide using scRNA-seq and scATAC-seq approaches. Neuronal differentiation trajectories showed three distinct groups of differentially expressed genes (DEGs). Notably, while some neurogenesis-related genes like VIM, MAPT, STMN2, and SLC17A6 were unaffected by Aß, others, such as PAX6, NEFL, NEFM, and CALB1, displayed significant alterations, particularly in transitions to mature neuronal states. Functional enrichment analysis highlighted disruptions in ATP synthesis, oxidative phosphorylation, synaptic signaling, and cytoplasmic translation. Regulatory transcription factors (TFs) such as EGR1, GATA2, PAX6, and SOX2 were also implicated in these changes. Translational characterization revealed that the gene expression changes observed in the Aß condition partially aligned with dysregulated gene patterns in hippocampal tissue from AD patients. Differential analysis across stages indicated that mature neuronal differentiation stages exhibited stronger concordance with AD-related gene expression. To understand the role of chromatin accessibility, scATAC-seq analysis identified dysregulation in gene regulatory networks (GRNs) driven by key TFs such as ASCL1, HOXB2, POU2F2, and ONECUT2. Notably, regulons promoting neurogenesis in control conditions were impaired or delayed under Aß exposure, with regulons specific to mature neuronal transitions emerging only in the presence of Aß. Functional analysis of these regulons revealed enrichment for pathways associated with differentiation, neuron migration, and synaptic regulation, suggesting Aß-mediated disruption of neurogenesis-associated regulatory programs. In summary, this study highlights Aß-induced transcriptional and regulatory disruptions during neuronal differentiation, linking these changes to AD pathology. It underscores the importance of transcriptional and epigenetic regulation in understanding AD and provides insights into how Aß interferes with neurogenic processes critical for neuronal maturation and function. Overall design: Neural progenitor cells (NPCs) derived from XCL1 DCXpGFP (ACS5005™, ATCC, Manassas, VA, USA) were cultured and differentiated to neurons following standardized protocols. NPCs were seeded at 0.30 × 106 cells per well in CellMatrix Basement Membrane Gel-coated 12-well plates and maintained in NPC expansion medium, consisting of DMEM/F-12 supplemented with the Growth Kit for Neural Progenitor Cell Expansion. Cultures were incubated in a humidified chamber at 37°C with 5% CO2. For neuronal differentiation, NPCs were seeded at 80,000 cells/cm² in 6-well plates. On Day 0, NPCs were cultured in expansion medium, transitioning to a differentiation medium from Day 1 onward, with half-medium changes every 2–3 days. The differentiation medium was BrainPhys™ Neuronal Medium, supplemented with NeuroCult™ SM1 (1:50), N2 Supplement-A (1:100), BDNF (20 ng/mL), GDNF (20 ng/mL), dibutyryl-cAMP (1 mM), and ascorbic acid (200 nM). Amyloid-ß peptide (Aß1?42; 50 nM) or DMSO (vehicle control) was added weekly. Cells were harvested on Days 0, 7, 13, and 20 for both Aß-treated and control conditions. Detachment was performed using Accutase, followed by washing in Dulbecco's phosphate-buffered saline (DPBS) and centrifugation at 13,000 rpm. Cell pellets were resuspended in a filtered buffer (0.05% BSA in PBS). Prior to single-cell RNA sequencing (scRNA-seq), viable cells were enriched using TO-PRO-3 dye and sorted using a MoFlo Astrios EQs Sorter. Dead cells were excluded, and single, viable cells were gated based on FSC/SSC and FSC width/area criteria. Between 20,000 and 70,000 viable cells were collected and processed immediately for scRNA-seq analysis.