Characterization of a Human Neuronal Culture System for the Study of Cofilin-Actin Rod Pathology

Cofilactin rod pathology, which can initiate synapse loss, has been extensively studied in rodent neurons, hippocampal slices, and in vivo mouse models of human neurodegenerative diseases such as Alzheimer's disease (AD). In these systems, rod formation induced by disease-associated factors, such as soluble oligomers of Amyloid-beta (A beta) in AD, utilizes a pathway requiring cellular prion protein (PrP (c)), NADPH oxidase (NOX), and cytokine/chemokine receptors (CCR5 and/or CXCR4). However, rod pathways have not been systematically assessed in a human neuronal model. Here, we characterize glutamatergic neurons differentiated from human-induced pluripotent stem cells (iPSCs) for the formation of rods in response to activators of the PrP (c)-dependent pathway. Optimization of substratum, cell density, and use of glial-conditioned medium yielded a robust system for studying the development of A beta-induced rods in the absence of glia, suggesting a cell-autonomous pathway. Rod induction in younger neurons requires ectopic expression of PrP (c), but this dependency disappears by Day 55. The quantification of proteins within the rod-inducing pathway suggests that increased PrP (c) and CXCR4 expression may be factors in the doubling of the rod response to A beta between Days 35 and 55. FDA-approved antagonists to CXCR4 and CCR5 inhibit the rod response. Rods were predominantly observed in dendrites, although severe cytoskeletal disruptions prevented the assignment of over 40% of the rods to either an axon or dendrite. In the absence of glia, a condition in which rods are more readily observed, neurons mature and fire action potentials but do not form functional synapses. However, PSD95-containing dendritic spines associate with axonal regions of pre-synaptic vesicles containing the glutamate transporter, VGLUT1. Thus, our results identified stem cell-derived neurons as a robust model for studying cofilactin rod formation in a human cellular environment and for developing effective therapeutic strategies for the treatment of dementias arising from multiple proteinopathies with different rod initiators.

Automated summary

This study demonstrates that neurons differentiated from human-induced pluripotent stem cells provide a robust model for studying the formation of cofilactin rods. The formation of rods in response to disease-associated factors like A beta depends on a pathway involving cellular prion protein, NADPH oxidase, and cytokine/chemokine receptors. The absence of glia allows for easier observation of rods, revealing that neurons mature and fire action potentials without forming functional synapses. These findings are important for understanding and developing treatments for dementias caused by various proteinopathies.