Neurodevelopmental alterations are key drivers of SPG56.

Biallelic pathological variants in CYP2U1 are associated with SPG56, a complex ultra-rare form of hereditary spastic paraplegia. CYP2U1 encodes a member of the cytochrome P450 family 2, which is highly expressed in the brain and involved in arachidonic acid metabolism and lipid hydroxylation. Little is known about the early stages of neurodevelopment in SPG56. To gain insight into the molecular mechanisms of the disease, we generated and characterized both a zebrafish knock-out model (cyp2u1-/-) and cortical neurons derived from induced pluripotent stem cells (iPSCs) of two patients affected by SPG56. cyp2u1-/- zebrafish showed increased mortality and locomotor impairment, and transcriptome analysis revealed defects in the cell cycle, neural development, and oxidative phosphorylation pathways, consistent with observed decreased cell proliferation, increased apoptosis, and bioenergetic impairment. To assess whether these molecular alterations impact network function, we performed local field potential recordings and whole-brain calcium imaging in mutant larvae. Local field potential analyses revealed prolonged high-frequency events despite reduced overall power, and calcium imaging showed increased skewness of neuronal activity distributions, findings indicating altered brain network dynamics in mutants. Transcriptional profiling of cortical cultures performed at neural progenitor, early-born, and mature neuron stages pointed to altered neural network development as one of the main pathogenic mechanisms of the disease and highlighted several similarities with the zebrafish model. Multiple features of the cyp2u1-/- zebrafish recall the human SPG56 phenotype, and combined transcriptomics profiling in cyp2u1-/- zebrafish and SPG56 patient iPSC-derived cortical neurons supports impaired neural network development as a key disease mechanism.