Modeling Spastic Paraplegia 4 using Human Corticospinal Motor Neuron enriched Cortical Organoids Uncovers Genotype–Phenotype Distinctions and HDAC6-Targetable Pathology

Spastic Paraplegia 4 (SPG4), the most common form of Hereditary Spastic Paraplegia, causes progressive gait deficiency due to corticospinal tract degeneration. SPG4 results from mutations in the SPAST gene, which encodes spastin, a microtubule-severing AAA-ATPase. To dissect genotype–phenotype relationships, we generated isogenic human induced pluripotent stem cell lines carrying either a SPAST missense (SPASTWT/C448Y) or truncation (SPASTWT/S245X) mutation and differentiated them into motor cortical organoids. These models revealed mutation-specific patterns of aberrant neuronal activity, microtubule hypoacetylation and axonal degeneration. We identified mutant M1-spastin-induced hyperactivation of histone deacetylase 6 (HDAC6), a major tubulin deacetylase as the key pathogenic culprit. Pharmacological inhibition of HDAC6 with Tubastatin A restored microtubule acetylation and rescued axonal degeneration in organoids, with corresponding improvements in corticospinal tract integrity and gait defects in SPG4 transgenic mice. Our study uncovers HDAC6 hyperactivation as a targetable mechanism for SPG4 and verifies human organoids as a platform for therapeutic discovery. Overall design: The generated corticospinal motor neuron enriched cortical organoids were dissociated and processed using 10X genomics, 3' NEXT-GEM kit. The wild-type organoids sc-RNAseq data was used to identify the different cell-populations.