Genotype-Phenotype Distinctions in Spastic Paraplegia 4 Reveal HDAC6 as a Therapeutic Target.

Spastic Paraplegia 4 (SPG4) is the most prevalent form of Hereditary Spastic Paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and debilitating gait impairment, primarily driven by axonal degeneration of corticospinal motor neurons (CSMNs). Caused by mutations in the gene encoding spastin, an AAA-ATPase involved in microtubule severing and intracellular organelle function, SPG4 accounts for 40-50% of autosomal dominant HSP cases, yet without effective treatments. Although reduced microtubule acetylation has emerged as a key pathological mechanism, whether and how distinct mutations lead to microtubule deacetylation and subsequent neurodegeneration remains unclear. To address this, we generated isogenic human induced pluripotent stem cell (hiPSC) lines with two distinct heterozygous mutations - SPAST (missense) and SPAST (truncation). Employing an innovative differentiation protocol, we created human motor cortical organoids enriched in CSMNs, providing a robust platform to study SPG4 pathophysiology. These organoids revealed striking genotype-phenotype distinctions, with mutation-specific variations in CSMN loss, axonal degeneration and neuronal activities, mirroring clinical heterogeneity. Mechanistic studies identified aberrant activation of histone deacetylase 6 (HDAC6), a major neuronal microtubule deacetylase, as a key driver of SPG4 pathology. This dysregulation was specifically attributed to mutant M1-spastin, the longer isoform of spastin. Remarkably, pharmacological inhibition of HDAC6 with Tubastatin A restored microtubule acetylation status and mitigated axonal degeneration in both SPAST-mutant organoids, with corresponding improvements in corticospinal tract integrity and gait deficits validated in SPG4-transgenic mice. Collectively, our study establishes isogenic hiPSC-derived motor cortical organoids as a robust human model for corticospinal motor neuron degeneration and identifies HDAC6 hyperactivation as a central pathogenic mechanism and viable therapeutic target in SPG4.