Reversing Autophagy Inhibition Ameliorates Neurodegeneration in Hereditary Spastic Paraplegia Caused by a Degradation‐Resistant SPAST Mutation

BACKGROUND: Hereditary spastic paraplegias (HSPs) are monogenic neurodegenerative disorders, and SPAST mutations causing spastic paraplegia type 4 (SPG4) represent the most common form of HSP. SPAST mutations elevate SPASTIN protein levels beyond haploinsufficiency, but the mechanisms and downstream consequences are unclear. METHODS: We identified a de novo SPAST missense variant (p.I344E) in a young Chinese female with SPG4. Wild-type (WT) and mutant (I344E/K) SPAST constructs were expressed in HEK293 for biochemical and functional assays including cycloheximide chase, ubiquitination analysis, and immunofluorescence. Leveraging somatic cell reprogramming and CRISPR-based gene editing, we generated patient-derived induced pluripotent stem cells (iPSCs) and their isogenic controls. Both lines were differentiated into cerebral organoids. RESULTS: I344E/K-SPASTIN exhibited markedly higher steady-state levels than WT-SPASTIN owing to impaired ubiquitin-proteasome-mediated degradation; the I344E variant showed the greatest accumulation. Mutant SPASTIN mislocalized in cells, displayed diminished microtubule-severing activity, and elevated acetylated tubulin-phenotypes that were rescued by overexpression of WT-SPASTIN. In patient cerebral organoids, the I344E mutation led to increased p62/SQSTM1 aggregates, reduced autophagic flux, and enhanced neuronal death. Rapamycin restored autophagy, decreased p62 levels, and reduced cell death. CONCLUSIONS: Our study provides evidence linking autophagy dysfunction to SPG4 pathogenesis and demonstrates that the I344E mutation acts through a gain-of-function mechanism. These findings challenge the prevailing haploinsufficiency model and implicate autophagy modulation as a potential therapeutic strategy for SPG4 and possibly other HSPs. © 2025 International Parkinson and Movement Disorder Society.