Intermediate filament dysregulation in astrocytes in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN)

Giant Axonal Neuropathy (GAN) is a pediatric-neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, which regulates intermediate filament turnover. Previous neuropathological studies and examination of postmortem brain tissue in the current study revealed involvement of astrocytes in GAN. To develop a clinically relevant model, we reprogrammed skin fibroblasts from seven GAN patients to pluripotent stem cells (iPSCs), which were used to generate neural progenitor cells (NPCs), astrocytes, and brain organoids. Multiple isogenic control clones were derived via CRISPR/Cas9 gene editing of one patient line carrying the G332R gigaxonin mutation. All GAN iPSCs were deficient for gigaxonin and displayed patient -specific increased vimentin expression. GAN NPCs had lower nestin expression and fewer nestin-positive cells compared with isogenic controls, but nestin morphology was unaffected. GAN brain organoids were marked by the presence of neurofilament and GFAP aggregates. GAN iPSC-astrocytes displayed striking dense perinu-clear vimentin and GFAP accumulations and abnormal nuclear morphology. In overexpression systems, GFAP oligomerization and perinuclear aggregation were augmented in the pres-ence of vimentin. GAN patient cells with large perinuclear vimentin aggregates accumulated significantly more nuclear KLHL16 mRNA compared with cells without vimentin aggregates. As an early effector of KLHL16 mutations, vimentin may be a potential target in GAN.