NKX6.2 related autosomal recessive hypomyelinating leukodystrophy, is rescue therapy via homeoprotein transfer a possibility?
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- NKX6.2 is a homeodomain transcription factor whose CNS expression is enriched in mature myelinating oligodendrocytes. Loss-of-function mutations in NKX6.2 cause Spastic Ataxia type 8 (SPAX8), a rare hypomyelinating leukodystrophy for which no curative treatment currently exists. Homeoproteins (HPs) constitute a superfamily of transcription factors capable of unconventional intercellular transfer via the third helix of their homeodomain, raising the possibility that wildtype NKX6.2 could rescue NKX6.2-deficient oligodendrocytes in SPAX8 patients through paracrine transfer. The aim of this study was to determine whether NKX6.2 behaves as a secretory homeoprotein and to evaluate its intercellular transfer in oligodendrocyte-relevant experimental models. Using a bicistronic pCAGIG-V5-NKX6.2-IRES-EGFP construct that allows discrimination between donor cells (V5+/GFP+) and putative transfer recipients (V5+/GFP−) by immunofluorescence, transfer was assessed in HEK293 cells, Human Oligodendroglioma (HOG) cells, and chick embryo neural tubes via in ovo electroporation. NKX6.2 intercellular transfer was detected in HEK293 cells at a mean frequency of 1.5 ± 1.3% across five independent experiments (6,831 cells total), demonstrating that NKX6.2 fulfils the criteria for a secretory homeoprotein. Transfer in oligodendrocyte-relevant systems could not be conclusively assessed: HOG cells proved refractory to lipofection-mediated transfection, precluding generation of sufficient donor cells; a conditioned medium co-culture approach produced no detectable V5 signal in HOG recipient cells, consistent with possible HSPG-mediated sequestration of secreted protein; and in ovo electroporation confirmed plasmid expression in the chick neural tube but revealed no detectable intercellular transfer. These results establish that NKX6.2 is competent for transfer in vitro, while highlighting the technical obstacles that must be resolved before the HP-based rescue hypothesis can be directly tested in oligodendrocyte-relevant models of SPAX8.
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Deward, T. (2026). NKX6.2 related autosomal recessive hypomyelinating leukodystrophy, is rescue therapy via homeoprotein transfer a possibility? https://dial-mem.test.bib.ucl.ac.be/handle/2078.2/47164