Myelin Changes in H-ABC Tubulinopathy Revealed by Nile Red Hyperspectral Imaging

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a recently described tubulinopathy caused by mutations in the tubulin beta-4a isotype. It is a severe disorder characterized by progressive myelin loss and neurological impairment. The taiep rat, a spontaneous TUBB4A mutant, is a model for investigating the cellular and molecular mechanisms underlying this disease. Homozygous taiep rats exhibit hallmark features of H-ABC, including early-onset hypomyelination, progressive demyelination, and microtubule accumulation in oligodendrocytes, whereas heterozygous animals appear to maintain normal myelin structure using conventional techniques. We combined hyperspectral imaging (HSI) and Nile Red (NR) staining to differentiate homozygous taiep and wild-type animals and determine whether subtle myelin alterations could be detected in heterozygous rats. Remarkably, heterozygous animals also showed changes in the Nile Red emission profile, with fluorescence shifted toward longer wavelengths compared with controls, uncovering previously unrecognized modifications in the myelin microenvironment. Using spectral phasor analysis, these alterations were localized and quantified without model assumptions, demonstrating the sensitivity of this approach for detecting subtle pathological changes. Together, our findings contribute to the characterization of myelin-associated spectral changes in this tubulinopathy and support the use of spectral microscopy to distinguish alterations in myelin properties, with potential applications in the study of other white matter disorders.