A Missense Mutation in the Gene Encoding Heat Shock Cognate Protein 70 Causes Neuroaxonal Dystrophy in Rats

Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-/ rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-/ rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-/ rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-/ rat genome. Furthermore, we developed the knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous -KI rats exhibited ataxia and axonal spheroids similar to those of F344-/ rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 , which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the gene caused NAD in mammals.