Mechanisms of Axonopathy in Krabbe Disease

In this work we describe the molecular mechanisms of axonal damage in Krabbe disease (KD). KD is a fatal genetic lysosomal storage disorder (LSD) caused by the loss of the lysosomal enzyme galactosyl-ceramidase (GALC). The deficiency causes the accumulation of galactosyl-sphingolipids. Psychosine, one of GALC substrates, is a lipid-raft associated neurotoxin believed to cause the death of myelinating cells in the CNS of affected patients. KD patients also suffer of neurodegeneration, due to axonal and neuronal deficiencies. Over the years, most studies have focused on the loss of myelin induced by psychosine, and have considered neurodegeneration as a consequence of demyelination. Recently, our laboratory showed that bone marrow transplantation (BMT) improved myelin preservation in Twitcher mice, the natural murine model for this disease, but it was insufficient to prevent neurodegeneration. There are various potential interpretations for this result. Neuronal loss and axonal degeneration in KD may start before oligodendroglia is affected, creating a progressive compounding factor contributing to the severity of the disease. Neurodegeneration may even be independent from demyelination. We hypothesized that GALC deficiency causes a cell-autonomous defect in the mutant neurons, leading to neurodegeneration. By studying the Twitcher mouse, we show that the Twitcher axons are affected by a decrease in axonal caliber and by morphological abnormalities from the first postnatal days. Importantly, we show that these defects are at least partially caused by changes in two critical components of neuronal function: the neurofilament (NF) cytoskeleton and fast axonal transport (FAT). In particular, we provide evidence that psychosine alters the activities of critical phosphotransferases, interfering with the phosphorylation state of NFs and kinesin, one of the molecular motors responsible for FAT. Finally, we identify protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and glycogen synthase kinase 3 β (GSK3β) as the phosphotransferases involved in psychosine toxicity. By injecting the newborn Twitcher mouse with an inhibitor of GSK3β, we rescue the functionality of the mutant nerve to a significant level, further demonstrating the relevance of altered phosphorylation in the development of axonopathy in the Twitcher mouse. In summary, these studies have shown that a sphingolipid can regulate NFs and FAT through specific phosphotransferases, and that this effect can cause axonal damage in KD. Pharmacological inhibition of these enzymes may provide new therapeutic alternatives to protect NFs and FAT in KD, therefore rescuing the mutant axons. Importantly, our studies also define a new mechanism by which psychosine, a substrate of a LSD, can cause axonopathy, raising the possibility that alterations in NFs and FAT might occur in other LSDs.