Most babies diagnosed with the rare Krabbe Disease (KD) die before their second birthday. KD is a white matter disease caused by the deficiency of the lysosomal galactosylceramidase (GALC) enzyme, which breaks down toxic psychosine and galactosylceramide. Psychosine accumulation leads a rapid loss of myelin and neuronal dysfunction. Although bone marrow transplantation (BMT) in KD children has resulted in significantly extended life spans, it has been limited in preventing subsequent neurological deficits. This suggests that neuroprotective approaches combined with BMT are necessary for a more effective therapy for KD, which is the focus of my proposal. We have evidence in an authentic mouse model of KD (twitcher mouse) that abnormal GSK3? activation leads to defects in fast axonal transport (FAT), which may be a pathogenic defect contributing to the neurological deficits. The experiments outlined in this proposal aim to test the overarching hypothesis that overactive GSK3? resulting from psychosine-induced PI3K/Akt/mTOR downregulation is a conserved target for neuroprotection in KD.
Aim 1 will investigate the mechanism of GSK3? activation in KD using in vitro and in vivo models. In preliminary experiments, I found that PI3K/Akt/mTOR pathway was downregulated in twitcher tissue. Using both twitcher mice and cell culture systems, I will test the hypothesis that accumulation of psychosine leads to activation of GSK3? by downregulating mainly the PI3K/Akt/mTOR pathway. To test this, I will use both in vitro and in vivo methods to monitor the levels of PI3K/Akt/mTOR signaling. I will investigate twitcher tissue at progressive stages of the disease (P7, P15, P30) and also test for a synergistic effect by adding varying concentrations of psychosine in VEGF-induced motorneuron-like cells (NSC34) by immunoblotting and immunohistochemistry. A low dose of psychosine will also be used in combination with specific inhibitors of PI3K and mTOR, and their ability to synergize on FAT defects in normal NSC34 cells will be measured using live imaging of mitochondrial movement as an indication of FAT.
Aim 2 will test the hypothesis that abnormal activation of GSK3? is conserved in KD in other species (such as monkey and human). This will be accomplished by immunoblotting and immunohistochemistry to measure for active GSK3? and PI3K/Akt/mTOR pathway components in frozen and fixed brain tissue from human KD and monkey KD and will be compared to normal tissue.
Aim 3 will test the hypothesis that GSK3? inhibitors can be used as neuroprotective agents that will synergize with BMT to improve disease symptoms in twitcher mouse. To assess the potential of GSK3? inhibition for a combined therapy, I will perform BMT on newborn twitcher pups at P2 and a group of them will be treated for 30 days with a GSK3? inhibitor (TDZD8) starting at P3. I will compare treated vs. untreated mutants in terms of survival, engraftment of donor cells, globoid cell count, nerve conduction velocity and muscle strength. Together, the aims of this proposal will add to our understanding of disease mechanism, and attempt to develop an improved treatment for KD.
Inherited white matter diseases of the brain, such as Krabbe Disease (KD), are associated with substantial morbidity and mortality in children. Current treatments using bone marrow cells from a healthy donor and transplanting them into affected children has had limited success, emphasizing the need for developing combination therapies. The proposed experiments using a well-established mouse model of KD will significantly improve our understanding of disease pathology and progression, and will attempt to develop an improved therapy for the treatment of KD using this knowledge.