Date of Award
Doctor of Philosophy (PhD)
Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a rapidly progressing, invariably fatal pediatric disorder first described in 1916. Krabbe disease is caused by a deficiency in the lysosomal enzyme, galactosylceramidase (GALC), and is characterized clinically by failure to thrive, limb stiffness, seizures, developmental regression, and death by 2-4 years of age. Galactosylceramidase degrades the cytotoxic glycolipid, galactosylsphingosine (psychosine). In the absence of GALC activity, psychosine accumulates primarily in oligodendrocytes and Schwann cells, resulting in profound demyelination. In 1972, psychosine was hypothesized to be responsible for the clinical signs associated with Krabbe disease. However, the ‘Psychosine Hypothesis’ has never been tested, due to the inability to dissociate GALC deficiency from psychosine accumulation. This is due, in part, to a limited understanding of psychosine biosynthesis. Two studies published in 1960 and 1973 provided complementary evidence suggesting that psychosine is synthesized via an anabolic pathway. However, neither the cDNA nor the enzyme catalyzing that reaction has been identified. In the first part of this dissertation, we overturn those studies and show that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction effectively dissociates GALC deficiency from psychosine accumulation, allowing us to test and confirm the ‘Psychosine Hypothesis. ’ These data also identify ACDase as a potential target for substrate reduction therapy (SRT). We show that pharmacological inhibition of ACDase activity significantly prolongs the lifespan of the Twitcher (Twi) mouse, a GALC-deficient model that faithfully mimics the biochemical, histological, and clinicobehavioral features of Krabbe disease. These data clarify our understanding of psychosine synthesis, confirm the long-held ‘Psychosine Hypothesis,’ and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease. Although Krabbe disease is a monogenic disorder, it is remarkably refractory to treatment, and single modality therapies are minimally effective. However, combining CNS-directed, adenoassociated virus (AAV) 2/5-mediated gene therapy, hematopoietic stem cell transplantation, and SRT greatly increases efficacy in the Twi mouse. In the second part of this dissertation, we incorporated a newer generation vector, AAV2/9, into this combination therapy regimen. This single change significantly increased the lifespan of Twi mice. Importantly, it also dramatically improved, and in some cases, normalized, the clinicobehavioral deficits that remained uncorrected in mice treated with the AAV2/5-combination therapy. Unfortunately, nearly all of the AAV2/9- combination-treated Twi mice and all combination-treated wild type control mice died from hepatocellular carcinoma (HCC). Integration site analysis confirmed AAV sequence incorporation into the mouse genome. These data demonstrate the value of targeting multiple pathogenic mechanisms for complex metabolic diseases, but highlight the potential risks associated with these approaches. Taken together, the data presented in this dissertation greatly advance the field of Krabbe research by increasing our fundamental understanding of Krabbe pathogenesis, and by making significant progress in the treatment of this fatal disease. Modification of the combination therapy regimen to include a safe, ACDase-inhibiting SRT to target psychosine synthesis, as well as a liver de-targeted gene therapy to minimize HCC penetrance will likely bring us even closer to a cure.
Chair and Committee
Mark S. Sands
Abhinav Diwan, Christina A. Gurnett, Robyn S. Klein, Daniel S. Ory,
Li, Yedda, "Pathophysiology and Treatment of Murine Globoid Cell Leukodystrophy" (2020). Arts & Sciences Electronic Theses and Dissertations. 2209.