The goal of this proposal is two-fold: 1) to understand why the same mutation in the galactosylceraminidase (Galc) gene results in ample clinical variability among Krabbe leukodystrophy patients, and 2) to understand if brain cells other than oligodendrocytes are directly targeted by GALC deficiency. Answers to these two questions are crucially important for accurate diagnosis and for the design of more effective therapies. This disease is due to autosomal recessive mutations in the lysosomal enzyme GALC, which cause severe demyelination and neurodegeneration. More than 85% of Krabbe patients show infantile-onset, progressive neurologic deterioration and death by two years of age, while others have a late onset, less severe disease. This disease is diagnosed by measuring GALC activity and confirmed by mutation analysis and clinical features. However clinical studies have revealed that the pathogenetic mutation that a patient carries in the GALC gene is not always predictive of outcome, and the reasons for such variability are unclear. Therefore, it is difficult to predict the disease course accurately. Unfortunately, the only available therapeutic option is hematopoietic cell transplantation (HCT) before symptoms occur, thus prognostic indicators are of utmost importance for disease management. HCT only partially improves survival and attenuates the disease course in the infantile phenotype, presumably by transfer of GALC from donor cells to myelinating cells of the patients. One unanswered question in Krabbe and other demyelinating disease is whether neurodegeneration is only secondary to demyelination, or if neurons or other brain cells are also direct targets of the disease process. I hypothesize that: 1) In addition to the mutation, cis-polymorphisms also affect the trafficking of GALC to the lysosome which is indispensable for GALC processing and activation, ultimately influencing the phenotype. 2) Any brain cell other than oligodendrocytes can produce toxicity which eventually damages neurons.
Two specific aims will test this idea: 1) Identify the effect of the combination of cis-polymorphisms with Galc mutations on GALC trafficking, processing, and activity in the ER, Golgi, and lysosome, 2) Identify the effect of the absence of GALC in each brain cell type on myelination and neurodegeneration. For the first aim, I will test if any of the cis-polymorphisms that have been reported to coexist with certain mutations affects the trafficking of GALC. For the second aim, I will purify each type of brain cell from the twitcher mouse model of Krabbe disease and coculture them in various combinations to elucidate in which cells GALC loss-of-function is important and which cells are the most important target of toxicity in demyelinating neurodegeneration. This approach is innovative because for the first time it considers cell autonomy and the effect of cis- polymorphisms in Krabbe disease. The project will have impact because it will allow us to better understand which factors determine the prognosis of Krabbe patients, and will provide information necessary to design rational and effective therapies.
Krabbe leukodystrophy is a progressive neurologic lysosomal storage disorder and affects babies causing death usually before two years of age. It is difficult to predict the disease course and to decide which cell type to target with therapy, because the mutation in the GALC gene that a Krabbe leukodystrophy patient carries is not always predictive of outcome for unknown reasons, and it is also not known which cell first generates the toxicity. In this proposal, I will study 1) how other DNA polymorphisms beside the disease mutation, affect the behavior of the protein and 2) which type of brain cells produce toxicity that kills neurons.