The Origin and Characteristics of Globoid Cells in Krabbe Disease

• Main Author: Mohammed Haseeb Nawaz
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2023
• Subjects: Cellular biology; Molecular biology; Neurosciences
• ISBN: 9798379734879

Krabbe disease (KD) is a lysosomal storage disorder caused by the mutation in the Galactosylceramidase (GALC) gene. GALC is a lysosomal hydrolase that catabolizes the myelin sphingolipid galactosylceramide (GalCer), a major lipid component of myelin sheath. More than 85% of KD is infantile-onset, untreatable, and manifests severe neurological symptoms such as irritability, failure to thrive, delayed development, decerebrate posturing, and eventually becoming fatal. Another GALC substrate, galactosylsphingosine (=psychosine), is a toxic lipid that accumulates in KD tissues and is thought to drive majority of the disease pathology causing extensive demyelination and neurodegeneration in the central and peripheral nervous systems (CNS and PNS). In addition, the giant multinucleated globoid cells, a pathological hallmark of the disease due to their prevalent distribution in KD tissues, have been presumed to form crystals with a swollen cytoplasm that becomes pro-inflammatory. However, questions about the mechanistic origin of globoid cells, their functional role in pathogenesis, and their importance in disease progression are largely speculative. A previous study in our lab has proved that GALC-deficient macrophages phagocytizing GalCer elicit a globoid cell phenotype associated with impaired phagocytosis, increased oxidative stress, and inflammation in the PNS. Nevertheless, there is a disagreement about whether the innate microglial cells solely or the perivascular macrophages are the origin of globoid cells in the CNS. To answer these questions in this study, we purified microglia from Galc-knockout brains along with wildtype control and treated them with GalCer and psychosine, or cocultured with Galc-knockout oligodendrocytes that are also purified from Galc knockout brains by Magnetically Activated Cell Sorting (MACS) method. We found that: 1) GalCer-treated microglia significantly induced the expression of Monocyte Chemoattractant Protein-1 (MCP-1), but the psychosine did not. 2) Neither GalCer nor psychosine treatment was able to induce globoid cell formation from the Galc-knockout microglial cells. 3) Galc-knockout microglia cocultured with Galc-knockout oligodendrocytes became multinucleated globoid cells as was confirmed by confocal microscopic analysis. Moreover, RT-qPCR analyses of the induced globoid cells revealed that MCP-1, Interleukin-6 (IL-6), and Secreted phosphoprotein 1 (SPP1) were significantly elevated in the globoid cells, indicating that globoid cells are the major contributor to Krabbe pathogenesis. Modulating the upregulated molecules in globoid cells should be further evaluated as a potential strategy to ameliorate KD.