Autophagy as a pathogenic mechanism and a therapeutic target in Krabbe disease

Globoid Cell Leukodystrophy (GLD), also known as Krabbe disease, is a rare hereditary neurodegenerative condition. This disorder affects about 1 in 100000 live births and the early-infantile form represents approximately 85-90% of all cases. GLD is rapidly progressive and results in neurological degeneration and death within the first 1-2 years from birth. The cause of this disorder has been attributed to the deficiency of a lysosomal enzyme called galactosyl-ceramidase (GALC). Normally, GALC degrades galactosyl-ceramide (GLC) and other sphingolipids, among which galactosylsphingosine (psychosine, PSY). GALC loss of function leads to PSY accumulation in the central and peripheral nervous system, which causes massive oligodendrocyte and Schwann cell death, with resulting demyelination. The accumulation of PSY afflicts myelinating cells and neurons as well, leading to a series of cellular and tissutal modifications. Unfortunately, the systemic administration of GALC is not an effective therapy, since the enzyme can not cross the blood brain barrier (BBB). Many therapeutic approaches have been attempted but, except for symptomatic and life-support treatments, there is no cure currently available. Owing to the poor general understanding of GLD physio-pathology, novel approaches to investigate the underlying mechanisms prove necessary. Lysosomal disorders are typically characterized by an abnormal accumulation of substances within lysosomes. Lysosomes are intracytoplasmic organelles endowed with the enzymes necessary to degrade all principal classes of biological molecules and capable of eliminating entire organelles, thus playing a fundamental role in metabolizing endogenous substances whose accumulation within the cell is potentially toxic. The process by which lysosomal enzymes make contact with the substances to be degraded is known as autophagy. The accumulation of un-degraded substrates interferes with different cellular functions and autophagic mechanisms in particular result frequently altered. As in other lysosomal storage disorders, autophagy might have a significant role also in GLD, but this process has never been investigated for this pathology. The aim of this thesis is to study autophagy as a pathogenic mechanism and a therapeutic target. Given that oligodendrocyte degeneration is among the major characteristics of Krabbe disease, a human oligodendrocytes hybrid cell line (MO3.13) has been used as a cellular model. MO3.13 have been treated with exogenous PSY alone or in conjunction with lithium acetate, an autophagy modulator, and the reaction of cells to this drug has been assessed by determining cellular modifications such as viability, different expression of autophagy-involved factors and Reactive Oxygen Species (ROS). Flow cytometry analysis of cells stained with both Annexin V FITC (AV), a marker for cell apoptosis, and Propidium Iodide (PI), an indicator for cell necrosis, revealed an increased mortality rate in cells treated with PSY, while the additional administration of lithium incremented cell viability, suggesting some sort of lithium-mediated betterment in oligodendrocytes disturbed by the presence of PSY. To confirm the hypothesis that lithium-induced autophagy may be the cause of such improvement, cells were treated with exogenous PSY alone, or else in conjunction with lithium, and then tested for LC3 and p62 as autophagic activity markers. LC3 localization within the cell was investigated through confocal fluorescence microscopy, whereas levels of LC3-II and p62 were examined through western blot. To further inspect whether the cause of the improved viability obtained with lithium could be a lithium-induced autophagy, in addition to the usual treatment, the cells were administered with prolyl endopeptidase inhibitor 2 (PEI), an inhibitor of prolyl oligopeptidase activity, which nullifies the effects of lithium. Here as well, cell viability was checked by staining the cells with AV-PI and examining the emitted fluorescence with a cell sorter. Moreover, to investigate whether the supposed lithium-induced autophagy may decrease PSY levels, cells treated with the usual treatments were lysed, lipids were extracted and levels of PSY were quantified by HPLC and mass spectrometry. Finally, since PSY-induced cell death is associated with ROS production, variation in ROS levels was examined by administering the cells with the usual treatment, staining them with a fluorogenic marker for ROS and detecting the emitted fluorescence with a cell sorter