Molecular mechanisms involved in krabbe s disease

Abstract

Krabbe disease is an autosomal recessive disorder resulting in the deficiency of galactosilcerebrosidase (GALC), a lysosomal enzyme involved in the catabolism of two lipids extremely represented during formation of myelin sheet: galactosylceramide and psychosine. Despite the GALC activity deficiency, galactosylceramide does not increase in the brain of patients, while psychosine accumulation is reported as cause of myelin-generating cells death and of severe neurodegeneration. The present work outlined a molecular explanation for how psychosine mediates a dose-dependent cell death in oligodendrocytes precursor and fibroblast cells with and without GALC mutation. In addition, it was analyzed how this mechanism may interfere in oligodendrocytes maturation. The connexins play essential roles in cell homeostasis, growth, differentiation and death. Among connexin family members, Connexin43 is recently reported as an active element in apoptosis by gap junction-dependent or -independent mechanisms. Still today, there are no scientific researches evaluating Connexin43 expression and localization in normal and in GALC mutated oligodendrocytes precursor cells. The results of this study established that the different effects of exogenous psychosine addition could be linked to different sensitivity of cell membrane. Furthermore, it was determined a primary involvement of cell proliferation and a relationship between caspase-3 activation and disturbance of membrane-activated protein involved in cellular growth and survival. Psychosine treatments resulted in proteolysis of procaspase-3 proportioned to PTEN up-expression and PI3K activity down-regulation, confirmed by Bad release, p53 enhanced levels and decrease of NF-kB. The intrinsic apoptosis activation was established. Moreover, psychosine treated oligodendrocyte precursors increased Connexin43, demonstrating preferential intracytoplasmatic localization and indicating that no gap- junction was involved. These in vitro findings were supported by examination of Connexin 43 levels in mouse brains: wild-type mouse brain proteins were compared to those ones of twitcher mouse, a natural occurring model of Krabbe disease. Reminding that Connexin43 never was found in mature oligodendrocytes, the second part of this work was pioneer in evaluating Connexin43 expression during wild-type- and GALC-mutated oligodendrocytes maturation steps without psychosine treatment. In experimental condition applied, it was verified that wild-type oligodendrocyte progenitors differentiate into a mature phenotype with loss of Connexin43. Under the same condition, GALC-mutated oligodendrocyte precursor did not express marker of maturity, they became multinuclear and Connexin43 levels remained higher than control, suggesting that psychosine is accumulating in early steps of oligodendrocyte precursor cell maturation. Taken together, these findings led to the general idea that psychosine may functionally impact cells through interference on the growth system and alteration in maturation programme, strongly related to Connexin 43 expression. Thus, the third part of this work probed the likelihood of using adipose tissue-derived mesenchymal stem cells (AT-MSCs) as source of oligodendrocytes. Two different conditioned media were used and the results demonstrated that AT-MSCs can be influenced by the environment versus a neural phenotype. For this reason, stem cells can furnish potential material for cell replacement therapy. In summary, this thesis provides a new and unexplored molecular-mechanism for understanding the appearance and maintaining Krabbe disease hallmark. This work is the first description of a potential Connexin43 involvement in oligodendrocytes maturation and psychosine-inducted toxicity.