• autophagy
• krabbe disease
• reverse micelles

Krabbe disease (KD), also known as globoid cell leukodystrophy (GLD), is a rare (one in 100,000 births), autosomal recessive and neurodegenerative disorder. It affects both central and peripheral nervous system (CNS and PNS) and belongs to the family of the lysosomal storage diseases (LSDs), a large group of disorders characterized by disrupted lysosomal homeostasis. It affects humans, but several animal species show clinical manifestations typical of the pathology as well. These species are widely used as animal models for the study of the disease, in particular a murine model called Twitcher (TWI). KD is caused by mutations in the gene encoding for the lysosomal enzyme β-galactosylceramidase (or galactocerebrosidase; GALC). GALC is involved in the degradation of some lipids during myelin turnover, and a GALC deficiency leads to impaired degradation of its substrates, galactosylceramide (GalCer) and psychosine (PSY). However, while GalCer can be degraded by another lysosomal enzyme (GM1 ganglioside β-galactosidase), the only enzyme capable of hydrolyzing PSY is GALC. Consequentially, PSY accumulates, primarily within oligodendrocytes, consequentially blocking downstream biochemical pathways and inducing myelin-forming cells to death. This condition, in the end, leads to a severe demyelination and consequent neurodegeneration of both KD CNS and PNS. Several experimental approaches are tested in the TWI mice, comprising both single and combination therapies. Concerning single-modality approaches, cell therapy (neural, hematopoietic and mesenchymal stem cell-based therapies), enzyme replacement therapy (ERT), gene therapy (GT) strategy with AAV or LV (systemic and/or intracerebral administration), anti-inflammatory drug-based therapy (anti-INF) and substrate reduction therapy (SRT) have been evaluated. Nevertheless, the more efficient strategy to ameliorate KD phenotype seems to be a combination of different experimental strategies, which aim to target different pathogenic mechanisms of the disease. Still, all these therapies present limitations and need further studies.
Therefore, one of the aims of this thesis is to study a new type of ERT for KD, based on brain-targeted polymeric reverse micelles (RMs) encapsulated with GALC enzyme and functionalized with a brain targeting peptide (Angiopep-2), with the purpose of restoring GALC activity in the brain of the TWI mice. In particular, post natal day (PND) 20 TWI mice were treated intravenously through retro-orbital injection with micelles and were sacrificed 4 or 24 hours after treatment. Then, GALC enzymatic assay was performed on the organs extracted from the sacrificed mice in order to evaluate the activity recovery, especially in the brain and sciatic nerve. Yet, recent evidence proved that the restoration of GALC activity in KD models is not sufficient to completely recover the WT phenotype; for this reason, therapies with different targets could be effective in improving the conditions of KD patients. Thus, on the other hand, my thesis aims also to exploit the effect of the modulation of the autophagic pathway in the TWI mice, achieved by the administration of an autophagy modulator, Lithium (Li). Autophagy is a cellular degradation and recycling process responsible for the delivery of cytosolic material to the lysosome in which is degraded. This process begins with the formation of a double membrane organelle called phagophore, which, following a trigger signal, is bent by microtubule-associated protein 1 light-chain 3 (LC3) that helps the phagophore to close up forming the autophagosome. Moreover, LC3 recruits and binds the ubiquitin-binding protein p62, which regulates the process by selecting the material to degrade, thereby making the autophagic pathway a selective process. Then, autophagosomes fuse with lysosomes, shaping the autolysosomes in which the material is degraded by hydrolytic enzymes. Previous studies proved that the autophagic pathway in KD is dysregulated but not impaired (because p62 regularly mediates its cargo delivery to autolysosomes), as instead occurs for other LSDs. Additionally, some molecules, such as PSY and alpha-synuclein, have been found accumulated in the nervous system of both KD patients and TWI mice. Thus, upregulation of autophagy could be a promising therapeutic strategy for KD. Moreover, since autophagy inducers (Li and RAP) have been shown to correctly induce autophagy in in-vitro KD models, our aim is to evaluate the stimulation of autophagy in-vivo, in the TWI mice. Thus, PND 20 TWI mice were treated with Li administered by drinking water and subsequently motor behavioral experiments (wire hang, grip strength and rotarod test) were performed, in order to evaluate the effect of Li on TWI mice motor skills. Moreover, autophagy markers such as LC3 and p62 were quantified in the brain and sciatic nerve lysates through western blot experiments to assess the ability of Li to stimulate autophagy in the treated mice. In addition to autophagy markers, proteins such as GFAP (marker for the activation of astrocytes) and MBP (marker for myelination) have also been quantified in the same lysates.
Overall, the final idea of the thesis is to develop a combined therapy for KD, that, on one hand aims to restore the enzymatic activity of GALC in the affected organs, and on the other hand aims to stimulate autophagy to facilitate the removal of lipid or protein aggregates in the nervous system, thereby helping to reach a complete KD phenotype rescue.