MENINGES AS AN OVERLOOKED TARGET FOR GLOBOID CELL LEUKODISTROPHY

Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a severe neurological disorder caused by the deficiency of galactocerebrosidase (GALC) enzyme, leading to the accumulation of toxic metabolites and ultimately resulting in progressive demyelination and neurodegeneration. The only treatment option currently available is the hematopoietic stem/progenitor cell transplant (HSPC-T), although this is not effective if administered after diseases onset. Thus, there is no cure for GLD emphasizing the need for novel approaches. While most of the GLD neurodegenerative clues in the brain have been clarified, the underlying mechanisms of neuroinflammation are not yet fully understood. GLD brain shows an important inflammatory infiltration including the accumulation of unique and often multinucleated macrophages (“globoid cells”). However, the role in GLD pathogenesis and progression of all other non-parenchymal components of the brain, potentially contributing to the inflammatory process and diseases progression, has not been elucidated yet. Recently, new and important interactions between brain and meninges have been described. Meninges appeared as a highly heterogeneous tissue with trophic, immune and neurogenic properties. Recently it has been identified a neurogenic stem/progenitor cell (NSPC) population present in meninges. In vivo, meningeal NPCs can migrate to the brain and, without proliferation, differentiate into functional integrated neurons of the cortex. Following brain pathological conditions, meninges are activated increasing their immune function and neural stem cell properties. However, the potential involvement of meninges during GLD pathogenesis and progression is completely unknown. In this project, I have studied the involvement of meninges in the pathogenesis and progression of GLD in the Twitcher mouse model that resembles the early infantile form in humans. I have used mice at four different time points that represent the disease stages, including early pre-onset, pre-onset, onset and disease progression. I have found an increase of immune cells, including neutrophils, macrophages and T cells in TWI meninges starting from very early pre-onset GLD stage and persisting throughout the disease. Moreover, neuronal committed NSPCs appeared to be present in meninges at pre-onset and onset stages of GLD. These findings reveal, for the first time, the early GLD-induced response of meninges that start perinatally, well before the pathological involvement of the brain and the appearance of the clinical symptoms are detectable in CNS. Then, I have exploited meninges as a new complementary and less invasive site for in vivo gene therapy (GALC lentiviral transduction). Post natal day 1 (P.1) pup mice have been injected in meninges with lentiviral vector (LV) overexpressing GALC gene and then analyzed at P.30 when the GLD is severe. Of note, GALC transduction in meninges at P.1 induced at P-30 a significant improvement on motor function and a large reduction of inflammatory infiltrate in the brain. Moreover, GALC overexpressing NSPCs at P.1 meninges have been found in P.30 mice with neuronal morphology into the second layer of the cortex expressing the neuronal marker NeuN, suggesting that they migrated from meninges to the cortex and differentiated into cortical neurons. Finally, I have assessed the proof of concept of efficacy and safety of LV-GALC overexpression in somatic adult human meningeal NSPCs as potential therapeutic target for meningeal-directed in vivo gene therapy. These results showed that GALC overexpression in human meningeal neurons derived from NSPCs showed no toxicity and alteration in stem cells and neuronal properties. Overall, these data highlight the importance of early meningeal involvement in GLD pathogenesis, opening the opportunity to find in meninges newly GLD-modifier therapeutic targets. Moreover, meninges could be exploited as novel, safe and effective site for gene therapy, which is less invasive than current intraparenchymal gene transfer approaches. This work paves the way for new therapeutic strategies for GLD, changing the focus from brain toward its stromal component.