Mechanotransduction Impairment in Primary Fibroblast Model of Krabbe Disease

Round 1

Reviewer 1 Report

Dear Authors,

congratulations on this interesting and elegant research on mechanotransduction and migration processes in primary fibroblasts collected from the Twitcher mouse, a model of Krabbe disease (KD). This analysis evaluated some of the mechanisms underlying the pathogenesis of KD that cannot be explained by the accumulation of cytotoxic sphingolipid psychosine accumulation alone. Besides the study of cellular models, could these results help to develop new therapeutic approaches for the treatment of KD disease?

Author Response

R1: congratulations on this interesting and elegant research on mechanotransduction and migration processes in primary fibroblasts collected from the Twitcher mouse, a model of Krabbe disease (KD). This analysis evaluated some of the mechanisms underlying the pathogenesis of KD that cannot be explained by the accumulation of cytotoxic sphingolipid psychosine accumulation alone. Besides the study of cellular models, could these results help to develop new therapeutic approaches for the treatment of KD disease?

We thank the reviewer for the comment and the interesting question. We would point out that, even if monogenic, KD is an extremely complex disease, and its molecular pathogenesis has not yet been completely understood. Recent research findings, in fact, show that psychosine accumulation is not the only players in the molecular pathogenesis of KD [Vantaggiato et al., 2022]. Molecular mechanisms other than PSY-related cell death might play an important role. Furthermore, it is also known that the reinstatement of the GALC activity in the nervous system is not enough to completely cure the disease [Heller et al., 2021]. New aspects of the KD pathogenesis are emerging and it seems that these aspects cannot be effectively rescued by the only GALC supplying, suggesting the need for specific complementary therapies.

Our work suggests that pharmacological approaches focused on the autophagy process might be a promising option for KD. In particular, the autophagy inducer Rapamycin is already present in the clinical practice for other diseases. Further exploitation of rapamycin such as therapeutic approach for the treatment of KD disease is promising and in our plans for preclinical testing.

Refs:

• Heller, G. J., Marshall, M. S., Issa, Y., Marshall, J. N., Nguyen, D., Rue, E, Bongarzone, E. R. (2021). Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease. Molecular Therapy, 29(5), 1883-1902.
• Vantaggiato, L., Shaba, E., Carleo, A., Bezzini, D., Pannuzzo, G., Luddi, A., et al. (2022). Neurodegenerative Disorder Risk in Krabbe Disease Carriers. International Journal of Molecular Sciences, 23(21), 13537.

Reviewer 2 Report

The manuscript by Mezzena et al describes an experimetal studies to characterise the mechanosensing mechamisms in fibroblasts in Krabbe disease. 

The manuscript is well written, experiments are clearly described in details. 

The figures are clear and easy to follow.

Could the authors mention how the work could be extrapolated to the clinical studies?

Author Response

R2 Could the authors mention how the work could be extrapolated to the clinical studies?

We thank the reviewer for this forward-looking question.

Our work suggest that pharmacological approaches focused on the autophagy process might be a promising option for KD. Rapamycin is especially promising, given it is an already FDA-approved drug and in the clinical practice for other diseases.

Autophagy dysfunctions have been identified in KD, by us and by others groups [Del Grosso et al, 2016 & 2019; Ribbens et al 2014; Lin et al., 2020], and autophagy inducers are being tested to help lysosomal storage disorders. We here and previously demonstrated that two differently acting autophagy inducers (lithium and rapamycin) can improve some KD hallmarks in-vitro. We already tested lithium carbonate in-vivo in the KD mouse model for KD, even if the results demonstrated that lithium has not a significant rescue effect on the TWI phenotype, although it can slightly and transiently improve muscle strength [Del Grosso et al, 2020]. Further studies on rapamycin at preclinical level are needed, as in our previous paper on lithium. Our results suggest that a boost to the autophagy process can help to counteract some KD pathological features, laying the foundation for rapamycin pre-clinical testing.

In our opinion, a composite therapy based on gene therapy or GALC-enzyme delivery together with autophagy inducers (to boost the cellular clearance process) might be an optimal strategy to cure KD.

Refs:

• Del Grosso A et al. Dysregulated autophagy as a new aspect of the molecular pathogenesis of Krabbe disease. Neurobiol Dis. 2019;129:195-207.
• Del Grosso A, Angella L, Tonazzini I, et al. Lithium improves cell viability in psychosine-treated MO3.13 human oligodendrocyte cell line via autophagy activation. J Neurosci Res. 2016;94:1246-1260.
• Lin DS, Ho CS, Huang YW, et al. Impairment of proteasome and autophagy underlying the pathogenesis of leukodystrophy. Cells. 2020;9(5):1124.
• Ribbens JJ, Moser AB, Hubbard WC, Bongarzone ER, Maegawa GHB. Characterization and application of a disease-cell model for a neurodegenerative lysosomal disease. Mol Genet Metab. 2014;111:172-183