ABSTRACT Krabbe disease (KD) is a rare disorder caused by a deficiency of the lysosomal enzyme galactosylceramidase (GALC), resulting in the accumulation of the cytotoxic metabolite psychosine (PSY) in the nervous system. This accumulation triggers demyelination and neurodegeneration. Despite ongoing research, the underlying pathogenic mechanisms remain incompletely understood, and there is currently no cure available. Previous studies from our lab revealed the presence of autophagy dysfunctions in KD pathogenesis, as evidenced by the presence of p62-tagged protein aggregates in the brains of KD mice and increased p62 levels in the KD sciatic nerve. We also demonstrated that the autophagy inducer Rapamycin (RAPA) can partially restore the wild-type (WT) phenotype in KD primary cells by reducing the number of p62 aggregates. In this study, we tested RAPA in the Twitcher (TWI) mouse, a spontaneous KD mouse model. We administered the drug ad libitum via drinking water (15 mg/L) starting from post-natal day (PND) 21-23. We longitudinally monitored the motor performance of the mice through grip strength and rotarod tests, along with various biochemical parameters related to KD pathogenesis (i.e. autophagy markers expression, myelination, astrogliosis, and PSY accumulation). Our findings demonstrate that RAPA significantly enhances motor functions at specific treatment time points and reduces astrogliosis in TWI brain, spinal cord, and sciatic nerves. Using western blot and immunohistochemistry, we observed a decrease in p62 aggregates in TWI nervous tissues, which corroborates our earlier in-vitro results. Furthermore, RAPA treatment partially reduces PSY levels in the spinal cord. In conclusion, our results support the consideration of RAPA as a supportive therapy for KD. Importantly, as RAPA is already available in pharmaceutical formulations for clinical use, its potential for KD treatment can be promptly evaluated in clinical trials.

Twitcher heterozygous (HET) mice (TWI+/− C57BL6 mice; the Jackson Laboratory, Bar Harbor, ME) were used as breeding pairs to produce homozygous TWI mice (TWI−/−, elsewhere in the Dataset abbreviated as TWI for simplicity). This breeding was carried out at the Center for Experimental Biomedicine of CNR in Pisa. The animals were housed under standard conditions and all procedures were performed in accordance with the protocols and ethical guidelines approved by the Ministry of Health (permit no. 535/2018-PR; official starting date: July 9, 2018). Starting from post-natal days (PND) 21-23, mice received chronic Rapamycin (RAPA) dissolved in their drinking water at a concentration of 15 mg/L. The treatment continued until they reached a body weight loss ≥ 25%, which was the predetermined ethical endpoint. At that stage, they were euthanized. Wild type (WT) mice were euthanized alongside the last sacrificed TWI mouse in the specific experiment. Behavioral tests were conducted at three time points (TP): TP 1 = PND 24-25; TP 2 = PND 29-30; TP 3 = PND 35-37. All procedures were carried out with the utmost care to minimize any potential suffering of the mice. Tissues (total brain, cerebrum, cerebellum, spinal cord, sciatic nerves, liver and kidneys) were extracted from each mouse and used to investigate various biochemical parameters related to KD pathogenesis (i.e. autophagy markers expression, myelination, astrogliosis and PSY accumulation) throught western blot, immunohistochemistry and Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC/HRMS).

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