Lysosomal regeneration as a therapeutic strategy for Hereditary Spastic Paraplegia

SPG4, SPG11 and SPG15 are forms of Hereditary Spastic Paraplegia (HSP), an uncurable neurodegenerative disorder marked by progressive corticospinal tract degeneration. Although genetically different, similar lysosomal defects have been reported in different preclinical models. SPG4 is due to mutations of Spastin, a microtubule-severing ATPase that regulates microtubules dynamics, affecting ER shaping and endosomal fission and trafficking. Spastin mutations lead to impaired lipid droplet (LD) biogenesis and accumulation of enlarged lysosomes. Spastizin and Spatacsin, mutated in SPG15 and SPG11 respectively, are proteins involved in the same endolysosomal processes. Spastizin and Spatacsin interact, they form a membrane complex necessary for the initiation of the Autophagic Lysosome Reformation (ALR). Mutations in these proteins can lead to the accumulation of autolysosomes and a block in ALR, resulting in fewer functional lysosomes. In this work we exploited Drosophila melanogaster as a model organism to further analyse the role of spastin, spastizin and spatacsin in vivo. Our studies were aimed at describing the function of these proteins in the endolysosomal system and specifically in lysosome regeneration. By using loss of function models and using endolysosomal markers we confirmed the presence of similar autophagic and lysosomal defects across all three models. Indeed, we detected severe lysosomal dysfunctions and block of the ALR process in larval muscles. By performing a pharmacological screening on SPG15 models we identified lysosomes as a valid target for SPG15 treatment. Moreover, SMER28 was the most efficient in inducing ALR in vivo and rescuing the locomotor deficit in SPG15 Drosophila model. Unfortunately, no clinical trials are registered for SMER28 and no information is available on its tolerability and pharmacokinetic. These results prompted the choice of other molecules—tideglusib, naringenin— able to promote lysosome regeneration, via ex novo biogenesis of through ALR to test on the SPG11 and SPG15 models. Tideglusib and naringenin can activate the nuclear translocation of TFEB and activates lysosomal and autophagy regulating genes, naringenin can also activate BKCa-channels and inhibit TPC2 lysosomal calcium channels, important regulators of lysosomal function. In addition, we tested miglustat, an inhibitor of glucosylceramide synthase in clinical trial for SPG11. However, miglustat yielded negative results (NCT04768166), helping us define the pharmacological predictability of the model. Miglustat had no positive effects, and long-term treatment worsened locomotor ability. Tideglusib showed mild effectiveness on lysosomal parameters, but failed to rescue the ALR process, resulting in a loss of its benefits over time. Naringenin, in contrast, was the most effective compared to SMER28 in the SPG11 and SPG15 models, improving all intracellular parameters and rescuing the defective ALR process, leading to a significant improvement in locomotor function. It was also the most effective molecule tested in the SPG4 model, enhancing locomotor performance, restoring lipid homeostasis, and—most importantly—restoring lysosomal tubulation under starvation conditions. Our preliminary data indicate that naringenin's mechanism involves the activation of lysosomal gene transcription, suggesting the activation of TFEB nuclear translocation. Additionally, our results imply that BKCa channels play a role in this mechanism, as selective inhibition of BKCa channels worsened the vitality defect in the SPG4 model. Furthermore, naringenin can integrate into the phospholipid bilayer and reduce membrane fluidity. Notably, reducing lysosomal membrane fluidity in control experiments produced results similar to those seen with naringenin. Our work underscores that lysosomal regeneration is a promising therapeutic approach not only for SPG4, SPG11, and SPG15 but also for other HSP subtypes with similar ALR defects.