Spastin elevating approaches and their validation in a Drosophila melanogaster model for Hereditary Spastic Paraplegia type 4 (SPG4-HSP)

Hereditary spastic paraplegias (HSPs) are a group of motor neuron disorders characterized by progressive spasticity in the lower limbs due to axonal degeneration. The most common form of HSP is due to SPG4 gene haploinsufficiency. SPG4 encodes the microtubule severing enzyme, spastin. Although, there is no cure for SPG4- HSP, strategies to induce a spastin recovery are emerging as promising therapeutic approaches. Our study aims to identify the molecular pathway regulating the turnover of spastin protein. Here, we show that the DDB1-Cullin-4-Ring ubiquitin ligase complex (CRL4) regulates spastin stability. We have generated a Drosophila melanogaster model of SPG4 (Dm-SPG4), in which we achieved RNAi-mediated downregulation of spastin in different fly tissues. Spastin downregulation in flies causes defects in locomotor activity, which is associated with alterations of neuromuscular junction morphology and function. Genetic inhibition of the CRL4 complex significantly affects several of these SPG4-like phenotypes in flies We show that the RNAi- mediated downregulation of Cullin 4 reduces the locomotor defects observed in the Dm-SPG4 model and ameliorates the alteration of synapse morphology. The downregulation of Cullin 4 rescues spastin protein levels in the same fly model. As a proof of concept, we have also pharmacologically inhibited the CRL4 complex with NSC1892. NSC1892 treatment elevates spastin levels and rescues pathological phenotypes in flies. Similar results were observed in patient-derived cells. Taken together, these findings show CRL4 involvement in spastin turnover and suggest the possibility of novel therapeutic interventions through the modulation of CRL4 activity.