Spastin elevating approaches and their validation in Drosophila melanogaster models of Hereditary Spastic Paraplegia type 4 (SPG4-HSP)

Hereditary spastic paraplegias (HSPs) are a group of motor neuron disorders, characterized by a progressive spasticity and weakness at the lower limbs. Although mutations in more than 85 genes were found to be associated with HSP, the most frequent affect SPG4 gene and display autosomal dominant inheritance. SPG4 encodes spastin, a microtubule severing ATPase which controls microtubule network dynamics affecting cell division, intracellular membrane traffick- ing and, axonal transport. There is no cure to alleviate motor neuron degeneration in HSP. Since SPG4 haploinsufficiency is thought to be responsible of disease, restoring spastin para- physiological levels in SPG4-HSP patients might represent a promising therapeutic approach. Thus, the aim of our work is to identify the molecular machinery involved in the regulation of spastin protein stability to find a way to slow down its turnover. Recently in our labora- tory, it has been shown that the kinase HIPK2 phosphorylates spastin at serine S268 and this prevents its polyubiquitylation and proteasomal-mediated degradation. Further, we observed in human cell line (preliminary results) that a Cullin 4-Ring-ubiquitin-Ligase complex may be involved in spastin degradation. Here, we have established a Drosophila melanogaster model of SPG4 haploinsufficiency, by RNAi-mediated downregulation of spastin in fly. By exploiting Drosophila genetics, we have studied the effects of spastin downregulation in different fly tissues and in different types of neurons. Accordingly, we show that the in vivo inhibition of Cullin 4-Ring-ubiquitin-Ligase complex, which is highly conserved in Drosophila, significantly affects spastin-mediated phenotypes. We have demonstrated that Cullin 4 silencing considerably res- cues alteration of neuromuscular junction morphology and locomotor defects of spastin-deficient flies. Finally, we are now assessing the possibility of using this Drosophila model of HSP to perform drug screening to identify drugs able to prevent spastin degradation.