Inhibition of Cullin 4 Ring Ubiquitin Ligase complex recovers spastin protein levels and reduces defects in preclinical Drosophila models of Spastic paraplegia type 4

Hereditary spastic paraplegias (HSPs) are a group of motor neuron disorders characterized by progressive spasticity and weakness in the lower limbs due to axonal degeneration. Spastic paraplegia type 4 (SPG4) is the most common form, it has an autosomal dominant inheritance pattern. Haploinsufficiency is the most plausible pathogenic mechanism behind the disease onset. SPG4 encodes spastin, an enzyme involved in microtubule remodeling and dynamics, which directly affects axonal trafficking. There is no cure for SPG4, although spastin-elevating strategies are emerging as promising therapeutic approaches. Our study aims to identify the molecular pathway regulating the turnover of spastin protein. It was already established that spastin levels are finely regulated by polyubiquitylation and proteasomal-mediated degradation, in a neddylation-dependent manner. Here, we show that the DDB1-Cullin-4-Ring ubiquitin ligase complex (CRL4) regulates spastin stability. Inhibition of CRL4 increases spastin levels by preventing its poly-ubiquitination and subsequent degradation. we have generated a Drosophila melanogaster model of SPG4 (Dm-SPG4), in which we achieved RNAi-mediated downregulation of spastin in different fly tissues. Genetic inhibition of the CRL4 complex, highly conserved in Drosophila, significantly affects several SPG4-like phenotypes in flies. We observed that RNAi-mediated downregulation of spastin in Drosophila causes defects in the locomotory activity, which is caused by alterations of neuromuscular junction morphology and function. Here we show the RNAi-mediated downregulation of Cul4 rescues the alteration of synapse morphology and function, and the locomotor defects observed in the Dm-SPG4 model. Further, silencing Cullin 4 rescues spastin protein levels in the same fly model. As a proof of concept, we have pharmacologically inhibited the CRL4 complex with NSC1892. NSC1892 elevates spastin levels and rescues pathological phenotypes in the fly model and patient-derived cells. To find a more exclusive way to inhibit CRL4-mediated degradation of spastin, we are screening candidate DDB1-CUL4 associated factors (DCAFs) to find those specifically involved in spastin turnover. Chromatin assembly factor 1/ p55 subunit (CAF1-55) is one of the candidates DCAFs that has caught our attention. The RNAi-mediated downregulation of CAF1-55 in Dm-SPG4 model partially rescued spastin protein levels and retina degeneration due to spastin downregulation. Taken together, these findings show CRL4 involvement in spastin turnover and suggest the possibility of novel therapeutic interventions through the modulation of CRL4 activity.