Spastin recovery in hereditary spastic paraplegia by preventing neddylation-dependent degradation.

Francesca Sardina,Marco Crescenzi,Marialuisa Casella,Andrew J Grierson,Carlo Casali,Silvia Soddu,Cinzia Rinaldo,Manuela Ferrara,Angelo Peschiaroli,Alessandra Pisciottani,Davide Valente

doi:10.26508/lsa.202000799

Abstract

Hereditary Spastic Paraplegia (HSP) is a neurodegenerative disease most commonly caused by autosomal dominant mutations in the SPG4 gene encoding the microtubule-severing protein spastin. We hypothesise that SPG4-HSP is attributable to reduced spastin function because of haploinsufficiency; thus, therapeutic approaches which elevate levels of the wild-type spastin allele may be an effective therapy. However, until now, how spastin levels are regulated is largely unknown. Here, we show that the kinase HIPK2 regulates spastin protein levels in proliferating cells, in differentiated neurons and in vivo. Our work reveals that HIPK2-mediated phosphorylation of spastin at S268 inhibits spastin K48-poly-ubiquitination at K554 and prevents its neddylation-dependent proteasomal degradation. In a spastin RNAi neuronal cell model, overexpression of HIPK2, or inhibition of neddylation, restores spastin levels and rescues neurite defects. Notably, we demonstrate that spastin levels can be restored pharmacologically by inhibiting its neddylation-mediated degradation in neurons derived from a spastin mouse model of HSP and in patient-derived cells, thus revealing novel therapeutic targets for the treatment of SPG4-HSP.

Highlights

Spastin is an AAA ATPase microtubule (MT)-severing enzyme that regulates cytoskeleton rearrangement associated with membrane remodeling
Spastin reduction was observed upon HIPK2 depletion in murine neuron-like NSC34 differentiated cells (Figs 1D and S1B and C), in human SH-SY-5Y neuron-like differentiated cells and in primary cortical neurons explanted from CRE-inducible Hipk2 KO mice upon infection with an adenovirus expressing the recombinase CRE (Figs 1E and S1D), showing that spastin levels are regulated by HIPK2 in postmitotic differentiated primary neurons
We have identified and characterized a pathway responsible for the stability of the spastin protein, which is mutated in the most common form of autosomal dominant Hereditary Spastic Paraplegia (HSP), and exploited this knowledge to propose a novel approach for corrective elevation of spastin levels in disease by preventing its neddylation-dependent degradation

Summary

Introduction

Spastin is an AAA ATPase microtubule (MT)-severing enzyme that regulates cytoskeleton rearrangement associated with membrane remodeling. It is a critical regulator in cytokinesis and nuclear envelope resealing during cell division and it is involved in intracellular traffic [1, 2, 3]. Spastin has key roles in axonal transport and regeneration [4, 5, 6] This enzyme functions as hexamers that drive the remodeling and severing of MT by tugging the C-terminal tail of tubulin through the central pore of the hexamer [7, 8, 9]. Splice variants are the least abundant isoforms, for which no specific functions have been reported far

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