Lentiviral Vectors Expressing Chimeric NEDD4 Ubiquitin Ligases: An Innovative Approach for Interfering with Alpha-Synuclein Accumulation.

Stefania Vogiatzis,Arianna Calistri,Claudia Del Vecchio,Kathleen Maguire-Zeiss,Cristina Parolin,Gloria Magro,Giorgio Palù,Deran Erdengiz,Marta Trevisan,Michele Celestino

doi:10.3390/cells10113256

Stefania Vogiatzis, Arianna Calistri + Show 8 more

Open Access

https://doi.org/10.3390/cells10113256

Copy DOI

Abstract

One of the main pathological features of Parkinson’s disease (PD) is a diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 Ub ligase promotes aS degradation by the endosomal–lysosomal route. Interestingly, NEDD4, as well as being a small molecule able to trigger its functions, is protective against human aS toxicity in evolutionary distant models. While pharmacological activation of E3 enzymes is not easy to achieve, their flexibility and the lack of “consensus” motifs for Ub-conjugation allow the development of engineered Ub-ligases, able to target proteins of interest. We developed lentiviral vectors, encoding well-characterized anti-human aS scFvs fused in frame to the NEDD4 catalytic domain (ubiquibodies), in order to target ubiquitinate aS. We demonstrate that, while all generated ubiquibodies bind to and ubiquitinate aS, the one directed against the non-amyloid component (NAC) of aS (Nac32HECT) affects aS’s intracellular levels. Furthermore, Nac32HECT expression partially rescues aS’s overexpression or mutation toxicity in neural stem cells. Overall, our data suggest that ubiquibodies, and Nac32HECT in particular, represent a valid platform for interfering with the effects of aS’s accumulation and aggregation in neurons.

Highlights

Parkinson Disease (PD) is the second most frequent neurodegenerative disease after Alzheimer’s, affecting more than 1% of people over 55 years old and more than 3% of people over 75 years old [1]
Tofaris and colleagues convincingly showed that NEDD4 is overexpressed in the brain regions presenting Lewy’s bodies (LBs), and that a single nucleotide polymorphism in its coding sequence is associated with a greater risk of Parkinson’s disease (PD) onset [17]
Selected scFvs were fused to a GSGSG spacer peptide, which provides suitable flexibility to the enzyme [34], followed by the NEDD4 homologous to E6-AP carboxyl terminus (HECT) catalytic domain and by an-HA tag

Summary

Introduction

Parkinson Disease (PD) is the second most frequent neurodegenerative disease after Alzheimer’s, affecting more than 1% of people over 55 years old and more than 3% of people over 75 years old [1]. Strong evidence suggest that aS exploits its unfolded structure to acquire alternative ordered conformations, enabling the protein to interfere with different cellular processes within the CNS [6]. This plasticity can lead to aS self-binding through its non-amyloid component (NAC) domain, resulting in a stable β-sheet structure. This happens, for instance, when aS intracellular levels raise above a certain threshold, or in the presence of specific NAC mutations

Objectives

Methods

Results

Conclusion