Inhibition of UCH-L1 in oligodendroglial cells results in microtubule stabilization and prevents α-synuclein aggregate formation by activating the autophagic pathway: implications for multiple system atrophy.

Katharina Pukaß,Christiane Richter-Landsberg

doi:10.3389/fncel.2015.00163

Abstract

α-Synuclein (α-syn) positive glial cytoplasmic inclusions (GCI) originating in oligodendrocytes (ODC) are a characteristic hallmark in multiple system atrophy (MSA). Their occurrence may be linked to a failure of the ubiquitin proteasome system (UPS) or the autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin, and deubiquitinated by deubiquitinating enzymes (DUBs) before proteolytic degradation is performed. The DUB ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a component of the UPS, it is abundantly expressed in neuronal brain cells and has been connected to Parkinson’s disease (PD). It interacts with α-syn and tubulin. The present study was undertaken to investigate whether UCH-L1 is a constituent of ODC, the myelin forming cells of the CNS, and is associated with GCIs in MSA. Furthermore, LDN-57444 (LDN), a specific UCH-L1 inhibitor, was used to analyze its effects on cell morphology, microtubule (MT) organization and the proteolytic degradation system. Towards this an oligodendroglial cell line (OLN cells), stably transfected with α-syn or with α-syn and GFP-LC3, to monitor the autophagic flux, was used. The data show that UCH-L1 is expressed in ODC derived from the brains of newborn rats and colocalizes with α-syn in GCIs of MSA brain sections. LDN treatment had a direct impact on the MT network by affecting tubulin posttranslational modifications, i.e., acetylation and tyrosination. An increase in α-tubulin detyrosination was observed and detyrosinated MT were abundantly recruited to the cellular extensions. Furthermore, small α-syn aggregates, which are constitutively expressed in OLN cells overexpressing α-syn, were abolished, and LDN caused the upregulation of the autophagic pathway. Our data add to the knowledge that the UPS and the autophagy-lysosomal pathway are tightly balanced, and that UCH-L1 and its regulation may play a role in neurodegenerative diseases with oligodendroglia pathology.

Highlights

A common pathogenic event in various neurodegenerative diseases is the failure to clear misfolded or aggregated proteins, which occur as protein inclusions in nerve cells or glia (Dohm et al, 2008; Jellinger, 2012)
We have recently shown that treatment of oligodendroglial cells with the broad range deubiquitinating enzymes (DUBs) inhibitor PR-619 caused the accumulation of ubiquitinated proteins and the formation of ubiquitin-positive protein aggregates, which colocalize with heat shock proteins, such as HSP70 and αB-Crystallin (Seiberlich et al, 2012)
The present study was undertaken to investigate whether ubiquitin carboxylterminal hydrolase L1 (UCH-L1) is a constituent of ODC and associates with glial cytoplasmic inclusions (GCI) in multiple system atrophy (MSA), and whether its pharmacological inhibition by LDN-57444 (LDN) affects cell morphology, MT formation and the proteolytic degradation system

Summary

Introduction

A common pathogenic event in various neurodegenerative diseases is the failure to clear misfolded or aggregated proteins, which occur as protein inclusions in nerve cells or glia (Dohm et al, 2008; Jellinger, 2012). K48-linked polyubiquitin chains generally target proteins for proteasomal degradation, while K63-linked protein chains are involved in various cellular processes These include autophagy, cell sorting, and DNA repair processes (Todi and Paulson, 2011). DUBs comprise a large family of enzymes opposing the function of E3 ligases and are important regulators of the ubiquitin system (Amerik and Hochstrasser, 2004; Komander et al, 2009; ReyesTurcu et al, 2009) They prevent ubiquitin degradation in conjunction with the substrate thereby maintaining ubiquitin homeostasis. PR-619 did not affect proteasomal activity directly, but chronic inhibition of DUBs led to an overload of ubiquitinated proteins, which exhausted the UPS and autophagy was activated possibly as a compensatory mechanism (Seiberlich et al, 2013). The present study was undertaken to investigate whether UCH-L1 is a constituent of ODC and associates with GCIs in MSA, and whether its pharmacological inhibition by LDN-57444 (LDN) affects cell morphology, MT formation and the proteolytic degradation system

Materials and Methods

Results

Discussion