The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury.

H Liu,M E Rose,S H Graham,B W Day,W Li,M Balasubramani,R W Hickey,K V Patel,J Chen,G T Uechi

doi:10.1038/cddis.2015.323

Abstract

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), are reactive prostaglandin metabolites exerting a variety of biological effects. CyPGs are produced in ischemic brain and disrupt the ubiquitin-proteasome system (UPS). Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain-specific deubiquitinating enzyme that has been linked to neurodegenerative diseases. Using tandem mass spectrometry (MS) analyses, we found that the C152 site of UCH-L1 is adducted by CyPGs. Mutation of C152 to alanine (C152A) inhibited CyPG modification and conserved recombinant UCH-L1 protein hydrolase activity after 15dPGJ2 treatment. A knock-in (KI) mouse expressing the UCH-L1 C152A mutation was constructed with the bacterial artificial chromosome (BAC) technique. Brain expression and distribution of UCH-L1 in the KI mouse was similar to that of wild type (WT) as determined by western blotting. Primary cortical neurons derived from KI mice were resistant to 15dPGJ2 cytotoxicity compared with neurons from WT mice as detected by the WST-1 cell viability assay and caspase-3 and poly ADP ribose polymerase (PARP) cleavage. This protective effect was accompanied with significantly less ubiquitinated protein accumulation and aggregation as well as less UCH-L1 aggregation in C152A KI primary neurons after 15dPGJ2 treatment. Additionally, 15dPGJ2-induced axonal injury was also significantly attenuated in KI neurons as compared with WT. Taken together, these studies indicate that UCH-L1 function is important in hypoxic neuronal death, and the C152 site of UCH-L1 has a significant role in neuronal survival after hypoxic/ischemic injury.

Highlights

UCH-L1 closely interacts with proteins of the neuronal cytoskeleton and may have an important role in axonal transport and maintaining axonal integrity.[2,3] UCH-L1 regulates synaptic function and long-term potentiation (LTP) under normal and pathological conditions and may be involved in memory function.[4]
To explore which cysteine residues in UCH-L1 are the primary targets for 15dPGJ2 adduction, Flag-tagged UCHL1 was overexpressed in the rat primary neurons by lentivirus (LV)-Flag UCHL-WT infection and cells were incubated with 15dPGJ2 (20 μM) for 24 h before harvest
The major findings of this study are (1) Cyclopentenone prostaglandins (CyPGs) such as 15dPGJ2 adduct the C152 of UCH-L1 and mutation of the C152 of UCH-L1 attenuates the loss of hydrolase activity after 15dPGJ2 treatment; (2) the UCH-L1 C152A mutation decreases 15dPGJ2-induced accumulation and aggregation of UCH-L1 and ubiquitinated proteins in primary neurons; and (3) primary neurons derived from UCH-L1-C152A mutant mice are resistant to cell death and neurite injury induced by treatment with 15dPGJ2

Summary

Introduction

UCH-L1 closely interacts with proteins of the neuronal cytoskeleton and may have an important role in axonal transport and maintaining axonal integrity.[2,3] UCH-L1 regulates synaptic function and long-term potentiation (LTP) under normal and pathological conditions and may be involved in memory function.[4]. UCH-L1 is a target of CyPG modification.[13,14,15] In the current study, mass spectrometry (MS)/MS was used to determine that cysteine[152] is the binding site of the CyPG 15dPGJ2 to UCH-L1. Primary neurons derived from KI and wild-type (WT) mice were used to determine the effect of CyPG binding to UCH-L1 on cell death and disruption of the UPS. These studies address a potential role for modification of UCH-L1 by CyPGs and other reactive lipid species in stroke and neurodegenerative diseases

Methods

Results

Conclusion